ABL Transcripts Research Articles

Chronic Myeloid Leukemia Presenting with Megakaryocytic Blast Crisis: a Case Report with a Favorable Outcome

Chronic myeloid leukemia (CML) belongs to the chronic myeloproliferative neoplasms, a clonal disorder of pluripotent hematopoietic stem cells, arising because of a hallmark genetic anomaly- reciprocal translocation between chromosomes 9 and 22, known as the Philadelphia chromosome, which generates the BCR::ABL fusion gene. The disease can have a biphasic/triphasic evolution, comprised of an accelerated phase and a terminal blast phase (usually biphasic when blastic phase is lymphoid in nature). Most cases present during the chronic phase of the disease, with only 10% of cases presenting in the blast phase. Megakaryocytic blast crisis is an unusual form of presentation, accounting for less than 3% of cases and it commonly carries an unfavorable prognosis compared to the classic myeloid, lymphoid or even mixed lineage blast crises. Aim: To bring forth an unusual case of CML presenting with megakaryocytic blast crisis and the therapeutic challenges associated with this particular presentation, unexpectedly ending with a favorable outcome. Matherial and methods: we hereby present the case of a 50 year old female patient, diagnosed in 2015 with CML- megakaryocytic blast crisis, who underwent treatment with a tyrosine kinase inhibitor (TKI)- imatinib and intensive chemotherapy regimen 3+7, without satisfactory response. A second induction chemotherapy regimen followed (MEC), alltogether with the same TKI and cytoreductive treatment (hydroxyurea/HU), but still without achieving a complete remission. Thus, the TKI was switched to dasatinib and complete normalisation of blood counts followed (including thrombocytes). However, there was still inadequate molecular and cytogenetic response. Therefore, TKI dosage was increased and after 4 months the patient achieved complete remission, with no detectable BCR::ABL transcript. She spent 9 years on the same TKI until important pleural effusion developed, which required switching to another 2nd generation TKI- bosutinib, based on the patients age, genetic profile and commorbidities. She is still in complete remission without detectable BCR::ABL transcript/Philadelphia chromosome to this day (2024). Results and conclusions: this is the case of a rare presentation of CMLmegakaryocytic blast crisis, who underwent both TKI and intensive chemotherapy induction regimens, marked by a favorable unexpected outcome.

Abstract 341: Co-detection of BCR::ABL1 common and atypical fusion and mutation using a novel NGS assay, Dup-Seq BCR-ABL1

Abstract The clinical management of CML and Ph+ ALL patients requires the identification of BCR::ABL1 transcript variants and drug resistance mutations. Currently these assessments need to be performed using separate assays resulting in higher sample requirements, longer turnaround times and higher costs. In this study a custom NGS assay (Dup-Seq BCR-ABL1) was developed and validated that enables co-detection and identification of transcript variants (common and atypical) and resistance mutations. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification of BCR-ABL1 from a single source of the 1st strand cDNA initiated by ABL1 specific priming. The custom data analysis pipeline enables overlapped mutation calling from duplicates as well as transcript determination. The assay minimizes the low-level artifacts that can be seen with other NGS-based BCR::ABL1 assays. This study demonstrates that the Dup-Seq BCR-ABL1 assay achieves high accuracy (PPA for fusion: 0.985; PPA and NPA for mutation at 0.978 and 0.9999, respectively) and sensitivity (LoD for mutation detection at 3% from 10,000 copies of BCR-ABL1 input). The use of the Dup-Seq BCR-ABL1 assay for both transcript variant identification and mutation detection provides comprehensive and accurate results for the clinical management of CML and Ph+ ALL patients while reducing sample requirements, testing costs and turnaround time. Citation Format: Jin Li, Zhenyu Yan, Lin Shi, Wei Li, Weihua Liu, Cynthia Spittle, Chad Galderisi. Co-detection of BCR::ABL1 common and atypical fusion and mutation using a novel NGS assay, Dup-Seq BCR-ABL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 341.

The Screening of microRNAs in Chronic Myeloid Leukemia: A Clinical Evaluation.

Chronic myeloid leukemia (CML) is a type of leukemia whose main genetic marker is the reciprocal translocation that leads to the production of the BCR::ABL1 oncoprotein. The expression of some genes may interfere with the progression and development of leukemias. MicroRNAs are small non-coding RNAs that have the potential to alter the expression of some genes and may be correlated with some types of leukemia and could be used as biomarkers in the diagnosis and prognosis of patients. Therefore, this project carried out an analysis of microRNA-type plasma biomarkers in patients with chronic myeloid leukemia at unique points, including follow-up analysis of patients from the Erasto Gaertner Hospital. 35 microRNAs were analyzed in different cohorts. Inside those groups, 70 samples were analyzed at unique points and 11 patients in a follow-up analysis. Statistically different results were found for microRNA-7-5p, which was found to be upregulated in patients with high expression of the BCR::ABL1 transcript when compared to healthy controls. This microRNA also had evidence of behavior related to BCR::ABL1 when analyzed in follow-up, but strong evidence was not found. In this way, this work obtained results that may lead to manifestations of a relationship between miR-7-5p and chronic myeloid leukemia, and evaluations of possible microRNAs that are not related to this pathology.

Chronic myeloid leukemia with e6a2 fusion gene: a case report and literature review

Chronic myeloid leukemia (CML) with e6a2 transcript type is very rare in clinic,which is usually related to disease aggressiveness. Its clinical characteristics and relationship with tyrosine kinase inhibitor efficacy are still unclear. In this paper, the clinical characteristics and related laboratory tests of a patient with e6a2 fusion gene positive CML characterized by multiple osteolytic bone destruction throughout the body and eosinophil infiltration in gastrointestinal tract, lymph nodes and other organs were retrospectively analyzed, and the relevant literature was reviewed. The patient was Ph chromosome positive with chromosome +8, and the common BCR::ABL1 transcript of CML was negative, but e6a2 transcript was positive detected by RT-PCR. The patient was treated with dasatinib 100 mg/d. Three months later, the patients achieved CHR, CCyR and MR4.0. However, the e6a2 transcript is very rare in clinical practice, and more cases of e6a2 transcript need to be studied to clarify its clinical characteristics and improve the treatment effect of these rare cases.

European Stop Tyrosine Kinase Inhibitor Trial (EURO-SKI) in Chronic Myeloid Leukemia: Final Analysis and Novel Prognostic Factors for Treatment-Free Remission.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.The European Stop Kinase Inhibitors (EURO-SKI) study is the largest clinical trial for investigating the cessation of tyrosine kinase inhibitors (TKIs) in patients with chronic myeloid leukemia in stable deep molecular remission (DMR). Among 728 patients, 434 patients (61%; 95% CI, 57 to 64) remained in major molecular response (MMR) at 6 months and 309 patients of 678 (46%; 95% CI, 42 to 49) at 36 months. Duration of TKI treatment and DMR before TKI stop were confirmed as significant factors for the prediction of MMR loss at 6 months. In addition, the type of BCR::ABL1 transcript was identified as a prognostic factor. For late MMR losses after 6 months, TKI treatment duration, percentage of blasts in peripheral blood, and platelet count at diagnosis were significant factors in multivariate analysis. For the entire study period of 36 months, multiple logistic regression models confirmed duration of treatment, blasts, and transcript type as independent factors for MMR maintenance. In addition to the duration of treatment, transcript type as well as blasts in peripheral blood at diagnosis should be considered as important factors to predict treatment-free remission.

Spotlight on the real-world treatment of CML pts in Germany: a retrospective survey in private oncology practices

Clinical trials in chronic myeloid leukemia (CML) are usually carried out in specialized centers whereas primary care for patients (pts) with CML is mainly provided by local oncology practices. The aim of this study was to assess treatment practices in pts with CML in the setting of private oncology practices in Germany. We collected data of 819 pts with a confirmed diagnosis (dx) of CML in 2013 or later from 43 practices. At dx, 84.2% (n=690) and 9.4% (n=77) of pts were in chronic or accelerated phase, 0.7% (n=6) had a blast crisis. Molecular monitoring was provided by EUTOS certified laboratories in 87.7% of pts. Typical BCR::ABL1 transcripts were detected in 86.6% (n=709). Molecular response was assessed after 2.8, 6.0, 9.4 and 12.9 m (mean) after start of treatment. Of the pts with available data, 11.1% did not achieve early molecular response and at 18 m, 83.7% had at least a major molecular response. 288 (35.2%) of pts switched to 2nd line (2L) treatment after a mean of 21.0 months. Reasons for 2L treatment were side effects in 43.4% and suboptimal response or failure in 31.4% of pts. 106 pts went on to third line (3L) treatment. 36.8 % of pts switched to and 92.8 % of pts still on 3L treatment achieved BCR::ABL1IS ≤1% at 12 m. In conclusion, in Germany pts with CML are routinely monitored by qPCR and good responses are achieved in the majority. Treatment changes are mainly due to adverse events rather than suboptimal responses.

Frequencies of BCR::ABL1 Transcripts in Patients with Chronic Myeloid Leukemia: A Meta-Analysis.

Chronic myeloid leukemia (CML) is associated with the Philadelphia chromosome and distinct BCR::ABL1 gene transcripts. We assessed the frequencies of these transcripts in Mexico, Latin America, and worldwide. We determined the prevalence of BCR::ABL1 transcripts in CML patients and intercontinental or regional variations using specialized databases and keywords. We analyzed 34 studies from 20 countries, encompassing 5795 patients. Keyword-based searches in specialized databases guided data collection. ANOVA was employed for transcript distribution analysis. The b3a2 transcript was most prevalent globally, followed by b2a2, with e1a2 being the least frequent. Interestingly, Mexico City exhibited a higher incidence of b2a2, while b3a2 predominated in the remaining country. Overall, no significant intercontinental or regional variations were observed. b3a2 was the most common BCR::ABL1 transcript worldwide, with b2a2 following closely; e1a2 was infrequent. Notably, this trend remained consistent in Mexico. Evaluating transcript frequencies holds clinical relevance for CML management. Understanding the frequency of transcript informs personalized CML treatments.

Beyond BCR::ABL1-The Role of Genomic Analyses in the Management of CML.

Chronic myeloid leukemia (CML) is a model of genomically based diagnosis and management where BCR::ABL1 is successfully targeted by tyrosine kinase inhibitor (TKI) therapy in most patients. The dynamics of BCR::ABL1 transcript decline during therapy is a dependable biomarker of response, relapse, and drug resistance. Missense mutations acquired within the BCR::ABL1 kinase domain that disrupt TKI binding can evolve during therapy and are frequently detected in patients for whom TKI treatment fails. Importantly, specific BCR::ABL1 missense mutations are targetable alterations and direct therapeutic decisions based on the individual mutant TKI sensitivity profile. Nevertheless, BCR::ABL1 mutations are only implicated in approximately half of the cases of acquired resistance. Furthermore, not all patients with a single BCR::ABL1 mutation that is predicted to be sensitive to a specific TKI will experience a response when switched to that TKI. Progression to blast phase heralds independence from BCR::ABL1, and this phase of the disease is notoriously difficult to treat. The independent drivers of resistance and disease progression have long been investigated to both predict progression and to find targets for therapeutic intervention. Recent data reaffirm that drug resistance and disease progression is a mutation-driven process in CML, and somatic variants in genes that are known to drive acute myeloid and lymphoid leukemia have been detected in patients in the advanced phases of CML. Genomic testing over the last few decades for patients with blood cancer has revealed of variety of genomic aberrations that drive disease. Consequently, incorporation of genomic factors into patient management for a range of blood cancers has led to the implementation of high-throughput gene testing to detect clinically actionable variants. Is it time to integrate broader genomic screening into clinical management strategies for patients with CML?

Expression of HOTAIR and PTGS2 as potential biomarkers in chronic myeloid leukemia patients in Brazil.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm in which all the patients has the translocation (9;22) that generates de BCR::ABL1 tyrosine kinase. Despite this disease possessing a good biomarker (BCR::ABL1 transcripts level) for diagnosis and prognosis, many studies has been performed to investigate other molecules, such as the long noncoding RNAs (lncRNAs) and mRNAs, as potential biomarkers with the aim of predicting a change in BCR::ABL1 levels and as an associated biomarker. A RNAseq was performed comparing 6 CML patients with high BCR::ABL1 expression with 6 healthy control individuals, comprising the investigation cohort to investigate these molecules. To validate the results obtained by RNAseq, samples of 87 CML patients and 42 healthy controls were used in the validation cohort by RT-qPCR assays. The results showed lower expression of HOTAIR and PTGS2 in CML patients. The HOTAIR expression is inversely associated with BCR::ABL1 expression in imatinib-treated CML patients, and to PTGS2 showing that CML patients with high BCR::ABL1 expression showed reduced PTGS2 expression.

Expression of genes potentially involved in loss of response in patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematological malignancy characterized by the presence of the BCR::ABL1 fusion gene, which leads to uncontrolled cell growth and survival. Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of CML, but a significant proportion of patients develop resistance or lose response to these drugs. Understanding the molecular mechanisms underlying treatment response and resistance is crucial for improving patient outcomes. This study aimed to analyze the expression patterns of genes involved in treatment response and resistance in CML patients receiving TKI therapy. The expression levels of MET, FOXO3, p15, p16, HCK, and FYN genes were examined in CML patients and compared to healthy donors. Gene expression levels were compared between optimal responders (OR) and resistant patients (R) vs. healthy donors (HD). The MET and FOXO3 OR group showed significant differences compared with the HD, (p < 0.0001) and (p = 0.0003), respectively. p15 expression showed significant differences between OR and HD groups (p = 0.0078), while no significant differences were found in p16 expression between the HD groups. FYN showed a statistically significant difference between R vs. HD (p = 0.0157). The results of HCK expression analysis revealed significant differences between OR and HD (p = 0.0041) and between R and HD (p = 0.0026). When we analyzed OR patients with undetectable BCR::ABL1 transcripts, a greater expression of HCK was observed in the R group. These findings suggest that monitoring the expression levels of MET and FOXO3 genes could be valuable in predicting treatment response and relapse in CML patients. Our study provides important insights into the potential use of gene expression analysis as a tool for predicting treatment response and guiding treatment decisions in CML patients. This knowledge may ultimately contribute to the development of personalized treatment strategies to improve patient outcomes in CML.

Correlation between BCR::ABL1 Transcript LEVEL in Circulating Extracellular Vesicles and BOTH the Molecular Response and the Ongoing Therapy: A Study on Adult CML Patients

Small extracellular vesicles (sEVs) have been shown to play a function in tumor growth and have been studied in Chronic Myeloid Leukemia (CML). Analysis of sEVs and their contents offers a non-invasive strategy that might identify messages transmitted by persisting leukemic cells. The sEVs recovered from CML patients may contain the BCR::ABL1 transcript, but its relationship to clinical and biological factors is still unclear. This study aims to connect the disease status and several clinical features with the BCR::ABL1 transcript levels in sEVs (BCR::ABL1-EV). sEVs were isolated by a commercial kit from 104 plasma samples of adult CML patients in molecular response: 18/104 (17%) in MMR and 86/104 (83%) in DMR assessed by RT-qPCR following the international scale. At sampling, 21/104 (20%) patients were treated with imatinib, 14/104 (13.5%) with nilotinib, 6/104 (5.5%) with dasatinib, 1/104 with bosutinib (1%), 27/104 (26%) with intermittent TKI therapy, and 24/104 (23%) were in treatment free remission (TFR). Taking advantage of digital PCR's (dPCR) sensitivity, it was used to detect the presence of BCR::ABL1 transcript in sEVs. The undetectable samples by RT-qPCR on cells were 38/104 (36.5%), while by dPCR on sEVs were 13/104 (12.5%). This difference resulted statistically significant (p&amp;lt;0.0001) (Fig 1A). In terms of sensitivity, dPCR was able to detect more BCR::ABL1 transcript in the sEVs than in the cells (p= 0.0052), suggesting the release of sEVS shuttling BCR::ABL1 transcript by CML cells resident in the bone marrow (Fig 1B). Considering the quantity of BCR::ABL1 transcript molecules, the BCR::ABL1-EV showed a decrease at the deepening of the MR classes and there was a significant difference between MR3.0 and MR4.5 and MR5.0 (p= 0.0226 and p=0.0101, respectively) (Fig 1C). By gathering overall samples according to MMR and DMR definition, the BCR::ABL1-EV resulted statistically different (p= 0.0017), presenting an increased significance when compared with the analysis on the different MR classes (Fig 1D). A significant difference between BCR::ABL1-EV and the ongoing therapy was observed. For this analysis, nilotinib, dasatinib, and bosutinib were considered together as “2 nd generation TKI”. The BCR::ABL1-EV were significantly higher in samples from patients in intermittent therapy compared to those from patients treated with imatinib (p=0.0398). No statistical difference resulted by other comparisons, even if a trend can be appreciated (Fig 1E). This evidence may be due to the therapy duration, since the mean therapy duration is 111 months and 45 months for imatinib and 2 nd generation TKI, respectively. Despite that, no linear regression between therapy duration and BCR::ABL1-EV. Another fascinating hypothesis is that the difference could be related with mechanisms activated by the different drugs. Further analysis will clarify this point. Interestingly, when comparing the results obtained by dPCR on cells and sEVs, a significant difference was observed in samples of patients under intermittent treatment. A trend can be appreciated in the other conditions, with except for imatinib (Fig 1F). In conclusion, dPCR associated with sEVs analysis confirmed its higher sensitivity to detect even low levels of target and it is able to identify the presence of not-circulating leukemic cells releasing BCR::ABL1 positive sEVs. Moreover, the analysis of the BCR::ABL1 transcript on sEVs revealed a transcript reduction at the deepening of the MR classes, discriminated between MMR and DMR, and correlates with the ongoing therapeutic strategy. Further analysis will clarify the mechanisms at the bases of this correlation. This is the first report about the application of this approach on a cohort of adult CML patients, but strongly suggests how a liquid biopsy approach is able to identify active leukemic cells not otherwise detectable. It opens to the identification of different sub-sets of patients presenting different vesicular BCR::ABL1 transcript levels. The different sub-sets could be related to the therapy response as well as the eligibility to TKI suspension, two of the main hot points in the present CML patients' management.

Predictive Value of BCR::ABL1 Transcripts Kinetics in the Molecular Relapse of Chronic Myeloid Leukemia Patients after Tyrosine Kinase Inhibitors Discontinuation: Results from the DES-CML Study

Duration of previous treatment, duration and depth of molecular response, previous resistance, BCR::ABL1 transcript, and lymphocyte subtypes are known predictive factors of molecular recurrence after tyrosine kinase inhibitors (TKI) discontinuation in chronic myeloid leukemia (CML) patients (pts). Few studies explored the kinetics of BCR::ABL1 during a dose de-escalation phase before discontinuation in predicting molecular relapse. This study aimed to evaluate the kinetics of BCR::ABL1 transcripts during the de-escalation phase of the DES-CML trial and to correlate with molecular relapse after TKI discontinuation. Methods: This prospective, multicenter, single-arm, phase 2 study included CML pts treated DES-CML Study. Inclusion criteria: Adult CML pts treated with TKI for a minimum of 3 years that achieved sustained deep molecular response (DMR), defined by a minimum duration of MR4.5 for two years, confirmed by four real-time quantitative reverse-transcription polymerase chain reaction (qPCR) tests with six months interval. Exclusion criteria: pts with atypical BCR::ABL1 transcripts, previous or current advanced stage CML, previous resistance to TKI or ABL mutations. The TKI dose was reduced by 50% for six months before discontinuation (de-escalation phase). Molecular monitoring was performed by qRT-PCR, using ABL as the control gene. The results were reported using the International Scale. BCR::ABL1 transcripts were evaluated monthly during the de-escalation phase. After discontinuation, assessments were performed monthly for six months, every two months at months 7-24, and then every three months. Therapy was restarted at the same dose before dose reduction if loss of MMR (qPCR&amp;lt;0.1%). Statistical analysis was performed by using SPSS software. Rates of MRFS were calculated by Kaplan-Meier analysis, and groups were compared using a log-rank test. P values of &amp;lt;.05 were considered statistically significant. This trial is registered at ReBEC (RBR-6f5xbq; UTN code: U1111-1252-7312) and was approved by the ethics committees of the participating centers. All pts signed informed consent. Results: This study is ongoing. Forty-one pts were enrolled from September 2020 to July 2023, with a median age of 61 years (23-85), 60% men, 33% Sokal low risk, 50% intermediate, 17% high; 78% with b3a2 BCR::ABL1 transcript type; 85% using imatinib, 7.5% dasatinib, 5% nilotinib, and 2.5% bosutinib. For 3 pts, it was the second attempt at stopping TKI. The median duration of TKI treatment until discontinuation was 11 years (3-20). The cutoff date was July 2023, with a median follow-up of six months (1-26) after TKI discontinuation. One patient had a screen failure; another was enrolled but has not started the de-escalation phase yet. Five patients are in the de-escalation phase, and 25 are in the discontinuation phase. Two patients did not complete the de-escalation phase due to a lack of medication and went to the discontinuation phase. One patient died from causes unrelated to CML in the discontinuation phase and was censored in the MRFS analysis. 15/33 (45%) patients lost MR4.5 at any time in the de-escalation phase, and 4/15 (31%) lost MMR after discontinuation. Three of the 18 patients who did not lose MR4.5 during de-escalation relapsed (16%, P=0.05). The two patients who did not undergo de-escalation were censored in this analysis. Nine pts lost MMR: one during de-escalation and 8/39 (20%) after discontinuation. The median time between discontinuation and loss of MMR was two months (0-10). The median time to MMR recovery was 1.5 months (1-5). The overall MRFS was 72% (95% CI: 56-88%)(Figure A) and 81% in the first-attempt discontinuation group. All pts in the second attempt relapsed (3 patients) (P&amp;lt;0.0001). There was no difference in MRFS by gender, BCR::ABL1 transcript type, Sokal, and ELTS. Molecular response status at discontinuation: 23 (74%) had MR4.5, 6 (19.5%) MR4.0, and 2 (6.5%) MMR. Patients with MR4.5 at discontinuation had a higher MRFS than those in MR4.0 and MMR (78% vs. 62% vs. 0%, respectively, P=0.012)(Figure B). No patient lost hematologic response or had disease progression. Conclusions: Loss of MR4.5 during the de-escalation phase or molecular status not in MR4.5 before discontinuation was predictive of subsequent loss of MMR. Treatment-free remission was not successful in the second attempt of discontinuation.

Highly Sensitive Chip-Based Digital PCR Platform for Quantitative Detection of BCR::ABL1 Transcripts throughout CML Treatment

Introduction: With various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease 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) is one of the prerequisite requirements. Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of the CML clone, it requires the development of more sensitive technologies. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and variations in sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods require the inclusion of one or more negative partitions to calculate the concentration using Poisson distribution. Consequently, this leads to a more than a 10-fold decrease in the maximum detectable concentration in a single test compared to conventional qPCR. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (so called HDR-drPCR) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner. Methods: For linearity assessment, we used BCR::ABL1 positive (e14a2) K562 cell line diluted to 7 points by 10-fold serial dilutions from 25 to 2.5x10 -5 %IS with BCR::ABL1 negative HL60 cells. In HDR-drPCR, the GUSB gene was employed as a control gene to determine %IS values. For this purpose, the 1 st WHO international genetic reference panel was utilized to calculate the correction factor (CF). With the results from cell lines, we also evaluated equivalence between conventional qPCR and HDR-drPCR using 219 clinical specimens. Among them, 100 samples were determined as DMR (&amp;lt;0.01%IS) based on the qPCR test results 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. For HDR-drPCR (1-step reverse transcriptase PCR), 10 6 to 10 7 copies per reaction of GUSB gene were applied to disposable cartridges of Genotizer™ HDR BCR::ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). Based on the results, correlation analysis between qPCR and HDR-drPCR was performed using correlation graphs and Blend-Altman plots. Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R 2 = 0.9972) at the range of BCR::ABL1/GUSB ratio of 10 1 to 10 -5(%). Notably, HDR-drPCR technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS) (Figure 1A). The correlation analysis for 209 clinical samples showed that measured values from both qPCR and HDR-drPCR were closely correlated with the R 2 of 0.973 1 (Figure 1B) . The Bland-Altman plot showed a mean bias of 0.159, and the limit of agreement at the 95% confidence interval (LOA95) was calculated as -0.387 to 0.705. Furthermore, the concordance rate between the two methods was 96.17%, as measured by the proportion of samples in which the difference between the two methods was within a factor of 5. This indicates a strong concordance between the two methods. HDR-drPCR reliably detected BCR::ABL1 transcripts in all 90 clinical specimens measured below 0.01%IS (DMR) using qPCR. Additionally, HDR-drPCR detected trace amounts of BCR::ABL1 transcripts in 6 out of 10 specimens that were not detected by qPCR, showcasing its improved sensitivity compared to the conventional qPCR method. Conclusion: Taken together, we believe our high dynamic range real-time digital PCR(HDR-drPCR) may minimize false positive results and maximize the analytical sensitivity of detecting BCR::ABL1 fusion transcripts and thus, would be useful for accurately assessing treatment in routine clinical practice and facilitating the application of treatment-free remission (TFR). We will include these results in further intensive studies focusing on DMR clinical specimens Study support: Funded by Korea Research Foundation

T rial of Imatinib after Ponatinib Induction (TIPI) in the Front-Line Treatment of Chronic Phase (CP) Chronic Myeloid Leukemia (CML) Setting. Report of the First Therapeutic Sequence

Introduction: The prevention ofdisease transformation which typically occurs during the first years of treatment, is one of the critical goals of the treatment of CP-CML with TKI. In addition, treatment free remission (TFR) represents now another major challenge. In vitro data show that ponatinib is one of the most powerful inhibitors of wild-type as well as mutated BCR::ABL1 TK and this has been translated in heavily pre-treated CML patients (pts) intolerant or resistant to other compounds. In this trial we assessed the safety and efficacy of a 6-months debulking strategy using ponatinib as front-line therapy in newly diagnosed CP-CML, followed by a maintenance with imatinib, in order to bring a high proportion of pts in TFR. Methods: This open-label phase II national academic trial (Clinical Trial: NCT04070443) enrolled newly diagnosed adult CP-CML pts ≤65 years, with a major BCR::ABL1 transcript and no significant underlying cardio-vascular disease, uncontrolled hypertension or diabetes with target organ damage, QTc prolongation and with adequate organ functions. They were stratified according to the ELTS score. The primary endpoint is the impact of ponatinib induction on the rate of pts reaching TFR criteria as defined by our group (Rea et al. Cancer, 2018) at M36. Secondary endpoints were the safety and clinical activity of this strategy, ponatinib pharmacokinetics (PK), QoL under both inhibitors and compliance. Pts were treated with ponatinib 30 mg QD for 6-months, followed by imatinib 400 mg QD until TFR criteria (i. e. MR4.5 ≥2 years) would been reached before M60. All molecular assessments were centralised and BCR::ABL1 transcripts were expressed in % on the international scale (IS). This analysis relies on the intention-to-treat principle. Results: One hundred and seventy-eight pts were screened, 170 enrolled and 169 treated between November 2019 and October 2022. Median age was 48 (18-65) years, 113 pts were males (67%). One pt had a cryptic Ph, and 15 (9%) pts harboured additional chromosomal abnormalities (ACA). Eighty-four (51.5%) pts had b2a2 and 99 (61%) b3a2 transcripts knowing that they could harbour both. ELTS were low for 67 (40%) pts, intermediate for 73 (44%) and high for 27 (16%). For 2 patients it could not be calculated (1 splenectomy, 1 no spleen assessment). European Society of Cardiology (ESC) score was &amp;lt;2% in 158/167 (93%) evaluable pts, 3-9% in 8 (5%) pts and &amp;gt;10% in 1 (2%) pt. One hundred and one (60%) pts never smoked, 46 (27%) were past smokers. The median follow-up at database lock was 18 (3-33) months. Only induction data are presented here. The median number of days on ponatinib is 172 (1-193) and the median dose of ponatinib delivered is 30 (16.5-32) mg daily. Overall, 135 grade 3-5 AEs occurred after a median of 51 (0-186) days: 123 (91%) grade 3, 11 (8%) grade 4 and 1 fatal AE; 49 (36%) were hematologic, and 86 (64%) non-hematologic, and 109 (81%) were considered as related to study treatment. Seven pts (5%) discontinued permanently ponatinib, and 56 (41.5%) received the full planned dose. Six (4.5%) Grade 3-5 cardiac events (1 fatal cardiac arrest) and 17 (12.5%) vascular events (15 newly hypertensive pts, 1 pulmonary embolism? 1 carotid stenosis) occurred. Their cumulative incidence is shown in Figure 1A. Digestive &amp; clinical hepato-biliary events occurred in 10 (7.5%), infections in 7 (5%), lipase elevation in 15 (11%) and liver enzymes elevation in 20 (15%) pts. Eye, skin and neurologic events occurred in less than 2% of pts. Regarding efficacy, 147 evaluable pts/158 (93%) were in CHR at M1, 115 in CCyR (70.5%) at M3 and 158/163 (97%) in EMR. The median halving time (calculated with ABL1 as a reference gene) was 13.5 (11.5-17.5) days. The cumulative incidence of molecular response is shown in Figure 1B. Fourty-seven pts over 157 assessable (30%) were in deep molecular response at M6. One (0.6%) pt transformed into myeloid blast crisis, alive in CR before transplant. PK data and their relationship with safety and efficacy will be presented. Univariate analysis identified the halving time as the only factor impacting on MR4.5 at M6. Conclusions: Ponatinib displays high anti-leukemic activity as front-line therapy in selected newly diagnosed CP-CML pts with high early molecular response rates with non-negligible severe -in particular cardio-vascular- toxicity at 30 mg QD. Whether or not this will be translated into substantial TFR rates will be determined at later time points.

Olverembatinib (HQP1351) Combined with Chemotherapy Is an Effective and Safe Treatment in Patients with Philadelphia Chromosome-Positive (Ph +) Acute Lymphoblastic Leukemia (ALL) and Chronic Myeloid Leukemia in Lymphoid Blast Phase (CML-LBP) That Failed TKI-Based Regimens

Background The prognosis of relapsed or refractory (R/R) Philadelphia Chromosome-Positive (Ph+) acute lymphoblastic leukemia (ALL) and TKI-resistant chronic myeloid leukemia in lymphoid blast phase (CML-LBP) remains poor, with lower rates of complete remission (CR) and shorter durations of response. Olverembatinib is a novel third-generation tyrosine kinase inhibitor (TKI) that has been demonstrated to effectively target a wide range of BCR::ABL1 kinase mutations, especially T315I, in patients with CML in the chronic or accelerated phase resistant to either imatinib or second-generation TKIs. However, data on olverembatinib-based regimens in R/R Ph+ ALL and CML-LBP are limited. Here, we report the efficacy and safety of olverembatinib, combined with chemotherapy, in patients with Ph+ ALL or CML-LBP that failed prior TKI-based therapies. Methods A retrospective study was conducted in adults with Ph + ALL or CML-LBP who failed imatinib- or second-generation TKI-based chemotherapy and subsequently received olverembatinib-based chemotherapy. Per NCCN 2023 guidelines: refractory disease was defined as CR not achieved at the end of induction; relapsed disease, reappearance of blasts in the blood or bone marrow (&amp;gt; 5%) or any extramedullary site after CR; molecular resistance, persistent or rising BCR::ABL1 transcript (&amp;gt; 1-log) by real-time quantitative polymerase chain reaction (RT-qPCR); and complete molecular response (CMR), BCR::ABL1 transcript &amp;lt; 0.01% by RT-qPCR. The primary endpoints of this study were CR rates in R/R patients and CMR rates in molecular-resistant patients. Results From February 2022 to May 2023, 31 patients were included in this study, including 24 (77%) with TKI-based chemotherapy-failed Ph + ALL; 7 (23%) with TKI-resistant CML-LBP; 15 (48%) with R/R disease; and 16 (52%) with molecular-resistant disease. A total of 17 (57%) patients were male, and the median (range; IQR) age was 46 (20-72; 30-64) years. Among 30 patients screened for BCR::ABL1 kinase domain mutations before administration of olverembatinib, 6 (19%) had no mutations; 15 (48%) had T315I; and 9 (29%) had other mutations, including E255K (n = 2), G250E(n = 2), D241E (n = 1), Y253H (n = 1), F317L (n = 1), F359C(n = 1), and Y253Fplus M351T (n = 1). The 15 R/R patients (11, Ph + ALL; 4, CML-BP) received olverembatinib 30 or 40 mg on alternate days combined with VP (vindesine 4 mg once per week for 4 weeks and prednisone 1 mg/kg for 3 weeks and tapered at the fourth). A total of 13 (86%) patients achieved CR after 4-week induction therapy. At a median (IQR) follow-up of 8 (4-13) months, 5 patients received allogeneic transplantation in CR, and were alive in CMR. A total of 8 patients received olverembatinib-based consolidation chemotherapy of either VP (n = 4) or hyper-CVAD (n = 4) after achieving CR. Six patients relapsed, of whom 4 died, and 1 remained in CMR. The 1-year relapse-free survival (RFS) and 1-year survival rates were 52% (95% CI, 21-83) and 68% (95% CI, 37-98), respectively. Among the 16 patients with molecular resistance to TKI-based chemotherapy (imatinib [n = 4]; dasatinib [n = 9]; flumatinib [n = 3]), 2 received olverembatinib monotherapy, 7 olverembatinib plus VP, and 7 hyper-CVAD, of whom 8 received subsequent allogeneic transplantation. At a median (IQR) follow-up of 9 (5-11) months, 1 of the patients receiving olverembatinib monotherapy progressed to hematologic relapse and was switched to CAR T-cell therapy; the other patient did not achieve CMR and underwent transplantation. Of the 14 patients receiving olverembatinib plus chemotherapy, 7 (50%) achieved CMR and 3 (21.4%) progressed to relapse, of whom 1 had hematological relapse and 2 developed central nervous system leukemia (CNSL) with bone marrow remission. A total of 7 patients received allogeneic transplantation, of whom 3 achieved and 4 did not achieve CMR before transplantation. After transplantation, all the 7 patients were in CMR except one who developed CNSL. The 1-year RFS and survival rates were 67% (95% CI, 42-91) and 93% (95% CI, 79-100), respectively. Among all patients, hematologic adverse events were readily manageable. Treatment-related nonhematologic severe adverse events were observed in 3 patients, including (each) stable angina pectoris, severe pneumonia, and fatal Klebsiella sepsis. Conclusions Olverembatinib-based chemotherapy is effective and safe in patients with R/R and molecular resistant Ph + ALL or CML-LBP.

Retrospective Study to Evaluate Probability of MR4 Maintenance Following Frontline Nilotinib Dose De-Escalation after MR4 Achievement in the Patients with Chronic Myeloid Leukemia in Chronic Phase

Introduction: Nilotinib (NIL) is a potent second-generation tyrosine kinase inhibitor (TKI) approved for the treatment of newly diagnosed chronic myeloid leukemia (CML) patients (pts) in chronic phase, but reported to increase long-term toxicity such as cardiovascular (CV) risks. The ENESTfreedom study (Leukemia 2021) has reported 55% of treatment-free remission (TFR) rate after front line NIL discontinuation with a median 43.5 months of NIL treatment duration. Question still remains how to improve TFR rate after NIL frontline therapy. A potential solution is dose de-escalation (DESC) after MR4 achievement which could reduce the CV risk while extending MR4 duration prior to TFR attempt, thus increasing the chance of successful TFR attempt. DESTINY trial (Lancet Haematology 2019) had attempted TKI dose DESC to half of the standard dose for 12 months before stopping TKI therapy, reporting only 3 pts (2.4%) lost molecular response after TKI dose DESC in 125 pts who achieved molecular response with 4 log or deeper (MR4). However, 85% of this cohort is mainly treated with imatinib, limiting applying this experience in NIL treated pts. Accordingly, the present study attempted to evaluate the efficacy of NIL dose DESC following MR4 achievement based on real-world experience. Patients &amp; Method: We have retrospectively analyzed the outcome of adult pts with CML in chronic phase treated with NIL frontline therapy and achieved sustained MR4 ( BCR::ABL1 transcript ≤0.01%IS). From the pts treated with NIL frontline therapy, we identified the cases attempted NIL de-scalation after achievement of MR4. The molecular response after NIL de-escalation was closely monitored every 3 months using BCR::ABL1 qPCR. Molecular recurrence was defined as an increase in BCR::ABL1 transcript levels above MR4 on at least two consecutive occasions or a single increase in BCR::ABL1 transcript level above MR3 ( BCR::ABL1 transcript ≤0.1%IS). At any time, pts who lost MR3 had returned Nilotinib dose to 300 mg BID. Primary endpoint of the study was event-free survival (EFS) following NIL de-escalation, which was defined from NIL DESC starts to molecular recurrence or death from any cause. Results: We have identified 52 pts who started NIL as first line therapy for CML-CP under clinical practice. With median follow-up duration of 9 years (range:2-12 years), 33 patient (64%) remains on NIL at the last follow-up visit. In 33 pts on NIL treatment, with a median follow up duration of 8 years (range;2-12), 25 (76%) had attempted NIL DESC at median of 6 years (range; 1-10 years) after frontline NIL therapy start, of whom the median time from NIL start to MR4 achievement was 12 months (range: 5-50 months). The median time from first achievement of MR4 to NIL DESC starts was 50 months (range: 3-105 months), while the median time from NIL start to NIL DESC was 77 months (range: 13-121 months). At the latest follow up, only one patient experienced molecular recurrence at 3 months after DESC but regained MR3 in 9 months after re-escalation of NIL to 300mg BID. One patient died of cardiac arrest at 80 months after NIL DESC with other comorbidities of coronary artery disease, hypertension and diabetes. One patient discontinued NIL at 51 months post DESC and has sustained MR4 till last follow up. In the DESC group, the EFS at 3 years, 95% (69.5-99.3%). There is another case developed myocardial infarction while on DESC dose of NIL, who switched the treatment to Asciminib. Conclusion: Achieving early and sustained MR4 with frontline NIL treatment facilitates DESC approach without compromising molecular response and might promote the TFR rate by extending MR4 duration. This DESC strategy can bridge CML pts on NIL frontline therapy prior to NIL discontinuation for TFR attempt.

A Phase II Study of Low-Intensity Chemotherapy (Mini-Hyper-CVD) and Ponatinib Followed By Blinatumomab and Ponatinib in Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Background Patients with Philadelphia chromosome (Ph)-positive B-cell acute lymphoblastic leukemia (ALL) treated with Hyper-CVAD and ponatinib achieve deep responses and long-term overall survival (OS). We aim to investigate if a reduced intensity regimen of mini-Hyper-CVD (mini-HCVD) with sequential blinatumomab (blina) in combination with ponatinib may further improve outcomes while mitigating the toxicity of intensive chemotherapy and the need for hematopoietic stem cell transplantation (HSCT). Methods Patients age ≥18 years with newly diagnosed (ND) or relapsed/refractory (R/R) Ph-positive ALL, or chronic myeloid leukemia in lymphoid blast phase (CML-LBP) were eligible. Other inclusion criteria included adequate liver, renal and cardiac function, and ECOG performance status ≤2. Patients with baseline central nervous system (CNS) involvement were not excluded. All patients had BCR::ABL1 transcripts and fluorescence in situ hybridization (FISH) for t(9;22) done at baseline. Morphological analysis of FISH patterns was performed in ND patients to identify if the BCR::ABL1 signal was observed in myeloid cells (e.g., segmented neutrophils). Four cycles (C) of mini-HCVD alternating with methotrexate (MTX) + cytarabine were followed by blina 28µg/d given for four weeks every six weeks during C5-8. Patients received ponatinib 30mg daily initially, with a dose reduction to 15mg daily once in complete molecular remission (CMR). CMR was defined as undetectable BCR::ABL1 with an assay sensitivity of 0.01%. Rituximab was given for CD20-positive disease. Maintenance ponatinib, vincristine and prednisone for 15 cycles with blina + ponatinib every three cycles was given, followed by ponatinib for at least five years. All patients without CNS disease received 12 intrathecal injections of MTX or cytarabine. Results 20 patients (12 ND, 4 R/R, 4 CML-LBP) were treated between November 2019 and May 2023. Baseline characteristics are shown in Table 1. One patient with CML-LBP had received prior dasatinib while in chronic phase. The predominant BCR::ABL1 transcript was p190 in 7 (58%) ND patients. All patients achieved a complete remission (CR). Among patients in the ND, R/R, and CML-LBP cohorts, CMR was achieved in 10/12 (83%), 3/4 (75%), and 4/4 (100%) patients, respectively. In the ND cohort, six (50%) patients achieved CMR after C1 and eight (67%) after C3. Five (42%) ND patients had documented BCR::ABL1 signal in myeloid cells by FISH. Four of them achieved CMR, after C1, C3, C4 and C3 maintenance respectively. With a median follow-up of 25 months (range, 2-42), the 2-year continuous remission duration (CRD) and OS rates were 93% and 83% for the entire cohort (Figure 1), and 90% and 82% in the ND cohort, respectively. In the ND cohort, one (8%) patient had isolated CNS relapse (during C4 of maintenance with blina and ponatinib), three (25%) patients died (two in CR due to COVID-19 and one of HSCT complications), and eight (67%) patients are in remission without HSCT. No patients relapsed in the R/R cohort; one patient underwent HSCT, one patient died in CR from MTX-associated disseminated necrotizing leukoencephalopathy, and two patients are in remission without HSCT. None of the CML-LBP relapsed; one patient underwent HSCT in CR. Ponatinib dose was reduced in two patients prior to obtaining CMR (one had pancreatitis, one had cardiomyopathy). One patient switched from ponatinib to dasatinib due to a pulmonary embolism in C2. No patients required dose modification of blina. The 60-day mortality rate was 0%. Conclusion In patients with Ph-positive ALL, the combination of mini-HCVD and ponatinib followed by sequential blina and ponatinib yielded excellent outcomes with durable remissions. The toxicity profile was favorable and HSCT was avoided in most patients with ND disease. Longer follow-up is needed to confirm the durability of responses.

Olverembatinib Combined with Venetoclax and Reduced-Intensity Chemotherapy for Patients with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Early Results from a Phase II Study

Background The combination of olverembatinib (HQP1351), a novel third-generation tyrosine kinase inhibitors (TKIs), with venetoclax generated high response rates in patients with relapsed/refractory Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL). However, the efficacy and safety of these two agents-based regimens as frontline treatment remains unknown. Methods This is a single-arm phase II study (NCT05594784) that enrolled patients (pts) ≥ 14 years (yrs) of age with newly diagnosed Ph + ALL. Pts were required to have an ECOG performance score ≤ 2 and adequate organ function. Pts with accelerated phase or blastic phase of chronic myeloid leukemia were excluded. Pts were treated with a combination of venetoclax (100 mg d1, 200 mg d2, 400 mg d3-28), olverembatinib 40 mg once every continuously other day, vincristine 1.4 mg/m 2 (maximum dose 2 mg) on day 1, 8, 15, 22, and prednisone 60 mg/m 2 on day 1-14; 40 mg/m 2 on day 15-28 in cycle 1. In cycle 2-3, oral treatment with venetoclax 400 mg × 7 days, olverembatinib once every other day continuously and prednisone 60 mg/m 2 × 7 days was administrated in the form of outpatient treatment (Figure 1A). Cycles were repeated every 28 days. Olverembatinib 40 mg once every other day was given during cycle 1 and was reduced to 30 mg once every other day for pts achieving a complete molecular response (CMR). Routine triple intrathecal injection (methotrexate 10 mg, cytarabine 50 mg, and dexamethasone 10 mg) was performed to prevent central nervous system involvement. The selection of subsequent treatment was determined based on the patient's molecular response at 3 months and the availability of donor. The primary end point of this study was the CMR rate at 3 months. CMR was defined as undetectable BCR:ABL1 transcripts by using RT-PCR method with sensitivity of 0.001%. Major molecular response (MMR) was defined as more than 3-log reduction of BCR:ABL1 transcripts. Results From August 2022 to April 2023, a total of 31 pts were enrolled. All pts completed 3 cycles of treatment and could be assessed for the primary end point. Th data cutoff date was 25 th July 2023 with a median follow-up time of 5.8 months. The median age was 40 years (range, 20-66 years) and males accounted for 58.1%. Twenty-three pts (74.2%) expressed the p190 transcript and 8 pts (25.8%) expressed the p210 transcript. The median expression level of BCR:ABL1 was 96.33% (range, 70.79%-175.39%). All patients achieved CR at the end of cycle 1 and no tumor lysis syndrome or treatment-related deaths occurred. Molecular response at the end of cycle 1 was CMR in 17 patients (54.8%), MMR in 8 (25.8%), and less than MMR in 6 (19.4%). Molecular response at 3 months was CMR in 19 patients (61.3%), MMR in 10 (32.3%), and less than MMR in 2 (6.5%) (Figure 1B). Of the 10 pts with MMR at 3 months, 8 pts had BCR:ABL1 transcript levels fluctuating between 0.001% and 0.01%. No patients developed relapses or deaths at the last follow-up. The regimen was well-tolerated and safe. Most side effects were grade 1-2. The demand for transfusion and the incidence of infections significantly decreased compared to our historic data treating with intensive chemotherapy plus TKIs. No patient discontinued olverembatinib or venetoclax due to toxicity. Conclusion The combination of olverembatinib and venetoclax with reduced-intensity chemotherapy is a safe and effective regimen in patients with newly diagnosed Ph + ALL. The regimen results in high rates of CMR in the absence of intensive chemotherapy or immunotherapy.

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.

Outcome of Patients with Chronic Myeloid Leukemia in Chronic Phase Showing Clinical Resistance to Frontline Treatment with Second Generation Tyrosine Kinase Inhibitors. an Italian Prospective Study from the CML Campus

BACKGROUND: Frontline treatment with second generation tyrosine-kinase inhibitors (2G-TKIs) in patients with chronic myeloid leukemia in chronic phase (CP-CML) demonstrated higher efficacy as compared to imatinib therapy in inducing deeper molecular responses. However, its ability to reduce treatment failure and progressive disease is debated. Furthermore, limited information is currently available on the outcome of those CML patients showing clinical resistance to first-line treatment with a 2G-TKI. AIM: To describe the clinical outcome of CP-CML patients showing resistance to a frontline 2G-TKI and that switched to alternative TKIs therapy. PATIENTS AND METHODS: We performed this investigation on patients drawn from a web-based database (www.epiclin.it/lmc) used to collect clinical and biological data of CML patients enrolled in 24 Italian Hematology Centers from January 2013 onwards. Definitive information about first-line TKI treatment was available in 1277 prospective and consecutive CP-CML patients: 607 received imatinib and 670 dasatinib or nilotinib (2G-TKIs) as frontline therapy. Main inclusion criteria were: i) diagnosis of CP-CML; ii) first-line treatment with a 2G-TKI; iii) switch to second-line treatment in case of clinical resistance (i.e. failure according to ELN 2020 recommendations). Exclusion criteria was switch to second-line treatment for intolerance or for low adherence to therapy. Patients were treated according to the Center policy and the molecular data were obtained in accordance to the IS% parameters. RESULTS: Fifty-four CML patients met the inclusion/exclusion criteria and were identified as failure according to the ELN 2020 recommendations. The median age of these patients was 48.5 (20-81) years; male sex in 30 (55.5%) and female 24 (44.4%), Sokal risk score was low in 16 (29.6%), intermediate in 22 (40.7%) and high in 16 (29.6%) patients respectively;ELTS score was low in 26 (48.1%), intermediate in 14 (25.9%) and high in 14 (25.9%) patients. Additional chromosomal abnormalities were present at diagnosis in 4 out of 54 patients (7.4%: 3 additional Ph-chromosome and 1 trisomy 8). Nilotinib was administered in 27 (50%) and dasatinib in 27 (50%) of the patients.At 6 months, 30 out of 50 evaluable patients (60%) had BCR::ABL1 transcript values &amp;gt;1% and at 12 months 9 of 33 evaluable patients (28%) had BCR::ABL1 values still &amp;gt;1%. Second-line treatment after 2G-TKIs frontline failure started in all patients after a median time of 12 months (3-83) with ponatinib (30 pts, 55.5%), dasatinib (8 pts, 14.8%), nilotinib (4 pts, 7.4%), bosutinib (7 pts, 12.9%) or imatinib (5 pts, 9.2%).At 12 months the switch to second-line treatment induced the achievement of BCR::ABL1 values &amp;lt;1% in 45% of the evaluable cases. Eighteen patients (in which no mutations were found) switched to third-line treatment after a median time of 11 months (0-45) (ponatinib, 8 pts; nilotinib, 1 pt; dasatinib, 3 pts; imatinib, 3 pts; bosutinib, 1 pts; asciminib, 2 pts). MMR or a better response was reached by 6 of these patients and allogeneic stem-cell transplantation (SCT) was performed in 7 patients.Progression to CML advanced phase occurred in 8 pts. After a median follow-up of 59.9 months (15.6 - 99.3) 44 patients were still alive and death occurred in 10 pts (18.5%) (7 CML-related and 3 unrelated). Overall survival (OS) at 5 years was 82% (see Figure below). Indeed, OS at 5 years was 94% for the total 670 patients treated frontline with 2G-TKIs. In this population, 12 out of 32 observed deaths were CML-related and all were recorded in patients who changed TKI therapy, whereas in 19 cases the death was CML-unrelated and in 1 case was not specified. However, those patients who maintained the same therapy for 5 years showed an OS of 96%, whereas those who had to change treatment for any cause showed an OS of 89% at 5 years. CONCLUSION: Our findings, collected from the prospective cohort of the CML Italian observational registry, show that CP-CML patients failing frontline 2G-TKI therapy, despite a complex management, may have still an acceptable prognosis and survival due to the availability of both multiple TKI options and SCT.