B3a2 Transcript Research Articles

Gender and BCR-ABL transcript type are correlated with molecular response to imatinib treatment in patients with chronic myeloid leukemia.

Achieving a major molecular response (MMR) is the goal of imatinib therapy for chronic myeloid leukemia. However, the association between gender, BCR-ABL transcript type, and age with MMR is not well understood and often controversial. We retrospectively analyzed 166 patients who have been treated with imatinib for up to 10 yr. Men had a lower MMR rate than women (63.3% vs. 81.6%, P = 0.006) and a shorter time to relapse (median 354 vs. 675 d, P = 0.049), while patients with b3a2 or with both b3a2 and b2a2 break point transcripts had higher MMR rate than those with b2a2 (81.8%, 77.1% vs. 60.7%, P = 0.023 for b3a2 vs. b2a2, P = 0.043 for both vs. b2a2). A striking difference was found between men with b2a2 and women with both b2a2 and b3a2 in terms of MMR rate (43.8% vs. 88.9%), MMR rate within 6 months (7.1% vs. 62.5%) and the time to MMR (median d 493 vs. 159, P = 0.036). Both gender and BCR-ABL transcript, but not age, were significantly associated with the molecular response. Men with b2a2 represent a less favorable group in their response to imatinib treatment and may need alternative therapy regimen and closer monitoring.

Simple multiplex RT-PCR for identifying common fusion BCR-ABL transcript types and evaluation of molecular response of the a2b2 and a2b3 transcripts to Imatinib resistance in north Indian chronic myeloid leukemia patients

Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, an abnormally shortened chromosome 22. It is the result of a reciprocal translocation of chromosomes 9 and 22, creating BCR-ABL fusion transcripts, b3a2, b2a2, and e1a2. The aim of our study was to determine the type of BCR-ABL fusion transcripts for molecular diagnosis and investigate the frequency of BCR-ABL fusion transcripts in CML patients by multiplex RT-PCR in CML. A single reaction with multiple primers multiplex PCR was used to detect and investigate the type and frequency in 200 CML patients among which 116, 33, and 51 were in CP, AP, and BC phase, respectively. The study included 200 CML patients, among whom breakpoints in b3a2, b2a2 transcripts were detected in 68% and 24%, respectively, while 8% of the patients showed both b3a2/b2a2. A statistically significant difference was seen between frequency of BCR-ABL fusion transcripts and gender (P = 0.03), molecular response (P = 0.04), and hematological response (P = 0.05). However, there was no correlation found between frequencies of BCR-/ABL fusion transcripts and other clinicopathological parameters like age, type of therapy, thrombocytopenia, and white blood cell count. Multiplex reverse transcriptase-polymerase chain reaction is useful and saves time in the detection of BCR-ABL variants; the occurrence of these transcripts associated with CML can assist in prognosis and treatment of disease.

Efficacy and Safety of Nilotinib in First Line Therapy in a Real Life Cohort of Chronic Myeloid Leukemia Patients

IntroductionConsistent data show that the real goal in CML therapy is to obtain a very rapid and deep response because the cinetic of molecular response (MR) is important for quickly reducing the tumor burden by reducing the BCR/ABL level.The second generation TKIs Nilotinib was designed to have enhanced selectivity and potency toward BCR-ABL compared with imatinib and was able to obtain very rapid and deeper response as shown by many clinical trials. Nilotinib has been approved in multiple countries for use in first line therapy in CML-CP Pts. In order to verify the efficacy and safety of Nilotinib used in first line treatment, we collected the results of a real life cohort of Pts treated in some italian hematological Institutions.Patients and Methods68 Pts affected by CML-CP were treated in 9 hematologic units with Nilotinib as first line therapy from 2010 to 2014. The median age was 47.7y with equal distribution by sex. Cytogenetic analysis at diagnosis showed in 66/68 Pts the presence of the typical Ph1 chromosome, 1 pt showed a Ph1 in a complex translocation and 1 pt showed the typical Ph1 chromosome plus der(17). The molecular analysis confirmed the presence of a BCR/ABL hybrid gene (55/68: b3a2 transcript; 12/68 b2a2 transcript; 1/68 both transcripts). The Sokal risk was: Low 24 Pts (35.3%), Intermediate 33 Pts (48.5%), High 11 Pts (16.2%); the Hasford risk was: Low 35 Pts (51.5%), Intermediate 27 Pts (39.7%), High 6 Pts (8.8%); the EUTOS risk was: Low 61 Pts (89.7%) and High 7 Pts (10.3%). All Pts have an ECOG 0/1 out of 1 with an ECOG 2. 45/68 pts received a cytoreduction by HU. All Pts received the Nilotinib standard dose of 600 mg/die except 1 pt who received 300 mg/die. At diagnosis some Pts showed comorbidities (21/68: hypertension; 6/68: diabetes; 5/68: dyslipidemia; 2/68: previous myocardial infarction; 1/68 was in treatment for HIV infection)Results66/68 Pts received at least 3 months of treatment and were considered for analysis. At 3 months, all Pts obtained hematological response; cytogenetic response was complete for 65/68 and partial in 3/68.At 6, 12 and 18 months cytogenetic response was complete in all Pts except for 1 that showed a partial response (pt not compliant); at 24 mo the cytogenetic response was complete for all 25 evaluable Pts.The molecular results at the different time points are summarized in table 1.6/68 Pts showed G1/G2 cutaneous side effects (pruritus, erithema, alopecia) , 6/68 a G1 increase of amylase and/or lipase, but only 1 developed clinical pancreatitis (at diagnosis: hypertension, hypercolesterolemia and cardiac disfunction). One pt underwent to leg amputation for PAOD (at diagnosis: hypertension and hypertriglyceridemia). He discontinued treatment in optimal response at 12mo. One pt had ischemic cerebral event (at diagnosis had hypertension and a previous heart attack). One pt discontinued treatment for pancitopenia in failure of response at 3mo and one pt died at 12mo in failure of response for IMF diagnosed during treatment.No pts developed mutations of BCR/ABL during treatment. In 24 months of observation no Pts lost the response.ConclusionThe response to Nilotinib treatment at 3 months according to ELN revealed that all evaluable Pts were in optimal response; at 6 mo 51/55 (92,7 %) Pts were in optimal response, while at 12 mo 38/45 (86,7%) Pts were in optimal response. At 18 mo 94,1% reached at least MMR with a very deep response (MR>= 4) in 55,9% of Pts. This kind of response was mantained at 24 mo (92% of Pts had at least an MR3 and 15/25 had an MR 4).Notabily starting from 12 months, at least 40 % of pts showed a very deep response (MR >= 4) and the number increased at the subsequent time points.The side effects were few and of low grade. Only 3 Pts discontinued the treatment because side effects. Only a severe side effect (PAOD) was reported in a pt who already had a vascular risk because affected from hypertension and hypertriglyceridemia. It is recommended to check at diagnosis and carefully follow during the treatment for all comorbidities to prevent toxicity.These data collected in a real life treatment with Nilotinib in first line in CML -CP patients, confirmed the results of the clinical trials. The rapid and deep response with a tolerable toxicity profile allow to consider Nilotinib as a good choice for first line treatment also in consideration of a stop therapy in a future.Table 1Time Pts MR1 MR2 MR3 MR4 MR4.5 MR5(mo) (N)3 60 10 31 13 4 2 -6 55 4 16 20 4 11 -12 45 1 6 20 4 13 118 34 0 2 13 6 12 124 25 0 2 8 6 8 1 DisclosuresNo relevant conflicts of interest to declare.

Dasatinib or Nilotinib Discontinuation in Chronic Phase (CP)-Chronic Myeloid Leukemia (CML) Patients (pts) with Durably Undetectable BCR-ABL Transcripts: Interim Analysis of the STOP 2G-TKI Study with a Minimum Follow-up of 12 Months – on Behalf of the French CML Group Filmc

Background: Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have revolutionized the prognosis of pts suffering from CML but these drugs are considered as non-definitively curative and current recommendation is to treat pts during their entire lifespan. However, prospective trials such as STIM, TWISTER and EUROSKI suggest that imatinib may be successfully stopped in pts with deep and sustained molecular responses. Here, we report on the feasibility of second generation TKIs discontinuation in the setting of the French STOP 2G-TKI study.Methods: Adult CP-CML pts on dasatinib or nilotinib first line or after imatinib without prior allogeneic transplantation or progression to advanced phase CML were proposed TKI discontinuation when presenting: (1) b2a2 or b3a2 BCR-ABL transcripts subtype, 2) TKI treatment duration for at least 36 months, (3) CMR4.5 achieved and maintained for at least 24 months. The primary objective was treatment-free survival without loss of major molecular response (MMR). After TKI discontinuation, BCR-ABL transcripts were monitored monthly during the first 12 months, every 3 months during the 2ndyear and every 3 to 6 months thereafter. Molecular relapse was defined by MMR loss on a single occasion and triggered TKI reintroduction. Data as of August 1, 2014 are reported in pts with at least 12 months of follow-up (n=52) and median follow-up was 32 months (12-56).Results: Median age was 60 years (34-81) and 61.5% of pts were female. Sokal risk group was low in 58%, intermediate in 23%, high in 13% and unknown in 6%. 2G-TKIs were given after imatinib intolerance in 67% of pts, suboptimal response or resistance to imatinib in 23% and upfront in 10%. Median duration of CML, TKI treatment, 2G-TKI treatment and CMR4.5 was 83 months (36-218), 78 months (36-136), 39 months (19-72) and 28 months (24-64), respectively. Twenty four pts lost MMR after a median time of 4 months (1-38) at last follow-up. Importantly, no loss of CHR or progression to advanced phase CML was observed. The 12- and 24-month probabilities of treatment-free survival without MMR loss were 61.4% (95% CI, 48.1-74.6) and 57% (95% CI, 43.3-70.6), respectively. The majority of relapses occurred within 6 months and in a landmark analysis, pts who were still in MMR without therapy at 6 months had 12- and 24-month probabilities of treatment-free survival without MMR loss of 91.2% (95% CI, 81.6-100) and 84.7% (95% CI, 72.2-97.1), respectively. All pts but 1 who lost MMR restarted 2G-TKI treatment and regained MMR after a median time of 3 months (1-8). Pts in MMR without any therapy (n=28) displayed varying patterns of spontaneous molecular response including stable CMR4.5 in 7 and fluctuations between CMR4.5 and MR4.5, CMR4.5 and MR4, CMR4.5 and MMR in 9, 4 and 4 pts, respectively. Gender, age, prior interferon exposure, 2G-TKI type, treatment duration and duration of CMR4.5 were not found to have any impact on outcome. By contrast, prior history of suboptimal response or resistance to imatinib was associated with a significantly lower chance of successful treatment discontinuation, with a 12-month probability of treatment-free survival without MMR loss of 41.7% (95% CI; 13.8%-69.6%), compared to 67.3% (95% CI, 52.6%-81.8%) in other patients (p=0.04).Conclusions: 2G-TKI could be safely and successfully discontinued in CP-CML pts with long-lasting undetectable BCR-ABL transcripts, especially in those without prior history of suboptimal response or resistance. Most of molecular relapses had an early onset and all were sensitive to 2G-TKI resumption. The recurrence of low levels of detectable residual disease below MMR after 2G-TKI withdrawal did not automatically herald CML relapse and did not preclude the possibility to remain treatment-free. DisclosuresNicolini:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Rousselot:Novartis: Research Funding. Gardembas:BMS: Honoraria. Legros:Novartis, BMS: Honoraria. Etienne:Novartis, BMS,Pfizer, ARIAD Pharmaceuticals, Inc.: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

A case of chronic myeloid leukemia with the m-bcr (p190) molecular rearrangement identified during treatment

Chronic myeloid leukemia (CML) is a hematological malignancy in which a high percentage of patients cytogenetically express the Philadelphia chromosome. This chromosome results from the reciprocal translocation, t(9;22)(q34;q11.2). During this exchange, the Abelson gene (abl) on chromosome 9 and the breakpoint cluster region gene (bcr) on chromosome 22 generate the breakpoint cluster region-Abelson murine leukemia (BCR-ABL) fusion gene. The most frequent breakpoint on chromosome 22, located between exons 12 and 16, is called the major breakpoint cluster region (M-bcr) and with its counterpart in exon 2 of chromosome 9 results in the b2a2 and b3a2 transcripts that code for a 210 kDa protein. Less frequently, a second rearrangement, minor breakpoint cluster

European Treatment and Outcome Study (Eutos) Score Predicts Early Molecular Response to Imatinib Therapy in Chronic Phase Chronic Myeloid Leukemia Patients (Cp-Cml)

rd month using quantitative RT-PCR. Results were expressed as BCR-ABL/ABL ratio. Early molecular response (EMR) was defined as a transcript level ≤ 10%. Univariate and multivariate analysis (Linear regression) was performed to identify potential factors associated with the achievement of EMR. Univariate analysis (Linear regression) with RQPCR value at 3 rd month as dependent variable in relation to the other continuous variables was performed. Variables showing statistically significant association with the outcome (EMR at 3 rd month) at p < 0.05 were considered as candidate variables for inclusion in the multivariate model. Multivariate analysis (linear regression) was performed with the potential candidate variables as the co-variates. SPSS software version 17.0 was used for analysis. Results: The study population included 68 newly diagnosed patients of CP-CML, of which 55.88% (38) were males. The median age at diagnosis in our cohort was 46 years. According to Sokal risk stratification 55.88% (38), 33.82% (23) and10.29% (07) were in high, intermediate and low risk respectively. As per Hasford risk stratification score 25% (17), 61.76% (42), and 13.24% (09) were in high,intermediate and low risk respectively. EUTOS score stratified 58.82% (40) of patients as high risk and 41.18% (28) as low risk. 69.12% (47) of patients had b3a2 transcript and 30.88% (21) had b2a2 transcripts. 32.35% (22) achieved EMR at 3 rd month. In univariate analysis, spleen size (p= <0.001),pretreatment basophil percentage(p= <0.001),WBC count at 3 rd month (p = 0.006),percentage of Ph+ metaphases at 3 rd month(p = 0.009) and EUTOS score (p = < 0.001) significantly predicted the achievement of EMR. In multivariate analysis spleen size(p = 0.01), pretreatment basophil percentage(p = < 0.001) and EUTOS score (p = < 0.001) significantly predicted the achievement of EMR. Conclusions: EUTOS score at diagnosis predicts early molecular response to imatinib therapy at 3 rd month.

Incidence of BCR-ABL transcript variants in patients with chronic myeloid leukemia: Their correlation with presenting features, risk scores and response to treatment with imatinib mesylate.

Context:The exact role of the different transcript variants of BCR-ABL in the pathogenesis of chronic myeloid leukemia (CML) and their impact on prognosis is yet to be definitely enumerated.Aims:In this study, we have tried to correlate the presenting features, risk scores and treatment response with the BCR-ABL variants detected in our patients.Settings and Design:A cross-sectional unicentric hospital-based study on 80 patients diagnosed to have CML by bone marrow cytogenetics and confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR).Materials and Methods:RT-PCR for BCR-ABL was performed on consecutive patients with CML attending the CML clinic from January 2010 to December 2010. The medical charts of these patients were analyzed after a follow-up of 18 months in a retrospective manner.Statistical Analysis:Box plot and histogram was used to see the distribution of variables. t-test was performed to enumerate the difference between risk scores in two populations of patients carrying two different BCR-ABL transcript variants.Results:Nearly 56.25% of patients had b3a2 (e14a2) while 41.25% of patients showed b2a2 (e13a2) transcripts. The rest 2.5% (two patients) expressed the rare e19b2 variant. Patients with b2a2 presented with higher Sokal, Hasford and European Treatment and Outcomes Study score than their b3a2 counterpart. Different parameters such as the platelet count, leukocyte count, hemoglobin and splenomegaly showed a minor difference between the groups. More patients in the b2a2 group achieved complete hematologic response at 3 months, but it was not significant.Conclusions:Patients with b2a2 variant CML tend to present with higher risk score, but do not behave in a vastly different manner than their b3a2 counterparts.

BCR-ABL Splice Variants In Three Phases Of CML: Association With Disease Biology and Response To Imatinib

BackgroundPhiladelphia chromosome, a characteristic chromosomal translocation (breakpoint cluster region-Abelson [BCR–ABL]; t [9; 22] [q34; q11]) in chronic myeloid leukemia (CML) encodes a product, which is the target of tyrosine kinase inhibitors (TKIs). In most CML patients, during translocation, the break takes place within a 5.8-kb region on both sides of BCR exons12-16 (originally referred to as exons b1-b5), described as themajor breakpoint cluster region (M-bcr)1. It can be either between exons b2 and b3, or between exons b3 and b4. The ABL exon which is mostly inserted in break points of BCR is a2. As a result of alternativesplicing, fusion transcripts with either b2a2 or b3a2 splicing variantcan be produced. Occasionally, breakpoints occur in unusual places, resulting in generation of rare BCR-ABL transcripts (b2a3, b3a3, e1a2, e8a2, e19a2). Association of BCR-ABL splice variants with clinical outcome in different phases of CML has rarely been studied2. We studied BCR/ABL splice variants and their association with disease biology and response to imatinib in 3 phases of CML in 70 patients. MethodsTotal RNA was extracted and reverse transcribed. PCR primers and nested PCR protocol for the detection of BCR-ABL splice variants in CML patients were adopted from van Dongen et al., 1999 3. Data was analyzed using SPSS Software version 17. ResultsMedian age of the patients was 34 years; 10 patients were 18 or younger and there were 40 females. 26 patients were in chronic phase (CP), 26 in accelerated phase (AP) and 18 in blast phase (BP). Overall, the frequencies of BCR/ABL splice variants b3a2, b2a2 and b3a2+b2a2 were found in 49 (70%), 15 (21.4%) and 6 (8.6%) patients, respectively. No rare BCR-ABL transcripts were identified. Percentage of BCR-ABL splice variant b2a2 was similar in patients with CP (31) and BP (28) of CML and low in AP (7.75). While the percentage of BCR-ABL splice variant b3a2 was more common in AP (84.5) and BP (72) as compared to CP (54). The combined expression of both transcripts (b3a2 and b2a2) was found in 4 patients with CP and 2 in AP of CML but none in BP of CML (Table 1). The co-existence of b2a2 and b3a2 transcripts was observed only in male patients. The BCR-ABL transcripts had no correlation with the age. Patients with the splice variant b2a2 had a better response to imatinib treatment and better survival as compared to patients with b3a2. The patients expressing both transcripts (b3a2 and b2a2) also showed better survival as compared to patients with only b3a2 transcript (figure 1). [Display omitted] Discussion and ConclusionsWe found a higher percentage of BCR-ABL splice variant b3a2 in AP & BP as compared to CP. Patients with the BCR-ABL splice variant b2a2 had a better response to imatinib and better survival, which is in contrast to previous reports 4. Co-existence of two transcripts b3a2 and b2a2 was observed mostly in CP, and in some patients in AP; it was also associated with a better response to imatinib. Larger studies are needed to further define the role of BCR-ABL splice variants in different phases of CML and their association with response to TKIs and prognosis. Studies of BCR-ABL splice variants can provide further insights into CML biology and new targets for BCR-ABL positive leukemia treatment 1, 5.

BCR-ABL Fusion Transcripts In Patients With Chronic Myeloid Leukemia In Chronic Phase (CML-CP): Experience With Ponatinib From MD Anderson Cancer Center

IntroductionThe presence of the BCR-ABL fusion gene, resulting from the Philadelphia chromosome is the hallmark of CML. The most commonly observed transcripts in CML are b3a2 (e14a2) and b2a2 (e13a2). It is not known whether the transcript type may impact response to newer TKI. We report our experience of ponatinib in patients with CML-CP with different BCR-ABL transcripts. MethodsA total of 85 pts with CML-CP [47 relapsed refractory (RR) and 38 frontline] who were treated at our institution between 2008 and 2013 on clinical trials with ponatinib were included in this analysis. Conventional cytogenetic analysis was done in bone marrow (BM) cells using a G-banding technique, and the BCR-ABL fusion transcripts were identified by the use of quantitative real-time polymerase chain reaction assay. ResultsAmong the 47 RR patients, 14 (30%) had the b2a2 transcript (median age 47 years; range 21–82), 23 (49%) had a b3a2 transcript (median age 61; range 24–94 years), and 10 (21%) co-expressed both transcripts (median age 45; range 49–79). Two pts with e1a2 transcripts were not included in the analysis. RR patients received a median of 3 prior therapies including interferon, imatinib, nilotinib, dasatinib, bosutinib, omacetaxine and SCT. Ten (71 %) of those with b2a2 were previously treated with ≥3 different TKI (imatinib, dasatinib, nilotinib and bosutinib); of the pts with b3a2, 12 (52%) were treated with ≥3 different TKI (imatinib, dasatinib, nilotinib and bosutinib) and in the group of pts with both transcripts 5 (50%) received 3 different TKI (imatinib, dasatinib, nilotinib). T315I mutation was present in 9 (19%) pts of which 2 were b2a2, 5 were b3a2 and 2 were both. After a median follow-up of 22.4 months, the rates of overall complete cytogenetic (CCyR) and major molecular responses (MMR) were 55% and 40%. Among these RR patients, the response with b2a2, b3a2 and both transcripts, were CCyR: 50% vs. 61% vs. 50% and MMR 29% vs. 52% vs. 30%, respectively. The 3-year probability of failure free survival (FFS) was 54%, 87% and 68% for pts with b2a2, b3a2 and both groups (P=0.08). The 3-year probability of overall survival (OS) was 62%, 100% and 100% for pts with b2a2, b3a2 and both groups (P=0.03).Among the 38 pts in the frontline setting, 17 (44%) had the b2a2 transcript (median age 50.7 years; range 21–74), 14 (36%) had b3a2 transcript (median age 49.7; range 29–72 years), and 7 (18%) presented with both transcripts (median age 56; range 38–63). The Sokal risk group for those with the b2a2 transcript was high in 2/17 (12%), intermediate in 4/17 (23%), and low in 11/17 (65%). For pts with b3a2 all 14/14 (100%) were low risk, and the group with both transcripts had 0%, 1/7(14%) and 6/7 (85%), respectively. There were two pts who went off study due to toxicity. For the evaluable pts with b2a2, b3a2 and both transcripts, the median levels of transcripts at 3 months were 0.098, 0.091 and 0.042 respectively. Similarly the median levels of transcripts at 6 months were 0.00525, 0.0035 and 0.0035 respectively. The magnitude of early CCyR was very high in all groups thus not allowing to discriminate among the transcripts. The median OS was 6.6 months and none of the pts have died. ConclusionsAlthough all pts have a good overall survival those receiving ponatinib after prior TKI failure with b3a2 transcripts have a higher rate of CCyR and MMR vs. patients with b2a2 transcripts. This mirrors what we have reported with other TKI. With early results, no difference is seen in the frontline setting perhaps because of the overall excellent results. The mechanism of differential responsiveness of pts with b2a2 transcripts and b3a2 transcripts warrants further investigation. Disclosures:Kantarjian:ARIAD, Novartis, BMS, Phizer: Research Funding. Jabbour:Ariad, BMS, Novartis, and Pfizer: Consultancy. Cortes:Ariad, Pfizer, Teva: Consultancy; Ariad, BMS, Novartis, Pfizer, Teva: Research Funding.

Deep Molecular Responses In Patients With Newly Diagnosed Chronic Myeloid Leukemia Receiving Nilotinib As Assessed Within The EUTOS Laboratory Network In The ENEST1st Study

Deep Molecular Responses In Patients With Newly Diagnosed Chronic Myeloid Leukemia Receiving Nilotinib As Assessed Within The EUTOS Laboratory Network In The ENEST1st Study

Predictive Factors For An Achievement Of 6-Month Early Molecular Response In New CP CML Patients Treated With Imatinib

BackgroundRecent studies have demonstrated that early molecular response (EMR) is predictive for long-term outcomes. However, the value of EMR has not been fully defined. Recently, European Leukemia Net (ELN) recommended that BCR-ABL1 ≤ 10%, and/or Ph+ <35% at 6 months of treatment was an optimal response. The aim of this study was to identify predictive factors for an achievement of 6-month EMR (BCR-ABL1 ≤ 1%) and to evaluate prognostic implications of 6-month EMR. MethodsCP CML patients who were newly diagnosed and receiving 400mg IM once daily with no prior treatment were eligible for this study. Molecular responses were monitored using qRT-PCR assay with 3 month intervals, and then 6 month intervals after achieving major molecular response (MMR). All qRT-PCR tests were performed in a single laboratory (Cancer Research Institute, The Catholic University of Korea, Seoul, Korea). Pharmacokinetics data of IM, drug adherence, and dose intensity as well as baseline biological characteristics were included as variables affecting the achievement of 6-month EMR. ResultsA total of 102 patients (including 61 men and 41 women) were enrolled. One patient changed IM treatment to second-generation TKI due to less than complete hematologic response before 3 months. At the time between 3 and 6 months, 9 patient were discontinued permanently from IM treatment due to progression (n = 1), ELN failure (n = 3), and intolerance (n = 5). Ninety-two patients' molecular responses were analyzed at 6 months. Day 29 trough IM level data were available from 99 patients and trough IM level data on the end of cycle 6 were available from 84 patients. Univariate analyses revealed that age of ≥ 40 years (P = 0.061), male sex (P = 0.042), b3a2 transcript type (P = 0.008), intermediate (P = 0.007) and high Sokal risk (P = 0.013), increased leukocyte count (P = 0.018), increased blast percentage (0.028), large splenic size (P = 0.020), and mean daily dose by 6 months of <350 mg/day (P = 0.004) were potential predictive factors for no achievement of 6-month EMR. Increased log reduction of BCR-ABL1 from baseline to 3 months (P <0.001) was associated with achievement of 6-month EMR. After adjusting for factors affecting relapse on univariate analyses, multivariate analyses showed that b2a2 transcript type (RR of 9.35, P = 0.013), increased log reduction of BCR-ABL1 from baseline to 3 months (RR of 9.58, P = 0.001), and mean daily dose by 6 months of ≥350 mg/day (RR of 13.10, P = 0.019) were independent factors for a achieving of 6-month EMR. In addition, patients with high Sokal risk had a lower 6-month EMR, compared with those with low Sokal risk (RR of 0.02, P= 0.035).In the current study, patients with BCR-ABL1 ≤1% at 6 months had a better MMR rates at 12 months (63% vs 10%, P<0.001), 3-year CI of CCyR (100% vs 76.1%, P<0.001) and MMR (100% vs 66.4%, P<0.001), EFS (78.9% vs 30.6%, P<0.001), and FFS (97.2% vs 68.7%, P<0.001). Patients with BCR-ABL1 >1% at 6 months showed a trend for lower 3-year PFS, compared with those who achieved ≤1% (100% vs 94.1%, P = 0.063). ConclusionsIn this study, we re-confirmed the prognostic significance of 6-month EMR and found that b2a2 transcript type, early decline of BCR-ABL1 transcript, mean daily dose by 6 months (≥350 mg/day), and Sokal risk were associated of the achievement of 6-month EMR. These predictive factors for 6-month EMR should be considered in the clinical decision of changing therapy at this time point. Further clinical investigations in a larger patient population with longer follow-up are needed. Disclosures:No relevant conflicts of interest to declare.

Clinical and laboratory investigation of pericentric inv(9)(p22q34) with the der(9)t(9;22)(q34;q11) in Ph-positive leukemia

To investigate clinical and molecule genetics features of four Ph-positive leukemia patients characterized by pericentric inv(9)(p22q34) with the der(9)t(9;22)(q34;q11). Cytogenetic analysis was carried out on bone marrow directly or after short-period culture. R banding was used for karyotype analysis. BCR/ABL fusion gene was detected with interphase fluorescence in situ hybridization (FISH), and chromosome painting was carried out using specific probes. RT-PCR was used to detect BCR/ABL chimeric transcripts. One patient with acute myeloid leukemia (AML) presented three clones, which included one with a normal karyotype, one with t(9;22)(q34;q11), and one with inv(9)(p22q34) involving the der(9)t(9;22) and additional t(8;12)(q12;p11). The inv(9)(p22q34) has always co-occurred with der(9)t(9;22)(q34;q11) accompanied by der(22)t(9;22)(q34;q11) in all metaphases from the three patients with chronic myeloid leukemia (CML). B3a2 transcript was detected in all patients by RT-PCR. Inv(9)(p22q34) was found in both CML and AML, and was associated with poor prognosis. Inv(9)(p22q34) is a novel, rare, but recurrent secondary chromosomal abnormality for Ph-positive leukemia. Leukemia with der(9)t(9;22) and inv(9)(p22q34) has unique clinical and laboratory characteristics.

Multiple copies of BCR-ABL fusion gene on two isodicentric Philadelphia chromosomes in an imatinib mesylate-resistant chronic myeloid leukemia patient

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. Amplification or duplication of the BCR-ABL gene has been found to be one of the key factors leading to drug resistance to imatinib mesylate (IM). In the present study, we identified the presence of isodicentric Ph chromosomes [idic(Ph)] in an IM-resistant patient. Fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes confirmed the heterogeneity and amplification of the fused BCR-ABL gene. FISH analysis superimposed on G-banding confirmed the presence of idic(Ph) chromosomes. Reverse transcription-polymerase chain reaction (RT-PCR) products revealed the presence of the BCR-ABL fusion transcript b3a2. The idic(Ph) chromosomes in CML were shown to be fused at the satellite regions of the short arms. The patient did not respond to IM chemotherapy and did not achieve remission. In this study, the impact of the idic(Ph) chromosomes on genomic instability, heterogeneity and amplification of the BCR-ABL gene in IM-resistant patients is discussed.

Analysis of the clinico-hematological relevance of the breakpoint location within M-BCR in chronic myeloid leukemia

The Philadelphia chromosome (Ph) derives from the balanced translocation between chromosomes 9 and 22. This chromosomal translocation results in the fusion between the 5' part of the BCR gene, normally located on chromosome 22, and the 3' part of the ABL gene on chromosome 9 giving origin to a BCR-ABL fusion gene which is transcribed and then translated into a hybrid protein. In general, three breakpoint cluster regions in the BCR gene have been described: major (M-BCR), minor (m-BCR) and micro (μ-BCR). Three main variants of the BCR-ABL gene have been described depending on the length of the sequence of the BCR gene included that encode for the P190, P210, P230 proteins. Most patients (95 %) were found to have P210 protein that resulted from rearrangement in the M-BCR region in the BCR gene and thus gives rise to b2a2 or b3a2 variants. The incidence of one or other rearrangement in chronic myeloid leukemia (CML) patients varies in different reported series. These two variants are associated with distinct clinical types of human leukemias. In this study, we report the frequencies of M-BCR-ABL fusion transcripts in 44 CML patients and we review the data on the correlations between the type of M-BCR/ABL variant and the corresponding sex, age and biological features. Forty-four untreated chronic phase CML patients were studied. The type of BCR-ABL fusion transcripts was determined by reverse transcriptase polymerase chain reaction (RT-PCR). More than half of them showed b3a2 fusion transcript (64 %), while (36 %) showed b2a2 transcript. No patients coexpressed b3a2/b2a2. Correlation between biological data demonstrated that: (a) M-BCR rearrangements were not associated with the sex of the patients. (b) Patients with b3a2 rearrangements were older than patients with b2a2 transcripts. (c) M-BCR rearrangements were influenced neither by the white blood count (WBC) nor with hemoglobin levels. However, platelet level is more elevated in patients with b3a2 transcript (681.2/L vs. 207/L; P = 0.001). In conclusion, we observed significant correlations between age, platelet level and M-BCR-ABL transcript, these observations deserve further investigations.

Response to Frontline Therapy with Second Generation Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia: Analysis of Outcome for b3a2 Vs. b2a2 Fusion Transcripts

AbstractAbstract 3781 Background:CML is characterized by the reciprocal translocation between chromosomes 9 and 22 [t(9;22)(q34;q11.2)], also known as the Philadelphia chromosome. This results in the formation of the BCR-ABL fusion gene, which is translated to a protein with increased tyrosine kinase activity. The breakpoint in the BCR gene can occur in different sites, most commonly between exons 13 and 15 which fuse to exon 2 of the ABL gene creating the b2a2 and b3a2 junctions. Previous studies have reported a better outcome for patients (pts) having a b2a2 transcript when treated with imatinib. To our knowledge there are no published reports analyzing the outcome of pts treated with a 2ndgeneration TKI as initial therapy for CML according to the fusion transcript. We analyzed the significance of these variations among pts with CML in chronic phase (CP) enrolled in 2 parallel trials of dasatinib and nilotinib as initial therapy. Patients and Methods:A total of 204 pts with CML in CP, treated at our institution between 2005 and 2012 with nilotinib or dasatinib as frontline therapy were included in this analysis. Among them 88 (43%) had the b2a2 transcript (median age of 47 years; range 18–80) at the start of therapy, 74 (36%) showed the b3a2 variant (median age of 47; range 17–82 years), and 42 (21%) presented both (median age 53; range 27–81). One patient had a b3a3 transcript and 2 pts showed an e1a2 transcript. Forty-two (48%) of those with b2a2 were treated with dasatinib and 46 (52%) with nilotinib; of the pts with b3a2 34 (46%) were treated with dasatinib and 40 (54%) with nilotinib, in the group of pts with both transcripts 24 (57%) received dasatinib 24, and 18 (43%) of them nilotinib (Table 1). Results:The Sokal risk group for those with the b2a2 transcript was high in 10%, intermediate in 29%, and low in 61%. For pts with b3a2 the risk classification was 5%, 25, and 70%, respectively. The group with both transcripts had 10%, 31% and 69%, respectively. Significant difference was observed in the time of molecular response and overall outcome between pts with b2a2 and those with b3a2 (CCyR 91% vs. 96%; MMR 75% vs. 91%, respectively). The 3-year probability of overall survival (OS) was 100% for both groups. The 3-year probability of event-free survival (EFS) was 93% and 99%, and the transformation-free survival (TFS) 99% and 98% for the b2a2 and the b3a2 group respectively (Table 2). On the 12 mo. follow-up one of the pts with e1a2 transcript achieved CCyR (treated with nilotinib), and the second patient (pt) major cytogenetic response (receiving dasatinib). The pt presenting with b3a3 was treated with dasatinib and achieved MMR by 3 mo. and CCyR at the 12 months follow-up. Conclusions:Although all pts whether they express b3a2 or b2a2 at diagnosis, have an excellent overall survival, those with b3a2 have a significantly higher rate of molecular responses and a trend for better EFS. These results mirror what we have previously reported for pts treated with imatinib as initial therapy. The biologic characteristics for this difference warrant further investigation.Table 1Treatment distribution in analyzed groupsTranscriptDasatinibNilotinibb2a242 (48%)46 (52%)b3a234 (46%)40 (54%)b2a2 and b3a224 (57%)18 (43%)Table 2Results of patients with variant BCR-ABL transcripts treated with 2ndgeneration TKIsb2a2b3a2b2a2+b3a2Overall CCyR90.4%95.7%95.1%Overall MMR75%91.5%95%12 mo. CCyR93.8%100%100%12 mo. MR66.7%98.184.7%3 years O.S.100%100%94%3 years EFS93%99%98%3 years TFS99%98%100% Disclosures:Kantarjian:Novartis: Research Funding; Bristol Myers Squibb: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Jabbour:Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Cortes:Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.

The BCR-ABL1 Transcript Type Does Not Influence the Response and the Outcome of Chronic Myeloid Leukemia Patients Treated Frontline with Nilotinib

AbstractAbstract 1680 Background.Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 hybrid gene. Different types of BCR-ABL1 fusion transcripts can be found, but the most frequent are the e13a2 (b2a2) and the e14a2 (b3a2). In the tyrosine kinase inhibitors (TKIs) era, few data about the prognostic significance of the transcript type in early chronic phase (ECP) CML are available. Three larger studies suggested that the e13a2 transcript may have an adverse prognostic impact in ECP CML patients treated with imatinib (IM): Vega-Ruiz et al. (251 patients, ASH 2007) reported inferior molecular responses; Lucas et al. (71 patients, Haematologica 2009) reported lower cytogenetic response rates and lower event-free survival (EFS); the GIMEMA CML WP (493 patients, EHA 2011) reported a slower time to major molecular response (MMR) with inferior EFS and progression-free survival (PFS). To our knowledge this is the first evaluation of the prognostic influence of the BCR-ABL1 transcript type on the responses and the outcome of ECP CML treated frontline with nilotinib (NIL). Methods.The CML Italian Registry of Nilotinib includes 215 patients treated with NIL-based regimens. The patients were enrolled within 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) or treated at the “S. Orsola-Malpighi” University Hospital (Bologna, Italy), with NIL 300 mg BID or 400 mg BID as initial treatment. All the registered patients were analyzed. Patients expressing rare transcripts and patients with both b2a2 and b3a2 transcripts were excluded: 201 out of 215 patients were evaluable, 81 (40%) with e13a2 transcript and 120 (60%) with e14a2 transcript. Differences between groups were tested using χ2 test, Fisher exact test or t-test, as appropriate. Response monitoring: conventional cytogenetic examination (bone marrow) and QPCR (peripheral blood). Definitions: MMR: BCR-ABLIS ratio <0.1% (International Scale); failures: according to 2009 ELN recommendations; events: failure or treatment discontinuation for any reason. The time-to-response and the outcome were estimated using the Kaplan-Meier method, and compared by log-rank test. Results.The baseline characteristics of the 2 groups were comparable (no significant differences in age, Sokal/Hasford/EUTOS score distribution, clonal chromosomal abnormalities in Ph+ cells, NIL dose), except for the percentage of basophils in the peripheral blood, higher in patients with e14a2 transcript (3.4% vs 2.3%, p=0.01). The median observation was 29 months (range 18–47); 92% of the patients had at least 2 year observation. The CCgR and MMR rates at 12 months were comparable in the 2 groups. The time to MMR was longer for patients with e13a2 transcript (6 months vs 3 months, p=0.04), but the overall CCgR rates (93.8 vs 91.7, p=0.79) and the overall MMR rates (85.1 vs 90.0, p=0.38) were not significantly different in patients with e13a2 or e14a2 transcript, respectively. The probability of Overall Survival (OS), Progression-Free Survival (PFS) and Failure-Free Survival (FFS) were comparable: 91.4% vs 95.8% (p=0.61), 90.7% vs 95.0% (p=0.51), and 90.7% vs 88.7% (p=0.40) in patients with e13a2 and e14a2 transcript, respectively. Conclusions.In our experience, based on 201 early CP CML patients treated frontline with NIL with a minimum follow-up of 18 months, the BCR-ABL transcript type did not show any relevant prognostic impact. The time to MMR was longer in patients with e13a2 transcript, but no response and outcome differences have been observed so far. The number of observed events was low and a longer observation is required. Acknowledgments.European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures:Castagnetti:Novartis Pharma: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Speakers Bureau. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria. Breccia:Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Cavazzini:Novartis Pharma: Honoraria; Bristol Myers Squibb: Honoraria. Turri:Novartis: Consultancy, Novartis Other; Bristol Myers Squibb: Bristol Myers Squibb, Bristol Myers Squibb Other, Consultancy. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy. Martinelli:Novartis: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers-Squibb: Consultancy, Honoraria, Speakers Bureau. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Rosti:Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

Improving the Accuracy of BCR-ABL Transcript Quantitation: Pitfalls of Using a Common Standard for b3a2 and b2a2 Transcripts

AbstractAbstract 4423Considerable variation is observed in BCR-ABL analysis by qPCR making it difficult to compare inter-laboratory results in monitoring therapy of CML. Many factors contribute to the variation (primers, probes, and standards). The combination of two primers plus a probe and b3a2 plasmid standard from the European Against Cancer (EAC) program is widely accepted to quantitate two major types of p210, b3a2 and b2a2.Since the amplicon size and sequences are different between b3a2 and b2a2, we used EAC primers plus probe with b3a2 and b2a2 standards to quantitate b3a2 and b2a2 respectively. It was found that with well determined copy numbers (CN) of both types of standards, the Ct (threshold cycle) values of b2a2 standards are smaller than b3a2 standards at each concentration point especially at lower CN (30000 CN, 0.26 cycle earlier; 300 CN, 0.42 cycle earlier; 30 CN, 0.56 cycle earlier compared to b3a2 standards from 32 experiments). In order to find out if these differences in Ct have any effect in quantitation of b2a2 samples, RNA samples pre-determined as b2a2 p210 were amplified and CN quantitated in the presence of b3a2 and b2a2 standards in the same qPCR plates. For b2a2 samples, a higher ratio of b2a2 to ABL using b3a2 standards was observed compared to the ratio using b2a2 standards (1.48 fold, p=0.0025, n=45). The data was further broken down into three International Scale (IS) ranges. For >1–10% IS range, the ratio by b3a2 standard is 1.30±0.08 fold (n=12) compared to b2a2 standard. Similarly, for > 0.1–1% IS range, the ratio is 1.46±0.08 fold (n=12) and for ≤ 0.1% IS range, the ratio is 1.60±0.22 fold (n=21).Equal numbers of b3a2 and b2a2 RNA samples were analyzed by reference lab and GHSU lab to establish IS conversion factor (CF) for GHSU lab. There was a small difference between b3a2 and b2a2 CF when their own respective standards were used (0.67 for b3a2, 0.57 for b2a2). Therefore, a common CF of 0.62 can be generated for both types of p210. With application of common CF (0.62) to convert b3a2 and b2a2 raw ratios to IS, only 7% is under-estimated for b3a2 and 9% is over-estimated for b2a2. However, when b2a2 and b3a2 quantitation uses only b3a2 standard, there is a big difference in CF value (0.67 for b3a2 vs. 0.39 for b2a2), and a common CF (0.51) can be still generated when two types of BCR-ABL samples are mixed equally. The accuracy of results in IS were jeopardized if the labs used only b3a2 standard for both b3a2 and b2a2 quantitation. For example if this CF (0.51) is used to convert b3a2 and b2a2 raw ratios, 24% is under-estimated for b3a2 and 31% is over-estimated for b2a2.These variations may look small but may be exaggerated if the labs have big variations in their assay and do not have equal numbers of b3a2 and b2a2 to establish the CF. Further, data from recent survey results were compared to see if the labs using only b3a2 standards for both types of p210 have higher ratios for b2a2 BCR-ABL than the labs using two individual standards. The results of commercially available standards (b3a2 only) were compared with GHSU results, which uses both types of standards (b3a2 and b2a2) to evaluate CN's of respective fusion gene.In one survey with b3a2 RNA, the means of original raw ratio and ratio in IS by using the commercial reagent were very similar to the results by GHSU using b3a2 results (1.13 - raw ratio; 1.34 - IS between two). In two other surveys where b2a2 RNA was used, the means of raw ratio in samples analyzed using commercial reagent (b3a2 Standards) were 1.46 and 1.48 fold, compared to means of same samples analyzed by GHSU using b2a2 standards. Similarly, 1.59 and 1.76 fold difference is observed between samples using commercial standards (b3a2) and GHSU standards (b2a2) in the second survey. Moreover the ratios in IS reported in the second survey by commercial reagent were 1.85 fold and 2.23 fold of GHSU results. These findings suggest that there is a statistically significant over-estimation of b2a2 to control gene ratio, especially at MMR range, when b3a2 standards are used for b2a2 analysis. This may be one of the important factors that contribute to the considerable variation in BCR-ABL analysis. The phenomenon may be explained by the sequences or structure of an extra exon in b3a2 that is missing in the b2a2 fusion gene, and this may result in the different amplification rates of two types of p210. It is suggested that in order to quantitate the ratio of b3a2 and b2a2 accurately, their own standards should be used in qPCR analysis. Disclosures:No relevant conflicts of interest to declare.

Equivalence of BCR-ABL Transcript Levels

AbstractAbstract 2495 Introduction:Response to therapy in patients with chronic myeloid leukemia (CML) is monitored by both cytogenetic assessment of bone marrow metaphases and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) of peripheral blood samples. Although standardization of BCR-ABL quantification according to the international scale (IS) provides results comparable between different laboratories, current treatment recommendations are still predominantly based on cytogenetic analyses. The aim of this work was to determine a BCR-ABL expression level according to the IS (BCR-ABLIS) which corresponds to complete cytogenetic remission (CCyR). Patients:1,329 paired cytogenetic and molecular assessments of 557 chronic phase CML patients on imatinib-based therapies from the German CML study IV, all during follow-up, have been considered. Median number of samples per individual was 3 (range 1–24). Only molecular evaluations derived from peripheral blood and from patients expressing b2a2 and/or b3a2 BCR-ABL transcripts were considered. Participating laboratories provided results standardized according to the international scale. For cytogenetic analyses at least 20 metaphases, evaluated by conventional chromosome banding analysis, were required. Paired molecular and cytogenetic analyses were performed within an interval of <3 days. Methods:Data was split randomly at the rate of 2:1 into learning and validation sample. 10,000 subsamples that contained no more than one observation per patient were repeatedly drawn randomly from the original learning sample, according to the bootstrap method Using these subsamples, the BCR-ABLIS expression level providing the best discrimination between CCyR and no CCyR was determined choosing the smallest p-value of the exact Fisher test. Only cut-off levels with p-values which were significant after adjustment for multiple testing were considered.To confirm these results, again 10,000 subsamples were randomly drawn from the original validation sample. For the cut-off points discovered in the learning sample, sensitivity and specificity for predicting a CCyR from the molecular data and the p-values from the Fisher test were calculated within each of these subsamples. Results:1329 cytogenetic analyses using a median of 25 metaphases (range, 20–30) of which 75% were in CCyR were compared with 1329 molecular data with a median BCR-ABLIS expression level of 0.055% (range, 0–237%). In the learning sample the most frequently found BCR-ABLIS value was 0.35%, but all values between 0.2% and 1.1% were similarly well-suited for separating between patients in CCyR and not in CCyR. Based on this range, two potential cut-off levels were chosen for validation, 0.35% and 1%. While the first one represents a value, where one would rather expect to underestimate the number of CCyRs, the latter represents a value, where one would rather expect to overestimate the number of CCyRs. For the level of 0.35% the p-value was below 0.05 for 67.2% of the 10,000 subsamples, the median p-value was 0.017. With a median value of 88.4%, CCyR cases were correctly identified while cases that were not in CCyR were correctly assessed with a median rate of 71.4%. For the 1% BCR-ABLIS level, the p-value was below 0.05 for 72.0% of the 10,000 subsamples with a median p-value of 0.011. At a median value of 96.0%, CCyR cases were correctly identified while cases that were not in CCyR were correctly assessed with a median rate of 60.0%. When restricting on BCR-ABLIS values between 0.1 and 10.0%, the median concordance rate was 76%. Conclusions:Compared to molecular analyses, cytogenetic assessment is imprecise and limited with regard to sensitivity. Thus, it is not surprising to find a range of molecular data equivalent to CCyR but not a one-to-one cut-off point. However, for practical reasons it seems desirable to have such a cut-off point. Since lack of CCyR is considered a failure criterion according to the ELN treatment recommendations, it is suggested to keep the proportion of patients small that are considered erroneously not to be in CCyR. Additionally, a considerable amount of patients in major molecular remission or even better is present in the sample, so there might be a slight bias towards lower cut-off values. Thus, BCR-ABLIS expression of <1% is considered to be equivalent to CCyR in chronic phase CML patients treated with imatinib with a median concordance rate of 89%. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis, BMS, MSD, Ariad, Pfizer: Consultancy Other, Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

A STUDY ON THE EXPRESSION OF BCR-ABL TRANSCRIPT IN MIXED PHENOTYPE ACUTE LEUKEMIA (MPAL) CASES USING THE REVERSE TRANSCRIPTASE POLYMERASE REACTION ASSAY (RT-PCR) AND ITS CORRELATION WITH HEMATOLOGICAL REMISSION STATUS POST INITIAL INDUCTION THERAPY

Introduction:The MPAL comprise 2–5% of all acute leukemia. The present WHO 2008 classification has separated two groups in MPAL based on t(9;22) positivity and MLL rearrangement.Aims & Objectives:The aim of the present pilot study is to note the frequency of BCR-ABL transcript in MPAL cases using the RT-PCR assay and to correlate the status with hematological remission post induction.Materials & Methods:A total of 10 MPAL cases classified on Flow-cytometry based on the current WHO 2008 criteria were enrolled. In all the cases Bone marrow or peripheral blood sample in EDTA was processed for molecular studies and the RT-PCR reaction carried out using primers specific to the t (9;22) and t(4;11) translocation. The post induction check marrow slides were also reviewed.Results:Out of the total 10 MPAL cases, 7/10 (70%) were adult and 3/10 (30%) pediatric cases. A total of 4/10 (40%) cases showed positivity for the t(9;22) transcript and none for t (4;11). Of the 4 positive cases, 3/10(30%) were adult cases and 1/10(10%) pediatric case. The BCR-ABL transcript type in adult cases was b3a2 (p210) in 2/3 (66%) and e1a2 (p190) in 1/3 (33.3%) case. The single pediatric case was positive for b3a2 transcript.Discussion & Conclusion:All the 4 positive MPAL cases presented with high TLC and low platelet count (p<0.05). The positive cases also showed hematological remission at post induction check marrow (blasts<5%). This could partly be explained due to good response to the imatinib added to the treatment protocol.

Cytogenetic &amp; molecular analyses in adult chronic myelogenous leukaemia patients in north India

Background & objectives:Chronic myelogenous leukaemia (CML) is the commonest leukaemia in Asia. There is a paucity data on cytogenetic and molecular analyses of Indian CML patients. This apparently reflects the low availability of cytogenetic and molecular techniques in our country. This study aimed to document various types of BCR-ABL fusion transcripts in different phases of CML and to compare the Ph chromosome positivity/negativity vis-a-vis BCR-ABL fusion transcripts in adult CML patients.Methods:Between June 2004 and February 2009, 208 patients were diagnosed as CML in chronic phase (CP), accelerated phase (AP) and blast crisis (BC), according to standard criteria. Cytogenetic and molecular genetic analyses were performed in all patients. Various types of BCR-ABL hybrid transcripts were compared with phases of CML and cytogenetic abnormalities.Results:Among 208 CML patients, b3a2 BCR-ABL transcripts were most commonly detected (66.82%) followed by b2a2 (28.84%), b3a2 + b2a2 (3.36%), b3a2 + e19a2 (0.48%) and b2a2 + e19a2 (0.48%). b3a2 transcripts were more frequently detected than b2a2 transcripts, in the whole group of 208 as well as in 183 CML-CP patients (P<0.0001). Ph chromosome was positive in 135 of 139 patients with b3a2 transcripts and 56 of 60 patients with b2a2 transcripts, difference not being significant. Additional cytogenetic abnormalities detected in 3.8 per cent patients in CML-CP and 44 per cent patients in CML-AP/BC, did not show predilection for any BCR-ABL transcript type.Interpretation & conclusions:This study documents higher Ph positivity (96.15%) by cytogenetic analysis among CML patients, as confirmed by qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in a large patient group from north India. Both the techniques contribute towards understanding the disease biology, and have important implications for diagnosis and management of CML patients.