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Philadelphia-negative chronic myelogenous leukemia in a patient with a unique complex translocation: 46,XY,t(9;12;15)(q34;q12;q21)
Leukemia Research 22 (1998) 645 – 648
Philadelphia-negative chronic myelogenous leukemia in a patient with a unique complex translocation: 46,XY,t(9;12;15)(q34;q12;q21) Alan L. Shanske a,*, Hans Grunwald b, Perry Cook c, Nora Heisterkamp d, John Groffen d a
Department of Pediatrics NW556, Center for Congenital Disorders, Montefiore Medical Center/Albert Einstein College of Medicine, 111 E. 210th Street, Bronx, NY 10467, USA b Department of Medicine, Queens Hospital Center, Jamaica, NY, USA c Department of Medicine, Brooklyn Hospital Center, Brooklyn, NY, USA d Department of Pathology, Children’s Hospital of Los Angeles, Los Angeles, CA USA Received 22 December 1997; accepted 17 February 1998
Abstract Chronic myelogenous leukemia (CML) is associated with an acquired karyotypic abnormality, the Philadelphia (Ph) chromosome, in 95% of cases. The Ph chromosome is the product of a balanced translocation that results in a hybrid gene that is considered essential for the pathogenesis of this disease. We have found a complex translocation involving chromosomes 9, 12, and 15 in a 42-year-old Haitian male with the clinical findings of CML. Complex translocations have been shown to result in the masking of the Ph chromosome. We used a mixture of two BCR-specific DNA probes for Southern blot analysis in order to test this hypothesis in our patient. High-molecular weight DNA was digested with the restriction enzymes BglII, BamHI and HindIII. The BglII digestion revealed the presence of two abnormal fragments of 3.9 and 3.0 kb and the BamHI digestion an abnormal 15-kb fragment. These data suggest there is a breakpoint in region 2 of M-bcr. The identification of this breakpoint confirms our hypothesis that a rearrangement involving 22q11 has occurred in the leukemic cells of our patient. A secondary translocation involving chromosomes 12 and 15 has hidden the effects of this translocation. Combined cytogenetic and molecular analysis establishes the karyotype of our patient as 46,XY,t(9;12;15;22)(q34;q12;q21;q11). © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Philadelphia-negative CML; Unique complex translocation
1. Introduction The Philadelphia (Ph) chromosome is found in greater than 95% of patients with chronic myelogenous leukemia (CML). The molecular correlate of the Ph chromosome is the translocation of the protooncogene c-ABL (Abelson) from band 9q34 to within the BCR (breakpoint cluster region) gene on band 22q11. The BCR/ABL recombinant gene is found in 91 – 100% of cases with a cytogenetically identified Ph chromosome and in many cases lacking a Ph chromosome [1 – 4]. The majority of breakpoints on chromosome 22 are located within a 5.8-kb subregion of the bcr which is referred to * Corresponding author. Tel.: +1 718 9204300; fax: + 1 718 9206506. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(98)00058-7
as the major breakpoint cluster region (M-bcr) [5]. The M-bcr (Fig. 1) contains four exons and most breakpoints are located between the second and fourth exons, usually within the intronal sequences [6]. Knowledge of the genetic basis of this disorder has permitted the application of molecular analysis utilizing Southern blotting and PCR. The use of several restriction enzymes and probes hybridizing to the 5% and 3% regions has shown evidence of a rearranged M-bcr in 30–100% of cases lacking a Ph chromosome by cytogenetic investigation [5]. The clinical features and the hematological parameters of Ph-negative but M-bcr rearrangement-positive cases of CML are comparable with those of cytogenetically positive cases. However, patients with morphological features of CML who are Ph-negative and BCR gene rearrangement-negative are
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A.L. Shanske et al. / Leukemia Research 22 (1998) 645–648
Fig. 1. A schematic diagram of chromosome 22 and M-bcr, indicating zones 1 – 5 delineated by restriction sites, and exons indicated by solid boxes.
clinically distinct and may have a clinical course similar to a different myeloproliferative disorder or myelodysplastic syndrome. It is thus imperative to supplement cytogenetic analysis with molecular technics, either Southern blotting or PCR. We have identified a patient with CML with an unusual complex translocation which has not been previously reported who is Ph-negative and have utilized Southern blotting to demonstrate a BCR/c-abl recombinant event.
2. Case report J.B. was a 42-year-old male Haitian electronics worker who was referred to our hematology service because of hematuria and bleeding from the oral cavity. He gave no history of abnormal bleeding in the past. He denied exposure to aromatic hydrocarbons, solvents, toxic chemicals or radiation. The physical examination revealed a thin male appearing somewhat older than his stated age. He had swelling on the left side of his face, and an ecchymotic area in the left side of the buccal mucosa. There were multiple small lymph nodes on palpation of the anterior cervical and inguinal areas bilaterally which were nontender and non-adherent. The spleen was palpable 3 cm beneath the left costal margin during inspiration. The CBC revealed a Hb of 11.5 am%, a Hct of 35%, and MCV of 0.99 fl, a WBC of 195000 ml − 1 and a platelet count of 184000 ml − 1. The differential was 38% segmented neutrophils, 245 bands, 10% metamyelo-
cytes, 17% myelocytes, 3% promyelocytes, 2% myeloblasts, 3% eosinophils, 2% basophils, 35% lymphocytes and 2% monocytes. Review of the smear showed moderate normochromic anisocytosis with a moderate number of target cells, one nucleated RBC per 100 WBC and occasional larger platelets. The bone marrow aspirate revealed an intensely hypercellular (packed) specimen, with intense myeloid hyperplasia (myeloid:erythroid ratio= 33:1) and with left-shifted maturation (11% promyelocytes, 7% myeloblasts). Megakaryocytes were adequate but not increased in number. There was no stainable iron, and no foreign cells were identified. The biopsy confirmed a densely hypercellular marrow with myeloid hyperplasia and normal proportion of megakaryocytes. The leukocyte alkaline phosphatase score was 7 Kaplow units. The patient was begun on therapy with allopurinol and hydroxyurea, on which his cell count decreased to 22500 ml − 1 by the following month. He did not, however, return to the clinic for his subsequent follow-up appointments, and stopped taking his medications. Six months after the diagnosis his spleen was 7 cm beneath the costal margin. A second bone marrow aspiration was essentially unchanged. He was restarted on hydroxyurea and allopurinol, but again failed to keep his follow-up appointments. He reappeared 1 year later complaining of fatigue and weight loss. The marrow at that time was hypercellular and the WBC was 2×105 ml − 1. He was again restarted on hydroxyurea but without benefit and was discharged on busulfan. He subsequently returned to Haiti and was lost to follow-up.
A.L. Shanske et al. / Leukemia Research 22 (1998) 645–648
647
Fig. 2. Representative G-banded karyotype obtained from bone marrow. Arrows indicate chromosomes involved in the translocation.
3. Materials and methods
3.2. Southern blot analysis
3.1. Cytogenetic analysis
High-molecular weight DNA was isolated from 10 ml of heparinized blood or 2 ml of bone marrow and prepared as described, digested with restriction enzymes and electrophoresed on an 0.7% agarose gel. Southern blotting was done on nitrocellulose (Schleicher and Schuell, pH 7.9). Nick translation of probes and filter hybridization was as described. Filters were exposed to XAR-2 film (Kodak) at − 70°C with Dupont Lightning Plus intensifying screens. A mixture of two bcr-specific DNA probes corresponding to zones 1 and 4 of Fig. 1 (a 2.0-kb BfIII/ HindIII and a 1.2-kb HindIII/BglII fragment, respectively) was used for Southern blot analysis of DNA. Both of these probes have been used to detect rearrangements in M-bcr. High-molecular weight blood and bone marrow DNA from the patient (lanes 1 and 2) and normal human DNA (lane 3) were digested with the restriction enzymes BglII (panel A), BamHI (panel B) and HindIII (panel C) (Fig. 4).
Bone marrow aspirates were collected in RPMI 1640 medium and were cultured for an additional 24 h in the same medium containing 10% fetal calf serum. A peripheral blood specimen was analyzed at the same time as the second bone marrow aspirate. Metaphase nuclei were analyzed using our modified trypsin Giemsa banding technique. A total of 22 metaphase nuclei from the first marrow, 40 nuclei from the second, 20 nuclei from the third and 20 cells from the peripheral blood were analyzed. An abnormal male karyotype was recorded in each cell: 46,XY,t(9;12;15)(q34;q12;q21) (Figs. 2 and 3). There was no apparent rearrangement involving chromosome 22.
4. Results
Fig. 3. Partial karyotypes of translocation chromosomes.
The BglII digestion revealed the presence of two abnormal fragments of approximately 3.9 and 3.0 kb, in panel B one abnormal BamHI fragment of approxi-
648
A.L. Shanske et al. / Leukemia Research 22 (1998) 645–648
Fig. 4. Southern blots of DNA obtained from patient blood and bone marrow (lanes 1 and 2) and normal DNA (lane 3) digested with BglII (panel A), BamHI (panel B) and HindIII (panel C). Abnormal fragments are seen in panels A and B.
mately 15 kb is visible. No abnormal fragments are visible upon digestion with HindIII (panel C). This would suggest that the breakpoint in this patient occurred in zones 2 or 3 of M-bcr; these regions are not covered by the probes and would explain the HindIII digestion results. Moreover, in panel B a relatively weakly hybridizing normal BamHI fragment of 3.2 kb is visible, suggesting that the breakpoint took place in this fragment (zones 3, 4 and 5 of Fig. 1) resulting in an abnormal BamHI fragment of approximately 15 kb. These data suggest there is a breakpoint in zone 3 (Fig. 1) of M-bcr.
5. Discussion These results demonstrate that our patient has a breakpoint in the M-bcr region indicating that a rearrangement involving 22q11 has occurred and that Abl has been translocated to the BCR gene of chromosome 22. As a result of a secondary translocation, the chimeric BCR/Abl gene could have been translocated to chromosome 12 or 15 resulting in a cytogenetically ‘normal appearing’ chromosome 22. Molecular analysis using Southern blotting, the polymerase chain reaction (PCR), and in situ hybridization have become common adjuncts to the cytogenetic investigation of the Ph chromosome. A BCR/c-abl rearrangement is found in 30 – 50% of Ph-negative patients with clinical features of CML [7]. The finding of BCR/ Abl rearrangements in all patients who demonstrate the Ph chromosome and in some who do not, but who have a clinical diagnosis of CML, suggests that the BCR/cabl analysis is a more sensitive test than the presence of the Philadelphia chromosome. In fact, the use of in situ .
hybridization studies have revealed a concealed complex translocation t(9;22;20) in a patient with Ph-negative, BCR-positive CML [8]. The PCR analysis in particular has added a high degree of sensitivity useful in following patients in remission and their response to therapy, in particular their response to bone marrow transplantation. Although, useful for diagnosis and disease monitoring, the role of molecular diagnosis is less clear in determining prognosis. For example, there is an inconsistent correlation of the length of the chronic phase or the onset of the blast phase with the position of the breakpoint in M-bcr. The molecular analysis of our patient confirmed the presence of a masked Ph chromosome. Our findings indicate a breakpoint in M-bcr in region 2 between the second and fourth exons, as has been the case in the majority of cases of CML. The M-bcr rearrangement in our patient is a consequence of a complex translocation. Combined cytogenetic and molecular investigations then indicate that the actual karyotype is 46,XY,t(9;12;15;22)(q34;q12;q21;q11), although the effects of a secondary translocation obscure the breakpoint on 22q11. These studies also suggest that he does not belong to the subgroup of patients with morphological features of CML and a clinically distinct biological course who lack both cytogenetic and molecular evidence of the Ph.
References [1] Saglio G, Guerrasio A, Tassinari A, et al. Variability of the molecular defects corresponding to the presence of a philadelphia chromosome in human hematologic malignancies. Blood 1988;72:1203. [2] Ayscue LH, Ross DW, Ozer H, et al. BCR/abl recombinant DNA analysis versus karyotype in the diagnosis and therapeutic monitoring of chronic myeloid leukemia. Am J Clin Pathol 1990;94:404. [3] Kantarjian HM, Kurzrock R, Talpaz M. Philadelphia chromosome negative chronic myelogenous leukemia and chronic myelomonocytic leukemia. Hemat Oncol Clin North Am 1990;4:389. [4] Min GL, Martiat P, Pu GA, et al. Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement. Leukemia 1990;4:650. [5] McClure JS, Litz CE. Chronic myelogenous leukemia: molecular diagnostic considerations. Hum Pathol 1994;25:594. [6] Mills KI, Benn P, Birnie GD. Does the breakpoint within the major breakpoint cluster region (M-bcr) influence the duration of the chronic phase in chronic myeloid leukemia? An analytical comparison of current literature. Blood 1991;78:1155. [7] Kantarjian HM, Shtalrid M, Kurzrock R, et al. Significance and correlations of molecular analysis results in patients with Philadelphia chromosome-negative chronic myelogenous leukjemia and chronic myelomonocytic leukemia. Am J Med 1988;85:639. [8] Morris SW, Daniel L, Ahmed CMI, Elian A, Lebowitz P. Relationship of the bcr breakpoint to chronic phase duration, survival and blast crisis lineage in chronic myelogenous leukemia patients presenting in early chronic phase. Blood 1990;75:2035.
Philadelphia-negative chronic myelogenous leukemia in a patient with a unique complex translocation: 46,XY,t(9;12;15)(q34;q12;q21) Alan L. Shanske a,*, Hans Grunwald b, Perry Cook c, Nora Heisterkamp d, John Groffen d a
Department of Pediatrics NW556, Center for Congenital Disorders, Montefiore Medical Center/Albert Einstein College of Medicine, 111 E. 210th Street, Bronx, NY 10467, USA b Department of Medicine, Queens Hospital Center, Jamaica, NY, USA c Department of Medicine, Brooklyn Hospital Center, Brooklyn, NY, USA d Department of Pathology, Children’s Hospital of Los Angeles, Los Angeles, CA USA Received 22 December 1997; accepted 17 February 1998
Abstract Chronic myelogenous leukemia (CML) is associated with an acquired karyotypic abnormality, the Philadelphia (Ph) chromosome, in 95% of cases. The Ph chromosome is the product of a balanced translocation that results in a hybrid gene that is considered essential for the pathogenesis of this disease. We have found a complex translocation involving chromosomes 9, 12, and 15 in a 42-year-old Haitian male with the clinical findings of CML. Complex translocations have been shown to result in the masking of the Ph chromosome. We used a mixture of two BCR-specific DNA probes for Southern blot analysis in order to test this hypothesis in our patient. High-molecular weight DNA was digested with the restriction enzymes BglII, BamHI and HindIII. The BglII digestion revealed the presence of two abnormal fragments of 3.9 and 3.0 kb and the BamHI digestion an abnormal 15-kb fragment. These data suggest there is a breakpoint in region 2 of M-bcr. The identification of this breakpoint confirms our hypothesis that a rearrangement involving 22q11 has occurred in the leukemic cells of our patient. A secondary translocation involving chromosomes 12 and 15 has hidden the effects of this translocation. Combined cytogenetic and molecular analysis establishes the karyotype of our patient as 46,XY,t(9;12;15;22)(q34;q12;q21;q11). © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Philadelphia-negative CML; Unique complex translocation
1. Introduction The Philadelphia (Ph) chromosome is found in greater than 95% of patients with chronic myelogenous leukemia (CML). The molecular correlate of the Ph chromosome is the translocation of the protooncogene c-ABL (Abelson) from band 9q34 to within the BCR (breakpoint cluster region) gene on band 22q11. The BCR/ABL recombinant gene is found in 91 – 100% of cases with a cytogenetically identified Ph chromosome and in many cases lacking a Ph chromosome [1 – 4]. The majority of breakpoints on chromosome 22 are located within a 5.8-kb subregion of the bcr which is referred to * Corresponding author. Tel.: +1 718 9204300; fax: + 1 718 9206506. 0145-2126/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved. PII: S0145-2126(98)00058-7
as the major breakpoint cluster region (M-bcr) [5]. The M-bcr (Fig. 1) contains four exons and most breakpoints are located between the second and fourth exons, usually within the intronal sequences [6]. Knowledge of the genetic basis of this disorder has permitted the application of molecular analysis utilizing Southern blotting and PCR. The use of several restriction enzymes and probes hybridizing to the 5% and 3% regions has shown evidence of a rearranged M-bcr in 30–100% of cases lacking a Ph chromosome by cytogenetic investigation [5]. The clinical features and the hematological parameters of Ph-negative but M-bcr rearrangement-positive cases of CML are comparable with those of cytogenetically positive cases. However, patients with morphological features of CML who are Ph-negative and BCR gene rearrangement-negative are
646
A.L. Shanske et al. / Leukemia Research 22 (1998) 645–648
Fig. 1. A schematic diagram of chromosome 22 and M-bcr, indicating zones 1 – 5 delineated by restriction sites, and exons indicated by solid boxes.
clinically distinct and may have a clinical course similar to a different myeloproliferative disorder or myelodysplastic syndrome. It is thus imperative to supplement cytogenetic analysis with molecular technics, either Southern blotting or PCR. We have identified a patient with CML with an unusual complex translocation which has not been previously reported who is Ph-negative and have utilized Southern blotting to demonstrate a BCR/c-abl recombinant event.
2. Case report J.B. was a 42-year-old male Haitian electronics worker who was referred to our hematology service because of hematuria and bleeding from the oral cavity. He gave no history of abnormal bleeding in the past. He denied exposure to aromatic hydrocarbons, solvents, toxic chemicals or radiation. The physical examination revealed a thin male appearing somewhat older than his stated age. He had swelling on the left side of his face, and an ecchymotic area in the left side of the buccal mucosa. There were multiple small lymph nodes on palpation of the anterior cervical and inguinal areas bilaterally which were nontender and non-adherent. The spleen was palpable 3 cm beneath the left costal margin during inspiration. The CBC revealed a Hb of 11.5 am%, a Hct of 35%, and MCV of 0.99 fl, a WBC of 195000 ml − 1 and a platelet count of 184000 ml − 1. The differential was 38% segmented neutrophils, 245 bands, 10% metamyelo-
cytes, 17% myelocytes, 3% promyelocytes, 2% myeloblasts, 3% eosinophils, 2% basophils, 35% lymphocytes and 2% monocytes. Review of the smear showed moderate normochromic anisocytosis with a moderate number of target cells, one nucleated RBC per 100 WBC and occasional larger platelets. The bone marrow aspirate revealed an intensely hypercellular (packed) specimen, with intense myeloid hyperplasia (myeloid:erythroid ratio= 33:1) and with left-shifted maturation (11% promyelocytes, 7% myeloblasts). Megakaryocytes were adequate but not increased in number. There was no stainable iron, and no foreign cells were identified. The biopsy confirmed a densely hypercellular marrow with myeloid hyperplasia and normal proportion of megakaryocytes. The leukocyte alkaline phosphatase score was 7 Kaplow units. The patient was begun on therapy with allopurinol and hydroxyurea, on which his cell count decreased to 22500 ml − 1 by the following month. He did not, however, return to the clinic for his subsequent follow-up appointments, and stopped taking his medications. Six months after the diagnosis his spleen was 7 cm beneath the costal margin. A second bone marrow aspiration was essentially unchanged. He was restarted on hydroxyurea and allopurinol, but again failed to keep his follow-up appointments. He reappeared 1 year later complaining of fatigue and weight loss. The marrow at that time was hypercellular and the WBC was 2×105 ml − 1. He was again restarted on hydroxyurea but without benefit and was discharged on busulfan. He subsequently returned to Haiti and was lost to follow-up.
A.L. Shanske et al. / Leukemia Research 22 (1998) 645–648
647
Fig. 2. Representative G-banded karyotype obtained from bone marrow. Arrows indicate chromosomes involved in the translocation.
3. Materials and methods
3.2. Southern blot analysis
3.1. Cytogenetic analysis
High-molecular weight DNA was isolated from 10 ml of heparinized blood or 2 ml of bone marrow and prepared as described, digested with restriction enzymes and electrophoresed on an 0.7% agarose gel. Southern blotting was done on nitrocellulose (Schleicher and Schuell, pH 7.9). Nick translation of probes and filter hybridization was as described. Filters were exposed to XAR-2 film (Kodak) at − 70°C with Dupont Lightning Plus intensifying screens. A mixture of two bcr-specific DNA probes corresponding to zones 1 and 4 of Fig. 1 (a 2.0-kb BfIII/ HindIII and a 1.2-kb HindIII/BglII fragment, respectively) was used for Southern blot analysis of DNA. Both of these probes have been used to detect rearrangements in M-bcr. High-molecular weight blood and bone marrow DNA from the patient (lanes 1 and 2) and normal human DNA (lane 3) were digested with the restriction enzymes BglII (panel A), BamHI (panel B) and HindIII (panel C) (Fig. 4).
Bone marrow aspirates were collected in RPMI 1640 medium and were cultured for an additional 24 h in the same medium containing 10% fetal calf serum. A peripheral blood specimen was analyzed at the same time as the second bone marrow aspirate. Metaphase nuclei were analyzed using our modified trypsin Giemsa banding technique. A total of 22 metaphase nuclei from the first marrow, 40 nuclei from the second, 20 nuclei from the third and 20 cells from the peripheral blood were analyzed. An abnormal male karyotype was recorded in each cell: 46,XY,t(9;12;15)(q34;q12;q21) (Figs. 2 and 3). There was no apparent rearrangement involving chromosome 22.
4. Results
Fig. 3. Partial karyotypes of translocation chromosomes.
The BglII digestion revealed the presence of two abnormal fragments of approximately 3.9 and 3.0 kb, in panel B one abnormal BamHI fragment of approxi-
648
A.L. Shanske et al. / Leukemia Research 22 (1998) 645–648
Fig. 4. Southern blots of DNA obtained from patient blood and bone marrow (lanes 1 and 2) and normal DNA (lane 3) digested with BglII (panel A), BamHI (panel B) and HindIII (panel C). Abnormal fragments are seen in panels A and B.
mately 15 kb is visible. No abnormal fragments are visible upon digestion with HindIII (panel C). This would suggest that the breakpoint in this patient occurred in zones 2 or 3 of M-bcr; these regions are not covered by the probes and would explain the HindIII digestion results. Moreover, in panel B a relatively weakly hybridizing normal BamHI fragment of 3.2 kb is visible, suggesting that the breakpoint took place in this fragment (zones 3, 4 and 5 of Fig. 1) resulting in an abnormal BamHI fragment of approximately 15 kb. These data suggest there is a breakpoint in zone 3 (Fig. 1) of M-bcr.
5. Discussion These results demonstrate that our patient has a breakpoint in the M-bcr region indicating that a rearrangement involving 22q11 has occurred and that Abl has been translocated to the BCR gene of chromosome 22. As a result of a secondary translocation, the chimeric BCR/Abl gene could have been translocated to chromosome 12 or 15 resulting in a cytogenetically ‘normal appearing’ chromosome 22. Molecular analysis using Southern blotting, the polymerase chain reaction (PCR), and in situ hybridization have become common adjuncts to the cytogenetic investigation of the Ph chromosome. A BCR/c-abl rearrangement is found in 30 – 50% of Ph-negative patients with clinical features of CML [7]. The finding of BCR/ Abl rearrangements in all patients who demonstrate the Ph chromosome and in some who do not, but who have a clinical diagnosis of CML, suggests that the BCR/cabl analysis is a more sensitive test than the presence of the Philadelphia chromosome. In fact, the use of in situ .
hybridization studies have revealed a concealed complex translocation t(9;22;20) in a patient with Ph-negative, BCR-positive CML [8]. The PCR analysis in particular has added a high degree of sensitivity useful in following patients in remission and their response to therapy, in particular their response to bone marrow transplantation. Although, useful for diagnosis and disease monitoring, the role of molecular diagnosis is less clear in determining prognosis. For example, there is an inconsistent correlation of the length of the chronic phase or the onset of the blast phase with the position of the breakpoint in M-bcr. The molecular analysis of our patient confirmed the presence of a masked Ph chromosome. Our findings indicate a breakpoint in M-bcr in region 2 between the second and fourth exons, as has been the case in the majority of cases of CML. The M-bcr rearrangement in our patient is a consequence of a complex translocation. Combined cytogenetic and molecular investigations then indicate that the actual karyotype is 46,XY,t(9;12;15;22)(q34;q12;q21;q11), although the effects of a secondary translocation obscure the breakpoint on 22q11. These studies also suggest that he does not belong to the subgroup of patients with morphological features of CML and a clinically distinct biological course who lack both cytogenetic and molecular evidence of the Ph.
References [1] Saglio G, Guerrasio A, Tassinari A, et al. Variability of the molecular defects corresponding to the presence of a philadelphia chromosome in human hematologic malignancies. Blood 1988;72:1203. [2] Ayscue LH, Ross DW, Ozer H, et al. BCR/abl recombinant DNA analysis versus karyotype in the diagnosis and therapeutic monitoring of chronic myeloid leukemia. Am J Clin Pathol 1990;94:404. [3] Kantarjian HM, Kurzrock R, Talpaz M. Philadelphia chromosome negative chronic myelogenous leukemia and chronic myelomonocytic leukemia. Hemat Oncol Clin North Am 1990;4:389. [4] Min GL, Martiat P, Pu GA, et al. Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement. Leukemia 1990;4:650. [5] McClure JS, Litz CE. Chronic myelogenous leukemia: molecular diagnostic considerations. Hum Pathol 1994;25:594. [6] Mills KI, Benn P, Birnie GD. Does the breakpoint within the major breakpoint cluster region (M-bcr) influence the duration of the chronic phase in chronic myeloid leukemia? An analytical comparison of current literature. Blood 1991;78:1155. [7] Kantarjian HM, Shtalrid M, Kurzrock R, et al. Significance and correlations of molecular analysis results in patients with Philadelphia chromosome-negative chronic myelogenous leukjemia and chronic myelomonocytic leukemia. Am J Med 1988;85:639. [8] Morris SW, Daniel L, Ahmed CMI, Elian A, Lebowitz P. Relationship of the bcr breakpoint to chronic phase duration, survival and blast crisis lineage in chronic myelogenous leukemia patients presenting in early chronic phase. Blood 1990;75:2035.