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Philadelphia chromosome in chronic myelogenous leukemia: Confirmation of cytogenetic diagnosis in Ph positive and negative cases by fluorescence in situ hybridization
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
MATERIALS AND METHODS
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 cytogenetic analysis is impossible. Recently, unicolor- and dual color- (DC) FISH have been shown to be useful in directly visualizing the BCRABL 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 cytogenetic results. Thus we analyzed Ph positive and negative patients with CML using abl and bcr cosmid probes with unicolor- and DC-FISH.
Samples Between 1991 and 1994, twelve cytogenetically Ph chromosome 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 analyzed by unicolor-FISH, were studied. Samples were prepared in accordance with current cytogenetic protocol. Metaphase spreads were also obtained from the same preparations as the ones used for interphase-FISH study.
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
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-11dUTP (Boehringer Mannheim), respectively, by nick translation kit (Gibco, BRL). Hybridization was performed as follows: The hybridization mixture was preincubated at 37°C for 60 rain after denaturing 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 performed and followed by washes at 43°C in 2 x SSC for 5 rain. For DC-FISH, the detection of the probes was performed according to the method described by Johnson et al. [11] with some modifications. Dual color signals were
0165-4608/97/$17.00 PII S0165-4608(96)00167-7
76 Table 1
H. Acar et al. Unicolor-FISH findings on metaphase spreads using the abl-bcr probe: a p p l i c a t i o n in 12 patients with Ph-negative CML
Case No.
Cell source
1
Blood
2
Marrow
3
Marrow
4
Marrow
5
Blood
6
Marrow
7
Marrow
8
Marrow
9
Marrow
10
Marrow
11
Marrow
12
Marrow
Metaphase FISH results (bcr-abl positive/total)
Probes abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr
0/9 0/5 0/15 0/5 0/10 0/4 0/16 0/17 0/11 0/9 0/15 ~
RESULTS
0/5
no metaphases no metaphases 0/8 0/4 0/4 0/8 0/12 0/6 0/7 0/4 6/6 0/3
°Seven out of fifteen metaphases have trisomy 9. visualized by treating the slides with a mixture of antidigoxigenin r h o d a m i n e and fluorescein isothiocyanate(FITC-) avidin (Vector Laboratories). For unicolor-FISH, the h y b r i d i z e d probes were detected w i t h only FITC-aviTable 2
din. The slides were stained w i t h counterstain m e d i u m containing either DAPI [4',6-diamidino-2-phenyl-indole) for DC-FISH or PI ( p r o p i d i u m iodide) for unicolor-FISH, in mounting solution. Slides were screened w i t h an epifluorescence microscope (Zeiss Axioplan) e q u i p p e d with a CCD camera (photometric camera Gr3) (Digital Scientific}. For each case, as m a n y metaphases as possible were analyzed. For DC-FISH analysis, fifty interphase nuclei hyb r i d i z e d with abl and bcr c o s m i d probes were analyzed.
The unicolor-FISH results from CML patients with Ph-negative are given in Table 1. One out of the twelve cases (case 12) s h o w e d the BCR-ABL fusion on the long arm of chrom o s o m e 22. The remaining cases were found to have normal 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 c h r o m o s o m e 9, w h i c h w o u l d suggest trisomy for chromosome 9. This finding was in agreement with the conventional cytogenetic results. In case 10, one metaphase was found to be tetraploid with normal abl signal location. DC-FISH results in control cases and cases w i t h CML are shown in Table 2. In the control group, the incidence of the red-green fusion signals in interphase nuclei as a false positive was observed to be 4%. The r e m a i n d e r of the nuclei had two i n d i v i d u a l red and two i n d i v i d u a l green signals. However, no metaphases bearing the red-green fusion on either c h r o m o s o m e 9 or c h r o m o s o m e 22 were observed from those controls. In the patient group, the DCFISH results on interphase nuclei s h o w e d that a red-green fusion signal was detected in at least a proportion of nuclei from all cases k n o w n to be positive, and in the case where
DC-FISH results in patients w i t h CML Interphase FISH results (% bcr-abl fusion)
Case No.
Cell source
1
Blood
46,XY,t(9;22)
98%
2
Marrow
46,XY,t(9;22)[30]
80%
Karyotypes
3
Marrow
46,XX,t(9;22)[22]
71%
4
Marrow
46,XY,t(9;22)[7]
not informative
5
Blood
46,XX,t(9;22)[20]
94%
6
Marrow
46,XY,t(9;22)[ll
68%
7
Marrow
Failed
32%
8
Marrow
46,XY
2%
9
Marrow
46,XX,t(9;22)[3]/46,XX[17]
60%
10
Marrow
not analyzed
88%
11
Marrow
46,XY,t(9;22}[14]/46,XY[1]
66%
12
Marrow
47,XYY[14]
84%
13
Marrow
46,XY
0%
Normal
4%
Controls (n = 3)
Philadelphia Chromosome in CML 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 significant 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 chromosomal 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 hybridization signal [7]. Therefore our FISH results and previous studies have demonstrated that the Ph-negative
Figure 1 Case no 12. DC-FISH analysis using abl (green) and bcr (red) probes showing the red-green fusion signals for representing of the BCR-ABL rearrangement, and single green and red signals representing the ABL and BCR genes.
77 patients with CML may have the BCR-ABL rearrangement either on chromosome 22 or on chromosome 9. This demonstrates 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 provides potential for identifying classic and variant Ph chromosomes. 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 nuclei. Comparison of the results from FISH and conventional cytogenetic analysis in the present study suggests that FISH is more sensitive in detecting submicroscopic rearrangements. 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 resuits 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 cooperation and Dr. C. Chu for critical reading of the manuscript.
REFERENCES 1. Rowley JD (1973): A consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence Giemsa staining. Nature 243:290-293. 2. Groffen J, Stephenson JR, Heisterkamp N, deKlein A, Bartram CR, Grosveld G (1984): Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93-99. 3. Van der Plas D, Grosveld G, Hagemeijer A (1991): Review of clinical cytogenetic and molecular aspects of Ph negative CML. Cancer Genet Cytogenet 52:143-156. 4. Arnoldus EPJ, Wiegant J, Noordermeer IA, Wessels JM, Beverstock GC, Grosveld GC, Van der Ploeg M, Raap AK (1990): Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet Cell Genet 54:108-111. 5. Calabrese G, Stuppia L, Franchi PG, Peila R, Morizio E, Liberrati AM, Spadano A, Di Lorenzo R, Donti E, Antonucci A, Palka G (1994): Complex translocations of the Ph chromosome and Ph negative CML arise from similar mechanisms, as evidenced by FISH analysis. Cancer Genet Cytogenet 78:153-159. 6. Chen Z, Morgan R, Berger CS, Pearce-Birge L, Stone JF, Sandberg AA (1993): Identification of masked and variant Ph (complex type) translocations in CML and classic Ph in AML
78
7.
8.
9.
10.
H. Acar etal. and ALL by fluorescence in situ hybridization with the use of bcr/abl cosmid probes. Cancer Genet Cytogenet 70:103-107. Nacheva E, Holloway T, Brown K, Bloxham D, Green AR (1993): Philadelphia-negative chronic myeloid leukaemia: Detection by FISH of bcr-abl fusion gene localised either to chromosome 9 or chromosome 22. British Haemat 87:409-412. Nylund ST, Ruutu T, Saarinen U, Knuutila S (1994): Metaphase fluorescence in situ hybridization (FISH) in the follow-up of 60 patients with haemopoietic malignancies. British Haemat 88:778-783. Tkachuk DC, Westbrook CA, Andreeff M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Render A, Gray l, Piukel D (1990): Detection of bcr-abl fusion in chronic myelogeneous leukaemia by in situ hybridization. Science 250:559-562. Zhao L, Kantarjian HM, Oort JV, Cork A, Trujillo JM, Liang JC
(1993): Detection of residual proliferating leukaemic cells by fluorescence in situ hybridisation in CML patients in complete remission after interferon treatment. Leukemia 7:168-171. 11. Johnson CV, McNeil Carter KC, Lawrance JB (1991): A simple, rapid technique for precise mapping of multiple sequence in two colors using a single optical filter set. GATA 8:75-76. 12. Lazaridou A, Chase A, Garicochea B, Diamond J, Goldman JM (1993): Lack of reciprocal translocation in bcr-abl positive Ph-negative CML. Blood 82:(suppl.) 40a. 13. Hagemeijer A, Buijs A, Smit E, Janssen B, Creemers GJ, Van der Plas D, Grosveld G. (1993): Translocation of BCR chromosome 9: a new cytogenetic variant detected by FISH in two Ph-negative BCR-positive patients with chronic myeloid leukaemia. Genes Chromosom Cancer 8:237-245.
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
MATERIALS AND METHODS
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 cytogenetic analysis is impossible. Recently, unicolor- and dual color- (DC) FISH have been shown to be useful in directly visualizing the BCRABL 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 cytogenetic results. Thus we analyzed Ph positive and negative patients with CML using abl and bcr cosmid probes with unicolor- and DC-FISH.
Samples Between 1991 and 1994, twelve cytogenetically Ph chromosome 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 analyzed by unicolor-FISH, were studied. Samples were prepared in accordance with current cytogenetic protocol. Metaphase spreads were also obtained from the same preparations as the ones used for interphase-FISH study.
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
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-11dUTP (Boehringer Mannheim), respectively, by nick translation kit (Gibco, BRL). Hybridization was performed as follows: The hybridization mixture was preincubated at 37°C for 60 rain after denaturing 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 performed and followed by washes at 43°C in 2 x SSC for 5 rain. For DC-FISH, the detection of the probes was performed according to the method described by Johnson et al. [11] with some modifications. Dual color signals were
0165-4608/97/$17.00 PII S0165-4608(96)00167-7
76 Table 1
H. Acar et al. Unicolor-FISH findings on metaphase spreads using the abl-bcr probe: a p p l i c a t i o n in 12 patients with Ph-negative CML
Case No.
Cell source
1
Blood
2
Marrow
3
Marrow
4
Marrow
5
Blood
6
Marrow
7
Marrow
8
Marrow
9
Marrow
10
Marrow
11
Marrow
12
Marrow
Metaphase FISH results (bcr-abl positive/total)
Probes abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr abl bcr
0/9 0/5 0/15 0/5 0/10 0/4 0/16 0/17 0/11 0/9 0/15 ~
RESULTS
0/5
no metaphases no metaphases 0/8 0/4 0/4 0/8 0/12 0/6 0/7 0/4 6/6 0/3
°Seven out of fifteen metaphases have trisomy 9. visualized by treating the slides with a mixture of antidigoxigenin r h o d a m i n e and fluorescein isothiocyanate(FITC-) avidin (Vector Laboratories). For unicolor-FISH, the h y b r i d i z e d probes were detected w i t h only FITC-aviTable 2
din. The slides were stained w i t h counterstain m e d i u m containing either DAPI [4',6-diamidino-2-phenyl-indole) for DC-FISH or PI ( p r o p i d i u m iodide) for unicolor-FISH, in mounting solution. Slides were screened w i t h an epifluorescence microscope (Zeiss Axioplan) e q u i p p e d with a CCD camera (photometric camera Gr3) (Digital Scientific}. For each case, as m a n y metaphases as possible were analyzed. For DC-FISH analysis, fifty interphase nuclei hyb r i d i z e d with abl and bcr c o s m i d probes were analyzed.
The unicolor-FISH results from CML patients with Ph-negative are given in Table 1. One out of the twelve cases (case 12) s h o w e d the BCR-ABL fusion on the long arm of chrom o s o m e 22. The remaining cases were found to have normal 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 c h r o m o s o m e 9, w h i c h w o u l d suggest trisomy for chromosome 9. This finding was in agreement with the conventional cytogenetic results. In case 10, one metaphase was found to be tetraploid with normal abl signal location. DC-FISH results in control cases and cases w i t h CML are shown in Table 2. In the control group, the incidence of the red-green fusion signals in interphase nuclei as a false positive was observed to be 4%. The r e m a i n d e r of the nuclei had two i n d i v i d u a l red and two i n d i v i d u a l green signals. However, no metaphases bearing the red-green fusion on either c h r o m o s o m e 9 or c h r o m o s o m e 22 were observed from those controls. In the patient group, the DCFISH results on interphase nuclei s h o w e d that a red-green fusion signal was detected in at least a proportion of nuclei from all cases k n o w n to be positive, and in the case where
DC-FISH results in patients w i t h CML Interphase FISH results (% bcr-abl fusion)
Case No.
Cell source
1
Blood
46,XY,t(9;22)
98%
2
Marrow
46,XY,t(9;22)[30]
80%
Karyotypes
3
Marrow
46,XX,t(9;22)[22]
71%
4
Marrow
46,XY,t(9;22)[7]
not informative
5
Blood
46,XX,t(9;22)[20]
94%
6
Marrow
46,XY,t(9;22)[ll
68%
7
Marrow
Failed
32%
8
Marrow
46,XY
2%
9
Marrow
46,XX,t(9;22)[3]/46,XX[17]
60%
10
Marrow
not analyzed
88%
11
Marrow
46,XY,t(9;22}[14]/46,XY[1]
66%
12
Marrow
47,XYY[14]
84%
13
Marrow
46,XY
0%
Normal
4%
Controls (n = 3)
Philadelphia Chromosome in CML 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 significant 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 chromosomal 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 hybridization signal [7]. Therefore our FISH results and previous studies have demonstrated that the Ph-negative
Figure 1 Case no 12. DC-FISH analysis using abl (green) and bcr (red) probes showing the red-green fusion signals for representing of the BCR-ABL rearrangement, and single green and red signals representing the ABL and BCR genes.
77 patients with CML may have the BCR-ABL rearrangement either on chromosome 22 or on chromosome 9. This demonstrates 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 provides potential for identifying classic and variant Ph chromosomes. 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 nuclei. Comparison of the results from FISH and conventional cytogenetic analysis in the present study suggests that FISH is more sensitive in detecting submicroscopic rearrangements. 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 resuits 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 cooperation and Dr. C. Chu for critical reading of the manuscript.
REFERENCES 1. Rowley JD (1973): A consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence Giemsa staining. Nature 243:290-293. 2. Groffen J, Stephenson JR, Heisterkamp N, deKlein A, Bartram CR, Grosveld G (1984): Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93-99. 3. Van der Plas D, Grosveld G, Hagemeijer A (1991): Review of clinical cytogenetic and molecular aspects of Ph negative CML. Cancer Genet Cytogenet 52:143-156. 4. Arnoldus EPJ, Wiegant J, Noordermeer IA, Wessels JM, Beverstock GC, Grosveld GC, Van der Ploeg M, Raap AK (1990): Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet Cell Genet 54:108-111. 5. Calabrese G, Stuppia L, Franchi PG, Peila R, Morizio E, Liberrati AM, Spadano A, Di Lorenzo R, Donti E, Antonucci A, Palka G (1994): Complex translocations of the Ph chromosome and Ph negative CML arise from similar mechanisms, as evidenced by FISH analysis. Cancer Genet Cytogenet 78:153-159. 6. Chen Z, Morgan R, Berger CS, Pearce-Birge L, Stone JF, Sandberg AA (1993): Identification of masked and variant Ph (complex type) translocations in CML and classic Ph in AML
78
7.
8.
9.
10.
H. Acar etal. and ALL by fluorescence in situ hybridization with the use of bcr/abl cosmid probes. Cancer Genet Cytogenet 70:103-107. Nacheva E, Holloway T, Brown K, Bloxham D, Green AR (1993): Philadelphia-negative chronic myeloid leukaemia: Detection by FISH of bcr-abl fusion gene localised either to chromosome 9 or chromosome 22. British Haemat 87:409-412. Nylund ST, Ruutu T, Saarinen U, Knuutila S (1994): Metaphase fluorescence in situ hybridization (FISH) in the follow-up of 60 patients with haemopoietic malignancies. British Haemat 88:778-783. Tkachuk DC, Westbrook CA, Andreeff M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Render A, Gray l, Piukel D (1990): Detection of bcr-abl fusion in chronic myelogeneous leukaemia by in situ hybridization. Science 250:559-562. Zhao L, Kantarjian HM, Oort JV, Cork A, Trujillo JM, Liang JC
(1993): Detection of residual proliferating leukaemic cells by fluorescence in situ hybridisation in CML patients in complete remission after interferon treatment. Leukemia 7:168-171. 11. Johnson CV, McNeil Carter KC, Lawrance JB (1991): A simple, rapid technique for precise mapping of multiple sequence in two colors using a single optical filter set. GATA 8:75-76. 12. Lazaridou A, Chase A, Garicochea B, Diamond J, Goldman JM (1993): Lack of reciprocal translocation in bcr-abl positive Ph-negative CML. Blood 82:(suppl.) 40a. 13. Hagemeijer A, Buijs A, Smit E, Janssen B, Creemers GJ, Van der Plas D, Grosveld G. (1993): Translocation of BCR chromosome 9: a new cytogenetic variant detected by FISH in two Ph-negative BCR-positive patients with chronic myeloid leukaemia. Genes Chromosom Cancer 8:237-245.