**Meet the editors**

Professor Dr. Margarita Guenova received her medical training at the Medical University of Sofia and her PhD degree at the National Center of Haematology in 2000. She is employed as Head of the Laboratory of Haematopathology and Immunology and Professor of Hematology and Blood Transfusion at the National Specialised Hospital for Active Treatment of Haematological Diseas-

es, Sofia, Bulgaria, teaching both undergraduate and graduate studies at the Sofia Medical University. She specializes in the field of leukemia and lymphoma diagnostics. In regard to her scientific interests, she worked on the elucidation of critical mechanisms of leukemia pathogenesis and progression, investigation of clinically relevant biomarkers and potential targets for therapy in leukemias and lymphomas, characterization of leukemic stem cell populations, minimal residual disease, and implementation of a multifaceted approach in oncohematology. Dr. Guenova has authored and coauthored many abstracts, articles in peer-reviewed journals, and book chapters. She serves on several editorial boards and is the President of the Bulgarian Society of Hematology as well as a member of several professional societies, such as the Bulgarian Society of Pathology, the Bulgarian Association of Clinical Immunology, the European Hematology Association, the European Association of Haematopathology, and the International Society of Haematology.

Professor Dr. Gueorgui Balatzenko received his medical training at the Medical University of Sofia and his PhD degree at the National Center of Haematology in 2002. He obtained additional qualifications at the Red Cross Blood Bank, Groningen-Drenthe, the Netherlands; Institute Paoli-Calmettes, Marseille, France; and Necker-Enfants Malades, Paris, France. He is Professor of

Hematology and Blood Transfusion at the Laboratory of Cytogenetics and Molecular Biology at the National Specialised Hospital for Active Treatment of Haematological Diseases, Sofia, Bulgaria, teaching both undergraduate and graduate studies at the Sofia Medical University. His major interest is in the field of molecular investigations in leukemias. In regard to his scientific achievements, he worked on the elucidation of the incidence and clinical relevance of molecular biomarkers in chronic and acute leukemias as well as on the molecular monitoring of treatment response. Dr. Balatzenko has authored and coauthored many articles in peer-reviewed journals and book chapters. He is a member of several professional societies, such as the Bulgarian Society of Hematology, the Bulgarian Society of Genetics, and the European Hematology Association; a life member of the UICC; and a referee for international journals.

## Contents

## **Preface XI**


Margarita Guenova


Isabel González Gascón y Marín, María Hernández-Sánchez, Ana-Eugenia Rodríguez.Vicente and José-Ángel Hernández-Rivas


## Preface

Chapter 7 **Perspective on Therapeutic Strategies of Leukemia Treatment**

Chapter 8 **New Insights in Prognosis and Therapy of Chronic Lymphocytic**

Bo Yuan, Noriyoshi Iriyama, Xiao-Mei Hu, Toshihiko Hirano, Hiroo

Isabel González Gascón y Marín, María Hernández-Sánchez, Ana-Eugenia Rodríguez.Vicente and José-Ángel Hernández-Rivas

Rositsa Vladimirova, Dora Popova, Elena Vikentieva and Margarita

**— Focus on Arsenic Compounds 191**

Chapter 9 **Chronic Lymphocytic Leukemia — Microenvironment**

Chapter 10 **Zinc Oxide Nanoparticles and Photodynamic Therapy for the Treatment of B-chronic Lymphocytic Leukemia 277** Sandra Loydover Peña Luengas, Gustavo H. Marin, Luis Rivera,

Adrian Tarditti, Gustavo Roque and Eduardo Mansilla

Toyoda and Norio Takagi

**Leukaemia 219**

**VI** Contents

**and B Cells 247**

Chapter 11 **Update on Leukemia in Pregnancy 315** Khalid Ahmed Al-Anazi

Guenova

**Leukemias** are a heterogeneous group of clonal diseases of different cell lineages that originate from a cell of the hematopoietic and lymphatic tissues with diverse incidence, etiology, pathogen‐ esis, and prognosis. A significant progress in the clinical management has been achieved, but de‐ spite that current therapeutic approaches often produce prolonged survival, the therapeutic responses are often partial, brief, and unpredictable. However, hematology has constantly been advancing in parallel with technological developments that have expanded our understanding of the phenotypic, genetic, and molecular complexity and extreme clinical and biological heteroge‐ neity of leukemias. This in turn allowed for developing more effective and less toxic alternative therapeutic approaches directed against critical molecular pathways in leukemic cells.

The continuous and rather extensive influx of new information regarding the key features and underlying mechanisms as well as treatment options of leukemias requires frequent updating of this topic. The primary objective of this book is to provide the specialists involved in the clinical management and experimental research of acute and chronic leukemias with comprehensive and concise information on some important theoretical and practical developments in the biology, clinical assessment, and treatment of patients with leukemias as well as on some molecular and pathogenetic mechanisms and their respective translation into novel therapies. Specific clinical scenarios such as pregnancy and age are also within the scope of the book. An international panel of experts provide novel insights into various aspects of leukemias and contribute their experi‐ ence to updates in the field.

Classically, leukemias have been classified according to their cellular origin, as myeloid or lym‐ phoid, or according to their course, as acute or chronic.

**Acute lymphoblastic leukemia (ALL)** remains among the greatest challenges in hematology, even if the road to curing most children with ALL, the most common childhood cancer, may be the greatest success story in the history of cancer. Approximately 80% of children with ALL are now cured however, for those children with unfavorable features, defined either by disease biolo‐ gy or by response to treatment, the outcomes remain poor. The first chapter addresses the great strides that have been made in effectively treating pediatric leukemia and presents data on the contribution of risk stratification combined with intensified therapies. The authors focus on role of the increasingly more precise genomic studies for defining specific subtypes of high-risk leuke‐ mia and identifying druggable targets in aberrant pathways complemented with the develop‐ ment of relevant preclinical models which are expected to fuel rapid advances in the future. Recent genomic and immunophenotypic studies underlie the identification of new clinically rele‐ vant ALL subtypes, such as the early T-cell precursor ALL (ETP-ALL), which has been recently recognized as a form with poor prognosis and reviewed in the second chapter. Recent advances in the biology, genetics, and clinical features of this aggressive disease are presented in a compre‐ hensive summary of the major characteristics and scoring systems in the reported series, further

contributing with two clinical cases with a particular clinical presentation. Various viruses have been claimed to be associated with lymphoid neoplasms, including HTLV-1 and HIV. The third chapter reviews the interaction of these two retroviruses within the immune system and more specifically examines the impact of HIV-1 infection on the leukemogenic process induced by HTLV-1 in coinfected individuals as well as the impact of HTLV-1 infection on HIV-1 disease.

**Acute myeloid leukemia (AML)** accounts for approximately 80% of cases of acute leukemia in adults. It is characterized by clonal proliferation of myeloid precursors and accumulation of leu‐ kemic blasts in the bone marrow, ultimately resulting in hematopoiesis failure. Although nowa‐ days AML is cured in 35–40% of adult patients who are 60 years of age or younger and in 5–15% of patients who are older than 60 years of age, the outcome in a significant proportion of patients who are unable to receive intensive chemotherapy without unacceptable side effects remains dis‐ mal. The cytogenetic and the enormous molecular heterogeneity of the disease have become in‐ creasingly apparent over the past years. The recent large-scale sequencing of AML genomes is now providing opportunities for patient stratification and personalized approaches to treatments that are based on individual mutation profiles. The fourth chapter addresses recent knowledge on genetic and molecular aberrations in AML, describing the incidence, prognostic influence, and association with other molecular markers of the most important recurrent mutations in AML. It is followed by a comprehensive update on non-M3 AML etiology, classification, risk stratification, emergencies, complications, disease in special circumstances, and current and future therapeutics provided in the fifth chapter of the book.

The unsatisfactory clinical outcomes of a significant proportion of AML patients urged the devel‐ opment of **new anti-AML therapy strategies**, one of which includes the implementation of new nucleoside analogs. The sixth chapter summarizes the available data in regard to clofarabine, which has offered new promising perspectives within induction and consolidation therapies. This chapter evaluates the efficacy and tolerability of the drug as a single agent and in combination therapy, including hematopoietic stem cell transplantation, for AML patients. On the other hand, the treatment of most cases of acute promyelocytic leukemia differs from the usual AML treat‐ ment. Initial treatment includes the non-chemotherapy drug all-trans-retinoic acid (ATRA), which is most often combined with chemotherapeutic drugs. Another option is to include another differ‐ entiating drug called arsenic trioxide (ATO). This is often used in patients who cannot tolerate an anthracycline drug, but it is an option for other patients as well. The seventh chapter highlights the pharmacokinetics of ATO and the detailed mechanisms underlying the cytocidal effects of arsenic compounds. A detailed insight into potential future clinical applications of those promis‐ ing candidates endowed with potent antitumor activities in view of combination with arsenic compounds is provided.

In regard to chronic leukemias, **B-cell chronic lymphocytic leukemia (B-CLL)** is the most com‐ mon type of leukemia in adults. CLL displays immense clinical heterogeneity, as many patients have an indolent disease that will not require intervention for many years, while others will present with an aggressive and symptomatic leukemia. While clinical staging systems have been used to stratify patients into risk categories, they lack the ability to predict disease progression or response to therapy. Therefore, the significant interest in identifying additional prognostic mark‐ ers that can be used to distinguish those patients who may have an aggressive form of CLL and might benefit from early intervention has pushed research toward unraveling the biology of the disease. Numerous cellular and molecular markers with potential prognostic and therapeutic sig‐ nificance have been identified. In parallel, an increasing number of therapeutic compounds and new targeted therapies are under development with promising results. The eightth chapter presents a concise overview of the new prognostic markers of CLL, the relationship they have with each other to build prognostic scores, the role they have in guiding treatment decisions, and the novel therapies that have emerged recently with immunologic, biochemical, and genetic tar‐ gets. However, conventional treatments are still not directed to the interactions between CLL cells and their microenvironment. The existence of a complex network of antiapoptotic and prosurviv‐ al molecules, including cell adhesion, proinflammatory, angiogenic, and proto-oncogenic mole‐ cules, is responsible for supporting the infiltrating malignant cells and for the maintenance of the neoplastic tissue in CLL. Many prosurvival signaling pathways potentially sustaining CLL cell maintenance interact with one another. Thus, it appears that developing new classes of drugs af‐ fecting simultaneously various signaling pathways, and therefore abrogating signaling redundan‐ cy-associated chemoresistance to classical drugs, is feasible. Based on these concepts, the ninth chapter comprises a review of recent studies on key biomarkers of intercellular interactions of the leukemic population, which enable clarification of key processes in the development of the dis‐ ease and can be the basis for defining a separate risk patient group to optimize the therapeutic approach. It is essential to have new treatment modalities in order to increase the anti-B-CLL ef‐ fects, providing greater biological activity and much more specificity for the disease. However, in general, in most anti-neoplastic therapies, the side effects are also frequent and the development of resistance or relapse is usually inevitable. The achieved efficiency might be associated with un‐ acceptable toxicity. Therefore, current scientific research efforts are focused on the development of anti-neoplastic agents, which might achieve maximum effect and also decrease the potential damage on normal cells. The tenth chapter presents a novel therapy using zinc oxide nanoparti‐ cles and photodynamic therapy for the treatment of CLL, claiming that this new therapeutic ap‐ proach is a very specific one with very low toxicity for non-leukemic cells and probably very useful not only for B-CLL but also for all the other indolent lymphomas as well as for all types of cancer.

contributing with two clinical cases with a particular clinical presentation. Various viruses have been claimed to be associated with lymphoid neoplasms, including HTLV-1 and HIV. The third chapter reviews the interaction of these two retroviruses within the immune system and more specifically examines the impact of HIV-1 infection on the leukemogenic process induced by HTLV-1 in coinfected individuals as well as the impact of HTLV-1 infection on HIV-1 disease. **Acute myeloid leukemia (AML)** accounts for approximately 80% of cases of acute leukemia in adults. It is characterized by clonal proliferation of myeloid precursors and accumulation of leu‐ kemic blasts in the bone marrow, ultimately resulting in hematopoiesis failure. Although nowa‐ days AML is cured in 35–40% of adult patients who are 60 years of age or younger and in 5–15% of patients who are older than 60 years of age, the outcome in a significant proportion of patients who are unable to receive intensive chemotherapy without unacceptable side effects remains dis‐ mal. The cytogenetic and the enormous molecular heterogeneity of the disease have become in‐ creasingly apparent over the past years. The recent large-scale sequencing of AML genomes is now providing opportunities for patient stratification and personalized approaches to treatments that are based on individual mutation profiles. The fourth chapter addresses recent knowledge on genetic and molecular aberrations in AML, describing the incidence, prognostic influence, and association with other molecular markers of the most important recurrent mutations in AML. It is followed by a comprehensive update on non-M3 AML etiology, classification, risk stratification, emergencies, complications, disease in special circumstances, and current and future therapeutics

The unsatisfactory clinical outcomes of a significant proportion of AML patients urged the devel‐ opment of **new anti-AML therapy strategies**, one of which includes the implementation of new nucleoside analogs. The sixth chapter summarizes the available data in regard to clofarabine, which has offered new promising perspectives within induction and consolidation therapies. This chapter evaluates the efficacy and tolerability of the drug as a single agent and in combination therapy, including hematopoietic stem cell transplantation, for AML patients. On the other hand, the treatment of most cases of acute promyelocytic leukemia differs from the usual AML treat‐ ment. Initial treatment includes the non-chemotherapy drug all-trans-retinoic acid (ATRA), which is most often combined with chemotherapeutic drugs. Another option is to include another differ‐ entiating drug called arsenic trioxide (ATO). This is often used in patients who cannot tolerate an anthracycline drug, but it is an option for other patients as well. The seventh chapter highlights the pharmacokinetics of ATO and the detailed mechanisms underlying the cytocidal effects of arsenic compounds. A detailed insight into potential future clinical applications of those promis‐ ing candidates endowed with potent antitumor activities in view of combination with arsenic

In regard to chronic leukemias, **B-cell chronic lymphocytic leukemia (B-CLL)** is the most com‐ mon type of leukemia in adults. CLL displays immense clinical heterogeneity, as many patients have an indolent disease that will not require intervention for many years, while others will present with an aggressive and symptomatic leukemia. While clinical staging systems have been used to stratify patients into risk categories, they lack the ability to predict disease progression or response to therapy. Therefore, the significant interest in identifying additional prognostic mark‐ ers that can be used to distinguish those patients who may have an aggressive form of CLL and might benefit from early intervention has pushed research toward unraveling the biology of the disease. Numerous cellular and molecular markers with potential prognostic and therapeutic sig‐ nificance have been identified. In parallel, an increasing number of therapeutic compounds and new targeted therapies are under development with promising results. The eightth chapter

provided in the fifth chapter of the book.

VIII Preface

compounds is provided.

Besides the specific characteristics of various leukemias, a diagnosis of a **blood cancer during preg‐ nancy** is a rare and traumatic experience which poses unique challenges for the mother and the unborn baby, as well as for the treating medical team in managing both the pregnancy and the blood disorder. The last chapter covers the available data about the consequences of maternal and fetal exposures to cytotoxic chemotherapy, radiotherapy, and targeted therapies; a detailed de‐ scription of coexistence between various types of leukemia and pregnancy; and the specific data obtained from the major studies and important case reports on pregnancy in different types of leu‐ kemia.

Each chapter is a separate publication that reflects each author's views and concepts. However, the book presents an update and introduces novel insights into our current understanding of the biology and clinical presentation, the risk assessment, and therapeutic challenges in patients with leukemias.

#### **Prof. Dr. Margarita Guenova, MD, PhD**

Laboratory of Haematopathology and Immunology, National Specialised Hospital for Active Treatment of Haematological Diseases, Sofia, Bulgaria

#### **Prof. Dr. Gueorgui Balatzenko, MD, PhD**

Laboratory of Cytogenetics and Molecular Biology, National Specialised Hospital for Active Treatment of Haematological Diseases, Sofia, Bulgaria

## **Pediatric High Risk Leukemia — Molecular Insights**

Chandrika Gowda, Olivia L. Francis, Yali Ding, Parveen Shiraz, Kimberly J. Payne and Sinisa Dovat

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61247

#### **Abstract**

Acute leukemia comprises of 31% of all cancers in children making it the most com‐ mon childhood malignancy. Significant strides have been made in treatment, partly through risk stratification and intensified therapy. A number of subtypes remain at high risk for relapse and poor outcome, despite current therapies. Here we describe risk stratification and molecular diagnosis used to identify high risk leukemias and guide treatment. Specific cytogenetic alterations that contribute to high risk B and T cell acute lymphoblastic leukemia (ALL), as well as infant leukemia are discussed. Particular attention is given to genetic alterations in IKZF1, CRLF2, and JAK, that have been identified by whole genome sequencing and recently associated with Phlike ALL. Ongoing studies of disease mechanisms and challenges in developing pre-clinical patient-derived xenograft models to evaluate therapies are discussed.

**Keywords:** Acute lymphoblastic leukemia, Pediatric Leukemia, high risk leukemia, Ikaros, IKZF1, CRLF2

## **1. Introduction**

Acute leukemia comprises 31% of all cancers in children, making it the most common child‐ hood malignancy. Acute lymphoblastic leukemia (ALL) makes up 80% of these cases and the remaining are leukemias of the myeloid lineage. Among the lymphoblastic leukemias, there are two immunophenotypic groups: B cell precursor ALL (B-ALL, 80% of all ALL) and T cell ALL (T-ALL, 20%). In the following sections, the further classification of each of these subtypes of leukemia, based on their molecular characteristics, is discussed. Also discussed are the clinical importance, prognosis, and new therapies available for each subtype.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Overview of classification of pediatric leukemia**

## **2.1. Definition of standard and high risk leukemias**

The National Cancer Institute (NCI) has classified acute lymphoblastic leukemia in children based on age at diagnosis, initial white blood count, and the presence of extra medullary disease.


The Children's Oncology Group (COG) has classified acute leukemia into four risk groups using prognostic factors that are strongly predictive of outcome such as: 1) age; 2) initial white count; 3) gender; 4) presence of extra medullary disease at diagnosis (CNS or testicular disease); and 5) blast cytogenetic findings and ploidy, and 6) response to induction therapy [1, 2].

Based on these factors, the four risk groups for COG classification of newly diagnosed B-ALL for the AALL08B1 study are low risk, average risk, high risk, and very high risk. In T-ALL, high white count does not have a prognostic significance. The above classification applies to B cell phenotype cases. The presence of CNS or testicular disease, age <1 year, and trisomy 21 are considered high risk. The importance of tumor cell characteristics will be discussed in the following sections.

## **2.2. Molecular diagnosis and its importance**

Immunophenotyping is used to classify leukemia into B-ALL and T-ALL [3, 4].

B-ALL: comprises 80% of ALL. B-ALL cells express cytoplasmic CD79a, CD19, HLA-DR. Surface CD10 (formerly known as common ALL antigen or CALLA) is seen in 90% of these cases. Subtypes are as follows:


T-ALL: These cells express cytoplasmic CD3, with CD7 plus CD2 or CD5 on leukemic blasts. T-ALL is associated with older age, male gender, high initial white count, and mediastinal mass.

Cytogenetic alterations: The presence of recurrent numerical and structural chromosomal abnormalities in both ALL and AML are very common and are associated with prognostic significance. Hence, cytogenetic characteristics are now used for risk stratification of patients with ALL [5, 6].

#### Pediatric High Risk Leukemia — Molecular Insights http://dx.doi.org/10.5772/61247 3

**Figure 1.** Frequency of Cytogenetic Abnormalities in ALL

**2. Overview of classification of pediatric leukemia**

The National Cancer Institute (NCI) has classified acute lymphoblastic leukemia in children based on age at diagnosis, initial white blood count, and the presence of extra medullary

**•** Standard risk: initial WBC count less than 50,000/μL and age 1 to younger than 10 years

The Children's Oncology Group (COG) has classified acute leukemia into four risk groups using prognostic factors that are strongly predictive of outcome such as: 1) age; 2) initial white count; 3) gender; 4) presence of extra medullary disease at diagnosis (CNS or testicular disease); and 5) blast cytogenetic findings and ploidy, and 6) response to induction therapy [1, 2].

Based on these factors, the four risk groups for COG classification of newly diagnosed B-ALL for the AALL08B1 study are low risk, average risk, high risk, and very high risk. In T-ALL, high white count does not have a prognostic significance. The above classification applies to B cell phenotype cases. The presence of CNS or testicular disease, age <1 year, and trisomy 21 are considered high risk. The importance of tumor cell characteristics will be discussed in the

B-ALL: comprises 80% of ALL. B-ALL cells express cytoplasmic CD79a, CD19, HLA-DR. Surface CD10 (formerly known as common ALL antigen or CALLA) is seen in 90% of these

**•** Common Precursor B Cell ALL (75% of cases): These cells are CD10 positive and express no

**•** Pro B ALL (5% of cases): Commonly seen in infants with MLL rearrangement. These cells

T-ALL: These cells express cytoplasmic CD3, with CD7 plus CD2 or CD5 on leukemic blasts. T-ALL is associated with older age, male gender, high initial white count, and mediastinal

Cytogenetic alterations: The presence of recurrent numerical and structural chromosomal abnormalities in both ALL and AML are very common and are associated with prognostic significance. Hence, cytogenetic characteristics are now used for risk stratification of patients

**•** High risk: initial WBC count 50,000/μL or greater and/or age 10 years or older

Immunophenotyping is used to classify leukemia into B-ALL and T-ALL [3, 4].

surface or cytoplasmic Ig. This group has the best prognosis.

are CD10 negative and express no surface or cytoplasmic Ig.

**•** Pre B ALL (20% of cases): These cells express cytoplasmic Ig.

**2.1. Definition of standard and high risk leukemias**

disease.

2 Leukemias - Updates and New Insights

following sections.

mass.

with ALL [5, 6].

cases. Subtypes are as follows:

**2.2. Molecular diagnosis and its importance**


**Table 1.** Common Genetic Alterations and Clinical Significance B-ALL.

Risk stratification based on cytogenetic characteristics has revealed many subgroups of NCI standard and high risk groups that are now treated differently. Many treatment strategies using targeted therapies directed at specific genetic alterations in the tumor cells have opened up a new era of leukemia therapy. Figure 1 and Table 1 show cytogenetic abnormalities seen in ALL [6, 7].

## **3. Infant leukemia and leukemia with MLL rearrangement**

## **3.1. Overview of infant leukemia**

Infant leukemia comprises only 1–2 % of childhood ALL. The cells often show an immature pro-B phenotype. They present with very high WBC count and large extra medullary disease burden which confer poor prognosis in this subset of children [8].

## **3.2. Biology of infant leukemia**

Rearrangements of the *KMT2A* (also known as mixed lineage leukemia (MLL)) gene located on chromosome 11q23 are seen in more than 80% of infant ALL, with increased frequency in patients under 6 months of age. They are also seen in ~50% of infant AML [8] and a majority of adults with therapy-related leukemia secondary to topoisomerase II inhibitors. Balanced translocations result in the fusion of *MLL* to a number of different partner genes, leading to the production of novel chimeric proteins with t(4;11)(q21;q23) *MLL-AFF1* (also called *MLL-AF4*) being the most common. Other rearrangements are t(11;19)(q23;p13.3) *MLL-/ELN*; t(6;11) (q27;q23) *MLL/MLLT4, also called MLL/AF6*; t(9;11)(p22;q23) *MLL/MFT3*, also called *MLL-AF9*; and t(10;11)(p12;q23) *MLL/MLLT10*, also called *MLL/AF10*. The *MLL gene product* plays a critical role in hematopoiesis by regulating the *HOX* group of genes, which sequentially influence hematopoietic stem cell renewal and leukemogenesis [9, 10].

#### **3.3. Risk stratification, current therapeutic approaches and outcome in infant leukemia**

Infant ALL is considered high risk for many reasons. The factors that confer poor prognosis are age younger than 6 months, extreme hyperleukocytosis (>300,000 white blood cells/μL), CD10 negativity, and the presence of *MLL* rearrangement. The type of the partner gene fused to *MLL* does not seem to influence outcome [9, 11, 12]. Studies by the BFM group and the international ALL protocol Interfant-99 have shown that patients who were early good responders to prednisone therapy had a better outcome [13, 14].

Infant ALL is currently treated with intensive chemotherapy including high dose cytarabine – "AML-like" therapy. The prednisone poor responders are classified as high risk and require intensification of therapy. Hematopoietic stem cell transplantation for infants with ALL in first remission remains controversial [15, 16].

## **4. FLT3 mutations in ALL**

Risk stratification based on cytogenetic characteristics has revealed many subgroups of NCI standard and high risk groups that are now treated differently. Many treatment strategies using targeted therapies directed at specific genetic alterations in the tumor cells have opened up a new era of leukemia therapy. Figure 1 and Table 1 show cytogenetic abnormalities seen

Infant leukemia comprises only 1–2 % of childhood ALL. The cells often show an immature pro-B phenotype. They present with very high WBC count and large extra medullary disease

Rearrangements of the *KMT2A* (also known as mixed lineage leukemia (MLL)) gene located on chromosome 11q23 are seen in more than 80% of infant ALL, with increased frequency in patients under 6 months of age. They are also seen in ~50% of infant AML [8] and a majority of adults with therapy-related leukemia secondary to topoisomerase II inhibitors. Balanced translocations result in the fusion of *MLL* to a number of different partner genes, leading to the production of novel chimeric proteins with t(4;11)(q21;q23) *MLL-AFF1* (also called *MLL-AF4*) being the most common. Other rearrangements are t(11;19)(q23;p13.3) *MLL-/ELN*; t(6;11) (q27;q23) *MLL/MLLT4, also called MLL/AF6*; t(9;11)(p22;q23) *MLL/MFT3*, also called *MLL-AF9*; and t(10;11)(p12;q23) *MLL/MLLT10*, also called *MLL/AF10*. The *MLL gene product* plays a critical role in hematopoiesis by regulating the *HOX* group of genes, which sequentially influence

**3.3. Risk stratification, current therapeutic approaches and outcome in infant leukemia**

Infant ALL is considered high risk for many reasons. The factors that confer poor prognosis are age younger than 6 months, extreme hyperleukocytosis (>300,000 white blood cells/μL), CD10 negativity, and the presence of *MLL* rearrangement. The type of the partner gene fused to *MLL* does not seem to influence outcome [9, 11, 12]. Studies by the BFM group and the international ALL protocol Interfant-99 have shown that patients who were early good

Infant ALL is currently treated with intensive chemotherapy including high dose cytarabine – "AML-like" therapy. The prednisone poor responders are classified as high risk and require intensification of therapy. Hematopoietic stem cell transplantation for infants with ALL in first

**3. Infant leukemia and leukemia with MLL rearrangement**

burden which confer poor prognosis in this subset of children [8].

hematopoietic stem cell renewal and leukemogenesis [9, 10].

responders to prednisone therapy had a better outcome [13, 14].

remission remains controversial [15, 16].

in ALL [6, 7].

4 Leukemias - Updates and New Insights

**3.1. Overview of infant leukemia**

**3.2. Biology of infant leukemia**

## **4.1. Incidence of** *FLT3* **alterations in ALL and its prognostic significance**

Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) plays an important role in the pathogenesis of hematopoietic malignancies. *FLT3* activating mutations, internal tandem duplications (ITD), and kinase domain (KD) mutations were initially discovered in acute myeloid leukemia (AML) and are associated with poor prognosis in both adult and pediatric AML. However, in lymphoblastic leukemia, FLT3 ITD did not cause significant change in overall survival and event free survival [17]. Recently, FLT3 overexpression has been seen in two types of ALL: 18% of infant ALL with *MLL* rearrangement and 21–25% of hyperdiploid ALL and in relapsed ALL samples [18, 19]. Studies have shown that high *FLT3* expression mutation identifies MLL-AF4+ (also called *MLL-AFF1+*) ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors [20–23].

## **4.2. FLT3 inhibitors in the treatment of leukemia with FLT3 alterations**

Recent gene expression studies have shown that in *MLL*-rearranged leukemia, FLT3, a receptor tyrosine kinase that plays a role in promoting cell proliferation and transformation is overex‐ pressed. This has led to the pursuit of FLT3 inhibitors as targeted therapy for this disease [22]. Currently, Lestaurtinib (CEP-701™), an oral, highly selective small-molecule FLT3 inhibitor, is being used in COG - phase III trial wherein infants with *MLL*-rearranged ALL are being randomized to intensive chemotherapy with or without Lestaurtinib.

## **5. BCR-ABL positive leukemia**

## **5.1. Incidence, current therapy, and outcome in BCR-ABL+ (Ph+) leukemia**

Chimeric BCR–ABL1 protein is encoded by *BCR-ABL1* fusion gene created by reciprocal t(9;22) (q34;q11) translocation on chromosome 22, also known as the Philadelphia chromosome. The *BCR–ABL1* fusion gene is generated by joining most of the coding region of the *ABL1* tyrosine kinase gene (Abelson murine leukemia) on chromosome 9 to the breakpoint cluster region (*BCR*) gene on chromosome 22. The molecular consequence of all BCR–ABL1 fusion proteins is a hyperactive ABL1 kinase domain and aberrant phosphorylation of a variety of targets [23, 24]. There are two gene product variants: p190, which is seen in >90% of Philadelphia+ (Ph+) ALL in children, and p210, which is seen in CML.

Ph+ ALL comprises 3–4% of pediatric ALL, and about 25% of adult ALL cases. Prior to tyrosine kinase inhibitors, these patients had dismal outcomes despite the use of inten‐ sive chemotherapy and hematopoietic stem cell transplant in the first remission was the best available option [24].

## **5.2. Tyrosine kinase inhibitors in BCR-ABL+ (Ph+) leukemia and current challenges**

Imatinib was the first generation of tyrosine kinase inhibitors that changed the face of treatment for Ph+ ALL and CML. The addition of Imatinib to intensive chemotherapy in childhood BCR-ABL1–positive ALL results in a 4-year event-free survival rate of 84%, more than double that of historical controls [25]. About 40% of the newly diagnosed Ph+ ALL patients carry point mutations within the kinase-binding domain of BCR-ABL that confers resistance to Imatinib.

Dasatinib and Nilotinib are second-generation tyrosine kinase inhibitors. Dasatinib is a multikinase inhibitor targeting several tyrosine kinases, including BCR-ABL and SRC kinases. It is 325 times more potent than Imatinib, binds to the active and inactive forms of BCR-ABL, and has excellent CNS penetration. These are effective in patients resistant to Imatinib, except those with the *T315I* mutation [26].

Currently, allogeneic hematopoietic stem cell transplant (HSCT) is the standard of care in second remission for patients with Ph+ ALL. It has been observed that most of the patients treated with tyrosine kinase inhibitor (TKI)-based therapy will eventually relapse without HSCT. However, benefits of allogeneic HSCT in first remission, after intensive chemotherapy and TKI therapy, has to be considered based on donor availability, minimal residual disease (MRD) status, and clinical status of the patient [27]. Recent studies in both adults and children have failed to prove clear benefit of allogeneic HSCT in the first remission for this group of patients mainly because of the small sample size [23, 28, 29].

## **6.** *Ikaros***-altered ALL**

## **6.1. Overview of** *Ikaros*

Ikaros is a DNA-binding zinc finger protein encoded by the *IKZF1* gene. Ikaros is a transcrip‐ tion factor that functions as a regulator of gene expression and chromatin remodeling [30]. Ikaros regulates the development and function of the immune system and acts as a master regulator of hematopoietic differentiation. Genomic profiling studies identified *IKZF1* as an important tumor suppressor in ALL, particularly in ALL that is associated with poor prognosis [31, 32]. The mechanism by which Ikaros suppresses malignant transformation and the development of ALL is largely unknown. In the past few years, experiments by several groups have shown that Ikaros regulates expression of its target genes by recruiting them to pericen‐ tromeric heterochromatin, resulting in their activation or repression [33, 34].

## **6.2. Clinical importance of Ikaros deletion**

*IKZF1* has been established as one of the most clinically relevant tumor suppressors in highrisk ALL. Genome wide analysis studies have shown that 15% of all cases of pediatric B-cell ALL show deletion of a single *IKZF1* allele or mutation of a single copy of *IKZF1*, resulting in haploinsufficiency of *IKZF1*. Haploinsufficiency occurs with expression of a functionally inactive Ikaros splice form that acts as a dominant negative. Genetic inactivation of *IKZF1* is more rare in T-ALL where *IKZF1* alterations occur in ~5% of cases [35]. Over 80% of BCR-ABL1 ALL and 66% of chronic myeloid leukemia (CML) patients during lymphoid blast crisis show mutation or deletion of an *IKZF1* allele. One-third of BCR-ABL1 negative ALL patients also show *IKZF1* deletion or mutation. This group of patients is at increased risk of poor outcome with more than threefold increase in relapse rate [30]. Poor outcome associated with *IKZF1* alteration is frequently independent of age, sex, white cell count, and level of minimal residual disease – factors which are commonly used for risk stratification. Testing for *IKZF1* status at the time of diagnosis is now being explored in prospective trials. In recent genome profiling studies, *IKZF1*-altered high risk pediatric ALL cases have shown marked similarity to BCR-ABL positive ALL giving rise to a new subset of cases now called "Ph-like " or "BCR-ABLlike" ALL. This will be discussed further later in the chapter.

## **6.3. Regulation of Ikaros function in T cell leukemia**

The role of Ikaros in normal T cell development is demonstrated by evidence that Ikaros regulates the expression of key genes in T cell differentiation. T cell differentiation is significantly impaired in Ikaros-deficient mice. Terminal deoxynucleotide transferase (*DNTT)* is a gene product critical for thymocyte differentiation that is regulated by Ikaros. Ikaros also regulates the expression of CD4, CD8, and IL-2 and plays a critical role in T cell differentiation [36, 37].

## *6.3.1. Regulation of Ikaros function*

**5.2. Tyrosine kinase inhibitors in BCR-ABL+ (Ph+) leukemia and current challenges**

those with the *T315I* mutation [26].

6 Leukemias - Updates and New Insights

**6.** *Ikaros***-altered ALL**

**6.1. Overview of** *Ikaros*

**6.2. Clinical importance of Ikaros deletion**

patients mainly because of the small sample size [23, 28, 29].

Imatinib was the first generation of tyrosine kinase inhibitors that changed the face of treatment for Ph+ ALL and CML. The addition of Imatinib to intensive chemotherapy in childhood BCR-ABL1–positive ALL results in a 4-year event-free survival rate of 84%, more than double that of historical controls [25]. About 40% of the newly diagnosed Ph+ ALL patients carry point mutations within the kinase-binding domain of BCR-ABL that confers resistance to Imatinib. Dasatinib and Nilotinib are second-generation tyrosine kinase inhibitors. Dasatinib is a multikinase inhibitor targeting several tyrosine kinases, including BCR-ABL and SRC kinases. It is 325 times more potent than Imatinib, binds to the active and inactive forms of BCR-ABL, and has excellent CNS penetration. These are effective in patients resistant to Imatinib, except

Currently, allogeneic hematopoietic stem cell transplant (HSCT) is the standard of care in second remission for patients with Ph+ ALL. It has been observed that most of the patients treated with tyrosine kinase inhibitor (TKI)-based therapy will eventually relapse without HSCT. However, benefits of allogeneic HSCT in first remission, after intensive chemotherapy and TKI therapy, has to be considered based on donor availability, minimal residual disease (MRD) status, and clinical status of the patient [27]. Recent studies in both adults and children have failed to prove clear benefit of allogeneic HSCT in the first remission for this group of

Ikaros is a DNA-binding zinc finger protein encoded by the *IKZF1* gene. Ikaros is a transcrip‐ tion factor that functions as a regulator of gene expression and chromatin remodeling [30]. Ikaros regulates the development and function of the immune system and acts as a master regulator of hematopoietic differentiation. Genomic profiling studies identified *IKZF1* as an important tumor suppressor in ALL, particularly in ALL that is associated with poor prognosis [31, 32]. The mechanism by which Ikaros suppresses malignant transformation and the development of ALL is largely unknown. In the past few years, experiments by several groups have shown that Ikaros regulates expression of its target genes by recruiting them to pericen‐

*IKZF1* has been established as one of the most clinically relevant tumor suppressors in highrisk ALL. Genome wide analysis studies have shown that 15% of all cases of pediatric B-cell ALL show deletion of a single *IKZF1* allele or mutation of a single copy of *IKZF1*, resulting in haploinsufficiency of *IKZF1*. Haploinsufficiency occurs with expression of a functionally inactive Ikaros splice form that acts as a dominant negative. Genetic inactivation of *IKZF1* is more rare in T-ALL where *IKZF1* alterations occur in ~5% of cases [35]. Over 80% of BCR-ABL1

tromeric heterochromatin, resulting in their activation or repression [33, 34].

Casein kinase has been shown to phosphorylate Ikaros at multiple sites, and indeed, CK2 kinase is responsible for the majority of Ikaros phosphorylation. Studies by the Dovat group showed that a single phosphomimetic mutation at amino acid 13 or 294 caused the redistrib‐ ution of Ikaros protein in the nucleus from pericentromeric localization to a diffuse nuclear staining pattern, while phosphoresistant mutations produced no changes in the subcellular localization of Ikaros. These data suggest that targeting of Ikaros to pericentromeric hetero‐ chromatin is regulated by its phosphorylation at specific amino acids [34, 38].

## *6.3.2. CK2 mediated phosphorylation of Ikaros impairs Ikaros function*

Recent studies have demonstrated that CK2-mediated phosphorylation of Ikaros controls essential functions of Ikaros including DNA-binding, subcellular localization, and chromatin remodeling, as well as the level of Ikaros protein in cells (via ubiquitination and degradation). Phosphorylation of Ikaros by CK2 kinase also regulates cell cycle progression and Ikaros function in T cell differentiation [39, 40]. Since the overexpression of CK2 kinase and the loss of Ikaros function have been strongly associated with leukemogenesis, it is proposed that increased CK2 kinase activity leads to impaired function and/or degradation of Ikaros, which results in malignant transformation and the development of leukemia.

## **6.4. Inhibition of CK2 as a potential therapeutic approach to treat high risk leukemia**

Casein Kinase II inhibition has been an attractive therapeutic strategy in several malignancies. A specific CK2 inhibitor, CX-4945, orally bioavailable small molecule is in Phase I clinical trial for solid tumors. The role of CK2 inhibitor as an antileukemic drug in ALL needs to be explored.

## **7. Hypodiploid ALL**

## **7.1. Subclassification and treatment outcome of hypodiploid ALL**

Hypodiploid ALL comprises 1–2% of all B-ALL cases and confers poor prognosis. Hypodi‐ ploid ALL is a chromosome number abnormality in the leukemic cells that results in 45 chromosomes or less. Hypodiploid ALL has been subdivided in various ways. In general, Hypodiploid ALL may be subclassified into the following four groups:


Patients with 44 or 45 chromosomes have a much better outcome than patients with fewer than 44 chromosomes [41].

## **7.2. Genomic profiles of hypodiploid ALL**

Recently, a large group of 124 pediatric patients with hypodiploid ALL were analyzed using microarray profiling of gene-expression and copy-number alteration, and next-generation sequencing. These analyses indicate that near-haploid and low-hypodiploid ALL are distinc‐ tive from each other and from other types of ALL. In near-haploid ALL, genetic alterations target RTK (receptor tyrosine kinase) signaling, Ras signaling, and the lymphoid transcription factor gene *IKZF3,* while in low-hypodiploid ALL, genetic alterations involve *TP53*, *RB1*, and *IKZF2* [42].

RTK and Ras signaling alterations were present in more than two-thirds (70.6%) of nearhaploid ALL cases, while they were much less common in low-hypodiploid (8.8%) and neardiploid ALL (31.8%). The RTK and Ras signaling alterations involve deletion, amplification, and/or sequence mutation of *NF1*, *NRAS*, *KRAS*, *MAPK1*, *FLT3*, or *PTPN11*.

The *TP53* alterations were present in 91.2% of low-hypodiploid ALL, but less than 5% of nonlow-hypodiploid ALL. Meanwhile, *TP53* alterations are also present in non-tumor cells in 43.3% of the mutation-carrying cases, suggesting that the *TP53* mutations are inherited and the low-hypodiploid ALL might be a manifestation of Li-Fraumeni Syndrome (LFS).

The *Ikaros* gene family is very important for lymphoid development and differentiation. *IKZF2* is highly expressed in common lymphoid progenitors and pre-pro-B cells, while *IKZF3* is mainly expressed in more mature lymphoid precursors. *IKZF2* and *IKZF3* alterations occur in low-hypodiploid and near-haploid ALL, respectively, suggesting that low-hypodiploid ALL may arise from the transformation of a lymphoid progenitor that is a less mature lymphoid than those from which near-haploid ALL arises. More importantly, both low-hypodiploid and near-haploid leukemic cells show activation of Ras-signaling and PI3K-signaling pathways. In addition, PI3K and mTOR inhibitors can inhibit proliferation of both low-hypodiploid and near-haploid leukemic cells ex vivo, which indicates that these drugs should be further investigated as a new therapeutic strategy for hypodiploid ALL [42].

## **8. Leukemias involving the** *E2A-PBX1* **translocation**

## **8.1. Introduction: Wild type E2A and PBX1**

**7. Hypodiploid ALL**

8 Leukemias - Updates and New Insights

44 chromosomes [41].

and *IKZF2* [42].

**•** near-haploid cases – 24–31 chromosomes

**•** near-diploid cases – 44–45 chromosomes

**7.2. Genomic profiles of hypodiploid ALL**

**•** low-hypodiploid cases – 32–39 chromosomes

**•** high-hypodiploid cases – 40–43 chromosomes

**7.1. Subclassification and treatment outcome of hypodiploid ALL**

Hypodiploid ALL may be subclassified into the following four groups:

Hypodiploid ALL comprises 1–2% of all B-ALL cases and confers poor prognosis. Hypodi‐ ploid ALL is a chromosome number abnormality in the leukemic cells that results in 45 chromosomes or less. Hypodiploid ALL has been subdivided in various ways. In general,

Patients with 44 or 45 chromosomes have a much better outcome than patients with fewer than

Recently, a large group of 124 pediatric patients with hypodiploid ALL were analyzed using microarray profiling of gene-expression and copy-number alteration, and next-generation sequencing. These analyses indicate that near-haploid and low-hypodiploid ALL are distinc‐ tive from each other and from other types of ALL. In near-haploid ALL, genetic alterations target RTK (receptor tyrosine kinase) signaling, Ras signaling, and the lymphoid transcription factor gene *IKZF3,* while in low-hypodiploid ALL, genetic alterations involve *TP53*, *RB1*,

RTK and Ras signaling alterations were present in more than two-thirds (70.6%) of nearhaploid ALL cases, while they were much less common in low-hypodiploid (8.8%) and neardiploid ALL (31.8%). The RTK and Ras signaling alterations involve deletion, amplification,

The *TP53* alterations were present in 91.2% of low-hypodiploid ALL, but less than 5% of nonlow-hypodiploid ALL. Meanwhile, *TP53* alterations are also present in non-tumor cells in 43.3% of the mutation-carrying cases, suggesting that the *TP53* mutations are inherited and

The *Ikaros* gene family is very important for lymphoid development and differentiation. *IKZF2* is highly expressed in common lymphoid progenitors and pre-pro-B cells, while *IKZF3* is mainly expressed in more mature lymphoid precursors. *IKZF2* and *IKZF3* alterations occur in low-hypodiploid and near-haploid ALL, respectively, suggesting that low-hypodiploid ALL may arise from the transformation of a lymphoid progenitor that is a less mature lymphoid than those from which near-haploid ALL arises. More importantly, both low-hypodiploid and near-haploid leukemic cells show activation of Ras-signaling and PI3K-signaling pathways. In addition, PI3K and mTOR inhibitors can inhibit proliferation of both low-hypodiploid and

the low-hypodiploid ALL might be a manifestation of Li-Fraumeni Syndrome (LFS).

and/or sequence mutation of *NF1*, *NRAS*, *KRAS*, *MAPK1*, *FLT3*, or *PTPN11*.

The *TCF3* gene (also known as *E2A)* encodes two proteins, E12 and E47, which are generated by differential splicing events. Both proteins contain a C-terminal basic helix-loop-helix (bHLH) domain and two activation domains called AD1 and AD2. E2A proteins function as transcriptional activators by binding to E-box DNA sequence and recruiting histone acetyl‐ transferase complexes. E2A proteins contribute to various aspects of lymphocyte differentia‐ tion and development. In E2A deficient mice, B cell development is arrested at the early pro-B stage [43].

*PBX1* was first described through its involvement in the translocation t(1; 19) [44]. *PBX1* itself is not expressed in lymphoid cells, though its related genes, *PBX2* and *PBX3*, are expressed in lymphocytes [45]. PBX1 contains a homeodomain involved in DNA binding and proteinprotein interaction. It can cooperate with other homeodomian containing proteins of HOX and MEINOX classes to regulate the transcription of target genes [46].

## **8.2. Structure and function of the** *E2A-PBX1* **translocation**

The chromosomal translocation between chromosomes 1 and 19 results in a fusion event between *TCF3* and *PBX1*, which is detected in 3–5% of all pediatric pre-B cell ALL cases [47, 48]. The E2A-PBX1 fusion proteins contain two-thirds of the N-terminal of E2A proteins (which retain the AD1 and AD2 domain, but lose the bHLH domain) and most of the PBX1 protein. Two forms of E2A-PBX1 proteins are detected, E2A-PBX1a and E2A-PBX1b, which differ at the C-terminus of PBX1 [49].

The E2A-PBX1 fusion protein is capable of transforming various cell types. Enforced expres‐ sion of E2A-PBX1 induces lethal lympho-proliferative diseases in transgenic mice and aggressive myeloproliferative diseases in a murine bone marrow transplantation model [50].

#### **8.3. Target genes of E2A-PBX1**

Many efforts have been made to find the mechanisms by which E2A-PBX1 mediates transfor‐ mation of pre-B cells. One important way is try to find potential target genes or pathways that are regulated by E2A-PBX1. Using ChIP-chip assay, Diakos et al. found 108 direct E2A-PBX1 targets [51]; however, few targets have been studied in detail. *Wnt16* and *EB-1* are two target genes of E2A-PBX1 that are well studied. Wnt16 belongs to the Wnt family, a group of signaling factors that plays important roles in many developmental processes including cell differen‐ tiation, proliferation, polarity, and migration [52]. Wnt16 is normally expressed in peripheral lymphoid organs but not in bone marrow. In contrast, Wnt16 is highly expressed in bone marrow and cell lines that are derived from pre-B ALL patients carrying E2A-PBX1 [52]. *EB-1* expression was much lower in marrow from pre-B ALL patients without t(1; 19) than marrow from pre-B ALL patients and pre-B cell lines that contain the t(1; 19). EB-1 is a signaling protein containing a phosphotyrosine binding domain, and may play a role in the regulation of cell proliferation. [53].

## **8.4. Clinical treatment and outcome for E2A-PBX1 ALL**

For pediatric B-ALL patients with the E2A-PBX1 fusion protein, the prognosis has been controversial. It was initially associated with poor outcome when treated with antimetabolitebased therapy [54, 55], but the recent development of intensified chemotherapy has much improved the outcome of this subgroup. However, in a trial conducted by St. Jude Children's Research Hospital, although patients with the t(1; 19) had an overall favorable outcome with intensified chemotherapy, this group had a significantly higher incidence of CNS relapse, suggesting intensive CNS-directed therapy is needed to further improve the outcome in this group of patients [56].

## **9. Ph-like ALL**

#### **9.1. Description and prevalence of Ph-like ALL**

Ph-like (BCR-ABL1-like) ALL defines a distinct subtype of high risk ALL that is characterized by a gene expression profile similar to that of Ph+ ALL but lacking the characteristic t(9;22) translocation. This subtype represents about 15–20% of all cases of ALL [57, 58]. The prevalence of Ph-like ALL increases with age: 10–13% of ALL in children and 21–27% of ALL in adolescents and young adults. Patients with Ph-like ALL have higher leukocyte counts at presentation and are more likely to have minimal residual disease at the end of induction chemotherapy. The survival rates of patients with Ph-like ALL is significantly lower when compared to non-Phlike ALL among all age groups, adults faring worse than children [58]. Genes that are involved in B cell development, including *IKZF1* (Ikaros), are deleted more frequently in Ph-like ALL as compared to non-Ph-like ALL (82% vs. 36%) [57, 58]. Genomic alterations involving kinase signaling have been observed in >90% of Ph-like ALL. These include alterations involving *ABL1, JAK, EPOR, CRLF2, IL7R, FLT3*, Ras, and others [58]. Subtypes of Ph-like ALL are shown in Figure 2.

#### **9.2. Translocations involving ABL1 in Ph-like B-ALL**

The *BCR-ABL1* translocation and the resulting Ph+ leukemia was described in Section 4. Other translocations involving the *ABL1* gene have been demonstrated in B-ALL as well as T-ALL. The *ETV6* gene encodes an Ets family transcription factor that is essential for hematopoiesis and vascular growth. The *ETV6*-*ABL1* fusion, t(9;12)(q34;p13) is a rare event that has been reported in 22 cases of hematological malignancies including 6 cases of B-ALL and one case of T-ALL [59]. The *RCSD1-ABL1* fusion, t(1;9)(q24;q34), has been identified in 3 cases of B-ALL

[60]. One case of B-ALL with *SFPQ-ABL1* fusion, t(1;9)(p34;q34), and another case of B-ALL with *ZMIZ1-ABL1* fusion, t(9;10)(q34;q22.3) have been reported [61, 62].

**Figure 2.** Ph-like ALL subtypes

marrow and cell lines that are derived from pre-B ALL patients carrying E2A-PBX1 [52]. *EB-1* expression was much lower in marrow from pre-B ALL patients without t(1; 19) than marrow from pre-B ALL patients and pre-B cell lines that contain the t(1; 19). EB-1 is a signaling protein containing a phosphotyrosine binding domain, and may play a role in the regulation of cell

For pediatric B-ALL patients with the E2A-PBX1 fusion protein, the prognosis has been controversial. It was initially associated with poor outcome when treated with antimetabolitebased therapy [54, 55], but the recent development of intensified chemotherapy has much improved the outcome of this subgroup. However, in a trial conducted by St. Jude Children's Research Hospital, although patients with the t(1; 19) had an overall favorable outcome with intensified chemotherapy, this group had a significantly higher incidence of CNS relapse, suggesting intensive CNS-directed therapy is needed to further improve the outcome in this

Ph-like (BCR-ABL1-like) ALL defines a distinct subtype of high risk ALL that is characterized by a gene expression profile similar to that of Ph+ ALL but lacking the characteristic t(9;22) translocation. This subtype represents about 15–20% of all cases of ALL [57, 58]. The prevalence of Ph-like ALL increases with age: 10–13% of ALL in children and 21–27% of ALL in adolescents and young adults. Patients with Ph-like ALL have higher leukocyte counts at presentation and are more likely to have minimal residual disease at the end of induction chemotherapy. The survival rates of patients with Ph-like ALL is significantly lower when compared to non-Phlike ALL among all age groups, adults faring worse than children [58]. Genes that are involved in B cell development, including *IKZF1* (Ikaros), are deleted more frequently in Ph-like ALL as compared to non-Ph-like ALL (82% vs. 36%) [57, 58]. Genomic alterations involving kinase signaling have been observed in >90% of Ph-like ALL. These include alterations involving *ABL1, JAK, EPOR, CRLF2, IL7R, FLT3*, Ras, and others [58]. Subtypes of Ph-like ALL are shown

The *BCR-ABL1* translocation and the resulting Ph+ leukemia was described in Section 4. Other translocations involving the *ABL1* gene have been demonstrated in B-ALL as well as T-ALL. The *ETV6* gene encodes an Ets family transcription factor that is essential for hematopoiesis and vascular growth. The *ETV6*-*ABL1* fusion, t(9;12)(q34;p13) is a rare event that has been reported in 22 cases of hematological malignancies including 6 cases of B-ALL and one case of T-ALL [59]. The *RCSD1-ABL1* fusion, t(1;9)(q24;q34), has been identified in 3 cases of B-ALL

proliferation. [53].

10 Leukemias - Updates and New Insights

group of patients [56].

**9. Ph-like ALL**

in Figure 2.

**8.4. Clinical treatment and outcome for E2A-PBX1 ALL**

**9.1. Description and prevalence of Ph-like ALL**

**9.2. Translocations involving ABL1 in Ph-like B-ALL**

The *NUP214* gene encodes a nuclear pore complex protein that is involved in nucleocytoplas‐ mic transport. *NUP214-ABL1* is the second most prevalent fusion gene involving *ABL1*. It has been reported only in T cell ALL with a frequency of approximately 5%. So far, 60 cases of this fusion have been reported in the literature involving both childhood and adult cases of T-ALL [63, 64]. In one study by Graux et al., extrachromosomal (episomal) amplification of *ABL1* within the *NUP214-ABL1* transcript was observed in 5 out of 90 cases of T-ALL. Episomes are extrachromosomal DNA that are not visible by conventional cytogenetics [65]. The above mentioned study utilized FISH and microarray-based comparative genomic hybridization techniques to map the episomes and confirm the *ABL1* amplification. Roberts et al. have demonstrated that leukemias with ABL class fusions are sensitive in vitro to the ABL class inhibitors Imatinib and Dasatinib [58].

## **9.3. Genetic alterations in Ph-like ALL that include EPOR and JAKs**

A number of *JAK* mutations have been identified in Ph-like B-ALL, specifically mutations in the *JAK1* pseudokinase domain, as well as the *JAK2* pseudokinase and kinase domain, and in *JAK3* [66]. However, the most common are those occurring in the *JAK2* pseudokinase domain – R683G [67]. Activating mutations in JAK kinases (*JAK1, JAK2*, and *JAK3*) have been reported in approximately 10% of Ph-like pediatric ALL. The JAK mutations were associated with alterations in *IKZF1* and *CDKN2A/CDKN2B,* and with poor outcomes [58, 67]. Approximately 50% of CRLF2 B-ALL patients show *JAK* mutations [66, 68, 69] while rearrangement or translocations involving the *JAK2* gene are characteristic of ~7% of Ph-like ALL [58].

Erythropoietin (EPO) is a hematopoietic growth factor for the erythroid lineage and regulates the production of red blood cells. The binding of EPO to its receptor EPOR leads to downstream activation of JAK2-STAT5, PI3 kinase, and MAP kinase pathways [70]. Rearrangements that involve EPOR have been found in ~4% of Ph-like ALL [58].

## **9.4. Ras pathway genetic alterations in Ph-like ALL**

Ras pathway mutations have been described in pediatric ALL cases. In a report from the Children's Oncology Group by Zhang et al., among 23 childhood B-ALL cases with a Ph-like gene expression profile, 9% had Ras pathway mutations. However, the proportion of Ras pathway mutated cases increased to 62% among ALL with focal *ERG* deletion [71]. Among Ph-like B-ALL cases, 4% are known to have Ras pathway genetic alterations. A majority of these involve missense mutations in *KRAS* and *NRAS* [58].

## **9.5. IKZF1 alterations in Ph-like ALL**

Aberrations in the *IKZF1* gene have been observed in 80% of pediatric B-ALL cases that harbor the *BCR-ABL* rearrangement [72] and a similar pattern is seen in Ph-like ALL. Patients with *IKZF1* deletions have increased risk for relapse and poor treatment outcomes, making *IKZF1* an independent predictor for treatment outcomes [73–75]. More recently, alterations of the *IKZF1* gene have been associated with other genetic defects including *JAK* mutations and *CRLF2* rearrangements. Mullighan et al. demonstrated that 70% of *JAK* mutated cases harbored *IKZF1* alterations [67]. Dorge et al. also performed a study which showed a higher number of Ikaros deletions in patients that have *P2RY8-CRLF2* rearrangement compared to those that are negative for the rearrangement [73]. Further, in another study of 29 pediatric B-ALL cases that showed high CRLF2 expression accompanied by *CRLF2* rearrangement, ~70% of the patients had *JAK* mutations and 80% of cases had deletions or mutations in *IKZF1* [66]. These findings provide evidence that *CRLF2* rearrangements are significantly associated with alterations in *IKZF1*, as well as *JAK* mutations. Moreover, in patients harboring *CRLF2* rearrangements, *JAK* mutations and *IKZF1* deletions/mutations have a poor survival outcome, suggesting that these three lesions cooperate to worsen prognosis and treatment outcome of high-risk B-ALL. Therefore, they can serve as therapeutic targets, as well as prognostic tools that can be used effectively in risk-stratification and treatment strategies to improve treatment outcomes in CRLF2 and other high-risk B-ALL patients.

## **9.6. Rearrangement of CRLF2 in Ph-like ALL**

Overexpression of CRLF2 is responsible for more cases of Ph-like ALL than any other genetic alteration [58]. The *CRLF2* gene is located in the pseudoautosomal region 1 (PAR1) of Xp/Yp. Three types of *CRLF2* gene alterations can result in overexpression of the CRLF2 protein on the surface of the leukemic cells. One involves the juxtaposition of Xp/Yp into the immuno‐ globulin heavy chain locus at 14q32 resulting in *IgH-CRLF2* rearrangement. The other involves a focal deletion of PAR1 that juxtaposes the regulatory elements of the purinergic receptor gene *P2RY8* to *CRLF2* resulting in a chimeric fusion gene *P2RY8-CRLF2.* Thirdly, a less common missense mutation in exon 6 of *CRLF2,* F232C, results in constitutive CRLF2 dimeri‐ zation [66, 76]. CRLF2 overexpression is present only in B-ALL cases without rearrangements in *ETV6* (*TEL), KMT2A (MLL), TCF3*, and *BCR-ABL.* It is also worth noting that more than half of Down syndrome associated ALL overexpress CRLF2 resulting in poor outcomes in these patients [69]. CRLF2 is overexpressed in approximately 8% of childhood and adult ALL and comprises ~50% of Ph-like ALL [58].

## **10. Biology of CRLF2 B-ALL – The most common form of Ph-like ALL**

## **10.1. CRLF2–TSLP cytokine receptor component in CRLF2 B-ALL**

CRLF2 is a type I cytokine receptor that, along with the IL7 receptor alpha (IL-7Rα), forms a heterodimeric receptor complex for the cytokine TSLP (thymic stromal lymphopoietin). In normal B-cell progenitors the expression of CRLF2 is undetectable by flow cytometry, although TSLP cytokine acts on these cells to induce proliferation [77]. In contrast, the high levels of CRLF2 on CRLF2 B-ALL cells can easily be detected by flow cytometry [68]. In CRLF2 B-ALL cells, TSLP has been shown to increase the phosphorylation of STAT5, AKT, and S6, indicating increased JAK-STAT and AKT/mTOR signaling [78]. Studies of TSLP effects in a range of cell types indicates that it can also indicate cell survival signals through the activation of several biological pathways including ERK, MAPK, PI3K/AKT/mTORC1, and NFκB, at least in some cell types [79]. The physiological significance of the signaling induced by TSLP in CRLF2 B-ALL cells remains to be determined [78].

## **10.2. Role of JAK mutations in CRLF2 B-ALL**

alterations in *IKZF1* and *CDKN2A/CDKN2B,* and with poor outcomes [58, 67]. Approximately 50% of CRLF2 B-ALL patients show *JAK* mutations [66, 68, 69] while rearrangement or

Erythropoietin (EPO) is a hematopoietic growth factor for the erythroid lineage and regulates the production of red blood cells. The binding of EPO to its receptor EPOR leads to downstream activation of JAK2-STAT5, PI3 kinase, and MAP kinase pathways [70]. Rearrangements that

Ras pathway mutations have been described in pediatric ALL cases. In a report from the Children's Oncology Group by Zhang et al., among 23 childhood B-ALL cases with a Ph-like gene expression profile, 9% had Ras pathway mutations. However, the proportion of Ras pathway mutated cases increased to 62% among ALL with focal *ERG* deletion [71]. Among Ph-like B-ALL cases, 4% are known to have Ras pathway genetic alterations. A majority of

Aberrations in the *IKZF1* gene have been observed in 80% of pediatric B-ALL cases that harbor the *BCR-ABL* rearrangement [72] and a similar pattern is seen in Ph-like ALL. Patients with *IKZF1* deletions have increased risk for relapse and poor treatment outcomes, making *IKZF1* an independent predictor for treatment outcomes [73–75]. More recently, alterations of the *IKZF1* gene have been associated with other genetic defects including *JAK* mutations and *CRLF2* rearrangements. Mullighan et al. demonstrated that 70% of *JAK* mutated cases harbored *IKZF1* alterations [67]. Dorge et al. also performed a study which showed a higher number of Ikaros deletions in patients that have *P2RY8-CRLF2* rearrangement compared to those that are negative for the rearrangement [73]. Further, in another study of 29 pediatric B-ALL cases that showed high CRLF2 expression accompanied by *CRLF2* rearrangement, ~70% of the patients had *JAK* mutations and 80% of cases had deletions or mutations in *IKZF1* [66]. These findings provide evidence that *CRLF2* rearrangements are significantly associated with alterations in *IKZF1*, as well as *JAK* mutations. Moreover, in patients harboring *CRLF2* rearrangements, *JAK* mutations and *IKZF1* deletions/mutations have a poor survival outcome, suggesting that these three lesions cooperate to worsen prognosis and treatment outcome of high-risk B-ALL. Therefore, they can serve as therapeutic targets, as well as prognostic tools that can be used effectively in risk-stratification and treatment strategies to improve treatment outcomes in

Overexpression of CRLF2 is responsible for more cases of Ph-like ALL than any other genetic alteration [58]. The *CRLF2* gene is located in the pseudoautosomal region 1 (PAR1) of Xp/Yp. Three types of *CRLF2* gene alterations can result in overexpression of the CRLF2 protein on

translocations involving the *JAK2* gene are characteristic of ~7% of Ph-like ALL [58].

involve EPOR have been found in ~4% of Ph-like ALL [58].

these involve missense mutations in *KRAS* and *NRAS* [58].

**9.5. IKZF1 alterations in Ph-like ALL**

12 Leukemias - Updates and New Insights

CRLF2 and other high-risk B-ALL patients.

**9.6. Rearrangement of CRLF2 in Ph-like ALL**

**9.4. Ras pathway genetic alterations in Ph-like ALL**

Overexpression of *CRLF2* is believed to cooperate with other genetic lesions leading to leukemogenesis and chemoresistance. In most patients, overexpression of CRLF2 and *CRLF2* gene rearrangements has been associated with deletions and/or mutations of the *IKZF1* gene. Mutations in *JAK1* or *JAK2* are present in half of all cases of CRLF2-overexpressing B-ALL and can result in constitutive activation of this pathway [66, 68, 69].

To understand the contributions of CRLF2 and JAK2 to leukemogenesis, several groups have conducted studies using *CRLF2* and *JAK* mutants. Results from cellular models that utilized murine Ba/F3 cells transduced with combinations of patient-derived mutant *JAK1* or *JAK2,* and *CRLF2* achieved cytokine-independent growth in cells that contained both *CRLF2* and *JAK2* mutants. This cytokine independent growth did not occur in cells that were transduced with wildtype CRLF2 alone or JAK2 mutants alone. Co-immunoprecipitation studies demon‐ strated that human CRLF2 and phosphorylated mutant JAK2 interact physically [76, 80]. These research findings provide evidence that supports the hypothesis that CRLF2 and JAK2 can cooperate to produce leukemogenesis. Indeed, Hertzberg et al. showed that almost all patients with *JAK* mutations harbor *CRLF2* gene rearrangements[80] This is in keeping with CRLF2 serving as a scaffold to facilitate signaling of mutant *JAK2* [81]. However, ~50% of CRLF2 B-ALL patients show no *JAK* mutations, suggesting that another factor might be inducing JAK activation. This is certainly consistent with the role of endogenous TSLP-induced CRLF2 mediated JAK phosphorylation in CRLF2 B-ALL. TSLP has been shown to increase both JAK-STAT and AKT/mTOR phosphorylation in human CRLF2 B-ALL cells – including those with activating *JAK* mutations [78].

## **10.3. Inherited background variability and susceptibility to CRLF2 B-ALL**

Aside from these acquired genetic variations that contribute to leukemogenesis, other studies using Genome Wide Association techniques are employed to determine whether inherited (germline) genetic variations increase susceptibility to the development of high-risk Ph-like ALL. In a major study, 75 Ph-like ALL patients were compared to 6,661 non-ALL controls and two Single Nucleotide Polymorphisms (SNPs) within the *GATA3* gene achieved genome-wide significance (rs3824662 and rs3781093). The results showed that both SNPs were overrepresented in Ph-like ALL and were associated with higher risk of relapse [82]. More importantly, the *GATA3* SNP (rs3824662) was associated with *CRLF2* rearrangement, *JAK* mutations, and *IKZF1* deletions [82]. *GATA3* is one of the six-membered *GATA* transcription factor family that is involved in the reprogramming of somatic cells to pluripotency[83]. More specifically, *GATA3* is critical for T cell development [84] and genetic alterations in *GATA3* have been observed in T-ALL patients [85] and in other hematopoietic disorders such as AML and Hodgkin Lymphoma [86, 87]. Taken together, these studies provide evidence that in addition to acquired genetic alterations such as *CRLF2*, inherited genetic variations such as *GATA3* SNPs may also contribute to increased susceptibility to developing CRLF2 B-ALL and increased risk of relapse. Thus, germline and somatic genetic alterations may play an important role in leukemogenesis.

## **10.4. CRLF2 B-ALL and health disparities**

CRLF2 B-ALL is five times more prevalent in patients of Hispanic/Latino origin and after initial treatment they experience a higher rate of relapse. This high prevalence of CRLF2 B-ALL in Hispanics is further supported by studies that have been conducted in regions where there is a high proportion of Hispanics. For example, the highest incidence of childhood ALL is in Hispanics compared to other ethnic groups in California [88]. In a study of pediatric B-ALL patients, 18 of 51 (35.3%) Hispanic/Latino patients harbored *CRLF2* rearrangements compared to 11 of 154 (7.1%) patients of other ethnicities [66]. Harvey et al. further demonstrated that in addition to *JAK* mutations and *IKZF1* deletions, *CRLF2* rearrangement was also significantly associated with Hispanic/Latino ethnicity [66]. It has been established that populations originating from different ancestries can be distinguished by genetic polymorphisms that are unique to their ancestors [89]. Therefore, the current approach to studying the mechanisms that contribute to the racial disparities relating to incidence and outcome of ALL is to evaluate ancestry-related genetic alterations in conjunction with external factors such as the environ‐ ment and socioeconomic status [90, 91]. Recent studies to identify ALL susceptibility genes in the Hispanic population demonstrated that *ARID5B* and *GATA3* SNPs are more frequent in Hispanics compared to other ethnicities [82, 92]. These SNPs have been associated with higher ALL incidence (ALL susceptibility), increased frequency of relapse, and poorer outcome [82, 92]. A possible explanation for the increased frequency of these SNPs in Hispanics is their shared genetic ancestry with Native Americans that also exhibit increased frequency of the SNPs [92]. While ancestry-related studies can provide a basis for disease, there is still a need for studies that will identify the biological factors that contribute to the health disparity. Thus, there is a need for the development of preclinical animal models that can be used to evaluate Hispanic patient samples, thereby allowing us to effectively study CRLF2 B-ALL in context of racial disparities.

## **10.5. Challenges of preclinical models of CRLF2 B-ALL**

cooperate to produce leukemogenesis. Indeed, Hertzberg et al. showed that almost all patients with *JAK* mutations harbor *CRLF2* gene rearrangements[80] This is in keeping with CRLF2 serving as a scaffold to facilitate signaling of mutant *JAK2* [81]. However, ~50% of CRLF2 B-ALL patients show no *JAK* mutations, suggesting that another factor might be inducing JAK activation. This is certainly consistent with the role of endogenous TSLP-induced CRLF2 mediated JAK phosphorylation in CRLF2 B-ALL. TSLP has been shown to increase both JAK-STAT and AKT/mTOR phosphorylation in human CRLF2 B-ALL cells – including those with

Aside from these acquired genetic variations that contribute to leukemogenesis, other studies using Genome Wide Association techniques are employed to determine whether inherited (germline) genetic variations increase susceptibility to the development of high-risk Ph-like ALL. In a major study, 75 Ph-like ALL patients were compared to 6,661 non-ALL controls and two Single Nucleotide Polymorphisms (SNPs) within the *GATA3* gene achieved genome-wide significance (rs3824662 and rs3781093). The results showed that both SNPs were overrepresented in Ph-like ALL and were associated with higher risk of relapse [82]. More importantly, the *GATA3* SNP (rs3824662) was associated with *CRLF2* rearrangement, *JAK* mutations, and *IKZF1* deletions [82]. *GATA3* is one of the six-membered *GATA* transcription factor family that is involved in the reprogramming of somatic cells to pluripotency[83]. More specifically, *GATA3* is critical for T cell development [84] and genetic alterations in *GATA3* have been observed in T-ALL patients [85] and in other hematopoietic disorders such as AML and Hodgkin Lymphoma [86, 87]. Taken together, these studies provide evidence that in addition to acquired genetic alterations such as *CRLF2*, inherited genetic variations such as *GATA3* SNPs may also contribute to increased susceptibility to developing CRLF2 B-ALL and increased risk of relapse. Thus, germline and somatic genetic alterations may play an important

CRLF2 B-ALL is five times more prevalent in patients of Hispanic/Latino origin and after initial treatment they experience a higher rate of relapse. This high prevalence of CRLF2 B-ALL in Hispanics is further supported by studies that have been conducted in regions where there is a high proportion of Hispanics. For example, the highest incidence of childhood ALL is in Hispanics compared to other ethnic groups in California [88]. In a study of pediatric B-ALL patients, 18 of 51 (35.3%) Hispanic/Latino patients harbored *CRLF2* rearrangements compared to 11 of 154 (7.1%) patients of other ethnicities [66]. Harvey et al. further demonstrated that in addition to *JAK* mutations and *IKZF1* deletions, *CRLF2* rearrangement was also significantly associated with Hispanic/Latino ethnicity [66]. It has been established that populations originating from different ancestries can be distinguished by genetic polymorphisms that are unique to their ancestors [89]. Therefore, the current approach to studying the mechanisms that contribute to the racial disparities relating to incidence and outcome of ALL is to evaluate ancestry-related genetic alterations in conjunction with external factors such as the environ‐

**10.3. Inherited background variability and susceptibility to CRLF2 B-ALL**

activating *JAK* mutations [78].

14 Leukemias - Updates and New Insights

role in leukemogenesis.

**10.4. CRLF2 B-ALL and health disparities**

Current strategies to treat Ph-like ALL and more specifically CRLF2 B-ALL include: 1) stratification of patients using prognostic and clinical factors (age, gender, race, white blood cell count, CNS involvement, testicular involvement, steroid pretreatment and MRD, cell morphology, immunotyping and genetic alterations); and 2) administer treatment regimen based on risk group. Since 91% of the Ph-like ALL subset of patients (including CRLF2 B-ALL) is characterized by activated kinase signaling, tyrosine kinase inhibitors are currently recom‐ mended as part of the treatment regimen for these patients [58]. Recent studies were aimed at evaluating the effects of tyrosine kinase inhibitors, more specially JAK inhibitors and PI3K/ mTOR inhibitors on CRLF2 B-ALL cells in vitro and in vivo [78, 93, 94]. Results from these studies have shown that *CRLF2* rearranged B-ALL cells are sensitive to these inhibitors in vitro and in vivo. While JAK inhibitors and all other tyrosine kinase inhibitors have shown some promise, they have not been effective in all Ph-like B-ALL and not all CRLF2 B-ALL harbor JAK mutations. Since TSLP has been shown to increase activation of the JAK-STAT, PI3K/AKT/ mTOR signaling pathways, it is important to conduct studies of disease mechanisms and evaluate therapeutic candidates in context of TSLP and other cytokines that are often present in the cancer microenvironment. Though some in vitro studies were performed in the presence of TSLP [78], in vivo studies in context of TSLP stimulation are lacking. Therefore systematic evaluation of the contributions of TSLP to CRLF2 B-ALL leukemogenesis and the assessment of therapies to treat these patients are required to gain a better understanding of the etiology and treatment outcome of the disease.

Human-mouse xenograft models are ideal in vivo models for studying disease mechanisms and for evaluating therapies for leukemia, particularly in context of inherited genetic varia‐ bility. NOD/SCID mice have been shown to be receptive to the engraftment of human leukemia cells [95]. The development of additional mouse strains, such as the NOD/Scid/IL- 2Rγ null (NSG) strain have significantly increased the ability of human ALL cells to engraft [96]. These animal models are effective because mouse cytokines act on the human leukemia cells by providing the necessary growth signals required to facilitate proliferation and maintenance of leukemia cells [97]. However, while most mouse cytokines can activate receptors on human cells, some mouse cytokines are species-specific, e.g., IL-3, GM-CSF, and TSLP [97]. This poses a challenge in effectively recapitulating the systemic or BM microenvironment present in patients. It is important to conduct studies as far as possible with the full complement of cytokines present under physiological conditions in order to truly identify disease mechanisms and evaluate therapies.

## **10.6. Novel pre-clinical xenograft models that provide human cytokines**

The lack of cross species cytokine activity has been partially addressed for studies of myeloid leukemia by the recent development of so-called "cytokine mice" [98]. These immune-deficient mice express human IL-3, SCF, and GMCSF, three cytokines that play important roles in the production of myeloid lineage cells and show no or poor cross species activity. Xenografts from cytokine mice showed enhanced AML engraftment [98]. Alterna‐ tive strategies have been used to produce mice that express human IL-15 and FLT3 and other cytokines, which have resulted in enhanced production of functional human natu‐ ral killer cells, dendritic cells, monocytes/macrophages, and erythrocytes [97]. However, human TSLP has not been included as part of the complement of cytokines used in these studies.

Current xenografts that evaluate therapeutic candidates for CRLF2 B-ALL do not include human TSLP [94], which is required to provide the human CRLF2-mediated signals that were demonstrated by previous groups in vitro [78]. There is a need to develop new preclinical animal models that include TSLP and other species-specific cytokines in order to accurately evaluate disease mechanisms. A model that includes TSLP and allows for modulating the levels of TSLP will be of great value to the study of CRLF2 B-ALL. The rationale for this is that CRLF2, the receptor for the endogenous ligand TSLP, is overexpressed on CRLF2 B-ALL cells and has contributed to increased pathway signaling in these cells. Additionally, the role of TSLP in the initiation and maintenance of CRLF2 B-ALL is unknown. Current studies in our laboratories are aimed at developing a human TSLP+ xenograft model that provides human TSLP to activate CRLF2-mediated signals in human CRLF2 B-ALL cells transplanted into xenograft mice. This model will be used to study disease mechanisms and identify therapies to effectively treat CRLF2 B-ALL.

## **11. Conclusion**

In summary, great strides have been made in effectively treating pediatric leukemia. Risk stratification based on clinical features combined with intensified therapies has contributed to this outcome. Increasingly, more precise molecular phenotyping available from whole genome analyses make it possible to identify and study specific subtypes of high risk leukemias. Mechanistic studies that identify druggable targets in aberrant pathways are likely to fuel rapid advances in the future. This progress will depend on the development of relevant preclinical models for studying disease mechanisms and for evaluating candidate therapies.

## **Author details**

a challenge in effectively recapitulating the systemic or BM microenvironment present in patients. It is important to conduct studies as far as possible with the full complement of cytokines present under physiological conditions in order to truly identify disease mechanisms

The lack of cross species cytokine activity has been partially addressed for studies of myeloid leukemia by the recent development of so-called "cytokine mice" [98]. These immune-deficient mice express human IL-3, SCF, and GMCSF, three cytokines that play important roles in the production of myeloid lineage cells and show no or poor cross species activity. Xenografts from cytokine mice showed enhanced AML engraftment [98]. Alterna‐ tive strategies have been used to produce mice that express human IL-15 and FLT3 and other cytokines, which have resulted in enhanced production of functional human natu‐ ral killer cells, dendritic cells, monocytes/macrophages, and erythrocytes [97]. However, human TSLP has not been included as part of the complement of cytokines used in these

Current xenografts that evaluate therapeutic candidates for CRLF2 B-ALL do not include human TSLP [94], which is required to provide the human CRLF2-mediated signals that were demonstrated by previous groups in vitro [78]. There is a need to develop new preclinical animal models that include TSLP and other species-specific cytokines in order to accurately evaluate disease mechanisms. A model that includes TSLP and allows for modulating the levels of TSLP will be of great value to the study of CRLF2 B-ALL. The rationale for this is that CRLF2, the receptor for the endogenous ligand TSLP, is overexpressed on CRLF2 B-ALL cells and has contributed to increased pathway signaling in these cells. Additionally, the role of TSLP in the initiation and maintenance of CRLF2 B-ALL is unknown. Current studies in our laboratories are aimed at developing a human TSLP+ xenograft model that provides human TSLP to activate CRLF2-mediated signals in human CRLF2 B-ALL cells transplanted into xenograft mice. This model will be used to study disease mechanisms and identify therapies

In summary, great strides have been made in effectively treating pediatric leukemia. Risk stratification based on clinical features combined with intensified therapies has contributed to this outcome. Increasingly, more precise molecular phenotyping available from whole genome analyses make it possible to identify and study specific subtypes of high risk leukemias. Mechanistic studies that identify druggable targets in aberrant pathways are likely to fuel rapid advances in the future. This progress will depend on the development of relevant preclinical

models for studying disease mechanisms and for evaluating candidate therapies.

**10.6. Novel pre-clinical xenograft models that provide human cytokines**

and evaluate therapies.

16 Leukemias - Updates and New Insights

studies.

to effectively treat CRLF2 B-ALL.

**11. Conclusion**

Chandrika Gowda1 , Olivia L. Francis2 , Yali Ding1 , Parveen Shiraz3 , Kimberly J. Payne2,3\* and Sinisa Dovat1

\*Address all correspondence to: kpayne@llu.edu

1 Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA, USA

2 Department of Pathology and Human Anatomy, Loma Linda University, School of Medicine, Loma Linda, CA, USA

3 Department of Medicine, Loma Linda University, School of Medicine, Loma Linda, CA, USA

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