Cytogenetic Study of Myelodysplastic Syndrome

Case Report

CYTOGENETIC STUDY OF MYELODYSPLASTIC SYNDROME S Dayakar*, S Vanajakshi**, Amina Sheik**, Srinivas Chakravarthy***, K Iravathy Goud*, SJ Babu* and K Vijaya Lakshmi* *Department of Molecular Biology and Cytogenetics, **Department of Haematology, ***Department of Oncology, Apollo Hospitals, Jubilee Hills, Hyderabad 500 033, India. Correspondence to: Dr K Iravathy Goud, Molecular Biology and Cytogenetics Laboratory, Apollo Health City, Jubilee Hills, Hyderabad 500 033, India. E-mail: [email protected] Myelodysplastic syndrome (MDS) represents a group of clonal hematological disorders characterized by progressive cytopenia reflecting defects in erythroid, myeloid and megakaryocytic maturation. Cytogenetic study plays an important role in the diagnosis of chromosomal changes ranging between 23-78% have been reported in MDS patients. The authors present a case suspected to have MDS of Refractory cytopenia with multi-lineage dysplasia (RCMD)/ refractory anemia with excess blasts (RAEB) -Type-1 on the bone marrow aspiration and was further studied for conventional cytogenetic analysis and deletions of 5q, 7q, 8 and 20q by and fluorescence insitu hybridization (FISH). The results of conventional cytogenetic analysis revealed t (1; 3) ((p36; q21), and FISH analysis showed a normal study for chromosomes 5, 7 and 20 suggesting that cytogenetic analysis should be performed in all MDS cases as cytogenetic analysis studies holistically all 46 chromosomes which can pick up numerical and structural anomalies and to study of micro deletions and other complex translocations it should be supplemented with FISH analysis. Key words: Chromosomal abnormalities, Myelodysplastic syndrome.

was referred for conventional cytogenetics and FISH analysis for further confirmation.

CASE REPORT A fifty two years old house wife was referred to us from oncology outpatient block for cytogenetics analysis. The patient was married for thirty years and has three children. There is no indication of medication used for long duration.

Detailed information like age, family history, diet, social habits, lifestyle, medication taken in last three months and reproductive history was collected in a well designed proforma. Cytogenetic analysis of the patient was performed at molecular biology and cytogenetics lab, Apollo hospitals, Hyderabad. About 0.5-1 mL of bone marrow aspirate was aspirated from iliac crest in a heparinized vacutainer and was divided into two parts one vial was processed for conventional cytogenetic analysis [1] and another vial was sent to external lab for FISH analysis. 24 and 48 hours unstimulated bone marrow cultures were set up in duplicates. Two set of slides were prepared from each culture. On these slides Giemsa banding (GTG banding) was performed [2]. At least 20 metaphases were scored. Three cells were karyotyped according to International System for Human Cytogenetic Nomenclature (ISCN) criteria (Mitelman 2005) [3].

The patient presented with complaints of weakness, severe chronic microcytic anemia with menorrhagia, loss of appetite, body pains, fever and cough on and off. The TLC counts were low (>3,000). A full blood count showed a hemoglobin of 4.8 g/dL, white cell count of 5.0 × l09/L. Bone marrow aspiration (left posterior iliac crest) shows neutrophils 38%, lymphocytes 32%, monocytes 30%, normoblasts: 13, blasts: 07, myelocytes: 15, metamyelocytes: 07, iron stain shows increased hemosiderin grade 3-4, occasional ring sideroblast (2%) was seen and a platelet count of 567 × 109/L. Serum lysozyme was 2.6 tig/mL (normal range: 2.5-7.5, zg/mL) and urinary lysozyme was nil. Bone marrow aspiration yielded a sample of slightly increased cellularity. Erythroid precursors were virtually absent. Myelopoiesis was depressed, but megakaryocytes were well represented (dyspoietic).Abnormal mononuclear cells were present (60%), Myelopoiesis was depressed, but megakaryocytes were well represented and bone aspiration was suggestive of MDS may be RCMD/RAEB –Type-1 and Apollo Medicine, Vol. 7, No. 1, March 2010

FISH analysis was outsourced to external lab for studying deletions of 5q, 7q, 8 and 20q. The FISH was performed on interphase cells by Vysis probes for Loci specific identifier (LSI) 5q EGR1-Spec orange and D5S23-Spec green control DNA probe, 7q31 LSI46

Case Report

British (FAB) consensus conference and a more recent WHO classification system [4].

D7S486- Spectrum Orange, CEP 7-7p11.1-q11.1 Alpha Satellite- Spec green control DNA probe and CEP 8 Spec orange DNA probe and 20q12 LSI D20S108-Spec orange DNA and spec green centromereic control probe. About 500 cells were counted and analysis was performed by Applied Imaging system.

Cytogenetic analysis is usually used as a best predicted clinical outcome in the myelodysplastic syndromes (MDS). A higher prevalence of chromosomal defects and distinct defects were observed in patients with multi-lineage dysplasia and a blast cell percentage >10% [6]..

RESULTS

The authors present a case suspected to have MDS of RCMD/RAEB-Type-1 on the bone marrow aspiration and further was studied for numerical and structural anomalies of chromosomes by conventional cytogenetics and structural anomalies of chromosome numbers 5q, 7q and 20q by FISH analysis.

The cytogenetic analysis with GTG banding showed two cell populations one cell line had normal female karyotype detected in five metaphases (46, XY) (Fig. 1) and another cell line showed structural anomaly of 46, XX, t (1; 3) (p36; q21) involving chromosome number 1 and 3 in fifteen metaphases (Fig. 2).The FISH analysis showed a normal study for chromosomes numbers 5, 7, 8 and 20. (Fig.3).

Many leukemia’s, lymphomas, myeloproliferative disorders and tumors are associated with specific cytogenetic abnormalities. Identifying these abnormalities by chromosome analysis can help to define the type of cancer and in some cases subsequently indicate treatment protocols and prognosis [7]. After chromosomal analysis has identified an abnormal clone, molecular analysis utilizing fluorescent in situ hybridization (FISH) with a specific DNA probe can identify low percentages of abnormal cells in the bone marrow or peripheral blood after treatment to monitor remission states [8]. FISH testing often detects abnormal cells present before clinical symptoms reappear. Molecular analysis can provide a

DISCUSSION The myelodysplastic syndromes (MDS) encompass a heterogeneous series of hematologic conditions characterized by chronic cytopenias and abnormal cellular maturation. Risks arising from MDS are infection, bleeding, anemia, and progression to an acute myeloid leukemia which is often refractory to standard treatments [4,5]. Patients with MDS have been classified into subgroups based in large part, upon the percentage of bone marrow blasts, according to a 1982 French, American, and

Fig. 1. A normal female karyotype showing 46, XY.

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Fig. 2. Female karyotype showing 46, XYt(1;3) (p36;q21)

chromosome 13q deletion [7,8]. However, in our study we could detect structural anomaly of 46, XX, t (1; 3) (p36; q21) involving chromosome number 1 and 3 in fifteen metaphases by conventional cytogenetic analysis [12-14] which is similar to the study who had shown that significance of specific translocations in the pathogenesis of neoplasia. In some cases, it appears that the translocation, and possible subsequent activation, of an oncogene is important. This has been well demonstrated in Burkitt’s lymphoma, where the oncogene, c-myc, has been shown to be translocated from its normal position on chromosome 8 to a site within the immunoglobulin coding region on chromosome 14 [12]. There is now some evidence to suggest that the activation of two oncogenes (transforming and immortalizing) may be necessary for complete tumor development. This may be of relevance when considering the chromosomal abnormalities in preleukemic states. It is possible that alternative combinations of activation may account for differences in clinical presentation. Recently, a new oncogene, c-N-ras, has been assigned to chromosome 1 and an oncogene, c-raf, is known to be localized to chromosome 3. The exact subregional localization of these oncogenes has not yet been established. Hence, it is further suggested that cytogenetics should be performed in all the MDS cases and in cases of conventional cytogenetic failure to yield metaphases or in case of normal study it is recommended to use FISH analysis in order to detect microdeletions and

better assessment of the ratio of abnormal to normal cells present in a specimen which does not grow well in tissue culture [8]. FISH testing can also assess graft acceptance / rejection in sex-mismatched bone marrow transplants [8]. Conventional cytogenetic analysis has identified chromosome abnormalities in approximately 40-70% of de novo MDS cases and in 95% of therapy-related MDS at diagnosis, with no abnormality specific for a particular MDS subtype with the exception of the chromosome 5q deletion [9,10]. Recurrent chromosome changes in MDS include loss of chromosomes 5 or 7, deletions of chromosomes 5q or 7q, trisomy 8, and chromosome 20q deletion [7]. Hence, in the present study FISH analysis for chromosomes 5q, 7q, 8 and 20q was performed (Fig. 3). The primary utility of FISH analysis in MDS is based on the finding that 15-20% of MDS patients demonstrate a normal karyotype, yet possesses one or more clonal abnormalities of prognostic and/or therapeutic significance when analyzed by FISH [8,11]. In addition, the subset of MDS patients positive for one or more abnormalities by FISH but with a normal karyotype has demonstrated an increase in bone marrow blasts, an increased rate of leukemic transformation, and a poorer prognosis. Based on this and other studies, it is advised to use MDS FISH panel on the diagnostic specimen. The MDS FISH panel includes probes to detect -5/5q-, -7/7q-, trisomy 8, chromosome 20q deletion, chromosome 11q deletion (MLL gene), and Apollo Medicine, Vol. 7, No. 1, March 2010

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(a)

(b)

(c)

(d)

Fig. 3 (a-d) FISH analysis of chromosome 5q, 7q, 8 and 20q showing normal cell without any deletion

complex rearrangements. In this study the patient was benefited by conventional cytogenetic study and it correlated with clinical symptoms and the patients has been treated as a case of MDS and she is on follow up.

analysis is a standard practice in the evaluation of a patient with suspected myelodysplastic syndrome (MDS) and is considered an independent predictor of clinical outcome, overall survival, and progression to acute leukemia. In the current study, the utility of performing a tailored FISH panel, in addition to G-band karyotyping was evaluated. It is suggested that FISH testing is informative only in MDS cases with karyotype failure. However, conventional cytogenetic analysis forms the Gold standard as it screens

CONCLUSION FISH is very useful in detecting chromosomal alterations in MDS and it is an important complement to conventional cytogenetics. Bone marrow cytogenetic 49


Case Report

all the 46 chromosomes and FISH can be used as a supplement to this tests rather than using any of these tests as a standalone to diagnose MDS.

7. Haase D, Germing U, Schanz J, et al. New insights into the prognostic impact of the karyotype in MDS and correlation with subtypes: evidence from a core dataset of 2124 patients. Blood 2007; 110: 4385.

ACKNOWLEDGEMENTS

8. Rigolin GM, Bigoni R, Milani R, Cavazzini F, Roberti MG, Bardi A, et al. Clinical importance of interphase cytogenetics detecting occult chromosome lesions in myelodysplastic syndromes with normal karyotype. Leukemia 2001; 15: 1841-1847.

The authors acknowledge the pateint for accepting to give the sample and sharing the clinical information and the management of Apollo Hospitals for their support.

9. Boni M, Cavigliano PM, Calatroni S, Giardini I, et al. Clinical relevance of cytogenetics in myelodysplastic syndromes. Ann NY Acad Sci 2006; 1089: 395-410.

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2. Seabright M. A rapid banding technique for human chromosomes. Lancet 1971; 2:971-972.

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3. Mitelman F. ISCN. An International System for human cytogenetic nomenclature. Basel Karger 2005; 1-115.

12. Yuko Sato , Tohru Izumi , Hirakazu Kanamori, et al. t(1;3) (p36;p21) is a recurring therapy-related translocation, Cancer; 34: 186-192.

4. Bennett JM, Catovsky D, Daniel MT, et al. FAB Cooperative Group: Proposal for the classification of the myelodysplastic syndromes. Br J Haematol 1982; 51:189199.

13. Moir DJ, Jones PA, Pearson J, Duncan JR, Cook P, Buckle VJ. A new translocation, t(1;3) (p36;q21), in myelodysplastic disorders. Blood 1984; 64: 553-555.

5. Greenberg P, Cox C, LeBeau MM, et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89: 2079-2088.

14. Christiane Charrin, Amine Belhabri, Danielle TreilleRitouet, Gianina Theuil, et al. Structural rearrangements of chromosome 3 in 57 patients with acute myeloid leukemia: clinical, hematological and cytogenetic features, The Hematology Journal 2002; 3: 21-31.

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