ELSEVIER

Identification of Variant Translocations in Chronic Myeloid Leukemia by Fluorescence In Situ Hybridization

Hasan Acar, Janet Stewart, Elizabeth Boyd, and Michael J. Connor

ABSTRACT: We studied two cases of chronic myeloid leukemia (CML) having variant complex translo- cations detected by trypsin G-banding and fluorescence in situ hybridization (FISH). Application of dual color- (DC-) FISH using abl and bcr cosmid probes permitted us to detect the bcr-abl fusion event on both interphase nuclei and metapbase spread. Furthermore, FISH using combinatorial hybridization (centromeric-library and library-library probes) demonstrated the content and the position of the trans- locations in CML patients with variant (complex type) Ph-positive rearrangements. FISH analysis appears to be superior than conventional cytogenetic analysis. © Elsevier Science Inc., 1997

INTRODUCTION

Chronic myeloid leukemia (CML) is one of the most com- mon myeloproliferative disorders (MPDs) and on cytogenetic analysis, approximately 90-95% of CML patients have the standard well documented t(9;22)(q34;q11) (Philadelphia chromosome, Ph) [1, 2]. About 3-4% of Ph-positive patients with CML have variant translocations [3-5], involving another chromosome t(22;C) where C can be any chromo- some except for the Y chromosome. Cytogenetically three-, four-, and five-way variant translocations have been reported [4, 6]. Cytogenetic analysis of complex variant translo- cations is often technically difficult due to the complex chromosomal pattern and low quality of metaphase spreads, and moreover, the interpretation of chromosomal banding patterns requires highly-skilled personnel.

Recent improvements in fluorescence in situ hybridization (FISH) techniques have provided an alternative sensitive method to conventional cytogenetic analysis for the detection of specific chromosome abnormalities in hematological ma- lignancies. DC-FISH has been shown to be successful in di- rectly visualizing the bcr-abl fusion gene in metaphase and interphase nuclei from CML patients [7-11]. In addition, li- brary probes have been widely used to detect rearrangements [12-14]. We have applied unicolor and DC-FISH to detect complex translocations on metaphase spreads using centro- meric and library probes and the bcr-abl fusion event using

From the Department of Medical Genetics, University of Selguk, (H. A.) Konya, Turkey; the Department of Medical Genet- ics, Royal Hospital for Sick Children, (J. S., E. R., M. J. C.) G21 8SJ, Glasgow United tO'ndom.

Address reprint requests to: Dr. Hasan Acar, Sakarya Mah. ~erbetli sok. 10-4, 42080 Konya, Tflrkiye.

Received August 25, 1995; accepted May 21, 1996.

Cancer Genet Cytogenet 93:115-118 (1997) © Elsevier Science Inc., 1997 655 Avenue of the Americas , New York, NY 10010

abl and bcr cosmid probes on interphase nuclei from CML patients. These results were compared with conventional cy- togenetic analysis of G-banded metaphase chromosomes.

MATERIALS AND METHODS

Conventional cytogenetic analysis was performed on bone marrow (BM) cells from the two patients with CML. Metaphases were analyzed with trypsin G-banding.

To determine the bcr-abl rearrangement, locus specific abl and bcr cosmid probes (a generous gift from Dr. G. C. Grosveld, Leiden University, Holland) for chromosomes 9 and 22, respectively, were used. To detect complex trans- locations, the a-satellite centromeric DNA probes for chro- mosomes 3 (CAMBIO), 8 (PJM 8), and 22 (D22Z2), whole chromosome specific DNA (library) probes for chromo- somes 6, 8, 22 (CAMBIO) and 9 labeled with fluorochrome red (GIBCO-BRL) were used. The labeling of unlabeled probes was achieved by either biotin-dUTP (Sigma) or digoxigenin-dUTP (Boehringer-Mannheim) using a nick- translation kit (GIBCO-BRL).

The hybridization protocol followed that of Pinkel et al. [15] with minor modifications. Detection of the probes was performed according to the method described by Johnson et al. [16] with some modifications. The hybridization signals were revealed by treating the slides with the mixture of anti digoxigenin rhodamine (Boehringer-Mannheim) and fluores- cein isothiocyanate (FITC) avidin (Vector Laboratories). Probes labeled with biotin-dUTP were detected only with FITC-avidin. The slides were stained with counterstain medium containing DAPI (4',6-diamidino-2-phenyl-indole) in mounting solution. Slides were screened with an epi- fluorescence microscope (Zeiss Axioplan) equipped with a cooled charge-coupled device (CCD) camera (photomet-

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116 H. Acar et al.

Figure 1 DC-FISH with centromeric probe for chromosome 8 (red) and chromosome 6 library (green) probe in a metaphase spread; blue DAPI counterstaining. The arrow indicates deriva- tive chromosome 6 (partially painted with green and unpainted on the distal part). The arrowhead indicates derivative chromo-

ric camera Gr3) (Digital Scientific). Metaphases were exam- ined after the hybridization with a combination of probes (centromeric and library probes) were examined. Fifty inter- phase nuclei hybridized with abl and bcr cosmid probes were analysed to detect the bcr-abl fusion on in terphase nuclei.

RESULTS

G-Banding Analysis Metaphases from both pat ients were analyzed with t rypsin G-banding and their karyotypes were defined as follows:

Figure 2 DC-FISH with chromosome 8 library (green) and chromosome 9 library (red) probes in a metaphase spread; blue DAPI counterstaining. The arrow indicates derivative chromo- some 9 (partially painted with red, and with green on the distal part). The arrow head indicates derivative chromosome 8 (par- tially painted with green, unpainted on the distal part).

Patient 1: 46,XX,t(6;8;9;22)(q25;q22;q34;q11) [13]/46,XX[2]; and in a subsequent specimen fIom her, all metaphases were found to have 46,XX,t(6;8;9;22)(q25;q22;q34;q11). Patient 2: 46,XX,t(3;22)(q13;q12),t(9;22;17)(q34;q11;p13). In addition, in this case, a dele t ion of the long arm of chromosome 20 (20q-) was observed.

Fluorescence in Situ Hybridization DC-FISH analysis to in terphase nuclei using abl and bcr cosmid probes revealed 84%, 98%, and 96% of nuclei show- ing the red-green fusion, indicat ing the bcr-abl fusion event in the first pat ient and the subsequent specimen of this pat ient and the second patient, respectively.

DC-FISH is used to reveal the chromosomal rearrangement on metaphase spreads from the patients. In the first case, a combinat ion of the centromeric probe for chromosome 8 (detected with rhodamine) and the library probe for chromo- some 6 (detected with FITC) showed a normal chromosome 6 pa in ted green but the other homologue was rearranged, because the long arm of one homologue of chromosome 8 was shown to be par t ia l ly pa in ted with green (with a red centromeric region) indicating the existence of chromosome 6 mater ia l (Fig. 1). Fur ther invest igat ion, using a combi- na t ion of the l ibrary probe for chromosome 9 (detected with rhodamine) and the l ibrary probe for chromosome 8 (detected with FITC) showed one chromosome 9 pa in ted red, and the other homologue with part of the long arm pain ted green indicat ing a rearrangement (Fig. 2). The re-

Figure 3 FISH with centromeric probe for chromosome 3 (yel- low) and chromosome 22 library (yellow) probes in a metaphase spread; red propidium iodide counterstaining. The arrow indi- cates derivative chromosome 3 (yellow centromere, and partially painted with yellow on the distal part). The big arrow head indi- cates derivative chromosome 17 (partially painted yellow on the distal part). The long arrows indicate the derivative chromosome 22s (partially painted yellow on near to the centromeric area of chromosome 22, and Ph chromosome). The small arrowhead indicates chromosome 22 material (yellow on the distal part of chromosome 9).

Variant Translocations in CML 117

sults also showed one chromosome 8 painted green and the other homologue with whole of the short arm and part of the long arm painted green, but a small area of un- painted material was seen on the distal end of the long arm indicating a rearrangement. These results would be consistent with the transfer of material from 8q to 9q and the attachment of additional material from another chro- mosome (specifically from the long arm of chromosome 8 shown in Fig. 1) to the breakpoint on 8q (Fig. 2). Hybrid- ization with the library probe for chromosome 22 was un- informative because of very high cross-hybridization with the centromeric areas of the other acrocentric chromosomes.

In the second case, based on conventional cytogenetic analysis two sets of combinatorial probe hybridizations were carried out. In the first set the library probe for chromo- some 3 (detected with FITC) and a satellite centromeric probe for chromosome 22 (detected with rhodamine) were used simultaneously. This hybridization revealed the trans- location from one homologue of chromosome 3 to chromo- some 22. The second set was used for further investigation. Simultaneous application of the library probe for chromo- some 22 (detected with FITC) and the satellite centromeric probe for chromosome 3 (detected with FITC) with uni- color FISH showed chromosome 22 material on the der(22) t(9;17;22)(q34;p13;q11.2) (Ph chromosome), on the distal part of the long arm der(9)t(9;17;22) (q34;p13;q11.2), on the short arm of the der(17)t(9;22;17)(q34;q11.2;p13), and also on the distal part of the long arm der(22) t(3;22)(q13;q12) and on part of the long arm of the der(3)t(3;22)(q13;q12) (Fig. 3). The nature of the involvement of chromosome 17 in the com- plex Ph chromosome translocation as well as the reciprocal translocation between the other homologue of chromosome 22 and chromosome 3 is shown in Figure 3.

DISCUSSION

Variant type Ph translocations have been described using cytogenetic analyses [2, 4, 17] and molecular techniques [18, 19]. However, the origin and breakpoints in variant Ph rearrangements have not always been clear because of low quality metaphases. In this study, the application of DC-FISH to interphase nuclei was able to demonstrate that the bcr- abl rearrangement occurred in variant Ph translocations as shown by red-green fusion. There is no doubt that the bcr and abl probes can be used to identify the BCR-ABL fusion, as reported in the present and previous studies [7-9]. Of course, this would not be expected to be useful in defining all the derivative products in variant translocations, even though the application of these probes (abl and bcr cosmid probes) has the obvious advantage of delineating complex rearrangement in metaphase cells by showing the localiza- tion of the abl and bcr signals, in addition to analysis of the bcr-abl fusion on interphase nuclei when there is lack of metaphase spreads. In the present study, to define the nature of the variant translocations, we also used a combination of centromeric probe-library probe, and library probe- library probe combinations to metaphase spreads. Combi- natorial probe hybridizations, in both cases, demonstrated the actual part of complex translocations involving chromo- somes 6, 8, 9, and 22 in the first patient, and chromosomes

9, 22, and 17 in the second patient. These results suggest that combinatorial probe hybridization is very useful for defining both the standard translocation, t(9;22), and the variant form of Ph translocations. All FISH findings were in broad agreement with the conventional cytogenetic results.

Comparison of the results from conventional cytogenetic analysis and FISH in the present study suggests that FISH is a more sensitive method to detect variant translocations in CML. In this respect, when sufficient numbers of good- quality metaphases are not available for analysis by con- ventional cytogenetic methods, available metaphases may be used for FISH analysis to identify the variant transloca- tions without the necessity of qualified persons to analyze cells. In addition to the aforementioned advantages, inter- phase-FISH allows detection of the bcr-abl fusion in cases where metaphase spread is insufficient for conventional cytogenetic analysis. We conclude that FISH is an effective screening technique for diagnostic and follow-up purposes.

This research was supported by The Scientific and Technical Research Council of Turkey (TUBITAK-BAYG. 331.2(90) 2501). We thank Dr. C. Chu for critical discussion of the manuscript.

REFERENCES

1. Rowley ID (1973): A new consistent chromosomal abnormal- ity in chronic myelogenous leukemia identified by quina- crine fluorescence and Giemsa staining. Nature 243:290-293.

2. Sandberg AA (1991): The Chromosomes in Human Cancer and Leukemia. 2nd Ed., Elsevier Science Publishing Co., New York, pp. 104-463.

3. De Braekeloer M (1987): Variant Philadelphia translocations in chronic myeloid leukemia. Cytogenet Cell Genet 44: 215-222.

4. Ishihara T, Minamihisamatsu M (1988): The Philadelphia chromosome. Consideration based on studies of variant translocation. Cancer Genet Cytogenet 32:75-92.

5. Zaccara A, Testoni N, Tassinari A, Celso B, Rassool F, Saglio G, Guerrasia A, Rosti G, Tura S (1989): Cytogenetic and molecular studies in patients with chronic myeloid leukemia and variant Philadelphia translocations. Cancer Genet Cyto- genet 42:191-201.

6. Hagemeijer A, Bartram CR, Smit EME, Van Agthhoven AJ, Bootsma D (1984): Is the chromosomal region 9q34 always involved in variations of the Ph' translocation? Cancer Genet Cytogenet 13: 1-16.

7. Amoldus EPJ, Wiegant J, Noordermeer IA, Wessels JM, Bev- erstock GC, Grosveld GC, Van der Ploeg M, Raap AK (1990): Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet Cell Genet 54:108-111.

8. Tkachuk DC, Westbrook CA, Andreeff M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Render A, Gray J, Pinkel D (1990). Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization. Science 250:559-562.

9. Chen Z, Morgan R, Berger CS, Pearce-Birge L, Stone JF, Sand- berg AA (1993): Identification of masked and variant Ph (complex type) translocation in CML and classic Ph in AML and ALL by fluorescence in situ hybridization with the use of bcr-abl cosmid probes. Cancer Genet Cytogenet 70:103-107.

10. Nacheva E, Holloway T, Brown K, Bloxham D, Green RA (1993): Philadelphia-negative chronic myeloid leukaemia: Detection by FISH of bcr-abt fusion gene localised either to chromosome 9 or chromosome 22. Br J of Haematol 87:409--412.

11. Zhang J, Meltzer P, Jenkins R, Guan X-Y, Trent J (1993): Application of chromosome microdissection probes for elu-

118 H. Acar et al.

cidation of bcr-abl fusion and variant Philadelphia chromo- some translocations in chronic myelogenous leukaemia. Blood 81:3365-3371.

12. Zhao L, Kontarjian HM, Oort JV, Cork A, Turjillo JM, Liang JC (1993): Detection of residual proliferating leukaemic cells by fluorescence in situ hybridization in CML patients in complete remission after interferon treatment. Leukemia 7:168-171.

13. Seong DC, Liu P, Siciliana J, Zhao Y, Cork A, Henske E, War- burton D, Yu M-T, Champlin R, Trujillo JM, Deisseroth A, Siciliano MJ (1993): Detection of variant Ph-positive chronic myelogenous leukemia involving chromosomes 1, 9, and 22 by fluorescence in situ hybridization. Cancer Genet Cytoge- net 65:100-103.

14. Calapse G, Stuppia L, Guanciali F, Peila R, Morizio E, Liber- ati AM, Spadana A, Lorenzo RD, Donti E, Antonucci A, Palka G (1994): Complex translocations of the Ph chromosome and Ph negative CML arise from similar mechanisms, as evi- denced by FISH analysis. Cancer Genet Cytogenet 78:153-159.

15. Pinkel D, Straume T, Gray JM (1986): Cytogenetic analysis

using high-sensivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2943-2938.

16. Johnson CV, McNeil Carter KC and Lawrance JB (1991): A sim~ pie, rapid technique for precise mapping of multiple sequence in two colors using a single optical filter set. GATA 8:75-76.

17. Sessarego M, Fuggazza G, Bruzzone R, Patrone F (1994): Vari- ant Philadelphia chromosome translocations are frequently associated with additional structural abnormalities. Cancer Genet Cytogenet 73:57-59.

18. Hagemeijer A, Buijs A, Smit E, Janssen B, Creemers G-J, Plas DVd, Grosveld G (1993): Translocation of BCR to chromo- some 9: A new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leu- kemia. Genes Chromosom Cancer 8:237-245.

19. Macro, Saab P, Lin JH, DeSalvo AT, Shah HO, Verma RS (1993): Direct visualization of the transposed abl gene in duplicated masked Ph chromosome. Genes Chromosom Can- cer 8:127-130.