presentation of leukemia

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Diseases & Conditions

Leukemia is cancer of the body's blood-forming tissues, including the bone marrow and the lymphatic system.

Many types of leukemia exist. Some forms of leukemia are more common in children. Other forms of leukemia occur mostly in adults.

Leukemia usually involves the white blood cells. Your white blood cells are potent infection fighters — they normally grow and divide in an orderly way, as your body needs them. But in people with leukemia, the bone marrow produces an excessive amount of abnormal white blood cells, which don't function properly.

Treatment for leukemia can be complex — depending on the type of leukemia and other factors. But there are strategies and resources that can help make your treatment successful.

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Children’s Book: My Life Beyond Leukemia

Leukemia symptoms vary, depending on the type of leukemia. Common leukemia signs and symptoms include:

Fever or chills
Persistent fatigue, weakness
Frequent or severe infections
Losing weight without trying
Swollen lymph nodes, enlarged liver or spleen
Easy bleeding or bruising
Recurrent nosebleeds
Tiny red spots in your skin (petechiae)
Excessive sweating, especially at night
Bone pain or tenderness

When to see a doctor

Make an appointment with your doctor if you have any persistent signs or symptoms that worry you.

Leukemia symptoms are often vague and not specific. You may overlook early leukemia symptoms because they may resemble symptoms of the flu and other common illnesses.

Sometimes leukemia is discovered during blood tests for some other condition.

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Parts of the immune system

The lymphatic system is part of the body's immune system, which protects against infection and disease. The lymphatic system includes the spleen, thymus, lymph nodes and lymph channels, as well as the tonsils and adenoids.

Scientists don't understand the exact causes of leukemia. It seems to develop from a combination of genetic and environmental factors.

How leukemia forms

In general, leukemia is thought to occur when some blood cells acquire changes (mutations) in their genetic material or DNA. A cell's DNA contains the instructions that tell a cell what to do. Normally, the DNA tells the cell to grow at a set rate and to die at a set time. In leukemia, the mutations tell the blood cells to continue growing and dividing.

When this happens, blood cell production becomes out of control. Over time, these abnormal cells can crowd out healthy blood cells in the bone marrow, leading to fewer healthy white blood cells, red blood cells and platelets, causing the signs and symptoms of leukemia.

How leukemia is classified

Doctors classify leukemia based on its speed of progression and the type of cells involved.

The first type of classification is by how fast the leukemia progresses:

Acute leukemia. In acute leukemia, the abnormal blood cells are immature blood cells (blasts). They can't carry out their normal functions, and they multiply rapidly, so the disease worsens quickly. Acute leukemia requires aggressive, timely treatment.
Chronic leukemia. There are many types of chronic leukemias. Some produce too many cells and some cause too few cells to be produced. Chronic leukemia involves more-mature blood cells. These blood cells replicate or accumulate more slowly and can function normally for a period of time. Some forms of chronic leukemia initially produce no early symptoms and can go unnoticed or undiagnosed for years.

The second type of classification is by type of white blood cell affected:

Lymphocytic leukemia. This type of leukemia affects the lymphoid cells (lymphocytes), which form lymphoid or lymphatic tissue. Lymphatic tissue makes up your immune system.
Myelogenous (my-uh-LOHJ-uh-nus) leukemia. This type of leukemia affects the myeloid cells. Myeloid cells give rise to red blood cells, white blood cells and platelet-producing cells.

Types of leukemia

The major types of leukemia are:

Acute lymphocytic leukemia (ALL). This is the most common type of leukemia in young children. ALL can also occur in adults.
Acute myelogenous leukemia (AML). AML is a common type of leukemia. It occurs in children and adults. AML is the most common type of acute leukemia in adults.
Chronic lymphocytic leukemia (CLL). With CLL , the most common chronic adult leukemia, you may feel well for years without needing treatment.
Chronic myelogenous leukemia (CML). This type of leukemia mainly affects adults. A person with CML may have few or no symptoms for months or years before entering a phase in which the leukemia cells grow more quickly.
Other types. Other, rarer types of leukemia exist, including hairy cell leukemia, myelodysplastic syndromes and myeloproliferative disorders.

Risk factors

Factors that may increase your risk of developing some types of leukemia include:

Previous cancer treatment. People who've had certain types of chemotherapy and radiation therapy for other cancers have an increased risk of developing certain types of leukemia.
Genetic disorders. Genetic abnormalities seem to play a role in the development of leukemia. Certain genetic disorders, such as Down syndrome, are associated with an increased risk of leukemia.
Exposure to certain chemicals. Exposure to certain chemicals, such as benzene — which is found in gasoline and is used by the chemical industry — is linked to an increased risk of some kinds of leukemia.
Smoking. Smoking cigarettes increases the risk of acute myelogenous leukemia.
Family history of leukemia. If members of your family have been diagnosed with leukemia, your risk of the disease may be increased.

However, most people with known risk factors don't get leukemia. And many people with leukemia have none of these risk factors.

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Kliegman RM, et al. The leukemias. In: Nelson Textbook of Pediatrics. 21st ed. Elsevier; 2020. https://www.clinicalkey.com. Accessed Oct. 16, 2020.
Niederhuber JE, et al., eds. Abeloff's Clinical Oncology. 6th ed. Elsevier; 2020. https://www.clinicalkey.com. Accessed Oct. 16, 2020.
Leukemia. American Society of Hematology. https://www.hematology.org/education/patients/blood-cancers/leukemia. Accessed Oct. 16, 2020.
Warner KJ. Allscripts EPSi. Mayo Clinic. July 9, 2020.
Pruthi RK (expert opinion). Mayo Clinic. Dec. 2, 2020.

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Overview of Leukemia

Classification of Leukemia |
Risk Factors for Leukemia |
More Information |

Leukemia is a malignant condition involving the excess production of immature or abnormal leukocytes, which eventually suppresses the production of normal blood cells and results in symptoms related to cytopenias.

Malignant transformation usually occurs at the pluripotent stem cell level, although it sometimes involves a committed stem cell with more limited capacity for self-renewal. Abnormal proliferation, clonal expansion, aberrant differentiation, and diminished apoptosis (programmed cell death) lead to replacement of normal blood elements with malignant cells.

The American Cancer Society estimates that in the United States in 2023 there will be about 60,000 new cases of leukemia (of all types) in adults and children and about 24,000 deaths.

Classification of Leukemia

The current approach to classifying leukemia is based on the 2016 World Health Organization (WHO) system ( classification for hematopoietic neoplasms ). The WHO classification is based on a combination of clinical features and morphology, immunophenotype, and genetic factors. Other less commonly used classification systems include the French-American-British (FAB) system, which is based on the morphology of the abnormal leukocytes.

Leukemias are commonly also categorized as

Acute or chronic: Based on the percentage of blasts or leukemia cells in bone marrow or blood

Myeloid or lymphoid: Based on the predominant lineage of the malignant cells

The four most common leukemias and their distinguishing features are summarized in the table Findings at Diagnosis in the Most Common Leukemias .

For 2023, the American Cancer Society estimates the distribution of new cases in the United States by leukemia type as follows ( 1 ):

Acute myeloid leukemia (AML): 34%

Acute lymphoblastic leukemia (ALL): 11%

Chronic myeloid leukemia (CML): 15%

Chronic lymphocytic leukemia (CLL): 31%

Other leukemias: 8%

Acute leukemias

Acute leukemias consist of predominantly immature, poorly differentiated cells (usually blast forms). Acute leukemias are divided into

Acute lymphoblastic leukemia (ALL)

Acute myeloid leukemia (AML)

Chronic leukemias

Chronic leukemias have more mature cells than do acute leukemias. They usually manifest as leukocytosis with or without cytopenias in an otherwise asymptomatic person. Findings and management differ significantly between

Chronic lymphocytic leukemia (CLL)

Chronic myeloid leukemia (CML)

Myelodysplastic syndromes

Myelodysplastic syndromes are a group of clonal hematopoietic stem cell disorders unified by the presence of distinct mutations of hematopoietic stem cells. They involve progressive bone marrow failure but with an insufficient proportion of blast cells ( < 20%) for making a definite diagnosis of acute myeloid leukemia; 40 to 60% of cases evolve into acute myeloid leukemia.

Leukemoid reaction

A leukemoid reaction is a neutrophil count > 50,000/mcL (> 50 × 10 9 /L) not caused by malignant transformation of a hematopoietic stem cell. It can result from a variety of causes, particularly other cancers or systemic infection. Usually the cause is apparent, but apparent benign neutrophilia can be mimicked by chronic neutrophilic leukemia or chronic myeloid leukemia.

General reference

1. American Cancer Society: Cancer Facts and Statistics. https://www.cancer.org/research/cancer-facts-statistics.html

Risk Factors for Leukemia

Risk of developing leukemia is increased in patients with

History of exposure to ionizing radiation (eg, post–atom bomb in Nagasaki and Hiroshima) or to chemicals (eg, benzene, some pesticides, polyaromatic hydrocarbons in tobacco smoke); exposure can lead to acute leukemias

Infection with a virus (eg, human T lymphotropic virus 1 or 2, Epstein Barr virus) can rarely cause certain forms of ALL; this is seen mainly in regions where such infections are common, such as Asia and Africa

History of antecedent hematologic disorders, including myelodysplastic syndromes and myeloproliferative neoplasms , which can lead to AML

Preexisting genetic conditions (eg, Fanconi anemia , Bloom syndrome, ataxia-telangiectasia , Down syndrome , xeroderma pigmentosum, Li-Fraumeni syndrome), which predispose to acute myeloid leukemia and acute lymphoblastic leukemia

More Information

The following English-language resource may be useful. Please note that THE MANUAL is not responsible for the content of this resource.

Leukemia and Lymphoma Society: Resources for Healthcare Professionals : Provides information on education programs and conferences and resources for referrals to specialty care

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StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-.

StatPearls [Internet].

Acute lymphocytic leukemia.

Yana Puckett ; Onyee Chan .

Affiliations

Last Update: August 26, 2023 .

Continuing Education Activity

Acute lymphocytic leukemia (ALL) is a malignancy of B or T lymphoblasts characterized by uncontrolled proliferation of abnormal, immature lymphocytes and their progenitors which ultimately leads to the replacement of bone marrow elements and other lymphoid organs resulting in a characteristic disease pattern. ALL accounts for approximately 2 percent of lymphoid neoplasms in the United States and occurs slightly more frequently in males than females and three times as frequently in Caucasians as in African Americans. Patients typically present with symptoms related to anemia, thrombocytopenia, and neutropenia due to the replacement of the bone marrow with the tumor. Symptoms can include fatigue, easy or spontaneous bruising and/or bleeding, and infections. Additionally, B-symptoms, such as fever, night sweats, and unintentional weight loss, are often present but may be mild, and hepatomegaly, splenomegaly, and lymphadenopathy can be seen in up to half of adults on presentation. Central nervous system (CNS) involvement is common and can be accompanied by cranial neuropathies or symptoms, predominantly meningeal, related to increased intracranial pressure. This activity examines when acute lymphocytic leukemia should be considered on differential diagnosis and how to properly evaluate it. This activity highlights the role of the interprofessional team in caring for patients with this condition.

Identify the epidemiology of acute lymphocytic leukemia.
Outline the exam findings typically seen in patients with acute lymphocytic leukemia.
Review the management of acute lymphocytic leukemia.
Explain modalities to improve care coordination among interprofessional team members in order to improve outcomes for patients affected by acute lymphocytic leukemia.
Introduction

Acute lymphocytic leukemia (ALL) is a malignancy of B or T lymphoblasts characterized by uncontrolled proliferation of abnormal, immature lymphocytes and their progenitors, which ultimately leads to the replacement of bone marrow elements and other lymphoid organs resulting in a typical disease pattern characteristic of acute lymphocytic leukemia. ALL accounts for approximately 2 percent of the lymphoid neoplasms diagnosed in the United States. Acute lymphocytic leukemia occurs slightly more frequently in males than females and three times as frequently in Whites as in Blacks. Patients with acute lymphocytic leukemia typically present with symptoms related to anemia, thrombocytopenia, and neutropenia due to the replacement of the bone marrow with the tumor. Symptoms can include fatigue, easy or spontaneous bruising/bleeding, and infections. B-symptoms, such as fever, night sweats, and unintentional weight loss, are often present but may be mild. Hepatomegaly, splenomegaly, and lymphadenopathy can be seen in up to half of adults on presentation. Central nervous system (CNS) involvement is common and can be accompanied by cranial neuropathies or symptoms, predominantly meningeal, related to increased intracranial pressure. [1] [2] [3]

The etiology of acute lymphocytic leukemia is unknown. However, certain environmental factors have been implicated in the etiology of Acute Lymphocytic Leukemia, such as exposure to benzene, ionizing radiation, or previous exposure to chemotherapy or radiotherapy.

Genomic studies have noted that somatic, polymorphic variants of ARD5B, IKZF1 (the gene encoding Ikaros), and CDKN2A are associated with an increased risk of ALL (odds ratio 1.3 to 1.9). Other rare germline mutations in PAX5, ETV6, and particularly p53 can also strongly predispose to the development of leukemia.

Acute lymphoblastic leukemia is not considered a familial disease, and no screening programs have been developed to test for it in childhood.

Epidemiology

It is diagnosed in about 4000 people in the United States each year, with the majority being under the age of 18. It is the most common malignancy of childhood. The peak age of diagnosis is between two and ten years of age. Acute Lymphocytic Leukemia is more common in children with Trisomy 21 (Down syndrome), neurofibromatosis type 1, Bloom syndrome, and ataxia telangiectasia. All are common in children between two and three years of age. Prognosis is diminished in children when diagnosed in infants less than one year of age and in adults. It is more favorable in children. The association of the MLL gene in children at the 11q23 chromosome is associated with poor prognosis. Acute lymphocytic leukemia is a disease with low incidence overall in population studies. The incidence of acute lymphocytic leukemia is about 3.3 cases per 100,000 children. Survival rates for ALL have improved dramatically since the 1980s, with a current five-year overall survival rate estimated at greater than 85 percent.

Pathophysiology

Acute lymphocytic leukemia is thought to occur after damage to DNA causes lymphoid cells to undergo uncontrolled growth and spread throughout the body. Splenomegaly and hepatomegaly occur due to sequestration of platelets and lymphocytes in the spleen and liver; as the white blood cells are not typical, the spleen reacts to them by trying to remove them from the blood. [4] [5] [6]

Histopathology

On peripheral blood smears of acute lymphocytic leukemia patients, lymphoblasts vary in size. Various CD cytokines must be tested to evaluate for what kind of acute lymphocytic leukemia the patient has developed

History and Physical

The most common presenting symptoms of acute lymphocytic leukemia are nonspecific and may be difficult to distinguish from common, self-limited diseases of childhood. In a meta-analysis, more than half of children with childhood leukemia had at least one of the following five features on presentation: palpable liver, palpable spleen, pallor, fever, or bruising. ALL patients typically present with symptoms of night sweats, easy bruising, skin pallor, unexplained lymphadenopathy, weakness, weight loss, hepatosplenomegaly, or difficulty breathing. Some patients may present with superior vena cava syndrome. Bone pain, mental changes, and oliguria may also be present. ALL can also present with testicular enlargement, musculoskeletal pain, mediastinal mass, and incidentally found peripheral blood cell abnormalities.

Acute Lymphocytic Leukemia diagnosis should be explored initially with a laboratory evaluation consisting of a CBC, electrolyte and renal panel, and LDH level. Additionally, imaging, such as a chest x-ray for symptoms of shortness of breath, may be obtained. If abdominal fullness, tenderness, or abdominal mass are symptoms, then a CT scan of the abdomen and pelvis should be obtained. This can also help with the staging of the disease.

NCCN diagnosis guidelines:

Have presence of more than 20% bone marrow lymphoblasts
Hematoxylin and eosin-stained bone marrow clot and biopsy sections
Morphology of bone marrow aspirate assessed with Wright/Giemsa
Complete flow cytometric immunophenotyping
Baseline evaluation of the leukemic clone

Lumbar puncture is used to evaluate CNS involvement. The fluid is checked for the presence of lymphoblasts.

Treatment / Management

Children who are suspected of having acute lymphocytic leukemia should be referred to a pediatric center that specializes in cancer for evaluation and treatment.

For children with Acute Lymphocytic Leukemia, induction therapy consists of anthracycline, vincristine, 1-asparaginase, and a corticosteroid.

Today consolidation therapy is widely used and includes therapy with a variety of chemotherapeutic drugs with good results.

Maintenance therapy utilizes oral 6-mercaptopurine or methotrexate delivered once weekly or once monthly. Successful treatment of children with acute lymphocytic leukemia involves the administration of a multidrug regimen that is divided into several phases (i.e., induction, consolidation, and maintenance) and includes therapy directed to the central nervous system (CNS).

Most treatment protocols take two to three years to complete.

CNS prophylaxis is done via an intrathecal approach. Patients often require 8 to 16 intrathecal treatments.

If the patient has Ph-chromosome positive ALL, the current treatment includes the use of tyrosine kinase inhibitors like imatinib, nilotinib, dasatinib, or ponatinib. Several trials have shown a good response to these agents.

Stem cell transplantation can sometimes be used as a treatment in which a patient's normal source of blood cells (bone marrow) is replaced by healthy young blood cells (stem cells) from a healthy well-matched donor. However, with improvements in chemotherapy, the role of transplantation is declining in ALL.

Recently CAR-T cell therapy has been investigated in ALL with excellent results. Several studies show high rates of remission. Unfortunately, CART is also associated with serious toxicity that includes cerebral edema and cytokine release syndrome, which can be fatal.

All blood products must be irradiated prior to transfusion to prevent transfusion-related graft versus host disease, which is universally fatal.

Splenectomy is rarely required for acute lymphocytic leukemia. Splenectomy can help boost platelet count but does not affect the outcome of leukemia itself. Splenectomy can be performed for severe symptoms that are not amenable to chemotherapy treatment, such as abdominal pain. Radiation can also be used in cases of enlarged spleen to try and reduce the size of the spleen in most cases. [7] [8] [9]

Tumor lysis syndrome is a life-threatening complication that occurs in patients receiving chemotherapy. It is characterized by hyperuricemia, elevated potassium and phosphate, and decreased levels of calcium. Renal failure is invariably present.

Differential Diagnosis
B cell lymphoma
Acute myeloid leukemia
Non-Hodgkin lymphoma

Current World Health Organization Classification of ALL [10]

B-lymphoblastic leukemia/lymphoma

B-lymphoblastic leukemia/lymphoma, NOS
B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities
B-lymphoblastic leukemia/lymphoma with t(9;22)(q34.1;q11.2); BCR-ABL1
B-lymphoblastic leukemia/lymphoma with t(v;11q23.3); KMT2A rearranged
B-lymphoblastic leukemia/lymphoma with t(12;21)(p13.2;q22.1); ETV6-RUNX1
B-lymphoblastic leukemia/lymphoma with hyperdiploidy
B-lymphoblastic leukemia/lymphoma with hypodiploidy
B-lymphoblastic leukemia/lymphoma with t(5;14)(q31.1;q32.3); IL3-IGH
B-lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3); TCF3-PBX1
Provisional entity: B-lymphoblastic leukemia/lymphoma, BCR-ABL1-like
Provisional entity: B-lymphoblastic leukemia/lymphoma with iAMP21

T-lymphoblastic leukemia/lymphoma

Provisional entity: Early T-cell precursor lymphoblastic leukemia
Provisional entity: Natural killer (NK) cell lymphoblastic leukemia/lymphoma

Only about 30% of adults with ALL can be cured today. Criteria for good prognosis include:

Age of less than 30
No abnormal cytogenetics
WBC count less than 30,000
Complete remission within 4 weeks
High hyperdiploidy with 51–65 chromosomes in children
t(12;21)(p13;q22) in children

Poor prognostic factors include:

Age of more than 60
Presence of abnormal cytogenetics (t(9:22), t(4:11)
Failure to achieve remission within 4 weeks
Precursor B-cells more than 100,000
Pearls and Other Issues

Despite improvements in supportive care, death resulting from treatment toxicity remains a challenge. It is important to watch out for tumor lysis syndrome, which occurs when chemotherapy causes cancer cells to lyse, releasing certain intracellular elements such as potassium, calcium, uric acid, and phosphorus. These elements, in large numbers, result in toxicity that can often lead to renal failure. Pretreatment with fluids and steroids typically prevents Tumor Lysis Syndrome. However, if it occurs, aggressive fluid therapy is the treatment.

Even after treatment, acute lymphocytic leukemia can relapse. Relapses can occur as far back as 21 years. It is important to address other issues associated with cancer treatment in a young child, including providing psychological support to the child, parents, and family.

Enhancing Healthcare Team Outcomes

Like all malignancies, the management of acute leukemia is with an interprofessional team dedicated to the management of cancer patients; an interprofessional team includes an oncologist, an internist, an infectious disease expert, and a hematologist. The primary care provider and nurse practitioner may be responsible for follow-up after treatment and report back to the interprofessional team. These patients need close monitoring as they are prone to infections, coagulation dyscrasias, and relapse. Team conferences should be held while the patient is being treated, and any problems should be conveyed to the team.

The pharmacist should educate the patient on chemotherapy medications, their adverse effects, and their benefits. The dietitian should encourage a healthy diet. To prevent infections, the nurse practitioner should encourage hand washing, washing of fruits and vegetables, and maintaining good personal hygiene.

The oncology nurses should monitor the patient for adverse effects of the drug, including tumor lysis syndrome. Patients should be given consistent messages, and one should avoid offering unrealistic expectations. To improve outcomes, the team should keep updated on the latest clinical trials.

Despite improvements in supportive care, death resulting from treatment toxicity remains a challenge. It is important to watch out for tumor lysis syndrome, which occurs when chemotherapy causes cancer cells to lyse, releasing certain intracellular elements such as potassium, calcium, uric acid, and phosphorus. These elements, in large numbers, result in toxicity that can often lead to renal failure. Pretreatment with fluids and steroids typically prevents tumor lysis syndrome. However, if it occurs, aggressive fluid therapy is the treatment.

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Disclosure: Yana Puckett declares no relevant financial relationships with ineligible companies.

Disclosure: Onyee Chan declares no relevant financial relationships with ineligible companies.

This book is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ), which permits others to distribute the work, provided that the article is not altered or used commercially. You are not required to obtain permission to distribute this article, provided that you credit the author and journal.

Cite this Page Puckett Y, Chan O. Acute Lymphocytic Leukemia. [Updated 2023 Aug 26]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-.

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Acute Lymphoblastic Leukemia (ALL)

(acute lymphocytic leukemia).

Pathophysiology |
Classification |
Symptoms and Signs |
Diagnosis |
Treatment |
Prognosis |
Key Points |
More Information |

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer; it also strikes adults of all ages. Malignant transformation and uncontrolled proliferation of an abnormally differentiated, long-lived hematopoietic progenitor cell results in a high circulating number of blasts, replacement of normal marrow by malignant cells, and the potential for leukemic infiltration of the central nervous system (CNS) and testes. Symptoms include fatigue, pallor, infection, bone pain, CNS symptoms (eg, headache), easy bruising, and bleeding. Examination of peripheral blood smear and bone marrow is usually diagnostic. Treatment typically includes combination chemotherapy to achieve remission, intrathecal and systemic chemotherapy and/or corticosteroids for CNS prophylaxis, and sometimes cerebral irradiation for intracerebral leukemic infiltration, consolidation chemotherapy with or without stem cell transplantation, and maintenance chemotherapy for up to 3 years to avoid relapse.

(See also Overview of Leukemia .)

The American Cancer Society estimates that in the United States in 2023 there will be over 6500 new cases of acute lymphoblastic leukemia (ALL) and almost 1400 deaths will have occurred. Sixty percent of all ALL cases occur in children, with a peak incidence at age 2 to 5 years; a second peak occurs after age 50. ALL is the most common cancer in children, and represents about 75% of leukemias among children ARID5B gene.

Pathophysiology of ALL

Similar to acute myeloid leukemia , acute lymphoblastic leukemia is caused by a series of acquired genetic aberrations. Malignant transformation usually occurs at the pluripotent stem cell level, although it sometimes involves a committed stem cell with more limited capacity for self-renewal. Abnormal proliferation, clonal expansion, aberrant differentiation, and diminished apoptosis (programmed cell death) lead to replacement of normal blood elements with malignant cells.

Classification of ALL

In acute lymphoblastic leukemia, the precursor lymphoid neoplasms are broadly categorized based on their lineage into

B-lymphoblastic leukemia/lymphoma (B-ALL/LBL)

T-lymphoblastic leukemia/lymphoma (T-ALL/LBL)

Disease can manifest as a leukemia when neoplastic cells (lymphoblasts) involve blood and bone marrow (defined as > 20% bone marrow blasts) or as a lymphoma when blasts infiltrate mainly extramedullary tissue.

The 2016 World Health Organization (WHO) classification of lymphoid neoplasms incorporates genetic data, clinical features, cell morphology, and immunophenotype, all of which have important implications for disease prognosis and management.

Symptoms and Signs of ALL

Symptoms and signs of acute lymphoblastic leukemia may be present for only days to weeks before diagnosis.

The most common presenting symptoms are due to disrupted hematopoiesis with ensuing

Thrombocytopenia

Granulocytopenia

Anemia can manifest with fatigue, weakness, pallor, malaise, dyspnea on exertion, tachycardia, and exertional chest pain.

Thrombocytopenia can cause mucosal bleeding, easy bruising, petechiae/purpura, epistaxis, bleeding gums, and heavy menstrual bleeding. Hematuria and gastrointestinal bleeding are uncommon. Patients can present with spontaneous hemorrhage, including intracranial or intra-abdominal hematomas.

Granulocytopenia or neutropenia can lead to a high risk of infections, including those of bacterial, fungal, and viral etiologies. Patients may present with fevers and a severe and/or recurrent infection.

Organ infiltration by leukemic cells results in enlargement of the liver, spleen, and lymph nodes. Bone marrow and periosteal infiltration may cause bone and joint pain, especially in children with ALL. CNS penetration and meningeal infiltration are common and can result in cranial nerve palsies, headache, visual or auditory symptoms, altered mental status, and transient ischemic attack/stroke.

Diagnosis of ALL

Complete blood count (CBC) and peripheral blood smear

Bone marrow examination

Histochemical studies, cytogenetics, and immunophenotyping

A diagnosis of acute lymphoblastic leukemia is made when blast cells of lymphoid origin are ≥ 20% of marrow nucleated cells or ≥ 20% of non-erythroid cells when the erythroid component is > 50%. If marrow cells are insufficient or unavailable, diagnosis can be made by the same criteria using a peripheral blood sample.

By permission of the publisher. From Chang K, Forman S. In Atlas of Clinical Hematology . Edited by JO Armitage. Philadelphia, Current Medicine, 2004.

CBC and peripheral smear are the first tests done; pancytopenia and peripheral blasts suggest acute leukemia. Blast cells in the peripheral smear may approach 90% of the white blood cell (WBC) count. Aplastic anemia , viral infections such as infectious mononucleosis , and vitamin B12 deficiency , and folate deficiency should be considered in the differential diagnosis of severe pancytopenia. Unlike in AML, Auer rods (linear azurophilic inclusions in the cytoplasm of blast cells) are never present in acute lymphoblastic leukemia.

Bone marrow examination (aspiration and needle biopsy) is routinely done. Blast cells in the bone marrow are typically between 25 and 95% in patients with ALL.

Histochemical studies, cytogenetics, and immunophenotyping studies help distinguish the blasts of ALL from those of AML or other disease processes. Histochemical studies include staining for terminal deoxynucleotidyl transferase (TdT), which is positive in cells of lymphoid origin. Detection of specific immunophenotypic markers such as CD3 (for lymphoid cells of T cell origin) and CD19, CD20, and CD22 (for lymphoid cells of B cell origin) is essential in classifying the acute leukemias. Common cytogenetic abnormalities in ALL include t(9;22) in adults and t(12;21) and high hyperdiploidy in children (see table Common Cytogenetic Abnormalities in ALL ).

Less common cytogenetic abnormalities include the following:

t(v;11q23) / MLL or KMT2A rearranged, including t(4;11)/ KMT2A-AF4

t(1;19)/ E2A-PBX1 ( TCF3-PBX1 )

t(5;14)/ IL3-IGH

t(8;14), t(8;22), t(2;8)/ C-MYC rearranged

BCR-ABL -like acute lymphoblastic leukemia overlaps phenotypically with ALL in which the Philadelphia chromosome [a reciprocal balanced translocation between chromosomes 9 and 22, t(9;22)] is present (Ph+ ALL).

Other laboratory findings may include hyperuricemia, hyperphosphatemia , hyperkalemia , hypocalcemia , and elevated lactate dehydrogenase (LDH), which indicate a tumor lysis syndrome . Elevated serum levels of hepatic transaminases or creatinine, and hypoglycemia may also be present. Patients with Ph+ ALL and patients with t( v ;11q23) involving MLL rearrangements often present with hyperleukocytosis.

CT of the head is done in patients with CNS symptoms. CT of the chest and abdomen should be done to detect mediastinal masses and lymphadenopathy and may also detect hepatosplenomegaly. Echocardiography or multi-gated acquisition (MUGA) scanning is typically done to assess baseline cardiac function (prior to administration of anthracyclines, which are cardiotoxic).

Treatment of ALL

Systemic chemotherapy

Prophylactic CNS chemotherapy and sometimes CNS radiation

For Ph+ ALL, also a tyrosine kinase inhibitor

Supportive care

Sometimes immunotherapy , targeted therapy , stem cell transplantation , and/or radiation therapy

Treatment for newly diagnosed acute lymphoblastic leukemia generally consists of 3 to 4 cycles of chemotherapy blocks of non–cross-resistant chemotherapy for the first 9 to 12 months, followed by 2.5 to 3 years of maintenance chemotherapy.

Chemotherapy

The 4 general phases of chemotherapy for acute lymphoblastic leukemia include

Remission induction

Postremission consolidation

Interim maintenance and intensification

Maintenance

The goal of induction treatment is complete remission, defined as 1000/mcL (> 1 × 10 9 /L), a platelet count > 100,000/mcL (> 100 × 10 9 /L), and no need for blood transfusion. In patients with complete remission, a low measurable residual disease (also known as minimal residual disease or MRD) is the most important prognostic factor ( 1 ). Measurable or minimal residual disease is microscopic disease that is not detected by standard assays but can be measured by more sensitive assays. A low measurable residual disease (MRD negativity) is defined variably (based on the assay used) as

Components of induction therapy include

The goal of consolidation is to prevent leukemic regrowth. Consolidation therapy usually lasts a few months and combines regimen-specific courses of non–cross-resistant drugs that have different mechanisms of action. For adults with Ph+ ALL, allogeneic stem cell transplantation is recommended as consolidation therapy.

Interim maintenance and late/delayed intensification therapy are used after consolidation therapy. These phases of therapy incorporate a variety of chemotherapeutic agents with different doses and schedules that are less intense than induction and consolidation.

Most regimens include maintenance therapy

CNS prophylaxis

Medically frail patients with ALL

About one third of patients with acute lymphoblastic leukemia are older adults (> 65). Older ALL patients are more likely to have precursor B-cell ALL and have higher risk and more complex cytogenetics, including Philadelphia chromosome positive (Ph+) or t( v ;11q23) MLL or KMT2A rearranged disease.

hematopoietic stem cell transplantation is an option.

Targeted immunotherapy drugs that are available for treatment of relapsed or refractory ALL are increasingly used for treatment of older patients with ALL in clinical trials or clinical practice.

Older patients with ALL probably tolerate asparaginase more poorly than younger patients do.

Relapsed or refractory ALL

Leukemic cells may reappear in the bone marrow, CNS, testes, or other sites. Bone marrow relapse is particularly ominous. Although a new round of chemotherapy may induce a second remission in the majority of children and about one third of adults, subsequent remissions tend to be brief. Chemotherapy helps only a few patients with early bone marrow relapse to achieve long disease-free second remissions or cure.

Chimeric antigen receptor T (CAR-T) cells, engineered and generated from the patient's T cells, induce remission in patients with relapsed ALL with remarkable efficacy, albeit with significant toxicity ( 2 ).

Available immunotherapies for relapsed or refractory ALL include

a biospecific CD19-directed CD3 T-cell engager, prolongs overall survival for children and adults with relapsed or refractory B-cell precursor ALL, whether Ph+ or Ph-. Life-threatening toxicities may include cytokine release syndrome 3 ).

4 ). Inotuzumab may cause hepatotoxicity, including fatal and life-threatening veno-occlusive disease and is associated with higher post-transplant non-relapse mortality.

a CD19-directed genetically modified autologous T-cell immunotherapy, is available for the treatment of patients up to 25 years of age with B-cell precursor ALL that is refractory or in a 2nd or later relapse. Life-threatening toxicities may include cytokine release syndrome and neurologic toxicities ( 5 ).

Brexucabtagene autoleucel , a CD19-directed genetically modified autologous T cell immunotherapy, can be used to treat adult patients with relapsed or refractory B-cell precursor ALL. Complications, including cytokine release syndrome and neurologic toxicities, may be life threatening.

Other agents have been available, but clinically meaningful outcomes have not been convincingly demonstrated (ie, the approvals were based upon response rate but there were no trials verifying an improvement in disease-related symptoms or increased survival) for these. Examples include:

Stem cell transplantation following reinduction chemotherapy or immunotherapy offers the greatest hope of long-term remission or cure if an HLA-matched sibling is available. Cells from other relatives or from matched, unrelated donors are sometimes used. Transplantation is rarely used for patients > 65 years because it is much less likely to be successful and because adverse effects are much more likely to be fatal.

CNS relapse

Testicular relapse may be evidenced clinically by painless firm swelling of a testis or may be identified on biopsy. If unilateral testicular involvement is clinically evident, the apparently uninvolved testis should undergo biopsy. Treatment is radiation therapy of the involved testis and administration of systemic reinduction therapy.

Supportive care is similar in the acute leukemias and may include

Transfusions

Antimicrobials

Hydration and urine alkalinization

Psychologic support

Transfusions of red blood cells and sometimes platelets are administered as needed to patients with bleeding or anemia. Prophylactic platelet transfusion is done when platelets fall to < 10,000/mcL ( 9 /L). Anemia (hemoglobin < 7 or 8 g/dL [

Antimicrobials are often needed for prophylaxis and treatment because patients are immunosuppressed; in such patients, infections can progress quickly with little clinical prodrome. After appropriate studies and cultures have been done, febrile patients with neutrophil counts < 500/mcL ( < 0.5 × 10 9 Pneumocystis jirovecii infection or a viral infection should be suspected and confirmed by bronchoscopy and bronchoalveolar lavage and treated appropriately.

Aspergillus and Candida P. jirovecii

Hydration, tumor lysis syndrome G6PD deficiency

Psychologic support may help patients and their families with the shock of illness and the rigors of treatment for a potentially life-threatening condition.

Treatment references

1. Berry DA, Zhou S, Higley H, et al : Association of minimal residual disease with clinical outcome in pediatric and adult acute lymphoblastic leukemia: A meta-analysis. JAMA Oncol 3(7): e170580, 2017. doi:10.1001/jamaoncol.2017.0580

2. Lee DW, Kochenderfer JN, Stetler-Stevenson M, et al : T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 385(9967) :517–528, 2015.

3. Kantarjian H, Stein A, Gökbuget N, et al : Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N Engl J Med 376(9):836–847, 2017.

4. Kantarjian HM, DeAngelo DJ, Stelljes M, et al : Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia. N Engl J Med 375(8):740–753, 2016.

5. Maude SL, Laetsch TW, Buechner J, et al : Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 378(5):439–448, 2018.

Prognosis for ALL

Prognostic factors help determine treatment protocol and intensity.

Favorable prognostic factors are

Age 3 to 9 years

WBC count < 25,000/mcL ( 9 /L) or 9 /L) in children

Leukemic cell karyotype with high hyperdiploidy (51 to 65 chromosomes), t(1;19), and t(12;21)

No CNS disease at diagnosis

Unfavorable factors include

Leukemic cell karyotype with 23 chromosomes (haploidy), with

Leukemic cell karyotype with t( v ;11q23) MLL ( KMT2A ) rearranged, including t(4;11)/ KMT2A-AF4

Leukemic cell karyotype t(5;14)/ IL3-IG

Leukemic cell karyotype t(8;14), t(8;22), t(2;8) C-MYC rearranged

Presence of the Philadelphia (Ph) chromosome t(9;22) BCR-ABL1

Increased age in adults

BCR/ABL1 -like molecular signature

Regardless of prognostic factors, the likelihood of initial remission is ≥ 95% in children and 70 to 90% in adults. Of children, > 80% have continuous disease-free survival for 5 years and appear to be cured. Of adults,

Less ability to tolerate intensive chemotherapy

More frequent and severe comorbidities

Higher risk ALL genetics that confer chemotherapy resistance

Poorer adherence to ALL treatment regimens, which include frequent (often daily or weekly) out-patient chemotherapy and doctor visits

Less frequent use of pediatric-inspired treatment regimens

Most investigatory protocols select patients with poor prognostic factors for more intense therapy because the increased risk of and toxicity from treatment are outweighed by the greater risk of treatment failure leading to death.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children but also occurs in adults.

Central nervous system (CNS) involvement is common; most patients receive intrathecal chemotherapy and corticosteroids and sometimes CNS radiation therapy.

Response to treatment is good in children, with cure possible in > 80% of children but in

Repeat induction chemotherapy, immunotherapy, and stem cell transplantation may be helpful for relapse.

More Information

The following English-language resource may be useful. Please note that THE MANUAL is not responsible for the content of this resource.

Leukemia and Lymphoma Society: Resources for Healthcare Professionals : Provides information on education programs and conferences and resources for referrals to specialty care

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AMANDA S. DAVIS, MD, ANTHONY J. VIERA, MD, MPH, AND MONICA D. MEAD, MD

A more recent article on leukemia is available.

Am Fam Physician. 2014;89(9):731-738

Patient information : See related handout on leukemia , written by the authors of this article.

Author disclosure: No relevant financial affiliations.

Leukemia is a clonal proliferation of hematopoietic stem cells in the bone marrow. The four broad subtypes most likely to be encountered by primary care physicians are acute lymphoblastic, acute myelogenous, chronic lymphocytic, and chronic myelogenous. Acute lymphoblastic leukemia occurs more often in children, whereas the other subtypes are more common in adults. Risk factors include a genetic predisposition as well as environmental factors, such as exposure to ionizing radiation. Symptoms are nonspecific and include fever, fatigue, weight loss, bone pain, bruising, or bleeding. A complete blood count usually reveals leukocytosis and other abnormally elevated or depressed cell lines. Patients with suspected leukemia should be referred promptly to a hematologist-oncologist. The diagnosis is confirmed by further examination of the bone marrow or peripheral blood. Treatment may include chemotherapy, radiation, monoclonal antibodies, or hematopoietic stem cell transplantation. Complications of treatment include tumor lysis syndrome and serious infections from immunosuppression. Leukemia survivors should be monitored closely for secondary malignancies, cardiac complications, and endocrine disturbances such as metabolic syndrome, hypothyroidism, and hypogonadism. Five-year survival rates are highest in younger patients and in patients with chronic myelogenous leukemia or chronic lymphocytic leukemia.

Leukemia is a common malignancy in children and adults that occurs when alterations in normal cell regulatory processes cause uncontrolled proliferation of hematopoietic stem cells in the bone marrow. The age-adjusted incidence rate of leukemia in the United States is 12.8 per 100,000 persons each year. 1 The prevalence of leukemia is generally higher in whites and in males, and increases with age. 1 Approximately one in 70 persons develops leukemia in his or her lifetime. 1 The four subtypes of leukemia most often encountered by primary care physicians are acute lymphoblastic, acute myelogenous, chronic lymphocytic, and chronic myelogenous. Family physicians should be able to recognize the common presentations of leukemia, perform the initial diagnostic evaluation, and understand how to care for leukemia survivors.

Risk Factors

Several genetic syndromes, including Down syndrome and neurofibromatosis, are associated with an increased risk of childhood acute lymphoblastic leukemia and acute myelogenous leukemia. 2 Persons exposed to ionizing radiation, such as atomic bomb survivors, medical radiation workers before 1950, and patients with cancer who are receiving radiation treatment, have an increased risk of developing acute lymphoblastic leukemia, acute myelogenous leukemia, and chronic myelogenous leukemia. 2 , 3 Evidence from epidemiologic studies suggests that the amount of radiation from two or three computed tomography scans is associated with a statistically significant increase in the risk of cancer, including leukemia, with a greater risk in younger persons. 4

Occupational and environmental exposure to benzene (a chemical used in the manufacturing of paints and plastics, and released with the combustion of petroleum and coal) is an established risk factor for leukemia in adults, particularly acute myelogenous leukemia. 5 , 6 Household pesticide exposure in utero and in the first three years of life has been associated with an increased risk of childhood acute lymphoblastic leukemia. 6 Obesity may also increase the risk. Aggregate data from a meta-analysis of cohort studies suggest that an increase of 5 kg per m 2 in body mass index is associated with a 13% relative increase in the risk of leukemia. 7 A history of hematologic malignancy is also a risk factor for developing a different subtype of leukemia later in life. 8

Clinical Presentation

Acute leukemia.

Children . According to three retrospective case reviews of childhood leukemia (in which 75% to 100% of the cases were acute lymphoblastic leukemia), common presenting signs and symptoms include fever (17% to 77%), lethargy (12% to 39%), and bleeding (10% to 45%). 9 – 11 About one-third of children had musculoskeletal symptoms, particularly in the spine and long bones, 9 – 11 75% had an enlarged liver or spleen, and nearly 60% had lymphadenopathy. 9 , 10 Central nervous system involvement is present in approximately 7% of children at diagnosis. 10

Adults . Acute myelogenous leukemia accounts for 80% of acute leukemia in adults. 12 Adults also present with constitutional symptoms such as fever, fatigue, and weight loss. They may have anemia-related symptoms, such as shortness of breath or chest pain, or symptoms related to thrombocytopenia, such as excessive bruising, nosebleeds, or heavy menstrual periods in women. Adults are less likely to present with bone pain. Hepatosplenomegaly and lymphadenopathy are rare in adults with acute myelogenous leukemia, but are present in about 50% of adults with acute lymphoblastic leukemia. 12 Central nervous system involvement occurs in approximately 5% to 8% of adults with acute lymphoblastic leukemia. 12

CHRONIC LEUKEMIA

The chronic leukemia subtypes occur almost exclusively in adults. Patients with chronic leukemia may be asymptomatic at the time of diagnosis. Approximately 50% of patients with chronic lymphocytic leukemia and 20% of patients with chronic myelogenous leukemia receive the diagnosis incidentally when marked leukocytosis is found on a complete blood count obtained for an unrelated reason. 13 , 14 Constitutional symptoms are less common, occurring in 15% of patients with chronic lymphocytic leukemia and in approximately one-third of patients with chronic myelogenous leukemia. 13 , 14 Hepatosplenomegaly and lymphadenopathy are common physical examination findings in persons with chronic lymphocytic leukemia. 14 Splenomegaly is common in those with chronic myelogenous leukemia; in one large, retrospective review, 75% of patients had a palpable spleen. 13 Bleeding and bruising are less common presenting features in the chronic leukemia subtypes. Characteristics of the major subtypes of leukemia are listed in Table 1 . 1 , 9 – 18

Laboratory Findings and Diagnosis

If leukemia is suspected, a complete blood count should be obtained. Marked leukocytosis, often greater than 100,000 white blood cells per μL (100.0 × 10 9 per L), is the hallmark laboratory finding in chronic myelogenous leukemia and chronic lymphocytic leukemia. More than 96% of patients with chronic myelogenous leukemia have white blood cell counts greater than 20,000 per μL (20.0 × 10 9 per L), compared with only 34% to 38% of patients with acute myelogenous leukemia or acute lymphoblastic leukemia. 9 , 10 , 13 Acute leukemia can also present with leukopenia, combined with anemia or thrombocytopenia. Other helpful initial laboratory tests include measurement of serum electrolyte and creatinine levels, liver function tests, and coagulation studies. If the patient appears ill or is febrile, the physician should evaluate for infection with urinalysis, urine culture, blood cultures, and chest radiography.

The next step in diagnosis involves a peripheral blood smear and usually a bone marrow specimen (an aspirate or core biopsy). Figure 1 details the initial steps in the evaluation of possible leukemia. 15 – 17

Acute leukemia should be suspected when a peripheral blood smear or bone marrow specimen is overpopulated with blast cells (the earliest form of hematopoietic precursor cells). Classically, acute myelogenous leukemia is characterized by the presence of Auer rods on a peripheral smear. However, because Auer rods are not commonly detected, immunophenotyping by flow cytometry and cytogenetic testing are required to distinguish between acute leukemia subtypes such as acute myelogenous leukemia or acute lymphoblastic leukemia. 15 , 16 Table 2 describes the current approaches to the laboratory diagnosis of the leukemia subtype.

The diagnosis of chronic lymphocytic leukemia is based on a clonal expansion of at least 5,000 B lymphocytes per μL (5.0 × 10 9 per L) in the peripheral blood, confirmed by immunophenotyping. A bone marrow specimen is not required for diagnosis of chronic lymphocytic leukemia, but can be obtained to determine the extent of marrow involvement for prognosis. 17 The diagnosis of chronic myelogenous leukemia requires cytogenetic or molecular testing of the bone marrow or peripheral blood for a specific abnormality called the Philadelphia chromosome, or the BCR-ABL1 fusion gene. 16 In chronic myelogenous leukemia, a reciprocal translocation between chromosomes 9 and 22 results in the formation of the BCR-ABL1 fusion gene that disrupts the normal cell regulatory processes in the bone marrow. The shortened chromosome 22 (Philadelphia chromosome) is found in 95% of patients with chronic myelogenous leukemia. 19 The remaining 5% of patients have a different chromosomal rearrangement, but still form the abnormal BCR-ABL1 fusion gene.

A patient with suspected leukemia should be referred to a hematologist-oncologist to confirm the diagnosis and initiate treatment. Treatment for acute leukemia may include chemotherapy, radiation, monoclonal antibodies, or hematopoietic stem cell transplantation. The type of treatment depends on the leukemia subtype, cytogenetic and molecular findings, patient age, and comorbid conditions.

Early-stage chronic lymphocytic leukemia (i.e., no anemia or thrombocytopenia and less than three areas of nodal involvement) can be monitored without treatment. Active-stage disease is defined as worsening thrombocytosis, thrombocytopenia, or anemia; progressive lymphadenopathy or splenomegaly; or the presence of constitutional symptoms. 17

The discovery of tyrosine kinase inhibitors revolutionized the treatment of chronic myelogenous leukemia. The abnormal fusion gene created by the translocation of chromosomes 9 and 22 codes for tyrosine kinase, an enzyme that activates signal transduction cascades that cause uncontrolled cellular proliferation. This targeted approach of inhibiting the tyrosine kinase enzyme is not curative but can maintain long-term control of the disease without the adverse effects of chemotherapy. Curative treatment consists of hematopoietic stem cell transplantation, which is usually reserved for younger patients or when the disease does not respond to tyrosine kinase inhibitors. 20

Treatment Complications

Tumor lysis syndrome occurs as a result of chemotherapy (or rarely, spontaneously) when widespread cellular destruction releases intracellular contents into the bloodstream. The result is high potassium, phosphorus, uric acid, and blood urea nitrogen levels. Treatment is aimed at preventing renal failure, and includes aggressive intravenous fluid administration plus allopurinol (Zyloprim) or rasburicase (Elitek), a recombinant urate oxidase that breaks down uric acid. 21

Immunosuppression from chemotherapy, hematopoietic stem cell transplantation, or the leukemia itself may increase the risk of serious infections. In patients with leukemia, fever with neutropenia (fewer than 500 neutrophils per μL [0.5 × 10 9 per L]) should prompt an evaluation for infection source and the initiation of empiric broad-spectrum antibiotic therapy, such as imipenem/cilastatin (Primaxin), meropenem (Merrem), piperacillin/tazobactam (Zosyn), or cefepime. 22

Prognosis and Long-Term Sequelae

Prognosis depends on factors such as age, comorbid disease, leukemia subtype, and cytogenetic and molecular characteristics ( Table 1 1 , 9 – 18 ) . Survivors of leukemia have an increased risk of subsequent cancers, likely because of the cellular damage caused by chemotherapy or radiation. In the Childhood Cancer Survivor Study (a cohort of more than 17,000 childhood cancer survivors in North America treated between 1970 and 1986), the 30-year cumulative incidence of neoplasm after leukemia was 5.6%, and the median time to occurrence of the subsequent cancer was nine years. 23 The most common second neoplasms in childhood leukemia survivors are different subtypes of leukemia, or lymphoma. Other second neoplasms include bone, soft tissue, or central nervous system tumors. Guidelines recommend age- and sex-specific cancer screening, routine complete blood count to monitor for relapse or occurrence of a subsequent hematologic malignancy, and a low threshold for brain imaging for neurologic symptoms in patients who have received cranial or craniospinal irradiation. 8 , 24 , 25

Childhood survivors of leukemia are at increased risk of osteonecrosis of joints such as the hip, shoulder, and knee. Adolescent survivors of acute lymphoblastic leukemia are at highest risk, with a 20-year cumulative incidence of 2.8%. 26 Guidelines recommend bone density testing one year after hematopoietic stem cell transplantation. 27 Treatment with certain chemotherapeutic agents or radiation can affect cardiac function, including ejection fraction and electrical conduction of the heart. For example, 20 to 30 years after treatment with anthracyclines (e.g., daunorubicin, doxorubicin [Adriamycin]), 5% to 10% of patients develop congestive heart failure. 8 Guidelines recommend periodic cardiac evaluation in leukemia survivors. 28 , 29 Endocrine abnormalities are also common after leukemia treatment, including metabolic syndrome, thyroid function abnormalities, and gonadal failure. Monitoring and treatment for these and other complications are summarized in Table 3 . 24 , 25 , 27 – 30

Data Sources : We searched PubMed using the key word leukemia. We also performed a search in Essential Evidence Plus. We accessed the National Cancer Institute's Surveillance Epidemiology and End Results Program online database of cancer statistics, and the National Comprehensive Cancer Network database. Search dates: September and December 2011, January 2012, and January 2014.

National Cancer Institute. SEER cancer statistics review 2006–2010. http://seer.cancer.gov/statfacts/html/leuks.html . Accessed January 9, 2014.

Bhatia S, Robison LL. Epidemiology of leukemia and lymphoma. Curr Opin Hematol. 1999;6(4):201-204.

Yoshinaga S, Mabuchi K, Sigurdson AJ, Doody MM, Ron E. Cancer risks among radiologists and radiologic technologists: review of epidemiologic studies. Radiology. 2004;233(2):313-321.

Brenner DJ, Hall EJ. Computed tomography—an increasing source of radiation exposure. N Engl J Med. 2007;357(22):2277-2284.

Khalade A, Jaakkola MS, Pukkala E, Jaakkola JJ. Exposure to benzene at work and the risk of leukemia: a systematic review and meta-analysis. Environ Health. 2010;9:31.

Buffler PA, Kwan ML, Reynolds P, Urayama KY. Environmental and genetic risk factors for childhood leukemia: appraising the evidence. Cancer Invest. 2005;23(1):60-75.

Lichtman MA. Obesity and the risk for a hematological malignancy: leukemia, lymphoma, or myeloma. Oncologist. 2010;15(10):1083-1101.

Diller L. Clinical practice. Adult primary care after childhood acute lymphoblastic leukemia. N Engl J Med. 2011;365(15):1417-1424.

Sinigaglia R, Gigante C, Bisinella G, Varotto S, Zanesco L, Turra S. Musculoskeletal manifestations in pediatric acute leukemia. J Pediatr Orthop. 2008;28(1):20-28.

Ma SK, Chan GC, Ha SY, Chiu DC, Lau YL, Chan LC. Clinical presentation, hematologic features and treatment outcome of childhood acute lymphoblastic leukemia: a review of 73 cases in Hong Kong. Hematol Oncol. 1997;15(3):141-149.

Rogalsky RJ, Black GB, Reed MH. Orthopaedic manifestations of leukemia in children. J Bone Joint Surg Am. 1986;68(4):494-501.

Cornell RF, Palmer J. Adult acute leukemia. Dis Mon. 2012;58(4):219-238.

Savage DG, Szydlo RM, Goldman JM. Clinical features at diagnosis in 430 patients with chronic myeloid leukaemia seen at a referral centre over a 16-year period. Br J Haematol. 1997;96(1):111-116.

Yee KW, O'Brien SM. Chronic lymphocytic leukemia: diagnosis and treatment. Mayo Clin Proc. 2006;81(8):1105-1129.

National Comprehensive Cancer Network. Clinical practice guidelines in oncology: acute myeloid leukemia. http://www.nccn.org/professionals/physician_gls/pdf/aml.pdf (subscription required). Accessed January 9, 2014.

Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114(5):937-951.

Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines [published correction appears in Blood . 2008;112(13):5259]. Blood. 2008;111(12):5446-5456.

National Cancer Institute. SEER fast stats 1975–2005. http://seer.cancer.gov/faststats/selections.php . Accessed January 22, 2014.

Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340(17):1330-1340.

Moen MD, McKeage K, Plosker GL, Siddiqui MA. Imatinib: a review of its use in chronic myeloid leukaemia. Drugs. 2007;67(2):299-320.

Coiffier B, Altman A, Pui CH, Younes A, Cairo MS. Guidelines for the management of pediatric and adult tumor lysis syndrome: an evidence-based review [published correction appears in J Clin Oncol . 2010;28(4):708]. J Clin Oncol. 2008;26(16):2767-2778.

National Comprehensive Cancer Network. Clinical practice guidelines in oncology: prevention and treatment of cancer-related infections. http://www.nccn.org/professionals/physician_gls/pdf/infections.pdf (subscription required). Accessed January 9, 2014.

Friedman DL, Whitton J, Leisenring W, et al. Subsequent neoplasms in 5-year survivors of childhood cancer: the Childhood Cancer Survivor Study. J Natl Cancer Inst. 2010;102(14):1083-1095.

Children's Oncology Group Nursing Discipline Clinical Practice Subcommittee/Survivorship; Late Effects Committee. Establishing and enhancing services for childhood cancer survivors: long-term follow-up program resource guide. http://www.survivorshipguidelines.org . Accessed January 22, 2014.

Tsimberidou AM, Wen S, McLaughlin P, et al. Other malignancies in chronic lymphocytic leukemia/small lymphocytic lymphoma. J Clin Oncol. 2009;27(6):904-910.

Kadan-Lottick NS, Dinu I, Wasilewski-Masker K, et al. Osteonecrosis in adult survivors of childhood cancer: a report from the childhood cancer survivor study. J Clin Oncol. 2008;26(18):3038-3045.

Rizzo JD, Wingard JR, Tichelli A, et al. Recommended screening and preventive practices for long-term survivors after hematopoietic cell transplantation: joint recommendations of the European Group for Blood and Marrow Transplantation, the Center for International Blood and Marrow Transplant Research, and the American Society of Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2006;12(2):138-151.

Steinherz LJ, Graham T, Hurwitz R, et al. Guidelines for cardiac monitoring of children during and after anthracycline therapy: report of the Cardiology Committee of the Childrens Cancer Study Group. Pediatrics. 1992;89(5 pt 1):942-949.

Smith LA, Cornelius VR, Plummer CJ, et al. Cardiotoxicity of anthracycline agents for the treatment of cancer: systematic review and meta-analysis of randomised controlled trials. BMC Cancer. 2010;10:337.

Sinisalo M, Aittoniemi J, Käyhty H, Vilpo J. Vaccination against infections in chronic lymphocytic leukemia. Leuk Lymphoma. 2003;44(4):649-652.

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Acute Leukemia Clinical Presentation

Submitted: 27 April 2012 Published: 15 May 2013

DOI: 10.5772/53531

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1. Introduction

Acute leukemias are highly malignant neoplasms and are responsible for a large number of haematopoietic cancer-related deaths ( Jemal et al 2006 ). Although the survival rates have improved remarkably in the younger age group, the prognosis in older patients is still poor ( Redaelli et al 2003 ).

The clinical presentation of acute leukemia results from infiltration of bone marrow or extramedullary sites by blasts. As a result, initial symptoms may be due to the presence of anemia, neutropenia, or thrombocytopenia. Patients generally present with nonspecific complaints including weakness, lethargy, fatigue, dyspnea, fever, weight loss, or bleeding. Blasts may also infiltrate organs or lymph nodes, resulting in hepatosplenomegaly or adenopathy. Bone marrow infiltration with blasts can result in bone pain. Mucosal bleeding, petechiae, ecchymosis, and fundal hemorrhages may occur as a result of thrombocytopenia.

Patients with acute promyelocytic leukemia (APL) characteristically present with coagulopathy and signs of disseminated intravascular coagulation (DIC). It should be noted, however, that rapid cell turnover can result in DIC in any form of acute leukemia.

In acute monocytic leukemia the common findings are weakness, bleeding and a diffuse erythematous skin rash. There is a high frequency of extramedulary infiltration of the lungs, colon, meninges, lymphnodes, bladder and larynx and gingival hyperplasia.

The clinical onset of acute lymphoblastic leukemia (ALL) is most often acute, although a small percentage of cases may evolve insidiously over several months ( Pui 2006 ). The presenting symptoms and signs correlate with the leukemic cell burden and the degree of bone marrow replacement, leading to cytopenias.

Pathophysiology of the clinical manifestations of acute leukemias

2. Signs, symptoms and laboratory features of Acute Myeloblastic Leukemia (AML)

Clinical manifestations of AML result either from the proliferation of leukaemic cells or from bone marrow failure that leads to decrease in normal cells. Leukaemic cells can infiltrate tissues, leading to hepatomegaly, splenomegaly, skin infiltrates and swollen gums. As an indirect effect of the leukaemic proliferation leading to high cell destruction, hyperuricaemia and occasionally renal failure may occur. The haematopoiesis suppression leads to clinical features of anaemia, neutropenia and thrombocytopenia. Signs and symptoms that signal the onset of AML include pallor, fatigue, weakness, palpitations, and dyspnea on exertion. They reflect the development of anemia; however, weakness, loss of sense of wellbeing, and fatigue on exertion may be disproportionate to the severity of anemia. ( Gur et al 1999 ). Easy bruising, petechiae, epistaxis, gingival bleeding, conjunctival hemorrhages, and prolonged bleeding from skin injuries reflect thrombocytopenia and are frequent early manifestations of the disease. Very infrequently gastrointestinal, genitourinary, bronchopulmonary, or central nervous system bleeding can occur at the onset of the disease. Neutropenia translates into infectious manifestations. Pustules or other minor pyogenic infections of the skin and of minor cuts or wounds are most common. Major infections such as pneumonia, pyelonephritis, and meningitis are uncommon as presenting features of the disease, in part because absolute neutrophil counts under 500/μl (0.5 × 10 9 /L) are uncommon until chemotherapy is begun. Anorexia and weight loss are frequent findings. Fever is present in many patients at the time of diagnosis. Myeloid (granulocyte) sarcoma (MS) is an extramedullary tumor that occurs in 2 to 14% of cases of AML ( John et al 2004 ); and is composed of immature and mature granulocytes or monocytes ( Brunning et al 2001 ). These neoplasms are known by a variety of names in the literature, including granulocytic sarcoma, monocytic sarcoma, extramedullary myeloid cell tumor, myelosarcoma, myeloblastoma, and chloroma ( Carneiro et al. 1984 , Valbuena et al 2005 ). Virtually any extramedullary site can be involved by MS. Most patients with MS have a history of a myeloid neoplasm, most often AML and less often a myelodysplastic or myeloproliferative disease ( Brunning et al 2001 ). Alternatively, MS can be the initial manifestation of AML that subsequently involves blood and bone marrow ( Schmitt-Graff et al 2002 ). Very rarely, MS can be the only site of disease. MS is relatively more common in patients who have leukemias with prominent monocytic differentiation, such as acute myelomonocytic or monocytic leukemia and chronic myelomonocytic leukemia ( Menasce et al 1999 , Elenitoba et al 1996). MS manifesting as a testicular mass is uncommon and only rarely has occurred as an isolated mass. The tumors are usually localized ; they often involve bone, periostium, soft tissues, lymph nodes, or skin. Common sites of myeloid sarcoma are orbit and paranasal sinuses. However, it should be noted that according to the WHO classification the infiltrates of any site of the body by myeloid blasts in AML patients are not classified as myeloid sarcoma unless they present with tumor masses in which the tissue architecture is effaced ( Pileri et al 2008 ).

Blasts may infiltrate organs or lymph nodes, resulting in adenopathy or hepatosplenomegaly. Palpable splenomegaly and hepatomegaly occur in about one third of patients. Testicular infiltration is less common in AML than ALL, with an incidence of 1 to 8 % ( Wiernik et al 2001). Meningeal involvement has been reported in 5 to 20% of children and up to 16% of adults with AML ( John et al 2004 ). Leukemic blast cells circulate and enter most tissues in small numbers. Occasionally biopsy or autopsy will uncover marked aggregates or infiltrates of leukemic cells, and less frequently collections of such cells may cause functional disturbances.

3. Signs, symptoms and laboratory features of Acute Promyelocytic Leukemia (APL)

Acute promyelocytic leukaemia (APL) is a distinctive sub-type of acute myeloid leukaemia that has distinct biologic and clinical features.

According to the older French-American-British (FAB) classification of AML, based solely on morphology as determined by the degree of differentiation along different cell lines and the extent of cell maturation ( Cheson et al 1990 ), APL is sub-typed as AML-M3. The new World Health Organization (WHO) classification of AML incorporates and interrelates morphology, cytogenetics, molecular genetics, and immunologic markers and is more universally applicable and prognostically valid ( Brunning et al 2001 ). APL exists as 2 types, hypergranular or typical APL and microgranular (hypogranular) APL. APL comprises 5% to 8% of cases of AML and occurs predominately in adults in midlife ( Büchner et al. 1999 ). Both typical and microgranular APL are commonly associated with DIC ( Karp et al. 1987 , Gollard et al 1996 , Davey et al 1986 , Tobelem et al 1980 ). The severe bleeding diathesis associated with APL has a specific sensitivity to treatment with all-trans retinoic acid (ATRA), which acts as a differentiating agent ( Licht et al 1995 ). High complete remission rates in APL may be obtained by combining ATRA treatment with chemotherapy ( Brunning et al 2001 ).

4. Signs, symptoms and laboratory features of Acute Myelomonocytic (AML-M4) and Acute Monoblastic/Monocytic Leukemia (AML-M5)

Acute myelomonocytic (M4) and monoblastic/monocytic leukemia (M5), are the morphologic subtype of acute myelogenous leukemia that are most commonly characterized by weakness, bleeding and a diffuse erythematous skin rash and frequently presents with extramedullary involvement, including liver, spleen, lymph nodes, gingiva, skin, eyes, larynx, lung, bladder, meninges and the central nervous system. Involvement of the gastrointestinal tract is rare, the mouth, rectum and anal canal being the most affected sites ( Lichtman et al 1995 ). By contrast, leukemic infiltration of the stomach has been very rarely described, and when it has, it has been mainly in children ( Kasantikul et al 1989 ; Kontny et al. 1995 ; Domingo-Domenech et al 2000 ). Serum and urinary muramidase levels are often extremely high.

Neurological symptoms may occur such as, headache, nausea, vomiting, photophobia, cranial nerve palsies, pupil edema and/ or nuchal rigidity. These symptoms may result from leukostasis, but may also reveal meningeal invasion by myeloblasts or be the presenting symptoms of a "chloroma". These chloromas often have an orbital or periorbital localization, or may arise around the spinal cords causing paraparesis or " Cauda equine" syndrome. CNS leukemic infiltration occurs in 6-16% of AML ( Abbott et al 2003 ), especially in AML-M4.

Renal insufficiency occurs seldom. It is caused by hyperuriccuria and / or hyperphosphaturia, leading to obstructing tubular deposits and oliguria/ anuria.

5. Signs, symptoms and laboratory features of Acute Lymphoblastic Leukemia (ALL)

The clinical presentation of ALL may range from insidious nonspecific symptoms to severe acute life-threatening manifestations, reflecting the extent of bone marrow involvement and degree of extramedullary spread ( Pui et al 2006 ) ( Table 2) . The symptoms at onset are primarily produced by the detrimental effects of the expanding cell population on bone marrow, and secondarily by the infiltration of other organs and by metabolic disturbances ( Henderson et al 1990 , Gur et al. 1999 ). In younger patients the anemia-induced fatigue may be the only presenting feature. Dyspnea, angina, dizziness, and lethargy may reflect the degree of anemia in older patients presenting with ALL. Approximately half of all patients may present with fever attributable to the pyrogenic cytokines, such as IL-1, IL-6, TNF, released from the leukemic cells, infections, or both. Arthralgia and bone pain due to bone marrow expansion by the leukemic cells and occasionally necrosis can be observed, although less commonly in adults compared to children. Pallor, petechiae, and ecchymosis in the skin and mucous membranes due to thrombocytopenia, DIC, or a combination of the above may be observed. ALL may present with either leukopenia (~20%) or moderate (50%–5–25 × 10 9 /L) and severe leukocytosis (10%–>100 ×10 9 /L) with hyperleukocytosis (>100 x 10 9 /L ) present in approximately 15% of the pediatric patients ( Pui et al 2006 ). Neutropenia (less than 500 granulocytes per mm 3 ) is a common phenomenon and is associated with an increased risk of serious infection. Hypereosinophilia, generally reactive, may be present at diagnosis. The majority of patients present with platelet counts less than 100 × 10 9 /L (75%), while 15% have platelet counts of less than 10 × 10 9 /L. Decreased platelet counts (median, 50x10 9 /L) are usually present at diagnosis and can be readily distinguished from immune thrombocytopenia, as isolated thrombocytopenia is rare in leukemia. Severe hemorrhage is uncommon, even when platelet counts are as low as 20x10 9 /L, and infection and fever are absent. Coagulopathy, usually mild, can occur in T-cell ALL and is only rarely associated with severe bleeding. More than 75% of the patients presents with anemia, which is usually normochromic and normocytic and associated with a normal to low reticulocyte count. Anemia or thrombocytopenia is often mild (or even absent) in patients with T-cell ALL. Pancytopenia followed by a period of spontaneous hematopoietic recovery may precede the diagnosis of ALL in rare cases and must be differentiated from aplastic anemia.

Clinical features of adult acute lymphocytic leukemias

Bone marrow is usually infiltrated with >90% blast cells. Infiltration with less than 50% blasts represents only 4% of cases. Though the distinction between lymphoblastic leukaemia and lymphoma is still arbitrary, for many treatment protocols 25% bone marrow blasts is used as threshold for defining leukaemia ( Borowitz & Chan 2008 ). Normal trilineage haematopoiesis is consequently decreased. The classical triad of symptoms related to bone marrow failure are the following: (1) fatigue and increasing intolerance to physical exercise (caused by anaemia), (2) easy bruising and bleeding from mucosal surfaces and skin (caused by thrombocytopenia especially when platelets are < 20 × 10 9 /L), and (3) fever with infections (40% of all cases, caused by absolute granulocytopenia). Hyperleukocytic leukaemias with >100 x 10 9 /L blast cells rarely lead to the leukostasis syndrome and catastrophic early bleeding ( Porcu et al 2000 ). Also malaise, lethargy, weight loss, fevers, and night sweats are often present but typically are not severe. Compared to AML, patients with ALL experience more bone and joint pain. Rarely, they may present with asymmetric arthritis, low back pain, diffuse osteopenia, or lytic bone lesions [ Gur et al 1999 ]. Children experience these symptoms more frequently than adults. Young children may have difficulties in walking due to bone pain [ Farhi et al 2000 ]. Lymphadenopathy, splenomegaly, and hepatomegaly are more common than in AML and affect half of the adults with ALL. CNS involvement is also more common in ALL compared to AML. Patients may present with cranial neuropathies (most often involving the 6th and 7th cranial nerves). Nausea, vomiting, headache, or papilledema may result from meningeal infiltration and obstruction of the outflow of cerebrospinal fluid (CSF) leading to a raised intracranial pressure. Testicular involvement, presenting as a painless, unilateral mass, is noted at diagnosis in approximately 2% of boys. It is associated with infant or adolescent age, hyperleukocytosis, splenomegaly, and mediastinal mass [ Farhi et al 2000 ]. The diagnosis of testicular involvement is made by wedge biopsies. Bilateral biopsies are necessary due to the high incidence of contralateral testicular disease [Amendola et al 1985.[

6. Central nervous system involvement

The incidence of CNS involvement in patients with AML is considerably less common than CNS involvement in both adults and children with ALL (Charles et al 2012). Early CNS leukemia occurs in 8% of patients at the time of the first diagnosis while the percentage of relapsing CNS leukemia is 10%. ( Hardiono et al 2001 ).

Patients with CNS involvement may be asymptomatic or may have symptoms related to increased intracranial pressure (headache, nausea, vomiting, irritability). All patients newly diagnosed with ALL should have a lumbar puncture for cytologic analysis of the cerebrospinal fluid; for AML, however, this is performed only in patients with symptoms indicative of CNS involvement ( Pavlovsky et al 1973 ). There is an association of central nervous system involvement and diabetes insipidus in AML with monosomy 7, abnormalities of chromosome 3 and inversion of chromosome 16. ( Glass et al 1987 ; Lavabre-Bertrand et al. 2001 ; Harb et al 2009 ).

Central nervous system hemorrhage and infection are reported to cause 80% ( Lazarus et al 2006 ) of all deaths in patients with leukemia. The intracerebral hemorrhages that are often related to intravascular leukostases and leukemic nodules, and associated with leukocyte counts more than 100x10 9 /L in peripheral blood ( Phair et al 1964 ).

6.1. Leukemic parenchymal tumor

CNS may be affected as a solid tumors consisting of myeloid leukemic blasts called granulocytic sarcomas or chloromas ( Recht et al 2003 , Teshima et al 1990 ). The term chloroma results from the greenish color of these tumors caused by the presence of myeloperoxidase. Chloromas usually have a dural attachment although parenchymal tumors have rarely been reported. These tumors are hypercellular and avidly enhance with either cranial magnetic resonance imaging (MRI) or cranial computed tomography (CT). Neurologic findings are dependent upon location. Chloromas most often occur in bone that may result in epidural spinal cord compression, the orbit that may result in proptosis and a restrictive ophthalmopathy, or dura, which may simulate a meningioma.

6.2. Intracranial hemorrhage

Hemorrhagic complications are common in patients with acute leukemia (approximately 20%) and constitute the second most common cause of death in such patients (20% of all leukemic deaths result from intracranial hemorrhage) ( Kim et al 2004 , Kawanami et al 2002 ). Intracranial hemorrhage (ICH) is the most common hemorrhagic complication in acute promyelocytic leukemia and is not infrequent in AML and ALL (ranging in occurrence from 2-18% of all patients with acute leukemia). ICH may occur at the time of diagnosis (early hemorrhage) or subsequent to diagnosis and following initial treatment (late hemorrhage) ( Cortes et al 2001 ). DIC, disseminated aspergillosis or mucormycosis, leukemic cell infiltration, thrombocytopenia or L-asparaginase chemotherapy-related consequences, are the most common etiologies for ICH. Both DIC (especially common in the M3 subtype of AML) and thrombocytopenia typically result in a solitary often-massive ICH whereas disseminated fungal infection and ICH occurring during neutropenia and is a result of hemorrhagic infarction. Leukemic cell infiltration occurs with extreme leukocytosis (defined as >300x10 9 leukemic cells/L and increase the risk of multiple intracranial hemorrhages in acute leukaemia( Bunin et al 1985 ). L-asparaginase may induce hyperfibrinogenemia and result in cortical vein or sinus thrombosis complicated with venous infarction. Fungal-related mycotic aneurysms may also lead to ICH and would be a consideration in a patient with blood culture positive for fungus. Topographically the majority of ICH is intraparenchymal with cerebral hemorrhage more common than cerebellar. ( Wolk et al 1974 ).

Subarachnoid hemorrhage occurs in the context of ICH, either in isolation or more frequently as more diffuse hemorrhage secondary to DIC. Spinal subarachnoid hemorrhage may occur in the context of DIC and acute promyelocytic leukemia and present primarily with back pain that migrates rostrocaudally.

Risk - factor analysis revealed that female gender, APL, leukocytosis, thrombocytopenia and prolonged PT were the risk factors for fatal intracranial hemorrhages, while other reports have suggested the significance of serum fibrinogen ( Wide et al 1990 ).

6.3. Leukemic meningitis

Meningeal leukemia appears more often in patients with ALL than in those with AML ( Lazarus et al 2006 ). The manner in which leukemia cells enter the CNS is a subject of controversy, but the likely source include hematogenous spread or direct spread from adjacent infiltrated bone marrow.

Meningitis in leukemia may result from leptomeningeal infiltration of tumor (LM), subarachnoid hemorrhage, chemical (treatment-related following intra-CSF instillation of chemotherapy) or infection (bacterial or fungal) ( Cash et al 1987 , Dekker et al 1985 ). The presence or absence of LM always needs to be ascertained as if diagnosed, prognosis is profoundly affected. Chemical meningitis (typically due to intra-CSF cytarabine or methotrexate and most often given intraventricularly) is temporally related to intra-CSF chemotherapy. Chemical meningitis begins one to two days after intra-CSF chemotherapy administration, It is transient typically lasting less than five days and demonstrates no evidence of infection by CSF culture. Like other meningitis syndromes, patients complain of headache, fever, nausea, vomiting, photophobia and meningismus. Notwithstanding an inflammatory CSF, chemical meningitis rapidly abates and is mitigated by oral steroids. Infectious meningitis occurs in leukemia due to immunosuppression both as a result of the underlying disease and its treatment. Listeria , Candida and Aspergillus are common infectious etiologies however clinical presentation differs. Listeria presents as a meningitise syndrome whereas Candida presents with a diffuse encephalopathy and multiple small brain abscesses and Aspergillus presents with progressive hemorrhagic stroke confined to a single vascular territory ( Gerson et al 1985 , Winston et al 1993 ).

6.4. Cerebrospinal fluid in leukemic patients

The cerebrospinal fluid findings in leukemic patients must be carefully evaluated since bacterial meningitis, abscess formation or fungal disease occur with increased frequency. Cerebrospinal fluid pleocytosis, chemical abnormalities (elevated protein and low sugar) and elevated pressure may be present in these potential complications of the disease or its therapy. Appropriate cultures and stains, are often helpful in diagnosis. Abscesses can often be detected by brain scans, electroencephalograms and arteriography.

6.4.1. Categories of CNS status at diagnosis of acute leukemia

Patients who have nontraumatic diagnostic lumbar punctures at diagnosis may be placed into 3 categories according to white blood cells (WBCs) per microliter and the presence or absence of blasts on the cytospin: central nervous system 1 (CNS1) refers to CSF with <5 WBCs per microliter with cytospin negative for blasts; Cxlink refers to CSF with <5 WBCs per microliter with cytospin positive for blasts; CNS3 refers to CSF with >5 WBCs per microliter with cytospin positive for blasts. Children with ALL who presents with CNS disease at diagnosis (CNS3) are at high risk for treatment failure compared with patients not meeting the criteria of the CNS disease at diagnosis. Patients with Cxlink may be at an increased risk of CNS relapse, although this may not apply to all treatment regimens and can be overcome by more intensive intrathecal treatment ( Burger et al 2003 ).

7. Testicular involvement

Involvement of the testis - one of the most common sites of relapse in acute lymphoblastic leukemia usually presents with painless enlargement of one or both testis. Testicular involvement occurs in 10% to 23% of boys during the course of the disease at a median time of 13 months from diagnosis. Occult testicular involvement is recognized in 10% to 33% of boys undergoing bilateral wedge biopsies performed during the first 3 years of treatment or at any time after cessation of the therapy (Lanzkowsky et al. 1985). In a study in which biopsies were done in boys with newly diagnosed ALL, microscopic testicular involvement was reported to be 21% ( Neimeyer et al 1993 ). Testicular involvement of the endothelial side of the interstitium of one or both testis, leads to increased testicular size and firmness [ Kay et al 1983 ]. Hydrocele resulting from lymphatic obstruction may also present with painless scrotal enlargement and is readily identified by ultrasonography. Overt testicular involvement may occur in any form of acute lymphoblastic leukemia, most commonly in common C-ALL, but also in T-ALL and B-ALL. Rarely it is present when ALL is first diagnosed, but most often it is a late complication and, as with meningeal leukemia, the higher the initial blood blast count is, the earlier the discovery of testicular disease is likely ( Nesbit et al 1980 ).

8. Superior vena cava syndrome

Superior vena cava syndrome comprises the signs and symptoms associated with compression or obstruction to the superior vena cava. Patients with ALL (particularly T-ALL), may present with symptoms of cough, dyspnea, stridor, or dysphagia from tracheal and esophageal compression by a mediastinal mass (15% of patients). Compression of the great vessels by a bulky mediastinal mass also may lead to the life threatening superior vena cava syndrome ( Marwaha et al 2011 ). A child with leukemia may experience anxiety, confusion, drowsiness and sometimes unconsciousness ( Salsali et al 1969 ). There is facial edema, plethora, cyanotic faces. Venous engorgement of neck, chest and arm with collateral vessel and some sign of pleural effusion and pericardial effusion may be present ( Rice et al 2006 ).

9. Skin involvement

Various cutaneous lesions can be observed in patients with acute leukemias. These include specific cutaneous lesions resulting from infiltration of the skin by the leukemic cells, characteristic diseases such as pyoderma gangrenosum and Sweet syndrome, cutaneous signs of infection or hemorrhage resulting from the bone marrow dysfunction induced by the malignant process or chemotherapy.

Skin involvement may be of three types: nonspecific lesions, leukemia cutis, or granulocytic sarcoma of skin and subcutis. Nonspecific lesions include macules, papules, vesicles, pyoderma gangrenosum, or vasculitis ( Bourantas et al. 1994 , Nambiar Veettil et al 2009 ), neutrophilic dermatitis (Sweet's syndrome) ( Cho K-H et al 1997 , Philip R Cohen 2007 ), cutis vertices gyrata, or erythema multiforme or nodosum ( Byrd et al 1995 ). Leukemia cutis lesions usually appear at the time of diagnosis of systemic disease or thereafter, but occasionally can occur before peripheral blood or bone marrow involvement (aleukemic leukemia cutis). ( Christos Tziotzios et al 2011 , Márcia Ferreira et al 2006 ). T-cell ALL may show epidermotropism and monocytic leukemia often involves the entire dermis and the superficial panniculus ( Yalcin et al 2004 ).

10. The gastrointestinal tract

Gastrointestinal (GI) manifestations of leukemia occur in up to 25% of patients at autopsy, generally during relapse. Its presence varies with the type of leukemia and has been decreasing over time due to improved chemotherapy. Gross leukemic lesions are most common in the stomach, ileum, and proximal colon. Leukemia in the esophagus and stomach includes hemorrhagic lesions from petechiae to ulcers, leukemic infiltrates, pseudomembranous esophagitis, and fungal esophagitis. ( Dewar et al. 1981 ) The mouth, colon, and anal canal are sites of involvement that most commonly lead to symptoms. Oral manifestations may bring the patient to the dentist; gingival or periodontal infiltration and dental abscesses may lead to an extraction followed by prolonged bleeding or an infected tooth socket. ( Dean et al. 2003 ). The gingival hyperplasia is most commonly seen with the AML subtypes acute monocytic leukemia M5 (67%), acute myelomonocytic leukemia M4 (18.5%) and acute myelocytic leukemia M1-M2 (3.7%) ( Cooper et al 2000 ). Enterocolitis, a necrotizing inflammatory lesion involving the terminal ileum, cecum, and ascending colon, can be a presenting syndrome or can occur during treatment. Fever, abdominal pain, bloody diarrhea, or ileus may be present and occasionally mimic appendicitis. Intestinal perforation, an inflammatory mass, and associated infection with enteric gram-negative bacilli or clostridial species are often associated with a fatal outcome. Isolated involvement of the gastrointestinal tract is rare.( Tim et al 1984 ).. Neutropenic enterocolitis (NE), which is a fulminant necrotizing process is a well-recognized complication of neutropenia in patients dying from hematologic malignancies especially acute leukemia as indicated by various autopsy series ( Steinberg et al 1973 ). Proctitis, especially common in the monocytic variant of AML, can be a presenting sign or a vexing problem during periods of severe granulocytopenia and diarrhea.( Christos Tziotzios et al. 2011 )

11. Respiratory tract involvement

Infectious and noninfectious pulmonary complications represent a critical problem for patients with leukemia, which itself can be the direct cause of pulmonary leukostasis, pulmonary leukemic infiltration (PLI), and leukemic cell lysis pneumopathy. These disorders are usually more frequent in patients with hyperleukocytic leukemia. Pulmonary leukostasis is characterized by occlusion of the pulmonary capillaries and arterioles by leukemic cells. Leukemic infiltration may lead to laryngeal obstruction, parenchymal infiltrates, alveolar septal infiltration, or pleural seeding. Each of these events can result in severe symptoms and radiologic findings ( Potenza et al 2003 , Wu et al 2008 ).

Pulmonary disease in leukaemia is frequent and often lethal. Lung involvement in leukaemia is primarily due to (a) leukostasis of vessels and (b) true leukaemic infiltration of interstitium and alveoli. ( Majhail et al 2004 , Porcu et al 2000 ) Clinically, leukostasis in leukaemia should be suspected in patients with unexplained fever and cardiopulmonary or cerebral dysfunction. Pulmonary leukostasis was found in about40% of autopsy series. ( Mark et al 1987 ). Maile et al 1983 noted parenchymal opacities on 90% of chest radiographs obtained shortly before death in adult patients with leukaemia. These radiologic opacities on autopsy were attributed to infections, haemorrhages, leukaemic infiltrations and edema. In addition, drug induced pulmonary infiltrates and leukoagglutinin transfusion reactions were also reported ( Mark et al 1987 ). In spite of the above data, pulmonary leukostasis in leukaemia has been mentioned only incidentally as a cause of abnormalities on chest radiography.

12. Cardiac complications

Cardiac complications of the patients with acute leukemia are common. Most of the cardiac complications may be due to chemotherapeutics such as antracyclins, besides anemia, infections, or direct leukemic infiltrations of the heart. Symptomatic pericardial infiltrates, transmural ventricular infiltrates with hemorrhage, and endocardial foci with associated intracavitary thrombi can, on occasion, cause heart failure, arrhythmia, and death. Infiltration of the conducting system or valve leaflets or myocardial infarction may occur. ( Ashutosh et al 2002 , Fernando et al 2004 ). Cardiac and other tissue damage as a consequence of release of eosinophil granule contents can occur in patients with leukemia, associated with eosinophilia ( Kocharian et al 2006 ). Cardiac damage is a major determinant of the overall prognosis.

13. Urogenital involvement

The urogenital organs can also be affected. The kidneys are infiltrated with leukemic cells in a high proportion of cases, but functional abnormalities are rare. Hemorrhages in the pelvis or the collecting system are frequent, however, cases of vulvar, bladder neck, prostatic, or testicular involvement have been described.. ( Quien et al 1996 ).

14. Musculoskeletal system

Musculoskeletal manifestations are the presenting complaint in up to 20% of patients with pediatric leukemia,( Andreas et al 2007 ). The main clinical osteoarticular manifestations in early leukemia include limb pain, nighttime pain, arthralgia, and arthritis. Skeletal manifestations of acute leukaemia (bone or back pain, arthritis or radiographical abnormalities of skeleton) are well described in children ( Barbosa et al 2002 ). Arthritis can occur at any time during the course of acute leukaemia. It may lead to delay in diagnosis and therapy and any delay in therapy is associated with poor prognosis ( Sandeep et al 2006 ). The most common clinical presentation of leukaemic arthritis is additive or migratory asymmetrical oligoarticular large joint arthritis and in some cases juvenile idiopathic arthritis. ( Evans et al. 1994 , Mirian et al. 2011 ). The joints most commonly involved are the knee, followed by the ankle, wrist, elbow, shoulder and hip. Onset of arthritis may be sudden or insidious, and parallel the course of acute leukaemia ( Sandeep et al. 2006 ).

Arthritis as the first manifestation of acute leukaemia is however extremely uncommon in adults.

15. Hyperleukocytosis and leukostasis

Leukostasis is a syndrome, caused by clumping of leukocytes in the vasculature of the lungs and brain, often resulting in hypoxia, dyspnea, confusion, and coma, and may be fatal.

Leukapheresis is indicated in the initial management of leukostasis in patients with hyperleukocytosis in acute leukemias, particularly myeloid leukemias, or in patients who are at high risk of developing such a complication.

Adult T-cell leukemia/lymphoma is a distinct form of ALL that presents with progressive lymphadenopathy, hepatosplenomegaly, and hypercalcemia. It involves the skin, lungs, bone marrow, intestinal tract, and CNS. This disease is associated with HTLV-1 and is endemic in the Caribbean, southeastern United States, Africa, and Japan. Circulating tumor cells have a characteristic “cloverleaf”-shaped nucleus.

The risk factors for leukostasis are acute the leukaemia itself, younger age (most common in infants), certain types of leukaemia like acute promyelocytic (microgranular variants), acute myelomonocytic, acute monocytic leukaemia and T cell type of ALL. Cytogenetic abnormalities – 11q23 translocations and presence of Philadelphia chromosome are also associated with leukostasis ( Porcu et al 2000 ). The pathogenesis of leukostasis is determined by: - 1) sluggish flow with stasis, 2) aggregation of leukaemic cells, 3) formation of microthrombi, 4) release of toxic granules, 5) endothelial damage, 6) oxygen consumption by leukocytes, 7) tissue invasion ( Litchman et al 1987 ). Leukostasis is usually associated with counts of >100 x 10 9 but acute monocytic leukaemia may present with leukostasis with counts of 50 x 10 9 /L. 5-13% of patients of AML and 10-30% of patients of ALL will manifest with hyperleukocytosis. Earlier leukostasis was thought to be due to the presence of critical leukocrit (fractional leukocyte volume) and increased viscosity. Although hyperleukocytosis is also common presenting feature in patients with ALL, particularly with T-cell phenotype, 11q23, and t(9;22) chromosomal rearrangements, symptomatic leukostasis is exceedingly rare [ Porcu et al 2000 ]. While WBC count is a major factor contributing to microvessel occlusion seen with leukostasis, other features, such as activation of adhesion cell surface markers and mechanical properties of the leukemic blasts, are likely to be important. For example, the stiffness of myeloid blasts, as measured by atomic force microscopy, is 18 times that of lymphoid blasts [ Rosenbluth et al 2006 ]. This difference in deformability of the cells may at least partially explain the increased frequency of leukostasis in AML compared to than in ALL. Presence of symptoms suggestive of leukostasis, such as headache, blurred vision, dyspnea, hypoxia, constitute a medical emergency and efforts should be made to lower the WBC rapidly. However, the role of leukapheresis to reduce tumor burden in patients with ALL and leukocytosis remains controversial.

16. Metabolic complications

Hyperuricemia and hyperphosphatemia with secondary hypocalcemia are frequently encountered at diagnosis, even before chemotherapy is initiated, especially in patients with B-cell or T-cell ALL with high leukemic cell burden. Severe metabolic abnormalities may accompany the initial diagnosis of ALL and AML ( Haralampos et al 1999 ). Patients with high leukemic burden are at risk of developing acute tumor lysis syndrome (ATLS). Such metabolic changes may lead to the development of oliguric renal failure due to the tubular precipitation of urate and calcium phosphate crystals, fatal cardiac arrhythmias, hypocalcemic tetany, and seizures ( Jeha 2001 ).

17. Lactic acidosis

Lactic acidosis (LA), as the presenting manifestation of acute leukemia, is rare, but potentially fatal complication of acute leukemia ( Grossman et al 1983 ), characterized by low arterial pH due to the accumulation of blood lactate. It has been suggested that LA occurring in the setting of hematological malignancy is associated with an extremely poor prognosis [ Sillos et al 2001 ]. Lactate, the end product of anaerobic glycolysis, is metabolized to glucose by the liver and kidneys. Because leukemic cells have a high rate of glycolysis even in the presence of oxygen and produce a large quantity of lactate, LA may result from an imbalance between lactate production and hepatic lactate utilization [ Sillos et al 2001 ]. Several factors may contribute to the high rate of glycolysis. Overexpression or aberrant expression of glycolytic enzymes, such as hexokinase, the first rate-limiting enzyme in the glycolytic pathway [ Mazurek et al 1997 ] allows leukemic blasts to proliferate rapidly and survive for prolonged periods [ Mathupala et al 1997 ]. Although insulin normally regulates the expression of this enzyme, insulin-like growth factors (IGFs) that are overexpressed by malignant leukemic cells, can mimic insulin activity [ Werner 1996 ,]. LA is frequently associated with acute tumor lysis syndrome (ATLS) and its extent is correlated with the severity of ATLS.

Typically, the patient with lactic acidosis presents with weakness, tachycardia, nausea, mental status changes, hyperventilation, and hypotension, which may progress to frank shock as acidosis worsens. Laboratory studies show a decreased blood pH (<7.37), a widened anion gap (>18), and a low serum bicarbonates.

Definitions of comorbidities and HCT-CI scores included in the HCT-CI

18. Comorbidity

Many factors have been studied to predict outcome and allocate treatment in acute leukemia. The best established prognostic factors are karyotype and age. However, comorbidity may play an important role in the outcome.

A comprehensive assessment including performance status, evaluation of comorbidities and abilities to perform activities of daily living, geriatric depression scale in elderly patients has been proven to be useful in detecting treatment-related changes in older cancer patients and has been recommended to be incorporated into clinical outcome analysis. An index developed specifically for patients with hematologic malignancies has been developed: the Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI) presented in Table 3 ( Sorror ML et al 2005 ). This index captures comorbidities that predict non-relapse mortality in patients considered for allogeneic transplant and also proved to be a helpful tool for defining comorbid conditions in elderly untreated AML patients. ( Novotny J et al 2009 ; Sorror ML et al 2007 ). Modifications such as modified EBMT risk score have been developed and evaluated for ALL patients ( Terwey T et al, 2010 ).

Comorbidity scoring is currently still under the investigation in many cooperative groups. It is important to bear in mind that when translating the results from clinical trials into treatment decision-making for the individual patient, many patients with e.g. „unacceptable“ renal, cardial or hepatic abnormalities are generally not included into clinical trials. By such approach at least 20-30% of younger patients and more than 50% of elderly patients with AML are excluded and have not been reported in any results. Because of that it would be important to propose comorbidity score for all leukemia patients and to evaluate how many of the patients are able to receive standard therapy and stem cell transplantation, how many of them are candidate for low-intensity treatment and supportive care.

While acute leukemia patients depend on the expert recommendations from their physicians, knowledge of clinical presentation and patient's related prognostic factors can help to improve treatment decision and to identify patients who would benefit most from either intensive or low-intensive treatment or even best supportive care alone.

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59. Márcia Ferreira Mónica Caetano et al, Leukemia cutis resembling a flare-up of psoriasis, Dermatology Online Journal 2006 12 3
66. Nambiar Veettil Joe THOMAS et al, Cutaneous vasculitis as a presenting manifestation of acute myeloid leukemia, International Journal of Rheumatic Diseases 2009 12 70 73
72. Philip R Cohen Sweet’s syndrome- a comprehensive review of an acute febrile neutrophilic dermatosis, Orphanet Journal of Rare Diseases 2007

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Clinical presentation of leukemia.

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Clinical Presentation of Leukemia. AAP Grand Rounds December 2016; 36 (6): 70. https://doi.org/10.1542/gr.36-6-70

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Clarke RT , Van den Bruel A , Bankhead C , et al . Clinical presentation of childhood leukaemia: a systematic review and meta-analysis . Arch Dis Child . 2016 ; 101 : 894 – 901 : doi: https://doi.org/10.1136/archdischild-2016-311251 Google Scholar

Investigators from multiple institutions conducted a systematic review to describe how leukemia presents in childhood. They searched MEDLINE and EMBASE databases, using the search terms “leukemia” and “diagnosis” to identify studies that included infants, children, and adolescents. Studies were considered acceptable quality if they defined leukemia according to bone marrow findings, defined at least 2 baseline characteristics of participants, involved a sample that comprised all consecutive cases over the study period, and used a standardized data collection form and/or objectively measured signs. Data on presenting signs and symptoms were then extracted from all included studies that met quality criteria, and pooled proportions of children presenting with each feature were calculated.

Of the 14,963 identified in the original search, 35 studies met eligibility criteria. Of these, 33 met quality criteria and were included in analysis. All included studies were retrospective cohorts, conducted in 21 countries, and described presenting signs and symptoms in a total of 3,084 children. In pooled analysis, the 5 most common presenting signs and symptoms were hepatomegaly (64%), splenomegaly (61%), pallor (54%), fever (53%), and bruising (52%). Other common presenting signs and symptoms were recurrent infections (49%), fatigue (46%), limb pain (43%), hepatosplenomegaly (42%), bruising/petechiae (42%), lymphadenopathy (41%), bleeding tendency (38%), and rash (35%). Only 6% of children were asymptomatic on diagnosis.

The investigators conclude that because >50% of children with leukemia have palpable livers or spleens, children with unexplained illness should receive a focused examination that includes abdominal palpation.

Dr. Hogan has disclosed no financial relationship relevant to this commentary. This commentary does not contain a discussion of an unapproved/investigative use of a commercial product/device.

Leukemia comprises approximately 30% of childhood cancers, affecting an estimated 4,000 children annually. 1 Innate risk factors for leukemia include syndromes of trisomy 21, monosomy 7, immunodeficiency, bone marrow failure, tumor suppressor gene, and DNA repair gene disorders. 2 , 3 Associated exposure risk factors are chemotherapy agents such as topoisomerase II inhibitors, anthracyclines, and alkylating agents for prior cancers. 1 , 3 , 4

Signs and symptoms at leukemia presentation overlap with indicators of infection, allergy, autoimmune disease, or injury. 1 , 3 , 4 Bone or joint pain, limping, adenopathy, fatigue, pallor, petechiae, excessive bruising, and hepatosplenomegaly with or without fever are common. 1 , 4 – 6 Rarer symptoms (which include headache, nausea, vomiting, abdominal pain, weight loss, mucosal bleeding, shortness of breath, or skin lesions) need to be taken in context of symptom duration and severity in addition to a thorough physical examination. 1 , 4 , 5 Initial investigations may include complete blood count with differential white blood cell count, comprehensive metabolic panel, sedimentation rate, lactic dehydrogenase, and uric acid. 1 Although not diagnostic, a mediastinal mass detected on chest radiograph or hepatosplenomegaly on abdominal ultrasound necessitates referral to an oncologist. 1 , 6

The authors of the current study followed a systematic, international search strategy with clear inclusion criteria of published retrospective cohort studies of presenting features of children with any type of leukemia. Their findings substantiate the importance of a careful history and examination of ill-appearing children to discover possible leukemia in a timely manner.

There are limitations of the systematic review, however, which include heterogeneity of terms used to describe signs and symptoms, sample sizes, income status of country, study periods, and leukemia subtypes. No controls were reported to determine diagnostic accuracy. 5 Inherent in any systematic review is publication bias due to lack of unpublished or individual patient data, selection bias due to strict inclusion criteria, and volunteer bias due to missing information from nonparticipants who may have reported other features. Confounding factors contributing to signs and symptoms of leukemia, such as parental reporting, developmental age of child, physician records, comorbidities, and symptom duration and severity, were not addressed in the current study. 1 , 2 , 5 , 6

Bottom Line: Health professionals need to consider leukemia in the differential diagnosis when an ill-appearing child presents with seemingly unrelated symptoms, such as bruising and fatigue, or pallor and limping. A thorough examination for skin lesions, adenopathy and hepatosplenomegaly is essential.

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Clinical Presentation and Diagnosis in Leukemia

Clinical presentation of leukemia.

The presentation of ALL in children typically includes symptoms such as anorexia, fatigue, irritability, bone or joint pain, and low-grade fever. Occasionally, the duration of symptoms prior to diagnosis can be several months and include joint swelling and significant bone pain that wakes the child at night. As bone marrow failure progresses, signs such as pallor, bruising, and mucosal bleeding develop, as well as lymphadenopathy and hepatosplenomegaly. 1

These signs and symptoms can also be present in children with AML. However, the presence of subcutaneous nodules, infiltration of the gingiva, disseminated intravascular coagulation, and masses (chloromas or granulocytic sarcomas) are more specific to AML than ALL. 1

Like pediatric patients, adults with acute leukemia present with symptoms resulting from bone marrow infiltration. 14,15 However, chronic leukemias in adults are often diagnosed incidentally on routine blood tests because initial symptoms are mild. 11,12

Patients with both CLL and CML are often asymptomatic early in the disease course and may slowly develop nonspecific symptoms such as weight loss, fever, and fatigue. Most patients will have lymphadenopathy that is either localized or generalized. Hepatosplenomegaly is less common than lymphadenopathy and skin involvement is uncommon. 16

Patients with CML most commonly present in the chronic phase with nonspecific symptoms like those in patients with CLL. At time of clinical presentation of leukemia, 60 to 70% of patients will have splenomegaly, which is sometimes extreme. The development of fever, significant lymphadenopathy, or skin involvement is concerning.

Diagnosis

Acute lymphoblastic leukemia.

As patients with ALL are typically symptomatic at presentation, a complete blood count (CBC) is usually the first test obtained. This will often reflect anemia and thrombocytopenia. Presentation often includes neutropenia, and 20 to 40% of patients may have profound neutropenia. Alternatively, leukocytosis can also be present at the time of diagnosis. 1,6,7

Leukemic cells may be reported as atypical lymphocytes before further evaluation. Most patients have circulating blasts at the time of diagnosis. 1,6,7

Bone marrow aspiration is required for diagnosis of ALL and is considered diagnostic if greater than 25% of the bone marrow cells are a homogenous population of lymphoblasts. Cerebral spinal fluid examination should also be performed to evaluate for leukocytosis and the presence of blast cells, which are poor prognostic indicators. 1

Additionally, cytochemical staining, assessment of immunological markers, cytogenetic analysis, and evaluation of molecular markers should be performed to distinguish B-cell from T-cell lineage and further identify the specific subgroup of disease. 1,6

Acute Myeloid Leukemia

Similar to patients with ALL, those with AML typically present with anemia and thrombocytopenia. Neutropenia, including profound neutropenia, or leukocytosis may be present at the time of diagnosis. 8

Diagnosis of AML can be made when greater than 20% of bone marrow cells consist of a relatively homogenous population of blast cells that appear to be in early stages of myeloid differentiation. The 2016 World Health Organization (WHO) classification of leukemias incorporates morphology, chromosomal abnormalities, and gene mutations in determination of AML subtype, so evaluation for all of these entities is also an essential part of the diagnostic process. Identification of AML subtype allows for both increased prognostic accuracy and improved selection of appropriate treatment. 1,12,13

Chronic Lymphocytic Leukemia

A routine CBC showing an elevated white blood cell (WBC) count with lymphocytic predominance or a normal WBC count with lymphocytosis on differential is often the first indication of an underlying diagnosis of CLL. 10

When these findings are noted on CBC, flow cytometry should then be performed for further investigation. Flow cytometry demonstrating greater than or equal to 5 × 10 9 /L clonal B cells is diagnostic of CLL. The typical immunophenotype identified in CLL includes B-cell markers CD19, CD20, CD22, CD23, T-cell marker CD-5, and dim surface immunoglobulin kappa or lambda type. Cells should additionally be negative for CD10, CD79b, and FMC7. There are also atypical phenotypes of CLL that are identified by their morphology, cytogenetics, or clinical presentation of leukemia. 10,11

Patients with CLL will often have low levels of immunoglobulins A, G, and M at the time of diagnosis, with the degree of hypogammaglobulinemia correlating to the progression of the disease. This hypogammaglobulinemia makes patients more susceptible to sinopulmonary infections with encapsulated organisms. 10,11

Chronic Myeloid Leukemia

Like CLL, CML is often identified incidentally on routine blood work. The granulocyte count is elevated on CBC in patients with CML and the degree of elevation varies between symptomatic and asymptomatic patients. Asymptomatic patients will typically have a granulocyte count of less than 50 × 10 9 /L, while symptomatic patients can have granulocyte counts of 200 to 1000 × 10 9 /L. Neutrophilia, basophilia, and eosinophilia may also be noted on CBC, as well as thrombocytosis and polycythemia. 17

Bone marrow examination should be undertaken in evaluation for CML. Identification of the Ph chromosome on cytogenetic or molecular studies confirms the diagnosis in the 95% of patients with this genetic abnormality. The 5% of patients with atypical CML require additional testing for diagnosis. CML has the potential to transform into an accelerated phase followed by a blast phase. 13,17

Stages in Leukemia

Because leukemia is a cancer of blood cells and bone marrow, it is generally considered disseminated at diagnosis. Therefore, traditional staging systems that incorporate lymph nodes and metastases are used in other types of cancer and are not applicable to leukemia. 18

The acute leukemias are not staged, but rather prognosis is determined by the genetic and immunophenotypic characteristics used to classify the subtype of disease. CML prognosis and classification are based on the phase of disease (chronic, accelerated, or blast). 18,19

CLL is the one form of leukemia that is classified using staging systems. The Rai system is most often used in the US to stage CLL and the Binet system is used more in Europe. 10,11,18

Rai Staging System

Stage 0 (low risk): Lymphocytosis >5,000 cell /mm and >40% of cells in the bone marrow
Stage 1 (intermediate risk): Lymphocytosis and lymphadenopathy
Stage 2 (intermediate risk): Lymphocytosis and hepatomegaly and/or splenomegaly
Stage 3 (high risk): Lymphocytosis and anemia
Stage 4 (high risk): Lymphocytosis and thrombocytopenia

Binet Staging System

A: <3 areas of lymphadenopathy
B: >3 areas of lymphadenopathy
C: Hemoglobin < 10g/dL and/or platelets <100,000/µL
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Clinical presentation of childhood leukaemia: a systematic review and meta-analysis

Affiliations.

1 Department of Primary Care Health Sciences, University of Oxford, Oxford, UK.
2 Department of Paediatric Oncology/Haematology, Children's Hospital, John Radcliffe, Oxford, UK.
3 Department of Paediatric Oncology/Haematology, Leeds General Infirmary, Leeds, UK.
4 Department of Primary Care Health Sciences, University of Oxford, Oxford, UK Department of Family Medicine, University of Washington, Seattle, USA.
PMID: 27647842
DOI: 10.1136/archdischild-2016-311251

Objective: Leukaemia is the most common cancer of childhood, accounting for a third of cases. In order to assist clinicians in its early detection, we systematically reviewed all existing data on its clinical presentation and estimated the frequency of signs and symptoms presenting at or prior to diagnosis.

Design: We searched MEDLINE and EMBASE for all studies describing presenting features of leukaemia in children (0-18 years) without date or language restriction, and, when appropriate, meta-analysed data from the included studies.

Results: We screened 12 303 abstracts for eligibility and included 33 studies (n=3084) in the analysis. All were cohort studies without control groups. 95 presenting signs and symptoms were identified and ranked according to frequency. Five features were present in >50% of children: hepatomegaly (64%), splenomegaly (61%), pallor (54%), fever (53%) and bruising (52%). An additional eight features were present in a third to a half of children: recurrent infections (49%), fatigue (46%), limb pain (43%), hepatosplenomegaly (42%), bruising/petechiae (42%), lymphadenopathy (41%), bleeding tendency (38%) and rash (35%). 6% of children were asymptomatic on diagnosis.

Conclusions: Over 50% of children with leukaemia have palpable livers, palpable spleens, pallor, fever or bruising on diagnosis. Abdominal symptoms such as anorexia, weight loss, abdominal pain and abdominal distension are common. Musculoskeletal symptoms such as limp and joint pain also feature prominently. Children with unexplained illness require a thorough history and focused clinical examination, which should include abdominal palpation, palpation for lymphadenopathy and careful scrutiny of the skin. Occurrence of multiple symptoms and signs should alert clinicians to possible leukaemia.

Keywords: Haematology; Oncology.

Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Publication types

Meta-Analysis
Systematic Review
Abdominal Pain / etiology
Child, Preschool
Contusions / etiology
Early Detection of Cancer
Exanthema / etiology
Fever / etiology
Gastrointestinal Diseases / etiology
Hemorrhage / etiology
Hepatomegaly / etiology
Infant, Newborn
Infections / etiology
Leukemia / complications
Leukemia / diagnosis*
Musculoskeletal Diseases / etiology
Skin Diseases / etiology
Splenomegaly / etiology

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B-cell prolymphocytic leukemia: an enduring bona fide entity

Case Report
Published: 26 May 2024

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Miguel S. Gonzalez-Mancera 1 ,
Jean Lopategui 1 ,
David Hoffman 2 ,
Sumire Kitahara 1 &
Serhan Alkan 1

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B-cell prolymphocytic leukemia (B-PLL) was recognized as a distinct entity in the fourth edition of the World Health Organization (WHO) classification for hematolymphoid neoplasms (WHO-HAEM4); however, its de novo presentation has been removed from the upcoming 5th edition classification (WHO-HAEM5). We present a case of a 65-year-old man with leukocytosis, fatigue, and no organomegaly by imaging. Bone marrow examination showed a prolymphocytoid population comprising 78% of the marrow elements. After thorough exclusion of other entities by clinical parameters and ancillary methods, we concluded that this case represents a de novo case of B-PLL.

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The data that support the findings of this report are available on request from the corresponding author.

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Miguel S. Gonzalez-Mancera, Jean Lopategui, Sumire Kitahara & Serhan Alkan

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Gonzalez-Mancera, M.S., Lopategui, J., Hoffman, D. et al. B-cell prolymphocytic leukemia: an enduring bona fide entity. Int J Hematol (2024). https://doi.org/10.1007/s12185-024-03774-4

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Case report
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Published: 27 May 2024

A rare incidence of severe dermatological toxicities triggered by concomitant administration of all-trans retinoic acid and triazole antifungal in patients with acute promyelocytic leukemia: a case series and review of the literature

Aisha Jamal ORCID: orcid.org/0000-0001-5022-7498 1 , 3 ,
Rafia Hassam 1 ,
Qurratulain Rizvi 1 ,
Ali Saleem 1 ,
Anum Khalid 2 &
Nida Anwar 1

Journal of Medical Case Reports volume 18 , Article number: 261 ( 2024 ) Cite this article

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All-trans retinoic acid (ATRA) is an indispensable part of the treatment of acute promyelocytic leukemia (APL). Although, mild cutaneous toxicities like mucocutaneous xerosis, rash, and pruritus are well reported, ATRA associated severe dermatological toxicities are extremely rare. ATRA is primary metabolized by cytochrome P450 (CYP450) enzyme system, and triazole antifungals are notorious for their strong inhibitory effect on CYP450.

Case presentation

Three Asian APL patients experienced rare ATRA-induced severe dermatological toxicities: exfoliative dermatitis (ED) in cases 1 and 2, and necrotic scrotal ulceration in case 3. Both case 1 (33-year-old female), and case 2 (28-year-old male) landed in emergency department with dehydration, generalized skin erythema and xerosis during their induction chemotherapy. Both of these patients also developed invasive aspergillosis and required concomitant triazole antifungals during their chemotherapy. For ED, intravenous fluids and broad-spectrum antibiotics were started along with application of local emollients to prevent transdermal water loss. Although their general condition improved but skin exfoliation continued with complete desquamation of palms and soles. Dermatology was consulted, and clinical diagnosis of ED was established. Discontinuation of ATRA resulted in complete resolution of ED. Case 3 (15-year-old boy) reported two blackish mildly tender scrotal lesions during induction chemotherapy. He also had mucocutaneous candidiasis at presentation and was kept on triazole antifungal. Local bacterial & fungal cultures, and serological testing for herpes simplex virus were reported negative. Despite adequate local care and optimal antibiotic support, his lesions persisted, and improved only after temporary discontinuation of ATRA. After a thorough literature review and considering the temporal association of cutaneous toxicities with triazole antifungals, we speculate that the concomitant use of triazole antifungals inhibited the hepatic metabolism of ATRA, resulting in higher serum ATRA concentration, and markedly accentuated cutaneous toxicities in our patients.

By highlighting this crucial pharmacokinetic interaction, we want to caution the fellow oncologists to be mindful of the inhibitory effect of triazole antifungals on CYP450. We propose using a non-myelosuppressive combination of ATRA and arsenic trioxide for management of APL hence, obliterating the need of prophylactic antifungals. However, in the event of invasive fungal infection (IFI), we suggest using alternative class of antifungals.

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Acute promyelocytic leukemia (APL) is a rare and potentially curable subtype of acute myeloid leukemia (AML), accounting for 5–8% of AML cases [ 1 ]. Genetically, APL is characterized by reciprocal translocation t(15:17) (q22;q11–12), with consequent fusion of promyelocytic (PML) gene on chromosome 15q22 to retinoic acid receptor-alpha (RAR-alpha) gene on chromosome 17q21. The resultant fusion oncoprotein, PML-RARA, induces transcriptional repression, chromatin condensation, maturation arrest, and accumulation of abnormal promyelocytes [ 2 ]. Advent of all-trans retinoic acid (ATRA) has revolutionized the treatment landscape of APL, and along with the backbone of anthracycline based chemotherapy, it is considered to be the standard of care for APL patients. Combination treatment with ATRA plus anthracycline based chemotherapy achieves an overall complete remission and cure rate of 95% and 80% respectively, rendering ATRA indispensable in the management of APL [ 3 ].

ATRA, an active metabolite of vitamin A, belongs to a class of retinoids. Although retinoids are well known for their dermatological side effects like xerosis, xerostomia, erythema, pruritis, and exfoliation; severe dermatological side effects of ATRA, especially in the dosage pertinent to APL (45 mg/m 2 ), are rare. So far, only a single case of exfoliative dermatitis (ED) and a few cases of scrotal ulceration have been reported in literature [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. We, here in, report a case series of three patients with serious and rare ATRA associated dermatological complications. We have also discussed upon the potentially precipitating pharmacokinetic interactions, as well as the detailed clinical course and management of our patients as simply withholding ATRA can jeopardize the outcome of this potentially curable malignant disorder.

In all three patients, ATRA was started as soon as abnormal promyelocytes were documented on peripheral smear/bone marrow aspirate examination (Figs. 1 , 2 , 3 ). Diagnosis was further confirmed through cytogenetic analysis as well as PML-RARA detection by polymerase chain reaction. Additionally, in all three patients, chemotherapeutic treatment was instituted according to European APL protocol, based on their risk-group classification.

Exfoliative dermatitis &Onychomadesis (CASE 1). a Peripheral smear. b Bone marrow aspirate. c Desquamation of soles. d Desquamation of palms. e Dry exfoliation of feet and shins. f Onychomadesis

Exfoliative dermatitis (CASE 2). a Peripheral smear. b Bone marrow aspirate. c and d Erythema and scaling of hands. e Cutaneous desquamation of soles

Scrotal lesions (CASE 3). a Peripheral smear. b Bone marrow aspirate. c and d Necrotic scrotal lesions with black eschar

Case 1: A 33-year-old Asian female presented in ER with history of fever, heavy menstrual bleeding and rash all over body. Induction chemotherapy and steroid prophylaxis was promptly started to prevent differentiation syndrome (DS). On Day-10 of induction chemotherapy, she developed high grade fever, cough and shortness of breath. High-resolution computerized tomography (HRCT) showed randomly scattered discrete nodular opacities with surrounding ground glass haze in both lung fields, suggestive of invasive fungal infection (IFI). Voriconazole was immediately started along with broad-spectrum antibiotics. She improved over the following 72 hour, and was discharged from hospital on Day-17. Subsequently, she landed in emergency department on Day-23 with severe dehydration, shivering, tachycardia, generalized skin erythema and discoloration of nail beds. Intravenous fluids and broad-spectrum antibiotics were started along with application of local emollients to prevent transdermal water loss. Over the next 24-36 hour, her general condition was stabilized however; skin exfoliation continued with complete desquamation of palms and soles (Fig. 1 ). Dermatology was consulted, and a clinical diagnosis of onychomadesis and exfoliative dermatitis (ED) was made. A review of her clinical case demonstrated no apparent cause for ED except for a rare association with ATRA. However, considering the curative potential of ATRA, it was continued till Day-28 as per protocol. Her skin condition gradually resolved over next 10–14 days after discontinuation of ATRA. She had recurrence of similar skin condition upon re-exposure to ATRA in her consolidation chemotherapeutic cycles, however, the exfoliation was mild and patchy that responded well to good oral hydration and local skin emollients.

Case 2: A 28-year-old Asian male presented in the out-patient clinic with the history of generalized weakness, high-grade-fever, productive cough and bruises over body. On examination, he had multiple ecchymosis and petechiae with coarse crepitations involving right-middle and left-lower lung fields. He was promptly started on broad-spectrum antibiotics. Additionally, as per protocol, induction chemotherapy and dexamethasone prophylaxis was also instituted. His fever and cough remained unresponsive despite broad-spectrum antibiotics. Voriconazole was instituted upon the identification of IFI on HRCT findings. By day-10, coagulopathy was normalized, and clearance of abnormal promyelocytes was documented by Day-18. On Day-20, he complained of skin dryness, itching and scaling; physical examination revealed generalized xerosis and erythema (Fig. 2 ). Despite aggressive skin care, generalized skin exfoliation, most pronounced on palms and soles, ensued. Clinical diagnosis of ED was established after obtaining dermatological consultation. However, in view of his clinical stability, ATRA was continued. Bone marrow aspirate on Day-28 showed morphological remission. Recurrence of erythema and exfoliation was documented during consolidation phase of chemotherapy, but the condition was responsive to local emollients and oral hydration.

Case 3: A 15-year-old Asian male presented in the out-patient clinic with complains of high-grade-fever, muco-cutaneous bleeding and pancytopenia. On presentation, patient was febrile and had oral thrush. After sending his baseline tests he was taken on broad-spectrum antibiotics and triazole antifungal (itraconazole). After completion of induction chemotherapy, patient was discharged with bi-weekly follow-ups.On Day15, he reported two blackish, mildly tender scrotal lesions with minimal serous discharge (Fig. 3 ). Antibiotic cover for soft tissue infection was commenced along with local wound care with topical steroids and antibiotics. He had no sign of systemic infection/sepsis. Local bacterial & fungal cultures and serological testing for herpes simplex virus were reported negative. Despite adequate local care and optimal antibiotic support, his lesions showed no sign of healing, and two new lesions were developed. Lesion biopsy for histopathological evaluation was declined by the patient. Keeping the rare but reported occurrence of ATRA-induced scrotal ulceration and fournier's gangrene; ATRA was transiently withheld for ten days and the lesions started to regress. However, considering the indispensable role of ATRA in APL, it was reinstituted. Scrotal lesions persisted without any worsening. ATRA was stopped after completion of protocol. Complete resolution of scrotal lesions was documented over the following two weeks. Afterwards, he received two cycles of consolidation chemotherapy, but no recurrence was reported.

Discussion and conclusion

The antineoplastic role of ATRA remains indispensable in the curative management of APL. It is considered a relatively safe drug with a well-known toxicity profile. Commonly reported adverse events include DS, pseudotumor-cerebri, hypertriglyceridemia, transaminitis, and headache. Although, mild cutaneous toxicities like muco-cutaneous xerosis, photosensitivity, rash, pruritus and sweet’s syndrome are well reported, severe dermatological toxicities are rarely reported in literature [ 18 , 19 ]. In this case series, we have discussed three cases of ATRA-induced rare dermatological complications in APL.

Case 1 and 2 developed ED during remission induction phase of chemotherapy. Literature review revealed only a single reported occurrence of ATRA-induced ED in APL by YonelIpek et al. [ 4 ]. ED is a potentially life-threatening cutaneous manifestation that is characterized by diffuse skin erythema and scaling. Various underlying disorders can trigger its onset through a complex interplay of inflammatory cytokines and phagocytes. In contrast to our cases, the case reported by Yonel Ipek et al. developed xerosis in consolidation phase, which akin to our cases started after two weeks of ATRA exposure and rapidly deteriorated to generalized erythroderma and scaling. In both cases, discontinuation of ATRA resulted in complete resolution of ED.

In case 3, we have reported ATRA-induced necrotic scrotal ulceration. Literature review revealed that over the last two decades, a total of twenty cases of ATRA-induced scrotal ulceration have been reported. Histopathological evaluation of these lesions revealed atypical granulocytic infiltration, pointing towards the possible etiological role of differentiated APL cells in the pathogenesis. Most of these cases, including ours, developed genital-lesions almost after two weeks of ATRA exposure and remained unresponsive to local and systemic antibiotics. ATRA had to be halted in most of the cases to prevent progression to fournier’s gangrene [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].

Scattered over the span of three years and considered in isolation, it was not initially apparent to us that all three cases had one striking similarity: concomitant use of ATRA and triazole antifungals. ATRA is primary metabolized by cytochrome P450 enzyme system. Triazole antifungals are notorious for their strong inhibitory effect on CYP450 enzyme system, resulting in supra-therapeutic drug levels and toxicity [ 20 , 21 , 22 ].

Potentiation of serum ATRA levels by inhibition of CYP450 system was first explored by Rigas et al. [ 23 ]. This study reported 1.8 times higher serum concentration of ATRA with concomitant use of ketoconazole. Since then a number of cases have reported the augmentation of ATRA-induced toxicities due to this pharmacokinetic interaction. Concomitant use of ATRA and triazole antifungals that is voriconazole and posaconazole has been implicated to cause severe hypercalcemia [ 24 , 25 , 26 , 27 ]. Similarly, combination with fluconazole has been reported to cause severe neurotoxicity and nephrotoxicity [ 28 , 29 ].

Considering the temporal association of dermatological complications with triazole antifungals in our patients, we speculate that the concomitant use of triazole antifungals inhibited the metabolism of ATRA, resulting in higher serum concentrations and markedly accentuated cutaneous toxicities. A study further strengthening our hypothesis was conducted by Kurzrock et al. to evaluate the maximum tolerable dose of ATRA in myelodysplastic syndrome. The study reported severe dose-limiting cutaneous toxicities, such as generalized desquamation and genital ulceration, at doses > 150 mg/m 2 /day, compared to mild xerosis and erythema in the dose range of 45–100 mg/m 2 /day. Akin to our cases, the study reported complete resolution of cutaneous toxicities within 1–2 weeks of ATRA discontinuation [ 30 ].

Another important point is the recurrence of ED in both case 1 and 2 during their consolidation chemotherapy cycles, whereas recurrent scrotal ulceration was not documented in case 3. The most likely explanation is the continuation of voriconazole as secondary prophylaxis in patients with invasive fungal infections (IFI) (case 1 and 2), whereas itraconazole was discontinued after remission induction in case 3. This once again underscores the pharmacokinetic potentiation of ATRA-induced cutaneous toxicities by triazole antifungals. An important limitation of our study is that, due to the unavailability of serum voriconazole testing, we couldn’t document serum voriconazole levels, something that could provide valuable insights into the effect of serum azole levels on the severity of cutaneous manifestations.

By highlighting this crucial pharmacokinetic interaction and its potentially severe implications, we urge our fellow oncologists to remain vigilant regarding the inhibitory effects of triazole antifungals on the metabolism of ATRA. We propose the use of a non-myelosuppressive combination of ATRA and arsenic trioxide for APL, thereby eliminating the need for prophylactic antifungals. In the case of invasive fungal infections (IFI), we recommend considering alternative classes of antifungals. However, if triazole antifungals are deemed unavoidable, we suggest close monitoring for potential side effects and implementing prophylactic measures as clinically necessary.

Availability of data and materials

Data sharing is not applicable to this manuscript as no datasets were generated or analyzed during the current study.

Abbreviations

Acute promyelocytic leukemia

All-trans retinoic acid

Cytochrome P450

Differentiation syndrome

Exfoliative dermatitis

High-resolution computerized tomography

Invasive fungal infections

Promyelocytic leukemia-retinoic acid receptor alpha

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Aisha Jamal, Rafia Hassam, Qurratulain Rizvi, Ali Saleem & Nida Anwar

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Anum Khalid

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Aisha Jamal: Conceptualization, writing-original draft, writing-review & editing. Rafia Hassam: Conceptualization, writing-original draft. Qurratulain Rizvi: Writing-review & editing. Ali Saleem: Data curation, writing—review & editing. Anum Khalid: Writing—review &editing. Nida Anwar: Writing—review & editing.

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Jamal, A., Hassam, R., Rizvi, Q. et al. A rare incidence of severe dermatological toxicities triggered by concomitant administration of all-trans retinoic acid and triazole antifungal in patients with acute promyelocytic leukemia: a case series and review of the literature. J Med Case Reports 18 , 261 (2024). https://doi.org/10.1186/s13256-024-04577-1

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Acute myeloid leukemia with neutrophilic differentiation in a 12-year-old African lion ( Panthera leo )

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To describe the clinical presentation, progression, and diagnosis of acute myeloid leukemia (AML) with neutrophilic differentiation in an African lion ( Panthera leo ).

A 12-year-old male African lion kept at a zoological institution in Colombia.

CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES

The lion presented for anorexia, pale mucous membranes, and a hind limb lameness of acute onset. Feline leukemia virus testing was negative, and repeated blood samples revealed severe anemia, intermittent thrombocytopenia, lymphopenia, and neutrophilia. Coinfection with Anaplasma and Mycoplasma spp and chronic kidney disease were diagnosed based on clinicopathological findings.

TREATMENT AND OUTCOME

The lion received symptomatic treatment, doxycycline, and methylprednisolone or prednisolone. Euthanasia was elected due to clinical deterioration and unresponsive anemia, despite the resolution of Anaplasma and Mycoplasma spp infections. AML with neutrophilic differentiation was diagnosed based on bone marrow cytology, histopathology, and immunohistochemistry.

CLINICAL RELEVANCE

AML is a rare, aggressive hematopoietic disorder in domestic cats, although it has not yet been reported in nondomestic cats. This is the first description of the clinicopathological, histological, and immunohistochemical features of AML with neutrophilic differentiation in an FeLV-negative African lion that lacked circulating blasts.

A 12-year-old 190-kg intact male African lion ( Panthera leo ) housed at the Cali Zoo in Cali, Colombia, presented for anorexia, pale mucous membranes, steatorrhea and a mild right hind limb lameness. A CBC and serum biochemical profile (SBP) 2 months prior revealed a mild normocytic normochromic anemia (Hct, 23.8%; reference range [rr], 26.8 to 44.1) and total hypercalcemia (12.7 mg/dL; rr, 5.29 to 9.75 mg/dL). The lion had been vaccinated against rabies, feline panleukopenia, calicivirus, and herpesvirus within the 2 years before presentation.

Diagnostic Findings and Interpretation

Initial physical examination revealed dental calculus, low body condition score (2.5/5), and diffuse alopecia. A CBC and SBP showed a severe macrocytic normochromic anemia (Hct, 7.2%) and mildly elevated direct (0.19 mg/dL; rr, 0 to 0.08 mg/dl) and total bilirubin (0.94 mg/dL; rr, 0 to 0.85 mg/dl). The symmetric dimethylarginine concentration was above the upper reference limit for this biomarker in cats (16 µg/dL; upper limit of rr for cats, 14 µg/dL). Ultrasonographic examination of the thorax and abdomen revealed pericardial effusion, heterogeneous liver echogenicity, splenomegaly, enlarged left adrenal gland (cranial pole width, 7.0 mm; caudal pole width, 9.6 mm), and dilated left ureter (width, 6.3 mm). A neoplastic process was suspected, so a fine-needle aspirate of the spleen was performed, revealing a mixed lymphoid population predominated by small and medium lymphocytes, a few large lymphocytes and plasma cells, and several activated macrophages exhibiting erythrophagocytosis. Numerous megakaryocytes and metarubricyte aggregates were found. These findings suggested extramedullary hematopoiesis in the spleen, suspected to be secondary to immune-mediated hemolytic anemia.

Initial treatment consisted of ceftazidime (20 mg/kg, IV), methylprednisolone (2 mg/kg, IM, q 12 h for 2 days), and furosemide (1 mg/kg, IM, q 12 h for 4 days) as well as symptomatic care including nutritional supplements, gastroprotectants, and pain medication. The lion tested negative for FeLV, FIV, feline herpesvirus-1, feline panleukopenia virus, and Babesia, Bartonella, Ehrlichia, Hepatozoon , and Mycoplasma spp via PCR. Blood samples tested positive for Anaplasma sp via PCR, and structures morphologically consistent with Mycoplasma sp were observed on peripheral blood smears on day 7. A CBC and SBP revealed severe macrocytic hypochromic anemia (Hct, 16.8%), mild reticulocytosis (69.9 X 10 3 cells/µL; rr, 3 X 10 3 to 50 X 10 3 cells/µL), moderate thrombocytopenia (92 X 10 3 cells/µL; rr, 226 X 10 3 to 368 X 10 3 cells/µL), mildly elevated creatinine (3.8 mg/dL; rr, 0.89 to 3.23 mg/dL), moderately increased ALT (370.5 U/L; rr, 6.6 to 91.4 U/L), and severe hyperphosphatemia (21.36 mg/dL; rr, 4.42 to 5.8 mg/dL). A dipstick urinalysis of a sample collected from substrate revealed 3+ for blood, proteinuria (1+), and low specific gravity (1,018). Based on the new clinicopathological findings, anaplasmosis, mycoplasmosis, and chronic kidney disease (CKD) were diagnosed.

Treatment and Outcome

Treatment was adjusted, and the lion was administered doxycycline (5 to 7.5 mg/kg, PO, q 12 to 24 h for 19 days) and prednisolone (0.6 to 2 mg/kg, PO, q 12 to 24 h for 26 days). On day 17, the macrocytic hypochromic anemia (Hct, 13.2%) and hypercreatinemia (3.9 mg/dL) persisted, and moderate neutrophilia (24.9 X 10 3 cells/µL; rr, 3.9 X 10 3 to 18.54 X 10 3 cells/µL) developed. On day 19, the lion presented a seizure and received 1 L of whole blood from a clinically healthy 5-year-old intact female lion, with an Hct of 48%; and that was negative for Anaplasma, Babesia, Bartonella, Borrelia, Ehrlichia, Hepatozoon , and Mycoplasma spp, FeLV, FIV, and for antibodies to heartworm. Severe normocytic hypochromic anemia (Hct, 13.53%), mild thrombocytopenia (182 X 10 3 cells/µL), severe lymphopenia (0.3 X 10 3 cells/µL; rr, 0.76 X 10 3 to 7.22 X 10 3 cells/µL), mild hyperphosphatemia (6.87 mg/dL), moderate hypercreatinemia (4.07 mg/dL), moderate direct hyperbilirubinemia (0.21 mg/dL), and severe hypokalemia (2.65 mmol/L; rr, 3.56 to 4.17 mmol/L) were identified on CBC and SBP. Polymerase chain reaction testing of blood samples for Anaplasma sp was negative. On day 44, the lion had developed ischemic necrosis of the distal tail and a body condition score of 1/5, weight loss (body weight, 140 kg), pale mucous membranes, oral ulceration, multiple dermal erosions and excoriations, multifocal alopecia, scrotal dermatitis, right hind limb dermatitis and edema, and foot pad ulceration were found on physical examination. A CBC and SBP revealed a persistent severe normocytic normochromic anemia (Hct, 17.1%), moderate neutrophilia (20.1 X 10 3 cells/µL) with a mild left shift, moderately elevated direct bilirubin (0.19 mg/dL), moderate hyperphosphatemia (7.12 mg/dL), and severe hypokalemia (2.9 mmol/L) and hypercreatinemia (5.35 mg/dL). Serum symmetric dimethylarginine concentration had increased to 48 µg/dL. Abdominal ultrasonography showed heterogeneous echogenicity of the liver, dilated intrahepatic biliary ducts, and loss of kidney corticomedullary distinction. Euthanasia was performed due to the lion’s clinical deterioration and unresponsive anemia. Because a hematopoietic disorder was suspected, bone marrow aspirates and a postmortem examination were performed immediately after euthanasia. Gross postmortem findings included gastric mucosal erosion, irregular, asymmetric kidneys with poor corticomedullary distinction, and areas of renal cortical narrowing and cortical hemorrhage.

Histologically, bone marrow appeared hypercellular (> 90%) with numerous immature precursors in a paratrabecular distribution and extending toward the center of the marrow ( Figure 1 ) . Megakaryocytes, metarubricyte aggregates, and neutrophil and eosinophil granulocytes were occasionally seen. Bone marrow cytology revealed a predominant myeloid population with increased myeloblasts and progranulocytes ( Figure 2 ) , increased myeloid-to-erythroid ratio, and reduced erythroid and myeloid maturation ( Table 1 ) . Numerous metarubricytes with contracted nuclei and irregular and angular cytoplasm were observed. Paraffin-embedded bone marrow sections were stained with myeloperoxidase, CD11c, CD3, CD79a, and CD14. Most cells (70% to 80%) were positive for myeloperoxidase ( Figure 1 ), while <30% were either CD3 or CD79a positive. The spleen exhibited severe multifocal erythrophagocytosis and proliferation of poorly differentiated blasts with occasional mitosis in the red pulp and marginal zone ( Figure 3 ) . Based on these findings, acute myeloid leukemia (AML) with neutrophilic differentiation in bone marrow and spleen was diagnosed.

Photomicrographs of sections of bone marrow from the African lion. A—High cellularity is evident (> 90%), with scattered megakaryocytes and no visible fat cells. H&E stain; bar = 100 µm. B—Most cells stain brown, indicating that these are positive for myeloperoxidase. Immunohistochemical stain specific for myeloperoxidase; bar = 100 µm.

Citation: Journal of the American Veterinary Medical Association 262, 3; 10.2460/javma.23.09.0530

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Cytology of the lion’s bone marrow aspirates performed immediately after euthanasia. A—Myeloid lineage is predominant, and neutrophilic differentiation is observed as immature myeloid precursors, metamyelocytes, bands, and segmented neutrophils. Wright-Giemsa stain. B—A myeloblast (1), myelocytes (2), metamyelocytes (3), and a rubriblast (4) are shown. Wright-Giemsa stain.

Bone marrow differential cell counts from a male African lion that was euthanized following clinical deterioration associated with acute myeloid leukemia with neutrophilic differentiation. Proportions of cell types were calculated in relation to 326 cells that presented adequate morphology for cell type classification.

Photomicrographs of sections of bone marrow and spleen from the African lion. A—Bone marrow cell population predominantly formed by blasts and undifferentiated round cells with occasional mitotic figures. H&E stain; bar = 20 µm. B—Detail of splenic marginal zone with similar undifferentiated round cells and blasts with scattered mitotic figures. Erythrophagocytosis, numerous siderophages, and a few neutrophils are also observed. H&E stain; bar = 20 µm.

Acute tubular necrosis, severe medullary nephrocalcinosis, and mild cortical fibrosis were found in both kidneys, along with acute interstitial nephritis of the left kidney. Focal biliary duct ectasia, diffuse cholestasis, mild multifocal hepatic centrilobular necrosis, a lung adenocarcinoma in situ, and thyroid follicular adenoma were also identified.

AML is a rare and aggressive myeloid neoplasm where clonal myeloid progenitor cells unable to mature accumulate in bone marrow and blood due to aberrant proliferation. 1

AML is characterized by moderate to marked, persistent cytopenia in blood and ≥ 20% blasts in blood or bone marrow. Although the myeloblast percentage was 11.6% in the case of this lion, promyelocytes (27.3%) were counted as blasts for the total blast percentage calculation, as it has been previously performed in humans for the diagnosis of AML. 2

AML generally results from acquired genetic abnormalities in stem cells, and cell type–specific mutations lead to a predominant cell lineage, allowing the classification of AML into subtypes. Neutrophilic (M2) differentiation has been most commonly reported in cats. 3

Hallmarks of the M2 subtype proposed by the Animal Leukemia Study Group in 1991 were present in this case, including > 30% to < 90% blasts (including promyelocytes) calculated in relation to nonerythroid cells, > 10% of differentiated granulocytes, and < 20% of monocytic cells. These findings along with the elevated myeloid-to-erythroid ratio and the immunohistochemical findings supported the diagnosis of AML with neutrophilic differentiation in this lion. 3 Even though hematopoietic tumors occur in captive wild felids, AML specifically has not been previously reported in nondomestic cats.

The diagnosis of AML is based on CBC findings, bone marrow cytology, and occasionally histopathology and immunohistochemistry. The bone marrow in AML is usually normo- to hypercellular, and there is a profound reduction in 1 or 2 cell lines with expansion of the neoplastic cells. Leukemic infiltration of the spleen may also occur, as described here. 1

Cytochemical, immunophenotypic, and genetic features of neoplastic cells are used to classify AML in humans; however, cytochemical heterogeneity is observed in M2 cases in cats, and AML-associated antigens and genetic abnormalities require further investigation in this species. 3 In our case, the markers CD3 and CD79a were used to rule out T-cell and B-cell leukemia/lymphoma, respectively, and CD11c and CD14 were assessed to rule out AML with monocytic differentiation. Myeloperoxidase staining confirmed neutrophilic differentiation in this lion.

AML carries a grave prognosis and rarely responds to chemotherapy protocols, and when present the response is commonly short-lived. 1 The lion presented in this report had a short survival time and underwent rapid clinical deterioration in agreement with previous reports of AML in domestic carnivores.

This lion concurrently presented with other conditions, such as CKD, anaplasmosis, and mycoplasmosis. Anemia, dehydration, and hyperphosphatemia can also be secondary to CKD and can contribute to the development and progression of CKD as well. Persistent anemia and thrombocytopenia, signs of myeloid neoplasia that are often present in AML, were initially attributed to mycoplasmosis and anaplasmosis, respectively. 3 Anemia, anorexia, and lameness are clinical signs of Anaplasma phagocytophilum infection in cats. However, a previous study 4 failed to find a correlation between a positive PCR test result for A phagocytophilum and the presence of anemia or thrombocytopenia in apparently healthy adult feral cats. Asymptomatic A phagocytophilum infection is described in young lions as well. Thrombocytopenia is reported in cats with Mycoplasma haemofelis , and it appeared to resolve in this lion after doxycycline treatment. 5

Nonspecific signs such as weakness, inappetence, and weight loss are associated with cytopenias and metabolic or paraneoplastic complications. 1 Although circulating abnormal leukocytes are frequently seen in cats, they can be absent in some AML subtypes as reported here. 3 Finally, acute leukemia is commonly associated with FeLV infection in cats; however, negative PCR testing did not indicate FeLV infection in this case. 1

AML is a rare, aggressive neoplasm of domestic cats that has not previously been reported in nondomestic cats. Although this is the first report of AML in a nondomestic cat, AML should be considered as a differential diagnosis in FeLV-negative wild felids that have persistent cytopenias and lack circulating myeloblasts. Additionally, published criteria for classification of this disorder in dogs and cats may be useful for evaluation in nondomestic cats, but further study is warranted.

Acknowledgments

Dr. Diego Gomez and Dr. Brodie Reinhart provided valuable comments on previous versions of this manuscript.

Disclosures

The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.

The authors have nothing to disclose.

Dobson J , Villiers E , Morris J . Diagnosis and management of leukaemia in dogs and cats . In Pract . 2006 ; 28 ( 1 ): 28 . doi: 10.1136/inpract.28.1.22

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Vardiman JW , Thiele J , Arber DA , et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes . Blood . 2009 ; 114 ( 5 ): 937 - 951 . doi: 10.1182/blood-2009-03-209262

Jain NC . Classification of myeloproliferative disorders in cats using criteria proposed by the animal leukaemia study group: a retrospective study of 181 cases (1969-1992) . Comp Haematol Int . 1993 ; 3 ( 3 ): 125 - 134 . doi: 10.1007/BF00186096

Galemore ER , Labato MA , O’Neil E . Prevalence of Anaplasma phagocytophilum infection in feral cats in Massachusetts . JFMS Open Rep . 2018 ; 4 ( 1 ): 2055116917753804 . doi: 10.1177/2055116917753804

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Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia

Alena Malyukova 1 na1 ,
Mari Lahnalampi 2 na1 ,
Ton Falqués-Costa 3 ,
Petri Pölönen 2 ,
Mikko Sipola 2 ,
Juha Mehtonen 2 ,
Susanna Teppo 4 ,
Karen Akopyan 1 ,
Johanna Viiliainen 1 ,
Olli Lohi 4 ,
Anna K. Hagström-Andersson 3 ,
Merja Heinäniemi ORCID: orcid.org/0000-0001-6190-3439 2 na1 &
Olle Sangfelt 1 na1

Genome Biology volume 25 , Article number: 143 ( 2024 ) Cite this article

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Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence.

Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers.

Conclusions

Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

The WEE1 checkpoint kinase is a key regulator of the G2/M and G1/S transitions during cell division cycle [ 1 , 2 ] through inhibitory phosphorylation of cyclin-dependent kinases 1/2 (CDK1/2) [ 3 , 4 ]. WEE1 supports genome integrity by suppressing excessive origin firing during DNA replication and premature entry into mitosis [ 5 , 6 , 7 ]. Consequently, inhibition of WEE1 removes the negative phosphorylation on CDK1/2 (in concert with CDC25) resulting in deregulation of CDKs, increased origin firing and replication fork degradation leading to fork collapse, DNA damage, and unscheduled mitosis [ 1 , 8 , 9 , 10 ]. Recent studies show that WEE1 inhibition by AZD1775 (Adavosertib), a potent and specific small-molecule inhibitor, alone or in combination with DNA damaging agents effectively kills cancer cells of various origins [ 8 , 11 , 12 ]. The targeting of DNA damage response-related proteins while also challenging the cell with genotoxic agents is a widely adopted concept in cancer therapy [ 13 ]. However, clinical application is restrained by a high rate of toxicity in combination with chemotherapy [ 13 , 14 , 15 , 16 ].

Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, typically of B-cell lineage. Approximately 75% of B-ALL cases contain chromosomal rearrangements involving alteration of lineage-specific transcription factors (TFs) that are critical regulators of B-cell development and serve as prognostic markers underlying clinical responses. Mixed lineage leukemia (MLL) refers to chromosomal translocations involving the gene KMT2A which encodes histone lysine N-methyltransferase 2, an important regulator of epigenetic maintenance of stem cell gene transcription through activating histone modification at target genomic loci [ 17 ]. Despite improved treatment protocols with higher response rates and better outcomes for children with ALL, KMT2A-r patients have poor prognosis due to frequent relapses upon conventional chemotherapy [ 18 , 19 ]. However, these leukemias typically harbor very few additional mutations that could explain drug resistance [ 20 ]. Recently, single-cell genomics-approaches have provided new insight into the aberrant gene regulatory programs that cause cell state instability, and hence diversification of cell states at cell fate decision points even in isogenic cell populations in response to treatment [ 21 ]. Such non-genetic heterogeneity has emerged as a central mechanism of drug resistance in cancer, including in leukemia [ 22 , 23 , 24 ]. A network of regulatory interactions exerted by TFs poises cells at fate decision points, primed for diversification into cell phenotypes with differential drug sensitivity [ 25 , 26 ]. Specifically, increased drug tolerance in leukemia and other cancers [ 21 ] has been attributed to “stemness” properties, due to shift into more immature (stem-like) states—a phenotype switch governed by gene regulatory networks as opposed to resulting from genomic mutations [ 21 , 24 , 27 , 28 , 29 ].

Chromosomal rearrangements of the KMT2A gene can produce many different chimeric TFs through in-frame fusions with various partner genes [ 30 ], in ALL most commonly resulting in KMT2A::AFF1 (AF4) and KMT2A::MLLT1 (ENL) fusions through t(4;11) and t(11;19) rearrangements, respectively. These fusion proteins lack the C-terminal SET domain which normally confers the KMT2A multiprotein complex histone H3K4 methyltransferase activity. KMT2A fusion proteins instead associate with the super elongation complex, resulting in differentiation arrest at the early hematopoietic progenitor state due to increased levels of activating H3K79 methylation by DOT1L at genomic loci such as HOXA9 and MEIS1 , which are associated with leukemic transformation [ 18 , 31 ]. However, KMT2A fusions also compromise the S-phase checkpoint by abrogating stabilization of wild-type KMT2A upon DNA damage, thereby preventing trimethylation of H3K4 and causing aberrant loading of CDC45 at late replication origins [ 32 ]. Recent work further indicates that other hematopoietic master regulators of stemness and development, notably RUNX proteins, also participate in cell cycle checkpoint control and DNA repair independent of their TF activities [ 33 , 34 , 35 , 36 ]. The impact of KMT2A fusion proteins at the convergence of two pivotal regulatory pathways, cell cycle checkpoint and cell fate control, represents a potential vulnerability; however, diversification of cell states upon targeted therapy may induce tolerance in distinct subpopulations, whose control would require combinatorial and sequential treatment.

Cancer cell responses to drug perturbations are often assayed using simple phenotypic readouts, such as proliferation or cell death. However, these end-point assays are insufficient to uncover cellular mechanisms that allow some cells to survive and that arise only in response to treatment within a subpopulation. Thus, distinguishing between selection of pre-existing drug-resistant cells and drug tolerance through drug-induced mechanisms is important to predict long-term drug efficacy. Importantly, targeted therapy that impinges on core regulatory circuits affects cell fate decisions which are poised to cause cell fate diversification. The latter generates non-genetic heterogeneity that has emerged as a major driver of treatment resistance [ 21 , 37 ]. In the present study, we characterize in detail the genome-wide response and cell state dynamics and diversification upon treatment with cell cycle checkpoint-targeted drugs in leukemic cells. We used single-cell resolution multi-omics and demonstrated that perturbing the cell cycle regulation directly impacts cell fate regulatory circuits and cell fate decision.

WEE1 inhibitor AZD1775 results in prolonged growth inhibition selectively in KMT2A-r B-ALLs

WEE1 expression was previously reported to be higher in primary ALL blasts compared to normal mononuclear cells [ 38 ]. To determine whether specific leukemia subtypes may rely more on WEE1 expression, we used our data resource Hemap ( http://hemap.uta.fi/ ) which contains curated genome-wide transcriptomic data from more than 30 hematologic malignancies [ 39 ]. Interestingly, WEE1 expression was significantly higher (FDR < 0.01, Wilcoxon test) in the KMT2A-r ALL subtype (Fig. 1 a, see also Additional file 1 : Fig. S1a comparing KMT2A::AFF1, KMT2A::MLLT1 and KMT2A::MLLT3). Higher WEE1 expression in KMT2A-r compared to other ALL subtypes raised the possibility that KMT2A-r cells may be more vulnerable to targeted inhibition of the WEE1 kinase. However, since WEE1 activity is regulated also at the protein level via phosphorylation, we performed further functional evaluation comparing different leukemic cell lines. First, to assess the sensitivity of leukemic cells that represent different ALL subtypes and lymphoid lineage differentiation states to WEE1 kinase inhibition, we treated a panel of 15 ALL cell lines with increasing concentrations of AZD1775 (Fig. 1 b, refer to Additional file 1 , Table S1 for GI50 data). Acute drug treatment induced apoptosis as measured by activation of caspase-3/7 and reduced cell viability in a dose-dependent manner in all cell lines (Fig. 1 b, Additional file 1 : Fig. S1b-c).

Characterization of AZD1775 response and WEE1 expression in patient cells and in vitro models. a WEE1 expression in primary ALL samples in HEMAP. tSNE-map: low (in white), high (in red) mRNA level. Boxplot: log2 normalized gene expression across subtypes. Wilcoxon test FDR: Hyperdiploid 6.1 -22 , t(9;22) 1.8 -18 , t(12;21) 6.0 -6 , t(1;19) 5.6 -6 , Hypodiploid 0.009, B-other 3.4 -11 . b AZD1775 dose response (relative to DMSO control) assessed by Alamar Blue assay in T-ALL and B-ALL cell lines. Percentage of viable cells at 72 h with increasing concentrations is shown. c Recovery of proliferation following removal of AZD1775 (relative to DMSO) analyzed by Alamar Blue assay. Cells were treated for 3 days (AZD1775 GI50 for each cell line, see Additional file 1 : Fig. S1b) and allowed to recover for 14 days without the drug. Box plot comparing recovery in KMT2A-r and non-KMT2A-r ALL cell lines (TCF3-PBX1, ETV6-RUNX1, TCF3-PDGFRB subtypes) is shown. ** denotes p = 0.0094 determined using two-tailed t -test. Data are represented as mean ± SD. d Kaplan–Meier survival curves of the MLL-7 patient-derived KMT2A-r ALL treated with AZD1775 in NSG mice. e Western blot analysis of the AZD1775 response dynamics in KMT2A-r RS4;11 cells. Whole cell lysates were immunoblotted with specific antibodies; Cleaved PARP1 (cell death), phosphorylation of CDK1-Y15 (WEE1 inhibition), and phosphorylation of γH2AX-S139 (DNA damage). β-actin: loading control. f Flow cytometry density plots showing distribution of EdU (replicating cells) and DNA content (Propidium Iodide, PI) to determine the proportion of cells in G1/S (green) and S/G2M (purple). RS4;11 cells were pulse-labelled for 30 min with EdU, treated with DMSO or AZD1775, and chased for the times indicated. g Overview of the experimental setup for characterization of drug response dynamics to distinguish pre-existing and induced drug tolerance

Next, we assessed whether the response to AZD1775 was sustained over time, by measuring recovery of proliferation post-treatment in leukemia cell lines, comparing the different chromosomal translocations, including ETV6-PDGFRB (Nalm-6), TCF3-PBX1 (697, RCH-ACV and Kasumi-2), ETV6-RUNX1 (REH), and KMT2Ar-ALLs (RS4;11, SEM, ALL-PO and KOPN8). Cells were treated with AZD1775 at sublethal dose (GI50 concentration) for 3 days, followed by drug washout and re-expansion in drug-free media. Although acute response to AZD1775 (GI50) did not associate with any specific ALL subtype (Additional file 1 : Fig. S1b), WEE1 inhibition markedly attenuated re-proliferation following drug washout in KMT2A-r cells as opposed to non-KMT2A-r cells ( p = 0.0094) (Fig. 1 c, Additional file 1 : Fig. S1d). Thus, the KMT2A-r subtype with the highest expression of WEE1 exhibits significantly reduced recovery, in line with the patient genomics cohort result (Fig. 1 a). To provide independent cell line evidence based on Crispr-Cas9 targeting across hematopoietic malignancies, we further analyzed WEE1 gene dependency scores using the DepMap portal ( https://depmap.org/portal/ ) and found a greater dependency on WEE1 in leukemia cell lines with higher WEE1 expression (Spearman r = − 0.336, Additional file 1 : Fig. S1e). However, characterization of the response to AZD1775 monotherapy cycles in vivo, using a primary KMT2A-r patient-derived xenograft (PDX) model (MLL-7, KMT2A::AFF1 ) [ 40 ] transplanted to NOD.Cg- PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice ( N = 3 per group), did not show a strong survival benefit (Fig. 1 d, AZD1775 median survival; 44 days, DMSO median survival; 42 days, see also Additional file 1 : Fig. S1f-j), warranting a therapy strategy informed by cellular properties that contribute to drug tolerance or acquired resistance.

To explore the molecular determinants of the AZD1775 response, we selected KMT2A-r RS4;11 cells and non-KMT2A-r Nalm-6 cells for an in-depth analysis. First, we confirmed that AZD1775 reduced phosphorylation of CDK1 on tyrosine 15 [ 41 ], increased PARP cleavage and triggered DNA damage rapidly, as measured by phosphorylation of γH2AX (Fig. 1 e, RS4;11 cells), according to the established mode of action of AZD1775. To confirm that RS4;11 cells treated with AZD1775 enter G2/M prematurely, cells were pulse-labelled with EdU and treated with DMSO or AZD1775 for different times, up to 32 h. As shown in Fig. 1 f, while DMSO-treated cells divided and re-entered G1-phase, a large proportion of AZD1775-treated RS4;11 cells stalled at G2/M and failed to proceed through the cell cycle.

Analogous to KMT2A-r patients who initially respond to treatment but then relapse, the potent acute response of KMT2A-r cells in vitro was followed by recovery of proliferation after removal of AZD1775 (Fig. 1 c). We reasoned that the cell characteristics associated with drug tolerance may originate through non-genetic heterogeneity from (1) a pre-existing subpopulation of drug-tolerant cells that are enriched during treatment, or alternatively these characteristics (2) are acquired during treatment through drug-induced cell state transition [ 21 , 42 , 43 ]. The first scenario would manifest as a gradual increase in cell phenotype/mutation frequency, limited by the cell doubling rate. The latter would involve shifts in gene expression programs induced by drug exposure, driven by rapid changes in TF activity, where only a fraction of the cells are expected to adapt to a new, possibly reversible cellular state that supports drug tolerance. Considering these two possibilities (Fig. 1 g), we characterized the early shifts in gene expression programs using a combination of genomics assays. Concomitantly, we quantified the recovery capacity over extended time periods to assess the phenotype of drug-tolerant cells post-treatment.

WEE1 kinase inhibition triggers a rapid transcriptional response and emergence of multiple cell states

We first performed global run-on sequencing (GRO-seq) to assess early effects on newly synthesized RNA at gene and enhancer regions at 4, 10, and 24 h (< population doubling time). The time course GRO-seq profiles identified 1239 significant gene transcription changes across time points (F-test FDR < 0.001). The temporal response to AZD1775 in bulk (Fig. 2 a) could distinguish (1) genes primarily upregulated at 4 h, exemplified by the GRO-seq signal at the PLK1 gene locus (Fig. 2 b), (2) genes peaking in upregulation at 10 h, (3) genes most strongly upregulated at 24 h, and (4) genes downregulated at all time points (see also Additional file 2 , Table S2). Pathway enrichment analysis revealed genes associated with regulation of the cell cycle (FDR 4 -17 ), DNA replication (FDR 1 -12 ), PLK1/AURORA signaling (FDR 1 -15 , 4 -12 ), FOXM1 TF network (FDR 3 -6 ), and M-phase pathways (FDR 2 -19 ) in the early (4 h) upregulated cluster (Fig. 2 c, Additional file 2 , Table S2). Genes functionally related to cell cycle checkpoints (FDR 2 -20 ), DNA-strand elongation (FDR 3 -19 ), activation of pre-replication complexes (FDR 7 -18 ), and S-phase (FDR 9 -18 ) were repressed in the bulk genomics data (Fig. 2 a bottom, “down”). B cell survival pathway was enriched (FDR 5 -2 ) at 10 h, preceding upregulation of apoptosis modulation and signaling (FDR 2 -3 ) related genes at 24 h.

WEE1 kinase inhibition triggers a rapid transcriptional response and emergence of multiple cell states. a Heatmap illustrating the magnitude and direction of changes in the GRO-seq signal ( z -score; tones of red indicate high level, tones of blue low level) for annotated gene transcripts. Gene clusters that correspond to regulation patterns 4 h up, 10–24 h up, 24 h up, and down are shown. b GRO-seq signal is illustrated at PLK1 gene region from replicate samples collected at 4, 10, and 24 h (top left). Signal from + / − stand is plotted above (in red) or below (in gray), respectively. c Enrichment of biological processes for GRO-seq gene clusters (right: upregulated with distinct time profile, left: downregulated) shown as dot plots. Size of the dot corresponds to the number of genes from the gene set and − log10 FDR value is shown in color. d–f Low-dimensional UMAP projection and scRNA-seq transcriptome-based clustering for the RS4;11 cells from DMSO- and AZD1775-treated cells are shown. Colors correspond to cell state assignment (in d ), treatment (in e ), and assigned cell cycle status ( f ). Quantification of cell proportions across categories is shown in e and f . g UMAP based on transcriptome and chromatin access profiles generated separately for DMSO- and AZD1775-treated cells. Graph connectivity (PAGA) is visualized on scRNA-seq maps where connecting lines indicate putative cell state transition paths. Multiome data from 10 h includes scATAC-seq from the same cells. The colors correspond to assigned cell states based on 24 h scRNA-seq data (in d ). Three treatment-specific cell fates that are reproducibly found in both time points and data modalities are indicated by roman numerals (cell fate I, II and III, see also Additional file 1 : Fig. S2a)

Since WEE1 inhibition interferes with a central decision point of cell fate control during the cell cycle, we anticipated that this perturbation would result in cells occupying distinct states. To expose the diversification and nature of responses of individual cells, we therefore performed single-cell transcriptome and chromatin accessibility measurements. scRNA-sequencing revealed that 24 h after treatment with AZD1775, the surviving cells analyzed alongside DMSO-treated cells formed 11 cell clusters (Fig. 2 d). We refer to these clusters as cell states hereafter. As determined by expression of cell cycle phase-specific markers, states 2, 3, and 4, correspond to different cell cycle phases present in DMSO-treated cells. State 1 corresponded to G0/G1 cells from both treatments, while clusters 5–11 were almost completely represented by AZD1775-treated cells (Fig. 2 e). This initial analysis supports the scenario that AZD1775 treatment rapidly impacts the gene regulatory network, leading to the emergence of new cell states with unique active transcription profiles. As noted in the bulk GRO-seq analysis, treated and surviving cells had reduced number of cells in the transcriptional state associated with S phase and an enrichment of cells in G1 or G2/M phase (Fig. 2 f), indicating an arrest at the G1/S checkpoint and a possible failure to progress through mitosis, consistent with the flow cytometry data of Fig. 1 f.

Since the bulk GRO-seq analysis indicated ongoing transition and possible pro-survival adaptation of cells arising already at 10 h, we performed multiome single-cell RNA- and ATAC-seq to assay simultaneously from the same cell how open chromatin sites influence the transcriptional response and cell state diversity. We also compared the cell states to the scATAC-seq profile acquired at 24 h from parallel cell cultures.

First, to investigate the correspondence between cell states identified from the single cell transcriptome and clustering of cells defined by their chromatin accessibility, we examined the transcriptome and chromatin dynamics by separating the cells by treatment from 10 and 24 h and performing RNA velocity and PAGA graph analysis (Fig. 2 g, transcriptome-based cell state labels from 24 h scRNA-seq are shown, see also Additional file 1 : Fig. S2a-d and Methods). The predominant cell state dynamics (Fig. 2 g, left) in DMSO-treated cells matched to active cell cycle transitions, as indicated by lines on the UMAP based on PAGA graph analysis (see Methods). By contrast, AZD1775-treated cells at 10 and 24 h (Fig. 2 g, middle and right, respectively) followed a trajectory along three distinct branches. The first branch is dominated by cell state 11 (cell fate I), the second branch corresponds to a succession of cell states from 5 to 7 (cell fate II), and a third smaller population of cells branches into cell state 9 (cell fate III) (Fig. 2 g). Each trajectory is unique to the AZD1775-treated cells and the distinct genome-wide changes are detectable as early as the 10 h time point, thus supporting a tolerance-inducing transcriptional response at the level of transcription and chromatin access.

Altogether, despite the highly heterogeneous response, we found a high concordance between the cell subpopulations analyzed using both RNA- and ATAC-seq analysis (Additional file 1 : Fig. S2a), and between bulk and single-cell pathway analyses (Fig. 2 c, Additional file 1 : Fig. S2e, Additional file 3 , Table S3).

Forced mitotic entry in response to AZD1775 treatment arrests cells from S-phase in a condensed mitotic-like chromatin state

Next, to elucidate whether the transcriptome and chromatin dynamics indeed reflect diversification into functionally different cell fates, we analyzed the cell states in more detail. We found that state 11 distinguished a distinct subpopulation (cell fate I) that was prevalent (20%) already after 10 h and constituted 32% of cells at 24 h of AZD1775 treatment (Fig. 2 g, middle and right, respectively). Based on scATAC-seq TF motif analysis, these cells had elevated S-phase (E2Fs, YY1, Additional file 4 , Table S4) and mitotic checkpoint (NRF1, Ronin, Sp1, Additional file 4 , Table S4) TF motif access (Fig. 3 a and Additional file 1 : Fig. S3a-b). Simultaneously, these cells showed evidence of mitotic entry downstream the forced activation of CDK1 based on the scATAC-seq fragment length distribution, with cells corresponding to cell fate I exhibiting a condensed chromatin state and elevated nucleosome signal metric (Fig. 3 b, Additional file 1 : Fig. S3c,d). Moreover, pathway enrichment from scRNA-seq cell cycle phase marker genes was consistent with a mitotic-like state (Additional file 1 : Fig. S2e and Additional file 3 , Table S3, FDR 1.23 -24 ). Premature entry into mitosis can extend mitosis and enhance cell death [ 44 ]. In agreement with delayed mitotic exit, the fraction of phosphorylated serine 10 histone 3 (pS10-H3) with 4N DNA content increased with treatment time, reflecting a mitotic-like arrest (Fig. 3 c).

Forced mitotic entry upon AZD1775 treatment arrests cells from S-phase in mitotic-like chromatin state. a TF motif activity score (chromVAR) is shown for NRF1, E2F, YY1, GFY, Ronin, and Sp1 with highest score in cell fate I. To match with scRNA-seq cell states, TF motif activities are visualized on the 10 h scRNA-seq UMAP from the 10 h multiome profile. The NRF1 motif with highest cluster-specific activity (summarized in Additional file 4 , Table S4) is shown on multiome scATAC-seq UMAP (10 h). Darker tones correspond to high motif activity score. b Typical scATAC DNA fragment length distribution in RS4;11 cells (24 h DMSO-treated cells, top left) compared to cells assigned to cell state 11 (bottom left). The nucleosome signal metric calculated from the ratio of fragments between 147 and 294 bp (mononucleosome) to fragments < 147 bp (nucleosome-free) across cell states is shown as violin plots (right panel, top: DMSO, bottom 10 h AZD1775-treated cells). c Three-dimensional (3D) flow cytometry dot plots showing the distribution of EdU (replicating cells, Y-axes) versus DNA content (X-axes) and pS10-H3 positive cells (Z-axes). RS4;11 cells were pulse-labelled for 30 min with EdU, treated with DMSO or AZD1775 and chased for the times indicated. Mitotic status was analyzed by labelling of phosphorylated S10-H3 (pH3) at the indicated time points. Graphs (right panel) represent the proportion of pS10-H3 positive cells in DMSO or AZD1775-treated cells

We also performed scRNA-seq in non-KMT2A-r Nalm-6 cells and in sharp contrast to RS4;11, the majority of AZD1775-treated Nalm-6 cells retained a population in the active cell cycle (matched to cell states 2–4), in line with their ability to rapidly recover and proliferate upon drug withdrawal (Additional file 1 : Fig. S3e). Accordingly, phosphorylation of S10-H3 was only transiently increased in Nalm-6 cells, while it was continuously high in RS4;11 cells treated with AZD1775 (Additional file 1 : Fig. S3f), supporting a reduced capacity of KMT2A-r cells to cope with AZD1775-driven CDK-activation and replication stress compared to non-KMTA2-r cells.

AZD1775 triggers unscheduled replication characterized by genome-wide p53 response

Comparison of the other cell clusters detected from scATAC-seq (referred to as “chromatin states”) revealed that AZD1775 induced genome-wide changes in chromatin accessibility with overall highly distinct TF motif activity patterns (Fig. 4 a) that could underlie the emergence of distinct cell fates upon replication stress.

p53-driven gene regulatory response initiates from transcriptional silent chromatin and anti-correlates with RUNX1 binding. a TF motif activity score (scATAC, 24 h) across chromatin states 1–5 (with uniform QC metrics, refer to Additional file 1 : Fig. S3d) in AZD1775-treated cells is shown as a heatmap, where TF motifs (in rows) are clustered into three main patterns. b Pattern 1 TF motifs with highest activity score in cell fate II (top panel). Selected pattern 2 TFs are shown for comparison (bottom panel). c p53, RUNX1, and MYC TF motif activity (10 h) visualized on UMAPs, as in Fig. 3 a. d Pseudobulk scATAC-seq signal at − / + 1 kb at 200 up- and downregulated enhancer regions most specific to AZD1775 chromatin state 1 (AZD1775 1) vs other cell states. In the heatmap (left), each row corresponds to one enhancer and brighter color tones to higher access level. The magnitude and direction of changes in the cell state-specific chromatin accessibility signal across chromatin states 1–5 are summarized as average signal histograms (right). e ChIP-seq signal profile of RUNX1 in RS4;11 cells (DMSO) and p53 from doxorubicin (Doxo) stimulated lymphoblastoid cells (LCL). TF occupancy signal heatmap and histogram at the same enhancer regions as shown in d . f GRO-seq signal heatmap (left) and histogram (right) at the same enhancer regions as shown in d . Line color corresponds to treatment time and condition. g GRO-seq signal histogram at ChIP-seq-based high-occupancy p53 binding sites from LCL shown as in f

Elevated accessibility at the binding motifs for p53 (and bZIP-factors JUN/AP, FOS, Atf) was characteristic of AZD1775-specific chromatin states 1 and 2 (Fig. 4 b, Additional file 4 , Table S4). The TF motif activity score, visualized on the scRNA-seq map (Fig. 4 c), reveals that these cells correspond to the branch leading to cell states 6–7 (26% 10 h, 32% 24 h, cell fate II) (Fig. 4 c, see also comparison of RS4;11 and Nalm-6 in Additional file 1 : Fig. S4a). Given that p53 modulates the senescence program in response to replication stress, we assessed the proportion of senescence following AZD1775 treatment. Indeed, we found a high gene set score for the senescence pathway in cell states 6–7 (Additional file 1 : Fig. S4a), and an increased proportion of SA-β-Gal-positive cells in AZD1775-treated KMT2A-r compared to non-KMT2A-r cell lines (Additional file 1 : Fig. S4b). This is in line with the increased mRNA expression of components related to the double-stranded DNA (dsDNA) sensor pathway ( cGAS , STING ) and factors of the senescence-associated secretory pathway (SASP, HMGB2 ) in RS4;11 cells (Additional file 1 : Fig. S4c). Accordingly, AZD1775-treated Nalm-6 cells used for comparison exhibited a much smaller (20.4% respective) cell cluster matching p53-regulated gene sets (Additional file 1 : Fig. S4a). Additionally, the activation of caspase 3/7 was higher in KMT2A-r cells compared to non-KMT2A-r cells (Additional file 1 : Fig. S1c).

Based on the scATAC-seq profiles of the distinct cell states, the elevated p53 TF motif access (Fig. 4 a, in pattern 1) anti-correlated with two other TF motif access patterns (Fig. 4 a, patterns 2 and 3). Closer inspection of pattern 2 revealed mutual exclusivity with the lymphoid progenitor TF RUNX1 (clustered together with bHLH motifs recognized, e.g., by AP4) (Fig. 4 b, Additional file 1 : Fig. S4d). Notably, RUNX1 is a key TF regulating target genes downstream of KMT2A::AFF1 in a feedforward loop [ 25 ] and was expressed at higher levels in KMT2A-r patients compared to other ALL subtypes (Additional file 1 : Fig. S4e).

To further explore the relationship between cell state-specific chromatin accessibility and transcriptional regulation, we visualized the scATAC-seq signal profile at enhancer regions with the highest and lowest chromatin accessibility in chromatin state 1 (Fig. 4 d, AZD1775-treatment). Next, we generated RUNX1 ChIP-seq profile from RS4;11 cells (basal state) and retrieved p53 ChIP-seq signals from doxorubicin-stimulated lymphoblastoid cells (Fig. 4 e). The average TF occupancy signals at these cell fate II-specific high chromatin access regions showed high p53 and low RUNX1 signal, while an opposite profile characterized the low accessible regions, providing further confirmation of their mutually exclusive TF activities. Notably, cells with high RUNX1 TF motif activity in AZD1775-treated cells (chromatin states 4 and 5) corresponded to transcriptome cell states 1–4 (also present in the basal state), and their relative proportion decreased from 35% at 10 h to 11% at 24 h.

Next, we quantified transcription from enhancers (enhancer RNA, eRNA) at these same regions (AZD1775, chromatin state 1). This regulatory region activity profile showed that enhancers in cell fate II-specific accessible chromatin were transcriptionally silent at 4 and 10 h (Fig. 4 f). This is in agreement with gene-level GRO-seq results that indicated delayed upregulation of the p53-signaling network and pathways regulating apoptosis (cluster active at 24 h, Fig. 2 a, Additional file 2 , Table S2). Enrichment of p53 ( p -value 1 -47 ) and bZIP ( p -value 1 -3 ) binding motifs in enhancer regions active at 24 h was independently validated in the bulk GRO-seq data (see “Methods,” Fig. 4 g, Additional file 1 : Fig. S4f, Additional file 5 , Table S5). Hence, inducing a p53 response from initially silent regulatory regions could allow cells time to adapt and initiate pro-survival responses.

CDK-mediated degradation results in loss of RUNX1 in response to AZD1775

To further characterize the decline in RUNX1 activity and other pattern 2 TFs, including the KMT2A::AFF1 TF target MYC that promotes leukemia survival [ 25 ], we performed immunoblot analysis (in bulk) and found that AZD1775 treatment decreased RUNX1 protein expression levels in KMT2A-r RS4;11 and SEM cells, and in several non-KMT2A-r ALL cell lines (Fig. 5 a, Additional file 1 : Fig. S5a). The hematopoietic stem cell TFs GATA2 and MYC similarly had declining protein expression following AZD1775 treatment (Additional file 1 : Fig. S5b). In comparison, ATF4 that acts as transcriptional activator of the integrated stress response was transiently induced at 4 h (Additional file 1 : Fig. S5b).

WEE1 inhibition promotes CDK-driven RUNX1 protein degradation. a RUNX1 protein levels in RS4;11, Nalm-6, and 697 treated with AZD1775 or DMSO for 24 h analyzed by immunoblotting. Phosphorylation CDK1 was detected with pY15-CDK1 antibodies. β-actin is a loading control. b Left panel: poly-ubiquitination of RUNX1 in RS4;11 cells treated with AZD1775 for 20 h (and with proteasome inhibitor MG-132 for the last 3 h). RUNX1 was immunoprecipitated (IP) under denaturing condition. RUNX1-ubiquitination was detected by immunoblotting with ubiquitin antibodies. Ten percent of whole cell lysate was used as input control for IP. Right panel: reciprocal pulldown of ubiquitin chains from whole cell lysates of Nalm-6 cells stably overexpressing RUNX1. Ubiquitin was pulled down using tandem ubiquitin binding entities (TUBEs), pulldown and input blotted and probed with RUNX antibody. Input for both experiments was probed with RUNX1, pY15-CDK1, and β-actin antibody. c RS4;11 and Nalm-6 cells were synchronized at different cell cycle stages (G1/S or G2) by double thymidine block (DT) or by treatment with RO3306 (RO) (CDK1 inhibitor), and released for the indicated time points. RUNX1 protein levels were analyzed by immunoblotting of whole cell lysates using RUNX1 antibodies. Asynchronous (Async) cells were treated with DMSO. Expression of cyclin E, cyclin B1, and phosphorylation of pS10-H3 was analyzed as indicated. β-actin and H3 are loading controls. d RS4;11 cells treated with AZD1775, RO3306 (RO), or their combination (AZD1775 + RO) for 24 h. Expression of RUNX1, cleaved PARP1, phosphorylation of Y15-CDK1, and phosphorylation of S139-γH2AX was analyzed by immunoblotting of whole cell lysates with the antibodies as indicated. Tubulin is a loading control. e Schematic illustration of mechanism linking CDK1 activity and RUNX1 protein degradation following WEE1 inhibition by AZD1775

RUNX1 was previously reported to be degraded in a CDK-dependent manner by the APC ubiquitin ligase complex during mitosis [ 45 , 46 ]. According to the scATAC-seq TF motif analysis in DMSO-treated cells, the RUNX1 and bHLH motifs exhibit the highest accessibility in DMSO chromatin cluster 1 (matched to G1 phase, or early S-phase in the multiome profile, Sankey plot Additional file 1 : Fig. S2a) and lowest accessibility in G2 phase (Additional file 1 : Fig. S5c). We hypothesized that in the absence of inhibitory phosphorylation by WEE1 (due to AZD1775 treatment, Fig. 5 a, pY15-CDK1), the hyperactivation of CDK1/2 may promote ubiquitination and proteasomal degradation of RUNX1 during G2/M phase. Supporting this, AZD1775 treatment did not alter RUNX1 mRNA expression levels in RS4;11 cells or in other B-ALL cell lines (Additional file 1 : Fig. S5d), but increased RUNX1 protein ubiquitination upon proteasomal inhibition (Fig. 5 b). We next verified that RUNX1 levels decreased following release from a G2/M-phase block as compared to cells released from a G1/S-phase block (Fig. 5 c). Concurrent treatment with the CDK1 inhibitor RO3306 and AZD1775 partially rescued downregulation of RUNX1 protein, and reduced PARP cleavage and γH2AX (Fig. 5 d), further supporting CDK1-driven ubiquitination and proteasomal degradation of RUNX1 in response to WEE1 inhibition (illustrated in Fig. 5 e).

Together, these results suggest that WEE1 inhibition may interrupt a core KMT2A-r transcriptional regulatory program involving RUNX1-MYC-GATA TFs through reduced protein expression.

Stress- and pre-B-state regulatory programs distinguish a drug-tolerant sub-population with pre-BCR and BCL6 gene loci activation

Functionally, RUNX1 is part of a core regulatory circuit that acts as a switch, governing cell fate commitment [ 25 ]. Consequently, perturbing RUNX1 expression and TF activity dynamics by WEE1 inhibition may facilitate a transition to an alternative cell state. In line with this, the cell fate III subpopulation (cell state 9, Fig. 2 g) had decreased RUNX1 motif activity (Fig. 4 a, AZD chromatin state 3), but significantly high chromatin access of pre-B cell state TF motifs, including PAX5, EBF, GR, and MEF2C (FDR 2.8 -35 , 8.6 -41 , 1.5 -29 , 1.4 -99 , respectively), and high motif activity of TFs regulating cellular metabolism (SREBF, THR, LXR) (Fig. 6 a, see also Additional file 1 : Fig. S6a-b, Additional file 4 , Table S4). As a distinct feature, cell state 9 (cell fate III)-specific open chromatin regions had highest NFkB (FDR 2.4 -69 ) and heat shock factor (HSF, FDR 3.9 -67 ) motif activity (Fig. 6 a). Supporting activation of a functional TF circuitry, cell fate III cells had high expression of the respective TF target genes of SREBF ( LDLR , HMGCS1 ), HSF ( HSPA1B ), GR ( TSC22D3 ), NFkB (target gene set activity scores shown), and pre-BCR signaling ( BCL6 , SOCS1 ) (Additional file 1 : Fig. S6c-e, Additional file 3 , Table S3). The combination of stress- and pre-B fate-specific TF activation signifies a cell state transition in response to AZD1775 treatment that may confer a more stress-resistant phenotype. In a similar fashion as examining cell fate II-specific chromatin (Fig. 4 d–f), we quantified enhancer regions that represent highest vs lowest sc-ATAC-seq signal in cell fate III (AZ chromatin cluster 3) and compared chromatin accessibility (Fig. 6 b, scATAC-seq signal) and enhancer activity (Fig. 6 c, GRO-seq signal). Interestingly, the regions with high access in cell fate III were transcribed, albeit at a low level, even in the basal state (gray lines, Fig. 6 c), suggesting a primed state. Following AZD1775 treatment, these enhancers were quickly further activated (4 and 10 h profiles in red, Fig. 6 c). Comparison to NFkB ChIP-seq signal in lymphoblastoid cells provided further confirmation that the activated chromatin regions in cell fate III-specific open chromatin harbor NFkB binding sites (Fig. 6 d, upper panel). In comparison, the ChIP-seq signal at enhancers with lowest chromatin access revealed that these sites were p53-bound in lymphoblastoid cells treated with doxorubicin (Fig. 6 d, lower panel), which could indicate active suppression of p53 chromatin recruitment in cell fate III [ 47 ]. The difference in the temporal dynamics of transcriptional activation of cell state II vs cell state III enhancers observed in RS4;11 cells, coupled with the reduced chromatin accessibility at p53 binding sites, may therefore contribute to the increased drug tolerance in cell fate III. Furthermore, chromatin accessibility increased at gene loci encoding MEF2D , SREBF1 , BCL6 , SOCS1 , and components of the pre-BCR signaling pathway, all of which play central roles in the cellular survival pathways of B-lymphoid cells (Fig. 6 e). Interestingly, this pattern resembles a previously recognized leukemia-associated TF circuitry that is active in the MEF2D-fusion subtype of ALL [ 48 ]. In line with the emergence of a drug-tolerant cell state through transcriptional regulation, the B cell survival pathway was significantly enriched (FDR < 0.05) in the activated gene cluster identified from bulk GRO-seq profile at 10–24 h (Fig. 2 c). These data are consistent with leukemic cells adopting a BCL6 + pre-BCR + cell state having higher tolerance to AZD1775, concordant with results obtained in pre-BCR + Nalm-6 cells (Additional file 1 : Fig. S4a-b).

Stress- and pre-B-state regulatory programs distinguish a drug-tolerant sub-population with pre-BCR and BCL6 activation. a TF motif activity scores across AZD1775 chromatin states (1–5) is shown for TFs with high score in cell fate III (Pattern 3, see Fig. 4 a) (left). NFkB, MEF2, and SREBP motif activity scores are visualized on the 10 h scRNA-seq UMAP (right). b scATAC-seq signal at − / + 1 kb at 200 up- and downregulated enhancer regions most specific to AZD1775 chromatin state 3 (AZD1775 3) vs other cell states is shown as average signal histogram, as in Fig. 4 d. c GRO-seq signal histogram at the same enhancer regions as shown in b . d ChIP-seq signal profile of NfKB in lymphoblastoid cells (LCL) and p53 from doxorubicin (Doxo) stimulated lymphoblastoid cells. TF occupancy signal histogram is shown at the same enhancer regions as shown in b . e Chromatin access at TF and pre-BCR signaling-related gene loci (gene body flanked by 2.5 kb up- and downstream) compared across AZD1775 chromatin states is shown as a heatmap (top). Aggregated scATAC-signal across cells assigned to each cluster is exemplified at PIK3CD gene region (bottom). The track colors correspond to 24 h AZD1775 chromatin state annotation. f Experimental setup for analysis of drug response and recovery dynamics. g Flow cytometry histograms of pre-BCR (upper panels) and BCL6 (lower panels) in RS4;11 cells treated with DMSO and AZD1775 for 72 h and following 6 and 14 days drug washout

To investigate whether the activation of cell fate III transcriptional program constitutes a potential escape mechanism that may support recovery following AZD1775 treatment, we analyzed pre-BCR and BCL6 protein expression in subsequent time points from cells treated with AZD1775 for 72 h, and after 6 and 14 days of recovery. Notably, the drug tolerant cells that persisted at 72 h matched the pre-BCR + /BCL6 + population (cell fate III) based on protein levels that were strongly upregulated in RS4;11 cells (Fig. 6 f, g). This activation of pre-BCR and BCL6 in response to AZD1775 was reversible, albeit with a delay: pre-BCR expression returned to baseline levels by day 6 and BCL6 by day 14 after the removal of the drug (Fig. 6 f,g, Additional file 1 : Fig. S7a-b). Accordingly, RUNX1 could be detected at day 14 in cells that had recovered from treatment (Additional file 1 : Fig. S7c). Consistent with this reversibility, the cells that recovered post-treatment (drug schedule shown in Additional file 1 : Fig. S7a) retained their sensitivity to AZD1775 (Additional file 1 : Fig. S7b, right), mirroring the response of the parental, untreated cells (Additional file 1 : Fig. S7b, left). Additionally, both RS4;11 (Additional file 1 : Fig. S7c-f) and Nalm-6 (Additional file 1 : Fig. S7g-j) cells recovering from AZD1775 remained sensitive to other conventional drugs used in ALL treatment protocols (L-asparaginase, cytarabine, doxorubicin). Although our findings do not definitively rule out the alternative possibility (Scenario 1, Fig. 1 g) that a pre-existing resistant cell subpopulation is selected for and expands during the recovery phase, they are more consistent with reversible cell state switching induced by WEE1 inhibition. Furthermore, treatment with L-asparaginase, cytarabine, or doxorubicin did not activate pre-BCR in KMT2A-r cells (Additional file 1 : Fig. S7d-f), highlighting the specificity of the AZD1775-induced phenotype shift.

To investigate markers associated with cell state III in vivo, MLL-7-engrafted NSG mice were treated with AZD1775 or control vehicle (as detailed in Additional file 1 : Fig. S8a). hCD45 + hCD19 + cells were isolated and subjected to scRNA-seq after 28 h and at day 6 post-treatment to identify early transcriptional changes. Gene enrichment analysis revealed significant over-representation of genes from the BCR signaling pathway in cluster 6 (Additional file 3 , Table S3) of AZD1775-treated MLL-7 cells (28 h treatment and matched cells from day 6; FDR 1.03 -8 and 1.15 -8 , respectively, Additional file 1 : Fig. S8b). Resembling the profile found in RS4;11 cell state 9, expression of pre-BCR or BCR signaling, pre-B TF genes, and NFkB targets was increased, while MYC levels declined (Additional file 1 : Fig. S8c-e). The expression pattern in vehicle-treated cells was more dispersed with only weak expression of MS4A1 (encoding the differentiation marker CD20), the most significant marker gene for cluster 6 (Additional file 1 : Fig. S8d). For comparative analysis, we also included an in vivo treatment profile from MEF2D-fusion ALL case (Additional file 1 : Fig. S9). Positive correlation of TF expression rate could be detected at a cluster level for MEF2D - BCL6 (Pearson r 0.8, p -value 0.056) and BCL6 - SREBF1 (Pearson r 0.9, p -value 0.016). Together, these data indicate that the cell phenotype with pre-BCR and pre-B fate TF expression is present in leukemia patient-derived cells.

Sequential drug treatment can target non-genetic evolution of leukemic cells

Drugs disrupting the regulatory network activated in cell fate III could have high efficacy in preventing recovery of the remaining leukemic cell population. Therefore, we selected drugs that target pre-BCR signaling (dasatinib and ibrutinib) [ 49 , 50 ]. Based on higher TF motif accessibility observed in cell state III for master regulators of cellular lipid metabolism, such as SREBF1, we also included inhibitors of metabolism (mTOR inhibitor AZD2014) and fatty acid synthesis (fatostatin) [ 51 , 52 ]. To assess the potential of repositioning these drugs as effective combination therapies with AZD1775, we initially measured efficacy of each drug as single agents in the drug recovery setting (illustrated in Fig. 7 a,b, Additional file 1 : Fig. S10a). When used as monotherapies, these drugs did not affect recovery of proliferation (Additional file 1 : Fig. S10a), and neither did they when administered concurrently in combination with AZD1775 (Fig. 7 a). However, the sequential administration of AZD1775 in conjunction with several of these drugs effectively inhibited the recovery of RS4;11 and ALL-PO cells in vitro (Fig. 7 b). This supports the strategy that targeting drug-induced adaptive survival pathways attenuated drug tolerance in KMT2A-r cells (Fig. 7 c). Notably, administration of AZD1775 followed by AZD2014, dasatinib, or ibrutinib also strongly attenuated recovery of non-KMT2A-r Nalm-6 cells (Fig. 7 b).

Targeting WEE1 and the drug tolerant cell state sequentially attenuates recovery of leukemic cells. a,b Left panels: Overview of the experimental setup. Cells were treated as indicated and allowed to recover in drug-free media for an additional 10 days. Right panels: a recovery of cell proliferation after concurrent treatment, following removal of the drugs. Cells were treated with 0.5 μM of AZD1775 and indicated drugs (1 μM) for 96 h. b recovery of cell proliferation after sequential treatment. Drug response relative to recovery of proliferation of cells treated with AZD1775 is shown. Regrowth was assessed using Alamar Blue stainings. ** denotes p < 0.01 and *** denotes p < 0.001 between AZD1775 alone and indicated drug combinations determined using Student’s t -test. Data are presented as mean ± SD. c Schematic model of the treatment strategy. d Schematic illustration of sequential treatment in vivo. Drugs were administered daily as outlined. e Proportion plot of immunophenotypic profiling of CD34 subpopulations in RS4;11 and MLL-7 xenograft models. f Proportion plots and UMAP visualization of treatment group in in vivo RS4;11 model (top). Treatment group data is shown for two distinct cell populations (p1 and p2, indicated on the UMAP). UMAP visualization of treatment group in MLL-7 cells across sample (below). g CD34 mRNA level shown on UMAP RS4;11 in vivo model (top) and MLL-7 (below). h Cluster assignment in RS4;11 in vivo model shown on UMAP (left). Barplots of corresponding cell proportions per treatment group (right). Clusters corresponding to G/2 M phase are indicated on the UMAP. Cluster 1, with majority of cells corresponding to the drug combination group is highlighted. i Label transfer score for cell state 9 shown on UMAP (RS4;11 in vivo model). j Flow cytometric analysis of pre-BCR expression under treatment in RS4;11 and MLL-7 xenograft models. k Proportion plots and UMAP visualization of treatment group and cell cycle phase data in RS4;11 (top) and MLL-7 (below) xenograft models

To corroborate these findings, we sorted and profiled CD45 + / CD19 + RS4;11 and MLL-7 cells following a sequential combination therapy (two doses of AZD1775 followed by daily doses with dasatinib combination started at 72 h) in xenograft mouse models (Fig. 7 d, Additional file 1 : Fig. S10b). The combination therapy resulted in a lower number of leukemia cells in both blood and spleen tissues (see Additional file 1 : Fig. S10b), indicating that the treatment reduces the proliferation of leukemia cells compared to the control vehicle. To characterize the effect of treatment on cell phenotypes, we first performed immunophenotype analysis by flow cytometry using the stemness marker CD34. In MLL-7, nearly all cells (99%) treated with the vehicle were CD34 + , while AZD1775 treatment, and even more so the combination treatment, decreased the number of CD34 + cells (to 97 and 87%, respectively) (Fig. 7 e, upper panels). In comparison, the RS4;11 cells exhibited higher immunophenotype diversity, with 30% CD34 + cells in vehicle, declining to 23% (AZD1775) and 16% (AZD1775 + dasatinib) (Fig. 7 e, lower panel).

To examine the gene expression phenotypes, we subjected the cells to scRNA-seq. RS4;11 cells grouped across the treatments into two separate, actively dividing, cell populations, while a more uniform cell population was present in MLL-7 (UMAPs, Fig. 7 f). Comparison of progenitor marker genes revealed higher CD34 levels in the smaller population of RS4;11 (p2, Fig. 7 f,g), referred to as stem-like from here on. In agreement with the immunophenotype analysis, cells obtained from vehicle-treated mice (62.4%) were more prevalent in the stem-like population, compared to cells from mice treated with AZD1775 alone (30.1%), or the combination with Dasatinib (7.5%) (Fig. 7 f). In MLL-7, the scRNA-seq also identified cells with decreased CD34 expression (Fig. 7 g).

In the RS4;11 cells with low CD34 expression (p1, Fig. 7 f), cluster 1 showed the greatest proportion of cells treated with the combination, as indicated in Fig. 7 h. Label transfer scoring for cell fate III was highest in this cluster that revealed a significant enrichment of pathways that control cell death (apoptosis, FDR 0.003, Additional file 3 , Table S3) and upregulation of TP53 , GADD45A , and BAX (Additional file 1 : Fig. S10c), with concomitant decrease in cellular mRNA levels (Additional file 1 : Fig. S10d,e, also visible in MLL-7 Additional file 1 : Fig. S10f). These data are consistent with early apoptosis [ 53 , 54 ] and imply that the combination treatment specifically affects and may trigger cell death in cells that resemble cell state III.

Subsequent analysis of cell state III markers, specifically pre-BCR and BCL6, revealed that AZD1775 increased protein levels of these markers in the two in vivo models (Fig. 7 i, Additional file 1 : Fig. S10g), confirming the treatment effects observed in vitro (Fig. 6 g). When combined with Dasatinib, there was a notable decrease in pre-BCR expression in RS4:11 cells (Fig. 7 j). In MLL-7, the combination resulted in a distinct population of pre-BCR-negative cells compared to treatment with AZD1775 alone (Fig. 7 j).

Finally, scRNA-seq and DAPI staining indicated changes in the proportion of actively cycling cells following the combination treatment (Fig. 7 k, Additional file 1 : Fig. S10h-i). The combination treatment consistently decreased the population of cells in the S-phase in both xenograft models, with concomitant increase in G1-phase in MLL-7 and expansion of G2/M cells in RS4;11. Notably, cells that exhibited an early apoptotic gene expression signature in RS4;11 corresponded to G2/M or G2/M-to-G1 transitioning cells (depleted by treatment in MLL-7, as denoted by arrows on the UMAP plots separated by treatment, Additional file 1 : Fig. S10h). This suggests that the observed effects may stem from cell death during the S and G2/M phases, arrest in the G1 or mitotic phase, or a combination thereof. Concordantly, subsequent Annexin V/PI staining on CD45 + /CD19 + RS4;11 and MLL-7 cells revealed increased cell death in both xenograft models post-treatment (Additional file 1 : Fig. S10j).

Taken together, these results show that perturbing cell cycle regulation through inhibition of WEE1 kinase in KMT2A-r cells triggers multiple distinct genome-wide responses that include rewiring of TF programs, resulting in cell state diversification. The sequential administration of AZD1775 together with drugs targeting metabolism and pre-BCR signaling can disrupt a non-genetic evolution toward a drug tolerant state, a discovery that potentially offers new opportunities for drug repositioning to prevent or delay the regrowth of leukemic cells.

Targeted therapy holds promise in cancer treatment to reduce the toxicity of long-term chemotherapy. However, cancer cells can develop resistance to targeted treatments not only through the selection of genetic mutations but also through non-genetic heterogeneity, namely drug-tolerant states with phenotypic adaptations that allow cells to evade drug-induced lethality. Herein, we characterized in detail the genome-wide response and cell state evolution upon cell cycle checkpoint-targeted therapy through inhibition of the WEE1 kinase in ALL. Data-driven analysis from the HEMAP and the DepMap genomics resources combined with drug response profiling in different ALL subtypes revealed higher expression and increased WEE1 gene dependency in KMT2A-r ALL as compared to other genetic subtypes. We show that WEE1 inhibition and CDK1 activation led to degradation of RUNX1 and repression of the KMT2A-r stemness phenotype, with concomitant repression of CD34, MYC, and GATA2. Thus, contrary to currently held views that stemness features critically underlie drug resistance in KMT2A-r leukemias, we found that upon targeting their cell cycle control vulnerability, the drug-tolerant cells corresponded to a more differentiated phenotype with expression of pre-BCR and BCL6. We show that WEE1 inhibition followed by drugs targeting the drug-tolerant cell state regulatory program prevents recovery in KMT2A-r and pre-BCR + ALL cells. This synergy was observed only in the sequential, not in concurrent treatment schedule.

Upon WEE1 inhibition, most of ALL cell lines, including KMT2A-r cells, rapidly enriched for cell cycle markers consistent with deregulated DNA replication and unscheduled transition into G2M-phase, leading to cell death. However, the cell lines showed capacity to resume proliferation post-AZD1775 treatment within 1–14 days after removal of AZD1775. Using KMT2A-r RS4;11 cells as a model, we show that AZD1775 treatment results in early diversification in cell states, as evidenced by significant alterations at both transcriptional and epigenetic levels. Specifically, the single-cell profiles mapped the majority of cell states with extended mitotic arrest distinguished by condensed chromatin status (cell fate I) and strong p53 activation (cell fate II), consistent with induction of apoptosis and senescence pathways. In comparison, cell fate III had higher chromatin access at BCL6 and pre-BCR gene loci and TF motifs of NfKB, MEF2D, and the metabolic regulators SREBF and LXR. A reversible shift to pre-BCR + /BCL6 + state was confirmed at protein level (day 3) that may be attributable to drug-induced steady state (attractor) switch [ 21 ], consistent with a non-genetic and drug-induced acquisition of a drug tolerant cell phenotype through altered TF activity that persists several weeks in vitro. The comparison of enhancer activation signals in the cell fate-specific open chromatin at p53, RUNX1, and NFkB bound sites provided insight into how the alternative cell fates could arise downstream TF activity changes: a progressive response activating p53-bound sites in cell fate II diverted cells with unscheduled S-phase entry away from cell cycle and towards cell death and senescence through p53 activation at initially transcriptionally silent regions. In contrast, NfKB and BCL6 both correspond to TFs known to counteract p53-driven apoptosis and repress genes involved in sensing or responding to DNA damage [ 55 , 56 , 57 , 58 ]. The loss of chromatin access at RUNX1 occupied sites upon WEE1 inhibition, and concomitant loss of stemness markers, induction of differentiation-related TFs, activation of cell fate III-specific chromatin co-localizing with NFkB sites, and pre-BCR expression represent, to our knowledge, a previously unrecognized connection between cell cycle and cell fate regulatory circuits.

RUNX1, GATA2, and MYC are crucial drivers of leukemogenesis that belong to the core KMT2A-r gene regulatory network and represent regulatory targets of KMT2A-r fusions [ 25 , 33 , 59 , 60 ]. Consequently, disruption of the RUNX1-GATA2-MYC progenitor program could potentially reroute cell fate decisions favoring survival in absence of RUNX1-MYC through the activation of alternative TFs. In particular KMT2A-r cells display strong addiction to RUNX1 for survival [ 33 , 36 ]. The loss of RUNX1 through ubiquitin-mediated protein degradation reported here may impinge upon multiple surveillance mechanisms that when disrupted enhance the sensitivity of KMT2A-r cells to AZD1775. Alternatively, perturbations in the epigenetic control that are a hallmark of KMT2A-r leukemias, such as the increased transcriptional variability reported upon histone acetyltransferase KAT2A loss in KTM2A-r acute myeloid leukemia [ 37 ], may facilitate stochastic cell diversification upon stress or injury. Here, we examined the shift to pre-BCR + state, comparing drug-induced stress upon WEE1 inhibition or conventional chemotherapy drugs, and contrary to this more general mechanism found that the cell state diversification occurred specifically upon AZD1775 treatment. In addition, since RUNX proteins have been reported to function directly as integral regulators of DNA repair [ 35 ], and previous studies demonstrated that KMT2A-r cells have compromised the ATR-mediated S/G2-phase checkpoint [ 32 ], the inhibition of WEE1 in KMT2A-r cells may not only override the G2/M-phase checkpoint but also cause disruption of the G1/S checkpoint [ 1 ], resulting in CDK1/2-driven unscheduled origin firing, further enhancing p53 activation and senescence/apoptosis. These findings are thus consistent with the increased sensitivity of KMT2A-r cells to AZD1775 and the dependency on RUNX1.

Although KMT2A-r leukemias resemble the early lymphoid/multipotent progenitors by immunophenotype, recent single-cell characterization of patient cells has revealed that the transcriptional programs of a subset of cells present in primary bone marrow tissue match more differentiated pre-B-like states [ 24 , 29 ]. Similarly, our previous single-cell genomics study deciphering features of drug resistance directly in ETV6-RUNX1 ALL patients during induction chemotherapy [ 23 ] demonstrated that although the diagnostic blasts matched a pro-B-like cell state, the persisting leukemic cells at day 15 are re-programmed towards a pre-B TF activity state. The new profiles acquired here from KTM2A-r drug response both in vitro and in vivo indicated increased chromatin accessibility of genes encoding pro-survival BCR − and PI3K signaling components and the co-expression of BCL6 and pre-BCR signaling, consistent with non-genetic adaptation contributing to treatment resistance. Recent research, including flow-sorted subpopulations and cellular barcoding studies [ 61 , 62 , 63 ], has shed light on the inherent capacity of leukemia blasts, across different stages of immunophenotypic maturation, to propagate the disease. This emphasizes the clinical importance of considering non-genetic pathways to drug tolerance in treatment strategies.

BCL6 and pre-BCR signaling can form an oncogenic feedback loop in ALL cells that promotes survival signaling via SRC family kinases, SYK, ZAP70, and downstream PI3K activation [ 64 ]. Due to the recent development of efficient drugs inhibiting downstream pathways (dasatinib, ibrutinib), this elevated drug tolerance could potentially be disrupted. Our results further implicate regulation of lipid metabolism (LXR, SREBF) as a concomitant change that has been recognized in immune cell activation and oncogenic PI3K signaling in different cancers [ 65 , 66 ]. By interfering with pro-survival signaling through targeting lipid metabolism or inhibition of BCR signaling, sequential administration of mTOR inhibitor AZD2014, fatostatin, and inhibitors of BCR-signaling (dasatinib, ibrutinib) strongly suppressed recovery of KMT2A-r cells in vitro. Notably, this strategy also potently blocked recovery in non-KMT2A-r Nalm-6 cells, indicating that sequential targeting of WEE1 along with pre-BCR or metabolic activity may be broadly effective in ALLs.

Our results, consistent with a number of ALL drug therapy studies, indicate that targeting specific cancer vulnerabilities using carefully designed drug scheduling may represent the most useful measures for improving leukemia treatment success and counteracting disease recurrence. Despite existing data of potent chemo-sensitizing activities of AZD1775 in combination with different cytotoxic agents [ 67 , 68 , 69 , 70 ], WEE1 inhibitors have not been tested, to our knowledge, in ALL clinical trials. To date, several ongoing efforts aim to introduce immunotherapy in B-ALL treatment which requires implementation of a successful chemotherapy regime to initially reduce the leukemia burden, followed by immunotherapy. Combination therapies with WEE1 inhibitors may be attractive in this setting, reducing leukemia burden by inducing cell death through the cell-intrinsic mechanism, as characterized here. In solid cancers, favorable responses have been reported in phase 3 trials, though concerns in toxicity remain when combined with strongly cytotoxic backbone drug therapies. Our results show that several low toxicity drugs, already in clinical use for ALL or other non-communicable disease, potentiate the efficacy of WEE1 inhibitors in leukemia to overcome this challenge. In the preclinical xenograft models that represent the more diverse in vivo microenvironment, we could recapitulate the decrease in stemness markers, expression of pre-BCR and BCL6 and elevated cell death, as an indication of treatment efficacy following a sequential scheme. In follow-up, optimizing the drug scheduling to the in vivo cell state dynamics and assessing the long-term efficacy of the drug combination across a broader panel of KMT2A-r leukemias should be conducted. Furthermore, AZD1775 was recently reported to additionally activate immune signaling through activation of STING and STAT1 pathways, or the double-stranded RNA viral defense pathway, and sensitize solid tumors to immune checkpoint therapy [ 71 , 72 ]. Pre-clinical testing in xenograft models lacking the immune system, as used here, may therefore only partially represent the efficacy of AZD1775 therapy.

In summary, the combination of biochemical assays and cellular resolution genomics analyses provided new insight on the remodelling of the gene regulatory landscape upon cell cycle checkpoint targeting. Based on the results, we propose a strategy for WEE1 inhibition in ALL that anticipates the TF network re-wiring and the transition to a pre-BCR + BCL6 + cell state. This strategy targets the drug-tolerant phenotype through novel sequential drug administration, establishing a proof-of-concept that could be utilized in the design of new targeted drug screens and clinical trials.

Cell culture

A panel of ALL cell lines consisting of T-ALL: T-ALL1, Peer, Molt-16, and B-ALL cell lines: Nalm-6 (ETV6-PDGFRB); 697, RCH-ACV, Kasumi-2, MHH-Call3 (TCF3-PBX1); REH (ETV6-RUNX1); RS4;11, SEM, ALL-PO, KOPN8 (MLLr-ALL); SupB15, TMD5 (BCR-ABL) were purchased from the Department of Human and Animal Cell Lines (DSMZ). Cell lines were maintained in RPMI1640 (Gibco, Thermo Fisher) supplemented with 10% FBS (Gibco, Thermo Fisher) and 2 mM L-glutamine (Gibco, Thermo Fisher) and 10 mM HEPES (Gibco, Thermo Fisher). To generate stable Nalm-6-RUNX1 cell lines, RUNX1 cDNA (NM_001754, originally from Origene Technologies Inc, MD, USA, cat #RG223809) was cloned into pLVX-Tight-Puro vector (Clontech) using NotI and EcoRI restriction sites. Nalm-6 cells were transduced with the regulatory vector TetOn and subsequently with pLVX response vectors (either without an insert or with RUNX1).

Drug and dose response assessment

AZD1775, AZD2014 (Astra Zeneca), Dasatinib, Ibrutinib, and Fatostatin (MedChemExpress, Monmouth Junction, NJ, USA) were reconstituted in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, Missouri, USA) for in vitro experiments. For in vivo experiments AZD1775 and Dasatinib were reconstituted in 5% DMSO, 40% PEG 300, 5% Tween 80, and 50% NaCl (all from Sigma-Aldrich) and stored in aliquots at − 20 °C. Alamar Blue assay was used to determine cell viability of the cells treated with increasing doses of drugs for 72 h. Cells (30,000 cells/well) were seeded into 96-well plates. For the recovery of proliferation assay, cells were treated for either 72 or 96 h and allowed to recover for an additional 10–14 days without the drugs. Regrowth was assessed by Alamar Blue staining. Fluorescence was measured at 570 nm by spectrophotometer. All experiments were repeated three times.

To assess the combined effects of AZD1775 and Dasatinib, Ibrutinib, Fatostatin, and AZD2014 (Drug 2) on cell viability, cells were treated with 0.5 μM of AZD1775 for 48 h, followed by the Drug 2 (1 μM) for an additional 48 h. As controls, cells were treated with AZD1775 or Dasatinib, Ibrutinib, Fatostatin, and AZD2014 alone for 96 h. Subsequently, the drugs were washed out and cells were allowed to recover for 10–14 days. Cell viability was measured using Alamar Blue assay. A reference point was established as the number of cells recovered after treatment with AZD1775 alone for 96 h, which was considered 100% recovery. The recovery of cells treated with the combination of AZD1775 and Dasatinib, Ibrutinib, Fatostatin, and AZD2014 was calculated relative to this reference, allowing for an assessment of the impact of the combined treatment on long-term cell viability. The experiments were performed in triplicate.

Analysis of apoptosis using fluorescence live cell microscopy

Cells were stained with Incucyte® Caspase-3/7 Dye for Apoptosis (Sartorius) according to the manufacturer’s instructions, followed by live cell microscopy performed with an IncuCyte S3 Live Cell Analysis System (Essen Bioscience). Nine planes of view were collected per well, using the × 20 objective. The obtained data were analyzed with the IncuCyte S3 Cell-by-Cell Analysis Software Module (Essen Bioscience).

Western blotting

For biochemical analyses of protein phosphorylation and total level, the cells were lysed in NP-40 lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP-40) supplemented with both protease (Complete mini, Roche) and phosphatase (PhosSTOP, Roche) inhibitors. Proteins were resolved in 12% Bisacrylamide gel under reducing-denaturing condition, blotted onto PVDF membrane, and detected by immunoblotting using the appropriate antibodies. The list of antibodies and their sources can be found in the appendix file (Additional file 6 , Table S6).

Isolation of RUNX1 ubiquitin conjugates by TUBE pulldown

To detect ubiquitinated RUNX1, Nalm-6 cells overexpressing RUNX1 were used. RUNX1 expression was induced by adding doxycycline to the media 24 h prior to the experiment, followed by treatment with either AZD1775 or DMSO for 20 h before TUBE pulldown. Cells were lysed in NP-40 lysis buffer containing protease and phosphatase inhibitors, along with 100 mM N-ethylmaleimide (NEM). Lysates were incubated with anti-ubiquitin Agarose-TUBE 2 (Lifesensors, cat. no. UM402) for 1 h, according to the manufacturer’s protocol. Eluted proteins were subjected to immunoblotting with an anti-RUNX1 antibody (Abcam).

In vitro ubiquitination assay

RS4;11 cells were treated with AZD1775 or DMSO for 20 h. Four hours before harvesting, cells were treated with 20 μM MG-132 to preserve multiubiquitin chains of RUNX1. Cell lysis was performed under denaturing conditions to disrupt non-covalent interactions. Total cell lysates were immunoprecipitated with 1 μg of anti-RUNX1 antibody (Abcam). Eluted proteins were then immunoblotted with an anti-ubiquitin antibody (Cell Signaling).

Analysis of bulk microarray gene expression data in HEMAP

Normalized and log-transformed gene expression levels were compared between different hematologic malignancies or ALL subtypes from the HEMAP resource [ 39 ] based on the Wilcoxon test.

PDX transplantation and in vivo drug treatments

In-house-bred NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) 8–9-week-old mice were sublethally irradiated (250 cGy) and 24 h later, transplanted through the tail vein with 2 × 10 6 cells from MLL-7, a PDX established from a diagnostic sample from a childhood ALL with KMT2A::AFF1 (courtesy of Dr. Richard Lock) [ 73 ]. For combination treatments, as a second cell model 2 × 10 6 RS4;11 cells were transplanted to non-irradiated animals. Ciprofloxacin (KRKA, Stockholm, Sweden) was given in the drinking water for the duration of the experiment. A 1% engraftment of hCD45 + hCD19 + cells was set as a starting point for the treatment and assessed by taking 60 µl of blood from vena saphena 14 days after transplantation and then every other day if the 1% of hCD45 + hCD19 + was not reached. Red blood cells were lysed with ammonium chloride (Stemcell technologies, Cambridge, UK), washed in PBS (GE Healthcare Life Sciences, Logan, UT, USA) + 2% FBS (Thermo Scientific, Waltham, MA, USA), blocked with 10% mouse serum (#M5905, Sigma Aldrich) and stained with the following antibodies: anti-human CD45-APC (clone HI30, #555485, BD Bioscience, Franklin Lakes, NJ, USA) (hCD45) and anti-human CD19-APC/Cy7 (clone HIB19, #302217, Biolegend, San Diego, CA, USA) (hCD19). Dead cells were excluded with Draq7 (Biolegend). Flow cytometric analysis was performed using the LSRFortessa (BD Biosciences) and analyzed using FlowJo (FlowJo, LLC, Ashland, OR, USA). Once a 1% engraftment of hCD45 + hCD19 + cells was achieved, mice ( N = 3 per group) were either treated with 120 mg/kg of AZD1775 (MedChemExpress) 5 days a week, for 21 days (5 days on, 2 days off) (Additional file 1 : Fig. S1d-h) for the survival experiment, or for 48 h with 120 mg/kg AZD1775 (MedChemExpress) every 24 h, and 4 h after the last dose, animals were sacrificed. For combination treatments, animals were treated with 6 days of AZD1775 (MedChemExpress) at 120 mg/kg alone or with Dasatinib for 3 days (MedChemExpress) at 50 mg/kg. Vehicle animals received 6 days of 5% DMSO, 40% PEG 300, 5% Tween 80, and 50% NaCl solution (all from Sigma-Aldrich). Animals were sacrificed by cervical dislocation 20 h after the last treatment. Before mice were sacrificed, a blood sample was taken from vena saphena . An autopsy was performed and bone marrow cells from the tibia, femur, and hip from both legs, as well as from the spleen, were collected and homogenized into single-cell suspensions by manual trituration and viably frozen in 10% DMSO (Sigma-Aldrich) in FBS (Thermo Scientific).

GRO-seq assay

To analyze the actively transcribing RNA polymerases genome-wide, GRO-seq assays were performed as in [ 74 ]. Five million nuclei were collected from RS4;11 cells after 4, 10, and 24 h AZD1775 or DMSO treatment. The nuclear run-on reaction buffer (496 mM KCl, 16.5 mM Tris–HCl, 8.25 mM MgCl 2 , and 1.65% Sarkosyl (Sigma-Aldrich, Steinheim, Germany) was supplemented with 1.5 mM DTT, 750 μM ATP, 750 μM GTP, 4.5 μM CTP, 750 μM Br-UTP (Santa Cruz sc-214314A, Biotechnology, Inc., Dallas, Texas, USA and Sigma-Aldrich B7166), and RNAse inhibitors (RNase Inhibitor (Thermo Fisher, Carlsbad, CA, USA, and RNasin® Plus RNase Inhibitor Promega). To each 100 μl of nuclei samples, 50 μl of the run-on reaction buffer was added and incubated for 5 min at 30 °C. The RNA was collected using Trizol LS, fragmented and run-on reaction products were immuno-purified two times using anti-Br-UTP antibody (ab-6326, Abcam) bound to 30 μl of magnetic beads (Protein G DynabeadsThermo Fisher Scientific) and washed with 300 μl of PBST wash buffer four times (refer to detailed protocol in [ 75 ]). The cDNA template was PCR amplified (Illumina barcoding) for 12 cycles and size selected to 225–350 bp length. The ready libraries were sequenced with Illumina Hi-Seq2000 (GeneCore, EMBL Heidelberg, Germany).

ChIP-seq assay

To distinguish the signal corresponding to active enhancer regions from the collected GRO-seq profiles, RS4;11 cells were similarly treated for 4, 10, and 24 h with AZD1775 or DMSO and cells were crosslinked with 1/10 of volume of 11% formaldehyde solution (37% formaldehyde (Sigma-Aldrich), 5 M NaCl, 0.5 M EDTA pH 8.0, 0.5 M EGTA, 1 M HEPES) for 10 min gently rotating at 2 mio/ml cell concentration in media. The reactions were quenched by adding glycine to a final concentration of 120 mM, and cells were washed twice with ice-cold PBS. Crosslinked lysate was flash frozen with liquid nitrogen and stored at − 80°C. Lysates were thawed, nuclei were extracted, and MNase-treated and antibody-bound chromatin collected by adding 25 μl of Dynabeads Protein G (Thermo Fisher Scientific) and rotating for 1 h at 4°C as previously described [ 74 ] with the following minor modifications: 5 U- 1U of MNase (Thermo Fisher Scientific) was used and 5 cycles sonicated then (Bioruptor® Plus and Bioruptor® Next Gen, Diagenode). Then supernatant was diluted to dilution buffer 2.5 times. Samples were pre-cleared with Protein G Dynabeads (Thermo Fisher Scientific) using 25 μl per sample (1 h 4°C rotating). Subsequently, input samples were taken. Five micrograms of the specific RUNX1 antibody (cat# ab23980, Abcam) for 5–16 million cells and 2.5 μg of specific H3K27ac antibody (cat# ab4729, Abcam) for 5–9 million cells were used. Beads were washed with 1 ml of cold wash buffers I, II, and III as in for 3 min [ 74 ]. (Finally, beads were washed twice with TE-buffer. Beads were eluted twice in elution buffer (1% SDS, 100 mM NaHCO 3 ). Immunoprecipitated chromatin was reverse crosslinked by adding NaCl for a final concentration of 0.2 M and 0.4 mg/ml of RNAse A (Invitrogen) was added to each sample for 16 h at 65 °C. Proteinase K treated by adding 31 μl of Proteinase K buffer (161 mM EDTA, 645 mM Tris pH7.4, Proteinase K 0.65 mg/ml (Thermo Fisher Scientific)) for 1 h at 50°C. For DNA purification, ChIP DNA Clean & Concentrator (ZymoResearch, Irvine, CA, USA) was used like manufacturer’s protocol. ChIP-Seq libraries were prepared as previously described [ 74 , 76 ] with samples PCR amplified for 13–14 cycles and size selected for 225–350-bp fragments by gel extraction. Single-end sequencing (75 bp) was performed with Illumina NextSeq 500/550 High Output Kit v2.5.

GRO-seq read processing

GRO-Seq reads were quality controlled (FastQC). Reads were adapter trimmed using HOMER (version 4.9 [ 76 ] and filtered (min 95% of positions have a min phred quality score of 10) using the FastX toolkit ( http://hannonlab.cshl.edu/fastx_toolkit/ ). Read mapping to rRNA regions (AbundantSequences as annotated by iGenomes) were discarded. The Bowtie software (version 1.1.2 [ 77 ]) was then used for alignment of remaining reads to the hg19 genome version, allowing up to two mismatches and no more than three matching locations. The best alignment was reported. Reads corresponding with so-called blacklisted regions that include unusual low or high mappability as defined by ENCODE, ribosomal and small nucleolar RNA (snoRNA) loci from ENCODE, and a custom collection of unusually high signal depth regions from GRO-seq was used to filter the data. GRO-seq tagDirectories were generated from aligned reads with fragment length set to 75 and data visualized using makeMultiWigHub.pl with strand-specificity HOMER (version 4.9).

ChIP-seq read processing

Reads with poor-quality bases scores were filtered (min 97% of positions were required to have a min phred quality score of 10) using the FastX toolkit ( http://hannonlab.cshl.edu/fastx_toolkit/ ). Duplicate reads were collapsed using fastx (collapse). Bowtie (version 1.2.3 [ 77 ]) was used to align the reads to the hg19 genome version, allowing up to two mismatches and no more than three matching locations. tagDirectories were generated with HOMER (version 4.9) tool. Histone peaks (GSE148195) [ 78 ] were identified using HOMER (version 4.9) findPeaks with setting -style histone. The peak signal profile across pooled tagdirs was then used to distinguish dips that correspond to nucleosome free regions that can be accessed by TFs (this was found to work well for peaks < 7.5 kb). To identify RUNX1 peaks, HOMER (version 4.9) findPeaks-style factor was used with the following less stringent settings: fold enrichment over input tag count 2, Poisson p -value threshold relative to input tag count 0.001, fold enrichment over local tag count 2, Poisson p -value threshold relative to local tag count 0.001, FDR 0.01, -tagThreshold 10. GM12878 NfKB (Tnfa treatment) from ENCODE SYDH ChIP-seq tagDirectory was generated from bam file using HOMER.

GRO-seq gene signal analysis

To compare the effect of AZD1775 on primary transcript levels across the collected time points, Refseq (genome_annot_hg19 refGene_2018) coding and non-coding gene coordinates were quantified without exon regions. A raw count matrix was generated using Homer (version 4.9.1) specifying as maximum read per position 3 not allowing combining quantified reads if the same transcripts is detected more than one time. The low expression transcripts were filtered based on cpm and rpkm values requiring a row sum greater than 0.5 in at least two samples. To detect differentially expressed transcripts, a generalize linear model was fit using functions available in the R/Bioconductor package edgeR. Genes with significant change between any of the conditions (FDR < 0.001) were reported based on one-way ANOVA-like the likelihood ratio tested with glmLRT function. The genes were then clustered using k-means (into six clusters) and plotted as a heatmap (ComplexHeatmap R package) with six clusters. One cluster profile was excluded as it reflected mainly changes in DMSO basal levels between time points. Pathway analysis was performed on the gene clusters with > 100 genes; otherwise, the genes were plotted and examined individually.

GRO-seq enhancer signal analysis

The nascent transcriptome result allows quantification of eRNA signal that can guide the analysis of TF activity. Enhancer regions are typically defined based on multiple genomic signal features. Here we started with DNA hypersensitive sites (DHS from Encode Duke narrow peaks, available across the ENCODE cell collection) and ATAC-seq peak coordinates (from RS4;11 cells in GSE117865 (GSM3681445) [ 79 ] and from bone marrow cell populations in [ 80 ]) for specifying candidate enhancer centers. The candidate regions were extended + / − 500 bp and the 1 kb size enhancer regions were quantified with Homer (homer/4.9.1) analyzeRNA. Coordinates with overlap with gene sense-transcription or the promoter region signal were excluded. Enhancers that passed a minimum cpm cutoff or intersected (bedtools2/2.27.1) with RS4;11 histone ChIP-seq peaks for H3K4me1 (GSE71616) [ 81 ] and H3K27ac (our own GSE148195 [ 78 ], GSE71616 [ 81 ] and GSE117865 (GSM3312817) [ 79 ]) were kept for analysis. Intergenic enhancers should have bidirectional and approximately equal signal from plus and minus strand. If the mean difference was over tenfold only the lower signal was considered (the stronger signal typically derives from transcribed gene region signal extending beyond annotated transcription termination site). For tightly clustered enhancers, the enhancer that overlapped best with RS4;11 ATAC or centered nuclear-free region (< 7.5 kb peak size) of H3K27ac peaks was selected, or otherwise the region with maximum signal was used. Quantified enhancers were filtered (expression cut-off 5 in minimum two samples) and normalization using RLE. A quasi-likelihood negative binomial generalized log-linear model was fitted using edgeR. The quasi-likelihood (QL) F-test was used to detect significant eRNA-level changes (FDR < 0.1). Clustering was performed using hierarchical clustering and distinct cluster profiles were visualized using the R package ComplexHeatmap. Denovo motif discovery was performed by extracting the DNA sequence for motif enrichment analysis with Homer (4.9.1) findMotifsGenome.pl (region size 200 bp, repeat masked genome, background all enhancers from statistical analysis). To further visualize enhancer signal, GRO-seq signal histograms were generated. For TF binding sites, intergenic region corresponding to motif-centered TF ChIP-seq peaks were used for signal summary. p53 ChIP peaks were retrieved from GSE46991 [ 82 ] and centered with TP53 motif (p63(p53)/Keratinocyte-p63-ChIP-Seq (GSE17611)/Homer). The histogram was generated with bin size 25 bp + / − 2000 bp from the ChIP-peak center. For chromatin state-specific enhancer analysis, regions that had high/low accessibility in each chromatin state were detected using the Seurat Findmarkers function, with a minimum fold change difference of 0.1 and FDR < 0.1. To further visualize top 200 chromatin state-specific enhancer signals the assay-specific signal matrixes were generated with annotatePeaks.pl tool of the HOMER package (version 4.11) [ 76 ]. The matrixes were generated with bin size 25 bp + / − 1000 bp from the enhancer center. Visualization was done with ImageJ (version 1.53t) [ 83 ] and average signal summary histograms were generated with the same parameters visualized with base R plots (R version 4.1.0).

scRNA-seq assay from in vitro cell cultures

To analyze the drug response at cellular resolution RS4;11 or Nalm-6 cells were treated for 24 h with 1 µM AZD1775 or DMSO. Cell line samples from both the DMSO and AZD1775 treatments, representing the same parental line, were processed simultaneously. Cell viability was checked using Trypan blue with Cellometer Mini Automated Cell Counter (Nexcelom Bioscience), and only viable cells were processed further. To deplete dead cells, the Dead Cell Removal Kit (#130–090-101, MACS miltenyi Biotech) was used and the column rinsed twice with 1 ml Binding Buffer to elute viable cells (97–98% viability). Single-cell suspension, loading, and library preparation was performed according to the ChromiumTM Chromium Single Cell 3´Reagent Kits v3 User guide CG000184 Rev A and libraries constructed using the 10x Genomics Chromium technology. Loading concentrations were 1700 cells/µl, 1000 cells/µl for RS4;11 DMSO, AZD1775 cells; 500 cells/µl, 1300 cells/µl Nalm-6 DMSO, AZD1775, respectively. Each lane was loaded with 10,000 cells as pools of human and mouse cells (not part of this study) processed in parallel. Control and treatment samples were loaded onto the 10x Genomics Chromium controller and processed into libraries in parallel. Sequencing was performed in FIMM Technology Center Sequencing Laboratory, Biomedicum, Helsinki, Finland, by using a NovaSeq S2 sequencer aiming 50,000 reads per cell depth.

scATAC-seq assay

RS4;11 cell nuclei were collected from parallel cell cultures as those used for the scRNAseq experiments to study chromatin accessibility at the single-cell level (scATAC-seq). Nuclei were isolated with 10x Genomics Nuclei Isolation for Single Cell ATAC Sequencing user guide CG000169 Rev D. Nuclei quality was determined with light microscoping with a × 40 focus. Nuclei concentration was determined with Trypan blue, and pools of human and mouse (not part of this study) cell lines were loaded together. scATAC-seq was performed using Chromium Single Cell ATAC Library & Gel Bead and Chip E Single Cell ATAC Kit (10 × Genomics), user guide CG000168 Rev C. Sequencing was performed in the FIMM Technology Center Sequencing Laboratory, Biomedicum, Helsinki, Finland, using NovaSeq S2 sequencer aiming 50,000 reads per cell depth.

scMultiome-seq assay

To analyze the drug response transcriptional and chromatin accessibility from the same cells, RS4;11 were treated for 10 h with 1 µM AZD1775 or DMSO. In scMultiome assay nuclei were first isolated. 10x Genomics protocol (User guide CG000365 RevB) was first optimized for the RS4;11 cell line with a 3-min cell lysis time. Nuclei were then washed 3 times with 600 g 7 min centrifugations steps. Nuclei quality was determined with light microscoping at × 40 focus. Nuclei count and lysis optimalization was determined with diluted Trypan blue solution with Countess 3 cell counter (Invitrogen). Each sample was loaded with 10,000 nuclei and library preparation performed according to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User guide CG000338 Rev E and libraries constructed using the 10x Genomics Chromium technology. Loading concentrations were 7200 nuclei/µl for DMSO cells and 4860 nuclei/µl for AZD1775 cells. Multiome ATAC Sequencing was performed in Novogene Cambridge, UK, by using a NovaSeq PE50 sequencer aiming 20,000 reads per cell depth. Multiome RNA samples were sequenced in the same place with NovaSeq PE150 aiming 50,000 reads per cell depth.

PDX cell staining and sorting for scRNA sequencing

Sample preparation for scRNA-sequencing involved thawing viably frozen bone marrow cells for WEE1 28 h time point inhibitor-treated mice and using fresh bone marrow cells for day 6 sequentially treated ones, which were stained with Draq7 (BioLegend), hCD45 (BD Bioscience), and hCD19 (BioLegend), together with a PerCP/Cyanine5.5 anti-mouse CD45 (BioLegend). Approximately 20,000 alive/mCD45 − /hCD45 + /hCD19 + cells were sorted into a PBS (GE Healthcare Life Sciences) + 2% FBS (Thermo Scientific) coated tube and prepared for sequencing using the 10x Genomics platform at the Center for Translational Genomics, Lund University.

Single-cell genomics from primary ALL cells during chemotherapy

Bone marrow or blood mononuclear cells collected at diagnosis and 1 day following treatment start were analyzed from viably cryopreserved samples. Cells were thawn in a 37 °C water bath, immediately after adding 0.5 ml RPMI 1640 media (Thermo Fisher Scientific) + 10% FBS (Gibco) + 20 µl DNAse (Roche 100 U/µl) on top, then moving cells to 15 ml Falcon and filling the volume drop-wise, gently swirling. For samples with < 1 million cells, 10 µl of DNAse was added, filling volume to 5 ml. Cells were washed two times, then centrifuged and re-suspended in 50 µl Cell Staining Buffer (BioLegend) for counting. Subsequently, 100,000–200,000 cells per sample were processed by blocking with 5 µl of Human TruStain FcX Blocking Solution (BioLegend) for 10 min at 4 °C. Antibody pool (0.25 µg per million cells per specific antibody, except 0.125 µg for CD45 and CD8; 0.1 µg per hashtag antibodies) was added and incubated for 30 min at 4 °C. Samples were washed three times with cell staining buffer, counted and assessed for viability using trypan blue staining, and then sample pools were prepared, centrifuged, and loaded to Chromium lanes (10x Genomics). If post-stain viability was > 70% for all samples, equal ratios were loaded aiming at 10,000 cells. Otherwise, loading ratio was adjusted such that half target cell count was used for samples with viability 50–70% and one fifth of viability was 20–50%. The samples analyzed in this study (diagnosis EG9, day 2 EG19) are part of a sample set processed in three batches and with hashtag-barcoding of two donors per Chromium lane. The 5′ gene expression, ADT- and VDJ (BCR and TCR) libraries were prepared following manufacturer instructions (10x Genomics) and cDNA qualities were assessed using Bioanalyzer. Libraries were index barcoded and sequenced using Illumina Novaseq (S1/S2).

scRNA-seq data processing and visualization

Cell line in vitro scRNA-seq data was processed and aligned with Cell Ranger (version 3.0.2) using hg19 and mm10 genome as reference. Human cell counts were used for downstream analysis. scRNA-seq data from mouse xenograft models were processed and aligned with Cell Ranger (version 6.0.1) using GRCh38-2020-A genome as reference. Primary ALL scRNA- and scADT-seq data was aligned with Cell Ranger 6.0 version to human reference (hg19) with default parameters. Donor (and singlet/doublet) assignment was carried out by DSB-normalized hashtag signals available in Seurat package and SNP-based donor assignment implemented in the tool vireo, keeping only concordantly assigned cells [ 84 ].

For downstream analysis, cells were log-normalized using a scaling factor of 10,000. Vst selection method was used to select 2000 highly variable features (genes) and from these PCA dimension 1:10 were used for neighborhood analysis (FindNeighbors function), clustering (FindClusters with resolution parameter varied from 0.5 to 2) and dimensionality reduction (UMAP). The respective parameters are specified in the accompanying code repository and resulting cell and cluster numbers in Additional file 3 , Table S3. At 24 h, RS4;11 DMSO and AZD1775 samples were initially merged and filtered in the same manner. From the RS4;11 cell line in vitro sample, this resulted in a discovery of 11 distinct cell clusters from the merged object with DMSO and AZD1775 treatment (24 h) that are referred to as “cell states” and used as reference sample for label transfer analysis (see below). For cell cycle characterization, a list of cell cycle markers was utilized from [ 85 ].

Several statistical comparisons between the cells assigned to these cell states were performed: (i) Genes that had high/low expression in each cluster were detected using the Seurat Findmarkers function, with a minimum fold change difference of 0.25 between cells assigned to the cluster and other cells. (ii) AZD1775-treatment specific cell states were compared with the cell state matched to normal cell cycle G2/M-phase (cell state 4), (iii) cluster markers were detected between AZD1775-treatment specific cell states. Pair-wise correlation was also calculated for percentage of expression data (obtained from R dittoSeq version 1.6.0 dittoDotPlot function) to analyze TF co-expression at cluster level. This analysis was performed with rcorr function with Pearson correlation. Violin plots were used to visualize gene expression distributions. For visualizing gene expression level per cell cluster dotplots were generated using the dittoSeq package. Alternatively, all unique genes from cluster comparisons were acquired and the mean scaled counts were summarized. These mean expression values were then used as input for hierarchical clustering with 1-Pearson correlation as a distance metric. ComplexHeatmap R package was used for visualizations, testing different cluster numbers (10–20) for row-wise clustering. Pathway analysis was performed for the gene clusters and for broader patterns distinguished based on the heatmap.

scATAC-seq data processing and visualization

The raw reads were processed and aligned with Cell Ranger atac workflow (version 1.2.0). Seurat ([ 86 ], version 3.1.1) package and Signac ([ 87 ] version 0.1.6) were used for downstream analysis, selecting only the human peak coordinates and cells. Peaks detected from DMSO- and AZD1775-treated cells were merged using bedtools (version 2.27.1), discarding < 10 bp wide peaks. Data across treatments was combined by re-quantifying counts from the fragment files based on the merged peak regions. Peaks detected in > 1% of cells were kept for downstream analyses. Nucleosome signal was quantified based on fragments mapping to chr 1 using the Signac package NucleosomeSignal function. TSS enrichment was calculated based on coordinate ranges retrieved from EnsDb.Hsapiens.v75. These quality metrics were used in combination with metrics to select good quality nuclei for downstream analyses (peak region fragments > 1000, peak region fragments < 100,000, percentage reads in peaks > 15, blacklist ratio < 0.05, nucleosome signal < 10, TSS.enrichment > 2). To analyze the atypical nucleosome signal manifest in AZD1775-treated sample, the above filtering criteria were used without the nucleosome signal cutoff to plot the signal profiles. Term frequency-inverse document frequency (TF-IDF) normalization of peaks by accessibility was performed using the LSI implementation used by [ 87 ]. To reduce dimensions and perform clustering of the data matrix, singular value decomposition (SVD) was run on the TD-IDF normalized matrix, keeping 150 dimensions. Based on elbow plot analysis 50 dimensions were kept for UMAP visualization and clustering.

To study more specifically chromatin activity responses at each treatment condition, separate UMAPs were created (reduction method lsi, dims = 1:50, resolution 0.4). The two smallest chromatin states found in the chromatin state clustering of DMSO- (c5, c6) and AZD1775- (c6, c7) treated cells differed based on quality control metrics. The smallest clusters had a low peak region fragment amount (low quality) and were discarded from all downstream analyses. The clusters with elevated nucleosomal signal were reproduced in the scMultiome result and could be matched using this data to cells with a good quality RNA-seq profile. Moreover, they had a distinct TF motif activity pattern in chromVAR analysis (unlike the smallest cluster). However, in visualizing TF activity scores specific to other clusters, we omitted the clusters with elevated nucleosome signal from heatmaps since they consistently showed low signal. For chromatin state-specific enhancer analysis, we started with intergenic enhancer defined from bulk genomics data and identified high/low accessibility regions in each chromatin state using the Seurat Findmarkers function, with a minimum fold change difference of 0.1 and FDR < 0.1. To further visualize top 200 chromatin state-specific enhancer regions, the assay-specific pseudobulk signal matrixes were generated by creating a combined tagDirectory for cells matched to each chromatin state, followed by signal quantification with annotatePeaks.pl tool of the HOMER package (version 4.11 [ 76 ]). The matrixes were generated with bin size 25 bp + / − 1000 bp from the enhancer center. Visualization was done with ImageJ (version 1.53t [ 83 ]) and average signal summary histograms were generated with the same parameters visualized with base R plots (R version 4.1.0).

scMultiome-seq data processing and visualization

For comparison with the 24 h data, multiome scRNA-seq data was processed with the same Cellranger preprocessing workflow, resulting in reads mapped to the hg19 genome version. DMSO- and AZD1775-treated samples were merged and analyzed using Seurat v4.1.1. The raw reads were also processed and aligned with Cell Ranger Multiome workflow (cellranger-arc-2.0.1, hg38 genome version). The cell assignment available from per barcode metrics output was used to select nuclei for downstream analyses. The scATAC-seq workflow based on Signac [ 87 ] 0.1.6 and Seurat 3.1.1. versions that was used for 24 h data was run with minor modifications (EnsDb.Hsapiens.v86, no blacklist filtering, atac_peak_region_fragments > 1000 & atac_peak_region_fragments < 100000 & pct_reads_in_peaks > 50 & nucleosome_signal < 10) to generate comparable results. Nucleosome signal histogram analyses were performed using Signac version 1.6.0. The matching cell barcodes between scRNA- and scATAC-seq profiles from the same cells were utilized to visualize data across modalities. For example, this allowed showing the scATAC-seq nucleosome signal on the UMAP generated from scRNA-seq data. Cell type labels were assigned based on label transfer analysis with the respective 24-h sample used as reference sample.

scRNA-seq RNA velocity and PAGA analysis

Dynamic changes in gene transcription can be modeled based on reads corresponding to both unspliced and splice mRNA (newly synthetized RNA vs processed RNA, respectively). Based on the dynamic RNA processing model [ 88 ], the future transcriptome state can be visualized together with the measured current state. This analysis was used to analyze cell state dynamics in the RS4;11 cell model. Velocyto CLI (version 0.17.17 [ 88 ]) was used to calculate the count matrices, masking expressed repetitive elements (available for hg19 from UCSC Genome Browser). The scVelo-package (version 0.2.1 [ 89 ]) was used in downstream analysis. The gene expression matrix was accompanied with the spliced and unspliced count matrices. The data was first filtered by removing genes with less than 20 shared counts in both spliced and unspliced data. The matrices were each then normalized by dividing the counts in each cell with the median of total counts per cell. The 2000 most variable genes were extracted based on the spliced count matrix and the data matrices were log-transformed. Top PCs (30) were used for neighborhood graph calculation, with the number of neighbors set to 30. Then the AZD1775 and DMSO samples were separated for RNA velocity analysis. Based on the neighborhood connectivities, the first-order moments for spliced and unspliced matrices were calculated and the velocity was estimated using the dynamical model. The velocities were embedded to a UMAP presentation, which was calculated with 20 PCs and 10 neighbors. A PAGA graph was used to visualize connectivities (dashed lines) and transitions (solid lines). Python Scanpy-package (version 1.5.1) was used for gene set scoring. Before scoring, gene sets used in the analysis (e.g. senescence gene sets) were filtered to include only genes that are expressed in the data, excluding G2M-phase markers (to remove cell cycle bias in the scores).

TF motif activity profile from scATAC-seq profile

To determine motif activity, i.e., variability in chromatin accessibility per cell, the chromVAR ([ 90 ], version 1.6.0) tool was applied separately to each sample. The known TF motifs available in the Homer tool were scored. Subsequently, cluster-specific motifs were detected using statistical analysis (Seurat FindMarkers function, log2 fold change threshold 1.5, FDR < 0.0001) comparing each chromatin cluster to other clusters. Redundant motifs recognized by similar protein complexes were grouped manually and the most significant motif kept for visualization. The scaled chromatin accessibility values were used to calculate mean access per chromatin cluster and visualized using the ComplexHeatmap R package. The Signac visualization functions were used to generate genome browser track plots comparing different clusters.

Data integration of single-cell genomics samples using label transfer

To characterize similar cells between two different sample sets, the canonical correlation analysis available in the Seurat (Seurat 4.1.1) package was used. First anchors between two different samples were identified following the user guide recommendations. For cell line and PDX data, the 24 h RS4;11 data cell states 1–11 were used as reference. These cell state labels were transferred to the 10 h multiome scRNA-seq data, or the PDX data. Similarly, the MLL-7 clusters from 28 h were transferred to day 6 sample data. Prediction scores were visualized for quality control purposes. The same analysis was performed to characterize chromatin levels with user guide recommendations for ATAC data. Twenty-four-hour RS4;11 DMSO ATAC clusters 0–5 were used as reference for 10 h DMSO multiome scATAC-seq data. Twenty-four-hour RS4;11 AZD1775 ATAC clusters 0–6 were used as reference for 10 h AZD1775 multiome scATAC-seq data. Cells with prediction scores < 0.3 were omitted from visualizations (assigned to category NA). Proportion plots of label assignments were visualized using the R Dittoseq package (version 1.6.0) with the dittoBarPlot command. To illustrate the correspondence between 24-h clusters and 10-h clusters, we generated Sankey diagrams using the 10-h multiome samples treated with DMSO and AZD1775. We employed the R package scmap (version 1.16.0) and getSankey function. Rare categories of labels (assigned to < 1.5% cells analyzed) were omitted from the plot for clarity.

Pathway enrichment analysis

Gene lists acquired bulk genome-wide and scRNA-seq studies were analyzed using the online web server Enrichr [ 91 ] for enrichment of ontology and pathway terms. The analysis was performed based on gene sets from BioPlanet 2019 and TF perturbations. Enriched terms were ranked based on the lowest FDR and top terms per group visualized as dot plots (R package ggplot2).

Cell proliferation, EdU incorporation, and senescence assessment by FACS

To study the effects of AZD1775 on cell proliferation, cells were incubated with 10 μM EdU for 1 h at 37°C to label proliferating cell population. EdU-labelling of the cells was done prior to the treatment for further processing according to the kit protocol (Thermo Fisher). Following cell fixation and permeabilization, the cells were stained with pH3 (1:25, BD Bioscience), and 50 μg / mL Propidium Iodide in 1% BSA/ TBS – 0.5% Tween 20 containing 10 μg/mL RNAseA overnight at 4°C. Finally, the click-iT reaction were carried out according to the manufacturer’s protocol prior to FACS analysis. To analyze senescence, treated cells were or CellEvent Senescence Green Flow Cytometry Assay Kit (Invitrogen, C10840) and Propidium Iodide according to the manufacturer’s protocol.

Quantitative real-time PCR (qRT-PCR)

To perform the qRT-PCR, RNA was prepared using RNeasy kit (Qiagen). Residual genomic DNA was eliminated from the total RNA fraction by DNase I treatment (Qiagen), according to the manufacturer’s protocol (Qiagen). Fifty nanograms of the purified RNA was then used for cDNA synthesis (SuperScript VILO cDNA Synthesis Kit, Invitrogen, Life Technologies). Quantitative PCR were carried out using SYBR green probes as described in the appendix file (Additional file 6 , Table S6). The data were analyzed with ΔΔCt method and presented as relative expression.

Statistical analysis

Data from biochemical assays are presented as the mean + SD from two or more independent experiments unless indicated otherwise. Statistical analyses were performed using the statistical package GraphPad Prism 8 (GraphPad Software, San Diego, CA, U.S.A., http://www.graphpad.com ) using either Student’s t test, Log-rank (Mantel-Cox), one-way analysis of variance (ANOVA), or two-way analysis of variance (ANOVA), as indicated. Genome-wide statistical analysis was performed using dedicated software (R or Python packages as indicated). Multiple testing correction was performed using Benjamini–Hochberg or Bonferroni’s post hoc test when appropriate.

Availability of data and materials

Processed data from genomics assays are available via the NCBI Gene Expression Omnibus database under the following accession codes: GSE220099 (bulk GRO-seq profiles in the RS4;11 cell line) [ 92 ], GSE148195 (bulk ChIP-seq data in RS4;11 cell line) [ 78 ], GSE218621 (scRNA-seq data in RS4;11 and Nalm-6 cell lines) [ 93 ], GSE220476 (scRNA-seq data from mouse xenografts of RS4;11 and MLL7 cells) [ 94 ], GSE230295 (scRNA-seq data from primary ALL patients) [ 95 ], GSE218805 (scATAC-seq data in RS4;11 cell line) [ 96 ], and GSE220112 (sc-Multiome data in RS4;11 cell line) [ 97 ]. Additional data used in enhancer and ChIP-seq peak analysis is available under the accession codes GSE148192 (RUNX1 in Nalm-6 cell line) [ 98 ], GSE71616 (histone markers in RS4;11 cell line) [ 81 ], GSE117865 (chromatin accessibility and histone marker data in RS4;11 and SEM cell lines) [ 79 ], and GSE46991 (p53 binding sites in LCL) [ 82 ]. Code related to analyses is available at GitHub [ 99 ] and Zenodo [ 100 ] under GNU General Public License 3.0.

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Acknowledgements

The authors thank Professor Richard Lock (Children’s Cancer Institute Australia) for providing KMT2A-r PDX samples and related clinical information for this study, Kuopio and Tampere university hospital pediatric oncology clinics for prospective sample collection of pediatric leukemias during induction chemotherapy and patients consenting to participate in these studies, Professor Sui Huang (Institute for Systems Biology USA), Arne Lindqvist and Andrä Brunner (Karolinska Institute) for helpful discussions, Magdalena Paolino (Department of Medicine, Karolinska Institute) for help with flow cytometry analysis, Bharadwaja Velidendla for help with bulk genomics data visualization, and Aleksi Kokko, Sini Hakkola, Janne Suhonen, and Jonne Nieminen for setting up bioinformatics workflows for single-cell genomics samples. This research was funded by The Swedish Childhood Cancer Fund, the Swedish Cancer Society, The Swedish Research Council, Karolinska Institute, Radiumhemmets Research Foundation, AstraZeneca-SLL-KI Open Innovation grant (#18122013), the Academy of Finland (321553, 310106), the European Union Horizon 2020 research and innovation program under grant agreements No 824110 (EASI-Genomics) and ERAPERMED2018-209 (JTC2018 ERA-NET ERA PerMed), Väre Foundation, Emil Aaltonen Foundation, Cancer Foundation Finland, Jane and Aatos Erkko foundation, and Sigrid Juselius foundation. The authors wish to acknowledge CSC – IT Center for Science, Finland and UEF Bioinformatics Center, University of Eastern Finland, Finland for computational resources. The authors would like to acknowledge Single Cell Genomics Core (Biocenter Kuopio) and Biocenter Finland for infrastructure support, GeneCore Sequencing Facility (EMBL, Heidelberg, Germany), FIMM Genomics NGS Sequencing, Technology Centre (Biomedicum, Helsinki), Clinical Genomics Lund, SciLifeLab and Center for Translational Genomics (CTG), Lund University and SNP&SEQ Technology Platform, SciLifeLab Uppsala, for providing expertise and service with genomics, sequencing, and analysis.

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Anahita Bishop and Kevin Pang were the primary editors of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Review history

The review history is available as Additional file 8 .

Author information

Alena Malyukova, Mari Lahnalampi, Merja Heinäniemi and Olle Sangfelt are contributed equally to this work.

Authors and Affiliations

Department of Cell and Molecular Biology, Karolinska Institutet, Biomedicum, Solnavägen 9, 171 77, Stockholm, Sweden

Alena Malyukova, Karen Akopyan, Johanna Viiliainen & Olle Sangfelt

The Institute of Biomedicine, School of Medicine, University of Eastern Finland, Kuopio, Finland

Mari Lahnalampi, Petri Pölönen, Mikko Sipola, Juha Mehtonen & Merja Heinäniemi

Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden

Ton Falqués-Costa & Anna K. Hagström-Andersson

Tampere Center for Child, Adolescent and Maternal Health Research, Faculty of Medicine and Health Technology, Tampere University and Tays Cancer Center, Tampere University Hospital, Tampere, Finland

Susanna Teppo & Olli Lohi

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Contributions

AM, ML, MH, and OS designed the study. AM, JV, TFC, ST, and KA performed experiments; ML and MH designed and performed the scRNA, scATAC, scMultiome-seq, and bulk GRO-seq analyses. PP participated in the GRO-seq data analysis. JH performed HEMAP data analysis. MS performed Cell Ranger, velocity and Gene set scoring, and PAGA analyses. AKHA and OL performed critical reading of the manuscript. AKHA supervised animal work. OL coordinated clinical sample collection. AM, ML, MH, and OS wrote the manuscript.

Corresponding authors

Correspondence to Alena Malyukova , Merja Heinäniemi or Olle Sangfelt .

Ethics declarations

Ethics approval and consent to participate.

Collection of blood and bone marrow samples during induction chemotherapy was approved by the Regional Ethics Committee in Pirkanmaa, Tampere, Finland (#R13109), and conducted according to the guidelines of the Declaration of Helsinki. A written informed consent was received by the patient and/or guardians. PDX studies had prior approval from the Human Research Ethics Committees of the University of New South Wales. Mice were bred and maintained in accordance with Lund University’s ethical regulations and approved by the local ethics committee of Lund, Sweden.

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Not applicable.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1..

Contains supplementary figures S1-S10 and their legends. Contains data set access codes and Table S1.

Additional file 2.

Table S2 GRO-seq summary of significantly differentially expressed genes. Differentially expressed genes based on one-way ANOVA-like statistical comparison of mean transcriptional activity levels across sample conditions and summary of pathway enrichment analysis.

Additional file 3.

Table S3 Summary of scRNAseq samples and cluster marker gene statistical analyses. Data filtering: scRNAseq sample filtering cutoffs and number of cells used in analysis. Differentially expressed genes from comparison of AZD1775-specific clusters (cell states 5-11) and DMSO cell state 4 (matching G2/M cell cycle phase) from 24 h time point in RS4;11 cells, heatmap cluster assignment (refer to Fig. S2e) and summary of pathway results. Marker gene lists for 24 h time point RS4;11 cell AZD1775-specific clusters. List of cell fate III top markers. In vivo samples: MLL-7 PDX AZD1775 treatment (28 h) cluster 6 marker genes and summary of pathway analysis; MLL-7 PDX AZD1775 treatment (day 6) corresponding pathway analysis for cells matched to 28 h cluster 6; RS4;11 in vivo marker genes for 11 clusters of data merged across treatments.

Additional file 4.

Table S4 scATAC summary of significantly different motif access across clusters. RS4;11 24 h AZD1775 and DMSO chromVar analysis results. Significantly differentially active motifs used for clustering. Based on statistical comparison of AZD1775 and DMSO chromatin states (data sheets chromatin state 1-6), and DMSO chromatin state 1-5, p-value from Wilcoxon Rank Sum test is shown.

Additional file 5.

Table S5 GROseq summary of enhancer analysis. Up-regulated enhancers at 24 h: top enriched TF motifs for the corresponding DNA sequences.

Additional file 6.

Table S6 Primer and antibody summary. List of RT-qPCR primers and antibodies used in the study.

Additional file 7.

Contains original images of uncropped western blots.

Additional file 8.

Review history.

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Malyukova, A., Lahnalampi, M., Falqués-Costa, T. et al. Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia. Genome Biol 25 , 143 (2024). https://doi.org/10.1186/s13059-024-03260-4

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Published : 31 May 2024

DOI : https://doi.org/10.1186/s13059-024-03260-4

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