CHAPTER 10 CYTOGENETICS AND GENE REARRANGEMENT
CHAPTER 10 CYTOGENETICS AND GENE REARRANGEMENT
MICHELLE M. LE BEAU
Genetic Consequences of Chromosomal Rearrangements
Chronic Myelogenous Leukemia
Other Myeloproliferative Disorders
Acute Myeloid Leukemia De Novo
Acute Myeloid Leukemia and Myelodysplastic Syndromes Associated with Prior Cytotoxic Treatment (T-MDS and T-AML)
Malignant Lymphoproliferative Disorders
T Cell Acute Lymphoblastic Leukemia
Chronic Lymphocytic Leukemia
Cytogenetic analysis provides pathologists and clinicians with a powerful tool for the diagnosis and classification of hematologic malignant diseases. The detection of an acquired, somatic mutation establishes the diagnosis of a neoplastic disorder and rules out a reactive hyperplasia or morphological changes due to toxic injury or vitamin deficiency. Specific cytogenetic abnormalities identify homogeneous subsets of various malignant diseases and enable clinicians to predict their clinical course and their likelihood of responding to particular treatments. In many cases, the prognostic information derived from cytogenetic analysis is independent of that provided by other clinical features. Patients with favorable prognostic features benefit from standard therapies with well-known spectra of toxicities, whereas those with less favorable clinical or cytogenetic characteristics may be better treated with more intensive or investigational therapies. The disappearance of a chromosomal abnormality present at diagnosis is an important indicator of complete remission following treatment, and its reappearance invariably heralds relapse of the disease. Pretreatment cytogenetic analysis can be useful in choosing among postremission therapies that differ widely in cost, acute and chronic morbidity, and effectiveness.
Acronyms and abbreviations that appear in this chapter include: ALCL, anaplastic large cell lymphoma; ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; AML-M2, AML with maturation; AMMoL-M4Eo, acute myelomonocytic leukemia with abnormal eosinophils; APL, acute promyelocytic leukemia; BL, Burkitt lymphoma; CD30, cluster of differentiation; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; CMMoL, chronic myelomonocytic leukemia; CNS, central nervous system; CTCL, cutaneous T-cell lymphoma; DLCL, diffuse large cell lymphoma; FAB, French-American-British; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate; LPL, lymphoplasmacytoid lymphoma; MALT, mucosa-associated lymphoid tumor; MCL, mantle cell lymphoma; MDS, myelodysplastic syndrome; EFS, event-free survival; PML, promyelocytic leukemia; RA, refractory anemia; RAEB, RA with excess blasts; RAEB-T, RAEB in transformation; RARS, RA with ringed sideroblasts; RT-PCR, reverse transcriptase polymerase chain reaction; SIg, surface immunoglobulin; SLL, small lymphocytic lymphoma; WBC, white blood cell.
The malignant cells in many patients who have leukemia or lymphoma have acquired clonal chromosomal abnormalities. A number of specific cytogenetic abnormalities have been recognized that are very closely, and sometimes uniquely, associated with morphologically and clinically distinct subsets of leukemia or lymphoma.1,2,3 and 4 The detection of one of these recurring abnormalities can be helpful in establishing the correct diagnosis, influencing selection of therapy, and providing prognostic information. The appearance of new abnormalities in the karyotype of a patient under observation often signals a change in the pace of the disease, usually to a more aggressive disorder. Further detailed information regarding recurring chromosomal rearrangements is contained in reviews.1,2,3,4 and 5
GENETIC CONSEQUENCES OF CHROMOSOMAL REARRANGEMENTS
The genes that are located at the breakpoints of a number of the recurring chromosomal translocations have been identified (Table 10-1). Alterations in the expression of the genes or in the properties of the encoded proteins resulting from the rearrangement play an integral role in the process of malignant transformation.5,6 The altered genes fall into several functional classes, including those that encode for tyrosine or serine protein kinases, cell surface receptors, and growth factors (see Table 10-1). However, the largest class are those that encode transcriptional regulating factors; these proteins are involved in the initiation of gene transcription, often functioning in a tissue-specific fashion to regulate growth and differentiation.
TABLE 10-1 EXAMPLES OF TRANSFORMING GENES FOUND AT TRANSLOCATION JUNCTIONS: FUNCTIONAL CLASSIFICATION
There are two general mechanisms by which chromosomal translocations result in altered gene function. The first is deregulation of gene expression. This mechanism is characteristic of the translocations in lymphoid neoplasms that involve the immunoglobulin genes in B lineage tumors and the T cell–receptor genes in T lineage tumors. These rearrangements result in inappropriate expression of an oncogene with no alteration in its protein structure. The second mechanism is the expression of a novel fusion protein, resulting from the juxtaposition of coding sequences from two genes that are normally located on different chromosomes. Such chimeric proteins are “tumor-specific” in that the fusion gene does not exist in nonmalignant cells; thus, the detection of such a fusion gene or its protein product can be important in diagnosis, or in the detection of residual disease or early relapse. Moreover, they may also be appropriate targets for tumor-specific therapies. An example is the chimeric BCR/ABL protein resulting from the t(9;22) in chronic myelogenous leukemia. All of the translocations cloned to date in the myeloid leukemias result in a fusion protein.
Chromosomal translocations result in the activation of genes in a dominant fashion. A number of human tumors are believed to result from homozygous, recessive mutations.7 These mutations lead to the absence of a functional protein product, suggesting that these genes function as “suppressor” genes whose normal role is to limit cellular proliferation. The hallmark of tumor suppressor genes is the loss of genetic material in malignant cells, resulting from chromosomal loss or deletion, as well as by other genetic mechanisms.
Cytogenetic analysis of malignant diseases is based upon the study of the tumor cells themselves. In leukemia, the specimen is usually obtained by marrow aspiration and is either processed immediately (direct preparation) or cultured for 24–72 hours. When a marrow aspirate cannot be obtained, a marrow biopsy (bone core specimen) or a blood sample, for patients who have circulating immature myeloid or lymphoid cells, can often be studied successfully. An involved lymph node or tumor mass may be sampled. The use of amethopterin to synchronize dividing cells in culture, combined with brief exposures to mitotic inhibitors such as colchicine or to DNA binding agents (ethidium bromide) are used by some laboratories to obtain elongated chromosomes that have an increased number of bands.
For specimen collection, 1 to 5 ml of marrow are aspirated aseptically into a syringe coated with preservative-free sodium heparin (preservatives in heparin can suppress cell growth) and transferred to a sterile 15-ml centrifuge tube containing 5 ml of culture medium (RPMI 1640, 100 units sodium heparin). If a marrow aspirate cannot be obtained, a bone marrow biopsy may be taken and placed into the collection tube. For blood specimens, 10 ml are drawn aseptically by venipuncture into a syringe coated with preservative-free heparin. Specimens should be maintained at room temperature and transported in culture medium. To avoid loss of cell viability, it is critical that the specimen be transported to the cytogenetics laboratory without delay. Overnight shipment of specimens frequently results in loss of cell viability, and most laboratories experience a high proportion (25–50%) of inadequate analyses using such specimens. For specimens handled optimally, 95 to 98 percent of all cases should be adequate for cytogenetic analysis. Those cases that are inadequate generally represent samples from patients with hypocellular bone marrows. Overall, approximately 75 percent of bone core biopsies will yield adequate numbers of metaphase cells for complete analysis.
To prepare metaphase cells, the sample is exposed sequentially to mitotic inhibitors to accumulate cells in mitosis, hypotonic KCl (0.075 M) to swell the cells, and fixative (absolute methanol:glacial acetic acid, 3:1). Slides are prepared by dropping the cell suspension onto precleaned glass microscope slides, and the slides are air dried. The most popular chromosomal banding techniques is trypsin-Giemsa banding. Using this technique, a consistent chromosome banding pattern is induced by exposing cells to a dilute trypsin solution (0.1–0.25 percent), followed by staining in phosphate-buffered Giemsa stain.
Cytogenetic analysis of human tumors is often technically difficult, due to the presence of multiple abnormalities and multiple cell lines, and requires highly skilled personnel. These factors have led investigators to seek alternative methods for identifying chromosomal abnormalities, such as Southern blot analysis of DNA, RT-PCR analysis of RNA from tumor cells, or FISH.8
The technique of FISH is based upon the same principle as Southern blot analysis, namely, the ability of single-stranded DNA to anneal to complementary DNA. In the case of FISH, the target DNA is the nuclear DNA of interphase cells, or the DNA of metaphase chromosomes that are affixed to a glass microscope slide (FISH can also be accomplished with bone marrow or blood films or with fixed and sectioned tissue). The test probe is labeled with biotin- or digoxigenin-labeled nucleotides and detected with fluorescein isothiocyanate (FITC)- or CY3-conjugated avidin or with rhodamine-labeled anti-digoxigenin antibodies. Probes that are directly labeled with fluorochrome are also available for hybridization, thereby simplifying the technique by eliminating the probe detection steps. With the development of dual- and triple-pass filters, most laboratories now have the capacity to hybridize and detect 2 to 3 probes simultaneously.
Several types of probes can be used to detect chromosomal abnormalities by FISH. Hybridization of centromere-specific probes has been used to detect monosomy, trisomy, and other aneuploidies in both leukemias and solid tumors (Plate XXIV-A). Chromosome-specific libraries, which paint the chromosomes, are particularly useful in identifying marker chromosomes (rearranged chromosomes of unidentified origin), or structural rearrangements such as translocations (Plate XXIV-B). Translocations and deletions can also be identified in interphase or metaphase cells by using genomic probes that are derived from the breakpoints of recurring translocations or within the deleted segment (Plate XXIV-C). The newest innovation in FISH technology is spectral karyotyping, or multiplex FISH.9 Using this approach 24 differentially labeled painting probes representing each chromosome are cohybridized; Fourier spectroscopy is used to distinguish each spectrally overlapping probe and imaging software assigns a unique color to each chromosome (Plate XXIV-D). Often referred to as “color karyotyping,” this method is applicable to the identification of numerical abnormalities as well as many structural abnormalities.
FISH techniques have a number of applications (Table 10-2). In some cases, FISH analysis provides more sensitivity, in that cytogenetic abnormalities have been identified by FISH in samples that appeared to be normal by morphological and conventional cytogenetic analyses. FISH is most powerful when the analysis is targeted towards those abnormalities that are known to be associated with a particular tumor or disease. An example of how FISH could be used in a clinical setting is as follows: Cytogenetic analysis could be performed at the time of diagnosis to identify the chromosomal abnormalities in an individual patient’s malignant cells. Thereafter, FISH with the appropriate probes could be used to detect residual disease or early relapse and to assess the efficacy of therapeutic regimens. For example, the use of FISH to detect the t(9;22) in CML patients following transplantation or interferon therapy and for the sex chromosome composition of cells in the recipient after a sex-mismatched transplant has become frequent.
TABLE 10-2 APPLICATIONS AND ADVANTAGES OF FISH
Chromosomal abnormalities are described according to the International System for Human Cytogenetic Nomenclature (Table 10-3).10 To describe the chromosomal complement, the total chromosome number is listed first, followed by the sex chromosomes, then by numerical and structural abnormalities in ascending order. The observation of at least two cells with the same structural rearrangement, e.g., a translocation, deletion, inversion, or gain of the same chromosome, or of three cells showing loss of the same chromosome, is considered evidence for the presence of an abnormal clone. However, one cell with a normal karyotype is considered evidence for the presence of a normal cell line. Patients whose cells show no alteration or nonclonal (single cell) abnormalities are considered to be normal. An exception to this is a single cell characterized by a recurring structural abnormality. In such instances it is likely that this represents the karyotype of the malignant cells in that particular patient.
TABLE 10-3 GLOSSARY OF CYTOGENETIC TERMINOLOGY
CHRONIC MYELOGENOUS LEUKEMIA
The first consistent chromosome abnormality in any malignant disease was identified in CML. The Philadelphia, or Ph chromosome, was initially thought to be a deletion of chromosome 22 but was later shown to be a translocation involving chromosomes 9 and 22 [t(9;22)(q34;q11.2)], Fig. 10-1). The t(9;22) occurs in a pluripotent stem cell that gives rise to both lymphoid and myeloid lineage cells. The standard t(9;22) is identified in about 92 percent of CML patients; another 6 percent have variant translocations that involve a third chromosome in addition to numbers 9 and 22. The genetic consequences of the t(9;22) or the complex translocations is to move a portion of the Abelson (ABL) proto-oncogene on chromosome 9 adjacent to a portion of the BCR gene on 22. Analyses of leukemia cells from rare patients with typical CML who lack the t(9;22) has revealed a rearrangement involving ABL and BCR that is detectable only at the molecular level (1–2% of cases).12
FIGURE 10-1 Partial karyotypes from trypsin-Giemsa-banded metaphase cells depicting recurring chromosomal rearrangements observed in myeloid leukemias. (a) t(9;22)(q34;q11), CML. (b) t(8;21)(q22;q22), AML-M2. (c) inv(16)(pl3q22), AMMoL-M4Eo. (d) t(15;17)(q22;q11–12), APL. (e) t(9;11) (p22;q23), AMoL-M5. (f) del(5)(ql3q33), t-AML. The rearranged chromosomes are identified with arrows.
Marrow cells from rare “CML” patients lack both a Ph chromosome and the BCR/ABL fusion but often have a normal karyotype or trisomy 8.13 These patients have a substantially shorter survival than do those whose cells have the t(9;22). Most of these patients have a myelodysplastic syndrome (MDS), most commonly chronic myelomonocytic leukemia or refractory anemia with excess blasts, or the poorly understood disorder of “atypical CML.” Thus, the t(9;22) and resultant BCR/ABL fusion is the sine qua non of CML.13
The BCR/ABL protein is located on the cytoplasmic surface of the cell membrane and acquires a novel function in transmitting growth-regulatory signals from cell surface receptors to the nucleus via the RAS signal transduction pathway.13,14 The BCR/ABL fusion gene can be detected with standard Southern blot analysis of DNA or with RT-PCR analysis of mRNA for diagnosis and detection of residual disease. FISH can also be used to detect the Ph chromosome in both metaphase and interphase cells (Plate XXIV-C).
As they enter the terminal acute phase, most CML patients (80%) show karyotypic evolution, with the appearance of new chromosomal abnormalities in very distinct patterns in addition to the Ph chromosome.11 A change in the karyotype is considered to be a grave prognostic sign.11 With the exception of an isochromosome of the long arm of chromosome 17, i(17)(q10), which is usually associated with myeloid blast transformation, there is no association of a particular karyotype with lymphoid or myeloid blast transformation. The additional abnormalities are not correlated with the response to therapy during the acute phase.11 The most common changes, a gain of chromosomes 8 or 19, or a second Ph (by gain of the first), or an i(17q), frequently occur in combination to produce modal chromosome numbers of 47 to 50.11
OTHER MYELOPROLIFERATIVE DISORDERS
A cytogenetically abnormal clone is present in 14 percent of untreated polycythemia vera patients compared with 39 percent of treated patients.2,11 When the disease transforms to acute myeloid leukemia (AML), 85 percent have an abnormal clone. The presence of a chromosome abnormality at diagnosis does not necessarily predict a short survival or the development of leukemia. A change in the karyotype may be an ominous sign. Marrow cells frequently contain additional chromosomes (+8 or +9); these abnormalities also occur together, which is otherwise rare.11 Structural rearrangements most often involve a deletion of the long arm of chromosome 20, noted in 30 percent of patients, or a duplication of the long arm of chromosome 1, in 20 percent of patients. Loss of chromosome 7 (20 percent of patients) and del(5q) (40 percent of patients) are often observed in the leukemic phase. These changes may be related to the prior cytotoxic treatment received by these patients.
Cytogenetic analysis of myeloid metaplasia with myelofibrosis has revealed clonal abnormalities in 35 percent of patients.2 These abnormalities are similar to those noted in other myeloid disorders; the most common anomalies are +8, –7, del(7q), del(11q), del(13q), and del(20q).1,2 A change in the karyotype may signal evolution to acute leukemia.
Fewer than 5 percent of patients with essential thrombocytosis have an abnormal clone. No consistent abnormalities have been identified.
The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hemopathies including chronic myelomonocytic leukemia (CMMoL), refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), and RAEB in transformation (RAEB-T). Clonal chromosome abnormalities can be detected in marrow cells of 40 to 70 percent of patients with MDS at diagnosis (RA, 30%; RARS, 27%; RAEB, 70%; RAEB-T, 70%; CMMoL, 30%).2,15,16 The proportion varies with the risk that a subtype will transform to AML, which is highest for RAEB and RAEB-T. The common chromosome changes, +8, –5/del(5q), –7/del(7q), and del(20q), are similar to those seen in AML. The recurring abnormalities that are closely associated with the distinct morphologic subsets of AML de novo are almost never seen in MDS. With the exception of the 5q– syndrome, the chromosome changes show no close association with the specific subtypes of MDS. The 5q– syndrome occurs in a subset of older patients, frequently women, with RA, generally low blast counts, and normal or elevated platelet counts.17 These patients have an interstitial deletion of 5q, typically as the sole abnormality. The deletions vary in size but are similar to those noted in AML. These patients can have a relatively benign course which extends over several years.17
Cytogenetic abnormalities in MDS are predictive of survival and progression to AML.15,16 Patients with a “good outcome” have normal karyotypes, –Y alone, del(5q) alone, or del(20q) alone; those with an “intermediate outcome” have other abnormalities; and those with a “poor outcome” have complex karyotypes (³3 abnormalities, typically with abnormalities of chromosome 5 and/or 7) or chromosome 7 abnormalities.16
ACUTE MYELOID LEUKEMIA DE NOVO
THE 8;21 TRANSLOCATION
With initial banding analyses, clonal abnormalities were detected in 50 percent of patients with AML. This percentage has increased with improved banding and culture techniques; many laboratories are currently finding that at least 80 percent of patients have an abnormal karyotype.2,4 The most frequent abnormalities are a gain of chromosome 8 and a loss of chromosome 7, which are seen in most subtypes of AML.2,4 Specific rearrangements are closely associated with particular subtypes of AML as defined by the FAB classification (Table 10-4).2,4,18,19 The 8;21 translocation [t(8;21)(q22;q22)] was described in 1973 and was the first translocation identified in AML (see Fig. 10-1). The t(8;21) is common and is observed in 18 percent of all AML cases with an abnormal karyotype and in 30 percent of those patients with AML with maturation (AML-M2).21 This translocation is the most frequent abnormality in children with AML, and occurs in 15 to 20 percent of karyotypically abnormal cases. Loss of a sex chromosome (–Y in males, –X in females) accompanies the t(8;21) in 75 percent of cases. Most cases with the t(8;21) are classified as AML with maturation, but some cases have been diagnosed as acute myelomonocytic leukemia. Although AML-M2 is heterogeneous, the presence of the t(8;21) identifies a morphologically and clinically distinct subset. AML-2 with the t(8;21) has a favorable prognosis in adults, but the outcome in children is poor.19
TABLE 10-4 RECURRING CHROMOSOME ABNORMALITIES IN MALIGNANT MYELOID DISEASES
At the molecular level, the t(8;21) involves the AML1 gene, which encodes a transcription factor that is critical in hematopoiesis.20,21 The AML1 gene on chromosome 21 is fused to the ETO gene on chromosome 8 and results in an AML1/ETO chimeric protein.20,21 Transformation by AML1/ETO likely results from altered transcriptional regulation of normal AML1 target genes, combined with the activation of new target genes that prevent programmed cell death or cause aberrant proliferation.
THE 15;17 TRANSLOCATION
The t(15;17)(q22;q11.2–12) (see Fig. 12) is highly specific for acute promyelocytic leukemia (APL) and has not been found in any other disease. Although the t(15;17) was believed to be present in all cases of APL initially, it is now recognized that there are rare variant translocations, which occur in less than two percent of cases. These include the t(11;17)(q23;q11.2–12) and t(5;17)(q34;q11.2–12).22 Establishing the diagnosis of APL with the typical t(15;17) is important, because this disease is sensitive to therapy with all-trans-retinoic acid and arsenic trioxide, whereas other cases of AML and the APL-like disorders associated with the variant translocations do not respond to retinoic acid. The t(15;17) results in a fusion retinoic-acid-receptor-alpha protein (PML/RARA), which is thought to interfere with the retinoic acid receptor pathway that regulates terminal differentiation of myeloid precursors.22
ABNORMALITIES OF CHROMOSOME 16
Another clinical-cytogenetic association involves acute myelomonocytic leukemia with abnormal eosinophils (AMMoL-M4Eo), including large and irregular basophilic granules and positive reactions with periodic-acid-Schiff and chloroacetate esterase. Most patients have an inversion of chromosome 16, inv(16)(pl3q22) (see Fig. 10-1), but some have a t(16;16)(pl3;q22); these aberrations are relatively common, occurring in 25 percent of AMMoL patients.2,4,19 They have a good response to intensive chemotherapy, with a complete remission rate of approximately 90 percent and an overall 5-year survival of 75 percent.19 The breakpoint at 16q22 occurs within the CBFB gene, which encodes one subunit of the AML1/CBFB transcription factor. Thus, like the t(8;21), the inv(16) disrupts the AML1 pathway regulating hematopoiesis.20,21
ABNORMALITIES OF CHROMOSOME 11
Recurring translocations involving 11q23 are seen in approximately 35 percent of AML-M5 patients, and are of interest for at least three reasons.2,23 First, there are over 30 different recurring rearrangements that involve 11q23, and thus, along with 14q32, 11q23 is one of the bands most frequently involved in rearrangements in human tumor cells.2,3,23 The breakpoints in the translocation partners include 1p32, 4q21, and 19p13.3 in acute lymphoblastic leukemia (ALL), and 1q21, 2q21, 6q27, 9p22, 10p11, 17q25, 19p13.3, and 19p13.1 in AML. Second, these translocations occur in both lymphoid and myeloid leukemias. One common translocation in infants, t(4;11)(q21;q23), has a lymphoblastic phenotype, whereas other translocations, such as the t(9;11) (q22;q23) (see Fig. 12) and t(11;19)(q23;p13.1), are common in monoblastic leukemias. Translocations involving 11q23 have a very unusual age distribution; they comprise about three-quarters of the chromosome abnormalities in leukemia cells of children under one year of age.23 With the exception of the t(9;11) which may have an intermediate outcome, translocations of 11q23 are associated with a poor outcome.19 Translocations of 11q23 involve MLL, a very large gene (>100 Kb) with multiple transcripts of 12 to 15 Kb.20 All breakpoints fall within an 8.3-Kb breakpoint cluster region encompassing exons 5–11; thus, MLL translocations can be detected by Southern blot analysis of DNA using a small cDNA probe containing these exons.23 The MLL protein is likely to be a transcription factor but may also function in chromatin remodeling; the translocations result in fusion proteins.
Trisomy 11 is a rare abnormality, noted as a sole aberration in one to two percent of all AML or MDS cases,2,20 and has an unfavorable outcome. Trisomy 11 is notable in that duplications of the MLL gene are detected in 90 percent of AMLs with +11 as the sole abnormality, and in 10 percent of AML cases with an apparently normal karyotype.20 The rearrangement is the result of a partial tandem duplication of MLL exons 2–6 or 2–8, mediated by recombination between Alu repetitive elements, and may produce a partially duplicated protein.
OTHER CHROMOSOME ABNORMALITIES
Each of the other recurring rearrangements in AML occurs in less than three percent of patients.3 A unique feature of abnormalities involving the long arm of chromosome 3, inv(3)(q2l;q26) or t(3;3) (q2l;q26) is the presence of platelet counts above 100,000/µl, sometimes over 106/µl, and an increase in bone marrow megakaryocytes, especially micromegakaryocytes.4 It is noteworthy that most of the structural rearrangements described above occur in younger patients with a median age in the thirties, whereas some of the other abnormalities, such as –5/del(5q) or –7/del(7q) occur in patients with a median age over 50. Moreover, many of the latter patients have occupational exposure to mutagenic agents such as chemicals, including solvents, petroleum, and pesticides.18
ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES ASSOCIATED WITH PRIOR CYTOTOXIC TREATMENT (T-MDS AND T-AML)
Therapy-related MDS and AML (t-MDS/t-AML) has been recognized as a late complication of cytotoxic therapy used in the treatment of both malignant and nonmalignant diseases.24 In patients who received alkylating agents the characteristic recurring chromosome abnormalities observed are loss of part or all of chromosomes 5 and/or 7 (–5/del[5q] or –7/del[7q]) (see Fig. 12). In our experience, 93 percent of t-MDS/t-AML patients had an abnormal karyotype and 75 percent had an abnormality of chromosome 5, chromosome 7, or both (Refs. 4 and 24 and unpublished data); this has been confirmed in other series. In contrast, only about 16 percent of patients with AML de novo have a similar abnormality of chromosomes 5 or 7 or both.2,18
By cytogenetic analysis of 177 patients with malignant myeloid diseases and a del(5q) we identified a small segment of 5q, consisting of band 5q31, that was deleted in each patient.25 This segment has been termed the commonly deleted segment. By molecular analyses we have narrowed the commonly deleted segment to a region of approximately 1 Mb containing the EGR1 tumor suppressor gene and the CDC25C G2 checkpoint gene.25 Five hematopoietic growth factor genes mapped to 5q31 (GM-CSF, IL3, IL4, IL5, and IL9) are excluded from this region. We have detected no mutations of the remaining EGR1 or CDC25C alleles, suggesting that a novel tumor suppressor gene in 5q31 is involved in the pathogenesis of t-AML.25
A second subtype of t-AML has been identified that is distinctly different from the more common leukemia that follows alkylating agents or irradiation. This type of t-AML is seen in patients receiving drugs known to inhibit topoisomerase II, e.g., etoposide, teniposide, and doxorubicin.24,26 Clinically, these patients have a shorter latency period (1–2 years); present with overt leukemia, usually with monocytic features rarely present with MDS; and have a more favorable response to intensive remission induction therapy. Balanced translocations involving the MLL gene at 11q23 or the AML1 gene at 21q22 are common in this subgroup.26
MALIGNANT LYMPHOPROLIFERATIVE DISORDERS
ACUTE LYMPHOBLASTIC LEUKEMIA
The most useful prognostic indicators in acute lymphoblastic leukemia (ALL)—the most frequent leukemia in children—are age, white cell count, immunophenotype, karyotype (including ploidy), and CNS status.2,27,28 Children who are between 2 and 10 years old, with a white cell count of less than 10,000/µl, and whose leukemia cells express the common ALL antigen (CALLA, CD10) have the best prognosis. A number of recurring cytogenetic abnormalities are associated with distinct immunologic phenotypes of ALL (Table 10-5) with distinct outcomes.2,3 and 4
TABLE 10-5 CYTOGENETIC-IMMUNOPHENOTYPIC CORRELATIONS IN MALIGNANT LYMPHOID DISEASES
The 9;22 Translocation The incidence of the t(9;22) in ALL is 30 percent in adults and 5 percent in children. Thus, the Ph chromosome is the most frequent rearrangement in adult ALL. About one-half of the patients show additional abnormalities, a frequency that is substantially higher than that observed in CML. Monosomy 7 is a common secondary abnormality in Ph+ ALL and is associated with a poorer outcome.27 A chromosomally normal cell line is frequently noted in the bone marrow of Ph+ ALL patients (70 percent), but is rare in untreated CML. Most cases have a precursor B phenotype; however, some cases have had both B cell and myeloid markers.29 The disease in both adults and children is characterized by high WBC counts, a high percentage of circulating blasts, and a poor prognosis. As in CML, the t(9;22) in ALL results in a BCR/ABL fusion gene; however, in over half of the patients the break in BCR is more proximal, resulting in a smaller fusion protein.29
Abnormalities of Chromosome 11 Translocations involving the MLL gene at 11q23 are observed in 5 to 7 percent of ALL patients.2,4,23 Of these, the most common is the t(4;11)(q21;q23) (see Fig. 10-2). The t(11;19)(q23;p13.3) is second in frequency; however, this rearrangement is not limited to ALL, in that approximately 50 percent of these cases have AML, usually AML-M5. Of note is the high frequency of translocations involving 11q23 in infant ALL (60–80%). A recent study using RT-PCR detected the t(4;11) in 80 percent of infant ALL cases.27,29 Patients with the t(4;11) have high leukocyte counts (median WBC 183,000/µl), L1 or L2 morphology, an immature precursor B phenotype (CD10–, CD19+), with coexpression of monocytic or, less commonly, T cell markers.27,28 Clinically, they have aggressive features with hyperleukocytosis, extramedullary disease, and a poor response to conventional chemotherapy. Adults with the t(4;11) have a CR rate of 75 percent, but a median event-free survival (EFS) of only 7 months.28 Children with the t(4;11) have a similar poor outcome.27 Rearrangements affecting MLL represent a major class of mutations in acute leukemia and identify patients with a poor outcome.
FIGURE 10-2 (A–D) Photomicrographs of metaphase and interphase cells following FISH. In panels A-C, the cells are counterstained with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). (A) Hybridization of a directly labeled centromere-specific probe for chromosome 8 (CEP8TM Spectrum Green, Vysis Inc.) to metaphase and interphase cells from an AML with trisomy 8. Centromere-specific probes hybridize to the repetitive DNA sequences that are present at the centromeres of human chromosomes. The chromosome 8 homologs are identified with arrows. (B) Hybridization of a directly labeled chromosome 8-specific painting probe (WCP8TM Spectrum Green, Vysis Inc.) to a metaphase cell with trisomy 8 from an AML. (C) Hybridization of a locus-specific probe for the detection of a recurring translocation, the t(9;22)(q34;q11.2) in CML. The probe is a mixture of digoxigenin-labeled DNA probes (detected with rhodamine-labeled antibodies) for the major breakpoint cluster region of the BCR gene at 22q11.2, and biotin-labeled probes (detected with FITC-avidin) for the ABL gene at 9q34 (M-bcr/abl probe, Oncor). In cells with the t(9;22), only one green signal (arrowhead) and one red signal (short arrow) is observed on the normal 9 and 22 homologs, and a yellow fusion signal (long arrow) is observed on the Ph chromosome as a result of the juxtaposition of the ABL and BCR sequences. (D) Spectral karyotyping analysis of a metaphase cell from an AML-M7. Twenty-four differentially labeled probes, each representing one human chromosome, are cohybridized, and imaging analysis software assigns a unique color to each. A complex karyotype was identified by conventional cytogenetic analysis, including a derivative chromosome 1 with additional material of unknown origin on 1p, a deletion of 8p, a derivative chromosome 11 resulting from an unbalanced translocation involving 1 and 11, and a derivative chromosome 12, consisting of 11q and 12q. The results of spectral karyotyping confirmed the identity of the rearranged chromosome 12 (arrowhead) and clarified the other abnormalities. The additional material on 1p was derived from chromosome 8 (long arrow, blue signal), and the der(11) actually consisted of material from chromosomes 1, 11, and 12 (short arrow, 11p white signal; chromosome 12 brown signal; 1p blue-pink signal).
The 12;21 Translocation A translocation, t(12;21)(p12;q22), has been identified in a high proportion (~25 percent) of childhood precursor B leukemia.27,30 The translocation is not easily detected by cytogenetic analysis due to the similarity in size and banding pattern of 12p and 21q. However, the rearrangement can be detected reliably using RT-PCR or FISH analysis. The t(12;21) defines a distinct subgroup of patients characterized by an age between 1 and 10 years, B lineage immunophenotype (CD10+, CD19+), and a favorable outcome.27,30 It is not seen in T cell ALL and is uncommon in adults (approximately four percent of ALL cases). In a recent series, patients with the t(12;21) had a 5-year EFS of 91 percent, as compared with 65 percent for patients without this rearrangement. One-half of these patients would have fallen into a high-risk group using standard risk factors; thus, the t(12;21) may identify a subset of patients within the high-risk group who would benefit from well-tolerated, less toxic antimetabolite therapy.
Hyperdiploidy The leukemia cells of some patients with ALL are characterized by a gain of many chromosomes. Two distinct subgroups are recognized: a group with one to four extra chromosomes (47–50), and the more common group with >50 chromosomes. Chromosome numbers usually range from 51 to 60, and a few patients may have up to 65 chromosomes. Hyperdiploidy (>50 chromosomes) is common in children (~30%) but is rarely observed in adults (<5%). Certain additional chromosomes are common (X chromosome and chromosomes 4, 6, 10, 14, 17, 18, and 21). Chromosome 21 is gained most frequently (100 percent of cases).2,4 Patients who have hyperdiploidy with >50 chromosomes have all of the previously recognized clinical factors that indicate a good prognosis, including age between one and nine years, low WBC count (median 6,700/µl), and favorable immunophenotype (early pre-B or pre-B).27,28 In a recent analysis of 186 children with hyperdiploidy ALL, it was observed that ALL defined by 51 to 55 chromosomes and ALL defined by 56 to 65 chromosomes may be distinct clinical entities.31 The 105 patients in the first group (51 to 55 chromosomes) had an EFS at 5 years of 72 percent, compared with 86 percent (P = 0.04) for those patients with >56 chromosomes (63 patients).
The 1;19 and 8;14 Translocations The t(1;19)(q23;p3) has been identified in about 25 percent of patients with a pre-B phenotype; that is, the leukemia cells have cytoplasmic immunoglobulin and are CD10+ (Fig. 10-2).27,30 A reciprocal translocation involving the long arms of chromosomes 8 and 14 [t(8;14)(q24;q32)] is observed in B cell ALL (L3) (see Fig. 10-2).2 These patients have a high incidence of CNS involvement and/or abdominal nodal involvement at diagnosis. Although the outcome for both children and adults with a t(8;14) has been poor, the use of high intensity chemotherapy has markedly improved the outcome (EFS of 80 percent in children).27
T CELL ACUTE LYMPHOBLASTIC LEUKEMIA
A distinct pattern of recurring karyotypic abnormalities in T cell neoplasms has emerged.2,29 Rearrangements involving 14q11 (see Fig. 10-2) and two regions of chromosome 7 (7q34–35 and 7p15) are particularly frequent in T cell malignancies (see Table 10-5). The most common are the t(11;14) (p13;q11) (~3%), t(10;11)(q24;q11) (~3%), and t(7;9)(q34–35;q34) (~2%).1,3,29 In addition to their occurrence in T cell leukemia, these abnormalities have also been observed in lymphomas of T cell origin. The genes that are located at the breakpoints of a number of these abnormalities have been identified (see also Table 10-5). Patients with T cell ALL are most often young males and often have a mediastinal tumor mass, high WBC count, and leukemia cells in the cerebro-spinal fluid. These same clinical characteristics are associated with lymphoblastic lymphoma, another T cell malignancy.
CHRONIC LYMPHOCYTIC LEUKEMIA
Unfortunately, only half of CLL patients have an adequate number of metaphase cells in unstimulated cultures for thorough evaluation. Trisomy 12 is the most common cytogenetic abnormality reported in patients with B cell CLL; it is found in 20 to 60 percent of those with a cytogenetic abnormality.32 Abnormalities involving band 14q32 are also common, e.g., t(14;19)(q32;q13) (see Table 10-5). FISH is a sensitive alternative method for detecting abnormalities in interphase CLL cells. Using FISH, 30 percent of patients are found to have trisomy 12, and this abnormality is associated with a poorer survival.32 Similarly, a del(13q) can be detected in 30 percent of CLL cases using FISH. T cell CLL and large granular lymphocytic leukemia are uncommon disorders in which the malignant mature lymphocytes have a T cell immunophenotype. Rearrangements involving band 14q11, with or without an accompanying break in 14q32, have been reported in T-CLL as well as in T cell lymphomas (Table 10-5).1,2 and 3 The most common of these rearrangements is an inv(14)(q11q32) (see Fig. 10-2).
Cytogenetic analyses of lymphoma have been reported in a number of large series.2,4,33,34 and 35 These investigations have demonstrated that over 90 percent of cases are characterized by clonal chromosomal abnormalities and, more importantly, many of the recurring abnormalities correlate with histology and immunophenotype (see Table 10-5). For example, the t(14;18) is observed in a high proportion of follicular small cleaved cell lymphomas (70–90%), most patients with a t(3;22)(q27;q11) or t(3;14)(q27;q32) have diffuse large cell lymphomas (B cell), and patients with a t(8;14)(q24;q32) have either small noncleaved cell or diffuse large cell lymphomas (DLCL). Band 14q32, the location of the Ig heavy chain gene (IGH) is frequently involved in translocations in B cell neoplasms (~70%). In contrast, a large proportion of T cell neoplasms are characterized by rearrangements that involve 14q11, 7q34–35, or 7p15, the locations of the T cell receptor genes.
In 1972 a consistent abnormality (14q+) was identified in the cells of fresh Burkitt lymphomas (BL) and in cultured cell lines. Several years later the rearrangement was shown to be a reciprocal translocation involving chromosomes 8 and 14, t(8;14)(q24;q32) (see Fig. 10-2). The t(8;14) is characteristic of both endemic and nonendemic Burkitt tumors, as well as Epstein-Barr virus (EBV) negative and EBV-positive tumors. The t(8;14) has also been observed in other lymphomas, particularly small noncleaved cell (non-Burkitt) and large-cell immunoblastic lymphomas, AIDS-associated BL (100%), and AIDS-related DLCL (30%).33,34 and 35 As additional Burkitt tumors were examined, it became apparent that at least two other related translocations occur, t(2;8)(p12;q24) and t(8;22)(q24;q11). All three translocations involve chromosome band 8q24. As discussed earlier, these same translocations have been seen in some patients with B cell ALL. The t(8;14) involves a break within the IGH locus on chromosome 14 and a break either 5′ or within MYC on chromosome 8, and it relocates the MYC coding exons to chromosome 14.5,33 MYC is a transcription factor that plays a role in a number of cellular processes including proliferation and apoptosis, and its oncogenic properties are due to its constitutive expression.
Between 70 and 90 percent of follicular lymphomas and 20 percent of diffuse B cell lymphomas have the t(14;18) (see Fig. 10-2), in which the BCL2 gene at 18q21 is juxtaposed to the IGH J segment, leading to the deregulated expression of BCL2.33,34 Other malignancies which overexpress BCL2 but do not harbor the t(14;18) include hairy cell leukemia and CLL. The BCL2 gene encodes a 26 kDa membrane protein that functions to increase cell survival (antiapoptosis). Thus, this class of oncogene contributes to the development of a neoplastic state by preventing programmed cell death, rather than by promoting proliferation.
The t(11;14) (q13;q32) is observed in a relatively new pathologic entity known as mantle cell lymphoma.36 In most series, the percentages of cases having the t(11;14) by molecular analysis have varied between 30 and 55 percent of cases, but these may be underestimates, as only the major breakpoint cluster regions have been looked for. Besides mantle cell lymphomas, the t(11;14) has also been reported in 3 percent of multiple myeloma and up to 20 percent of prolymphocytic leukemias.36 Mantle cell lymphomas are currently regarded as a poor prognostic group, with a median survival from diagnosis of three years. This translocation results in the activation of the cyclin D1 (CCND1) gene by the IGH gene.33 Interestingly, the CCND1 gene is located 100 to 130 Kb away from the breakpoint on 11q13. The D-type cyclins act as growth factor sensors, causing cells to go through the restriction start point of the cell cycle at G1 and committing them to divide.
The BCL6 gene was cloned from the recurring breakpoint at 3q27 in cells characterized by a t(3;22)(q27;q11), t(3;14)(q27;q32), or rarely t(2;3)(p12;q27).33 BCL6 rearrangements occur in 40 percent of DLCL and, in some series, up to 10 percent of follicular lymphomas. The translocations lead to the truncation of the BCL6 gene within the first exon or the first intron, substitution of its promoter sequences with an IG promoter, and deregulated expression. The BCL6 gene product is a 96 kDa nuclear protein that acts as a potent transcriptional repressor. It is predominantly expressed in the B cell lineage, particularly in mature B cells, but not in immature bone marrow precursors or the more mature plasma cell.
A number of recurring chromosomal abnormalities have been recognized in T cell leukemias and lymphomas (see Table 10-5). Similar to B cell neoplasms, in which rearrangements frequently involve the chromosomal bands containing the immunoglobulin gene loci, T cell neoplasms often have rearrangements involving band q11 of chromosome 14, the site of the T cell receptor a-chain and d-chain genes (TCRA, TCRD) or, less often, one of two regions of chromosome 7 (7q34–35 or 7p15) to which the T cell receptor b-chain (TCRB) and g-chain (TCRG) genes have been localized respectively.1,3 These translocations result from aberrant recombination events during V-D-J recombination. With few exceptions, the involved gene on the partner chromosome encodes a transcription factor, whose expression is deregulated or activated as a result of the rearrangement (see Table 10-5).3,4 A chromosomal rearrangement which brings an oncogene under the controlling influence of promoters or enhancers active for immunoglobulin synthesis in B cells or T cell–receptor synthesis in T cells may, as a consequence, impart a proliferative advantage to the affected cell and result in malignant clonal expansion.
A distinctive subtype of lymphoma, namely, Ki-1(CD30)-positive anaplastic large cell lymphoma (Ki-1+ ALCL) has been characterized during the past few years. Patients with Ki-1+ ALCL tend to be young, and they present with skin and/or lymph node infiltration by large, often bizarre lymphoma cells, which preferentially involve the paracortical areas and lymph node sinuses. The majority of such tumors express one or more T cell antigens, a minority express B cell antigens, and some express both T and B cell antigens (the null phenotype). A reciprocal translocation, t(2;5)(p23;q35), appears to be restricted to ALCL of either T cell or null phenotype and is present in a high percentage of these cases.33 This translocation has also been found in CD30+ primary cutaneous lymphomas.
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn