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bcr Fusion Gene on Chromosome 9 in Ph-Negative Chronic Myelogenous Leukemia
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The ABL/BCR Fusion Gene on Chromosome 9 in Ph-Negative Chronic Myelogenous Leukemia: A Case for Vigilance in Fluorescence In Situ Hybridization Interpretation Wei-Tong Hsu, Harvey Preisler, Katarina Szego, Rita Sprudzs, and Xue-Zhi Gao
ABSTRACT: We report cytogenetic, fluorescence in situ hybridization (FISH), and molecular analysis in a case of Ph-negative chronic myelogenous leukemia patient with ABL/BCR fusion gene on chromosome 9 and a disparate FISH signal pattern using two commercially available bcr/abl probes (Vysis, Inc. and Oncor, Inc.). Cytogenetic analysis revealed a 46,XX normal female karyotype. FISH studies using Vysis LSI bcr/abl probe in interphase cells demonstrated a BCR/ABL fusion pattern, similar to that of m-BCR/ABL fusion found in acute lymphoblastic leukemia. However, examination of metaphases revealed the ABL/BCR fusion signal on one of the chromosomes 9, an ABL signal on the other chromosome 9, and two BCR signals of different sizes on each of the chromosomes 22. Subsequently, a FISH study with the Oncor major (M)-bcr/abl translocation probe confirmed the ABL/BCR fusion signal on chromosome 9 in addition to an ABL signal and a BCR signal located on chromosomes 9 and 22, respectively. Molecular studies (RT-PCR) revealed a rearrangement of the M-BCR region and expression of a chimeric bcr/abl mRNA of b3a2 configuration. This case suggests that it is imperative to have a full understanding of both the capabilities and the limitations of bcr/abl translocation probes and that FISH interphase signals should be confirmed on metaphase spreads for accurate diagnosis. © Elsevier Science Inc., 1998
INTRODUCTION The Philadelphia chromosome (Ph), due to t(9;22)(q34.1; q11.2), is the cytogenetic hallmark of chronic myelogenous leukemia (CML) and is observed in more than 90% of the cases. In 5–10% of CML patients, the Ph chromosome is derived from rearrangements, either simple or complex variant translocations, other than the standard t(9:22) [1]. In simple variant translocation, the segment lost from 22q is translocated to a chromosome other than chromosome 9, whereas three or more chromosomes take part in complex variant translocations. Molecular studies
From the Department of Pediatrics (W.-T. H.), the Rush Cancer Institute (H. P., X.-Z. G.), and the Rush Medical Laboratory (W.-T. H., K. S., R. S.), Rush-Presbyterian-St. Luke’s Medical Center, Rush Medical College, Chicago, Illinois, USA. Address reprint requests to: Dr. Wei-Tong Hsu, Section of Genetics, Department of Pediatrics, Rush-Presbyterian-St. Luke’s Medical Center, 1750 W. Harrison, Room 1509 Jelke Building, Chicago, IL 60612. Received April 25, 1997; accepted October 20, 1997. Cancer Genet Cytogenet 104:57–60 (1998) Elsevier Science Inc., 1998 655 Avenue of the Americas, New York, NY 10010
have shown that in both classic and the variant Ph translocations, the ABL oncogene from chromosome 9q34 is relocated to 22q11 adjacent to the BCR gene [2], giving rise to a chimeric BCR/ABL fusion gene on the derivative chromosome 22 [3]. The BCR/ABL fusion gene is transcribed as a large chimeric RNA that is spliced into an 8.5-kb mRNA with b2a2 and b3a2 configurations [4]. This mRNA is translated to a 210-kD BCR/ABL protein that plays a crucial role in the pathogenesis of CML. A minority (5–10%) of CML patients show no Ph chromosome cytogenetically and are classified as having Ph-negative CML. In a substantial subgroup of Ph-negative patients, molecular investigations have demonstrated the presence of the BCR/ABL fusion gene on chromosome 22 that is indistinguishable at the molecular level from the BCR/ABL gene found in classical Ph-positive CML [5–7]. This BCR/ABL gene rearrangement and its localization can be visualized microscopically by fluorescence in situ hybridization (FISH) by dual-color BCR/ABL translocation probes [8]. Recently, four cases of Ph-negative CML patients with a fusion between the ABL and BCR genes lo-
0165-4608/98/$19.00 PII S0165-4608(97)00430-5
58 cated on chromosome 9 rather than on chromosome 22 have been reported [9–11]. Here, we report a detailed cytogenetic and molecular analysis in a case of Ph-negative CML with the ABL/BCR fusion gene on chromosome 9 and a disparate FISH signal pattern obtained with the use of commercially available dual-color bcr/abl translocation probes from Vysis (Downers Grove, IL) and Oncor (Gaithersburg, MD).
W.-T. Hsu et al. cis-retinoic acid in induction and maintenance phases. In the course of the treatment, follow-up FISH studies revealed the ABL/BCR genes on chromosome 9 in all 20 metaphases examined. Her disease was stable and remained in chronic phase for 8 months. Then she became intolerant to cytosine arabinoside and retinoic acid therapy. A peripheral blood stem-cell transplant was performed with prior administration of G-CSF and GM-CSF to mobilize CD 341 cells. Currently, this patient remains well 3 months after transplant.
CASE REPORT A 55-year-old woman was referred to our clinic for a second opinion and treatment. The patient complained of left upper quadrant pain, fever, and weight loss. Physical examination revealed her spleen to be 9 cm below the left costal margin. At that time, she was found to have a white blood cell (WBC) count of 270,000 with a marked left shift showing all levels of myeloid maturation, including 7% blasts on the peripheral blood smear. Bone marrow biopsy showed marked hypercellularity consisting of immature and mature myeloid cells. There were clusters of immature cells constituting 10–20% of the marrow. The biopsy was considered to be consistent with CML in an accelerated phase. Cytogenetic analysis at that time revealed a 46,XX normal female karyotype. The patient was placed on hydrea and allopurinol treatment initially. One week after the hydrea was stopped, the patient was given 6 million units of interferon per day for 10 days. The patient was evaluated in our clinic 3 weeks after the initial diagnosis. Her prior medical history consisted of repeated hospitalizations for back pain and many years of treatment with lopid and niacin for lipid disorder. Physical examination revealed the presence of a palpable spleen 2 cm below the left costal margin. Peripheral blood showed hemoglobin 9.1 g %, platelets, 337 3 109/L, and WBC 27.6 3 109/L. A bone marrow biopsy showed marked hypercellularity with granulocytic hyperplasia, consistent with chronic myelogenous leukemia in chronic phase. Cytogenetic study of 20 bone marrow metaphases showed a 46,XX normal female karyotype. FISH analysis was initially performed with a Vysis LSI bcr/abl translocation probe. Examination of interphases demonstrated one ABL, two BCR, and one BCR/ ABL fusion signals (Fig. 1A), similar to that of m-BCR/ABL fusion found in acute lymphoblastic leukemia. However, examination of metaphases revealed a fusion signal consisting of the ABL and BCR genes located on one of the chromosomes 9, an ABL signal on the other chromosome 9, and two BCR signals of different sizes on each of chromosomes 22 (Fig. 1B). Subsequently, FISH was performed with an Oncor M-bcr/abl translocation probe. The results confirmed the localization of the ABL/BCR fusion gene on chromosome 9 in addition to an ABL signal and a BCR signal located on chromosomes 9 and 22, respectively (Fig. 1C and 1D). Reverse transcriptase–polymerase chain reaction analysis revealed a rearrangement of the M-bcr region and expression of a chimeric BCR/ABL mRNA of b3a2 configuration. A clinical diagnosis of CML in chronic phase was made. This patient was treated alternatively with either cytosine arabinoside and hydroxyurea or a-interferon and 13-
DISCUSSION Fluorescence in situ hybridization with dual-color bcr/abl translocation probe is a highly efficient tool for the study of CML patients, particularly in Ph-negative patients. It allows the detection of gene fusion, the determination of the type of the translocation, and a quantitative follow-up of the disease. Four cases of a fusion ABL and BCR gene located on chromosome 9 in Ph-negative CML patients have been documented in the literature [9–11]. We report another case here. Of the five CML patients with the ABL and BCR fusion gene on chromosome 9, two had a rapid clinical course and progressed to blastic crisis in approximately 1 to 1.5 years [9, 11]. Another patient had a highly variable WBC count that was very difficult to stabilize with cytotoxic therapy [9]. The remaining two patients were in chronic phase, but cytogenetic response to therapy was not observed [10]. Eventually, our patient underwent stem-cell transplant owing to the development of a toxic reaction to the treatment. Although the clinical significance of such reverse BCR/ABL arrangement has not been well defined, owing to the limited number of patients studied, it seems that patients with bcr translocation to chromosome 9 either have a rapid course or are unresponsive to treatment. The mechanism of the localization of the ABL and BCR fusion gene on chromosome 9 instead of chromosome 22 is not entirely clear. Our observations in this case and those of others [9, 10] suggest that the translocation of BCR to chromosome 9 in Ph-negative CML patients could be generated by an interstitial insertion of the BCR gene to the ABL gene on chromosome 9 or, alternately, by two successive translocations: a classic t(9;22)(q34.1;q11.2) followed by a second translocation between the long arms of the derivatives 9q1 and 22q2, masking the first chromosome exchange. Further investigation at the genomic level may delineate the mechanism of such gene rearrangement. In our case, FISH revealed a disparate hybridization signal pattern with the use of Vysis and Oncor dual-color bcr/abl translocation probes. This discrepancy is due to the different extend of the regions that the two bcr/abl probes cover. The Vysis bcr probe is a larger probe, which begins between BCR exons 13 and 14 (M-BCR exons 2 and 3) and extends centromerically on chromosome 22 for approximately 300 kb, covering beyond the m-BCR region. The abl probe begins between exons 4 and 5 and continues for about 200 kb toward the telomere of chromosome 9. This probe is able to detect BCR/ABL gene fusions involving M and m breakpoint regions on chromosome 22. The
59
ABL/BCR on Chromosome 9 in Ph-Negative CML
Figure 1 Dual-color FISH analysis on interphase and metaphase with the use of Vysis LSI bcr/abl probe and Oncor M-bcr/abl probe. (A) Interphase FISH with LSI bcr/abl probe consists of one ABL (red), two BCR (green), and one ABL/BCR fusion signals (yellow). (B) Metaphase FISH with LSI bcr/abl probe reveals an ABL/BCR signal on one of the chromosomes 9 (yellow), and ABL signal (red) on the other chromosome 9, and two BCR signals (green) on each of the chromosomes 22. (C) Interphase FISH with Oncor M-bcr/abl probe demonstrates one ABL signal (green), one BCR signal (red), and one ABL/BCR fusion signal (yellow). (D) Metaphase FISH with Oncor M-bcr/abl probe reveals the localization of an ABL/BCR fusion signal (yellow) on chromosome 9 in addition to an ABL signal (green) and a BCR signal (red) located on chromosomes 9 and 22, respectively.
cells having m-BCR/ABL fusions in ALL should appear similar to those with M-BCR/ABL fusions except that, in addition to one ABL, one BCR, and one fusion signal, another small BCR signal may be observed. The latter BCR signal is derived from the region of chromosome 22 between m-BCR and M-BCR that is translocated to chromosome 9. In our case, examination of interphases demonstrated an additional BCR signal, similar to that of the m-BCR/ABL fusion pattern in ALL. However, examination of metaphases revealed that a region of bcr probe remained on chromo-
some 22 after a segment of the BCR gene had moved to chromosome 9, through either interstitial insertion of BCR into the ABL gene or two successive translocations between chromosomes 9 and 22. These gene rearrangements resulted in two BCR signals of different sizes on each of chromosomes 22, respectively. The Oncor M-bcr probe consists of three overlapping cosmids that contain part of the 5.8-kb M-BCR region and adjacent regions extending to the centromeric side of M-BCR on chromosome 22, It recognizes only the M-BCR breakpoint on chromosome 22. The
60 abl probe consists of two overlapping cosmids that contain the 200-kb breakpoint region of ABL between exons Ib and II telomerically on chromosome 9 [12]. Therefore, the hybridization pattern with Oncor bcr/abl probe in interphases showed a M-BCR/ABL rearrangement, as expected. As demonstrated here, the ABL/BCR fusion gene on chromosome 9 was discovered only on metaphases by using both Vysis and Oncor probes. The case reported here should make cytogeneticists aware that it is imperative to have a full understanding of both the capabilities and the limitations of bcr/abl translocation probes and that FISH interphase signals should be confirmed on metaphase spreads at the time of diagnosis. Incomplete understanding and analysis may yield erroneous interpretation in more complex cases.
W.-T. Hsu et al.
6.
7.
8.
9.
REFERENCES 1. Heim S, Billstrom R, Kristofferson U, Mandahl N, Strombeck B, Mitelman F (1985): Variant Ph translocations in chronic myeloid leukemia. Cancer Genet Cytogenet 18:215–227. 2. De Klein A, Geurts van Kessel A, Grosveld G, Bartram CR, Hagemeijer A, Bootsma D, Spurr NK, Heisterkamp N, Groffen J, Stephenson JR (1982): A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature 300:765–767. 3. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G (1984): Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93–9. 4. Shtivelman E, Lifshitz B, Gale RP, Canaani E (1985): Fused transcripts of abl and bcr genes in chronic myelogenous leukemia. Nature 315:550–554. 5. Costello R, Lafage M, Toiron Y, Brunel V, Sainty D, Arnoulet
10.
11.
12.
C, Mozziconacci MJ, Bouabdallah R, Gastaut JA, Maraninchi D, Gabert J (1995): Philadelphia chromosome-negative chronic myeloid leukaemia: a report of 14 new cases. Br J Haematol 90:346–352. Kantarjian HM, Shtalrid M, Kurzrock R, Blick M, Dalton WT, LeMaistre A, Stass SA, McCredie KB, Gutterman J, Freireich EJ, Talpaz M (1988): Significance and correlations of molecular analysis results in patients with Philadelphia chromosomenegative chronic myelogenous leukemia and chronic myelomonocytic leukemia. Am J Med 85:639–644. van der Plas DC, Grosveld G, Hagemeijer A (1991): Review of clinical, cytogenetic, and molecular aspects of Ph-negative CML. Cancer Genet Cytogenet 52:143–156. Tkachuk DC, Westbrook CA, Andreef M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Redner A, Gray J, Pinkel D (1990): Detection of bcr/abl fusion in chronic myelogenous leukemia by in situ hybridization. Science 250:559–562. Hagemeijer A, Buijs A, Smit E, Janssen B, Creemers GJ, van der Plas D, Grosveld G (1993): Translocation of BCR to chromosome 9: a new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia. Genes Chromosom Cancer 8:237–245. Abelivoich D, Yehuda O, Krichevsky S, Nagler A, Ben-Neriah S, Werner M, Ludkovsky O, Ben-Yehuda D (1995): Reversed BCR/ABL rearrangement detected by FISH in Philadelphianegative chronic myelocytic leukemia. Cancer Genet Cytogenet 81:115–117. Brunel V, Sainty D, Costello R, Mozziconacci M-J, Simonetti J, Arnoulet C, Coignet L, Bouabdallah R, Gastaut J-A, Gabert J, Lafage-Pochitaloff M (1995): Translocation of BCR to chromosome 9 in a Philadelphia-negative chronic myeloid leukemia. Cancer Genet Cytogenet 85:82–84. Dewald GW, Schad CR, Christensen ER, Tiede AL, Zinsmeister AR, Spurbeck JL, Thibodeau SN, Jalal SY (1993): Application of fluorescence in situ hybridization to detect M bcr/abl fusion in variant Ph chromosomes in CML and ALL. Cancer Genet Cytogenet 71:7–14.
The ABL/BCR Fusion Gene on Chromosome 9 in Ph-Negative Chronic Myelogenous Leukemia: A Case for Vigilance in Fluorescence In Situ Hybridization Interpretation Wei-Tong Hsu, Harvey Preisler, Katarina Szego, Rita Sprudzs, and Xue-Zhi Gao
ABSTRACT: We report cytogenetic, fluorescence in situ hybridization (FISH), and molecular analysis in a case of Ph-negative chronic myelogenous leukemia patient with ABL/BCR fusion gene on chromosome 9 and a disparate FISH signal pattern using two commercially available bcr/abl probes (Vysis, Inc. and Oncor, Inc.). Cytogenetic analysis revealed a 46,XX normal female karyotype. FISH studies using Vysis LSI bcr/abl probe in interphase cells demonstrated a BCR/ABL fusion pattern, similar to that of m-BCR/ABL fusion found in acute lymphoblastic leukemia. However, examination of metaphases revealed the ABL/BCR fusion signal on one of the chromosomes 9, an ABL signal on the other chromosome 9, and two BCR signals of different sizes on each of the chromosomes 22. Subsequently, a FISH study with the Oncor major (M)-bcr/abl translocation probe confirmed the ABL/BCR fusion signal on chromosome 9 in addition to an ABL signal and a BCR signal located on chromosomes 9 and 22, respectively. Molecular studies (RT-PCR) revealed a rearrangement of the M-BCR region and expression of a chimeric bcr/abl mRNA of b3a2 configuration. This case suggests that it is imperative to have a full understanding of both the capabilities and the limitations of bcr/abl translocation probes and that FISH interphase signals should be confirmed on metaphase spreads for accurate diagnosis. © Elsevier Science Inc., 1998
INTRODUCTION The Philadelphia chromosome (Ph), due to t(9;22)(q34.1; q11.2), is the cytogenetic hallmark of chronic myelogenous leukemia (CML) and is observed in more than 90% of the cases. In 5–10% of CML patients, the Ph chromosome is derived from rearrangements, either simple or complex variant translocations, other than the standard t(9:22) [1]. In simple variant translocation, the segment lost from 22q is translocated to a chromosome other than chromosome 9, whereas three or more chromosomes take part in complex variant translocations. Molecular studies
From the Department of Pediatrics (W.-T. H.), the Rush Cancer Institute (H. P., X.-Z. G.), and the Rush Medical Laboratory (W.-T. H., K. S., R. S.), Rush-Presbyterian-St. Luke’s Medical Center, Rush Medical College, Chicago, Illinois, USA. Address reprint requests to: Dr. Wei-Tong Hsu, Section of Genetics, Department of Pediatrics, Rush-Presbyterian-St. Luke’s Medical Center, 1750 W. Harrison, Room 1509 Jelke Building, Chicago, IL 60612. Received April 25, 1997; accepted October 20, 1997. Cancer Genet Cytogenet 104:57–60 (1998) Elsevier Science Inc., 1998 655 Avenue of the Americas, New York, NY 10010
have shown that in both classic and the variant Ph translocations, the ABL oncogene from chromosome 9q34 is relocated to 22q11 adjacent to the BCR gene [2], giving rise to a chimeric BCR/ABL fusion gene on the derivative chromosome 22 [3]. The BCR/ABL fusion gene is transcribed as a large chimeric RNA that is spliced into an 8.5-kb mRNA with b2a2 and b3a2 configurations [4]. This mRNA is translated to a 210-kD BCR/ABL protein that plays a crucial role in the pathogenesis of CML. A minority (5–10%) of CML patients show no Ph chromosome cytogenetically and are classified as having Ph-negative CML. In a substantial subgroup of Ph-negative patients, molecular investigations have demonstrated the presence of the BCR/ABL fusion gene on chromosome 22 that is indistinguishable at the molecular level from the BCR/ABL gene found in classical Ph-positive CML [5–7]. This BCR/ABL gene rearrangement and its localization can be visualized microscopically by fluorescence in situ hybridization (FISH) by dual-color BCR/ABL translocation probes [8]. Recently, four cases of Ph-negative CML patients with a fusion between the ABL and BCR genes lo-
0165-4608/98/$19.00 PII S0165-4608(97)00430-5
58 cated on chromosome 9 rather than on chromosome 22 have been reported [9–11]. Here, we report a detailed cytogenetic and molecular analysis in a case of Ph-negative CML with the ABL/BCR fusion gene on chromosome 9 and a disparate FISH signal pattern obtained with the use of commercially available dual-color bcr/abl translocation probes from Vysis (Downers Grove, IL) and Oncor (Gaithersburg, MD).
W.-T. Hsu et al. cis-retinoic acid in induction and maintenance phases. In the course of the treatment, follow-up FISH studies revealed the ABL/BCR genes on chromosome 9 in all 20 metaphases examined. Her disease was stable and remained in chronic phase for 8 months. Then she became intolerant to cytosine arabinoside and retinoic acid therapy. A peripheral blood stem-cell transplant was performed with prior administration of G-CSF and GM-CSF to mobilize CD 341 cells. Currently, this patient remains well 3 months after transplant.
CASE REPORT A 55-year-old woman was referred to our clinic for a second opinion and treatment. The patient complained of left upper quadrant pain, fever, and weight loss. Physical examination revealed her spleen to be 9 cm below the left costal margin. At that time, she was found to have a white blood cell (WBC) count of 270,000 with a marked left shift showing all levels of myeloid maturation, including 7% blasts on the peripheral blood smear. Bone marrow biopsy showed marked hypercellularity consisting of immature and mature myeloid cells. There were clusters of immature cells constituting 10–20% of the marrow. The biopsy was considered to be consistent with CML in an accelerated phase. Cytogenetic analysis at that time revealed a 46,XX normal female karyotype. The patient was placed on hydrea and allopurinol treatment initially. One week after the hydrea was stopped, the patient was given 6 million units of interferon per day for 10 days. The patient was evaluated in our clinic 3 weeks after the initial diagnosis. Her prior medical history consisted of repeated hospitalizations for back pain and many years of treatment with lopid and niacin for lipid disorder. Physical examination revealed the presence of a palpable spleen 2 cm below the left costal margin. Peripheral blood showed hemoglobin 9.1 g %, platelets, 337 3 109/L, and WBC 27.6 3 109/L. A bone marrow biopsy showed marked hypercellularity with granulocytic hyperplasia, consistent with chronic myelogenous leukemia in chronic phase. Cytogenetic study of 20 bone marrow metaphases showed a 46,XX normal female karyotype. FISH analysis was initially performed with a Vysis LSI bcr/abl translocation probe. Examination of interphases demonstrated one ABL, two BCR, and one BCR/ ABL fusion signals (Fig. 1A), similar to that of m-BCR/ABL fusion found in acute lymphoblastic leukemia. However, examination of metaphases revealed a fusion signal consisting of the ABL and BCR genes located on one of the chromosomes 9, an ABL signal on the other chromosome 9, and two BCR signals of different sizes on each of chromosomes 22 (Fig. 1B). Subsequently, FISH was performed with an Oncor M-bcr/abl translocation probe. The results confirmed the localization of the ABL/BCR fusion gene on chromosome 9 in addition to an ABL signal and a BCR signal located on chromosomes 9 and 22, respectively (Fig. 1C and 1D). Reverse transcriptase–polymerase chain reaction analysis revealed a rearrangement of the M-bcr region and expression of a chimeric BCR/ABL mRNA of b3a2 configuration. A clinical diagnosis of CML in chronic phase was made. This patient was treated alternatively with either cytosine arabinoside and hydroxyurea or a-interferon and 13-
DISCUSSION Fluorescence in situ hybridization with dual-color bcr/abl translocation probe is a highly efficient tool for the study of CML patients, particularly in Ph-negative patients. It allows the detection of gene fusion, the determination of the type of the translocation, and a quantitative follow-up of the disease. Four cases of a fusion ABL and BCR gene located on chromosome 9 in Ph-negative CML patients have been documented in the literature [9–11]. We report another case here. Of the five CML patients with the ABL and BCR fusion gene on chromosome 9, two had a rapid clinical course and progressed to blastic crisis in approximately 1 to 1.5 years [9, 11]. Another patient had a highly variable WBC count that was very difficult to stabilize with cytotoxic therapy [9]. The remaining two patients were in chronic phase, but cytogenetic response to therapy was not observed [10]. Eventually, our patient underwent stem-cell transplant owing to the development of a toxic reaction to the treatment. Although the clinical significance of such reverse BCR/ABL arrangement has not been well defined, owing to the limited number of patients studied, it seems that patients with bcr translocation to chromosome 9 either have a rapid course or are unresponsive to treatment. The mechanism of the localization of the ABL and BCR fusion gene on chromosome 9 instead of chromosome 22 is not entirely clear. Our observations in this case and those of others [9, 10] suggest that the translocation of BCR to chromosome 9 in Ph-negative CML patients could be generated by an interstitial insertion of the BCR gene to the ABL gene on chromosome 9 or, alternately, by two successive translocations: a classic t(9;22)(q34.1;q11.2) followed by a second translocation between the long arms of the derivatives 9q1 and 22q2, masking the first chromosome exchange. Further investigation at the genomic level may delineate the mechanism of such gene rearrangement. In our case, FISH revealed a disparate hybridization signal pattern with the use of Vysis and Oncor dual-color bcr/abl translocation probes. This discrepancy is due to the different extend of the regions that the two bcr/abl probes cover. The Vysis bcr probe is a larger probe, which begins between BCR exons 13 and 14 (M-BCR exons 2 and 3) and extends centromerically on chromosome 22 for approximately 300 kb, covering beyond the m-BCR region. The abl probe begins between exons 4 and 5 and continues for about 200 kb toward the telomere of chromosome 9. This probe is able to detect BCR/ABL gene fusions involving M and m breakpoint regions on chromosome 22. The
59
ABL/BCR on Chromosome 9 in Ph-Negative CML
Figure 1 Dual-color FISH analysis on interphase and metaphase with the use of Vysis LSI bcr/abl probe and Oncor M-bcr/abl probe. (A) Interphase FISH with LSI bcr/abl probe consists of one ABL (red), two BCR (green), and one ABL/BCR fusion signals (yellow). (B) Metaphase FISH with LSI bcr/abl probe reveals an ABL/BCR signal on one of the chromosomes 9 (yellow), and ABL signal (red) on the other chromosome 9, and two BCR signals (green) on each of the chromosomes 22. (C) Interphase FISH with Oncor M-bcr/abl probe demonstrates one ABL signal (green), one BCR signal (red), and one ABL/BCR fusion signal (yellow). (D) Metaphase FISH with Oncor M-bcr/abl probe reveals the localization of an ABL/BCR fusion signal (yellow) on chromosome 9 in addition to an ABL signal (green) and a BCR signal (red) located on chromosomes 9 and 22, respectively.
cells having m-BCR/ABL fusions in ALL should appear similar to those with M-BCR/ABL fusions except that, in addition to one ABL, one BCR, and one fusion signal, another small BCR signal may be observed. The latter BCR signal is derived from the region of chromosome 22 between m-BCR and M-BCR that is translocated to chromosome 9. In our case, examination of interphases demonstrated an additional BCR signal, similar to that of the m-BCR/ABL fusion pattern in ALL. However, examination of metaphases revealed that a region of bcr probe remained on chromo-
some 22 after a segment of the BCR gene had moved to chromosome 9, through either interstitial insertion of BCR into the ABL gene or two successive translocations between chromosomes 9 and 22. These gene rearrangements resulted in two BCR signals of different sizes on each of chromosomes 22, respectively. The Oncor M-bcr probe consists of three overlapping cosmids that contain part of the 5.8-kb M-BCR region and adjacent regions extending to the centromeric side of M-BCR on chromosome 22, It recognizes only the M-BCR breakpoint on chromosome 22. The
60 abl probe consists of two overlapping cosmids that contain the 200-kb breakpoint region of ABL between exons Ib and II telomerically on chromosome 9 [12]. Therefore, the hybridization pattern with Oncor bcr/abl probe in interphases showed a M-BCR/ABL rearrangement, as expected. As demonstrated here, the ABL/BCR fusion gene on chromosome 9 was discovered only on metaphases by using both Vysis and Oncor probes. The case reported here should make cytogeneticists aware that it is imperative to have a full understanding of both the capabilities and the limitations of bcr/abl translocation probes and that FISH interphase signals should be confirmed on metaphase spreads at the time of diagnosis. Incomplete understanding and analysis may yield erroneous interpretation in more complex cases.
W.-T. Hsu et al.
6.
7.
8.
9.
REFERENCES 1. Heim S, Billstrom R, Kristofferson U, Mandahl N, Strombeck B, Mitelman F (1985): Variant Ph translocations in chronic myeloid leukemia. Cancer Genet Cytogenet 18:215–227. 2. De Klein A, Geurts van Kessel A, Grosveld G, Bartram CR, Hagemeijer A, Bootsma D, Spurr NK, Heisterkamp N, Groffen J, Stephenson JR (1982): A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature 300:765–767. 3. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G (1984): Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93–9. 4. Shtivelman E, Lifshitz B, Gale RP, Canaani E (1985): Fused transcripts of abl and bcr genes in chronic myelogenous leukemia. Nature 315:550–554. 5. Costello R, Lafage M, Toiron Y, Brunel V, Sainty D, Arnoulet
10.
11.
12.
C, Mozziconacci MJ, Bouabdallah R, Gastaut JA, Maraninchi D, Gabert J (1995): Philadelphia chromosome-negative chronic myeloid leukaemia: a report of 14 new cases. Br J Haematol 90:346–352. Kantarjian HM, Shtalrid M, Kurzrock R, Blick M, Dalton WT, LeMaistre A, Stass SA, McCredie KB, Gutterman J, Freireich EJ, Talpaz M (1988): Significance and correlations of molecular analysis results in patients with Philadelphia chromosomenegative chronic myelogenous leukemia and chronic myelomonocytic leukemia. Am J Med 85:639–644. van der Plas DC, Grosveld G, Hagemeijer A (1991): Review of clinical, cytogenetic, and molecular aspects of Ph-negative CML. Cancer Genet Cytogenet 52:143–156. Tkachuk DC, Westbrook CA, Andreef M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Redner A, Gray J, Pinkel D (1990): Detection of bcr/abl fusion in chronic myelogenous leukemia by in situ hybridization. Science 250:559–562. Hagemeijer A, Buijs A, Smit E, Janssen B, Creemers GJ, van der Plas D, Grosveld G (1993): Translocation of BCR to chromosome 9: a new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia. Genes Chromosom Cancer 8:237–245. Abelivoich D, Yehuda O, Krichevsky S, Nagler A, Ben-Neriah S, Werner M, Ludkovsky O, Ben-Yehuda D (1995): Reversed BCR/ABL rearrangement detected by FISH in Philadelphianegative chronic myelocytic leukemia. Cancer Genet Cytogenet 81:115–117. Brunel V, Sainty D, Costello R, Mozziconacci M-J, Simonetti J, Arnoulet C, Coignet L, Bouabdallah R, Gastaut J-A, Gabert J, Lafage-Pochitaloff M (1995): Translocation of BCR to chromosome 9 in a Philadelphia-negative chronic myeloid leukemia. Cancer Genet Cytogenet 85:82–84. Dewald GW, Schad CR, Christensen ER, Tiede AL, Zinsmeister AR, Spurbeck JL, Thibodeau SN, Jalal SY (1993): Application of fluorescence in situ hybridization to detect M bcr/abl fusion in variant Ph chromosomes in CML and ALL. Cancer Genet Cytogenet 71:7–14.