Variant Ph Chromosome Research Articles

Novel Four-Way Variant Translocation, t(1;9;22;16)(q21;q34;q11.2;q24), in a Patient with Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is characterized by the Philadelphia (Ph) chromosome resulting from the translocation of t(9;22)(q34;q11), producing the BCR::ABL1 fusion gene. Variant Ph chromosome translocations, involving rearrangements in chromosomes other than 9 and 22, occur in 5–10% of CML cases. Herein, we report a unique case of a 36-year-old male with a four-way variant Ph chromosome. Conventional chromosomal analysis performed on bone marrow aspirate samples showed 46, XY, t(1;9;22;16)(q21;q34;q11.2;q24). Nested RT-PCR of the BCR::ABL1 gene revealed a major BCR::ABL rearrangement. The treatment with nilotinib achieved a complete hematologic, cytogenetic, and molecular response after 12 months.

PB1891: VARIANT PHILADELPHIA CHROMOSOME IN CML

Background: Chronic myeloid leukemia is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the Philadelphia chromosome and the reciprocal translocation t(9:22)(q34;q11). Almost 5 to 10 percent of chronic myeloid leukemia cases demonstrate variant Philadelphia translocations. Aims: To contribute to the description of patients’ clinical characteristics and prognosis with variant Ph chromosome-positive leukemia, we report a Ph chromosome-positive CML in the chronic phase characterized by an unusual variant Ph chromosome. Methods: An elderly male Libyan patient was suspected to have chronic myeloid leukemia. Cytogenetic and molecular exploration were conducted using bone marrow and blood samples. Cytogenetic analysis was performed on lymphocytes and bone marrow cells after short-term culture (48 hours). The cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Multiplex reverse transcription-PCR (RT-PCR) was used to identify the BCR-ABL1 transcript. RT-PCR was performed according to European recommendations, as described previously. Results: Clinical assessment confirmed the first chronic phase of a chronic myeloid leukemia. Chromosomal formula showed an homogeneous structural translocation: t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Molecular investigation confirmed the Ph nature of the chromosomal abnormality by showing the BCR-ABL1 transcript. The patient was treated by using Imatinib molecule with a favorable therapeutic response. Summary/Conclusion: A new variant Ph chromosome-positive CML in the chronic phase is reported. It is a four-way Ph translocation t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Imatinib, the first-generation TKI treatment was efficient.

The molecular-cytogenetic characterization and tyrosine kinase inhibitors efficacy in newly diagnosed chronic phase CML patients with variant Philadelphia chromosomes

目的探究伴Ph染色体变异易位（vPh）初诊慢性髓性白血病慢性期（CML-CP）患者的分子、细胞遗传学特征及酪氨酸激酶抑制剂（TKI）的疗效。方法收集32例伴vPh且一线应用伊马替尼（IM）治疗、BCR-ABL转录本为P210的初诊CML-CP患者临床资料，并与同期相同条件的703例伴典型Ph染色体的患者进行对比分析。结果两组人口统计学与初诊时血液学特征比较差异均无统计学意义（P值均>0.05）。32例vPh组患者中3例（9.4%）为简单变异易位，29例为复杂变异易位。29例复杂变异易位患者中，28例（87.5%）累及3条染色体，1例（3.1%）累及4条染色体，除8、15、18及X、Y染色体外，其余染色体均被累及，其中12q（15.5%）与1p（12.1%）累及频率较高。FISH信号以2绿2红1黄（2G2R1Y）（74.1%）为主，其次为1G1R2F（14.8%）、2G1R1Y（3.7%）、1G2R1Y（3.7%）、1G1R1Y（3.7%），简单机制型与复杂机制型亚组比较，完全细胞遗传学反应（CCyR）率（P=0.269）、主要分子学反应（MMR）率（P=0.391）差异无统计学意义。与典型Ph组相比，vPh组具有更高的原发IM耐药率（χ2=3.978，P=0.046），尤其是原发血液学耐药率（χ2=7.870，P=0.005）；但两组CCyR（χ2=0.192，P=0.661）、MMR（χ2=0.822，P=0. 365）、无事件生存（χ2=0.509，P=0.476）、总生存（χ2=3.485，P=0.062）比较差异均无统计学意义。通过多因素分析显示vPh的存在并不影响患者总生存（RR=0.692，95%CI 0.393~1.765，P=0.658）、无事件生存（RR=0.893，95%CI 0.347~2.132，P=0.126）及无进展生存（RR=1.176，95%CI 0.643~2.682，P=0.703）。结论CML-CP vPh患者与典型Ph患者具有相似的人口学及血液学特征，除22q11、9q34外，vPh累及的染色体以12q、1p较多，FISH信号类型以2G2R1Y为主，不同的形成机制对TKI疗效无明显影响。vPh与较高的IM原发耐药率，尤其是与原发血液学耐药率相关，但换用二代TKI后患者能迅速获得深层次分子学反应，长期疗效并不受影响。

Prognostic Value of Clonal Evolution at the Time of Diagnosis in Patients with Chronic Myeloid Leukemia Treated with Frontline Tyrosine Kinase Inhibitors

Introduction:Additional cytogenetic clonal evolution (CE) is a known risk factor for a poor prognosis in chronic myeloid leukemia (CML). However, its prognostic significance in the setting of new tyrosine kinase inhibitor (TKI) remains unclear. We sought to analyze the baseline characteristics and clinical outcome in chronic phase (CP) CML pts with or without CE treated on frontline TKI clinical trials in a single institution.Methods:Patients (pts) with Ph-positive CML in CP with or without CE at the time of diagnosis receiving initial therapy with imatinib 400 mg/d, imatinib 800 mg/d, dasatinib 100 mg/d, nilotinib 800 mg/d or ponatinib 30 or 45 mg/d in consecutive or parallel clinical trials at a single institution were analyzed. Overall survival (OS), transformation free survival (TFS), event free survival (EFS), failure free survival (FFS) were analyzed from the start of therapy by Kaplan-Meier method. Clonal evolution (CE) was defined by the presence of any cytogenetic abnormality other than a single Ph, variant Ph chromosome or loss of Y chromosome. Also we analyzed CML pts with CE with regard 'major route' abnormalities vs other. The major route abnormalities includes trisomy 8 (+8), trisomy 19 (+19), isochromosome 17q10 (i17q) and additional Ph chromosome.Results:A total of 603 pts were analyzed including 579 pts in CP without CE and 24 pts with CE. Pts in CP without CE received initial therapy with imatinib-400 (n=70), imatinib-800 (n=200), dasatinib (n=138), nilotinib (n=122), or ponatinib (n=49), and pts with CE received imatinib-400 (n=2), imatinib-800 (n=7), dasatinib (n=10), nilotinib (n=4), and ponatinib (n=1). Pts with CP were usually older, female and have a higher WBC (P < 0.001). There was a statistically significant higher Complete cytogenetic response (CCyR) at 6 mo in pts without CE (P = 0.012), however the cumulative and 3-month rates of complete hematologic response (CHR), and the cumulative rates of CCyR and MMR were not different (Table). Similarly, the rates of MR4.0 and MR4.5 were similar for the two groups. At 5 years, the presence of additional cytogenetic findings at diagnosis does not seem to affect the rate of transformation, failure-free, event-free and overall survival. Acknowledging the small sample size for subset analysis, response rates and survival outcomes were comparable in CP with CE irrespective of whether chromosomal abnormalities were ’major route’ or other (n=12 in each arm).Conclusion:Additional cytogenetic CE at the time of diagnosis among patients with CML in CP is associated with a similar favorable outcome as those without CE when treated with TKI. The type of additional CE (major route vs other) does not seem to impact outcome. Patients with CML-CP with CE at the time of diagnosis can thus be treated with TKI as all other pts with CP with no need for consideration for SCT unless there is clear evidence of failure. DisclosuresJabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Ravandi:BMS: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding. DiNardo:Abbvie: Research Funding; Agios: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Novartis: Other: advisory board, Research Funding; Celgene: Research Funding. Daver:Ariad: Research Funding; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Research Funding; Kiromic: Research Funding; Otsuka: Consultancy, Honoraria. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Very Long-Term Follow-up (> 80 months) of Imatinib (IM) First-Line for Chronic Phase (CP) CML Patients

Very Long-Term Follow-up (> 80 months) of Imatinib (IM) First-Line for Chronic Phase (CP) CML Patients

Three-way Philadelphia translocation t(9;10;22)(q34;p11.2;q11.2) as a secondary abnormality in an imatinib mesylate-resistant chronic myeloid leukemia patient

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome created by the reciprocal translocation t(9:22)(q34;q11), resulting in the chimeric gene breakpoint cluster region (BCR)-Abelson (ABL). Variant Ph chromosome translocations involving chromosomes other than 9 and 22 occur in 5–10% of CML cases. In the present study, a novel case of a Ph chromosome-positive CML in the chronic phase (CP) is reported, with a three-way Ph translocation involving three chromosomal regions, 9q34, 10p11.2 and 22q11.2, in addition to the loss of the Y chromosome, where the latter was a secondary abnormality. Since the majority of CML cases are currently treated with imatinib, variant rearrangements generally have no specific prognostic significance, although the mechanisms involved in resistance to therapy have yet to be investigated. The underlying mechanisms and prognostic implications of these cytogenetic abnormalities are discussed in the present study.

A novel cytogenetic abnormality t(7;8)(p11.2:q11.2) and a four-way Philadelphia translocation in an imatinib mesylate-resistant chronic myeloid leukemia patient

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome, created by a reciprocal translocation t(9:22)(q34;q11) which forms the chimeric gene, BCR-ABL. Variant Ph chromosome translocations involving chromosomes other than 9 and 22 have been identified in 5–10% of CML cases. Four-way Ph chromosome translocations are an extremely rare event in myeloid malignancies and the phenotypic consequences of such rearrangements have not been investigated. Deletions in chromosome 9 are known to be associated with a poor prognosis. In the present study, a novel case of Ph chromosome-positive CML in blast crisis is reported. A four-way Ph translocation was identified, involving five chromosomal regions, 9p21, 9q34, 12p13.3, 20q11.2 and 22q11.2, as well as an unbalanced translocation, der(7)t(7;8)(p11.2;q11.2). Since the majority of CML cases are currently treated with imatinib, variant rearrangements in general have no specific prognostic significance, although the mechanisms involved in resistance to therapy have yet to be investigated. In the present case, multiple partial deletions, including ABL and ASS genes on chromosome 9, the region 7p11.2 to 7pter, 8q11.2 to 8pter and two regions on chromosome 12, were identified. An additional Ph chromosome was also detected. Immunophenotyping indicated that the patient had biphenotypic leukemia. The patient did not respond positively to imatinib chemotherapy and died for unknown reasons, one month after diagnosis. The underlying mechanisms and prognostic implications of these cytogenetic abnormalities are discussed.

Results of Second Generation Tyrosine Kinase Inhibitor Frontline Therapy in Chronic Myeloid Leukemia with Variant Philadelphia Chromosome

AbstractAbstract 3774 Background:Variant Philadelphia (Ph) chromosomes are observed in 5–10% of patients (pts) with CML, involving one or more additional chromosomes besides 9 and 22. Deletions of derivative chromosome 9 (del der (9)) occur in 10–15% of pts, and frequently these abnormalities coexist. Prior to the advent of TKIs pts with variant Ph or del der (9) had a worse outcome. With imatinib the adverse prognosis conferred by these abnormalities has been minimized. To our knowledge there are no published reports regarding the outcomes of pts with variant Ph or del der(9) while on treatment with a 2nd generation TKI as initial therapy for CML. We analyzed the significance of these abnormalities among patients with CML in chronic phase (CP) enrolled in 2 parallel trials of dasatinib or nilotinib as initial therapy. Patients and Methods:A total of 205 pts with CML in CP treated between 2005 and 2012 at our institution with frontline dasatinib or nilotinib based protocols were included in this analysis. Among them 193 (94%) had classic Ph chromosome, and 12 (6%) had a variant Ph chromosome. Nineteen of the 205 pts (9%) had del der (9). Their characteristics and outcomes were compared. Results:Only one additional chromosome was present in the Ph rearrangement in each of the variant Ph-positive cases. The additional chromosomes found were 17 in three patients and chromosomes 1, 3, 4, 5, 9, 11, 14, 19, 21 in one patient each. Del der (9) was found in 2 of 12 pts with variant Ph and in 17 with classic Ph. Five (50%) of the pts with variant Ph were treated with dasatinib and 5 (50%) with nilotinib. Of those with classical Ph 93 (48%) were treated with dasatinib and 100 (52%) with nilotinib. The median age was 48 yrs (range 17 to 86) for those with classic Ph, and 53 yrs (24 to 79) for those with variant Ph. The Sokal risk group for those with classic Ph was high in 16 (8%), intermediate in 53 (28%), and low in 124 (64%). For the patients with variant Ph risk classification was 10%, 50% and 40%.No significant differences were observed in response to therapy between pts with variant Ph and those with classic Ph (CCyR 100% vs. 93%; MMR 100% vs. 85%, respectively), or between those with del der (9) and those without it (CCyR 100% vs. 93%; MMR 100% vs. 84%). The 3-year probability of overall survival (OS) was 99% for pts with classic Ph and 89% for those with variant Ph. Corresponding values for 3-year event-free survival (EFS) were 96% and 89%, and transformation-free survival (TFS) 99% and 100% respectively. 3-year OS, EFS and TFS were also similar between pts with del derv (9) and those without it (Table 1). Among the 2 pts with concomitant variant Ph and del der (9), one achieved CCyR at 3 months and has maintained it with 12 months of follow up. The second one achieved CCyR but became pregnant during the course of treatment; the TKI was temporarily discontinued and she lost cytogenetic response, but regained response after 3 months of reinitiating TKI therapy and delivering a healthy baby. Conclusion:The presence of variant Ph chromosome or del der (9) does not affect the clinical characteristics or outcome of pts with CML CP treated with second generation TKI as initial therapy.Table 1Outcomes in Variant Ph-positive patients treated withVariant PhClassic PhDel. Der. 9No Del. Der. 9Overall CCyR100%93%100%93%Overall MMR100%85%100%84%12 mo. CCyR88%97%100%96%12 mo. MMR81%50%79%93%3 years O.S.89%99%99%100%3 years EFS89%96%96%100%3 years TFS100%99%99%100% Disclosures:Kantarjian:Pfizer: Research Funding; Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding. Jabbour:Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Cortes:Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding.

BCR/ABL Fusion Gene On Constitutional Der(14;22) Roberstsonian Translocation in a Case of Chronic Myeloid Leukemia with Masked Philadelphia Chromosome

AbstractAbstract 4822Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of the BCR/ABL chimeric gene. The remaining 5–10% of CML cases exhibit a variant Ph translocation generally involving a third or even a fourth chromosome in addition to chromosome 9 and 22, potentially leading to masked Ph chromosome or reveal cryptic translocations that remains undetected under conventional cytogenetic analysis. These chromosome rearrangements can be disclosed by means of fluorescence in situhybridization (FISH) or polymerase chain reaction (PCR) procedures. A very few Ph positive CML cases were reported with constitutional robertsonian translocations, i.e. translocation between two acrocentric chromomosomes (13–15, 21–22), with breakpoints in the short arms, leading to a dicentric chromosome and thus to 45 instead of 46 chromosomes Case Report.42 year-old woman presenting with asthenia. Physical examination: Grade 1 splenomegaly. Peripheral blood count showed: hemoglobin concentration 117g/L, platelet count: 329×109/L and white blood cell count (WBC): 199×109/L. Peripheral blood smear: myelemia exhibiting 3% of myeloid blasts. Cytogenetic analysis by G-banding performed on bone marrow metaphase cells afforded the following karyotype: 45, XX, der(14;22)(q10;q10)c?, t(9;22;11)(q34;q11;q13) [20]. The analysis of the BCR-ABLfusion gene according to standard protocols detected the presence of the b3a2 isoform. FISH studies using dual color dual fusion probes in metaphases showed a 1F2G2R signal pattern. We detect a normal ABL signal on chromosome 9 and BCR signal on chromosome 22; the fusion signal was present on the der(14;22);extra-signals BCR and ABL with reduced intensities were present on der(11) and der(9) respectively: ish der(9)(ABLdim+), der(11)(BCRdim+), der(14;22)(BCR+,ABL+) [10]. FISH analysis on interphase nuclei (n=200) presented the same signal pattern. Nuc ish (ABL, BCRx3)(BCR con ABL x1) [200]. Chromosome analysis of bone marrow cells after six months of Imatinib therapy showed the following karyotype: 45, XX, der(14;22)(q10;q10)c [20] thus demonstrating complete cytogenetic remission and that der(14;22) is a robertsonian constitutional abnormality that could be inherited and thus necessitate a familial genetic councelling to inform about the familial risk of congenital malformations and miscarriage. Discussion.To explain the formation of variant chromosome Ph translocations one-step, two-step and multi-step mechanisms have been proposed. In our case complex translocations involving four chromosomes and the participation of two acrocentric chromosomes, led to the hypothesis of the presence of a constitutional or acquired Robertsonian translocation. Karyotype analysis six months after treatment confirmed the presence of a constitutional Robertsonian translocation. According to the FISH pattern, this variant Ph chromosome was formed in one step.The occurrence of Philadelphia positive CML in a patient with a constitutional Robertsonian translocation is probably coincidental. The role of constitutional chromosomes abnormalities in hematologic malignancies is well known in Down syndrome patients and in chromosome breakage syndromes such as Fanconi anemia. In the literature, only one case of CML patients with Robertsonian t(14;22) have been described.To our knowledge this is the first report showing a Robertsonian t(14;22) in a variant Ph involving four chromosomes and exhibiting the fusion FISH signal in a derivative chromosome 14, with masked Ph. Disclosures:No relevant conflicts of interest to declare.

A Four-Way Translocation t (1;17; 9; 22) (p?35; q24; q34;q11) in a Newly Diagnosed Chronic Phase CML Patient: A Case Report

AbstractAbstract 4459Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disorder characterized by the translocation t(9,22)(q34;q11), resulting in the Philadelphia chromosome (Ph) and the bcr-abl fusion gene. A small proportion of Ph+ CML patients (5–10%) show variant Ph translocations involving one or more chromosomes in addition to chromosomes 9 and 22. Its generation mechanism and clinical significance remains unclear. We report a novel four-way translocation detected in a newly diagnosed chronic phase CML patient. Case presentationA 51 year-old white women with no significant past medical history presented with a two-week upper left abdominal pain. She denied weight loss, fever, night sweats, fatigue or thrombosis. She has 11 siblings. On physical examination an enlarged spleen was detected, 7 cm below left costal margin. No hepatomegaly was found.Peripheral blood counts on admission showed WBC 286 × 109/l, with immature myeloid forms (21%bands, 31%neutrophils, 3%lymphocytes, 5%basophiles, 1%eosinophils, 16%metamyelocites, 14%myelocytes, 6% promyelocytes, 1% blast cells), hemoglobin 11,8 g/dl and platelet count of 986 ×109/l. LDH= 1094 IU/L. Uric acid, renal and hepatic functions were normal. Abdominal ultrasonography showed homogeneous splenomegaly (185 mm) with no focal lesions. Bone marrow aspirate demonstrated increased cellularity with myeloid hyperplasia without remarkable basophilia. Trephine demonstrated hypercellularity without fibrosis and 1% blast cells. Conventional cytogenetic analysis by GTG-banding revealed a variant Ph chromosome positive in 100 % of metaphases analysed involving chromosomes 1 and 17 inaddition to chromosomes 9 and 22: 46 XX, t (1;17; 9; 22) (p?35;q24;q34;q11)[20]. No normal cells nor additional numerical and structural chromosomal changes were detected at diagnosis. A fragment of 1605 bp was amplified with primers BCR-P1F and ABL-P1R. The sequence analysis of the amplified PCR product showed a complex BCR-ABL rearrangement formed by exon 8 of the BCR (NM_004327) and exon 2 of the ABL genes (NM_005157), with a 148 bp insertion corresponding to the whole exon 2 of the MAST2 (NM_015112, Homo sapiens microtubule associated serine/threonine kinase 2) gene.Bone marrow cytogenetical evaluation at four months do not show Philadelphia chromosome on 20 cells analysed. Interphase FISH with DUAL Colour Dual Fusion probe has a normal pattern on 300 nuclei: 2R2G. It corresponds to a complete cytogenetic response. Chronic phase CML was diagnosed and cytoreductive treatment with hydroxyurea 1,5 g/day, fluids, thromboprophilaxis and allopurinol was initiated. A good control of blood counts was obtained, as well as a complete reduction of the enlarged spleen and regression of left abdominal pain. Once cytogenetic/molecular confirmed diagnosis, treatment with Imatinib 400 mg/day was started, and hidroxiurea was discontinued. She achieved complete hematologic response at 1 month. Splenomegaly was no longer detected. At 2 months she developed neutropenia (1100/mm3) and thrombocytopenia (55000/mm3) so Imatinib was held. Two weeks later, after hematologic recovery, Imatinib was re initiated but in the following control neutropenia and thrombocytopenia were again detected. DiscussionAbout 90–95% of CML have the characteristic t(9;22)(q34;q11.2) reciprocal translocation that results in the Ph chromosome [der(22q)], the cytogenetical hallmark of this entity. The remaining cases can have variant translocations that involve 3, 4 or more chromosomes and its occurrence is not associated with disease evolution. This patient shows a four way translocation involving chromosomes 1 and 17 in addition to chromosome 9 an d 22. It is controversial whether variant translocations result in a different disease phenotype. There is no impact on the cytogenetic or molecular response or outcome, so a special approach is not recommended. ConclusionThis is a novel four way translocation described in a chronic phase CML patient, with a good response to standard therapy. Complete haematological and cytogenetic response at 4 months of treatment with Imatinib was achieved. Up to now, a differential approach does not appear to be mandatory in CML with variant translocations. However, in this patient alternative tyrosine kinase inhibitor may be of benefit due to hematologic toxicity attributable to Imatinib. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Frequency and Diversity of Variant Philadelphia Chromosome In Chronic Myeloid Leukemia Patients

Abstract 4903The Philadelphia chromosome (Ph), t(9;22), is detected in around 90% of the chronic myeloid leukemia (CML) patients, but in the remaining 5–10% cases a variant type may be present. Variant Ph-chromosome is characterized by the involvement of another chromosome in addition to the 9 and 22. It can be a simple type of variant when one more chromosome is involved, or complex, in which two or more chromosomes take part in the translocation. The aim of this report is to relate the diversity of variants Ph found in CML patients at the Cytogenetic service of Fleury Lab (São Paulo, Brazil) during the past 12 years. All Ph-positive CML patients (ALL&AML were excluded) were surveyed and the variant Ph ones were listed. All the karyotype analysis were from non-stimulated short term cultures of bone marrow or peripheral blood and classified according to ISCN (1999-2009). Of the 1,071 Ph-positive cases, 967 were classic Ph, while 50 (5.17%) were variants. Regarding the variant form, 44 (88%) were simple type and 6 (12%) complex type (table 1), similar to Marzocchi et al (2011), who found 93% and 7% of each type, respectively. Some of the variant Ph translocations we detected are listed in table 1. In agreement with what Marzocchi et al reported, the most involved chromosome was the 17 (eleven times in the present report and four cases in theirs), followed in frequency by the chromosome 1 (six times), 20 (five times), 6, 11 (four times each), and 2, 10, 12 and 15 (three times each). The chromosomes not involved in the Ph translocation were: 8, 9, 18, 21, 22, X and Y (Figure 1). Among all breakpoints seen in this survey, six were repeated: 11p15, 14q32, 15q11.2, 16p13.1, 17p13 e 17q21, differing from Marzocchi et al who denied finding any recurrent breakpoint. Reid et al. (2003) listed nine recurring breakpoints in variant Ph translocations most reported by other authors, who also assert that most of breakpoints related to variant Ph occur in regions of known oncogenes or typical secondary breakpoints in other cancers. Indeed we endorse this statement, since we also found some breakpoints related to other cancers. Of the list Reid et al reported, three were found in this study as well: 12p13, 17p13 and 17q21. Some of the genes located in these regions are related to other cancers (i.e. ETV6, CD9, RARA, GAS7). However, the gene TP53, located in the region 17p13 was the only one previously reported as related to CML. Oliver M. (2007) states that this gene is involved in 20–30% of cases of blast crisis CML. Regarding the prognosis, evolution of the disease and response to the treatment, it seems that variant type of Ph translocation has no implication for survival (Marzocchi et al 2011 and Maha et al 2004. In conclusion, as we presented a large variety of variant Ph translocations, it becomes clear that there are lots of possible interactions to form variant type and most of them occur in regions already reported as oncogenes or related to cancers, indicating that there must be local phenomena favoring the predisposition to these breakpoints involvement.Table 1–Examples of variant Ph karyotypes found in this survey, showing a little of the diversity of interaction.Karyotype46,XY,t(1;9;22;16)(q32;q34.1;q11.2;p13)[2]/46,XY46,XX,t(9;22;1;13)(q34.1;q11.2;p12;q34)[17]/46,XX[3]46,XY,t(9;22;1;?3)(q34.1;q11.2;q36.3;?q26)[20]46,XX,t(1;9;22;14)(p21;q34.1;q11.2;q32)[20]46,XY,t(9;22;2)(q34.1;q11.2;q24)[8]/46,XY[12]46,XY,t(9;22;4)(q34.1;q11.2;p16)[20]46,XY,t(9;22;5)(q34.1;q11.2;p13)[20]46,XY,t(9;22;6)(q34.1;q11.2;q22)46,XX,t(9;22;7)(q34.1;q11.2;p22)[20]46,XY,t?(9;22;10;11)(q34.1;q11.2;q22;q25)[20]46,XY,t(9;22;12)(q34.1;q11.2;p13)[20]46,XY,t(9;22;14)(q34.1;q11.2;q32)[19]/46,XY[1]46,XY,t(9;22;15)(q34.1;q11.2;q11.2)[20]46,XX,t?(9;22;16)(q34.1;q11.2;p13.3)[20]46,XY,t(9;22;17)(q34.1;q11.2;p13)[20]46,XY,t(9;22;19)(q34.1;q11.2;q11.2)[20]46,XY,t(1;7)(p22;q22),t(9;22;20)(q34.1;q11;q11.2)[2]/46,XY[18] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Variant Philadelphia translocations with different breakpoints in six chronic myeloid leukemia patients.

The Philadelphia (Ph) chromosome, consisting of the t(9;22)(q34;q11) translocation, is observed in ~90% of patients with chronic myeloid leukemia (CML). Variant Ph translocations are observed in 5%-10% of CML patients. In variant translocations 3 and possibly more chromosomes are involved. Herein we report 6 CML patients with variant Ph translocations. Bone marrow samples were examined using conventional cytogenetic meth ods. Fluorescence in situ hybridization (FISH) with whole-chromosome paints and BCR-ABL 1D probes were used to confirm and/or complement the findings, and identify rearrangements beyond the resolution of conventional cytogenetic methods. Variant Ph translocations in the 6 patients were as follows: t(7;22)(p22;q11), t(9;22;15)(q34;q11;q22), t(15;22)(p11;q11), t(1;9;22;3)(q24;q34;q11;q21), t(12;22)(p13;q11), and t(4;8;9;22)(q11;q13;q34;q11). Among the patients, 3 had simple and 3 had complex variant Ph translocations. Two of the presented cases had variant Ph chromosomes not previously described, 1 of which had a new complex Ph translocation involving chromosomes 1, 3, 9, 22, and t(1;9;22;3)(q24;q34;q11;q21) apart from a clone with a classical Ph, and the other case had variant Ph translocation with chromosomes 4, 8, 9, and 22, and t(4;8;9;22)(q11;q13;q34;q11) full complex translocation. Number of studies reported that some patients with variant Ph translocation were poor responders to imatinib. All of our patients with variant Ph translocations had suboptimal responses to imatinib, denoting a poor prognosis also. Variant Ph translocations may be important as they are associated with prognosis and therapy for CML patients.

Additional chromosome abnormalities in chronic myeloid leukemia

The Philadelphia (Ph) chromosome and/or Breakpoint cluster region-Abelson leukemia virus oncogene transcript are unique markers for chronic myeloid leukemia (CML). However, CML demonstrates heterogeneous presentations and outcomes. We analyzed the cytogenetic and molecular results of CML patients to evaluate their correlation with clinical presentations and outcome. A total of 84 newly diagnosed CML patients were enrolled in the study. Patients were treated according to disease status. Bone marrow samples were obtained to perform cytogenetic and molecular studies. Clinical presentations, treatment courses, and survival were reviewed retrospectively. Among 84 patients, 72 had chronic phase and 12 had accelerated phase CML. Cytogenetic study showed 69 (82.1%) with the classic Ph chromosome, 6 (7.2%) with a variant Ph chromosome, and 9 (10.7%) with additional chromosome abnormalities. Fifty-four (64.3%) cases harbored b3a2 transcripts, 29 (34.5%) had b2a2 transcript, and 1 had e19a2 transcript. There was no difference in clinical presentations between different cytogenetic and molecular groups; however, additional chromosome abnormalities were significantly associated with the accelerated phase. Imatinib therapy was an effective treatment, as measured by cytogenetic response, when administered as first- and second-line therapy in chronic phase patients. Survival analysis showed that old age, additional chromosome abnormalities, high Sokal score, and no cytogenetic response in second-line therapy had a significant poor impact ( p < 0.05). In conclusion, we presented the cytogenetic and molecular pattern of CML patients and demonstrated that the additional chromosome abnormality was associated with poor outcome.

Successful treatment with nilotinib after imatinib failure in a CML patient with a four-way Ph chromosome translocation and point mutations in BCR/ABL gene

Chronic myelogenous leukemia (CML) is characterized by Philadelphia (Ph) chromosome with a chimeric gene BCR-ABL created by reciprocal t(9:22) (q34;q11) translocation. Variant Ph chromosome translocations involving chromosomes other than 9 and 22 are found in 5-10% of CML cases. We here report a CML patient who carries a four-way Ph chromosome translocation, t(9;22;15;19) (q34;q11;q15;q13). The patient was diagnosed in 1997 and initially treated with hydroxyurea. In 2002, treatment with imatinib, a selective BCR-ABL tyrosine kinase inhibitor (TKI), was started but Ph-positive chromosomes remained at the levels of 42-65%, indicating imatinib failure. In 2006, the point mutations of F359I and L387M were detected in BCR/ABL gene, which may be related to imatinib failure. Treatment with nilotinib, a TKI with high target specificity, was then started which resulted in durable major molecular response. Administration of nilotinib offered an effective treatment in a CML patient with variant Ph chromosome translocations and BCR-ABL point mutations after imatinib failure.

Variant Philadelphia Translocation with BCR/ABL Fusion Gene Site In 10q24 In a Case of Chronic Myeloid Leukemia

AbstractAbstract 4839 Introduction.Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures:No relevant conflicts of interest to declare.

Application of fluorescence in situ hybridization technique in diagnosis of variant Ph chromosome translocation and Ph-negative chronic myelocytic leukemia

To explore the value of fluorescence in situ hybridization (FISH) technique in diagnosis of variant Ph chromosome translocation (VT) and Ph chromosome-negative chronic myelocytic leukemia (CML). Nine CML patients with VT and 2 Ph chromosome-negative CML patients confirmed by R banding were assayed with dual color-dual fusion BCR/ABL probe by FISH. The 9 patients with VT involved chromosomes 1, 3, 5, 12, 13, 15, 17 and 21 besides chromosomes 9 and 22, and some of them showed recurrent aberrations; FISH results were positive and the signal feature was 2R2G1Y. The 2 Ph-negative CML patients had normal karyotypes; FISH was positive and the signal feature was 1R1G2Y and 1R1G1Y respectively. FISH can provide better diagnosis for CML with VT and Ph-negative CML. Abnormal karyotype and marker gene changes can be assessed based on the signal feature of the positive cell. So FISH is a complementary method to banding technique in diagnosis of CML.

Variant Philadelphia-Positive Chronic Myeloid Leukemia, der(9) Deletions and Response to Imatinib Mesylate: A Retrospective Multicentric Analysis from the French Fi-LMC Group.

Purpose: Major initial prognostic factors predicting chronic myeloid leukaemia (CML) response to imatinib mesylate (IM) therapy are represented by hema-tological status, Sokal score and duration of disease before IM. Before the era of IM, CML patients harbouring der(9) deletion classically had a worse outcome when compared to non deleted pts, but some preliminary studies suggest that this adverse factor might be reversed by IM. Additionally der(9) deletion seems to be more frequent in Variant Ph chromosome (vPh) patients (up to 30%) whereas its frequency is about 10% in classical Ph pts. We evaluated in this retrospective multicentric study the response to IM in vPh population and the possible impact of der(9) deletion on this response. 92 vPh patients treated by IM were selected from 11 hematology departments within the country. vPh chromosome was assessed by conventional karyotyping analysis and der(9) deletion by FISH. IM resistant patients were screened for ABL kinase site mutation by direct sequencing. Statistical analysis of survival and descriptive parameters were performed according to Kaplan Meyer method, log-rank test, Chi-2 and non parametric Kruskal Wallis tests.Results: among the vPh population 75% pts had a simple translocation and 25% had a more complex one (chromosomes 9, 22 and at least two other break-points). Initially 95% pts were in chronic phase (CP), 2.5% in accelerated phase (AP) and 2.5% in blastic crisis (BC).19 pts (33.3%) were scored in each Sokal score category (low, intermediate and high). At IM initiation: 83% were in CP (>35% Ph+ ), 8% were in major cytogenetic response (after interferon) and 9% were either in AP or in BC. 74 pts were screened for der(9) deletion, which was present in 16.2% of them. IM response and survival data were available for 53 pts with CP (with > 35% Ph+ ) at IM initiation. Median age of this CP population at diagnosis was 50.8 (19.5–79.4), median disease duration before IM initiation : 2 Mo (0–92.4), median follow-up after IM initiation : 24.8 Mo (6.1–69.9). 71.7% achieved RcyM including 67.9% in RcyC. 19 pts presented IM failure or suboptimal response (European LeukemiaNet consensus). 1 pt stopped IM for liver intolerance. We observed 9 (16.9%) primary resistance and 10 (18.9%) loss of response. 12% pts progressed towards AP/BC. 12 resistant pts were screened for ABL kinase site mutations : 3 of them harboured mutations. We found no difference for the overall survival between the populations with (n=9) and without (n=33) der(9q) deletions (median of survival not reached ). Respectively 1 pt (11%) and 3 pts (9%) died in these two cohorts. We found a trend for an increased rate of resistance in the der(9) deletion cohort (55.6% vs 30.3%) but the difference was not significative (p=0.16). We only found significant differences in the repartition of the Sokal score between these two cohorts with more frequent high scores for vPh patients (p=0.03). Resistance in vPh IM treated pts is high (35.8%) but the disease duration before IM was heterogenous. Der(9) deletions slightly increases the rate of IM resistance but we found no difference of survival between the two cohorts.

Interpretation of submicroscopic deletions of theBCR orABL gene should not depend on extra signal-FISH: Problems in interpretation of submicroscopic deletion of theBCR orABL gene with extra signal-FISH

Several groups have demonstrated that a submicroscopic gene deletion in Ph+ chronic myelogenous leukemia (CML) is associated with a poor prognosis and reduced response to treatment. To assess the variation between detection methods in the interpretation of a submicroscopic gene deletion, we performed an extra signal (ES)-FISH BCR/ABL and double-FISH (D-FISH) BCR/ABL on frozen bone marrow cells from 79 patients with CML (63 in the chronic phase, 6 in the accelerated phase, and 10 in blast crisis) and 30 patients with a BCR/ABL-negative myeloproliferative disorder as determined by RT-PCR. The normal cutoff values were 0.22% for ES-FISH and 0.25% for D-FISH. The cutoff values for false-positive signals from a juxtaposition of the BCR and ABL gene were 11% in ES-FISH and 13% in D-FISH. Of the 14 patients who showed an ABL gene deletion by ES-FISH, 5 had an ABL deletion only, 5 had both a BCR and an ABL deletion, but 4 proved to have a classic BCR/ABL rearrangement without a submicroscopic deletion, as determined by D-FISH. Discrepant results between ES- and D-FISH were observed in 12 of the 79 patients (15.8%), and the main causes of a discrepancy were a false-positive ABL deletion (4 of 12, 33%), a variant Philadelphia chromosome (3 of 12, 25%), an inversion of derivative chromosome 9 at the very breakpoint of the ABL gene (9q32) (1 of 12, 8.3%), a cryptic variant Ph chromosome (1 of 12, 8.3%), and a marker chromosome (1 of 12, 8.3%). Although there was no significant difference in the sensitivity for the detection of the fusion signal between ES- and D-FISH, ES-FISH showed a high percentage of cells with false-positive fusion signals (1 orange, 1 green, 1 yellow), which makes it difficult to interpret the submicroscopic ABL deletion. In conclusion, an interpretation of the submicroscopic deletions of the BCR or ABL gene should not depend on ES-FISH.

Interphase fluorescence in situ hybridization studies for the detection of 9q34 deletions in chronic myelogenous leukemia: a practical approach to clinical diagnosis

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph) in more than 90% of cases. Recent studies using fluorescence in situ hybridization (FISH) have shown that in a subset of patients with CML, deletions of 9q34 involving the argininosuccinate synthetase region occur at the time of the Philadelphia translocation and are associated with a poor prognosis. We performed interphase FISH studies in 152 cases of CML using a dual-color, dual-fusion probe system with a third probe directed at 9q34. Cytogenetic studies showed a simple (typical) Ph in 124/152 (82%), a cryptic Ph in 11/152 (7%), and a variant Ph chromosome with a complex translocation in 17/152 (11%) of cases. Interphase FISH studies showed single BCR/ABL fusion patterns in 48/152 (32%) of cases. Deletions of 9q34 were observed in 14% of all the cases and were present in 46% of cases with single BCR/ABL fusion pattern. All the 9q34 deletions occurred in cases with single BCR/ABL fusion signal. However, a single-fusion pattern is not specific for 9q34 deletions, and cases should be routinely screened for the presence of this prognostically significant abnormality by using a third probe directed specifically at 9q34.

Amplification of BCR-ABL Rearrangement in Atypical Philadelphia Chromosome : A New Variant Detected by Fluorescence In Situ Hybridization in One Case of Acute Lymphoblastic Leukemia

We report on a 67-year-old woman with acute lymphoblastic leukemia (ALL) who was supposed to have a variant Philadelphia (Ph) translocation identified by conventional cytogenetic techniques. Fluorescence in situ hybridization (FISH) analysis demonstrated the presence of an amplification of BCR-ABL rearrangement at locus 22q11. This is the first observation, to our knowledge, of a duplicated BCR-ABL chimeric gene within the derived chromosome 22 in ALL. Our observation supports the possibility of detecting a variant Ph chromosome at the single-cell level by FISH analysis.