E1a2 Transcripts Research Articles

Quantitative BCR::ABL1 Assay Using Digital Real-Time PCR System: Clinical Applications and Insights

Background: Accurate quantification of BCR::ABL1 transcripts is paramount for the management of chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Despite advancements in real-time reverse transcription PCR (RT-qPCR), the need for more sensitive and precise assays remains. This study evaluated the diagnostic utility of digital reverse transcription PCR (dRT-PCR) for BCR::ABL1 quantification in assessing minimal residual disease (MRD). Material and methods. Twelve patients with CML or Ph+ ALL were included. Peripheral blood or bone marrow samples, collected in PaxGene RNA preservation tubes, were extracted with the automated QIASymphony SP instrument. dRT-PCR conducted on the LOAA analyzer, utilized the Dr. PCR BCR-ABL1 Detection Kit CE_IVD (OPTOLANE Technologies, South Korea). By employing a dual-labeled hydrolysis probe and target-specific primers, this protocol enabled the simultaneous amplification and detection of BCR::ABL1 transcripts e13a2 and e14a2 and ABL1. The resulting copy numbers/reactions were used to calculate the International Scale percentage (%IS) and the molecular reduction (MR) value and analyzed for clinical correlations. Results. Digital RT-PCR demonstrated high precision in quantifying BCR::ABL1 transcripts, the lowest %IS value being 0.02%. A consistent quality RNA sample totalizing 110 ng/reaction was crucial for successful reactions. This assay proved faster and easier than conventional RT-PCR and did not require standards or calibrators. Conclusion. This study supports the feasibility of digital RT-PCR for quantitative analysis. Future objectives for our laboratory include expanding our diagnostic repertoire using this platform, for enhancing patient care.

Variant-specific BCR::ABL1 quantification discrepancy in chronic myeloid leukemia.

Accurate quantification of the BCR::ABL1 fusion gene in whole blood is pivotal for the clinical management of chronic myeloid leukemia (CML) patients. The fusion protein encoded by BCR::ABL1 can vary in size, depending on the BCR and/or ABL1 gene breakpoint. The vast majority of CML patients have a p210 BCR::ABL1 fusion gene (M-BCR), which can be attributed to the presence of either e14a2 (b3a2) or e13a2 (b2a2) mRNA transcript junctions. Twenty-five CML samples were analyzed in two different ISO15189-accredited centers that both use an Europe Against Cancer-based quantitative polymerase chain reaction (qPCR) protocol. Reanalysis of the sample set with transcript-specific standard curves and digital droplet PCR (ddPCR) were performed. qPCR quantification revealed a significant (up to 1 log) difference specifically for the e13a2 transcript variant in contrast to e14a2 transcripts (Hodges-Lehman 4.29; p < 0.001). Reanalysis of the sample set with transcript-specific standard curves abolishes the initial transcript-specific difference (Hodges-Lehman 0.003; p = 0.8192). Comparison of transcript-specific qPCR results of both centers with ddPCR, an absolute quantification method, showed a statically significant association, especially in the lower range, indicating the clinical utility of transcript-specific or absolute quantification methods. Our data show that differences between transcript-specific quantification might exist between centers, leading to potential clinical impact on the follow-up of CML patients. The use of transcript-specific standard curves for qPCR quantification, or absolute quantification, can significantly reduce these differences. Specific attention should be applied to the interpretation of quantification differences of CML patients that switch between diagnostic centers.

High Level of CD8+PD-1+ Cells in Patients with Chronic Myeloid Leukemia Who Experienced Loss of MMR after Imatinib Discontinuation.

Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8+PD-1+ cells in patients losing TFR. The level of CD8+PD-1+ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8+PD-1+ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8+PD-1+ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib.

E14a2 Transcript Favors Treatment-Free Remission in Chronic Myeloid Leukemia When Associated with Longer Treatment with Tyrosine Kinase Inhibitors and Sustained Deep Molecular Response

e13a2 and e14a2 are the most frequent transcript types of the BCR::ABL1 fusion gene in chronic myeloid leukemia (CML). The current goal with tyrosine kinase inhibitors (TKI) is to achieve sustained deep molecular response (DMR) in order to discontinue TKI treatment and remain in the so-called treatment-free remission (TFR) phase, but biological factors associated with these goals are not well established. This study aimed to determine the effect of transcript type on TFR in patients receiving frontline treatment with imatinib (IM) or second-generation TKI (2G-TKI). Patients treated at least 119 months with IM presented less post-discontinuation relapse than those that discontinued IM before 119 months (p = 0.005). In addition, cases with the e14a2 transcript type treated at least 119 months with IM presented a better TFR (p = 0.024). On the other hand, the type of transcript did not affect the cytogenetic or molecular response in 2G-TKI treated patients; however, the use of 2G-TKI may be associated with higher and earlier DMR in patients with the e14a2 transcript.

Age-Related Clonal Hematopoiesis Mutations Detected at the Time of Stopping Tyrosine Kinase Inhibitor Therapy Predict the Achievement of Treatment-Free Remission for Patients with CML

Background The gain of mutations with a competitive advantage in hematopoietic cells of healthy individuals is common with aging and termed clonal hematopoiesis of indeterminate potential (CHIP). Through sequencing studies we detected CHIP related mutants in chronic myeloid leukemia (CML) patients in deep molecular response. CHIP mutants may provide a selective growth advantage to non-leukemic cells, driving their clonal expansion in remission and potentially influencing treatment-free remission (TFR) if CHIP clones outcompete an emerging BCR:: ABL1 clone. Aim To determine if CHIP mutants detected at the time of stopping tyrosine kinase inhibitor (TKI) therapy could influence achievement of TFR. Methods An RNA-based sequencing method targeting 17 of the most frequently mutated CHIP genes was developed and applied to blood samples of 150 patients who attempted TFR. The median follow up of patients in TFR was 71 months. CHIP mutants met strict criteria for pathogenicity based on their potential to confer growth and survival advantages, i.e. the same criteria as cancer driver mutations. Variant allele frequency (VAF) of ≥0.4% was reproducibly detected. CHIP at the time of cessation, termed CHIP TOC, was defined as 1) ≥1 CHIP mutant with VAF ≥2.0% (n=30 patients) or 2) a novel criterion for patients with multiple CHIP mutants where the combined VAF was ≥2.0% (n=7). Overall, 25/37 patients (68%) with CHIP TOC had multiple mutations, range 2-7. Results CHIP TOC was detected in 37/150 patients (25%). The average age of patients with CHIP TOC was 66.3 years vs 58.0 for those without, P=.0004, which is consistent with the age related nature of CHIP. No patient aged &amp;lt;40 had CHIP TOC, whereas 44% of patients ≥70 had CHIP TOC. Twelve genes were mutated and the most frequent were TET2, DNMT3A, ASXL1 and PPM1D. The overall probability of TFR was 60.4% at 12 months and 53.0% at 84 months. CHIP TOC was associated with TFR. At 12 months, 83.6% of patients with CHIP TOC maintained TFR versus 52.9% of patients without CHIP TOC, P=.002. At 84 months, 62.9% of patients with CHIP TOC maintained TFR versus 49.6% of patients without CHIP TOC, P=.014. We previously demonstrated that e13a2 transcripts and a slow initial decline of BCR:: ABL1 after starting frontline TKI therapy, measured as the number of days over which BCR:: ABL1 halved, were our strongest predictors of relapse. Prognostic factors for the achievement of TFR at 12 and 84 months were examined in multivariable analysis. Candidate prognostic factors were CHIP TOC, BCR:: ABL1 halving time, BCR:: ABL1 transcript type, age at diagnosis, gender, duration of MR4.5 and time on TKI before TKI stop. The independent predictors of TFR at 12 and 84 months were the presence of CHIP TOC at TKI stop, a shorter BCR:: ABL1 halving time (indicating a more rapid initial BCR:: ABL1 decline) and the e14a2 (plus e14a2/e13a2) BCR:: ABL1 transcript type (Table 1). The rate of late relapse, defined as loss of MMR &amp;gt;12 months after cessation, was higher in patients with CHIP TOC. Overall, 85 patients had TFR at 12 months and late relapse occurred in 8. A landmark analysis demonstrated that the probability of maintaining TFR at 84 months for these 85 patients was 93.8% for those without CHIP TOC (n=57) and 75.2% for those with CHIP TOC (n=28), P=.007. The only variable associated with late relapse among the patients with CHIP TOC was fluctuation of BCR:: ABL1 within the first 12 months after cessation, defined as irregular rise and fall comprising 2 consecutive BCR:: ABL1 ratios with loss of MR4.5, or a single loss of MR4. Overall, 13 patients had fluctuation within the first 12 months and 5 of these had late relapse. All 5 had CHIP TOC. Patients with CHIP TOC and stable MR4.5 by 12 months had a 93.8% probability of TFR at 84 months (Table 2). Conclusion The presence of CHIP TOC at TKI cessation, a shorter BCR:: ABL1 halving time and e14a2 BCR:: ABL1 transcripts were independent predictors of TFR at 12 and 84 months. Delayed loss of MMR that occurred for a minority of patients with CHIP TOC may be associated with competition between CHIP clones and a residual leukemic clone. These patients had fluctuating BCR:: ABL1 over the first 12 months after cessation and prior to late relapse. Novel strategies may be warranted to reduce the risk of late relapse for these patients. Patients who sustained TFR by 12 months in stable MR4.5 had a reassuringly low risk of late relapse. CHIP status could be a key variable to guide TFR decision-making and monitoring.

Comparable b2a2/e13a2 and b3a2/e14a2 Reporting in BCR-ABL Assay When Calibrated By Who Is and By IVT-RNA Copy Number

Introduction: Xpert® BCR-ABL Ultra, automated cartridge-based assay for monitoring BCR-ABL transcript levels, is calibrated by WHO IS to standardize the %reporting for both b2a2/e13a2 BCR-ABL(e13a2) and b3a2/e14a2 BCR-ABL(e14a2) relative to control ABL gene in peripheral blood of patients as a standard management of Chronic Myeloid Leukemia (CML) [1]. Studies have shown differences in patients carrying e13a2 or e14a2 in molecular response to Tyrosine Kinase Inhibitor (TKI) treatment [2-4]. Therefore, it is necessary to understand if there is %reporting differences between two transcripts calibrated by WHO IS[5] and explore the calibration method using breakpoint specific RNA input copy number (CN) for accurate TKI treatment monitoring. Objectives: To establish % reporting method calculated with known CN of e13a2, e14a2 and ABL IVT-RNA within a wide CN range and compare % e13a2 and % e14a2 to WHO IS quantification system. Methods: Three IVT-RNAs, containing e13a2-ABL-BCR, e14a2-ABL-BCR and ABL-BCR, were synthesized as shown in Figure 1. Serial diluted e13a2 RNA, e14a2 RNA and ABL RNA panels were tested on Xpert BCR-ABL Ultra assay to generate individual standard curve of e13a2 Ct, e14a2 Ct and ABL Ct as a function of CN for % reporting calculation. Four levels of IVT-RNA panels with same CN of e13a2 and e14a2 were tested for breakpoint specific % reporting comparison. K562(e14a2), BV173 (e13a2) cell lysates and CML clinical samples carrying e13a2 or e14a2 transcripts were tested to evaluate the %CN reporting compared to WHO IS. Result: Good linearity demonstrated in Ct vs CN input for e13a2, e14a2 and ABL IVT-RNA (Figure2) with comparable efficiency (E) between e13a2 (E=0.992) and e14a2 (E=0.986). %e13a2 reporting was ~1.50-fold (by WHO IS) and ~1.46-fold (by %CN) higher than %e14a2, by testing IVT-RNA panel (Table1). Minor differences in %reporting observed between % CN and WHO IS for e13a2 (84.5%~110.8%) vs e14a2 (82.5%~89.5%) from cell lysates (Table2) and e13a2 (92.6%~105.5%) vs e14a2 (88.1%~92.2%) from clinical samples (Table3). Conclusions: A %CN e13a2/ABL and e14a2/ABL reporting method with known CN of IVT-RNAs has been successfully established in Xpert BCR-ABL Ultra. Both WHO IS and %CN showed very minor differences between e13a2 and e14a2 for % reporting. %CN method demonstrated comparable %reporting to WHO IS in Xpert BCR-ABL Ultra. Recent Publications 1. Dominy KM, Simon IM, Khorashad JS (2021) Evaluation of Xpert® BCR-ABL Ultra for the confirmation of BCR-ABL1 international scale conversion factors for the molecular monitoring of chronic myeloid leukaemia, Int J Lab Hematol, Feb;43(1):e31-e34. 2. Hanfstein B, Lauseker M, Hehlmann R, Saussele S, Erben P, Dietz C (2014) Distinct characteristics of e13a2 versus e14a2 BCR-ABL1 driven chronic myeloid leukemia under first-line therapy with imatinib. Haematologica.99:1441-7. 3. Jain P, Kantarjian H, Patel KP, Gonzalez GN, Luthra R, Shamanna RK (2016) Impact of BCR-ABL transcript type on outcome in patients with chronic-phase CML treated with tyrosine kinase inhibitors. Blood.127:1269-75. 4. Castagnetti F, Gugliotta G, Breccia M, Iurlo A, Levato L, Albano F (2017) The BCR-ABL1 transcript type influences response and outcome in Philadelphia chromosome-positive chronic myeloid leukemia patients treated frontline with imatinib. Am J Hematol. 92:797-805. 5. Matthew S, White HE, Hana Z, Nicholas C.P C (2022) Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy. Leukemia 36(7):1-8

Genetic alterations in the BCR-ABL1 fusion gene related to imatinib resistance in chronic myeloid leukemia

Use of the potent tyrosine kinase inhibitor imatinib as the first-line treatment in chronic myeloid leukemia (CML) has decreased mortality from 20% to 2%. Approximately 30% of CML patients experience imatinib resistance, however, largely because of point mutations in the kinase domain of the BCR-ABL1 fusion gene. The aim of this study was to use next-generation sequencing (NGS) to identify mutations related to imatinib resistance. The study included 22 patients diagnosed with CML and experiencing no clinical response to imatinib. Total RNA was used for cDNA synthesis, with amplification of a fragment encompassing the BCR-ABL1 kinase domain using a nested-PCR approach. Sanger and NGS were applied to detect genetic alterations. HaplotypeCaller was used for variant calling, and STAR-Fusion software was applied for fusion breakpoint identification. After sequencing analysis, F311I, F317L, and E450K mutations were detected respectively in three different participants, and in another two patients, single nucleotide variants in BCR (rs9608100, rs140506, rs16802) and ABL1 (rs35011138) were detected. Eleven patients carried e14a2 transcripts, nine had e13a2 transcripts, and both transcripts were identified in one patient. One patient had co-expression of e14a2 and e14a8 transcripts. The results identify candidate single nucleotide variants and co-expressed BCR-ABL1 transcripts in cellular resistance to imatinib.

Effect of BCR::ABL1 transcript type and droplet digital polymerase chain reaction on successful treatment-free remission in chronic myeloid leukemia patients who discontinued tyrosine kinase inhibitor.

Droplet digital polymerase chain reaction (ddPCR) is an exact method of measurement. We conducted this study to identify the prognostic factors for successful treatment-free remission in patients with chronic-phase chronic myeloid leukemia who discontinued tyrosine kinase inhibitors (TKIs). We also aimed to validate ddPCR for predicting molecular relapse. This is a prospective, multicenter study. We enrolled patients treated with TKIs for at least 3 years with a confirmed sustained deep molecular response (DMR) for at least 1 year. TKI was re-administered in patients who experienced the loss of major molecular response (MMR). A total of 66 patients from five institutions in South Korea were enrolled. During a median follow-up period of 16.5 months, 29/66 (43.9%) patients experienced molecular relapse; the probability of molecular relapse-free survival (RFS) at 6 or 12 months after TKI discontinuation was 65.6% or 57.8%, respectively, with most molecular relapses occurring within the first 7 months. All patients who lost MMR were re-treated with TKI, and all re-achieved MMR at a median of 2.8 months. E14a2 transcript type (p = 0.005) and longer DMR duration (⩾48 months) prior to TKI discontinuation (p = 0.002) were associated with prolonged molecular RFS and with sustained DMR. Patients with both e13a2 transcript type and detectable BCR::ABL1 (⩾MR5.0) by ddPCR at the time of TKI discontinuation showed shorter duration of molecular RFS (p = 0.015). Our data suggest that transcript type and BCR::ABL1 transcript levels on ddPCR should be taken into consideration when deciding whether to discontinue TKI therapy.

The Pattern of Chromosome Aberrations and Molecular Markers in the Population of Hematological Patients Diagnosed at the University Clinical Center Tuzla

Introduction: Besides cardiovascular, malignant diseases are one of the leading causes of death in Bosnia and Herzegovina. At the top of this list are hematological diseases. This research aimed to identify cytogenetic and molecular biomarkers in patients treated for different types of hematological neoplasms. Methods: The retrospective study included 1600 samples of patients with different hematological diseases in the period from January 2006 to May 2022. The Polymerase Chain Reaction (RT-PCR) method was used to determine the presence of genetic rearrangements and to confirm the findings of conventional cytogenetic analysis. Results: Chromosomal aberrations were found in 739 (46,18%) patients. Using the RT-PCR technique, positive cases were increased by 1,5%. The BCR-ABL fusion gene was present in e14-a2 transcript form in 73% of samples, e13-a2 isoform in 21%, e1-a2 in 2%, while e14-a2/e1-a2 transcript coexpression was present in a percentage of 4% of the samples. The PML-RARa fusion gene was found in the form of bcr 1 transcripts in 21%, bcr2 32% and bcr3 59% of the samples. In twelve cases A type of the CBFB-MYH11 fusion transcript was detected. The MLL-AF4 fusion was found in only one case. Conclusion: The obtained percentages of frequency of individual molecular gene isoforms are in accordance with the results of most other researchers. This refers to the Balkan population and the Caucasian ethnic group.

Chip-Based Digital PCR Platform, Lab on an Array, the Newly Developed Highly Accurate and Sensitive Method for the Detection of BCR-ABL1 Transcripts in Chronic Myeloid Leukemia

Introduction: With the development of various BCR-ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable hematologic malignancy and current therapeutic goal is treatment free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) has become one of the prerequisite requirements. Since undetectable BCR-ABL1 by conventional TaqmanTM-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of CML clone, more precise methods should be developed. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and various sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods such as nanofluidic or droplet dqPCR have been developed for accurate and precise detection of various genes and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods may have limitations due to partition-based amplification and end-point interpretation. Therefore the establishment of clinical guideline is crucial for wide application on TFR in CML. Therefore, we have developed a novel technology, a chip-based real-time dqPCR platform (LOAA; Lab On An Array; so called Dr. PCR™) to achieve more stable, accurate and precise monitoring of BCR-ABL1 in a timely manner. Methods: To assess the equivalence between Dr. PCR and conventional qPCR, we evaluate the efficacy of BCR-ABL1 detection using cell lines and clinical specimens. For linearity assessment, we used BCR-ABL1 positive (e14a2) K562 cell line diluted to 9 points by 10-fold serial dilutions from 100 to 10-8 with BCR-ABL1 negative HL60 cells. With the results from cell lines, we also evaluated linearities for both e13a2 and e14a2 major BCR-ABL transcripts using 7 clinical specimens obtained from Leukemia Omics Research Institute of Eulji University. In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR. Total 800ng of RNA per test was used for qPCR (QS5, Thermo Fisher, USA) with 8 replicates and duplicate for cell line and clinical specimens, respectively. For Dr. PCR (1-step reverse transcriptase PCR), 400ng (for cell lines) or 500ng (for clinical specimens) of RNA per test was applied to disposable cartridges of Dr. PCR™ BCR-ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). The copies of BCR-ABL1 and ABL1 were analyzed by LOAA analyzer S/W (Optolane Technologies, South Korea). Based on the results, correlation analysis between qPCR and Dr PCR was performed using correlation graphs and Blend-Altman plots. Results: Correlation analysis for cell line dilution assay showed that measured values from both qPCR and Dr. PCR were closely correlated with the R2 of 0.984. LOA95 (limit of agreement at 95% confidence interval) analyzed by Blend-Altman plot was -0.711 ~ 0.303 (mean = 0.02), indicating the two methods are closely correlated (Figure 1). The linearity assessment for e13a2 and e14a2 sub-genotypes using primary cells from 7 clinical specimens of CML patients again showed a close correlation with the R2 of 0.984 and 0.985, respectively. LOA95 values were -0.516 to 0.384 (mean = -0.066) and -0.407 to 0.361 (mean = -0.023), respectively. Interestingly, Dr PCR efficiently detected clinical specimens with low copy number of BCR-ABL1 compared to conventional qPCR, which is beneficial for the determination of MDR levels in patients especially those who are required to monitoring after cessation of TKIs. Comparison between conventional qPCR, droplet dqPCR and Dr PCR is listed (Table 1). Results with more specimens from CML patients and additional information will be updated at ASH presentation. Conclusion: Taken together, we believe our novel chip-based real-time dqPCR (Dr. PCR™) may minimize false positive results and maximize the analytical sensitivity of detecting BCR-ABL1 fusion transcripts and thus, would be useful for the selection of therapeutic option in routine clinical practice and TFR. We will include the result for further intensive study using more clinical specimens including samples of low copy number of BCR-ABL1. Study support: Funded by Korea Research FoundationFigure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A new aberrantly spliced BCR-ABL1 transcript variant (e13a1) identified in routine monitoring using different quantitative reverse transcription polymerase chain reaction techniques in a patient with chronic myeloid leukemia.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of BCR-ABL1 transcript level is an essential part of routine disease monitoring in patients with chronic myeloid leukemia. One patient sample (e13a2 transcript detected by nested PCR) attracted attention by revealing an aberrantly spliced BCR-ABL1 transcript variant e13a1. The last 38 base pairs (bp) of BCR exon 13 were replaced by a 37 bp insertion of the ABL1 intron 1-2/exon 1 sequence. The rare aberrant BCR-ABL1 fusion transcript can cause discrepancies in molecular diagnostics. This scenario highlights the importance of an individual characterization of the BCR-ABL1 fusion sequence in case of unclear qRT-PCR results.

Assessment of quantitative polymerase chain reaction for BCR-ABL1 transcripts in chronic myeloid leukaemia: Are improved outcomes in patients with e14a2 transcripts an artefact oftechnology?

The clinical outcome of chronic myeloid leukaemia patients has vastly improved since the introduction of tyrosine kinase inhibitor treatment, with a significant proportion of patients able to achieve treatment-free remission. However, studies have shown that patients with the e13a2 transcript were less likely to achieve major molecular response compared to those with e14a2 transcripts. Most quantitative polymerase chain reaction (PCR) assays for detection of the BCR-ABL1 fusion gene do not differentiate between the two transcripts and we therefore hypothesised that technical bias linked to the qPCR assay could partially explain the discrepancy in outcomes. We designed an e14a2-specific assay and identified no difference in results compared to an e13a2standard assay. We then demonstrated that the commercial e14a2standards were causing a significant overestimation of the e13a2 transcripts. Finally, we reviewed patient management after the qPCR values were corrected, using our new evaluation. We concluded that despite statistically significant differences in qPCR results, there was no impact on patient management or outcome. We conclude that, at least in our institution, it would be inappropriate to perform separate assays for patients with e13a2 or e14a2.

Clinical Effect of Tyrosine Kinase Inhibitors in the Treatment of P230 Chronic Myeloid Leukemia

To observe the curative efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of e19a2 transcript (P230) CML chronic phase (CML-CP) patients. The clinical data of 11 P230 CML-CP patients were collected from July 2008 to December 2019. Blood routine examination, bone marrow cytology, chromosome, and BCR-ABL qualitative and quantitative tests were performed at initial diagnosis. After TKIs treatment, BCR-ABL (P230)/ABL in peripheral blood was regularly detected to evaluate molecular response by real-time quantitative PCR. There were 11 patients (7 males and 4 females) in chronic phase from 6 domestic hospitals enrolled, their median age was 46 years old (range from 19 to 56 years old). Among 4 patients treated with imatinib (400 mg, qd) firstly, 3 cases switched to nilotinib (400 mg, bid) and 1 case switched to dasatinib (100 mg, qd) due to failure to achieve best molecular response at the landmark time or mutation of ABL kinase. Then major molecular response (MMR) was obtained within 1 year. In addition, 5 patients were treated with nilotinib (300 mg, bid) and 2 patients with dasatinib (100 mg, qd) as first-line treatment, all of them got MMR within 6 months. For intolerance or resistance to imatinib, second-generation TKIs can enable P230 CML patients to achieve deeper molecular response, and MMR in a short time.

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

IntroductionThe 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]MethodologyWe 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).ResultsPatients 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 responsesThe 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.ConclusionIn the patient-cohort studied there were no significant differences in molecular response based on transcript type or body weight/BMI. [Display omitted] DisclosuresNo 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.

Alterations in Sialylation Patterns are Significantly Associated with Imatinib Mesylate Resistance in Chronic Myeloid Leukemia

The study examined sialylation changes for their potential predictive value in assessment of imatinib mesylate (IM) resistance, alone and/or with BCR-ABL1 transcript variants among chronic myeloid leukemia (CML) cases. A total of 98 CML cases (un-treated cases, IM non-responders and IM responders) were enrolled in the study. Total sialic acid (TSA) and total protein (TP) levels were estimated spectrophotometrically, the expression profiles of BCR-ABL1, ST3GAL1 and ST3GAL2 were evaluated using qRT-PCR assays and BCR-ABL1 transcript variants were identified through subjecting PCR products to agarose gel electrophoresis. The results manifested increase in e14a2 transcript and decrease in co-expression of both transcripts (e13a2 and e14a2) in IM non-responders than un-treated CML cases. Notably, TSA/TP ratio was higher, whereas ST3GAL1 and ST3GAL2 expressions were lower in un-treated CML cases and IM non-responders as against IM responders. Further, ST3GAL2 expression was lower in un-treated CML cases than IM non-responders. Receiver operating characteristic curves also proved their discriminatory efficiencies. Decisively, the rise in TSA levels and the fall in ST3GAL1 and ST3GAL2 levels were evidently related to CML progression and clinical indicators of treatment failure (high BCR-ABL1 ratio, high WBC count, high platelet count and low Hb levels). The alterations in TSA, ST3GAL1 and ST3GAL2 levels were remarkably associated with each other. The altered levels of TSA, ST3GAL1 and ST3GAL2 are, to a significant extent, associated with IM resistance in CML, which have clinical relevance in treatment monitoring and IM resistance treatment.

P50-3 Treatment-free remission in e19a2 BCR-ABL1 chronic myeloid leukemia

Chronic myeloid leukemia (CML) has become a chronic disease, for which the chronic phase is manageable with tyrosine kinase inhibitor (TKI) therapy. Several TKI discontinuation studies in patients with durable deep molecular response demonstrate that stopping TKI therapy is feasible, and this situation is called treatment-free remission (TFR). In present, The NCCN guidelines and the ESMO guidelines provide guidelines for TKI discontinuation. In these guidelines, quantifiable BCR-ABL1 transcript and monitoring using international scale (IS) are involved in the criteria of TKI discontinuation. Because IS can quantify only e13a2 or e14a2 transcripts, other transcript variants does not match the TFR criteria. Here we describe an e19a2-positive CML patient who maintain TFR. A 77-year old woman was diagnosed e19a2-positive CML, and achieved deep molecular response by nilotinib. Since 66 months of nilotinib treatment, e19a2 transcript has been undetected. Since 84 months of nilotinib treatment, she has refused subsequent medication for CML and therefore careful monitoring by QT-PCR has been done. By 40 months since nilotinib discontinuation, she has been well without any sign of withdrawal syndrome and e19a2 transcript has been undetected. In literature search, there is no report of the e19a2-positive CML who obtained TFR. As in the case we presented here, TFR may be achievable in patients with transcript variants other than e13a2 or e14a2. The problem to be solved is the standardization of transcript quantification. Both the ELN guidelines and the NCCN guidelines are created on the premise that they are monitored by the IS method which can only use for e13a2 or e14a2 transcript, meaning there is no treatment guidelines for other variants. Establishing standardized quantification of variant transcripts based on global clinical trials may contribute to prepare the treatment guideline including TFR as similar to e13a2 or e14a2 transcript.

Comparison of Molecular Responses between e14a2 and e13a2 BCR-ABL1 Transcripts in Patients with Chronic Myeloid Leukemia in Chronic Phase Treated with Front-Line Second Generation Tyrosine Kinase Inhibitors: Taiwan CML Study

Background and Purpose: e14a2 (b3a2) and e13a2 (b2a2) are the two major BCR-ABL1 transcripts of CML. Our previous study (EHA 2016, abs P609) showed that patients with e13a2 transcripts received imatinib as front-line therapy had a significant higher treatment failure rate at 6 and 12 months, a lower cumulative incidence of MMR by 2 years, a higher frequency of lymphoid crisis, and a trend of inferior progression-free survival (PFS) as compared to those with e14a2 patients. The prognostic relevance of the two transcript subtypes following front-line second generation tyrosine kinase inhibitors (TKIs) therapy remains to be determined. We aimed to compare the molecular responses between e14a2 and e13a2 transcripts and their impact on outcomes of CML patients treated with front-line nilotinib or dasatinib.Patients and Methods: Between March 2008 and April 2017, there were 260 newly diagnosed CML patients in chronic phase with age older than 18 years, received 2nd generation TKIs as front-line therapy. BCR-ABL1 transcripts included 170 e14a2, 67 e13a2, 19 both e14a2 and e13a2, 2 e1a2, and one each of e14a3 and e13a3. The 237 patients carrying either e14a2 or e13a2 transcripts but not both were enrolled in this study, 143 patients received nilotinib and 94 patients received dasatinib. The e14a2 subtype accounted for 76.9% (n = 110) in the nilotinib-treated group and 63.8% (n = 60) in the dasatinib-treated group. The BCR-ABL1 levels expressed as International Scale (IS) were measured by TaqMan RQ-PCR assay with the lab-specific conversion factor every 3 months in a central laboratory.Results: Of the 237 patients with e14a2 or e13a2 transcripts, there was no difference in age, gender and Sokal score between the two transcript subtypes in each treatment group. In the nilotinib-treated group, patients with e14a2 achieving optimal molecular responses based on the ELN definition at 3, 6, and 12 months were significantly higher than those with e13a2 subtype, and had a significantly lower failure rate at 6 months (P < 0.001). In the dasatinib-treated patients, no difference was observed between the two transcript subtypes in the molecular responses at these early landmark time points. The median time to MMR or MR4.5 wasnotdifferent between the two transcript subtypes in both treatment groups. With a median follow-up of 27.5 months in the nilotinib-treated patients, 76% of e14a2 patients achieved MMR compared with 49% of e13a2 patients (P= 0.004) whereas no difference was observed for the frequency of MR4.5 between the two subtypes; 5 patients had disease progression and died. With a median follow-up of 20.8 months in the dasatinib-treated patients, there were no differences in MMR and MR4.5 rates, disease progression occurred in 4 patients and 3 of them died. In patients treated with nilotinib, the cumulative incidence of CCyR by 12 months and MMR by 24 months were significantly lower in e13a2 subtype compared with e14a2 subtype (P= 0.022, Fig. 1A and P= 0.037, Fig. 1B, respectively) but no difference in the cumulative incidence of MR4.5 between e14a2 and e13a2 by 36 months (P= 0.434). There were no difference in the cumulative incidence of CCyR by 12 months (P= 0.658, Fig. 1C), MMR by 24 months (P= 0.735, Fig. 1D) and MR4.5 by 36 months (P= 0.871) between the two transcript subtypes in the dasatinib-treated group. PFS and overall survival (OS) were not different between the two transcript subtypes in both nilotinib- and dasatinib-treated groups. Sokal risk group did not influence molecular responses, PFS or OS with respect to transcript subtypes in both treatment groups. The molecular responses and outcomes of e14a2 and e13a2 CML patients treated with front-line nilotinib and dasatinib are summarized in Table1.Conclusions: The results of Taiwan CML study demonstrated that patients with e13a2 transcripts had a significantly inferior early molecular responses compared with e14a2 when treated with front-line nilotinib. There was no difference in molecular responses between the two transcripts in patients treated with dasatinib. Sokal risk group had no impact on the treatment responses, irrespective of transcript subtypes. [Display omitted] DisclosuresShih:Novartis (Taiwan) Co., Ltd.: Research Funding.

An e13a2 Type of BCR-ABL Transcript Has a Significant Adverse Impact on the Achievement of a Sustained Deep Molecular Response and on the Maintenance of a Treatment Free Remission after Stopping Tyrosine Kinase Inhibitors

An e13a2 Type of BCR-ABL Transcript Has a Significant Adverse Impact on the Achievement of a Sustained Deep Molecular Response and on the Maintenance of a Treatment Free Remission after Stopping Tyrosine Kinase Inhibitors

E14a2 and e14a2+e13a2 BCR-ABL Transcripts Are Associated with Earlier Cytogenetic and Molecular Response Rates in CML-CP Treated with Imatinib

Background: The prognostic significance of BCR-ABL1 transcripts in chronic myeloid leukemia (CML) is still controversial. Methods: All consecutive CML patients in chronic phase treated with Glivec between January 2010 and December 2011 and generic imatinib between January 2015 and February 2017 from 8 Brazilian centers were analyzed. BCR-ABL1 transcripts were evaluated by multiplex qualitative RT-PCR. Only patients with BCR-ABL transcripts e13a2 and/or e14a2 were included in this analysis. All patients started imatinib less than six months from diagnosis. Study data were collected and managed using REDCap electronic data capture tools. Demographic data were collected at diagnosis: age, gender, blood cell counts, Sokal, Hasford and EUTOS score, cytogenetics and BCR-ABL transcript type. The definition of the responses followed the European Leukemia Net 2013 criteria. Event-free survival (EFS) was measured from starting of treatment until loss of complete hematologic remission, loss of complete cytogenetic response (CCyR), loss of major (MMR), progression to accelerated (AP) or blast phase (BC), or death from any cause at any time after initial therapy. Overall survival (OS) was measured from starting of imatinib until last follow-up or date of death from any cause at any time. Progression-free survival (PFS) was measured from date of imatinib starting to transformation to AP or BC or deaths while on therapy. SPSS 21.0 software (IBM Corp., Armonk, NY, USA) was used for chi-square, t-test, ANOVA, when adequate, considering p-value <0.05 as significant.Results191 CML-CP patients treated with imatinib (121 Glivec and 70 generic imatinib) were analyzed. Patient's characteristics are described in Table 1. The median age of patients was 46 (18-89) years. Sokal score: 52.1% low; 33.8% intermediate and 14.1% high risk. BCR-ABL transcripts: 109 (57%) presented e14a2 transcripts, 74 (38.8%) e13a2 and 8 (4.2%) both transcripts. There was no difference between the groups concerning age, gender, Hasford, EUTOS scores, whereas white blood cell counts at diagnosis was higher in patients with e13a2 transcripts (P=0.009). Higher rates of CCyR at 6 months were observed in e14a2 and both transcripts group, compared to e13a2 (58.4%, 60% and 30%, respectively - P=0.003); e14a2 group had higher rates of BCR-ABL transcripts <10% measured by real-time quantitative polymerase chain reaction (RQ-PCR) at 3 months (82.9%, 85.7% and 59.3%; P=0.007) and BCR-ABL <1% at 6 months (78.4% e14a2; 61.7% e13a2 and 43% both transcripts; P=0.039). The groups presented similar OS (89 %, 87% and 83%, P=0.49) and PFS (88%, 84% and 71%, P=0.08). EFS was inferior in the e13a2 group (45% vs. 65% e14a2 vs. 54% both transcripts; P=0.02).Conclusion: Patients with BCR-ABL transcripts e14a2 presented higher rates of CCyR at 6 months and higher rates of optimal molecular response at 3 and 6 months when compared to e13a2 transcripts, but there was no difference in overall and progression-free survival by 5 years. DisclosuresPagnano:Bristol-Meirs Squibb: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Amgen: Consultancy; Novartis: Consultancy.