Viruses

Human T cell lymphotropic virus type 1 (HTLV1) is an enveloped, single-strand RNA virus that is associated with the development of a distinct form of adult T cell leukemia found in southwestern Japan, the Caribbean basin, and Africa. The virus can be spread vertically from mother to fetus or horizontally by sexual contact or blood products. Although endemic in the areas noted above, the virus only causes leukemia in 2% to 4% of those infected, and has a very long latency period, estimated at 30 years or more.⁶ Epstein-Barr virus, a DNA herpes family virus, is associated with the endemic African form of Burkitt lymphoma/leukemia.

Radiation

Ionizing radiation is leukemogenic. An increased incidence of AML, ALL and chronic myelogenous leukemia (CML) was seen in survivors of the atomic bombings of Hiroshima and Nagasaki, which began approximately 1.5 years after exposure, peaked at 7 years, and returned to baseline 25 years later. The risk of leukemia was also increased in individuals treated with radiation for ankylosing spondylitis in the 1940s. Although most of the data from humans showing the leukemogenic potential of ionizing radiation come from individuals exposed to high dose rates, occupational exposure to low dose-rate radiation has more recently been associated with the development of leukemia.⁷

Environmental Carcinogens

Extensive exposure to benzene or benzene-containing solvents can lead to marrow damage culminating in the development of aplastic anemia, myelodysplasia, or acute leukemia. Most studies examining the issue have found a small but consistent increase in AML among cigarette smokers.

Prior Therapy

Although the exact percentage is uncertain, as much as 10% of AMLs and a smaller percentage of ALLs are thought to be the consequence of prior exposure to chemotherapy or radiation for a primary malignancy or autoimmune disorder.⁸ Treatment-related leukemias can be grouped into several syndromes. AML developing after alkylating agent therapy has a latency of 5 to 7 years, often first appears as a myelodysplastic syndrome (MDS), and is frequently associated with abnormalities involving the long arms of chromosomes 5 or 7. All alkylating agents are likely leukemogenic, with an increased incidence of leukemia observed after prolonged exposure, following the use of dose-intense regimens, or when used in certain combinations. Treatment-related AML is also seen following exposure to topoisomerase II inhibitors, such as etoposide, teniposide or anthracyclines. These leukemias develop relatively rapidly, often within 2 years of exposure, are not generally preceded by a myelodysplastic phase, and frequently have rearrangements involving 11q23, the locus for MLL (the mixed-lineage leukemia gene) or 21q22. A number of other drugs have also been implicated in the subsequent development of acute leukemia, including bimolane, an agent used to treat psoriasis, and recently, lenalidomide, an agent used in the treatment of myeloma or myelodysplasia.⁹ Patients with lymphoma who receive autologous hematopoietic cell transplants are at increased risk for the development of leukemia, which may, in part, be predicted by the therapy received before the transplant.

Not all treatment-related leukemias fall into the discrete categories described here. Secondary leukemias with inv(16), t(9;22), and abnormalities involving 3q21 have been reported. Although there remains some debate, in general, patients with treatment-related leukemias do not fare as well as patients with de novo leukemia, even after adjusting for known risk factors.¹⁰

Antecedent Hematologic Malignancies

A number of hematologic malignancies can eventually evolve into a disease indistinguishable from acute myeloid leukemia. This is a common occurrence for the myelodysplastic syndromes, and sometimes occurs with polycythemia vera, idiopathic myelofibrosis, and rarely with essential thrombocythemia. Antecedent hematologic malignancies are present in 3% to 4% of cases of AML in patients younger than age 60, and in as many as 15% of cases in patients older than age 60. The term “secondary AML” is sometimes used to describe both therapy-related AMLs and those arising from an antecedent hematologic malignancy. There are certain common features, including tri-lineage dysplasia and a high incidence of unfavorable cytogenetic changes. The current WHO classification schema distinguishes between the two, with separate definitions for therapy-related myeloid neoplasms and AML with myelodysplasia-related changes.

The Leukemic Stem Cell

Within the clonal populations of cells in patients with acute leukemia, there must be a subpopulation of cells capable both of self-renewal and proliferation. Such cells have been termed “leukemia stem cells.” Although there is no gold standard for their identification, the ability of human leukemic cells to engraft long-term in immunodeficient NOD/SCID mice is generally accepted as a marker for a leukemia stem cell. In human AML, the subpopulation of cells capable of long-term engraftment in immunodeficient mice is within the CD34++CD38– CD 123+ fraction and comprises only a small percent (0.2 to 100/10⁶) of the leukemic cells present.¹³ However, many cases of AML do not engraft even in the more permissive NOD/SCID/IL2R immunodeficient mouse, including almost all cases of acute promyelocytic leukemia (APL), and thus the ability of leukemic cells to grow in an immunodeficient mouse is not a true assay for the leukemia stem cell.

Marrow Failure

The persistent growth of the leukemic clone leads to the failure of the marrow to produce normal red cells, white cells, and platelets, but the mechanisms by which this happens are not well understood. Although marrow failure may result at least in part by leukemia cells physically crowding out normal progenitors, frequently peripheral blood counts start to fall weeks or months before the appearance of leukemic blasts in the marrow, and cases of hypoplastic acute leukemia are not uncommon.

Morphology

AML cells are typically 12 to 20 µm in diameter with large nuclei, multiple nucleoli, and azurophilic granules in the cytoplasm. ALL cells are typically smaller than AML blasts and are devoid of granules (Figure 98-2). For more than 3 decades, the French-American-British (FAB) system was used to describe and classify morphologic subtypes of AML and ALL. Morphologic subclassification of acute leukemia adds little prognostic or therapeutic information beyond that provided by cytogenetic and mutational categorization, but still remains part of the overall World Health Organization (WHO) classification schema.

Immunophenotyping

Acute leukemias can be categorized according to the cell surface antigens they express as determined by their reactivity to a panel of monoclonal antibodies (Table 98-1). The cell surface antigens expressed largely reflect the level of differentiation of the leukemic cell, and this information provides some prognostic information, especially in ALL. In addition, the antigens on leukemia blasts are almost always present in abnormal combinations or concentrations, allowing for the detection of as few as 0.01% abnormal cells in an otherwise normal looking bone marrow using multidimensional flow cytometry.

Most cases of AML express antigens found on normal immature myeloid cells. CD34, CD117, and HLA-DR are expressed on the surface of the more immature forms of AML. CD13 and CD33 are pan-myeloid markers and are present on the surface of the majority of cases of AML. CD14, CD15, and CD11b are more associated with monocytic differentiation. Erythroid leukemias express CD36, CD71, and the blood group H antigen, whereas megakaryocytic leukemias express CD41a and CD61. In 10% to 20% of AML cases, the blasts express antigens usually restricted to lymphoid cells, including CD4, CD7, or CD19. Expression of lymphoid antigens in AML does not appear to influence the clinical behavior or prognosis of the disease. The European Group for the Immunologic Classification of Leukemias has designated five markers as classical myeloid: myeloperoxidase, CD13, CD33, CD65, and CD117. AML cases expressing all five (a so-called pan-myeloid phenotype) have a better clinical outcome than cases that express only a subset of these.

Adult acute lymphocytic leukemia can be divided into B-cell ALL (about 75% overall) and T-cell ALL (about 25%). Approximately 10% of all ALLs express the early B-cell antigens CD19 or CD22 without any more mature markers, and are defined as pro-B-cell ALL. In about 50% of cases, the ALL blasts express CD10 (the common ALL antigen or CALLA). In about 10% of cases, the B-cell ALLs express cytoplasmic immunoglobulin but not surface immunoglobulin (pre-B cell ALL), whereas in 5% of cases surface immunoglobulin is also present (mature B-cell or Burkitt leukemia). T-cell ALL can be categorized as early T-precursor ALL, expressing CD7 but not CD1a or CD3, thymic (or cortical) T-ALL, expressingCD1a, but not cell-surface CD3, and mature T-ALL, expressing surface CD3. Thymic T-ALL makes up about half of T-ALLs and has a more favorable prognosis than either early-precursor or mature T-ALL, which each comprises about 25% of T-ALLs.

A small percentage of cases of acute leukemia express characteristics of both lymphoid and myeloid cells, and have recently been defined by the WHO as mixed-phenotype acute leukemia (MPAL).¹⁴ To fulfill the WHO criteria, the leukemia must express the myeloid-specific marker myeloperoxidase (either by flow cytometry or cytochemistry), and either a strictly specific T lymphoid marker (cytoplasmic CD3) or a B lymphoid marker (strong expression of CD19 or weak expression of CD19 together with expression of at least three other B-cell-associated markers). Many of these patients will have rearrangements of BCR/ABL or MLL or have complex cytogenetics. The median survival for this rare group of patients appears to be short, in the range of 18 months, and they should be considered as a high-risk group when selecting appropriate therapy.

Cytogenetics

Cytogenetic analysis of human leukemia has been central to the identification of the genetic events involved in leukemogenesis. In addition, cytogenetics remains the most important diagnostic factor in acute leukemia. Conventional cytogenetics involves the staining of metaphase cells and thus requires dividing cells. Because malignant cells in the marrow are more frequent and have a higher mitotic rate, marrow is preferred over peripheral blood as the source of cells for analysis. Typically 20 metaphases are examined. Fluorescent in situ hybridization (FISH) techniques involve hybridization of single-stranded DNA probes to homologous single-stranded sequences in chromosomes of metaphase or interphase cells. FISH has the advantage of being able to analyze large numbers of dividing (metaphase FISH) or nondividing (interphase FISH) cells with relatively little effort. Only those abnormalities targeted by the specific probe being applied will be detected. Thus FISH is very useful for monitoring the disappearance or reappearance of a known specific abnormality, but is not a substitute for conventional cytogenetics in the initial evaluation of acute leukemia. Polymerase chain reaction (PCR) is a method capable of amplifying selected regions of DNA through repeated cycles of DNA synthesis, denaturation, and hybridization. Standardized PCR analyses of several of the more common translocations have been developed and are being used for monitoring minimal residual disease.

For AML, the Southwest Oncology Group (SWOG) and the Medical Research Council (MRC) each developed and adopted cytogenetic risk classifications over two decades ago based on the outcomes of large prospective clinical trials.15,16 The resulting classifications, though similar, are not identical. For example, as shown in Table 98-2, in the SWOG categorization, patients with clonal abnormalities not recognized as within one of the three major groups are assigned to a fourth category with a predicted treatment outcome somewhere between the intermediate- and unfavorable-risk groups. And there are minor differences between classification schemas in the distinction between intermediate and unfavorable risk cytogenetics. More recently, in 2010, the MRC revised their classification system, with the major change being the movement of patients with various translocations involving chromosome 11 (with the exception of t(9;11) and t(11;19)) from the intermediate-risk group into the unfavorable group.¹⁷ Although these classification schemas differ slightly, they are each able to distinguish three major groups of patients. Among patients age <65 treated with standard chemotherapy, those with a favorable karyotype have complete response (CR) rates in the 85% to 90% range and a 5-year overall survival (OS) of 50% to 60%. Those with intermediate-risk cytogenetics have CR rates of 65% to 75%, and a 5-yr OS of 35% to 45%. And for those with unfavorable-risk cytogenetics, a CR rate of 45% to 55% and a 5-yr OS of 10% to 20% can be expected.

In some cases of AML, multiple unrelated cytogenetic abnormalities will be seen in a single karyotype. If there are a sufficient number of abnormalities, such cases are defined as having “complex” cytogenetics. If these multiple abnormalities accompany one of the favorable-risk translocations (inv 16 or t(8 : 21)), CR rates and overall survival are slightly poorer than seen with the favorable-risk translocations alone, but still better than would be expected with intermediate risk or unfavorable risk. Thus cases with inv(16) or t(8;21) and multiple abnormalities remain within the favorable-risk group and are not considered as “complex” in most discussions. All other cases with complex cytogenetics are defined as unfavorable, and make up between a third and half of all cases of unfavorable-risk disease, or 10% overall. In the recent MRC revision of their classification system, they found that those with four abnormalities did worse than those with three, and those with five or more did worse than those with four. In the SWOG analysis, they further found that those with complex cytogenetics but without involvement of either chromosomes 5 or 7 had a CR rate of 50% and an overall survival of 20% compared with those with complex cytogenetics and involvement of chromosomes 5 and/or 7 who had a CR rate of 37% and only 3% OS.

The finding of a single monosomy is a relatively common finding in AML, with monosomy 7 being the most frequent. The presence of a single monosomy (excluding loss of a sex chromosome) is associated with a negative outcome, with a 12% OS at 4 years in the Dutch experience.¹⁸ Patients with two or more distinct monosomies, or a single monosomy but with an additional structural abnormality, have an even worse prognosis, with a predicted 4-year survival of less than 4%.¹⁸ Thus this category of patient (two monosomies or one plus an additional structural abnormality) has been defined as having a “monosomal karyotype” and identifies a group of patients with a very dismal prognosis if treated with conventional chemotherapy. There is obvious overlap among patients with unfavorable, complex, and monosomal karyotypes. All patients with complex karyotypes fall within the unfavorable-risk group and comprise between a third and half of the total, and almost all patients with a monosomal karyotype are within the complex category, accounting for about two-thirds of those patients.

As in AML, cytogenetic analysis plays an important role in the evaluation of patients with ALL (Table 98-3). The most common abnormality in ALL, seen in 20% to 30% of cases, is t(9;22)(q34;q11), which results in the Philadelphia chromosome. Other common translocations in adult B-cell ALL include t(4;11)(q21;q23), which is seen in 7% of cases, involves the MLL and AF4 gene and is associated with a poor prognosis, and t(8;14)(q24.1;q32), which is seen in 2% to 4% of adult B-cell ALL, involves c-MYC and the immunoglobulin heavy chain, and is the translocation associated with Burkitt lymphoma. T-cell ALLs often have translocations involving chromosomes 7 or 14 at T-cell receptor enhancer gene sites. The other most common cytogenetic changes seen in adult ALL involve del(9p) seen in 5% to 9% of cases, del(6q) seen in 5% to 7% and del(13q) seen in 3% to 5%. Abnormalities in chromosome number are also common. High hyperdiploidy (51 to 65 chromosomes) is seen in as many as 10% of cases, whereas low hypodiploidy (30 to 39 chromosomes)/near triploidy (60 to 78 chromosomes) is seen in 4%. Approximately 5% will have a complex karyotype. Compared with those cases of ALL with normal cytogenetics, those with del(9q) or high hyperdiploidy appear to have a more favorable outcome, whereas those with t(4;11), t(8;14), and del(6q), low hypodiploidy/near triploidy, or complex cytogenetics do worse (Figure 98-3).¹⁹

Mutational Events

The identification of recurrent chromosomal abnormalities in acute leukemia, including translocations, point mutations, and gene duplications, followed by the cloning of the involved genes, has provided important insights into the pathogenesis of the disease. These initial studies have since been supplemented by a number of techniques, including whole genome sequencing, which have allowed for the identification of additional nonrandom mutations not recognizable by conventional cytogenetics. In AML, along with routine cytogenetic analysis, assays for the mutational status of CEBPA, NPM1, and FLT3 have entered standard clinical practice.

AML-Associated Mutations

Retinoic Acid Receptor Alpha Translocations

Acute promyelocytic leukemia (APL), which accounts for approximately 8% of adult AML cases, is almost always associated with t(15;17)(q22;q11.2), a translocation that fuses the promyelocytic leukemia gene (PML) on chromosome 15 to the retinoic acid receptor alpha (RARa) gene on chromosome 17. The resultant PML/RARa fusion protein acts as a dominant negative inhibitor of normal PML function and of the function of RXRa, an important heterodimeric partner of RARa. PML/RARa recruits a nuclear co-repressor (NCoR) and the molecules sin3 and histone deacetylase (HD) (Figure 98-4). HD deacetylases histones, a process that in turn inhibits binding of transcription factors, thus inhibiting the expression of genes required for hematopoietic differentiation. The unique activity of all-trans-retinoic acid (ATRA) in APL appears to be explained by its ability to bind to the PML/RARa fusion, changing its configuration and releasing the attached nuclear co-repressor. This then allows subsequent transcription and gene expression with subsequent maturation of the APL blasts. A number of other translocations, including t(5;17) and t(11;17), involve RARa and result in an APL-like phenotype. These leukemias are, however, generally unresponsive to ATRA because clinically achievable concentrations of the drug do not result in release of the NCoR-HD complex.

Core Binding Factor Translocations

Core binding factor is a heterodimeric transcription factor made up of two subunits, CBFα (also known as RUNX-1) and CBFβ. CBF plays a role in the transcriptional activation of a number of genes required for normal hematopoietic differentiation. The t(8;21) abnormality seen in approximately 8% of adult AML cases results in the fusion of the RUNX1 gene on chromosome 21 to the MTG8 gene on chromosome 8. The RUNX1/MTG8 fusion protein acts as a dominant negative inhibitor of the wild-type RUNX-1 gene (Figure 98-5). AML characterized by t(8;21) has a favorable prognosis. Similarly, inv(16)(p13;q22) and t(16;16)(p13;q22) both result in the fusion of CBFβ at 16q22 to the smooth-muscle myosin heavy chain (MYH11) gene at 16p13. As in the case of t(8;21), the resultant fusion protein acts as a dominant negative regulator of transcription. AML cases with involvement of 16q22 account for approximately 9% of adult AML cases, have a unique myelomonocytic morphology, and like t(8;21) leukemias have a favorable prognosis.

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