B3a2 Transcript Research Articles

Distribution of common BCR-ABL fusion transcripts and their impact on treatment response in Imatinib treated CML patients: A study from India.

Philadelphia chromosome (Ph): Hallmark of CML is caused by reciprocal translocation between chromosomes 9 and 22 resulting in BCR-ABL fusion protein. Most commonly associated breakpoint with CML is M-bcr in exon 13 or exon 14, producing splice variant b2a2 or b3a2 respectively. The distribution of these transcripts and their influence on clinico-hematological parameters is variable. Impact of the fusion transcripts on treatment outcome in Imatinib treated CML patients is still a matter of debate. We conducted this study on 400 CML-CP patients to look for the distribution of fusion transcripts i.e. b3a2 and b2a2, their clinico-hematological profile and impact on treatment response in patients treated with Imatinib. CML-CP was diagnosed by reverse transcriptase PCR (RT-PCR) for the BCR-ABL fusion transcript. Real-time quantitative PCR (RQ-PCR) was performed on peripheral blood every 3-6 monthly to look for treatment response. The overall frequency of b3a2 transcript was observed in 288 (72%) followed by b2a2 in 104 (26%) and hybrid fusion transcript (b3a2 + b2a2) was seen in 8 (2%) cases. MMR was attained in 198/288 (68.7%) patients with b3a2 transcript and 90/288 (31.3%) patients failed to achieve MMR after 12 months of Imatinib therapy. Among the patients with b2a2 transcript, 44/104 (42.3%) patients achieved MMR and 60/104 (57.7%) patients failed to achieve MMR after 12 months of Imatinib therapy. In conclusion, the frequency of b3a2 transcript was more as compared to b2a2 transcript. MMR was significantly higher in patients with b3a2 transcript as compared to patients with b2a2.

Comparison of BCR-ABL Transcript Variants Between Patients With Chronic Myeloid Leukaemia and Leukaemia Cell Lines.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by the presence of breakpoint cluster region-Abelson murine leukemia (BCR-ABL1) gene fusion as a hallmark that is expressed as two major transcripts b2a2 and b3a2. The aim of this study was to compare the BCR-ABL transcripts in the blood cells of patients with CML, and in chemoresistant and chemosensitive CML cell lines to validate their use as a good method to elucidate CML biology. Twelve patients with CML and CML cell lines (K562, K562-LUCENA and FEPS) were analyzed by real-time polymerase chain reaction to evaluate gene expression of BCR-ABL transcripts. All patients had the same expression levels of b2a2 and b3a3 transcripts, however, CML cell lines presented only b3a2 expression. There were no significant differences in absolute b3a2 expression between patients and CML cell lines. CML cell lines provide a good in vitro alternative in that they have the same BCR-ABL expression as patients.

B3a2 Transcript Is an Independent Factor for the Achievement of a Deep Molecular Response in Chronic Phase - Chronic Myeloid Leukemia Patients Treated with Imatinib in First-Line

Introduction: The achievement of a deep and sustained molecular response after treatment with tyrosine kinase inhibitors (TKI) is one of the requirements for therapy discontinuation in chronic myeloid leukemia (CML) patients.Objectives: This study aimed to evaluate the proportion of CML patients treated with imatinib in first-line that achieve MR4.5 (BCR-ABL transcripts ≤ 0.0032% in the international scale) after imatinib treatment and the predictive factors for this response. The secondary objectives were to evaluate the rate of MMR (PCR ≤0.1%), MR4.0 (PCR ≤ 0.01%), time to molecular responses, event-free survival, progression-free and overall survival.Patients and methods: This is a retrospective analysis of CP-CML patients treated in first-line with imatinib in a single center. Patients diagnosed after 2006 were managed according to the European Leukemia Net recommendations. All patients started CML imatinib within six months from diagnosis. The type of BCR-ABL1 transcript was determined by multiplex RT-PCR from cDNA synthesized from total leukocytes RNA at diagnosis. BCR-ABL transcripts by quantitative real-time polymerase chain reaction were assessed at baseline, and then every three months for the first year until reaching a stable major molecular response, then every 3-6 months. The SPSS software was used for the Cox Regression model for univariate and multivariate analysis, using backward Wald. The cumulative incidence was calculated with the R Program, with Gray test for curve comparisons. Event-free, progression-free and overall survivals were calculated with the Kaplan-Meier method, and the curves were compared with the log-rank test.Results: From January 2005 to December 2015 172 patients were treated with imatinib, median age 48 years (18-93), 55.8% male. Sokal score: 31% low-risk, 40% intermediate-risk and 29% high-risk. The median follow-up was 56 months (0-157). The Cumulative Incidence (CI) of MMR was 48% in 24 months and 84% in 5 years, while the CI of MR4.0 and MR4.5 was 63% and 51%, respectively, in 5 years. The median time to MMR, MR4.0 and MR4.5 was 16.7 (2-102), 31 (5-150) and 36 months (7-153), respectively. The univariate regression factors related to MR4.5 achievement were: age >48 years (HR 1.79; 95%CI: 1.08-2.98; P=0.023); days between diagnosis and imatinib start (HR=0.99; 95%CI: 0.98-0.99, P=0.020); e14a2 (b3a2) transcript (HR= 3.37; 95%CI: 1.67-6.81; P=0.001), which was the only factor in the multivariate analysis. Imatinib was discontinued in 96/172 (55.8%) patients due to resistance (36.5%), intolerance (20.8%), clinical trials (25%), death (15.6%) and adherence (2.1%). Eighteen out of 67 (26.8%) patients that achieved MR4.5 are currently participating in a discontinuation trial. Overall survival, PFS and EFS were 92%, 87% and 61% in 5 years.Conclusion: Approximately half of the patients that start imatinib in first-line obtain deep molecular responses within five years and could be candidates for treatment discontinuation. B3a2 transcript was an independent factor for MR4.5 achievement in the multivariate analysis, confirming the results of other studies that have reported earlier and deeper molecular responses in patients with b3a2 transcripts. DisclosuresDe Paula:Hematology and Transfusion Medicine Center, University of Campinas: Employment. Pagnano:Abbvie: Consultancy; EMS: Other: Financial support for participation in congress; Shire: Other: Lecture; Novartis: Consultancy.

Efficacy and Safety of Generic Imatinib Compared to Glivec in Chronic Phase - Chronic Myeloid Leukemia - a Multicenter, Observational Study

Glivec was the first tyrosine kinase inhibitor (TKI) approved for chronic myeloid leukemia (CML) treatment and its efficacy has been demonstrated in a large number of trials. Generic formulations have been used recently as a more cost effective treatment, but there are few studies that have prospectively evaluated the efficacy and safety of these drugs.Aims: The present study aimed to evaluate the efficacy and safety of generic imatinib in CP-CML in first line therapy. Methods: This is a multicenter, observational, cohort-type study. The prospective cohort consisted of patients who initiated treatment with generic imatinib between January 2015 and September 2017; whereas the retrospective cohort was treated with Glivec between January 2010 and December 2011. All patients started imatinib in CP, less than six months from diagnosis. Patients were managed according to European Leukemia Net (ELN) 2009 and 2013 recommendations. The definition of the responses followed the ELN 2013 criteria. Adverse events were assessed based on the Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0.3, 2010. Event-free survival (EFS) was measured from starting date of treatment until loss of complete hematologic response, loss of major cytogenetic response, progression to accelerated (AP) or blast crisis (BC) or death from any cause at any time during initial therapy or discontinuation of imatinib by any cause. Patients that switched from Glivec to generic where censored. Overall survival (OS) was measured from starting date of imatinib until to the date of death from any cause while on therapy or last seen. Progression-free survival (PFS) was measured from starting date of imatinib to transformation to AP or BC or deaths while on therapy. Survival curves were calculated by the Kaplan-Meier method and compared with the log-rank test. Cox regression was used with Backward Wald Method. SPSS version 21.0 was used applying the chi-square and t-test, when adequate. All analysis considered p-value <0.05 as significant.Results:A total of 445 patients were analyzed (Glivec=285; Generic=160). The median age was 54 years (18-87) in Glivec and 50 (18-89) in the Generic group (P=0.027). Sokal score stratification in Glivec and generic groups, was respectively: low risk 14.4%/25.7%; intermediate risk 42.3%/38.2% and high risk 43.3%/36.1% (P=0.02). B3a2 frequency was 51.5% vs. 37.7% in Glivec and Generic group, respectively, and b2a2 41.4% vs. 53.8% (P=0.017). There was no significant difference in gender, Hasford, EUTOS scores and ECOG. The median follow-up was 25 months (0-71) and 11 months (0-31) for Glivec and Generic groups, respectively (P<0.0001). The median time between diagnosis and imatinib starting was 18 days (0-119) in Glivec and 27 days (0-168) in the Generic group (P= 0.015). The rate of treatment failure at 3 months according to the ELN 2013 criteria was 6.6% and 14.7% in Glivec vs. Generic (P=0.04); whereas at 6 months there were no significant differences (12.3% vs. 18.9%; P=0.09). There was no significant difference in grade 3 and 4 hematological and non-hematological toxicity during the follow-up. There were 5 and 3 cases of progression in the Glivec and Generic group, respectively. In the Cox regression multivariate analysis, the independent factors for EFS were age at diagnosis (HR=1.03; CI95% 1.00-1.07; P=0.039) and toxicity grade 3-4 (HR 3.37; CI95% 1.08-10.4; P=0.036). The initial therapy was discontinued for the following reasons, in Glivec and generic group respectively: resistance (19.7% vs. 47.5%), intolerance (15.3% vs. 23.7%), non-adherence (4.4% vs. 3.4%), death (2.0% vs. 6.8%), clinical trial (0.5% vs. 10.2%), progression (2.0% vs. 5.0%), pregnancy (0 vs. 3.4%), switch from Glivec to generic (56.1%). OS, PFS and EFS at 24 months were higher in Glivec group in comparison to Generic (99% vs. 96%, P=0.016), (98% vs. 95%, P= 0.026) and (73% vs. 58%; P<0.0001 respectively.Conclusion: The group treated with generic imatinib presented higher rate of failure at 3 months and lower OS, PFS and EFS at 24 months. Differences between the groups included a longer time to initiate treatment in the Generic group, a higher proportion of patients with b2a2 transcripts, which were related to an inferior rate of molecular responses and survival in other studies. There was no difference in the safety profile. The long-term impact in prognosis will be evaluated after a longer follow-up. DisclosuresPagnano:Shire: Other: Lecture; Abbvie: Consultancy; EMS: Other: Financial support for participation in congress; Novartis: Consultancy. Magalhaes:Novartis: Consultancy, Other: Lecture. Clementino:EMS: Other: Financial support for congress. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Centrone:EMS: Other: Financial support for congress, Speakers Bureau; Bristol Meiers-Squibb: Other: Financial support for congress, Speakers Bureau; Novartis: Other: Financial support for congress, Speakers Bureau; Astra Zeneca: Speakers Bureau; Janssen: Other: Financial support for congress, Speakers Bureau. Fogliatto:Novartis: Consultancy; Roche: Consultancy, Speakers Bureau; Janssen: Honoraria, Research Funding. Giai:Pfizer: Consultancy; Novartis: Consultancy. Bortolheiro:Abbvie: Consultancy, Speakers Bureau; Sanoffi: Speakers Bureau; Novartis: Consultancy, Speakers Bureau.

Overall Survival and Response Rates after a 10-Year Follow-up of Chronic Myeloid Leukemia Patients in Chronic Phase Treated with Imatinib in a Real-Life Practice

Background. Imatinib, the first BCR/ABL kinase inhibitor, has drastically changed the chronic myeloid leukemia (CML) scenario improving the rate of responses and long-term outcome. The aim of our analysis was to assess, after a median follow-up of 10.2 years (range 5.8-14.8), the long-term efficacy and safety of imatinib treatment in a monocentric cohort of CML patients treated outside of clinical trials.Methods. We retrospectively analyzed a series of 458 CML patients treated with imatinib frontline or after interferon (IFN) failure at a single center between 2000 and 2016. Long-term analysis included overall survival (OS), progression-free survival (PFS), event-free survival (EFS), response to treatment and adverse events.Results. The median age was 55.1 years (range 18.8-91.2). Seventy-one % of patients received imatinib frontline and 29% after IFN failure. Median time of prior IFN treatment was 5.6 years (range 2.4-8.9); the 10-year OS of patients treated with imatinib after IFN and with imatinib frontline was 75.3% (CI 95%: 66.9-81.9) and 77.8% (CI 95%: 72.2-82.5) (p=0.468), respectively. The 10-year-OS of the whole cohort was 77.1% (CI 95%: 73.1-81.5); the 10-year probability of dying due to CML and of other causes was 7.8% (CI 95%: 5.1-10.3) and 16% (CI 95%: 11.5-18.8), respectively (Figure 1). When patients were stratified into 4 groups according to age (18-35, >35-<50, >50-<65 and >65 years), no difference was found in the 10-year OS considering only the CML-related deaths (p=0.472), whereas a difference was observed when all other causes of death were considered (p<0.001) (Figure 2). The b2a2 transcript (detected in 34% of patients) at baseline had a negative impact on the 10-year PFS (p=0.035), but not on the 10-year OS (p=0.151). The Eutos long-term survival (ELTS) better stratified the baseline risk of patients in terms of 10-year OS compared to the Sokal and Hasford scores. The 10-year PFS was 77.1% (CI 95%: 72.6-81), while the 10-year EFS was 55% (CI 95%: 50-59.6), considering as event anything that could lead to treatment discontinuation: intolerance (10.4%), lack of cytogenetic response (17.5%), no major molecular response over time (MMR, 22.6%), lack of molecular response (6.1%), evolution into blast phase (13.7%), death (29.7%). After a median time of 10.2 years, 83 patients (18.1%) achieved as their best molecular response a MMR after a median time of 2.4 years (range IQ 1.36-4.01), 74 patients (16.1%) a MR4.0 after a median time of 4.57 years (range IQ 2.08-7.33), 162 (35.3%) a MR4.5 after a median time of 8.23 years (range IQ 4.14-11.53) and 60 (13.1%) never achieved a MMR during treatment. The achievement of an early molecular response (EMR) at 3-months (BCR/ABL1 <10%) and a MMR at 12-months was associated with a significant advantage in terms of 10-year OS (p=0.006 and p=0.032, respectively) and of 10-year PFS (p=0.004 and p=0.009, respectively). One-hundred and twenty eight patients (27.9%) switched to a second generation inhibitor (2TKI) after a median time of treatment of 3.48 years (range IQ 1.47-5.89); 23 patients (17.9%) were intolerant, while 105 (82.1%) were resistant. Among resistant patients, 81 (77.1%) never achieved a MMR during imatinib, while 24 who had achieved at least a MMR lost it after a median of 14 months (range 6-22). Furthermore, 11 patients (10.4%) experienced a blast phase evolution. The 5-year OS after starting a 2TKI was 85.2% (CI 95%: 73.5-94.3). We collected also the long-term safety with a particular attention to cardiovascular (CV) events and development of solid tumors. In 27 patients (5.8%), a grade 3 CV event occurred during treatment. Eight (29.6%) experienced a myocardial infarction and 3 patients died, 4 (14.8%) a heart failure that required hospitalization, 4 (14.8%) a stroke with 3 deaths, 5 (18.6%) an atrial fibrillation controlled with specific therapy, 3 (11.1%) a venous thrombotic event that required anticoagulant treatment and 3 (11.1%) a peripheral arterial occlusive event with 1 pulmonary embolism. Thirty-nine patients (8.5%) developed a solid tumor during the course of treatment: the most frequent sites involved were the prostate (20.5%), gastrointestinal (15.3%), bladder (12.8%), lung (10.2%) and breast (10.2%).Conclusions. A 10-year real-life follow-up of CML patients demonstrates that imatinib maintains efficacy over time and is associated with a low rate of CV events and second neoplasias. [Display omitted] DisclosuresRizzo:Sapienza University, Rome: Other: Resident in Hematology. Foà:GILEAD: Speakers Bureau; INCYTE: Other: ADVISORY BOARD; AMGEN: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; NOVARTIS: Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau. Breccia:Novartis: Honoraria; BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria.

P210 and P190 BCR-ABL fusion transcripts variants frequencies among Philadelphia chromosome-positive chronic myeloid leukemia in Sudan

Breakpoint cluster region-abelson (BCR-ABL) leukemic fusion gene types in chronic myeloid leukemia (CML) correlate with the disease clinical course and outcome. There are variations in the reports of previous studies about the frequencies and distribution of BCR-ABL transcripts in chronic myelogenous leukaemia among Sudanese patients. This research aims to determine the frequencies of BCR-ABL fusion transcript variants in Sudan. One hundred (informed consent) Philadelphia positive chronic myeloid leukaemia patients, in chronic phase, were enrolled in this study. EDTA anticoagulated peripheral blood samples were collected from each participant, RNA was extracted from mononuclear cells by (TRIzol) reagent. BCR-ABL transcripts were detected by qRT-PCR technique with specific primers forP190 and P210 BCR-ABL transcript variants. The typical p210 BCR-ABL transcripts (b3a2 or b2a2) were detected in all patients (100%) the b3a2 transcript was detected in 96/100 (96%) and the b2a2 transcript was detected in 4/100 (4%).co-expression of p210/p190 (b2a3/e1a2) was detected in 6/100 (6%). p190 variant was not detected independently.

Assessment of BCR-ABL1 Fusion Transcripts and Their Association with Response to Imatinib Treatment in Chronic Myeloid Leukemia Patients

Abstract Objectives: BCR-ABL1 fusion transcripts with contrasting data on response to imatinib therapy have been reported from different parts of the world. Hence, the present study aimed to determine the frequencies of transcripts and their association with response to imatinib therapy in chronic myeloid leukemia (CML) patients. Methods: A total of 170 (76 follow-up and 94 imatinib-resistant) CML samples were included in the study. BCR-ABL1 fusion transcripts and expression status were analyzed in all cases using multiplex reverse transcriptase PCyR and real-time PCyR. Sanger sequencing was used for tyrosine kinase domain (TKD) mutation screening in imatinib mesylate-resistant patients. Results: Of 170 CML patients, 36.36% showed b2a2, 63.53% had b3a2, and 2.94% had b2a2 + b3a2 isoforms. Mean platelet counts and blasts were significantly lower in b2a2 carriers (P = 0.0092; P ≤ 0.0001). Patients with b2a2 transcript were found to be more in responders group (both hematological and cytogenetic), whereas b3a2 patients were more in partial responders group and death (P = 0.763; P = 0.309). In follow-up patients, mean baseline BCR-ABL1 expression levels are significantly higher in b2a2 versus b3a2 carriers (P = 0.0351). Of 94 imatinib-resistant patients, 36 (38.29%) had acquired TKD mutations. Among 36 patients, mean BCR-ABL1 levels are significantly higher in b2a2 and b2a2 + b3a2 group (P = 0.0002; P ≤ 0.0001). TKD mutation frequency was more in b3a2 (61.11%) compared to other types. With respect to follow-up status in 36 patients, 17 patients died while 19 were on imatinib higher doses or 2nd-generation tyrosine kinase inhibitors. Of 17 patients, 41.66% had b2a2 transcript and 54.54% had b3a2 transcript. Conclusion: Patients with b3a2 transcripts might be associated with poor response and worse prognosis in CML with imatinib treatment.

The Biology of Chronic Myelogenous Leukemia in Childhood and Young Adolescents: An Indian Perspective

Abstract Objective: The purpose of this study was to determine the clinical, biological, and molecular characteristics at diagnosis in children and adolescents with chronic myelogenous leukemia (CML) in the Indian scenario at our tertiary patient care center. Subjects and Methods: We evaluated 51 children and adolescents with CML registered at our clinic, from January 2007 to December 2015. The mean and median of various parameters were calculated using a Microsoft excel sheet and SPSS software version 16. Results: The median age of presentation in children was 16 years; 92.2% of them were older than 10 years, with a higher prevalence in boys than girls (gender ratio 2.6:1). The symptoms at presentation were fatigue, fever, awareness of mass due to splenomegaly, and bleeding manifestations. One patient presented with Bell's palsy. Markedly raised leukocyte counts were present in 29.4% patients (median white blood cell count &gt;400 × 109/L). Most of the patients presented in the chronic phase of the disease, four each were in accelerated phase and blast crisis, respectively. Majority of patients were categorized as intermediate risk as per Sokal and Hansford score. About 60.7% of these pediatric patients fell in low-risk category as per European Treatment and Outcome Study score at baseline. A predominance of transcript P210-b3a2 (68%) was observed in the children who were studied for the type of chimeric BCR-ABL mRNA. Conclusions: This is one of the most recent reported series of CML in children and adolescents from India highlighting the difference in presentation from adults; mainly hepatomegaly, bleeding manifestations, and higher leukocyte count. Presence of b3a3 transcript of p210 breakpoint of BCR-ABL was more common in children (68%) than b2a2 transcript (32%) when compared to adults as recently described in a study from India, which may explain the differences at presentation.

Types of bcr-abl and their correlations with the blood count in chronic myeloid leukemia (CML) in Togo

Décrire les différents types de transcrits de fusion moléculaire bcr-abl afin d'évaluer leur fréquence et étudier leurs influences sur les paramètres de l'hémogramme au diagnostic. Il s'agit d'une étude prospective de 34 patients porteurs d'une leucémie myéloïde chronique au Togo. La recherche des transcrits de fusion a été réalisée au laboratoire d'hématologie biologique de l'hôpital Henri Mondor à Créteil (France). L'âge moyen des patients était de 42,32±14,87 ans avec des extrêmes de 9 et 65ans. On notait une prédominance masculine avec un sex- ratio de 1,61 (21 hommes et 13 femmes). L'étude moléculaire révélait deux types de transcrits b3-a2 et b2-a2. Dix-neuf patients (55,88%) avaient le transcrit b3-a2, 13 (38,24%) le transcrit b2-a2 (32,10 %) et deux patients possédaient les deux types de transcrits b3-a2 et b2-a2 (5,88%). Au diagnostic, le taux d'hémoglobine moyen, le nombre moyen de globules blancs, le chiffre moyen des plaquettes chez les patients porteurs du transcrit b3-a2 étaient respectivement de 99,2g/l ; 207,63G/l et 451,28G/l. Pour les patients porteurs du transcrit b2-a2 les valeurs sont 104,6g/l, 114,32G/l et 486,11G/l. Les patients ayant les deux types de transcrit de fusion avaient respectivement 67g/l, 867G/l et 780G/l. Les paramètres de l'hémogramme sont significativement plus perturbés chez les patients porteurs des deux types de transcrits b3-a2 et b2-a2.

Prognostic Implication of BCR-ABL Fusion Transcript Variants in Chronic Myeloid Leukemia (CML) Treated with Imatinib. A First of Its Kind Study on CML Patients of Kashmir

Background:The prognostic significance of the common BCR-ABL transcripts like e13a2 (b2a2) and e14a2 (b3a2) in Chronic myeloid leukemia (CML) has been reported from patients treated with different tyrosine kinase inhibitors but its impact on clinical response and overall survival remains still unexplored. The aim of this study was to evaluate the prognostic significance of different transcript types in a cohort of CML patients treated with imatinib.Methods:A total 42 confirmed cases of Chronic Myeloid Leukemia (CML) patients were recruited into our cohort study and a multiplex Reverse Transcriptase-Polymerase Chain Reaction technique (RT-PCR) was used to detect 3 main transcript types ‘e1a2’, ‘e13a2’, and ‘e14a2’ found in CML.Results:Only two types of transcripts e13a2 (b2a2) and e14a2 (b3a2) were detected in our CML patients and none had the e1a2 type. All the patients were RT-PCR positive for either e13a2 or e14a2 fusion transcript demonstrating 100% concordance with their Ph+ve cytogenetic status at baseline. TLC count (range of 201-600x103/µl) and platelet count (range of 201-900x103/µl) at baseline were found to be associated more with the e14a2 (b3a2) than the e13a2 (b2a2) transcript type (p-value: 0.001). The two transcripts found did not relate significantly towards sex, age-group or indicated spleen size ranges as well as percentage ranges of blast cells.Conclusion:We conclude that there is no overall prognostic implication of either the e13a2 or the e14a2 transcript type across the spectrum of indicated clinical parameters evaluated. Even the overall survival analysis of the two transcript types revealed no prognostic association whatsoever.

BCR-ABL fusion genes and laboratory findings in patients with chronic myeloid leukemia in northeast Iran.

Background:A specific chromosomal abnormality, the Philadelphia chromosome (BCR-ABL fusion), is present in all patients with chronic myeloid leukemia (CML). The b2a2 and b3a2 fusion mRNAs encode p210 fusion protein p210 and e1a2 encode p190. The aim of this study was to evaluate the frequency of BCR-ABL fusion transcript variants in Northeast of Iranian CML patients and to compare the laboratory results of our patients.Methods:This study was conducted in 85 peripheral blood and bone marrow samples of CML patients. Ribonucleic acid (RNA) was extracted by a commercial kit, RT- PCR for identifying BCR-ABL fusions was carried out by using designed primers and the PCR products were electrophoresed in agarose gels. Finally, statistical analysis was performed for variant frequency identification and their comparison was performed.Results: All patients examined were positive for BCR/ABL rearrangement. Fusion of b3a2 was detected in 53 (62.35%) patients, b2a2 in 25 (29.41), e1a2 in 1 (1.17%) and coexpression of b3a2 and e1a2 in 6 (7.05%) patients. There were significant differences between the mean age in patients with b3a2 positive ( 44.07 years) and in b3a2 negative group (50.35 years) however, no significant differences were seen between sex and b2a2 (P=0.61), b3a2 (P=0.79) and e1a2 (P=0.20).Conclusions:This study showed higher frequency b3a2 than b2a2 and e1a2 transcripts in CML patients in Northeast Iran and there was no association between e1a2 transcripts frequencies and monocytosis in peripheral blood.

In Elderly Patients with CML Second Generation TKIs Are Very Effective and Can be Given Long Term Particularly in the Absence of Comorbidities: A Single Center Experience

INTRODUCTIONSecond and third generation tyrosine kinase inhibitors (TKIs) are available for Philadelphia positive chronic myeloid leukemia (Ph+ CML) treatment, but though median age at diagnosis is 60-65 years, elderly patients are under-represented in clinical trials concerning 2G-TKIs and a very small number of papers has been published with series of CML patients > 65 years treated with 2G-TKIs or ponatinib. AIMwe have reviewed our single-center experience with 2G-TKIs in chronic-phase CML (CP-CML) patients > 65 years, in first or subsequent therapeutic lines. PATIENTS AND METHODSthe patients were 10 females and 13 males; CML diagnosis had been made between 1994 and 2015 at a median age of 67 years (61-77). According to Sokal score, 3 patients (13%) were classified as low, 15 (65%) as intermediate and 5 (22%) as high-risk respectively. B3a2 transcript was detected in 13 (56%), b2a2 in 10 (44%) cases.Overall, 30 therapies with 2G-TKIs were prescribed in 23 patients CP-CML > 65 years old, from 2007 to 2015. At 2G-TKI therapy start the median age was 68 years (65-79). Charlson Comorbidity Index (CCI) score was 0 in 11 patients, 1 in 4 patients, 2 in 4 patients, 3 in 3 patients and 4 in 1 patient.Thirteen of 23 patients received second-generation TKIs (2G-TKIs) up-front (6 nilotinib, 7 dasatinib), 10/23 patients as 2nd line therapy (6 nilotinib, 4 dasatinib) after imatinib failure (8/10) or intolerance (2/10), median time from imatinib start to 2G-TKIs being 18 months (9-120). Five of 23 patients needed a TKI as 3rd therapeutic line (4 dasatinib, 1 bosutinib), 1 patient also as 4th (ponatinib) and 5th line (bosutinib). RESULTSall 23 patients are alive after a median follow up of 45 months (12-259) from diagnosis; median follow up from the first 2G-TKI treatment is 36 months (12-110), in details 37 months (12-110) for patients that received 2G-TKIs up front and 34 months (12-70) for patients that received first 2G-TKI as second line therapy.Seventeen of 23 patients (74%) are still on 2G-TKI therapy, with a median treatment of 37 months (12-110) -table 1-. At the last visit 15/17 patients had MMR or best results (1 MR4.5, 9 MR4, 4 MR3).Six patients of 23 (26 %) stopped definitively 2G-TKI therapy, after a median treatment duration of 31 months (6-68) from the first 2G-TKI (table 2), in all cases for non-hematologic toxicity: 1 recurrent pleural effusion (grade 2), 1 geriatric syndrome, 1 carotid atherosclerotic disease, 1 PAOD, 1 vomiting (grade 3) and 1 pulmonary hypertension. At discontinuation, the disease response was MMR (2 patients), MR4 (3 patients), MR 4.5 (1 patient).Hematologic toxicity occurred during 2G-TKI treatment in 7/23 patients (30%), grade 1-2 in 4, grade 3-4 in 3. Sixteen of 23 (69%) patients experienced non-hematologic toxicity, grade 1-2 in 7 (30%) -5/7 pleural effusion-, and grade 3-4 in 9 (39%) -table 3-.There were no significant differences between patients still on therapy and patients that completely discontinued 2G-TKIs regarding Sokal score risk and b2a2/b3a2 transcripts. Conversely, CCI differed in these 2 categories, because CCI was > 1 in 5/6 (83%) of "discontinued" patients and in 7/17 (41%) of "ongoing" patients. Among the eleven patients with CCI = 0, only 1 discontinued 2G-TKI treatment. CONCLUSIONSOur experience shows that 2G-TKIs are very effective in elderly CP-CML, not only as first line therapy. Non-hematologic toxicity is high and it is the main reason for 2G-TKI discontinuation, that appears to be related to comorbidities. The vast majority of patients without comorbidities remain on 2G-TKI treatment long term. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Treatment-free remission (TFR) in patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP) treated with frontline nilotinib: Results from the ENESTFreedom study.

7001Background: In prior clinical trials, ≈40-60% of pts with CML-CP and sustained deep molecular response (MR) maintained TFR after stopping long-term imatinib (median duration: ≈ 5-7 y). The single-arm phase 2 ENESTfreedom study (NCT01784068) is the first to specifically investigate TFR following frontline nilotinib. Methods: Pts with CML-CP with typical b2a2 or b3a2 BCR-ABL1 transcripts, ≥ 2 y of frontline nilotinib, and MR4.5 (BCR-ABL1IS ≤ 0.0032%) at prescreening were eligible. Upon enrollment, pts continued nilotinib for 1 y, with RQ-PCR assessments every 12 wk (consolidation [CONS] phase) at a central standardized laboratory. Pts with no assessment worse than MR4 (BCR-ABL1IS ≤ 0.01%), ≤ 2 assessments between MR4 and MR4.5, and MR4.5 in the last assessment of the CONS phase were eligible to stop treatment (TFR phase). Loss of major MR (MMR [BCR-ABL1IS≤ 0.1%]) triggered re-initiation of nilotinib (ReRx phase). The data cutoff for this analysis was 30 Nov 2015, when all pts who entered the TFR phase h...

Frequency of BCR-ABL Transcript Types in Syrian CML Patients.

Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs) and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%), 3 (14.3%), 2 (9.5%), and 1 (4.8%), respectively. Three samples (14.3%) contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P = 0.047). Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

Impact of the Type of BCR-ABL Fusion Transcripts on Response and Survival in Patients with Chronic Phase CML Treated with Four Frontline TKI Modalities

Introduction: Most commonly observed BCR-ABL transcripts in CML are b3a2 (e14a2) and b2a2 (e13a2). The significance of transcript type regarding r outcome after frontline therapy with different TKI is not well known.Methods: 481 pts with newly diagnosed CML-CP treated in single institution clinical trials with imatinib 400, imatinib 800, dasatinib or nilotinib were analyzed. Cytogenetic and molecular response, and survival endpoints were analyzed by transcript type.Results: 200 (42%) expressed b2a2, 196 (41%) b3a2 and 85 (18%) coexpressed both transcripts. Pts with b2a2 had significantly lower platelets (Median 288 K/µL; range 15-1906 K/µL) compared to b3a2 (405 K/µL; 77-1476 K/µL) or both transcripts (358 K/µL; 100-2928 K/µL) (p<0.001). Other baseline characteristics (Sokal score, hemoglobin, WBC, blast%, spleen size and serum LDH) were not significantly different. The proportion of b2a2, b3a2 and both transcripts pts achieving complete cytogenetic response (CCyR) at 3 months was 59%, 67% and 63%, and at 6 months 73%, 81% and 82%, respectively. MMR rates were 27%, 49% and 50% at 3 months, 42%, 67% and 70% at 6 months, and 55%, 83% and 76% at 12 months, respectively, and MR4.5 at 6 and 12 months were 10%, 29%, and 30%, and 19%, 42% and 41%, respectively. At 3-months, BCR-ABL levels ≤10% were achieved in 75% with b2a2, 79% with b3a2 and 83% with coexpression. The median levels of transcripts (b2a2, b3a2 and both) at 3 months were 0.2004, 0.056 and 0.0612 respectively. Similarly the median levels of transcripts at 6 months were 0.091, 0.0109 and 0.0130 respectively. Pts with b2a2 transcripts demonstrated a slower rate of decline over time (3, 6, 12, 18, 24, 36 and 60 months) after treatment with TKI's as compared with b3a2 and coexpressed b2a2 and b3a2 transcripts (Figure-1A). Furthermore, median time to CCyR was similar in pts expressing b2a2, b3a2 and coexpressed b2a2 with b3a2 - 3 months in each transcript while time to MMR and MR4.5 was much longer in pts with b2a2 transcripts compared to b3a2 and coexpressed b2a2 with b3a2 (6, 3.5 and 3.5 months for MMR and 20, 10 and 11 months for MR4.5 respectively) (Figure-1B). Decline in b3a2 and coexpressed transcript levels over time was faster compared to b2a2 transcripts. For b2a2 transcripts, only the pts treated with nilotinib achieved reduction in levels similar to b3a2 and coexpressed transcripts. Overall, 5 year survival outcomes for b2a2, b3a2 and both transcripts cohorts were 78%, 89%, 87% for event-free survival (EFS; p=0.09); 91%, 97% and 99% for transformation-free survival (TFS; p=0.01) (Figure-1C-D); and 89%, 95% and 98% for overall survival (OS; p=0.34), respectively. In multivariate analysis (MVA), b3a2 and coexpressed transcripts predicted for significantly superior CCyR and MMR at 6 and 12 months. Similarly, type of transcripts significantly predicted for longer EFS (p=0.043; b3a2) and TFS (p=0.04 for coexpressed and 0.07 for b3a2) compared to b2a2.Conclusions: Patients with b3a2 and coexpressed b2a2 and b3a2 achieve earlier and deeper and more durable responses compared to those with b2a2. This translates into improved TFS but not OS. The mechanism of differential responsiveness of pts with b2a2 and b3a2 warrants further investigation. [Display omitted] DisclosuresEstrov:incyte: Consultancy, Research Funding. O'Brien:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding. Cortes:BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BerGenBio AS: Research Funding; Teva: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

Molecular Response with Nilotinib in Patients with Philadelphia Negative (Ph-) Chronic Myeloid Leukemia in Chronic Phase (CML-CP): ENEST1st Sub-Analysis

Molecular Response with Nilotinib in Patients with Philadelphia Negative (Ph-) Chronic Myeloid Leukemia in Chronic Phase (CML-CP): ENEST1st Sub-Analysis

Development of Xpert® BCR-ABL Ultra, an Automated and Standardized Multiplex Assay with Required Performance Characteristics for BCR-ABL1 Quantitative Measurement on an International Reporting Scale

Monitoring BCR-ABL transcript levels in patients on TKI therapy using real-time quantitative PCR is standard of care in the management of CML, and achieving sensitivity to 4.5 logs below baseline is important in patients being considered for TKI discontinuation. In order to accurately assess treatment response, it is essential to ensure that variability in testing methodologies is tightly controlled. This has resulted in substantial efforts to standardize BCR-ABL molecular measurement across laboratories by introducing an International Scale (IS). Here we describe the development and analytical evaluation of Xpert® BCR-ABL Ultra, an automated cartridge-based BCR-ABL monitoring assay incorporating lot-specific standardization to the International Scale as defined by WHO.The BCR-ABL Ultra cartridge simplifies the generation of highly accurate and reproducible assay results by integrating nucleic acid extraction, target amplification, and quantification of the BCR-ABL b3a2 or b2a2 transcripts relative to ABL in peripheral blood specimens. The assay turn-around-time is approximately 2.5 hours and has a %BCR-ABL/ABL(IS) dynamic range of ~6 logs, including two key milestones for the assessment of clinical response at ~10% (IS) (SD=~0.08Log) and ~0.0033% (IS) (SD=~0.25Log) (CMR4.5). Lot-to-lot standardization is achieved using a secondary standard panel of controls derived from and aligned to the first WHO international genetic reference panel. The secondary standards are produced at Cepheid as part of the assay kit manufacturing process and applied to every production lot of cartridges in order to improve assay reliability and reporting comparability. Analytical sensitivity studies using spiked-in CML cell lines demonstrated a limit of detection (LoD) and limit of quantification (LoQ) of 0.003% BCR-ABL/ABL (IS) with a limit of blank (LoB) of 0.00017%. In an analytical specificity study, assay results were negative in 100% of 70 known CML-negative samples, including 20 AML or ALL patients and 50 healthy donors. A study was further conducted to estimate the lower limit of clinical sensitivity using blood from 12 CML patients on TKI therapy who had achieved and maintained MMR with reporting below 0.05% (IS). Eleven out of twelve low CML subjects were detected in at least 19 out of 20 replicates tested per subject over a range of 0.038% (SD=~0.17Log) to 0.0011% (IS) (SD=~0.4Log).In conclusion, the Xpert BCR-ABL Ultra assay demonstrates sensitivity in the range necessary to make clinical decisions regarding the continuation of TKI therapy in patients achieving CMR4.5, and every production lot of cartridges is assigned a unique conversion factor guaranteeing alignment to the WHO International Scale. DisclosuresUy:Novartis: Research Funding.

Impact of Age on Efficacy and Toxicity of Nilotinib in Patients with Chronic Myeloid Leukemia in Chronic Phase (CML-CP): ENEST1st Sub-Analysis

Impact of Age on Efficacy and Toxicity of Nilotinib in Patients with Chronic Myeloid Leukemia in Chronic Phase (CML-CP): ENEST1st Sub-Analysis

Predictive Value of 3-Month Early Molecular Response in New Chronic Phase Chronic Myeloid Leukemia Patients Treated with Radotinib

Background: Recent studies have demonstrated that early molecular milestones were able to identify high-risk chronic myeloid leukemia patients treated with frontline imatinib (IM) and second generation tyrosine kinase inhibitors (2G TKIs) such as nilotinib and dasatinib. However, whether a single measurement of BCR-ABL1 transcripts level after 3 months of treatment is sufficient to define failure necessitating a change of treatment is not confirmed. Radotinib (RAD) is a 2G TKI for BCR-ABL1 tyrosine kinase, which was approved by the Korea FDA for the second-line therapy, and the phase 3 study comparing the efficacy and safety of RAD 300 and 400 mg twice daily and IM 400 mg once daily in patients with newly diagnosed CP CML was performed. The aim of this study was to identify the predictive value of 3-month molecular milestone for an achievement of major molecular response (MMR) by 12 months to RAD therapy. Additionally, in the same population, predictive factors for achieving MMR by 12 months were analyzed.Methods: Among 241 patients who were enrolled in the randomized, open-label, phase 3 study of RAD, 236 patients with available 3-month qRT-PCR on study therapy [RAD 300 mg twice (n = 79), RAD 400 mg twice (n = 79), IM 400 mg once (n = 78)] were evaluated. Molecular responses were monitored using a qRT-PCR assay in 3-month intervals by 12 months. All qRT-PCR were tested with at least 4.5-log sensitivity in the central laboratory (Cancer Research Institute, The Catholic University of Korea, Seoul, Korea) and MMR was defined as a BCR-ABL1 transcript level of 0.1% or lower on the international scale (IS).Results: 236 patients (including 149 men and 87 women) with available 3-month qRT-PCR on study therapy were evaluated. With a median age of 45 years (range, 18-84 years), the distribution of low, intermediate and high Sokal risk scores were 27%, 47% and 26%, respectively. At 3 months, BCR-ABL1 ≤10% [RAD 300 mg twice (n = 68), RAD 400 mg twice (n = 69), IM 400 mg once (n = 55)] and >10% [RAD 300 mg twice (n = 11), RAD 400 mg twice (n = 10), IM 400 mg once (n = 23)] were observed. In the IM 400 mg once group, patients with BCR-ABL1 ≤10% at 3 months showed a significant higher rate of MMR by 12 months compared with that of patients with BCR-ABL1 >10% (38.2% vs 13.0%, P = 0.028). In the RAD 300 and 400 mg twice group, an achievement of 3-month EMR was associated with a higher rate of MMR by 12 months [57.4% vs 18.2%, P = 0.016 (RAD 300 mg twice) and 50.7% vs 10.0%, P = 0.018 (RAD 400 mg twice)]. After adjusting for factors affecting achievement of MMR by 12 months on univariate analyses, multivariate analyses showed that b2a2 transcript type (RR of 0.46, P = 0.023), large spleen size (RR of 0.91, P = 0.001), and no achievement of 3-month EMR (RR of 0.24, P = 0.004) were predictor for not achieving MMR by 12 months. Significance of 3-month EMR for achieving MMR by 12 months was observed in the separated treatment groups: RR of 0.24, P = 0.037 in the IM 400 mg once group, RR of 0.17 P = 0.028 in the RAD 300 mg twice group, and RR of 0.11, P = 0.040 in the RAD 400 mg twice group.Conclusions: Our results suggest that 3-month EMR can play key roles for 12-month MMR achievement in CP CML patients treated with IM and RAD. In addition, some factors for achieving 12-month MMR were detected. To evaluate the long-term prognostic value of 3-month EMR, further clinical investigations in a larger patient population with longer follow-up are needed. DisclosuresKim:IL-YANG Pharm.Co.Ltd: Research Funding. Chung:Alexion Pharmaceuticals: Research Funding.

Performance of two commercially available BCR-ABL1 quantification assays that use an international reporting scale.

Quantifying the BCR-ABL1 rearrangement is important for monitoring chronic myelogenous leukemia (CML). To standardize BCR-ABL1 quantification, the World Health Organization (WHO) established the first international genetic reference panel. Here, we compared the BCR-ABL1 levels determined using international scale (IS)-based commercially available assays. BCR-ABL1 transcripts were quantified using two IS-based assays. 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 dilutions of the b3a2 positive RNA were used for evaluating linearity, precision, and limit of detection. Correlation of the assay was evaluated by using DNA obtained from CML patients carrying the BCR-ABL1 b3a2 and b2a2 types. Both Ipsogen and Asuragen assays showed fine linearity with reasonable %CV. LOD of each assay was calculated as 0.003% for Ipsogen, and 0.005% for Asuragen. By comparing the results that were lower than 10% by either one of the assay, Ipsogen and Asuragen results showed an overall good linear correlation with a tendency for the Ipsogen assay to show slightly higher levels than the Asuragen assay for b3a2 transcript. For b2a2, the tendency was opposite, with Asuragen showing higher values than the Ipsogen. Two commercially available IS-based BCR-ABL1 assays showed an overall good quantitative correlation. It should be taken into consideration that each assay tended to produce higher values than the other, depending on the BCR-ABL1 subtypes, suggesting that a separate conversion factor for each subtype can be more helpful when BCR-ABL1 transcript levels are converted into IS.