ELSEVIER

Philadelphia Chromosome in Chronic Myelogenous Leukemia: Confirmation of Cytogenetic Diagnosis in Ph Positive and Negative Cases by Fluorescence In Situ Hybridization

Hasan Acar, Janet Stewart, and Michael J. Connor

ABSTRACT: In this study, patients with Philadelphia (Ph) negative and positive chronic myelogenous leukemia (CML) were analyzed by unicolor- and dual color- (DC-) fluorescence in situ hybridization (FISH) using abl and bcr cosmid probes. Unicolor- and DC-FISH analysis revealed a BCR-ABL fusion in a Ph-negative patient. DC-FISH to interphase nuclei revealed the BCR-ABL fusion, even though no metaphases were available for metaphase-FISH and conventional cytogenetic analysis. In the remaining cases, the findings of FISH were in agreement with the cytogenetic results. © Elsevier Science Inc., 1997

INTRODUCTION

At least 90-95% of patients with chronic myelogenous leukemia (CML) are characterized by the Philadelphia (Ph) chromosome, which arises from a translocation between chromosomes 9 and 22 [1], The ABL protooncogene, which is normally located at band 9q34, moves next to the BCR gene at band 22qll as a consequence of the translocation [2]. Approximately 2-4% of patients with morphological CML have a normal karyotype but the BCR-ABL rearrangement has been reported by molecular studies [3]. Unfortunately, identification of this rearrangement using conventional cyto- genetic analysis is impossible.

Recently, unicolor- and dual color- (DC) FISH have been shown to be useful in directly visualizing the BCR- ABL rearrangement in patients with CML, both in metaphase and interphase nuclei [4-10]. The aim of the present report was to investigate the usefulness of FISH in detecting the BCR-ABL fusion, and to confirm the cytoge- netic results. Thus we analyzed Ph positive and negative patients with CML using abl and bcr cosmid probes with unicolor- and DC-FISH.

From the Department of Medical Genetics, University of Sel~uk, (H. A.) Konya, Turkey; the Department of Medical Genetics, Royal Hospital for Sick Children,'(], S., M. J. C.) Glasgow, United Kingdom.

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

Received June 20, 1995; accepted May 20, 1996.

Cancer Genet Cytogenet 94:75-78 (1997) © Elsevier Science Inc., 1997 655 Avenue of the Americas, New York, NY 10010

MATERIALS AND METHODS

Samples Between 1991 and 1994, twelve cytogenetically Ph chromo- some negative patients with a provisional diagnosis of CML were monitored by using unicolor-FISH with the abl and bcr cosmid probes. In DC-FISH analysis of interphase nuclei and metaphase spreads, three control cases with normal karyotypes and thirteen CML cases including five cases (cases 1, 5, 7, 11, and 12), which had also been ana- lyzed by unicolor-FISH, were studied. Samples were pre- pared in accordance with current cytogenetic protocol. Metaphase spreads were also obtained from the same prep- arations as the ones used for interphase-FISH study.

Fluorescence In Situ Hybridization Locus specific abl and bcr cosmid probes [a generous gift from Dr. G. C. Grosveld, Leiden University, Holland) were labeled with biotin-ll-dUTP (Sigma) and digoxigenin-11- dUTP (Boehringer Mannheim), respectively, by nick trans- lation kit (Gibco, BRL).

Hybridization was performed as follows: The hybridiza- tion mixture was preincubated at 37°C for 60 rain after dena- turing the probe at 65°C for 10 min. The slides were denatured at 65°C for 3 rain and then the prehybridization mixture was applied to the denatured samples (slides) under a coverslip. After hybridization, two 3-rain post hybridization washes at 43°C in 2 x SCC and 50% formamide/2 × SSC were per- formed and followed by washes at 43°C in 2 x SSC for 5 rain.

For DC-FISH, the detection of the probes was per- formed according to the method described by Johnson et al. [11] with some modifications. Dual color signals were

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76 H. Acar et al.

Table 1 Unicolor-FISH findings on metaphase spreads using the abl-bcr probe: appl ica t ion in 12 pat ients wi th Ph-negative CML

Metaphase Case Cell FISH results No. source Probes (bcr-abl positive/total)

1 Blood abl 0/9 bcr 0/5

2 Marrow abl 0/15 bcr 0/5

3 Marrow abl 0/10 bcr 0/4

4 Marrow abl 0/16 bcr 0/17

5 Blood abl 0/11 bcr 0/9

6 Marrow abl 0/15 ~ bcr 0/5

7 Marrow abl no metaphases bcr no metaphases

8 Marrow abl 0/8 bcr 0/4

9 Marrow abl 0/4 bcr 0/8

10 Marrow abl 0/12 bcr 0/6

11 Marrow abl 0/7 bcr 0/4

12 Marrow abl 6/6 bcr 0/3

°Seven out of fifteen metaphases have trisomy 9.

visual ized by treating the slides with a mixture of anti- digoxigenin rhodamine and f luorescein isothiocyanate- (FITC-) avidin (Vector Laboratories). For unicolor-FISH, the hybr id ized probes were detected wi th only FITC-avi-

Table 2 DC-FISH results in pat ients wi th CML

din. The sl ides were s tained wi th counters ta in m e d i u m containing either DAPI [4 ' ,6-diamidino-2-phenyl- indole) for DC-FISH or PI (propid ium iodide) for unicolor-FISH, in mount ing solution. Sl ides were screened wi th an epif- luorescence microscope (Zeiss Axioplan) equ ipped with a CCD camera (photometr ic camera Gr3) (Digital Scientific}. For each case, as many metaphases as possible were ana- lyzed. For DC-FISH analysis, fifty in terphase nuclei hy- br id ized with abl and bcr cosmid probes were analyzed.

RESULTS

The unicolor-FISH results from CML patients wi th Ph-nega- tive are given in Table 1. One out of the twelve cases (case 12) showed the BCR-ABL fusion on the long arm of chro- mosome 22. The remaining cases were found to have nor- mal abl signal localization. In case no. 7 no metaphases were found. In case no. 6, the abl signal was detected in seven out of fifteen metaphases on the long arm of three copies of chromosome 9, which would suggest t r isomy for chromosome 9. This finding was in agreement with the con- ventional cytogenetic results. In case 10, one metaphase was found to be te t raploid wi th normal abl signal location.

DC-FISH results in control cases and cases wi th CML are shown in Table 2. In the control group, the inc idence of the red-green fusion signals in interphase nucle i as a false posi t ive was observed to be 4%. The remainder of the nucle i had two ind iv idua l red and two ind iv idua l green signals. However, no metaphases bearing the red-green fusion on either chromosome 9 or chromosome 22 were observed from those controls. In the pat ient group, the DC- FISH results on interphase nucle i showed that a red-green fusion signal was detected in at least a proport ion of nuclei from all cases known to be posit ive, and in the case where

Case Cell No. source Karyotypes

Interphase FISH results (% bcr-abl fusion)

1 Blood

2 Marrow

3 Marrow

4 Marrow

5 Blood

6 Marrow

7 Marrow

8 Marrow

9 Marrow

10 Marrow

11 Marrow

12 Marrow

13 Marrow

Controls (n = 3)

46,XY,t(9;22) 98%

46,XY,t(9;22)[30] 80%

46,XX,t(9;22)[22] 71%

46,XY,t(9;22)[7] not informative

46,XX,t(9;22)[20] 94%

46,XY,t(9;22)[ll 68%

Failed 32%

46,XY 2%

46,XX,t(9;22)[3]/46,XX[17] 60%

not analyzed 88%

46,XY,t(9;22}[14]/46,XY[1] 66%

47,XYY[14] 84%

46,XY 0%

Normal 4%

Philadelphia Chromosome in CML 77

cytogenetic analysis had failed (in case 7). The red-green fusion signals in interphase nuclei were also observed in one of the three Ph negative cases where cytogenetic analysis showed a 47,XYY karyotype (Fig. 1). This finding was confirmed on metaphase spreads, by exhibiting nine out of nine metaphases with the red-green fusion on the long arm of chromosome 22. Thus DC-FISH results in metaphase spreads and interphase nuclei were in agreement with the findings of unicolor-FISH analysis. The other Ph negative cases showed results in the control range.

DISCUSSION

Rapid detection and reliable assessment of abnormalities are very important in cancer genetics, because certain chromosomal abnormalities have been shown to be signifi- cant in terms of diagnosis and prognosis of the disease. The development of the FISH technique has allowed the use of chromosome specific DNA probes to detect chromo- somal abnormalities and rearrangements of genes on metaphase spreads and interphase nuclei.

In this study, the finding observed by unicolor-FISH generally confirmed the cytogenetic results. In contrast, both unicolor- and DC-FISH analysis on metaphase spreads and interphase nuclei were able to demonstrate that the BCR-ABL rearrangement on chromosome 22 also occurred in one Ph-negative patient with CML. Similarly, Lazaridou et al. [12] reported the localization of the bcr-abl fusion gene on chromosome 22 in Ph-negative patients with CML. In contrast, Hagemeijer et al. [13] reported two patients with Ph-negafive CML and localization of the bcr-abl fusion gene on chromosome 9. Nacheva et al. (1993), [7] found that one abl cosmid signal located to chromosome 9 and the other located to chromosome 22, but only one chromosome 22 gave a signal with the bcr cosmid, the other showed no hy- bridization signal [7]. Therefore our FISH results and pre- vious studies have demonstrated that the Ph-negative

patients with CML may have the BCR-ABL rearrangement either on chromosome 22 or on chromosome 9. This demon- strates that FISH has sensitivity equal to that of molecular techniques in showing rearrangements of the BCR and ABL genes in Ph-negative patients.

The simultaneous detection of dual probe hybridization has several advantages; including increased efficiency, smaller sample requirement, and potential for analysis of a larger number of chromosomal abnormalities [4, 9]. Tkachuk et al. [9] and Chen et al., [9] reported that application of DC-FISH on interphase nuclei is fast and sensitive, and pro- vides potential for identifying classic and variant Ph chro- mosomes. In this study, in case 7, although both the metaphase FISH study and the conventional cytogenetic analysis failed, the DC-FISH on interphase nuclei revealed the red-green fusion signals in a significant number of nu- clei.

Comparison of the results from FISH and conventional cytogenetic analysis in the present study suggests that FISH is more sensitive in detecting submicroscopic rearrange- ments. The other advantage of using FISH over conventional cytogenetic techniques is that when a sufficient number of good-quality metaphases is not available for analysis by conventional cytogenetic techniques, metaphases can be analyzed by FISH to determine the bcr-abl fusion.

In conclusion, we have found that unicolor-FISH analysis is very helpful in confirming conventional cytogenetic re- suits and have reaffirmed its important role in detecting the bcr-abl fusion event. DC-FISH analysis has additional advantages in its ability to detect such rearrangement in interphase nuclei in cases where it is impossible to obtain metaphase spreads.

This research was supported by The Scientific and Technical Research Council of Turkey (T~IBITAK-BAYG. 331.2(90) 2501}. We thank Dr. I. McNeil and Dr. I. M. Franklin for their coopera- tion and Dr. C. Chu for critical reading of the manuscript.

Figure 1 Case no 12. DC-FISH analysis using abl (green) and bcr (red) probes showing the red-green fusion signals for repre- senting of the BCR-ABL rearrangement, and single green and red signals representing the ABL and BCR genes.

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