M E T H O D S I N M O L E C U L A R M E D I C I N E TM Edited by Guy B. Faguet, MD Hematologic Malignancies Methods and Techniques Edited by Guy B. Faguet, MD Hematologic Malignancies Methods and Techniques Humana Press Humana Press Cytogenetics Analysis 3 3 From: Methods in Molecular Medicine, vol. 55: Hematologic Malignancies: Methods and Techniques Edited by: G. B. Faguet © Humana Press Inc., Totowa, NJ 1 Cytogenetics Analysis Avery A. Sandberg and Zhong Chen 1. Introduction 1.1. Principles The establishment of reliable and meaningful chromosomal (cytogenetic, karyotypic) changes in hematological disorders, primarily the leukemias and lymphomas, must be based on the examination of the involved cells or tissues. Thus, in the case of the leukemias bone marrow (BM) aspirations yield optimal results in the preponderant number of patients, whereas in the lymphomas affected tissues, usually lymph nodes, are the best source of cells carrying cytogenetic anomalies. Generally, BM is not a good source of cells for cytoge- netic analysis in lymphoma. Not only is the marrow often not affected by the lymphoma, but also when it is, the number of abnormal cells is relatively small and/or the abnormal cells are not in division and, hence, do not yield a suffi- cient number of metaphases for cytogenetic analysis. In some situations, blood cells can be utilized as a source of metaphases affected by karyotypic changes, e.g., in cases with about 10% immature cells in the peripheral blood (PB), in chronic lymphocytic leukemia (CLL), in cases where the marrow is fibrotic or extremely hypocellular, or in determining the presence of Ph+ cells in estab- lished cases of chronic myelocytic leukemia (CML) (1,2). Paramount for a successful cytogenetic study is the presence of metaphases suitable for analysis. In the normal BM, a significant number of dividing cells, and hence metaphases, are usually present in sufficient number for cytogenetic analysis without having to resort to culture or lengthy incubation. However, in some leukemias the number of dividing cells (especially the leukemic ones) is very low and, hence, incubation of the marrow specimen for a number of days (2–5 d) may be necessary to generate a significant number of metaphases for 4 Sandberg and Chen cytogenetic study. The statements just made apply in particular to acute promyelocytic leukemia (APL) and following chemotherapy and/or radiation therapy for the leukemia (1,2). In cases where cytogenetic analysis reveals only abnormal metaphases, especially those with a balanced translocation, it may be necessary to rule out a constitutional chromosomal anomaly. This is best established through the cytogenetic examination of phytohemagglutinin (PHA) stimulated lympho- cytes of the PB. A number of mitogenic agents capable of stimulating lymphoid or, less fre- quently, myeloid cells have been introduced over the years. Outstanding among these has been PHA capable of stimulating the growth and division of lympho- cytes of T-cell origin. However, PHA is not routinely added to BM or PB cul- tures in acute leukemias because PHA may interfere with the evaluation of spontaneously dividing malignant cells. The quality of chromosome preparations has been significantly improved with some new techniques, such as the use of amethopterin for cell synchroni- zation, the use of short exposures to mitosis-arresting agents, the use of DNA- binding agents to elongate chromosome (3,4), improved staining procedures, and the use of conditioned culture medium containing hematopoietic growth factors [e.g., GCT (giant cell tumor)-conditioned medium primarily for myeloid disorders (4,5) and PHA/IL-2 (interleukin-2) for both B- or T-cell lymphoid diseases (6,7)]. The rate of successful cytogenetic analysis varies with the specific type of disease, and is also related to the adjustment of variables in each laboratory, such as serum concentration, medium pH, and cell concentration. 1.2. Clinical Applications The common and recurrent chromosome changes seen in the leukemias and lymphomas are shown in Tables 1–3 and Figs. 1 and 2. CML is a pluripotent stem cell disorder characterized cytogenetically by the Philadelphia chromosome (Ph), the first consistent abnormality observed in a human cancer. The Ph arises from a reciprocal translocation, t(9;22)(q34;q11) (8). It is characterized molecularly by the fusion of parts of the C-ABL gene (at 9q34) and the BCR gene (at 22q11), generating an abnormal BCR/ABL fusion gene (9). Cytogenetically, more than 85% of patients with CML are found to have the Ph in the CML cells, even during remission, unlike the Ph in acute leukemia, which is not seen during complete remission. When CML progresses, additional changes, such as +8, +Ph, i(17q), +19, and +21 are noted in 75–80% of cases. These changes may precede hematologic progression by 2–6 mo or occur at the blast phase; therefore, they are valuable prognostic indices. How- ever, there is no evidence that these additional changes correlate with response Cytogenetics Analysis 5 to presently used therapy during the acute phase of CML. Clinically, treatment strategies for CML should include, in addition to the hematologic criteria, the cyto- genetic findings and the molecular genetic criteria of the BCR/ABL fusion gene obtained using Southern blotting or polymerase chain reaction (PCR) techniques. Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by dysplastic and ineffective hematopoiesis and a high risk of transformation to ANLL. Clonal chromo- somal abnormalities can be detected in 40–70% of MDS patients at presenta- tion (see Table 4). Additional aberrations may evolve during the course of MDS and appear to portend its transformation to leukemia. To confirm the diagnosis of MDS, morphologic examination of BM aspirate and cytogenetic analysis should be performed. Moreover, the chromosomal findings have been shown to be an independent prognostic indicator second only to the French- American-British (FAB) classification subtype as a predictor of progression to leukemia and survival (see Table 5). Table 1 Common Chromosome Changes in Acute Nonlymphocytic Leukemia (ANLL) (1 , 12) der(1;7)(q10;p10) a t(9;22)(q34;q11) M1(M2) t(1;22)(p13;q13) M7 t(11;V)(q23;V) b M5(M4) ins(3;3)(q26;q21q26) a,d M1(M7) del(11)(q23) M5(M4) inv(3)(q21q26) a,d M1(M7) +11 t(3;3)(q21;q26) a,d M1(M7) del(12)(p11p13) a t(3;21)(q26;q22) a +13 a +4 M2, M4 +14 a –5 or del(5)(q12–13 or q31–35) a M1–M4 t(15;17)(q22;q11–21) M3 +6 del(16)(q22) c M4EO t(6;9)(p23;q34) a M2(M4) (basophilia) inv(16)(p13q22) c M4EO –7 or del(7)(q22) a M1–M5 t(10;16)(p13;q22) c M4EO +8 a t(16;21)(p11;q22) t(8;16)(p11;p13) M5b (erythrophagocytosis) i(17)(q10) a t(8;21)(q22;q22) M2 (Auer rods+) +19 +9 del(20)(q11–13) a del(9)(q22) +21 idic(X)(q13) a a Change also seen in myelodysplastic syndromes. b V = chromosomes 6, 9, 17, and 19. c Associated with marrow eosinophilia. d Associated with platelet and/or megakaryocytic anomalies. Where appropriate, the type of ANLL or other information associated with a particular chro- mosome change is also shown. 6 Sandberg and Chen The acute leukemias, which are classified either as lymphoblastic (ALL) or nonlymphocytic (ANLL), result from neoplastic transformation of uncommit- ted or partially committed hematopoietic stem cells. About two thirds of ANLL and ALL patients have recognizable clonal chromosomal anomalies. These Table 2 Common Chromosome Changes in B-Lineage Acute Lymphocytic Leukemia (ALL) (1,12) t(1;9)(q23;p13) Pre-B-cell t(2;8)(p12;q24) L3 (B-cell) t(4;11)(q21;q23) Biphenotypic, early B-precursor t(5;14)(q31;q32) del(6)(q13–14 or q21–27) t(8;14)(q24;q32) L3 (B-cell) t(8;22)(q24;q11) L3 (B-cell) del(9)(p13–22) t(9;22)(q34;q11) B-lineage del(11)(q14–23) t(11;19)(q23;p13) Mixed, biphenotypic t(12;V)(p12;V) a B-lineage t(14;19)(q32;q13) t(14;22)(q32;q11) a V = Chromosomes 7, 9, and 17. Table 3 Common Chromosome Changes in B-Cell Non-Hodgkin Lymphoma (NHL) (1,12) Chromosome changes Histology t(2;3)(p12;q27) Diffuse large cell t(2;8)(p12;q24) (Burkitt) small noncleaved cell t(3;14)(q27;q32) Diffuse large cell t(3;22)(q27;q11) Diffuse large cell +3 Follicular large cell, immunoblastic del(6q) Follicular small cleaved cell t(8;14)(q24;q32) (Burkitt) small noncleaved t(8;22)(q24;q11) (Burkitt) small noncleaved t(11;14)(q13;q32) Centrocytic (variable zone) with CD5 + cells +12 Diffuse small cell t(14;18)(q32;q21) Mixed, small cleaved, and large cell follicular t(18;22)(q21;q11) Follicular Cytogenetics Analysis 7 Fig. 1. G-banded karyotype of a marrow cell showing the Philadelphia (Ph) translo- cation, t(9;22)(q34;q11) (arrows point to breakpoints). This was the only change present in the affected cells of this case with CML. Fig. 2. G-banded karyotype showing trisomy 12 (+12) as the only change in a case of CLL. This change (+12) is seen in a significant number of CLL cases and is usually associated with a poor prognosis. 8 Sandberg and Chen may fall into a specific category that characterizes unique clinical and cytoge- netic entities. Survival, as a function of cytogenetic findings in ANLL and ALL, is shown in Table 6. The determination of the chromosomal changes in acute leukemia serves a number of practical purposes, for example, the estab- lishment of the exact diagnosis, prediction of prognosis, and as a guide to the treatment and monitoring phases of therapy or BM transplantation, as well as some basic purposes, such as supplying the molecular biologist with possible information on the location or nature of the genes affected by translocations, dele- tions, and inversions. A case in point is the t(15;17)(q22;q21), seen in APL, which has been shown to affect a gene related to the α-retinoic acid receptor. This has led to the use of retinoic acid in the therapy of APL with remarkable results. More than 90% of non-Hodgkin lymphomas (NHL) have clonal chromo- somal changes; t(8;14)(q24;q32), t(8;22)(q24;q11) and t(2;8)(p12;q24) have Table 4 Frequency of Chromosomal Changes and Evolution to ANLL in Myelodysplasia Evolution to Chromosomal FAB subtypes and distribution ANLL changes mo Refractory anemia (30%) 11% 48% 37 Refractory anemia With ring sideroblasts (18%) 15% 12% 49 With excess blasts (25%) 25% 57% 19 With excess blasts & transformation (12%) 50% 93% 16 Chronic myelomonocytic leukemia (15%) 15% 29% 22 Table 5 Prognosis in Myelodysplastic Syndromes According to Cytogenetic Findings Prognostic category Karyotype Median survival (mo) Good Normal >24 Deletion 5q Intermediate Trisomy 8 >18 Poor Monosomy 7 Deletion 7q Isochromosome 17q <12 Deletion 20q Complex changes Cytogenetics Analysis 9 been found in 75–80%, 10–17%, and 5–8% of Burkitt lymphomas (BL) of both African and non-African origin, respectively (10). Molecularly, fusion of the MYC gene to immunoglobulin genes has been identified in all BL cases. In non-Burkitt NHL, a 14q+ marker characterizes about 50% of the cases. Many of the nonrandom anomalies correlate with histology and immunologic pheno- type, such as t(14;18)(q32;q21) with follicular (nodular) B-cell lymphomas, del(6q) with large-cell lymphomas, and t(8;14)(q24;q32) with either small, noncleaved cell or diffuse large-cell lymphomas. Approximately 50% of CLL patients have chromosomal abnormalities, the most common of which are trisomy 12, 14q+, 13q, and 11q abnormalities. An abnormal karyotype is a poor prognostic sign in CLL, and trisomy 12 and pos- sibly 14q+ are the least favorable abnormalities. Three factors are of impor- tance in CLL: lymphocyte doubling time, diffuse lymphocyte infiltration of BM and lymph nodes, and the chromosomal pattern. Combining these three factors with the current clinical staging of CLL may optimize therapeutic decisions. Table 6 Chromosomal Abnormalities and Survival in ANLL and ALL Chromosomal abnormality Median survival (mo) ANLL Rearrangements of 16q22 18 Translocation (8;21)(q22;q22) (see Fig. 3)14 Normal karyotype 10 Abnormal 11q 18 Abnormal 5 and/or 7 13 Translocation (15;17)(q22;q21) a (see Fig. 4) a ALL More than 50 chromosomes 58 Deletion 6 (q15q21) 30 Normal karyotype 29 Translocation (9;22)(q34;q11) 12 Less than 46 chromosomes 12 Rearrangements of 14q32 18 Translocation (4;11)(q21;q23) 17 Rearrangements 15 a Survival for patients with a 15;17 translocation is markedly improved with aggressive treat- ment and trans-alpha retinoic acid therapy compared with the median survival previously reported (2 mo) 10 Sandberg and Chen 2. Materials 2.1. Specimens 1. BM aspirate or bone core biopsy: One to 3 mL of BM should be aseptically aspi- rated into a sodium-heparinized syringe and transferred to a sterile sodium vacutainer tube. The specimen can be transported with or without culture medium (RPMI 1640 + 5–10% fetal calf serum [FCS] + 1% penicillin[pen]/ streptomycin[strep]). If the specimen cannot be delivered immediately, it may be stored at room temperature or in a refrigerator overnight. Do not freeze the specimen. Cell viability drops off sharply by 72 h after collection. If a marrow aspirate cannot be achieved, a BM biopsy may be accepted for cytogenetic analysis. 2. Peripheral blood: Five to 10 mL of PB should be aseptically collected and trans- ferred, transported, and preserved in the same way as for a BM specimen. 3. Lymph node and spleen: Lymph node and spleen biopsies or samples should be collected aseptically and transferred to a sterile sodium vacutainer tube contain- ing culture medium (RPMI 1640 + 5–10% FCS + 1% penicillin/streptomycin). The specimen should be transported and preserved in the same manner as for BM specimen. Fig. 3. G-banded karyotype with the translocation (8;21)(q22;q22) as the only anomaly. This change is seen almost exclusively in M2 type of ANLL associated with a relatively good prognosis, Auer bodies in the cells, and a high remission rate. Cytogenetics Analysis 11 2.2.Reagents and Instruments 1. RPMI 1640/MEM Alpha/FBS complete media: 100 mL RPMI 1640, 70 mL MEM alpha, 30 mL fetal bovine serum (15%), 2 mL 3% L-glutamine (1%), 2 mL penicillin/streptomycin (1%) (10,000 units pen/mL; 10,000 µg strep/mL). 2. Colcemid (Gibco Karyomax colcemid solution—10 µg/mL): a. 1/20 Solution: Dilute 10 mL of the stock solution with 10 mL of sterile deion- ized water to a final concentration of 5 µg/mL). Store at 4°C. b. 1/200 Colcemid solution: Dilute 5 mL of 1/20 colcemid with 45 mL of sterile deionized water to a final concentration of 0.5 µg/mL). Store at 4°C. 3. Potassium chloride—0.068 M: a. Stock solution: 5.6 g KCl in 100 mL of deionized water. b. Working solution: 10 mL KCl of stock solution in 100 mL of deionized water. Prewarm to 37°C before use. Fig. 4. R-banded karyotype of a cell from a patient with APL (M3) containing the translocation (15;17)(q22;q11–21) as the only chromosome change. This transloca- tion is characteristic of APL (M3). [...]... Karger, Basel, Switzerland 12 Heim, S and Mitelman, F (1995) Cancer Cytogenetics, 2nd ed., Wiley-Liss New York FISH Analysis 19 2 FISH Analysis Avery A Sandberg and Zhong Chen 1 Introduction 1.1 History and Principles In situ hybridization of specific DNA or RNA sequences to cellular targets was developed over 20 yr ago (1,2) The early techniques employed isotopically labeled probes and subsequent autoradiographic... DNA is tagged with a hapten (such as biotin or digoxigenin) or is directly labeled with a fluorescent dye Detection From: Methods in Molecular Medicine, vol 55: Hematologic Malignancies: Methods and Techniques Edited by: G B Faguet © Humana Press Inc., Totowa, NJ 19 20 Sandberg and Chen Fig 1 Schematic presentation of some of the essential steps involved in FISH analysis of the hapten can be achieved... which has degraded because of improper handling and/ or shipping It is important that FISH probes be stored at –20°C and handled with gloves and autoclaved pipet tips A change in sample type and sample degradation may also influence probe signal intensity 2 Cross-hybridization (nonspecific fluorescent signals): Because exact pairing of DNA sequences is achieved and maintained under certain conditions,... Wilker, S., Stone, J F., and Sandberg, A A (1995) The PHA/IL2 “COCKTAIL” is an effective cytogenetic mitogen in blood and BM cells for revealing abnormal clonal karyotypes in lymphoid diseases Appl Cytogenet 21, 66 7 Morgan, R., Chen, Z., Richkind, K., Roherty, S Velasco, J., and Sandberg, A A (1999) PHA/IL2: an efficient mitogen cocktail for cytogenetic studies of nonHodgkin lymphoma and chronic lymphocytic... quinacrine fluorescence and Giemsa staining Nature 243, 290–293 9 deKlein, A and Hagemeijer, A (1984) Cytogenetic and molecular analysis of the Ph1 translocation in chronic myeloid leukemia Cancer Surv 3, 515–529 10 Zech, L., Haglund, V., Nilsson, K., and Slein, G (1976) Characteristic chromosomal abnormalities in biopsies and lymphoid cell lines from patients with Burkitt and non-Burkitt lymphomas... described in Subheading 3.1 for BM and PB specimens (lymphoid disorders) 3.3 Analysis 1 In general, a minimum of 20 cells must be counted and analyzed for each case, such that a rearrangement affecting one band of any chromosome can be detected in any given cell The cells must be selected to represent at least two culture conditions The individual morphology and band-by-band structure of each chromosome... of the patient These clots may be broken up by the aspiration through a needle or pipet and/ or minced Clots can trap cells and interfere with the harvesting procedure 4 Mislabeling is a common source of error This can be prevented by labeling and handling only one specimen at a time and by double checking labels and numbers 5 Avoid using the same pipet during harvest on two different patients 6 Cell... sequence probes, has cogent and practical application in myeloid malignancies, including acute nonlymphocytic leukemia (ANLL), chronic myelocytic leukemia (CML), myeloproliferative disorders (MPD), and myelodysplastic syndromes (MDS), where it can be used to characterize these disorders, e.g., monosomy 7 (–7) and trisomy 8 (+8) in MDS, +8 and +9 in MPD, t(9;22) in CML, and t(15;17) in ANLL (11,12)... monosomy 18 in 43% of 24 Sandberg and Chen cases with CLL, and 28% of those with small-cell lymphocytic lymphoma, trisomy, or tetrasomy 17 in 27% of NHL patients, and X-chromosome aneuploidy in patients with NHL (21) 2 Materials 2.1 Specimens Due to the high stability of DNA, FISH can be performed on most specimens, ranging from blood and bone marrow smears, buccal smears, cytospins, and touch print preparations... pathology specimens and epithelial cells in bladder washings and urine Logically, any nucleus can be evaluated with FISH methods as long as the DNA in the cell is not degraded (see Note 1) For hematological disorders, bone marrow (BM) and peripheral blood (PB) are usually the specimens submitted for FISH analysis Often these samples are first processed for chromosomal analysis and FISH is performed . Faguet, MD Hematologic Malignancies Methods and Techniques Edited by Guy B. Faguet, MD Hematologic Malignancies Methods and Techniques Humana Press Humana Press Cytogenetics Analysis 3 3 From: Methods. Molecular Medicine, vol. 55: Hematologic Malignancies: Methods and Techniques Edited by: G. B. Faguet © Humana Press Inc., Totowa, NJ 1 Cytogenetics Analysis Avery A. Sandberg and Zhong Chen 1. Introduction 1.1 collected and trans- ferred, transported, and preserved in the same way as for a BM specimen. 3. Lymph node and spleen: Lymph node and spleen biopsies or samples should be collected aseptically and