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Mechanism behind Philadelphia chromosome translocation?

Mechanism behind Philadelphia chromosome translocation?



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I was reading up on causative factors of leukemia on medicinenet and I came across the following statement:

… an exchange between chromosomes 9 and 22 leads to what is known as the Philadelphia chromosome.

What is the mechanism behind this? Is it purely mechanical? Would the locations on both chromosome's be equidistance from the end (for lack of biology vocabulary.) In other words, would each segment involved in the swap have to be similarly sized?


Chronic myeloid leukemia

Chronic myeloid leukemia is a slow-growing cancer of the blood-forming tissue (bone marrow). Normal bone marrow produces red blood cells (erythrocytes) that carry oxygen, white blood cells (leukocytes) that protect the body from infection, and platelets (thrombocytes) that are involved in blood clotting . In chronic myeloid leukemia, the bone marrow produces too many white blood cells. Initially, these cells function relatively normally. However, as the condition progresses, immature white blood cells called myeloblasts (or blasts) accumulate in the blood and bone marrow. The overgrowth of myeloblasts impairs development of other blood cells, leading to a shortage of red blood cells (anemia ) and platelets.

Chronic myeloid leukemia usually begins after age 60. Common features include excessive tiredness (fatigue), fever, and weight loss. Many affected individuals develop an enlarged spleen (splenomegaly), which can cause a feeling of fullness in the abdomen and a loss of appetite. About half of people with chronic myeloid leukemia do not initially have any signs and symptoms and are diagnosed when a blood test is performed for another reason.

The condition consists of three phases: the chronic phase, the accelerated phase, and the blast phase (or blast crisis). In the chronic phase, the number of mature white blood cells is elevated, and myeloblasts account for less than 10 percent of blood cells. Signs and symptoms of the condition during this phase are typically mild or absent and worsen slowly. The chronic phase can last from months to years. In the accelerated phase, the number of myeloblasts is slightly higher, making up 10 to 29 percent of blood cells. The signs and symptoms continue to worsen. The accelerated phase usually lasts 4 to 6 months, although it is skipped in some affected individuals. In blast crisis, 30 percent or more of blood or bone marrow cells are myeloblasts. Signs and symptoms are most severe in this phase, including a massively enlarged spleen, bone pain, and weight loss. Serious infections and uncontrolled bleeding can be life-threatening.


Introduction

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the dysregulated production and uncontrolled proliferation of mature granulocytes with normal differentiation. CML is associated with the BCR-ABL1 fusion gene that results from a translocation between chromosomes 9 and 22, t(922)(q34q11), called the Philadelphia (Ph) chromosome [1]. BCR-ABL1 is capable of autophosphorylation and uncontrolled signaling to multiple downstream oncogenic proteins in CML [2]. Tyrosine kinase inhibitors (TKIs) inhibit the initiation of the BCR-ABL1 pathway and are effective, frontline therapies for chronic phase (CP) CML (CML-CP) [3].

Ph chromosome is present in more than 90% of CML patients, and only about 5% of CML patients show complex variant translocations, which is due to the participation of one or more chromosomes other than 9 and 22 [4]. The mechanisms of the generation of the variant translocations are not fully understood. While some previous studies have suggested that CML patients with variant Ph translocations may have a worse outcome than those with classic translocations, other studies have shown that patients with variant Ph translocations have an outcome similar to those with classic Ph translocations when treated with imatinib mesylate [4, 5].

Herein, we describe a unique case of CML-CP with a three-way Ph chromosome variant t(4922)(q21q34q11.2). This was the 14th case of t(4922), in particular, a new variant Ph translocation involved in chromosome 4q21 and the first case treated with TKIs. In addition, we summarize previous case reports regarding three-way variant chromosome translocation t(4922) and discuss how this rare translocation is linked to pathogenesis, disease characteristics, treatment responses, and prognosis.


Contents

The way CML presents depends on the stage of the disease at diagnosis as it has been known to skip stages in some cases. [4]

90%) are diagnosed during the chronic stage which is most often asymptomatic. In these cases it may be diagnosed incidentally with an elevated white blood cell count on a routine laboratory test. It can also present with symptoms indicative of hepatosplenomegaly and the resulting upper quadrant pain this causes. The enlarged spleen may put pressure on the stomach causing a loss of appetite and resulting weight loss. It may also present with mild fever and night sweats due to an elevated basal level of metabolism. [4]

Some (<10%) are diagnosed during the accelerated stage which most often presents bleeding, petechiae and ecchymosis. [4] In these patients fevers are most commonly the result of opportunistic infections. [4]

Some patients are initially diagnosed in the blast phase in which the symptoms are most likely fever, bone pain and an increase in bone marrow fibrosis. [4]

In most cases no obvious cause for CML can be isolated. [5]

Risk factors Edit

CML is more common in males than in females (male to female ratio of 1.4:1) and appears more commonly in the elderly with a median age at diagnosis of 65 years. [5] Exposure to ionising radiation appears to be a risk factor, based on a 50 fold higher incidence of CML in Hiroshima and Nagasaki nuclear bombing survivors. [5] The rate of CML in these individuals seems to peak about 10 years after the exposure. [5]

CML was the first cancer to be linked to a clear genetic abnormality, the chromosomal translocation known as the Philadelphia chromosome. This chromosomal abnormality is so named because it was first discovered and described in 1960 by two scientists from Philadelphia, Pennsylvania, USA: Peter Nowell of the University of Pennsylvania and David Hungerford of Fox Chase Cancer Center. [6]

In this translocation, parts of two chromosomes (the 9th and 22nd) switch places. As a result, part of the BCR ("breakpoint cluster region") gene from chromosome 22 is fused with the ABL gene on chromosome 9. This abnormal "fusion" gene generates a protein of p210 or sometimes p185 weight (p210 is short for 210 kDa protein, a shorthand used for characterizing proteins based solely on size). Because abl carries a domain that can add phosphate groups to tyrosine residues (a tyrosine kinase), the bcr-abl fusion gene product is also a tyrosine kinase. [7] [8]

The fused BCR-ABL protein interacts with the interleukin 3beta(c) receptor subunit. The BCR-ABL transcript is continuously active and does not require activation by other cellular messaging proteins. In turn, BCR-ABL activates a cascade of proteins that control the cell cycle, speeding up cell division. Moreover, the BCR-ABL protein inhibits DNA repair, causing genomic instability and making the cell more susceptible to developing further genetic abnormalities. The action of the BCR-ABL protein is the pathophysiologic cause of chronic myelogenous leukemia. With improved understanding of the nature of the BCR-ABL protein and its action as a tyrosine kinase, targeted therapies (the first of which was imatinib) that specifically inhibit the activity of the BCR-ABL protein have been developed. These tyrosine kinase inhibitors can induce complete remissions in CML, confirming the central importance of bcr-abl as the cause of CML. [8]

CML is often suspected on the basis of a complete blood count, which shows increased granulocytes of all types, typically including mature myeloid cells. Basophils and eosinophils are almost universally increased this feature may help differentiate CML from a leukemoid reaction. A bone marrow biopsy is often performed as part of the evaluation for CML, and CML is diagnosed by cytogenetics that detects the translocation t(922)(q34q11.2) which involves the ABL1 gene in chromosome 9 and the BCR gene in chromosome 22. [9] As a result of this translocation, the chromosome looks smaller than its homologue chromosome, and this appearance is known as the Philadelphia chromosome chromosomal abnormality. Thus, this abnormality can be detected by routine cytogenetics, and the involved genes BCR-ABL1 can be detected by fluorescent in situ hybridization, as well as by PCR. [10]

Controversy exists over so-called Ph-negative CML, or cases of suspected CML in which the Philadelphia chromosome cannot be detected. Many such patients in fact have complex chromosomal abnormalities that mask the (922) translocation, or have evidence of the translocation by FISH or RT-PCR in spite of normal routine karyotyping. [11] The small subset of patients without detectable molecular evidence of BCR-ABL1 fusion may be better classified as having an undifferentiated myelodysplastic/myeloproliferative disorder, as their clinical course tends to be different from patients with CML. [12]

CML must be distinguished from a leukemoid reaction, which can have a similar appearance on a blood smear. [10]

Classification Edit

CML is often divided into three phases based on clinical characteristics and laboratory findings. In the absence of intervention, CML typically begins in the chronic phase, and over the course of several years progresses to an accelerated phase and ultimately to a blast crisis. Blast crisis is the terminal phase of CML and clinically behaves like an acute leukemia. Drug treatment will usually stop this progression if started early. One of the drivers of the progression from chronic phase through acceleration and blast crisis is the acquisition of new chromosomal abnormalities (in addition to the Philadelphia chromosome). [7] Some patients may already be in the accelerated phase or blast crisis by the time they are diagnosed. [10]

Chronic phase Edit

Approximately 85% of patients with CML are in the chronic phase at the time of diagnosis. During this phase, patients are usually asymptomatic or have only mild symptoms of fatigue, left side pain, joint and/or hip pain, or abdominal fullness. The duration of chronic phase is variable and depends on how early the disease was diagnosed as well as the therapies used. In the absence of treatment, the disease progresses to an accelerated phase. [10] Precise patient staging based on clinical markers and personal genomic profile will likely prove beneficial in the assessment of disease history with respect to progression risk. [13]

Accelerated phase Edit

Criteria for diagnosing transition into the accelerated phase are somewhat variable the most widely used criteria are those put forward by investigators at M.D. Anderson Cancer Center, [14] by Sokal et al., [15] and the World Health Organization. [12] [16] The WHO criteria [17] are perhaps most widely used, and define the accelerated phase by the presence of ≥1 of the following haematological/cytogenetic criteria or provisional criteria concerning response to tyrosine kinase inhibitor (TKI) therapy

  • Haematological/cytogenetic criteria
    • Persistent or increasing high white blood cell count (> 10 × 10 9 /L), unresponsive to therapy
    • Persistent or increasing splenomegaly, unresponsive to therapy
    • Persistent thrombocytosis (> 1000 × 10 9 /L), unresponsive to therapy
    • Persistent thrombocytopenia (< 100 × 10 9 /L), unrelated to therapy
    • ≥ 20% basophils in the peripheral blood
    • 10―19% blasts in the peripheral blood and/or bone marrow
    • Additional clonal chromosomal abnormalities in Philadelphia (Ph) chromosome-positive (Ph+) cells at diagnosis, including so-called major route abnormalities (a second Ph chromosome, trisomy 8, isochromosome 17q, trisomy 19), complex karyotype, and abnormalities of 3q26.2
    • Any new clonal chromosomal abnormality in Ph+ cells that occurs during therapy
    • Haematological resistance (or failure to achieve a complete haematological response d) to the first TKI
    • Any haematological, cytogenetic, or molecular indications of resistance to two sequential TKIs
    • Occurrence of two or more mutations in the BCR-ABL1 fusion gene during TKI therapy

    The patient is considered to be in the accelerated phase if any of the above are present. The accelerated phase is significant because it signals that the disease is progressing and transformation to blast crisis is imminent. Drug treatment often becomes less effective in the advanced stages. [12]

    Blast crisis Edit

    Blast crisis is the final phase in the evolution of CML, and behaves like an acute leukemia, with rapid progression and short survival. [10] Blast crisis is diagnosed if any of the following are present in a patient with CML: [18]

    • >20% blasts in the blood or bone marrow
    • The presence of an extramedullary proliferation of blasts

    The only curative treatment for CML is a bone marrow transplant or an allogeneic stem cell transplant. [19] Other than this there are four major mainstays of treatment in CML: treatment with tyrosine kinase inhibitors, myelosuppressive or leukopheresis therapy (to counteract the leukocytosis during early treatment), splenectomy and interferon alfa-2b treatment. [19] Due to the high median age of patients with CML it is relatively rare for CML to be seen in pregnant women, despite this, however, chronic myelogenous leukemia can be treated with relative safety at any time during pregnancy with Interferon-alpha hormones. [20]

    Chronic phase Edit

    In the past, antimetabolites (e.g., cytarabine, hydroxyurea), alkylating agents, interferon alfa 2b, and steroids were used as treatments of CML in the chronic phase, but since the 2000s have been replaced by Bcr-Abl tyrosine-kinase inhibitors [21] drugs that specifically target BCR-ABL, the constitutively activated tyrosine kinase fusion protein caused by the Philadelphia chromosome translocation. Despite the move to replacing cytotoxic antineoplastics (standard anticancer drugs) with tyrosine kinase inhibitors sometimes hydroxyurea is still used to counteract the high leukocyte counts encountered during treatment with tyrosine kinase inhibitors like imatinib in these situations it may be the preferred myelosuppressive agent due to its relative lack of leukemogenic effects and hence the relative lack of potential for secondary hematologic malignancies to result from treatment. [22] IRIS, an international study that compared interferon/cytarabine combination and the first of these new drugs imatinib, with long-term follow up, demonstrated the clear superiority of tyrosine-kinase-targeted inhibition over existing treatments. [23]

    Imatinib Edit

    The first of this new class of drugs was imatinib mesylate (marketed as Gleevec or Glivec), approved by the US Food and Drug Administration (FDA) in 2001. Imatinib was found to inhibit the progression of CML in the majority of patients (65–75%) sufficiently to achieve regrowth of their normal bone marrow stem cell population (a cytogenetic response) with stable proportions of maturing white blood cells. Because some leukemic cells (as evaluated by RT-PCR) persist in nearly all patients, the treatment has to be continued indefinitely. Since the advent of imatinib, CML has become the first cancer in which a standard medical treatment may give to the patient a normal life expectancy. [24]

    Dasatinib, nilotinib, radotinib and bosutinib Edit

    To overcome imatinib resistance and to increase responsiveness to TK inhibitors, four novel agents were later developed. The first, dasatinib, blocks several further oncogenic proteins, in addition to more potent inhibition of the BCR-ABL protein, and was initially approved in 2007 by the US FDA to treat CML in patients who were either resistant to or intolerant of imatinib. A second new TK inhibitor, nilotinib, was also approved by the FDA for the same indication. In 2010, nilotinib and dasatinib were also approved for first-line therapy, making three drugs in this class available for treatment of newly diagnosed CML. In 2012, Radotinib joined the class of novel agents in the inhibition of the BCR-ABL protein and was approved in South Korea for patients resistant to or intolerant of imatinib. Bosutinib received US FDA and EU European Medicines Agency approval on 4 September 2012 and 27 March 2013 respectively for the treatment of adult patients with Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) with resistance, or intolerance to prior therapy.

    Treatment-resistant CML Edit

    While capable of producing significantly improved responses compared with the action of imatinib, neither dasatinib nor nilotinib could overcome drug resistance caused by one particular mutation found to occur in the structure of BCR-ABL1 known as the T315I mutation (in other words, where the 315th amino acid is mutated from a threonine residue to an isoleucine residue). Two approaches were developed to the treatment of CML as a result:

    In 2007, Chemgenex released results of an open-label Phase 2/3 study (CGX-635-CML-202) that investigated the use of a non BCR-ABL targeted agent omacetaxine, administered subcutaneously (under the skin) in patients who had failed with imatinib and exhibited T315I kinase domain mutation. [25] [26] This is a study which is ongoing through 2014. [27] In September 2012, the FDA approved omacetaxine for the treatment of CML in the case of resistance to other chemotherapeutic agents. [28] [29]

    Independently, ARIAD pharmaceuticals, adapting the chemical structures from first and second-generation TK inhibitors, arrived at a new pan-BCR-ABL1 inhibitor which showed (for the first time) efficacy against T315I, as well as all other known mutations of the oncoprotein. The drug, ponatinib, gained FDA approval in December 2012 for treatment of patients with resistant or intolerant CML. Just as with second generation TK inhibitors, early approval is being sought to extend the use of ponatinib to newly diagnosed CML also. [ citation needed ]

    Vaccination Edit

    In 2005, encouraging but mixed results of vaccination were reported with the BCR/ABL1 p210 fusion protein in patients with stable disease, with GM-CSF as an adjuvant. [30]

    Before the advent of tyrosine kinase inhibitors, the median survival time for CML patients had been about 3–5 years from time of diagnosis. [3]

    With the use of tyrosine kinase inhibitors, survival rates have improved dramatically. A 2006 followup of 553 patients using imatinib (Gleevec) found an overall survival rate of 89% after five years. [31]

    A 2011 followup of 832 patients using imatinib who achieved a stable cytogenetic response found an overall survival rate of 95.2% after 8 years, which is similar to the rate in the general population. Fewer than 1% of patients died because of leukemia progression. [24]

    United Kingdom Edit

    CML accounts for 8% of all leukaemias in the UK, and around 680 people were diagnosed with the disease in 2011. [32]

    United States Edit

    The American Cancer Society estimates that in 2014, about 5,980 new cases of chronic myeloid leukemia were diagnosed, and about 810 people died of the disease. This means that a little over 10% of all newly diagnosed leukemia cases will be chronic myeloid leukemia. The average risk of a person getting this disease is 1 in 588. The disease is more common in men than women, and more common in whites than African-Americans. The average age at diagnosis is 64 years, and this disease is rarely seen in children. [33]


    From genes to therapy: the case of Philadelphia chromosome-positive leukemias

    The Philadelphia chromosome (Ph-chromosome) has long represented the only cytogenetic abnormality known to be associated with a specific malignant disease in humans, being present in more than 95% of patients with chronic myelogenous leukemia. This abnormality is the result of a reciprocal translocation between the long arms of chromosome 9 and 22, t(922)(q34q11), and its presence is not restricted to chronic myelogenous leukemia, but can also be found in 30% of cases of acute lymphoblastic leukemia in adults. In the 1980s, the molecular counterpart of the chromosomal rearrangement was identified to consist of the juxtaposition of parts of the BCR and ABL genes to form a BCR-ABL hybrid gene. The resulting chimeric proteins (P210 and P190), which retain constitutively activated tyrosine kinase activity, have demonstrated a causative role in the genesis of the leukemic process. Although many aspects of the BCR-ABL driven transformation remain unsolved, great advances in understanding the molecular pathology of Ph-positive leukemias resulted in meaningful improvement in the clinical setting. Molecular tools to diagnose disease (PCR, FISH, and southern blot) and to monitor minimal residual disease after potential curative treatment are now in current practice, and new powerful therapeutic tools have emerged that target the molecular oncogenic pathways activated in Ph-positive cells. Among them, specific ABL tyrosine kinase inhibitors recently obtained extraordinary results in many clinical protocols. This review summarizes the most recent advances in this field with special focus on the putative mechanisms of the transformation and progression of chronic myelogenous leukemia and on the major impact that understanding the molecular biology of these diseases is having in clinical practice.


    Translocation: Origin, Types and Effects | Genetics

    (ii) They may be induced by mutagens, viz., ionizing radiations and many chemical mutagens, since they induce chromosome breakage.

    (iii) Translocations may be induced by growing plants in calcium-deficient media, as reported by Nilan and Phillips in 1957.

    (iv) Translocations may be induced by oxygen applied at a high atmospheric pressure, as reported by Kronstad et al., in 1959 and Moutschen-Dahmen et al., in 1959.

    (v) Translocations can be recovered from certain interspecific crosses since the concerned species differ for chromosomal rearrangements, including translocation, which become observable in their interspecific hybrids.

    (vi) Genetically controlled breakage in the chromosomes may also produce translocations, such as, sticky gene (st) and DS-AC system in maize.

    In 1914, Belling reported 50% pollen abortion and 50% seed set in crosses of Florida velvet bean which he termed as semi-sterility. Later in 1924, Belling and Blakeslee, working with Datum stramonium, concluded that non-homologous chromosomes could exchange segments.

    The breeding behaviour of semi-sterility in Stizolobium deeringianum was explained in 1925 by Belling on the basis of “segmental interchange between non-homologues”.

    In maize plant, semi-sterility was reported by Brink in 1927. In 1930, Burnham reported a ring of 4 chromosomes in the semi-sterile plant of maize. In the same year, McClintock showed that translocation heterozygotes produced a “cross-shaped configuration” at pachytene. In Drosophila, the first translocation where a piece of X chromosome was attached to the Y chromosome was reported by Stern in 1926.

    Certain genes have been reported to induce chromosome breaks leading to the production of translocations. Genetically controlled systems of chromosome breakage have been observed in some cases. In maize, chromosome breaks occurred at AI of meiosis due to stickiness of chromosomes aberrations.

    The DS-AC system in maize first described by McClintock in 1950 also causes structural changes by inducing chromosome breaks.

    Interlocking of bivalents which takes place in certain species, such as, Tradescantia, also causes chromosome breakage leading to various aberrations. Translocations have been induced through various physical and chemical mutagens in several plant and animal species.

    Translocations originate through chromosome breakage and reunion. It can also be interpreted on the basis of exchange model. The unit of translocation may be a chromosome (chromosomal translocation) or a chromatid (chromatid translocation).

    Types of Translocation:

    Translocation may be classified on the basis of the trans-located segment being present in the same, homologous or non-homologous chromosome, and the number of breaks involved in the translocation.

    A. Classification on the basis of involvement of the same or different chromosomes:

    1. Intra-chromosomal (internal) translocation or shift:

    A segment of a chromosome is shifted from its original position to some other position within the same chromosome. It is of two types:

    The shift occurs in the same arm (Fig. 14.1).

    The shift occurs from one arm to the other arm (Fig. 14.1)

    2. Inter-Chromosomal translocation:

    A chromosomal segment is transferred from one chromosome to another one. It may be either fraternal or external.

    The chromosome segment is trans-located into the homologous (Fig. 14.1).

    The chromosome segment is trans-located into a non-homologous chromosome (Fig. 14.2).

    The inter-chromosomal translocation may be divided into the following three groups.

    I. Transposition:

    Transfer of a chromosome segment from one chromosome to another chromosome is called transposition. It may be of the following types.

    (i) Intercalation or insertion or insertional translocation:

    The transposition occurs in an intercalary position.

    (ii) Terminal transposition:

    The segment is attached to the chromosomal end. However, terminal translocation is not possible so long as the telomere of the chromosome remains intact. Therefore, terminal translocation can occur only when the chromosome end is deleted or trans-located.

    II. Reciprocal translocation or interchange:

    Exchange of segments between two or more non-homologous chromosomes is called reciprocal translocation or interchange. It is of two types: asymmetrical or aneucentric and symmetrical or eucentric.

    (i) Asymmetrical or aneucentric translocation:

    After breakage, the broken acentric segments fuse to form a trans-located acentric chromosome, while the two chromosomes with centromeres fuse to produce a trans-located chromosome with two centromeres (dicentric). The dicentric chromosome will produce bridge at anaphase if the two centromeres move to opposite poles (Fig. 14.2).

    (ii) Symmetrical or eucentric translocation:

    Broken segments are exchanged between the two non-homologous chromosomes so that both the chromosomes involved in translocation possess only one centromere each (mono-centric) (Fig. 14.2).

    III. Whole-Arm translocations or whole-arm transfers:

    These are the special types of translocations where almost the entire chromosome arms are transposed or interchanged.

    Such translocations are of three types:

    (i) Centric fusion or Robertsonian translocation:

    The long arms of two acrocentric chromosomes may fuse due to translocation to produce a metacentric chromosome, while their short arms fuse to form a very small chromosome (Fig. 14.2).

    (ii) Dissociation:

    Two metacentric chromosomes, one with long arms and other with short arms may produce two acrocentric chromosomes through translocation (Fig. 14.2).

    (iii) Tandem fusion:

    Such type of interchange is produced when the break in one chromosome occurs near the centromere and in the other chromosome, it occurs near the end. The result of such breakage and reunion may be a large acrocentric chromosome and a small metacentric chromosome, if both the chromosomes were originally acrocentric.

    If one chromosome is a metacentric, the result o the interchange will be two acrocentric chromosomes, one being small and the other being large (Fig. 14.2).

    B. Classification on the basis of the number of breaks involved:

    According to this system Schulz-Schaeffer in 1980 divided the translocations into four classes:

    (4) Complex (more than three breaks) translocations.

    1. Simple translocation:

    In such a translocation, a segment of a chromosome becomes attached to the end of a non-homologous chromosome. In 1929, Painter and Muller reported such type of translocations in Drosophila. In view of the stability of telomere, intact chromosomal end cannot fuse with a chromosomal segment.

    Therefore, cases of simple translocations are either reciprocal translocation in which a very small telomeric segment of one chromosome (apparently devoid of a detectable gene) is involved in a reciprocal translocation, or the telomeric region of the concerned chromosome gets deleted during the translocation.

    2. Reciprocal translocation or Interchange:

    In this type of translocation, segments are exchanged between two non-homologous chromosomes, therefore, it involves one break in each of the involved chromosomes (Fig. 14.2). Most of the translocations are reciprocal translocations. Such translocations have been extensively studied in various plant and animal species.

    3. Shift type of translocation or Transposition:

    It involves three breaks, and the broken segment is shifted (transposed) in the intercalary position (Fig. 14.1).

    According to whether same or different chromosomes involved, shift is of two types:

    (a) Intra-chromosomal shift:

    Shift is confined to the same chromosome the broken segment gets inserted either (i) within the same arm, or (ii) in the other arm of the chromosome.

    (b) Inter-chromosomal shift:

    A broken piece of a chromosome is inserted into an intercalary position of a non-homologous chromosome (Fig. 14.2).

    4. Complex Translocations:

    In such translocations, more than three breaks are involved. Mostly, such translocations are naturally occurring.

    Phenotypic Effects of Translocation:

    In general, no phenotypic effects of translocations are visible. But in case there is damage to the DNA during translocation, recessive mutations may arise. Translocations may also act as recessive lethals. Position effect may be produced by some translocations in certain organisms, such as, Drosophila, Oenothera etc.


    Cellular events

    Decreased apoptosis

    Apoptosis or programmed cell death can be considered a protective mechanism against cancer and its derangement has significance in many malignancies. Cells harboring or acquiring chromosomal errors by DNA damaging agents undergo apoptosis. Signals instructing a cell to undergo apoptosis are multiple, complex and highly redundant. The final decision for a cell to initiate apoptosis rather than cell cycle arrest, or a failure to respond by either method may be dependent on the magnitude and duration of the damage stimulus.

    In CML, multiple mechanisms contribute toward resistance against apoptosis. Phosphorylation and activation of the PI(3)-K/Akt pathway 59,71 is a major pathway by which BCR-ABL exerts its antiapoptotic effect. PI(3) kinase activation also via protein kinase B, results in the phosphorylation and inactivation of the pro-apoptotic protein BAD a member of the Bcl-2 family. 60 Increased expression of BCL-2 the prototype member of the BCL family of antiapoptotic proteins 72 has been described in cell types harboring the BCR-ABL oncogene and contributes to decreased apoptosis. 61 BCR-ABL-mediated activation of STAT5 and subsequent increases in BCLxl levels may also increase resistance to apoptosis. 62,63,73

    Most but not all studies using p210 BCR/ABL expressing cell lines have demonstrated that BCR-ABL expression protects from apoptosis induced by physical and chemical stresses. 74,75,76,77,78 Additionally, chronic phase CML CD34 + cells undergo delayed apoptotic death upon cytokine withdrawal when compared with normal progenitors. 79,80,81 The use of antisense oligonucleotides against BCR-ABL could reverse the delay in apoptosis in CML cell lines. 80,81 P210 BCR/ABL may protect cells from cytotoxic agent-induced apoptosis by preventing the release of cytochrome-c 82 and preventing the activation of caspases especially caspase 3. 83

    The redox-sensitive transcription factor NF-κB translocates to the nucleus upon cellular activation and its activity is generally associated with protection from apoptosis. There is evidence for activation of NF-κB in transgenic models of CML which is yet another mechanism that may protect against apoptosis. 84 Finally, the RAS pathway may be involved in BCR-ABL-mediated inhibition of apoptosis 75,85,86 and elevated cytokine production 87,88 may have an inhibitory effect on apoptosis.

    Interestingly, c-ABL is thought to have a pro-apoptotic function in the cytoplasm by inhibiting the survival pathway mediated by PI(3)K and as discussed later, methylation of the only normal ABL allele seen during disease progression may contribute towards resistance against apoptosis. Nuclear ABL is also believed to have a role in cell death. More evidence to support this theory has emerged with the demonstration that nuclear import of BCR-ABL in a CML cell line by treatment with STI-571 (gleevec, a new specific tyrosine kinase inhibitor that inhibits the BCR-ABL TK at micromolar concentrations 89 ) and its subsequent nuclear entrapment by leptomycin B induced apoptosis. 90 Therefore, the abnormal cytoplasmic location of BCR-ABL and the reduction in nuclear ABL protein may enhance resistance to apoptosis.

    Protection against apoptosis is relatively minimal in chronic phase CML but there is evidence that BCR-ABL mediates resistance to apoptosis in a dose-dependent fashion. 91 Increased expression of the BCR-ABL mRNA, often associated with disease progression 92 may therefore lead to increased resistance to apoptosis in accelerated phase and blast crisis. 91 Despite being a characteristic feature of CML decreased apoptosis alone is not sufficient for disease transformation and disease progression likely requires additional abnormalities such as for instance, loss of a tumor suppressor gene.

    Differentiation block

    During the process of hematopoeitic differentiation, pluripotent stem cells become lineage committed and eventually differentiate morphologically and functionally into distinct blood cell types. Although the p210 BCR/ABL itself does not significantly affect terminal cell differentiation in chronic phase, differentiation is blocked in blast phase CML. 93,94,95 The role of BCR-ABL itself or other events in this phenomenon is not fully elucidated. Additional mutations, formation of new oncogenes, elevated cytokine levels, inactivation of tumor suppressor genes are hypothetical reasons for this phenomenon. As exemplified in acute promyelocytic leukemia, tumor progression can be suppressed by inducing differentiation with cytokines or factors that regulate normal hematopoiesis. 96,97 Further studies elucidating the mechanisms underlying the differentiation block seen in blast crisis CML are warranted to help define a role for differentiating agents in the therapeutic armamentarium against blast crisis.

    Decreased immune surveillance

    MHC-restricted and MHC-unrestricted mechanisms play an important role in the natural control of the Ph clone in chronic phase as well as during progression of CML. 98 Among the MHC-restricted mechanisms, T lymphocyte-mediated killing of target cells via Fas-receptor triggering plays an important role in elimination of malignant CML cells. CML progenitor cells also express functional Fas-ligand, which may be an important immune surveillance escape factor. In comparison to the chronic phase, CML cells derived from patients in blast crisis are refractory to Fas-mediated apoptosis, regardless of the expression levels of Fas, suggesting that an immune-mediated selection pressure could result in acquisition of Fas resistance. 99,100 Natural killer (NK) and activated killer (AK) cells mediate MHC-unrestricted cytotoxicity. There is evidence for declining NK cell function in blast crisis CML but it is unclear if this is a cause rather than an effect of disease progression. 101,102

    Drug resistance

    CML progenitors obtained from patients during chronic phase and blast crisis are equally sensitive to STI-571 by in vitro assay methods. 103 Despite this observation there is a lower response rate in accelerated phase and blast crisis compared with chronic phase CML indicating development of drug resistance in vivo. The other possibility to consider is the presence of additional genetic errors that can stimulate the malignant clone in the absence of BCR-ABL. The observation that some patients who initially respond to STI-571 redevelop Ph-positive hematopoiesis may also indicate development of drug resistance. Additionally, resistance to interferon α or hydroxyurea is thought to be a herald of disease transformation. Drug resistance may be contributory to the change in character of chronic phase CML. There are multiple mechanisms involved in resistance to therapy, which could impact on disease progression. These include expression of the MDR-1 gene, 104,105 AGP-1, 106 reduplication of BCR-ABL or its overexpression, 104,107 decreased apoptosis 74 and possibly defective drug transport.


    Conclusion leukemogenesis is an outcome of the Ph combined with other genetic variations

    Expression of BCR-ABL1 (p210 transcript) has been detected at very low levels in the peripheral blood cells of some healthy individuals but not in umbilical cord blood cells [193]. In addition, BCR-ABL1-specific T cells are detected in healthy donors and in CML patients after allogeneic stem cell transplantation [194]. These results indicate that normal cells evolve progressively to a neoplastic state, and they may acquire a succession of genetic abnormalities and gain the ability to maintain proliferation, inhibit differentiation, and resist cell death [195].

    The Ph bearing the BCR-ABL1 fusion gene is the key initiator of different phenotypes of leukemia with diverse prognoses. The translocation leads to persistent TK activation and genomic instability during leukemogenesis. Disorders in multiple signaling pathways and genetic abnormalities combined with the Ph are essential for the evolution of different types of leukemia however, why cells possessing the Ph should evolve specifically into CML, AML, ALL, or MPAL is currently unclear and under investigation. Evidence shows that there are characteristics exclusive to specific leukemias, including deletion of BTG1 in B-cell leukemia, loss of IKZF1 with monosomy 7 in AML, and deletions involving IGH, TCR, IKZF1, and CDKN2A/B in CML-AP/CP. Greater understanding of leukemogenesis and the effect of treatment on clonal evolution will provide novel insights into the design of future therapeutic strategies for Ph-positive leukemia [196, 197].


    The Biology of Philadelphia Chromosome-Positive ALL

    BCR�L1-Induced Leukemia

    BCRABL1 translocations are associated with two distinct clinical hematologic malignancies, CML and ALL. For CML, three discrete clinical stages have been defined: chronic phase, accelerated phase, and blast crisis. Genomic instability, the accumulation of additional cytogenetic (trisomy 8, isochromosome 17) and molecular (p53 pathway mutations, loss of p16 INK4A/ARF ) abnormalities, and BCR�L1-independent activation of downstream signaling pathways (LYN, AKT, STAT5) are all associated with – and likely contribute to – the progression to blast crisis (3). In about 30% of the cases, the predominant lineage in blast crisis is B-lymphoid rather than myeloid, speaking to the likely hematopoietic stem-cell origin of the disease. This presumed stem-cell origin may also explain the inability to achieve any durable remissions using conventional chemotherapy. Prior to the advent of tyrosine kinase inhibition, temporary disease stabilization was often achieved using hydroxyurea, low-dose cytarabine, and/or interferon, but the only curative approach was an allogeneic hematopoietic stem-cell transplantation (HSCT).

    In addition to CML, BCR�L1 translocations are found in a distinct subtype of ALL, called Ph + ALL. The clinical presentation is indistinguishable from ALL with other cytogenetic abnormalities, and the diagnosis relies on the presence of the BCRABL1 translocation (cytogenetics and FISH) and/or fusion transcript (PCR). Outcomes for Ph + ALL were exceptionally poor when treated with chemotherapy, and HSCT in first remission was usually considered to be the best therapy (4). The frequency of BCR�L1 rearrangement in ALL increases with age (Figure 1) (5) and has been reported as high as 50% in the elderly (6). A greater percentage of patients with adverse cytogenetics contributes substantially to the overall worse outcome in adult compared to pediatric ALL (Figure 1).

    Figure 1. Frequency of BCR/ABL1 rearrangement in pediatric and adult ALL. Cytogenetic abnormalities in pediatric (ϡ year) and adult patients with ALL are shown (5). The majority of children ρ year of age carry a rearrangement of the MLL-gene and are not included in this graph. Favorable cytogenetic abnormalities are represented in green, neutral in blue, and unfavorable cytogenetics are represented in yellow/red. Favorable cytogenetics (high hyperdiploidy, ETV6–RUNX1) decrease, while the frequency of BCR/ABL1 rearrangement increases with age. The higher percentage of unfavorable cytogenetics substantially contributes to inferior outcomes in adult versus pediatric ALL.

    The first indication that the BCR�L1 fusion protein is indeed the crucial driver of CML came from mouse studies showing that expression of BCR�L1 in the bone marrow causes a CML-like disease (7𠄹). Studies that utilized a mutant BCR�L1 protein with an inactive tyrosine kinase domain defined the tyrosine kinase activity of ABL1 as absolutely required for transformation (10). This suggested that targeted inhibition of the ABL kinase domain might be an effective therapeutic strategy in BCR�L1-driven hematologic malignancies. The pioneering work of Brian Druker spearheaded the clinical development of the first tyrosine kinase inhibitor (TKI), Imatinib (11�). Imatinib gained FDA approval in 2000 and revolutionized CML therapy, converting a near universally fatal disease requiring HSCT into a chronic condition controlled with monotherapy of a targeted agent (14). In the years since the initial success of imatinib, second [nilotinib, dasatinib, bosutinib (15�)] and third [ponatinib (18)] generation ABL1 class TKIs have been developed, which are active against multiple imatinib-resistant BCR�L1 mutants.

    Early studies using imatinib as monotherapy in Ph + ALL were disappointing, with initial responses rapidly progressing to TKI-resistant disease. However, the integration of TKIs into a high-risk ALL chemotherapy backbone fundamentally changed our approach to Ph + ALL as well. Overall survival (OS) using this strategy more than doubled compared to chemotherapy-only treated historic controls (2), and HSCT is no longer universally recommended for Ph + ALL. Despite these advances, the survival of Ph + ALL still lags behind most other cytogenetic subgroups in pediatric ALL. A better understanding of the biology of Ph + ALL may help to refine therapy and develop rational combinations of targeted agents that will further improve outcomes for patients with this disease.

    The Philadelphia Chromosome and BCR�L1 Fusion

    Ph + ALL derives its name from the presence of the Philadelphia (Ph) chromosome, named after the city where it was first described in the leukemia cells of a CML patient by Nowell and Hungerford in 1960 (19). In 1973, Janet Rowley reported that the Philadelphia Chromosome was the der(22) product of the reciprocal t(922)(q34q11.2) translocation (20). The BCR�L1 fusion gene is generated by joining almost the entire coding region of the ABL1 tyrosine kinase gene (Abelson murine leukemia virus homolog, exons 2�, chromosome 9) to the breakpoint cluster region (BCR) gene on chromosome 22 (Figure 2) (21). There are two main regions where breakpoints cluster within the BCR gene. The 𠇌ML” breakpoint region lies between exons 12 and 16 in a region called the major breakpoint cluster region (M-BCR). Translocations involving the M-BCR produce the larger p210 BCR�L1 protein, which derives its name from its molecular size of 210 kDa. Translocations that occur within the minor 𠇊LL” BCR (m-BCR) yield a smaller p190 gene product that retains only the first exon of BCR. A rare p230 fusion protein (with a “micro”-BCR breakpoint between exons 19 and 20) has also been described. Both p210 and p190 transform primary human and murine bone marrow cells (8, 9, 22). Of the two, the 𠇊LL-type” p190 is the stronger transforming agent (7, 23). About 90% of pediatric Ph + ALL patients have the classic ALL-type p190 translocation (24) with some variability reported in the literature as CML in B-lymphoid blast crisis can sometimes be hard to distinguish from Ph + ALL.

    Figure 2. Structure of the most common BCR�L1 fusion genes. Domain structure of wild type BCR and wild type ABL1 protein, as well as retained domains in the three most common BCR�L1 variants, p230, p210, and p190. OD: oligomerization domain (coiled-coil domain) mediating oligomerization, Tyr177: tyrosine 177, which, when phosphorylated, serves as a docking site for the adaptor protein GRB-2 SH2-domain: SRC homology 2 (binding to phosphorylated tyrosine residues, including BCR exon 1), SH3-domain: SRC homology 2 (binding to proline rich peptides). SH1-domain: SRC homology 1 (ABL1 catalytic domain) GEF-domain: guanine nucleotide exchange factors (G-protein signaling) E1: exon 1 of ABL1, contains the inhibitory N-terminal �p” that binds the catalytic domain (SH1) of ABL1 and prevents autophosphorylation NLS: nuclear localization signal.

    Wild type ABL1 is a ubiquitously expressed but tightly regulated non-receptor tyrosine kinase that is present throughout hematopoietic development, with declining levels during myeloid maturation. It is predominantly located in the cytoplasm in hematopoietic cells, but can shuttle to the nucleus. In the cytoplasm, ABL1 is found mostly bound to actin, and functions include signaling and modulation of the cytoskeleton. Nuclear ABL1 has been implicated in cell cycle control. The N-terminus of ABL1 negatively regulates ABL1 kinase activity, allowing for tight titration of ABL1 kinase activity under physiologic conditions. Loss of the N-terminus as a result of the BCR�L1 translocation results in high constitutive kinase activity. Thus loss of this important regulatory domain is a major contributor to ABL1-mediated leukemogenesis (25) [reviewed in Ref. (26)].

    The fusion partner of ABL1, BCR, is a complex locus that is transcribed into two major proteins, both with multiple functional domains implicated in a variety of fundamental biological processes. These include G-protein signaling pathways, cytoskeletal organization, growth, and development. The only exon of BCR that is consistently retained in all fusions is exon 1, which encodes a coiled-coil domain facilitating dimerization and autophosphorylation (amino acids 1�) (27, 28), a docking site for the adaptor protein GRB-2 (phosphorylated tyrosine 177) (28, 29), and a tyrosine kinase domain (amino acids 298�) (30). The exact role of the BCR-tyrosine kinase domain is unclear, and in a murine model of CML utilizing retroviral introduction of p210BCR�L1 deletion mutants into bone marrow cells, it appeared to be dispensable. On the other hand, the consistent inclusion of the BCR-tyrosine kinase domain in human BCR�L1-driven malignancies suggests that it may play a functional role in leukemogenesis (30�).

    Downstream Pathways Activated by BCR�L1 Fusion Proteins

    The molecular consequence of all BCR�L1 fusion proteins is a hyperactive ABL1 kinase domain and aberrant phosphorylation of a variety of targets. Activation results from lack of autoinhibition due to loss of the N-terminal regulatory domain of ABL1, and homodimerization and autophosphorylation of the fusion protein (27). The importance of the homodimerization and autophosphorylation for BCR�L1 signaling is underscored by promising in vitro results of peptides and small-molecule inhibitors that cause allosteric inhibition of BCR�L1 (33�). BCR�L1 kinase activity leads to direct and indirect activation of multiple pathways (37), including PI3K (38), AKT (39�), MTOR (42, 43), RAS (39, 44), EGFR, MAP-kinase (40, 43, 45), JNK/SAPK (43), JAK1𠄳 (46), the SRC-family kinases LYN, HCK, and FGR (47), PTPN11, NF-kB, phospholipase C, and, as a common downstream effector of many of these pathways, STAT5 (Figure 3) (46, 48�). Most of these pathways have been worked out in CML, but the relevant binding sites or kinase domains are preserved in the p190 fusion protein. Activation of JAK1𠄳 (50) and STAT1, 3, 5, and 6 (48, 50) has been experimentally confirmed for p190. Work in CML suggests that JAK1𠄳 activation is mediated through the interaction of BCR�L1 with cytokine receptors rather than direct phosphorylation (51). On the other hand, JAK2 appears to directly phosphorylate BCR�L1 at the critical tyrosine-177 residue and increase BCR�L1 protein stability, thus enhancing BCR�L1 signaling (52). Another important downstream pathway that has been confirmed specifically in Ph + ALL is the PI3K𠄺KT–MTOR pathway. Deletion of PI3K inhibited leukemogenesis in a murine model of p190 Ph + ALL. A dual PI3K/MTOR inhibitor was effective on Ph + ALL patient samples (53) and showed synergy with Imatinib (54). The activation of AKT and MTOR signaling also plays a critical role in steroid resistance in ALL (55, 56), and multiple agents targeting the PI3K/AKT/MTOR axis are currently in clinical trials for pediatric ALL [reviewed in Ref. (57)].

    Figure 3. BCR�L1 signaling pathways. Downstream signaling pathways activated by BCR�L1. Numerical references 1𠄶 denote classes of inhibitors in Table 1. I: imatinib D: dasatinib, N: nilotinib.

    Table 1. BCR�L1 downstream and parallel pathways as drug targets.

    Of particular importance appears to be the adaptor proteins GRB-2 (58�) and GAB-2 (60), which interact with, and participate in the activation of nearly all of the signaling pathways cited above. GRB-2 has been shown to bind phosphorylated tyrosine-177 (Figures 2 and 3). The importance of this interaction is demonstrated by the impaired in vivo leukemogenesis in murine models of p210BCR�L1 constructs with an engineered inactivating mutation of tyrosine-177 (29, 45, 61). Peptide-inhibition of the SH3 domain of the adaptor protein GRB-2 reduced growth and induced apoptosis in the BCRABL1-positive K562 cell line (7). Similarly, genetic inactivation of GAB-2 impairs p210BCR�L1-mediated transformation in mice (62).

    Recently, overexpression of the epidermal growth factor ERBB was found to be specifically elevated in Ph + ALL (56 versus 4.8% of Ph − ALL) (63). The molecular details of how this pathway intersects with BCR�L1 signaling requires further study preliminarily, p70S6 kinase (MTOR target) has been implicated. From a translational perspective, the ERBB/HER2/NEU inhibitor lapatinib was synergistic with imatinib and nilotinib (but not dasatinib) on Ph + ALL cell lines.

    The reported activation of the SCR-family kinases LYN, HCK, and FGR by BCR�L1 has important implications. BCR�L1 has been shown to interact with and activate SRC-family kinases, and inhibition of SRK-family kinases decreased growth and survival of BCRABL1-positive myeloid cell lines in vitro (64�). In addition to being activated by BCR�L1, the Src-family kinases Lyn (72) and Hck (73) have been reported to in turn phosphorylate BCR�L1 at several sites, including the critical residue for the interaction with the adaptor proteins Grb-2 and Gab-2 (Tyrosine-177). Expression of p210BCR�L1 in murine lymphoid progenitors negative for all three kinases (Lyn −/− Hck −/− Fgr −/− ) near completely prevented leukemogenesis in a mouse model of Ph + lymphoid leukemia (47). There was considerable redundancy between Lyn, Hck, and Fgr in this model, and genetic inactivation of at least two kinases was required to protect mice from leukemia. Somewhat surprisingly, given that CML cell lines responded to Src inhibition in vitro (67�), Lyn, Hck, and Fgr were not required to induce CML in vivo (47). A small-molecule inhibitor of SRC-family kinases improved the survival of mice with lymphoid but not myeloid leukemia. Lack of inhibition of Src-family kinases by imatinib, and dual inhibition of Src-kinases and BCR�L1 with dasatinib were proposed to underlie the improved efficacy of dasatinib in the p210 lymphoid leukemia model (74). Under normal physiologic conditions, Lyn −/− Hck −/− FGR −/− mice display defects in B-cell maturation and autoimmune features suggesting a specific role for these kinases in B-cell development, but the early B-cell compartments appear to be preserved (47). Dependency of Src-family kinases may thus be a specific feature of Ph + ALL. This is highly relevant from a clinical–translational standpoint as it provides a compelling rationale to investigate the dual BCR�L1–SRC-family kinase inhibitor dasatinib in Ph + ALL, and suggests that this agent may be more effective in Ph + ALL than imatinib or nilotinib.

    Many of these downstream pathways – particularly the JAK–STAT pathway, are also targeted by several newly described leukemogenic fusion proteins that induce a disease similar to Ph + ALL, but without a BCRABL1 rearrangement. These “Ph-like” leukemias share with Ph + ALL a transcriptional signature indicative of kinase activation, co-occurring mutations in the B-cell transcription factor IKZF1, and a poor outcome. Initially identified based solely on transcription profiling, improved molecular techniques have allowed identification of a tyrosine kinase mutation in many of these patients. These include rearrangements of JAK2, ABL1, PDGFRB, CRLF2, and EPOR, deletion of SH2B3 encoding the JAK2-negative regulator LNK, and activating mutations of FLT3 and the IL7 receptor (IL7R) (75, 76). The presence of these mutations opens the door for potential therapeutic impact using targeted inhibitors (77). This fascinating subgroup of ALL is the topic of a dedicated review in this issue.

    Differences between p210 and p190 – Lessons from Mouse Models

    As mentioned earlier, p190BCR�L1 has stronger transforming activity than p210BCR�L1, both in fibroblast transforming assays (78) and in mouse models (7, 79). One possible reason may be a higher specific kinase activity and possibly broader substrate range of the p190BCR�L1 fusion protein (78). In transgenic animals, p190BCR�L1-induced exclusively B-lymphoid leukemia with a short latency, while p210BCR�L1 led to development of both lymphoid and myeloid leukemias with a longer latency (79). When introduced into stem cell and progenitor enriched mouse bone marrow, both p210BCR�L1 and p190BCR�L1 cause a myeloproliferative disease with expansion of granulocytic, myelomonocytic, and lymphoid compartments however, the disease induced by p190BCR�L1 has a significantly shorter latency (7). p190BCR�L1 induces stronger STAT1 and STAT5 phosphorylation in Baf3 cells than p210BCR�L1 (48), and also induces phosphorylation of STAT6 (50).

    Co-Occurring Genetic Abnormalities

    Next generation sequencing studies have revealed that many leukemia genomes are remarkably stable – particularly when compared to epithelial cancers. Nevertheless, Ph + ALL cells have been shown to carry several recurrent mutations that commonly co-occur with BCRABL1 fusions and contribute to leukemogenesis. The most frequent co-occurring genetic abnormalities are deletions of the lymphoid-specific transcriptional regulators IKAROS (IKZF1), PAX5 (paired box 5), and EBF1 (early B-cell factor 1). Deletions involving CDKN2A/B are also common. In addition, one of the first examples of 𠇌onvergent clonal evolution” within the same leukemia was described in Ph + ALL: one patient’s leukemia contained two cytogenetically distinct subclones that independently acquired a duplication of 8q, corroborating the crucial role of co-occurring mutations (80). Interestingly, GWAS studies have identified genetic polymorphisms of IKZF1 (81�), PAX5 (88), and CDKN2A/B (81, 89�) as susceptibility loci that mediate a genetic predisposition to childhood ALL. However, subgroup analysis, when performed, revealed no specific association with Ph + ALL. This may in part be due to a low number of Ph + ALL patients in these studies, and a targeted evaluation of IKZF1, PAX5, and CDKN2A/B susceptibility alleles specifically in Ph + ALL patients may be warranted.

    IKZF1 Deletions and Point Mutations in Ph + ALL

    A review of BCRABL1 in ALL requires discussion of its most frequent partner in crime, IKZF1 (86). BCRABL1 and IKZF1 mutations are strongly linked: about 70�% of Ph + ALLs have somatic mutations in IKZF1 (about 90% deletions and 10% point mutations), which is much higher than the rate of IKZF1 mutations in Ph − ALL (92�). There are three functional types of IKZF1 mutations: haploinsufficiency or near haploinsufficiency (due to monoallelic null mutations such as inactivating point mutations, premature stop codons, and deletions, 55%), complete absence of Ikaros due to bi-allelic deletions (12%) (92, 93, 95), and alterations that create a dominant-negative (DN) form of Ikaros, IK6 (33% of all IKZF1 mutations). The IK6 Ikaros mutant is produced by an in-frame deletion of exons 4𠄷, which deletes the DNA-binding domain and leads to cytosolic accumulation of the mutant protein (92, 96, 97). The resulting hematopoietic phenotype in a mouse model mimicking this mutation (a smaller deletion that phenocopies the loss of the DNA-binding and nuclear export) is more severe that monoallelic null mutations, as Ik6 associates with the wild type Ikaros and probably traps it in the cytoplasm together with other complex members such as Helios, Aiolos, and Eos (98). The mutations associated with a more profound reduction in Ikaros function (bi-allelic deletion and Ik6) are particularly common in Ph + ALL (92, 93, 95�). This underscores the remarkably tight link between Ikaros and Ph + ALL. In the closely related “Ph-like” ALL subset characterized by a gene-expression profile highly similar to that of Ph + ALL, but without BCRABL1 fusion, IKZF1 mutations are also common. However, the majority of mutations result in a less severe reduction of Ikaros function [i.e., haploinsufficiency in 55�% of all IKZF1 mutations in non-BCRABL1 ALL (95, 99)]. Twin studies and tracking of subclonal populations suggests that BCRABL1 fusion is the first hit, and IKZF1 mutations occur later during leukemogenesis (95, 100, 101). There are also reported cases of 𠇌onvergent” evolution of IKZF1 mutations, with different subclones within the same patient carrying different IKZF1 mutations, underscoring the importance of this locus for Ph + ALL (72, 80, 101). Much work has been dedicated to understanding the molecular mechanism of loss of Ikaros alone and in the context of Ph + ALL. Complete loss or expression of DN Ikaros in normal hematopoiesis causes a mild (Ikzf1 −/− ) to severe (DN) reduction in the number of hematopoietic stem cells (HSC), complete loss of the B-cell and dendritic cell compartments, a skewing toward the T-lymphoid lineage (98, 102�), and ultimately T-cell malignancies in mice (109). Despite this, IKZF1 mutations are much more common in B-lymphoid than in T-lymphoid malignancies (110). On the surface, the combination of BCRABL1 fusion and loss of IKZF1 neatly fits the paradigm proposed by Gilliand for acute myeloid leukemia (AML), which hypothesized that leukemia development requires a combination of class I (signal transduction pathway mutation leading to uncontrolled growth, such as FLT3, or RAS), and class II mutations (aberrant transcription factors resulting in differentiation block, such as PML–RAR, AML𠄾TO, MLL-translocations, or point mutations in C/EBPα) (111). According to this model, BCRABL1 is the class I mutation and IKZF1, the class II mutation. A possible reason for the frequent occurrence of at least the Ik6 mutation in B-cell precursor ALL could lie in the fact that the exons 4 and 7 are flanked by genomic regions that can function as off-target sites for recombination activated gene (RAG) proteins, which mediate VDJ recombination in this cell population (93, 112). Whether the particularly common co-occurrence of BCR�L1 and Ik6 is solely a function of the developmental stage of the cell of origin, or whether the presence of the BCRABL1 translocation predisposes to aberrant RAG activity is not known. In addition, if and how BCR�L1 and mutant Ikaros cooperate on a molecular level is still not fully understood. The normal function of Ikaros suggests that one of its main contributions to B-cell leukemogenesis is a differentiation block in the B-lymphoid lineage at the pro- to pre-B-cell transition. While complete loss of Ikaros results in a complete absence of the entire B-cell compartment, a severely reduced expression of Ikaros allows development up to the Pro-B-cell stage but not beyond (113). However, additional mechanisms likely play a role. Ikaros has been shown to downregulate Myc, thus loss of Ikaros may result in increased Myc activity and increased proliferation (114). Gene-expression profiling suggests that IKAROS mutated B-ALL has a more prominent “stem-cell signature” (99, 115), and a larger leukemia initiating cell pool [LIC, defined by CD34 expression rather than functionally (96)], suggesting that some of the functions of Ikaros in silencing stem-cell programs in HSCs may play a role (105, 116). Finally, it has been suggested that loss of Ikaros may either synergize with or enhance Jak–Stat signaling. This hypothesis is mostly based on the circumstantial evidence that the other main subtype of ALL with frequent IKZF1 mutations are the Ph-like leukemias. Ph-like leukemias share a transcriptional profile with Ph + ALL and frequently carry mutations that, like BCR�L1, activate Stat5 (75, 117). Modulation of this pathway could both provide a competitive advantage at a subclonal level of IKZF1 − clones, and provide an escape pathway for BCR�L1 inhibition. Loss of Ikaros predicts a poor prognosis even within Ph + ALL (94, 99).

    PAX5 Mutations in Ph + ALL

    Recurrent mutations of PAX5 occur in about one-third of B-ALL cases (99, 118, 119), and in up to 50% of Ph + ALL (92, 120, 121). PAX 5 is a transcription factor that is expressed specifically during B-cell development, and controls lineage identity and commitment (107, 122). Like loss of Ikaros, loss of Pax5 leads to a differentiation block at the pro- to pre-B-cell stage (122�). Loss of Pax5 also allows trans-differentiation of already lineage-committed pro-B cells into other lineages, confers a certain degree of self-renewal onto this population (125, 126), and can cause B-cell lymphomas (127). Unlike Ikaros, however, the physiologic expression of PAX5 is limited to B-cell precursor stages, and its loss is not associated with an adverse prognosis (99, 120, 121). It has been speculated that a lack of an effect of PAX5 on hematopoietic stem-cell transcriptional programs may be responsible for the different prognostic implications of PAX5 and IKZF1 mutations (120, 128). In addition to driving B-lymphoid development, Ikaros has been reported to repress hematopoietic stem-cell specific gene-expression programs during early lineage specification, a function not shared with the other two major regulators of B-cell development that are found mutated in Ph + ALL, PAX5, and EBF1 (Figure 4) (105, 116, 128).

    Figure 4. B-cell development and transcription factors mutated in Ph + ALL. Differentiation stage-dependent expression (blue bars) and function of the three major B-cell developmental regulators mutated in Ph + ALL, Ikaros, Pax5, and EBF1 (106, 107). Ikaros expression is detected early in hematopoietic development and appears to have a role in shutting down stem-cell programs and nudging cells toward lymphoid development. Ikaros expression is maintained through B-cell development. Complete loss of Ikaros in murine models leads to a differentiation block at the LMPP stage and complete absence of the entire B-cell lineage (red block). A severe reduction allows the development of B-cell progenitors, but maturation is blocked at the Pro-B stage (orange block). EBF1 is turned on in common lymphoid progenitors (CLPs) and controls lineage specification to the B-cell lineage. Loss of EBF1 in murine models leads to a differentiation block at the Pro-B stage (red block). Pax5 is turned on the latest and maintains lineage commitment. Loss of Pax5 causes a differentiation block at the Pro-B-cell stage. Neither Pax5 nor EBF1 appear to have a role in silencing hematopoietic stem-cell programs, which may explain why IKZF1 mutations are associated with a poor prognosis, while PAX5 and EBF1 mutations do not predict adverse outcomes. HSC: hematopoietic stem cells LMPP: lymphoid-primed multipotent progenitors CLP: common lymphoid progenitors.

    EBF1 Mutations in Ph + ALL

    Early B-cell factor 1 is the predominant transcription factor mediating B-cell lineage commitment (107). It has been shown to co-regulate target genes with PAX5. In mouse models, complete loss of Ebf1 leads to a differentiation block at the pre–pro-B-cell stage (129). In contrast to Ikzf1 and Pax5, Ebf1 −/− mice do not develop spontaneous hematologic malignancies (129). However, combining loss of one allele of Ebf1 or Pax5 with a constitutively active Stat5 allele (the downstream effector in both Ph + and Ph-like ALL) results in B-cell precursor leukemia in all animals (130). EBF1 mutations occur in about 14% of Ph + ALL.

    CDKN2A/B in Ph + ALL

    The CDKN2A/B locus is frequently altered in ALL. The products of the CDKN2A and CDKN2B genes, p16 INK4A and p15 INK4B , are inhibitors of cyclin-dependent kinases. In addition, transcription of an alternate reading frame of the CDKN2A locus produces p14 ARF , which antagonizes the p53 ubiquitin ligase, HDM2. Silencing of the CDKN2A/B locus in HSC has been implicated in HSC self-renewal (131�). The distribution of CDKN2A/B alterations within cytogenetic subgroups is non-random. CDKN2A/B is rarely deleted in ALL with translocations of E2A [E2A–PBX1 in t(119)(q23p13) and E2AHLF in t(1719)(q21�p13) ALL] (135). In contrast, increased rates of CDKN2A/B deletions are found in Ph + ALL with a reported frequency of 繐% (80, 92, 136) compared to around 30% in non-Ph + B-ALL (137, 138). CDKN2A/B deletions are rare in CML in chronic phase but frequently associated with the transformation to lymphoid blast crisis, suggesting a specific role in B-lymphoid leukemia (139). Similar to IKZF1, the mechanism of deletion of p16 in lymphoid malignancies may involve RAG-mediated recombination (140). Experimental overexpression of BCR�L1 induces expression of Arf, which, if unopposed, leads to apoptosis (141). Introduction of p190BCR�L1 into Arf -null murine bone marrow decreases the latency and increases resistance to imatinib in the lymphoid malignancy that develops in recipient mice. In most clinical studies that have assessed the prognostic significance of CDKN2A/B loss of function in ALL, CDKN2A/B deletion or hypermethylation do not appear to be associated with changes in outcome for pediatric ALL, while silencing or inactivation of the locus predicts a worse outcome in adults (99, 136, 138, 142�). In a recent study mapping clonal evolution in adult patient-derived Ph + ALL cells grown in immunodeficient mice, the loss of CDKN2A/B was associated with increased competitive advantage on a subclonal level, more aggressive growth in xenografts, a higher leukemia initiating frequency, and a trend toward inferior outcome in patients (80). In both children and adults, deletions as well as epigenetic silencing through promoter hypermethylation are found at increased frequencies in relapsed specimens as opposed to those from initial diagnosis, suggesting a role in mediating relapse and resistance to therapy (147�).

    Epigenetic Abnormalities in Ph + ALL

    In addition to genetic abnormalities, Ph + ALL has a characteristic DNA methylation profile. Ph + ALL can be distinguished from other subtypes of ALL by hierarchical clustering of DNA methylation profiles. A recent study that quantified differentially methylated regions (DMRs) in all major ALL subtypes (compared to B-cell precursors, i.e., using a developmentally matched control) revealed about 350 DMRs in Ph + ALL samples (151). This was remarkably different from only about 50 DMRs identified in CRLF2-rearranged ALL samples, many of which were “Ph-like” ALL samples that share a major transcriptional program with Ph + ALL but are negative for the BCRABL1 translocation, and instead commonly have activating Jak1/2 mutations. Whether differential methylation is a consequence of the BCR�L1 fusions or co-occurring genetic abnormalities, and whether it plays a role in malignant transformation, resistance or relapse is unknown. However, an active role for DNA methylation in malignant transformation (rather than a mere reflection of the transcriptional landscape) is supported by the dependence of several experimental tumor models on functional DNA methyltransferase Dnmt1 (152, 153), including MLL� and Myc�l2-driven leukemia (154, 155). Reactivation of silenced CDKN2A/B by demethylating agents may have therapeutic benefit in a subgroup of ALL patients where this locus is intact (148), and demethylating agents are currently in clinical trials for relapsed and refractory ALL. Very little is known about a potential involvement of other epigenetic mechanisms in the biology of Ph + ALL, such as covalent modifications of histones or nucleosome positioning. A better understanding of Ph + ALL biology, including associated genetic and epigenetic abnormalities, should facilitate the development of rational synergistic combinations of targeted agents with TKIs.

    Mechanisms of Resistance in Ph + ALL – BCR�L1 Mutations

    One of the primary mechanisms of resistance and treatment failure in CML is the acquisition of BCRABL1 mutations that render the fusion protein completely or relatively unresponsive to TKIs. A plethora of different mutations mediating imatinib-resistance have been described. Most of these mutant BCR�L1 proteins are still sensitive to the second generation ABL kinase inhibitors dasatinib and nilotinib. In addition, the recently approved ponatinib is active against the most common mutation that causes resistance to both first and second generation ABL TKIs, the “gatekeeper” T315I mutation (Table 2 Figure 5). Kinase domain mutations develop even more frequently in Ph + ALL treated with TKI monotherapy despite initial sensitivity (156�) (161, 163, 164). A rate of BCR�L1 kinase domain (TKD) mutations of 㺀% has been reported in (adult) patients with Ph + ALL at relapse (165�), with the most common mutations being T315I, Y253H, and E255K/V (42, 168). There has been considerable debate in the field whether these mutations occur during treatment, or whether TKIs select for pre-existing resistant subclones. Several studies suggest that a substantial percentage of patients harbor subclones with TKD mutations prior to the initiation of therapy (42, 169�). BCRABL1 mutations known to cause resistance have been identified in minor subclones in as many as 40% of Ph + patients at initial diagnosis (169). Tyrosine kinase domain mutations may be less common in patients treated with a combination of intensive chemotherapy and TKI. In addition, the kinetics of emergence of resistant mutants – when they do develop – is not well-studied. Sequencing of 10 evaluable pediatric patients treated with imatinib and highly intensive chemotherapy on AALL0031 revealed two known resistance causing BCR�L1 mutations at relapse, none of which were detected in the initial diagnostic sample (38). Both mutations are responsive to nilotinib or dasatinib (M244V and H396P) (53). In contrast, in the GIMEMA LAL1205 study, the T315I mutation was discovered in four out of seven patients who relapsed after induction with dasatinib + steroids only, followed by intensive consolidation (intensive chemotherapy + TKI + auto HSCT, or allo HSCT) (172). It is possible that the combination of TKIs with an up-front intensive chemotherapy backbone serves to reduce selective pressure on TKI-resistant subclones. However, more in depth analysis will be required to define the respective roles of resistance to TKI and standard chemotherapy, and their interdependence. The fact that BCR�L1 has been found mutated at relapse also raises the important question how aggressively patients with Ph + ALL who receive TKIs on a backbone of standard chemotherapy should be screened for the emergence of TKD mutations. Since patients may still respond to the chemotherapy portion, early warning signs of TKI failure may be missing. If resistant mutants develop or emerge with similar kinetics to what is observed with monotherapy, patients may receive months or years of ineffective TKIs only to ultimately relapse, when early detection of an emergent mutant clone could have prompted switching to another agent active against the mutant BCR�L1. A major technical difficulty of such studies is the limit of detection to reliably assess and follow clonal heterogeneity in a minimal residual disease (MRD) setting. The decreased cost of sequencing and novel techniques such as MRD-sort combined with high throughput single cell sequencing may be able to provide answers in the near future. However, the complexity and cost of such an approach would first require a more in depth study of whether BCR�L1 mutations are a substantial contributor to resistance and relapse in Ph + ALL treated with intensive chemotherapy plus TKI.

    Table 2. Activity (IC50) of imatinib, dasatinib, nilotinib, and ponatinib against selected BCR�L1 mutants.


    Conclusion

    Though this be madness, yet there is method in it.(Shakespeare W., Hamlet. Act 2, scene 2.)

    There are 2 ways to conclude this review after going through the vast amount of data presented. Surely one could argue that despite all these data, there is still no clear picture emerging and each piece of additional information adds only more confusion. Alternatively, what might help us against capitulation in the face of complexity is to try to simplify without oversimplification.

    Can we build a model of CML that incorporates all the scientific data available but still retains clarity? In other words, could we explain how Bcr-Abl works in a few sentences to somebody who has never heard of it? Perhaps the most promising approach might be to try to link the biologic behavior of a CML cell to the underlying molecular events (Figure 5). Crucially, we should be able to picture this scenario relying on BCR-ABL alone because, at least until now, there is no unequivocal evidence that additional genetic lesions are present during chronic phase. We do not know how long it takes to move from the initial genetic event to fully established chronic-phase CML, but there is good reason to believe that the proliferative advantage of CML over normal cells is limited. Together with the largely normal differentiation capacity and function of CML blood cells, one feels that Ph-positive hematopoiesis cannot be so much different from normal hematopoiesis until the disease accelerates. Thus, Bcr-Abl is likely to hijack pathways that normally increase blood cell output in response to physiologic stimuli rather than to interrupt or replace them with pathways that are not normally used in hematopoietic cells. Indeed, there is plenty of experimental evidence to support this notion. Importantly, Bcr-Abl is capable of activating survival pathways along with proliferative stimuli without the need for a second cooperating genetic lesion in this way, the apoptotic response that would otherwise follow an isolated proliferative stimulus is avoided. The sustained dependence on growth factors is an indication that Bcr-Abl is not a complete substitute rather, it tips the balance to provide a limited growth advantage in vivo. This growth advantage is also dependent on specific survival conditions: transient regeneration of Ph-negative hematopoiesis is often observed after autografting, even when the autograft seems to be comprised exclusively of Ph-positive stem cells, and long-term cultures initiated from patients with chronic-phase CML become dominated by BCR-ABL–negative cells after some time.177 Thus, there appears to be a specific interaction (or noninteraction) of CML progenitor cells with their microenvironment that is crucial to maintain their proliferative advantage. Whether this interaction is stimulatory for CML over normal progenitor cells or inhibitory for normal over CML progenitor cells remains to be seen. Similarly, we can look at extramedullary hematopoiesis as a loss of function (ie, loss of the capacity to respond to negative signals) or a gain of function (ie, acquisition of a capacity to respond to positive signals that are not provided in the bone marrow) phenomenon. Much of the evidence implicates integrins in mediating these abnormal interactions, but other proteins may also play a role. Overall, it appears that the organization of cell membrane and cytoskeleton is more profoundly perturbed in CML progenitor cells than might be anticipated from the largely normal function of their progeny. Furthermore, Bcr-Abl may interfere with the “wiring” between integrin receptors on the cell surface and the nucleus and so disturb the communication of the cell with its environment. Another mechanism may also be important: Bcr-Abl appears to induce the degradation of certain inhibitory proteins. This might thwart cellular counter-reactions that would otherwise be activated, rather like cutting the telephone cable before the police can be called in.

    Many questions remain unanswered. Why is there a predominantly myeloid expansion when all 3 lineages carry the translocation? What is the biologic basis for the extraordinary variability in the clinical course of a disease that appears to carry just a single genetic lesion? What is the molecular basis for the genomic instability that we see clinically as relentless progression to blast crisis?

    Where do we go from here? The more we learn about the pathogenesis of CML, the more we realize its extraordinary complexity. Perhaps one should not be too surprised because it has become clear that cellular processes tend to rely on integrated networks rather than on straight unidirectional pathways. Only in this way can the cell achieve the flexibility required to respond to the various stimuli within a multicellular organism. Clearly, some components must be more important, and some less so, in the transformation network operated by Bcr-Abl. Absolutely essential features may be restricted to functional domains and to certain residues of the Bcr-Abl protein itself, and downstream effectors may be able to substitute for each other, at least to some extent. In this respect, the use of knockout mice that lack specific downstream molecules will allow one to define their precise relevance for Bcr-Abl–mediated cellular transformation. It may turn out that the combined elimination of several components abrogates transformation by Bcr-Abl, whereas each component individually is of limited significance. Chronic phase CML operates very much by exploiting physiologic pathways, perhaps by gently “coaxing” hematopoiesis toward the classical CML phenotype nevertheless it prepares the ground for blast crisis. Thus, to understand CML, we must study its chronic phase. We must move away from artificial systems, such as transduced fibroblasts, and take on the demanding task of studying signal transduction in primary progenitor cells.

    Supported by grants from Leukaemia Research Fund (UK) and the Dr Ernst und Anita Bauer Stiftung (Germany).


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