Highly Sensitive Droplet Digital PCR to Identify CML Patients with a High Probability of Achieving Treatment-Free Remission

Introduction: Treatment Free Remission (TFR) is the ultimate goal of therapy for most CML patients. Despite adopting consensus eligibility criteria of a sustained deep molecular response and more than 4 years of TKI therapy, the relapse rate after TKI cessation is still around 50%. More sensitive detection of residual leukaemia has the potential to improve our capacity to predict TFR outcomes for individual patients. Aim: To correlate droplet digital PCR (ddPCR) assay results with TFR outcome, especially in the setting of undetectable levels measured by qRT-PCR. Method: ddPCR was performed on blood samples from 51 TFR-eligible CML patients at the time of TKI cessation. 5 µg RNA per sample was used in 8 wells/sample using the BioRad QXDx BCR-ABL %IS kit on QX200 ddPCR system which yielded BCR-ABL1% (IS) directly. All these patients achieved MR4.5 that was sustained for ≥ 2 years. Results: 100% of patient were in MR4.5 via qRT-PCR at the time of stopping. 61% of the 51 patients evaluated relapsed within 12 months. Median duration of TKI therapy for the whole group was 5.8 years (range 2.2- 14 years). 20 patients achieved TFR success with a median follow up of 24 months (TFR group; sustained BCR-ABL1 <0.1% (IS) after TKI discontinuation for ≥12 months), while 31 patients relapsed (Relapse group; BCR-ABL1 >0.1% (IS) after stopping; median time of relapse 3 months, range 1-10 months). A ROC curve analysis correlated TFR outcome with ddPCR results, with BCR-ABL1 level ≥0.003% via ddPCR at the time of stopping identified as an optimal cut-off. Kaplan-Meier analysis showed that 89% of the patients with ddPCR ≥0.003 relapsed after TKI cessation, whereas the ddPCR <0.003 demonstrated a significantly reduced relapse rate to 54% (p=0.01, Figure 1A). In addition, the TFR group (median BCR-ABL1 0.00065%) demonstrated approximately two-fold lower levels of BCR-ABL1transcript level compared to the relapse group (median 0.0012%). Interestingly, 7/31 (23%) of the relapsed group had undetectable BCR-ABL1 transcript even with the current highly sensitive method, while this undetectable level was only observed in 35% of the TFR group. We next assessed other known predictors of TFR success relative to ddPCR results in a Cox proportional hazard model. We have previously demonstrated that the BCR-ABL1 halving time after commencing therapy is highly predictive of TFR. At a univariate level, transcript type (e13a2 versus e14a2, p=0.01), BCR-ABL1 halving time (p>0.0001), and mRNA quantitation by ddPCR ≥ 0.003% (p=0.02) were all significantly associated with clinical outcome. Other variables including gender, age, ELTS score, Sokal score, MR4.5 duration and TKI duration were not associated with clinical outcome in this cohort (Figure 1B). In the multivariate analyses (Figure 1C), ddPCR remained an independent predictor after adjusting for ELTS, TKI duration and MR4.5 duration. Interestingly, ddPCR was not an independent predictor after adjusting for BCR-ABL1 transcript type or halving time. Conclusion: QXDx ddPCR assay is a promising tool for molecular residual disease monitoring in CML, especially when the BCR-ABL1 is undetectable by conventional method. The CML patients with levels of detectable BCR-ABL1 ≥0.003% measured by ddPCR have a significantly higher probability of relapse compared to patients with lower levels of the transcript. The ≥0.003% BCR-ABL1 level cut-off value could be a potential tool to aid decision-making when attempting TKI discontinuation in CML. However, even though a measurable level of BCR-ABL1 above 0.003% via ddPCR identified patients at high risk of relapse after a TFR attempt, it does not rule out the possibility of TFR; and a negative ddPCR result does not exclude the risk of molecular relapse. ddPCR may be most useful where other TFR predictive factors including BCR-ABL1 transcript type and halving time are not available. In-kind support was received from Bio-Rad for this study. Figure 1 Figure 1. Disclosures Branford: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cepheid: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes: Novartis: Honoraria, Research Funding; Incyte: Honoraria; BMS: Honoraria, Research Funding. Yeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

Effect of BCR-ABL1 Transcript Type and Body Weight on Outcome in Chronic Myeloid Leukemia

Introduction The hallmark of CML is BCR-ABL1 (breakpoint cluster region gene-Abelson murine leukemia viral oncogene homolog 1) on Philadelphia chromosome, which is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22 (t[9;22][q34;q11]) [1]. Chromosome 22 breakpoints influence the BCR portions preserved in the BCL-ABL1 fusion mRNA and protein and are mainly localized to one of three BCRs, namely major-BCR (M-BCR), minor BCR (m-BCR) and micro-BCR (µ-BCR). In comparison, breaks in chromosome 9 arise most frequently by alternative splicing of the two first ABL1 exons, and can also be generated in a large genetic region, upstream of exon Ib at the 5' end, or downstream of exon Ia at the 3' end. In the majority of CML cases, the breakpoint lies within the M-BCR and gives rise to e13a2 or e14a2 fusion mRNAs (previously denoted as b2a2 and b3a2) and a p210BCR-ABL fusion protein [2]. [3] Methodology We conducted a retrospective analysis of the files of 79 patients being treated in our center for CML with known BCR-ABL1 breakpoints; there were few more patients with known transcript type but excluded because either travelled immediately on diagnosis or had a failure due to confirmed compliance issues. Patients' management and response assessment was done based on ELN 2013 guidelines. The analysis is done based on two main groups, obese versus normal BMI, and then based on BCR-ABL1 transcripts: e13a2 versus e14a2. Ethical approval was obtained from Medical Research Center for Hamad Medical Corporation (MRC-01-18-337). Results Patients included 62 males (78.5%) and 17 females (21.5%) with the mean age at diagnosis 38.8±11.8 years (median, 38; range 21 to 69 years). The characteristics (demographics, anthropometric, hematological and clinico-pathological) of the patients and their association with transcript types and obesity are summarized in Table 1. Patient outcomes, cytogenetic and molecular responses The median follow-up was 30 months (range 6 to 196 months) and 38 months (range 3 to 192 months) in normal weight and obesity groups, respectively. The median follow-up was 28 months (range 3 to 196 months) and 39 months (range 10 to 192 months) in e14a2 and e13a2 patients, respectively. A total of 22 patients distributed among different groups ended up leaving the country (censored) after a variable duration of follow-up (6 - 196 months), 18 of them CML-CP, and 4 CML-AP. 3 patients died in our cohort, all of them had e14a2 transcript, one of them was in the normal weight/BMI group, two were in the obesity group. In e14a2 group, more patients were on imatinib at the time of analysis (15 (39.5%) vs 7 (17.1%) in e13a2 group, p = 0.026). The percentage of patients of had to switch TKI was similar in both groups (47.4% vs 53.7%, p = 0.576). However, less patients in e14a2 group had to switch TKI because of failure/progression (10 (55.6%) vs 17 (77.3%), p = 0.145); however, this didn't translate into a significant difference of achieving MMR at 1 year, where in e14a2 group, 10 patients achieved MMR at 1 year (31.3%), same as in e13a2 group (10 patients = 29.3%) p 0.331 (all shown in table 1). When comparing long-term outcomes, there was also no significant difference between groups based on transcript type with regards to MMR (44.7% vs 46.3% in e14a2 vs e13a2 respectively) or DMR (26.3% vs 22% respectively) as shown in figure. In the obesity group, there were 2 patients using ponatinib due to T315I mutation, compared to none in normal weight group. However, there were no significant differences in TKI used, switch of TKI, or reason for switch. Same applies for achieving MMR at 1 year, as 11 patients in the obesity group achieved MMR (28.2%) compared to 9 patients in normal weight group (33.3%), p = 0.778 (as shown in table 1). Regarding the long-term outcomes, more patients in the obesity group achieved MMR (53.2%) compared to normal weight group (34.3%), and this response was faster, but not statistically significant. This difference was less clear with regards to DMR (25.5% in the obesity group compared to 21.9% in normal weight group) as shown in figure. Conclusion In the patient-cohort studied there were no significant differences in molecular response based on transcript type or body weight/BMI. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Impact of BCR-ABL1 Transcript Type on Response, Treatment-Free Remission Rate and Survival in Chronic Myeloid Leukemia Patients Treated with Imatinib

The most frequent BCR-ABL1-p210 transcripts in chronic myeloid leukemia (CML) are e14a2 and e13a2. Imatinib (IM) is the most common first-line tyrosine–kinase inhibitor (TKI) used to treat CML. Some studies suggest that BCR-ABL1 transcript types confer different responses to IM. The objective of this study was to correlate the expression of e14a2 or e13a2 to clinical characteristics, cumulative cytogenetic and molecular responses to IM, acquisition of deep molecular response (DMR) and its duration (sDMR), progression rate (CIP), overall survival (OS), and treatment-free remission (TFR) rate. We studied 202 CML patients, 76 expressing the e13a2 and 126 the e14a2, and correlated the differential transcript expression with the above-mentioned parameters. There were no differences in the cumulative incidence of cytogenetic responses nor in the acquisition of DMR and sDMR between the two groups, but the e14a2 transcript had a positive impact on molecular response during the first 6 months, whereas the e13a2 was associated with improved long-term OS. No correlation was observed between the transcript type and TFR rate.

Clinicopathological Variables and Outcome in Chronic Myeloid Leukemia Associated With BCR-ABL1 Transcript Type and Body Weight: An Outcome of European LeukemiaNet Project

Objective It is debatable whether BCR-ABL1 transcript type has an impact on outcome of treatment of patients with CML, and it is not widely studied whether body weight influences response to treatment. In this study, we tried to find out if any of these factors has an impact on response to treatment and outcome. Methodology We conducted a retrospective analysis of the files of 79 patients being treated in our center for CML with known BCR-ABL1 breakpoints, and patients’ management and response assessment was done based on ELN 2013 guidelines. The analysis was performed based on two main groups, obese vs. normal BMI, and then based on BCR-ABL1 transcripts: e13a2 vs. e14a2. Cumulative incidence of MMR, CCyR, and DMR were estimated using the Kaplan–Meier survival curve method, and comparisons between groups were performed by the Log-rank/Gray test methods. Results/conclusion In the patient-cohort studied, there was no statistically significant difference in molecular response between patients with CML based on body weight or transcript type although patients in the obesity group achieved higher and faster MMR with no statistical significance.

Clinicopathological Variables and Outcome in Chronic Phase Chronic Myeloid Leukemia Associated with BCR-ABL1 Transcript Type and Obesity

Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the dysregulated production and uncontrolled proliferation of mature and maturing granulocytes with fairly normal differentiation. The hallmark of CML is BCR-ABL1 (breakpoint cluster region gene-Abelson murine leukemia viral oncogene homolog 1) on Philadelphia chromosome, which is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22 (t[9;22][q34;q11]). With rare exceptions, breaks in chromosome 22 localize to one of three BCRs and determine the portions of BCR retained in the BCR-ABL1 fusion mRNA and protein. In contrast, the chromosome 9 breaks can occur over a large genetic region, 5′ of ABL1 exon Ib, 3′ of ABL1 exon Ia, or most commonly between the two alternative first ABL1 exons. In an overwhelming majority of CML patients, the break occurs in the major BCR (M-BCR), generating e13a2 or e14a2 fusion mRNAs and a p210BCR-ABL fusion protein. p230BCR-ABL, the largest of the fusion proteins, corresponds to a break in the micro BCR (μ-BCR), an e19a2 fusion mRNA, and is associated with neutrophilic predominance and possibly less aggressive disease. Molecular monitoring of BCR-ABL1 transcript levels following treatment with tyrosine kinase inhibitors (TKIs) is central to the effective clinical management of patients with CML. BCR-ABL1 transcripts measured at standardized time points is used to define responses at key milestones in treatment allowing early signs of poor adherence or resistance to treatment to be detected and allow for early, effective clinical intervention. Objective The aim of this study is to evaluate response to treatment with standard dose TKI in obese and non-obese CML patients together with BCR-ABL1 transcript type Methodology A retrospective analysis of clinicopathological variables and response to treatment was performed for 37 chronic phase CMLs to compare, obese vs normal weight, and BCR-ABL1 transcript type determined at diagnosis. Patients' management and response assessment was done based on ELN 2013 guidelines. Response to treatment was assessed using RT-qPCR analysis of blood calculated on the International Scale (IS). Various statistical methods used, all Statistical analyses were done using statistical packages SPSS 22.0 (SPSS Inc. Chicago, IL) and Epi-info (Centers for Disease Control and Prevention, Atlanta, GA) software. Results The study cohort included 26 males (70.3%) and 11 females (29.7%) with mean age at diagnosis 36.8 years. 59.5% (n=22) expressed an e14a2 transcript, and 40.5% (n=15) an e13a2 transcript, most patients were started on imatinib, then switched either due to toxicity or failure. Median follow-up was 18 months for both transcript types. WBC, platelet counts, spleen size and Sokal scores at diagnosis, both median and Inter-quartile range (IQR) were observed to be higher in e14a2 compared to e13a2 transcript group, and, lower in obese patients compared patients with normal weight. At one year, patients with e13a2 transcript had higher percentage of CCyR (or better) 60% (95% CI 36.6, 80.3%) compared to e14a2 group 46.7% (95% CI 24.8, 69.9%), however this difference was statistically insignificant (odds ratio =1.71, 95% CI 0.40, 7.29; P=0.464) Overall, there was higher and faster achievement of CCyR and MMR in patients with e13a2 transcript compared to e14a2 transcript, and in the obese vs normal-weight patients. Patients in e13a2 group and obesity group had a lower rate of treatment failure and fewer indications to switch TKI. Of note MMR was observed to be significantly higher in patients of African origin (n=10) compared to patients with Asian ethnicity (50% vs 16%; P=0.038), which could be reflect differences in disease biology. Conclusion In the patient cohort studied an e14a2 BCR-ABL1 transcript type / normal body weight was associated with an inferior outcome when compared to e13a2 transcript / obesity groups Disclosures No relevant conflicts of interest to declare.

Clinicopathological Variables and Outcome in Chronic Phase Chronic Myeloid Leukemia Associated with BCR-ABL1 Transcript Type and Body Weight

Background: It has been reported that general adiposity in adulthood and early adulthood, and greater height may increase the risk of almost all types of lympho-haematopoietic cancers while a study done in MD Anderson found that obesity and adult weight gain are independent risk factors for CML however no study evaluated the role of obesity in the disease progression while more studies investigate the impact of translocation types. Method: We conducted a retrospective analysis of the files of 37 patients being treated in our center for CML in chronic phase (CMP-CP) with known BCR-ABL1 breakpoints, Results: patients’ management and response assessment was done based on ELN 2013 guidelines. Analysis is done based on two main groups, obese vs normal BMI, and then based on BCR-ABL1 transcripts: e13a2 vs e14a2. Although the number of cases is limited, in the patient-cohort studied an e14a2 BCR-ABL1 transcript type / normal body weight was associated with an inferior outcome when compared to e13a2 transcript / obesity groups Conclusion: our finding suggest the need to enlarge the series to better evaluate a potential role of altered metabolism and/or specific transcripts in the response to TKI in CML.

Novel multiplex RT-PCR assay to detect BCR/ABL mRNA variants

The detection of the t(9;22) translocation, which results in the formation of the BCR/ABL oncoprotein, in patients diagnosed with chronic and acute leukaemias, allows for the administration of highly effective tyrosine kinase inhibitors such as imatinib and nilotinib. For the effective management of these patients it is important to monitor for minimal residual disease, allowing early therapy intervention prior to haematological relapse. Currently this is achieved through very sensitive quantitative real-time PCR assays. Unfortunately, these assays are highly specific to the BCR/ABL variant expressed: p210, p190 or p230 and require identification of the transcript type prior to selection of the correct monitoring assay. We have developed a novel multiplex BCR/ABL variant PCR assay that can identify the variant in a single qualitative assay, creating both a cost and time effective way to identify the most appropriate monitoring methodology for each patient.

R-Interferon Treatment before Imatinib Reverses the Negative Impact of e13a2 Transcript Type on Treatment Free Remission Duration after Tyrosine Kinase Inhibitors Discontinuation in Chronic Myeloid Leukemia Patients

BACKGROUND Tyrosine Kinase Inhibitor (TKIs) discontinuation has become, nowadays, under proper conditions, a feasible option for chronic myeloid leukemia (CML) also in "real life" setting. Different papers investigated which factors (age, sex, type of TKI, previous r-interferonα -r-INFα- therapy, line of therapy at stop, type of transcript, duration of TKI therapy and of sustained deep molecular response -sDMR-, Sokal risk score) could predict a successful TKIs discontinuation either within protocols or outside of clinical trials, and the results are not unique. AIM We retrospectively evaluated our CML pts who stopped TKI after sDMR in order to assess the variables that could influence the probability of a durable TFR. METHODS BCR-ABL transcripts were determined by RQ-PCR performed according to EAC protocol (Gabert et al, 2003) and to the standards of the Italian National Network Labnet. Criteria for TKI discontinuation was sustained DMR (MR4 or better) for at least 2 years. After TKI withdrawal, RQ-PCR for BCR-ABL was performed every month during the first year and every 2 months thereafter. TKI treatment was reintroduced if DMR loss occurred.TFR was assessed using the Kaplan-Meier method; potential prognostic factors were considered for multivariable analyses at a level less than .20. RESULTS Between October 2010 and January 2019, 68 patients discontinued TKIs, 18 of them after less than 5 years of treatment because of pregnancy desire (3), intolerance (6), patient's desire/non compliance (5), enrollment in study protocols (4). At discontinuation median age was 63 years (30-85), median time from TKI start 85 months (30-190), median duration of sustained DMR 48 months (24-153). Sokal distribution was 48%, 31% and 18% for low, intermediate and high risk respectively (2 patient was not evaluable). E14a2 transcript type was present in 52 pts and e13a2 in 16 pts. Thirty-eight patients stopped imatinib, 25 nilotinib (19 in 1st line, 6 in 2nd line), 5 dasatinib. Before imatinib 15 patients received r-IFNα, for a median time of 60 months (3-256). Median follow up after TKI stop was 39 months (5-105, >24 in 61, <12 in only 2 patients). Twenty-eight (41%) patients lost DMR. Median time off-therapy for these patients was 3 months (1-19), only 2 lost DMR after 6 months (at +16 and +19 months). One patient aged 87 years has not yet resumed therapy but remains in stable MR3 at 34 months after TKI discontinuation. Therapy was restarted in 27 patients (1 in MR1, 11 in MR2, 15 in MR3), 24 achieved a second DMR after a median interval of 2 months (1-18); 3/27 patients are in M3 after 2, 22 and 26 months. Neither cytogenetic relapses, nor progressions were documented. One patient died in DMR for pancreatic cancer. Univariate analysis showed no difference in relapse risk according to age, gender, type of TKI (imatinib vs 2nd generation TKIs), and Sokal score, while the e14a2 vs e13a2 transcript type (p = 0.011), duration of TKI therapy > 60 months (p = 0.025) and previous r-IFNα therapy (p=0.021) were significantly linked to better outcome after TKI discontinuation; sDMR > 72 months is very close to be a significant variable (p=0.055). At multivariate analysis only the type of BCR-ABL transcript (p=0.027) and previous r-IFNα ( p=0.016) remained independent significant prognostic factors -figure A and figure B-. CONCLUSION e14a2 transcript type was confirmed as a robust favorable prognostic factor for TFR maintenance. In our experience, 2GTKIs didn't impact favorably TFR duration after TKIs discontinuation, conversely r-IFNα treatment before TKI improved the probability of maintaining DMR after TKI withdrawal, particularly in e13a2 patients. In fact r-IFNα before imatinib reversed the negative prognostic impact on TFR maintenance of the e13a2 transcript type. Disclosures D'Adda: Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Rossi:Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Daiichi-Sankyo: Consultancy.