in vivo 19: 979-982 (2005)

Hidden Abnormalities Detected by FISH in Adult Primary Myelodysplastic Syndromes

ANNA D. PANANI and VASILIKI PAPPA

The Critical Care Department, Medical School of Athens University, Research Unit, ∂vangelismos ∏Ôspital, Athens, Greece

Abstract. Acquired clonal chromosomal abnormalities are found gains or losses of genetic material may lead to a gene dosage in about 30-50 % of primary myelodysplastic syndromes (MDS). effect or to the loss of tumor suppressor genes. 8 These abnormalities are predominantly characterized by represents the most common chromosomal gain, found in total/partial chromosomal losses or gains and rarely by balanced about 8-10% of cytogenetically abnormal MDS patients, while structural aberrations. Trisomy 8 represents the most common it is observed in all FAB subgroups (1-5). The significance of chromosomal gain. In the present study, the numerical aberration +8 as a risk factor in MDS is not well established. The of chromosome 8 was evaluated by the fluorescence in situ International Prognostic Scoring System (IPSS) (6) ranked hybridization (FISH) technique in MDS, and the results compared this abnormality in the intermediate-risk group, while other with those of conventional cytogenetics. Thirty adult patients with studies showed that +8 may carry a poor prognosis (7-9). primary MDS, 17 with a normal and 13 with several Conventional cytogenetics sometimes, due to technical chromosomal abnormalities except chromosome 8, were included difficulties, cannot be informative in MDS patient evaluation. in this study. On comparing the results of FISH and conventional With regard to numerical chromosomal aberrations, a number cytogenetics, a superiority of FISH over the karyotype was detected of studies have postulated the superiority of fluorescence in in 3 cases. In one of them, further cytogenetic analysis confirmed situ hybridization (FISH) over the karyotype. Using the FISH the FISH results. Nevertheless, the FISH technique has limitations, technique, a large number of cells can be examined and detecting only abnormalities specific for the target FISH probe specific chromosomal aberrations can be detected in used. In clinical practice, conventional cytogenetics continues to interphases as well as in metaphases (10-14). be the basic technique for MDS patient evaluation. However, a In the present study, the numerical aberration of large number of metaphases, even those of poor quality, must be chromosome 8 was evaluated by the FISH technique in analyzed in each case. The FISH technique could be considered MDS and the results compared with those of conventional to be complementary to achieve a more accurate analysis. cytogenetics.

Acquired clonal chromosomal abnormalities are found in Materials and Methods about 30-50 % of adult primary myelodysplastic syndromes (MDS) and no specific cytogenetic abnormality is associated Thirty adult patients with primary MDS were included in this study, with a particular MDS French American British (FAB) for whom a cytogenetic analysis by conventional cytogenetics had been subtype. These abnormalities are predominantly characterized performed. Clinical data for the patients were not available. As many by total/partial chromosomal losses or gains and rarely by cells as possible and not fewer than 15, were cytogenetically analyzed balanced structural aberrations. Balanced aberrations, mainly in each case. Seventeen patients had a normal karyotype, while in 13 chromosomal abnormalities were detected. Patients with chromosome translocations, have been shown to result in the production of 8 abnormalities were not included in this study. FISH was applied on fusion genes that are implicated in tumorigenesis, whereas recently made slides from the methanol/acetic acid-fixed cells of all patients, as described elsewhere (15). The ·-satellite DNA probe D8Z2 specific for chromosome 8 (Cytocell Technologies, Cambridge, UK) (chromosome region 8p11.1 – q11.1) was used. The hybridization Correspondence to: Anna D. Panani, Critical Care Department, of the probe with the cellular DNA site was visualized by fluorescence Medical School of Athens University, ∂vangelismos ∏Ôspital, microscopy NIKON E600 with a triple filter DAPI/FITC/TEXAS Ipsilandou 45-47, Athens 106 76, Greece. Tel: 030-210-7259307, RED. Positive chromosome signals appeared as red spots in the Fax: 030-210-7259307, e-mail: [email protected] nuclei. A minimum of about 150-200 cells from each slide were evaluated for each case. The signals were scored using the criteria of Key Words: Fluorescence in situ hybridization, chromosome 8 Hopman et al. (16). A case was counted as aberrant if more than 10% aberrations, conventional cytogenetics, myelodysplastic syndrome. of the cell nuclei showed a loss or gain of signals for chromosome 8.

0258-851X/2005 $2.00+.40 979 in vivo 19: 979-982 (2005)

Results

In all patients with a normal karyotype, the FISH results were in accordance with cytogenetic analysis. In two cases clonal trisomy 8 was detected by FISH (Figure 1). A re-evaluation of the cytogenetic analysis, by studying a larger number of metaphases, revealed in one of the patients with FISH abnormality two cells with trisomy 8. In the second case, there was no material for further cytogenetic analysis. Among patients with an abnormal karyotype, two presented cytogenetically an extra chromosome of unidentified origin. FISH analysis in one of them showed trisomy 8. In all the remaining cytogenetically abnormal patients, no FISH abnormalities of chromosome 8 were observed.

Discussion

The role of cytogenetic analysis in the diagnosis, prognosis and follow-up of MDS patients has been well defined. Several studies have shown the prognostic value of chromosomal abnormalities in predicting survival and risk of leukemic transformation during the MDS patient’s clinical course. Patients with single trisomy 8 according to the IPSS are in the intermediate-risk cytogenetic subgroup. However, several studies showed that patients with this abnormality have a significantly increased risk of leukemic evolution, suggesting that single trisomy 8 might be segregated from the intermediate-risk IPSS cytogenetic category (6-9, 17). In clinical practice, because of the significant prognostic value of trisomy 8, it is important to detect MDS patients Figure 1. Copy number of chromosome 8 (red spots) detected by FISH in bone marrow cells of a MDS patient with a normal karyotype. Note the carrying this abnormality. Despite its importance, presence of three copies. sometimes conventional cytogenetics, because of technical problems, cannot lead to an accurate analysis. The detection of clonal chromosomal abnormalities occurring in a minority of cells might be hampered by the poor quality of the metaphases. Moreover, due to nonrandom selection of prognosis. However, a subset of them progress rapidly to the best metaphases, some chromosomal abnormalities acute myeloid . In such cases, hidden clones might occurring within bad-quality metaphases with overlapping be involved in the progression of MDS (18-22). chromosomes could be missed. Interphase FISH with Among 13 of our patients with an abnormal karyotype, specific probes provides a tool to study non-dividing cells, two presented an extra unidentified chromosome. Trisomy 8 revealing numerical as well as structural chromosomal was detected by FISH in one of them, suggesting that at abnormalities, which could otherwise remain undetected. least the pericentromeric area of chromosome 8 This study was conducted to detect, by FISH, the presence participated in the formation of this chromosome. All the of trisomy 8 in MDS patients, who presented either a normal remaining cytogenetically abnormal cases had two copies of or an abnormal karyotype but without chromosome 8 chromosome 8. abnormalities. The FISH results were compared with those of Several studies have compared conventional cytogenetics conventional cytogenetics. Among 17 patients with a normal and FISH in patients with a normal or an abnormal karyotype. karyotype, two patients presented clonal trisomy 8. However, They showed that sometimes an abnormal clone can be in one of them a further cytogenetic analysis with a larger detected in patients with a normal karyotype, while in high-risk number of analyzed metaphases confirmed the FISH results. MDS patients, according to the IPSS, there is a greater MDS patients with a normal karyotype constitute a probability for an abnormal karyotype to be detected by FISH. heterogeneous group from the biological point of view. A good correlation of the two methods has also been described According to the IPSS, these patients have a good (18-21). However, the FISH technique has limitations. Thus,

980 Panani and Pappa: Chromosome 8 in MDS chromosomal abnormalities involving any other chromosome 9 Fernandez TS, Ornellas MH, de Carralho LO, Tabak D and or chromosomal region out of the specified FISH probe target Abdelhay E: Chromosomal alterations associated with evolution can not be detected by the FISH technique. Thus, each from myelodysplastic syndrome to acute myeloid leukemia. Leuk Res 24: 839-848, 2000. method has its advantages and limitations, though in 10 Kibbelaar RE, Mulder JW, Dreef EJ, Van Kamp H, Fibbe WE, combination they may lead to a more accurate diagnosis, but Wessels JW, Beverstock GC, Haal HL and Kluin PM: Detection cost/benefit must be taken into account. of 7 and trisomy 8 in myeloid neoplasia: a comparison In our study, a superiority of FISH over the karyotype was of banding and fluorescence in situ hybridization. Blood 82: 904- detected in three cases. In one of them further cytogenetic 913, 1993. analysis confirmed the FISH results. Nevertheless, in clinical 11 Jenkins RB, Le Beau MM, Kraker WJ, Borell TJ, Stalboerger PG, practice conventional cytogenetics continues to be the basic Davis EM, Penland L, Fernald A, Espinosa R III, Schaid DJ et al: Fluorescence in situ hybridization. A sensitive method for trisomy technique for MDS patient evaluation, detecting the 8 detection in bone marrow specimens. Blood 79: 3307-3315, 1992. overwhelming majority of cytogenetic aberrations, whereas it 12 Arif M, Tanaka K, Damodaran C, Asou H, Kyo T, Dohy H and also has the potential to reveal chromosome anomalies that are Kamada N: Hidden monosomy 7 in acute myeloid leukemia and not detected by most panel FISH tests used. However, a large myelodysplastic syndrome detected by interphase fluorescence number of metaphases in each case must be studied and an in situ hybridization. Leuk Res 20: 709-716, 1996. effort must be made to karyotype all the metaphases, even 13 Flactif M, Lai JL, Preudhomme C and Fenaux P: Fluorescence those of poor quality. The FISH technique can be used in situ hybridization improves the detection of monosomy 7 in myelodysplastic syndromes. Leukemia 8: 1012-1018, 1994. whenever conventional chromosome study is unsuccessful. Also, 14 Tanaka K, Arif M, Eguchi M, Shintani T, Kumaravel TS, Asaoku in cases with a suspected cytogenetic abnormality, the FISH H, Kyo T, Dohy H and Kamada N: Interphase fluorescence in technique is complementary, leading to an accurate evaluation. situ hybridization overcomes pitfalls of G-banding analysis with special reference to underestimation of chromosomal aberration Acknowledgements rates Cancer Genet Cytogenet 115: 32-38, 1999. 15 Panani A, Ferti A, Avgerinos A and Raptis S: Numerical The technical assistance of Mrs Athanasia Babanaraki is gratefully aberrations of chromosome 8 in gastric cancer detected by acknowledged. fluorescence in situ hybridization. Anticancer Res 24: 155-160, 2004. 16 Hopman AHN, Ramaekers FCS, Raap AK, Beck JLM, Deville References P, Ploeg M and Vooijs GP: In situ hybridization as a tool to study numerical aberrations in solid bladder tumors. 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Leukemia 15: 1841-1847, 2001. for evaluating prognosis in myelodysplastic syndromes. Blood 21 Ketterling RP, Wyatt WA, VanWier SA, Law M, Hodnefield 89: 2079-2088, 1997. JM, Hanson CA and Dewald GW: Primary myelodysplastic 7 Pfeilstoker M, Reisner R, Nosslinger T, Gruner H, Nowotny H, syndrome with normal cytogenetics: utility of "FISH panel Tuchler H, Schlogl E, Pittermann E and Heinz R: Cross- testing" and M-FISH. Leuk Res 26: 235-240, 2002. validation of prognostic scores in myelodysplastic syndromes on 22 Trost D, Hidebrandt B, Müller N, Germing V and Royer- 386 patients from a single institution confirms importance of Pokora B: Hidden chromosomal aberrations are rare in primary cytogenetics. Br J Haematol 106: 455-463, 1999. myelodysplastic syndromes with evolution to acute myeloid 8 Sole F, Espinet B, Sanz GF, Cervera J, Calasanz MJ, Luno E, leukemia and normal cytogenetics. Leuk Res 28: 171-174, 2004. Prieto F, Granada I, Hernandez JM et al: Incidence, characterization and prognostic significance of chromosomal abnormalities in 640 patients with primary myelodysplastic Received June 22, 2005 syndromes. Br J Haematol 108: 346-356, 2000. Accepted August 29, 2005

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