BCR-ABL Transcript Research Articles

Long-Term Follow up for SWOG 1318: Combination of Dasatinib, Prednisone, and Blinatumomab for Older Patients with Philadelphia-Chromosome (Ph) Positive Acute Lymphoblastic Leukemia (ALL)

Tyrosine kinase inhibitors (TKIs) have improved the outcomes of patients with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Many older patients (pts) are not good candidates for intensive chemotherapy and are treated with TKIs plus corticosteroids or low-intensity chemotherapy. Although the remission rates with this approach have been high, the median disease-free survival (DFS) has been short. Therefore, novel treatment strategies are needed. This trial evaluated the feasibility of combining the TKI dasatinib with prednisone and blinatumomab in older pts with Ph+ ALL. Here we present updated results (median follow-up, 4.3 years). Methods: This trial was activated through the NCTN in January 2015 and closed to accrual in April 2021. Pt eligibility included: age ≥ 65 years; Ph+ or Ph-like ALL (with dasatinib-sensitive fusions or mutations); newly diagnosed or relapsed/ refractory; no evidence of central nervous system (CNS) disease; and adequate organ function. Treatment: For induction, pts received dasatinib 140 mg/d orally (PO) Days 1-56 along with prednisone 60 mg/m 2/d PO Days 1-24. Pts achieving complete remission (CR) or CR with incomplete count recovery (CRi) (Day 28 or Day 56) continued dasatinib until Day 84, followed by 3 cycles of post-remission therapy (PRT) with blinatumomab/ dasatinib. Pts not achieving CR or CRi by Day 56 received re-induction with blinatumomab. The response rate was assessed on Day 35 of blinatumomab. Pts not achieving CR/ CRi could receive a second cycle of blinatumomab. This was followed by 3 cycles of PRT with blinatumomab/ dasatinib. Maintenance therapy consisted of prednisone 60 mg/m 2/d x 5 days every 28 days for a total of 18 cycles, along with dasatinib 140 mg po qd indefinitely. CNS prophylaxis included intrathecal (IT) methotrexate every 4-6 weeks x 8 doses. IT methotrexate was given at least 2 days apart from blinatumomab. Response was assessed at Days 28, 56, and 84, and additional time points were dependent on response. Minimal residual disease (MRD) was assessed centrally by multi-color flow cytometry on Day 28. Statistics: A total of 24 eligible patients were accrued. The primary objective was the feasibility of the combination therapy. The combination was to be deemed feasible if the dose-limiting toxicity (DLT) was < 33%. DLT included: > Grade 3 non-hematologic toxicities (excluding nausea, vomiting, diarrhea) and Grade 4 neutropenia lasting > 42 days. The primary results were reported in 2022 with a median of 2.7 years of follow-up. Herein, we report long-term outcomes for these patients. Results: The median age was 73 years (range 65-87). All pts had newly diagnosed Ph+ ALL; 79% of Ph+ pts had additional cytogenetic abnormalities. During induction, 2 pts experienced treatment-related non-hematologic Grade 4 toxicities. No Grade 4 or higher treatment-related non-hematologic toxicities occurred during post-remission therapy or maintenance. The toxicity of the combination was deemed acceptable. Twenty-two of 24 pts (92%) achieved a CR during dasatinib and prednisone induction therapy. Four did not receive PRT (2 due to adverse events, 1 to receive a transplant, and 1 because of insurance issues). Sixteen pts who achieved CR had MRD data. Six of 16 pts (38%) were MRD undetectable by multi-color flow cytometry on Day 28. Most sites also monitored patients' blood or bone marrow with real-time quantitative PCR to quantify the BCR-ABL1 transcript for molecular response. Of 19 patients analyzed, 17 (89%) were in a major or complete molecular remission at some time point after treatment, with at least 12 of these patients achieving complete molecular remission. Four patients remain on maintenance. The median follow- up for pts who are alive is 4.3 years (range 2.6-6.5 years). The median overall survival (OS) is 6.5 years (95% CI 3-NA) and the median DFS has not been reached as of June 29, 2023. Kaplan-Meier 3-year estimates of OS and DFS are 75% (95% CI 52%-88%) and 72% (95% CI 49%-87%), respectively (Figure). Of the 8 relapses, 4 patients were CD19+ and 3 had T315I mutations. Conclusions: Median DFS and OS with dasatinib and blinatumomab in this older group of Ph+ ALL patients remain excellent despite only 1 patient proceeding to an allogeneic transplant. Further correlative studies (single cell transcriptomics and DNA methylation sequencing) are being planned to evaluate predictors of relapse and will be presented at the meeting.

Flumatinib Versus Nilotinib for Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia

Introduction Tyrosine kinase inhibitors (TKIs) have considerably improved the long-term clinical outcomes in patients with chronic phase chronic myeloid leukemia (CML-CP). Most of these patients receive 1 st generation TKI imatinib as first-line therapy; however, an increasing number of patients are now receiving 2 nd generation TKIs as first-line therapy, because of the more potent BCR-ABL1 inhibition with proven efficacy in patients resistant or intolerant to imatinib. Flumatinib is a novel 2 nd generation BCR-ABL1 TKI with promising efficacy and manageable safety in newly diagnosed CML-CP. Compared to imatinib, patients treated with flumatinib have achieved significantly higher rates of 12-month major molecular response (MMR) and complete cytogenetic response were observed in patients with CML-CP and that too within a shorter time duration. Though there have been many studies comparing 2 nd generation TKIs vs imatinib in CML-CP, studies comparing clinical outcomes between different 2 nd generation TKIs are scarce. Here, we report the real-world effectiveness and safety of flumatinib and nilotinib in patients with newly diagnosed CML-CP. Methods In this study, adult patients (≥18 years old) with newly diagnosed Philadelphia chromosome-positive (Ph+) CML-CP who received either 300 mg nilotinib twice daily or 600 mg flumatinib once daily, were included, with a follow-up duration of roughly 3 years (NCT04739826). The primary endpoint was the rate of MMR at 12 months as defined by European LeukemiaNet 2020 recommendations (BCR-ABL1 transcript level ≤0.1% in peripheral blood on RT-PCR assay on International Scale [IS]).The secondary endpoints were the rate of early molecular response (EMR) at 3 months (BCR-ABL1 IS ≤10%), the rate of molecular response at 6 months (BCR-ABL1 IS ≤ 1%) and safety (adverse events [AEs] reports in accordance to Common Terminology Criteria for Adverse Events, version 4.03). Results A total of 446 patients were enrolled in the study between November 2020 and August 2022, of which 150 and 296 patients were enrolled in nilotinib and flumatinib groups, respectively. The baseline characteristics were comparable between the two groups (Table 1). No significant difference in the rate of MMR was observed at 12 months between nilotinib and flumatinib (78% vs 80%; P=0.68) . Similarly, no significant difference was observed in the rate of EMR at 3 months (85% vs 89%; P=0.26) and the rate of optimal molecular response at 6 months (93% vs 92%; P=0.70; BCR-ABL1 is ≤1%) between the two groups. The overall safety profile was similar between the two groups. However, hyperbilirubinemia (47% vs 7%), increased ALT (33% vs 12%), increased AST (24% vs 9%), rash (30% vs 18%) and anemia (19% vs 10%) were more frequent in nilotinib group than flumatinib group, whereas diarrhea was lower in nilotinib than flumatinib (2.7% vs 8.8%). The incidence rate of grade 3-4 AEs were reported to be 13% in nilotinib group and 10% in flumatinib group. The safety of flumatinib was more favorable compared to nilotinib in terms of hepatic toxicities, skin toxicities, etc. (Figure 1). Conclusion The efficacy of flumatinib is comparable to nilotinib in Chinese patients with newly diagnosed Ph+ CML-CP. The overall incidences of grade 3-4 AEs were also accessible for the two drugs, nevertheless, a few AEs inclusively hepatic toxicity, anemia and skin toxicity were remarkably lower with flumatinib intervention.

Evaluating Effectiveness and Safety of Flumatinib for Chronic Phase Chronic Myeloid Leukemia in (CML-CP) without Optimal Response (Warning, Failure) to Imatinib or/and Dasatinib

Introduction Flumatinib is a novel second-generation BCR-ABL1 targeted kinase inhibitors (TKI) with better efficacy and safety than first generation TKIs in first-line treatment of chronic myeloid leukemia in chronic phase (CML-CP). In the previous phase III study, newly diagnosed CML-CP patients receiving flumatinib achieved higher treatment responses and safety than imatinib. When it comes to the patients not achieving the optimal response to TKIs, flumatinib is potentially a better option, nonetheless the robust evidence is unavailable. This study reports the effectiveness and safety of switching to flumatinib in patients who have not achieved the optimal molecular response to imatinib or/and dasatinib. Methods This prospective, multicentre study enrolled Philadelphia Chromosome-Positive (Ph+) CML-CP patients aged ≥18 years and who failed to achieve the optimal response with imatinib or/and dasatinib between March 2021 to October 2022. All patients were treated with flumatinib 600 mg once a day and followed up to 24 months. The primary endpoint of the study was rate of major molecular response (MMR) at 12 months as defined by European LeukemiaNet 2020 recommendations (BCR-ABL1 transcript level ≤0.1% in peripheral blood on RT-PCR assay on International Scale [IS]) and safety was the secondary endpoint assessed with CTCAE 4.03 version. In addition, the optimal molecular response in either the warning or failure patients was evaluated at 3, 6 and 12 months respectively, after switching to flumatinib, and the difference in effectiveness was also analyzed from the available mutation subtypes. Results Overall, 94 patients with Ph+ CML-CP were included in the study. The median (range) age of the patients were 53 (19.0-84.0) years old and 61.7% of the patients were males. A total of 38 (40.4%) patients reported warning response from the previous TKIs whereas 56 (59.6%) of patients reported treatment failure to the previous TKIs. Seventy-seven (81.9%) patients have been treated with one TKI, either imatinib or dasatinib, and 17 (18.1%) have been treated with both prior to the study. Overall, 25 (54%) patients achieved MMR at 12-months follow-up compared to the baseline (Figure 1). The rates of optimal molecular response at 3- and 6-months were also improved compared to the baseline. The optimal molecular response was 76(88%) at 3 months, and 52(73%) at 6 months. Subgroup analysis with previous response to TKIs revealed that flumatinib improved the rates of optimal molecular response in both warning and failure groups. Switching to flumatinib from imatinib or/and dasatinib have been reported with an enhanced optimal molecular response in patients who experienced warning compared with those who had treatment failure after the TKIs with 38 (100%) vs 38 (79%), 29 (91%) vs 23 (59%) and 15 (68%) vs 10 (42%) at 3-, 6- and 12-months respectively (Figure 2). Similarly, the rates of optimal molecular response were improved in patients without mutations compared to those having mutations, 65 (90%) vs 9 (75%) at 3 months, 46 (77%) vs 5 (56%) at 6 months and 23 (59%) vs 1 (20%) at 12 months respectively. The most common adverse events (AEs) were diarrhea in 17 (18%), rash in 11 (12%), bloating in 11 (12%), and thrombocytopenia in 11 (12%) patients. Common hematological AEs of grade 3/4 were thrombocytopenia in 7 (7%), anemia in 3 (3%) and leucopenia in 1 (1%) patients. Conclusion For Ph+ CML-CP patients without the optimal molecular response in previous imatinib or/and dasatinib, flumatinib is a better alternative to the existing treatments. In addition, if the patients were switched to flumatinib earlier, the optimal response was achieved faster. The grade 3/4 AEs were mainly hematologically related, whereas the gastrointestinal events were mostly of grade 1-2.

Multiomic Single-Cell Analysis Identifies Von Willebrand Factor and TIM3-Expressing BCR-ABL1+ Chronic Myeloid Leukemia Stem Cells

Characterization of the leukemic stem cell (LSC) population in chronic myeloid leukemia (CML) may identify therapeutic targets for sustained elimination of leukemic cells. High-throughput single-cell RNAseq (scRNAseq) has paved the way for detailed transcriptional assessment of CML LSC. Building on this technique, multiomic CITE-seq approaches carry additional benefit in that the paired protein expression information may enable identification of associated cell surface marker profiles, subsequent FACS-based isolation and functional characterization. However, detection of BCR-ABL1 transcripts, which is arguably the only unequivocal marker to distinguish CML LSC from healthy hematopoietic stem cells (HSC), remains challenging using current high-throughput 3' end capture-based scRNAseq methods. For the present study, we performed single-cell CITE-seq analysis of the expression of 597 genes and 51 proteins in >70,000 stem and progenitor cells from 16 chronic phase CML patients and five healthy donors. Furthermore, we developed and employed a strategy allowing parallel detection of BCR-ABL1 transcripts at the single-cell level. In bone marrow samples from CML patients, we observed pronounced expansion of erythroid and myeloid progenitors within the CD14 -CD34 + compartment, and substantial heterogeneity within the traditionally defined CD34 +CD38 -/low LSC compartment. In-depth analysis of the latter identified a group of immature BCR-ABL1 +cells displaying a CD45RA -cKIT -CD26 + TKI resistance phenotype sitting atop a hierarchy of myeloid progenitors. Within this group of potential LSC, expression of both previously reported ( e.g. CD25 and CD26) and unreported gene and protein markers was found to distinguish these cells from their healthy counterparts. Unlike HSC, the immature LSC showed high surface expression of TIM3 and high transcription of the von Willebrand factor gene ( VWF). While overexpression of VWF within the stem cell population may be linked to the aberrant myeloid-biased hematopoiesis characterizing the disease, the cell surface upregulation of TIM3 suggests that its targeting may have merit in CML. In conclusion, we here performed detailed multiomic characterization of CML stem and progenitor cells, paired with BCR-ABL1 detection at the single-cell level. Our findings revealed LSC-specific expression patterns that may have implications for the phenotypic definition of CML LSC. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML.

Development and evaluation of a rapid one-step high sensitivity real-time quantitative PCR system for minor BCR-ABL (e1a2) test in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL).

Minimal residual disease assessment of BCR-ABL messenger ribonucleic acid levels is crucial in Philadelphia chromosome-positive acute lymphoblastic leukemia for prognosis and treatment planning. However, accurately quantifying minor BCR-ABL transcripts, which comprise 70% of Philadelphia chromosome-positive acute lymphoblastic leukemia cases, lacks a national-approved method. We developed the "Otsuka" minor BCR-ABLmessenger ribonucleic acid assay kit with exceptional precision (0.00151%). Minor BCR-ABL messenger ribonucleic acid levels were analyzed in 175 adults, 36 children with acute lymphoblastic leukemia and 25 healthy individuals to evaluate the kit's performance. The "Otsuka" kit showed high concordance with a commonly used chimeric gene screening method, indicating reliable detection of positive cases. Quantitative results demonstrated a robust correlation with both a laboratory-developed test and a diagnostic research product. The "Otsuka" kit performs comparably or even surpass to conventional products, providing valuable insights into Philadelphia chromosome-positive acute lymphoblastic leukemia pathology. The 'Otsuka" minor BCR-ABL messenger ribonucleic acid assay kit exhibits excellent performance in quantifying minor BCR-ABL transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia patients. Our results align well with established screening methods and show a strong correlation with laboratory-developed tests and diagnostic research products. The "Otsuka" kit holds great promise as a valuable tool for understanding Philadelphia chromosome-positive acute lymphoblastic leukemia pathology and guiding effective treatment strategies.

Cytogenetic and Molecular Remission in Chronic Myeloid Leukemia in Togo

The availability of tyrosine kinase inhibitors (TKIs) in Togo through the GIPAP program has revolutionized the management of chronic myeloid leukemia and increased patient life expectancy. However, diagnosis and molecular monitoring of therapeutic efficacy remain a real challenge due to the inaccessibility of cytogenetic and molecular biology tools. We’ve conducted, a cross-sectional, descriptive study that ran from December 21, 2022, to January 20, 2023, with the aim to evaluate the cytogenetic and molecular remission in CML patients followed at the CHU Campus de Lomé in Togo. Patients diagnosed with chronic-phase CML who had been treated for at least three months with imatinib or dasatinib and had achieved complete hematological remission were included. Dosage of BCR-ABL transcript levels was performed by RQ-PCR in Seattle, USA, using Dried Blood Spot. The transcript detection limit was 0.003% i.e., MR4. A total of 38 patients were included, 68.4% of them were treated with imatinib and 31.6% with dasatinib. In terms of remission, 28.9% had not achieve a partial cytogenetic remission while 15.8% of patients had achieved a MR4 remission and 7.9% a major molecular remission. Major molecular remission was achieved at a mean follow-up time of 95 months for patients taking imatinib and 22 months for those taking dasatinib. Patients in MR4 were on imatinib after a mean of 68 months. All in one, our study showed a high rate of treatment failure. Better access to cytogenetic and molecular biology tools is needed to improve management.

Discordance between Next Generation Sequencing and BCR-ABL PCR Measurable Residual Disease in Adult Patients with Ph+ Acute Lymphoblastic Leukemia

Background: It remains unclear whether measurable residual disease (MRD) detected by next-generation sequencing (NGS) or BCR-ABL RT-PCR is more accurate in predicting relapse and guiding clinical management in patients with Ph+ acute lymphoblastic leukemia (ALL). In this multi-center retrospective study, we examine the discordance between BCR-ABL PCR and NGS MRD to better characterize practice patterns for disease monitoring for patients with Ph+ ALL. Methods: We included Ph+ ALL patients from five academic hospitals in the United States who were monitored with both BCR-ABL PCR and NGS-based clonoSEQ (Adaptive Biotechnologies) between 2015 and 2023. Patients were included if they had both baseline BCR-ABL PCR and NGS MRD assessments and at least 1 follow-up MRD assessment using both modalities. BCR-ABL PCR and NGS MRD results were only included if they were taken less than 1 month apart. Patient demographics, BCR-ABL isoform (p190 or p210), and IKZF1, CDK2NA, and PAX5 copy number alterations were also collected. We used frequency counts, median, and interquartile range (IQR) to describe our data. Univariable logistic regression was performed to quantify associations between diagnostic patient features and any post-diagnosis MRD discrepancy (i.e., MRD positive vs. MRD negative) between the two modalities. Results: 93 patients had both BCR-ABL PCR (p190 n=61 [66%], p210 n=21 [23%], both n=8 [9%]) and NGS MRD assessments. Descriptive statistics are shown in Table 1. Figure 1 is a Sankey diagram illustrating the trajectory of BCR-ABL PCR and NGS concordance at 3, 6, and 12 months following diagnosis. 60 patients (64%) had a discrepancy between qPCR and NGS at any time after Ph+ ALL diagnosis while 24 patients (26%) had a discrepancy within the first 3 months. For the 54 patients (58%) receiving transplant, 38 (70.4%) had a post-diagnosis MRD discrepancy. Among all patients, median time from diagnosis to first discrepancy was 3.9 (IQR, 2.3-7.3) months. The most common discrepancy was BCR-ABL PCR detectable / NGS undetectable (n=39, 42% of included patients). In 12 patients (20% of patients with an MRD discrepancy at any time), clinical decision making was changed due to this observed discrepancy in MRD status. While a persistently positive PCR result with negative NGS led to a change in treatment regimen for 8 of these patients (75%), one patient's positive PCR with negative NGS led to the decision to continue current therapy. Further, a positive NGS with negative PCR led to change of treatment in 3 patients. BCR-ABL isoform and copy number variation of target genes were not associated with increased risk of MRD discrepancy, although sample size was limited for the genetic alterations (Table 1). Conclusion: We found a considerable degree of discordance between BCR-ABL PCR and NGS in Ph+ ALL patients. The most common discrepancy was PCR detectable / NGS undetectable, and in case of discordance, PCR positive results are most frequently given precedence in guiding clinical management. In the discordant patients, the presence of MRD positivity via either test drove changes to treatment rather than MRD negativity in one of the two tests. Future studies should investigate whether BCR-ABL PCR or NGS is a better predictor of outcome in Ph+ ALL.

CYP Inhibition and Low-Dose Dasatinib for Chronic Myeloid Leukemia: Preliminary Results of a Promising, Cost-Effective Treatment Option

Introduction The standard treatment for newly diagnosed (ND)-CML is tyrosine kinase inhibitors (TKIs) such as dasatinib. However, the cost of treatment with these drugs can be prohibitively high. The CYP inhibition strategy involves intentionally inhibiting a specific enzyme, in this case CYP3A4, to increase plasma concentration of dasatinib and reduce the required dose from 100mg to 25mg daily. Patients and methods Five consecutive patients older than 18 years, with ND-CML in chronic phase and Eastern Cooperative Oncology Group (ECOG) of 0-2.The diagnosis and classification of CML followed the ESMO guidelines. After diagnostic confirmation, patients continuously received half pill of dasatinib (25mg orally daily) and a strong CYP3A4 inhibitor (ketoconazole, 200mg orally twice daily). Patients were monitored weekly during the first month and then monthly after that. The response to treatment was established according to the ESMO guidelines. Statistical analysis We examined the distribution of variables using the Shapiro-Wilk test, and descriptive analyses were conducted for demographic and clinical characteristics. The proportion of hematological and non-hematological side effects and the proportion of hematological response were obtained. To demonstrate the effect of dasatinib on hematological cells, we constructed a line plot showing the values of hemoglobin, leukocytes, and platelets in each visit. We compared basal hemoglobin, leukocytes, and platelets vs. follow-up counts at each visit with the Kruskal-Wallis test Results The median age of the patients was 42 years. All patients had a median ECOG score of 0. Three patients (60%) presented with thrombocytosis, three (60%) with splenomegaly, and two patients (40%) had B symptoms. Basal median (range) hemoglobin, leukocytes, and platelets counts were 9.9 g/dL , 272×10 9/L and 451×10 9/L, respectively. Three patients (60%) had low-risk CML, and two patients (40%) had high-risk disease, with a median EUTOS score of 71 points. Dasatinib treatment was initiated within a median of 9 days from diagnosis. In the first 28 days of treatment, median leukocytes and platelets were significantly reduced compared to baseline, with a reduction in leukocytes of 93.7% (125×10 9/L vs. 7.9×10 9/L, p=0.005) and in platelets of 64.6% (407×10 9/L, vs. 144×10 9/L, p=0.027). Hemoglobin levels significantly increased by the second month of treatment, with an increase of 26.4% from baseline (10.2 g/dL vs. 12.9 g/dL, p=0.023). All patients (100%) achieved major hematological response within a median of 22 days. Patient number 1 achieved the 3-month and 6-month treatment goals with 6.4% and <1% reduction in BCR-ABL transcript levels, respectively. After 3 months of treatment, 60% of patients achieved the BCR/ABL1 transcript level on the IS <10%, which is the optimal response according to guidelines, in the rest of the patients the time of evaluation has not been reached. Mild and self-limited adverse events were reported in 40% of patients at some point in their treatment. Non-hematological toxicity occurred in one patient (20%) with nausea and galactorrhea due to ketoconazole. Hematological toxicity occurred in (20%) of patients; the platelet count returned to normal after suspending ketoconazole for one week. No serious adverse events were reported. Conclusion Apart from this preliminary study, there is currently no evidence supporting the intentional use of CYP inhibition to reduce the dosage and cost of dasatinib for patients with ND-CML. Dasatinib is primarily metabolized using the CYP3A4 isoenzyme, and studies have shown that ketoconazole, an accessible antifungal, can strongly inhibit CYP3A4, increase dasatinib's plasma concentration and AUC by 5-fold and 385%, respectively, and allow for a reduction in dosage while maintaining therapeutic efficacy. The CYP inhibition strategy can decrease the cost of dasatinib to a point where it is cheaper than full-dose imatinib -the cheapest TKI- and represent a treatment option for patients who cannot afford full-dose dasatinib in low income countries. The CYP inhibition strategy can be of added value to the lower effective dose concept and give rise to ultra-low-dose schemes. This strategy could significantly increase dasatinib accessibility in many countries by reducing costs from $1,343.7 to $40.3-$100.8 monthly

Frequency and prognostic influence of ASXL1 mutations and its potential association with BCR-ABL1 transcript type and smoke in chronic myeloid leukemia patients

BackgroundHeterogeneous response to tyrosine kinase inhibitors (TKIs) and progress to advance phases, still is a significant clinical problem. These are attributed to additional mutations in mutated non-ABL1 genes. we aimed to determine prognostic effects of ASXL1 mutations as a biomarker for diverse treatment response and disease progression to aid clinical management. MethodsWe performed ASXL1 gene mutational screening in 80 Ph+CML patients at different phases and 10 healthy control by direct sequencing method. Multiplex and qRT-PCR, standard chromosome banding analysis were used to determine BCR-ABL1 transcript type, molecular and cytogenetic responses respectively. Resultsoverall, four type mutations were identified in 11.25% of the patients. There was significant difference regarding mutation frequency between chronic and advance phases (P = 0.0002), sokal risk score (P = 0.0001), smoking (P = 0.02) and mean of during time of imatinib treatment (P = 0.009) between patients with and without ASXL1 mutations. ASXL1 mutations frequency had a bias toward younger than older and women than men, but no significant (P > 0.05). ASXL1 mutations were found more recurrently in patients carrying ABL1 KD mutations (P = 0.003). The risk of increasing resistance and disease progression in patients with ASXL1 mutations was 32 and 63 fold higher than those without mutations respectively (P = 0.01; P = 0.0002). The risk of ASXL1 mutations presence in patients with b2a2 transcript type was much higher than b3a2 type (P = 0.02, OR = 10). ConclusionOur findings suggest that ASXL1 mutations may be favorable predictive biomarkers to determine the best TKI for each patient, and to prevent CML progression.

Safety and efficacy of Vitamin D3 supplementation with Imatinib in Chronic Phase- Chronic Myeloid Leukaemia: an Exploratory Randomized Controlled Trial

ObjectivesThe study aimed to compare early molecular response (EMR) rates at 3 months of imatinib therapy with and without vitamin D3 supplementation in patients newly diagnosed with chronic-phase chronic myeloid...

Concomitant BCR-ABL and JAK2 V617F in a Patient with Myeloproliferative Neoplasm: A Case Report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.

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.

Association of pre-treatment bone marrow morphology and achievement of BCR-ABL1 transcript milestones in CML

Chronic myeloid leukemia (CML) is characterized by the fusion gene BCR-ABL1 which encodes aberrantly functioning tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKI) is a landmark of CML management and the main goal is to achieve major molecular response (MMR) which is defined as BCR-ABL1IS ≤ 0.1 % at 12 months of therapy. The aim of this study is to analyze histologic features of bone marrow (BM) in CML patients at the time of diagnosis and compare it to the level of BCR-ABL1IS transcript at 3 (BCR-ABL1IS ≤10 % early molecular response; EMR) and 12 months (MMR) as well as to so called molecularly undetectable leukemia (MUL) to see weather bone marrow morphology can be of value in predicting achievement molecular response milestones. Thirty-two bone marrow biopsies of CML patients, prior TKI therapy, were re-evaluated and CD34 immunohistochemistry was performed to examine microvessel density (MVD) and microvessel area (MVA) and subsequently compared it to the level of BCR-ABL1IS transcript. This study showed statistically significant association between BM hypercellularity and EMR (p = 0.048) and MUL (p = 0.034), peri-trabecular adipocyte distribution and EMR and MUL (p = 0.027 and p = 0.011, respectively), MMR and bone marrow fibrosis (p = 0.029), loose megakaryocyte clustering and EMR and MUL (p = 0.004 and p = 0.018, respectively), absence of naked nuclei and MUL (p = 0.033) but there was no statistically significant association with vascular parameters. These results suggest that some bone marrow morphologic features prior TKI therapy might be indicators of favorable molecular response.

The role of miRNA- 96 and miRNA-150 between different BCR-ABL p210 transcript levels and between different levels of imatinib optimal response in CML patients

BackgroundDysregulation of miRNA expression patterns is one of the several effects developments of cancer. MiRNA has been found to express abnormally in hematological neoplasia such as chronic myeloid leukemia and solid malignancies. Resistance and the degree of response following tyrosine kinase inhibitor (TKI (treatment are correlated with miRNA expression. Hence this study has evaluated the correlation between miRNA- 96 and miRNA-150 with different p210 BCR-ABL transcript levels and the role of miRNA- 96 and miRNA-150 at different levels of imatinib optimal response in CML patients. MethodThis study is based on 60 CML patients divided into two groups based on response to imatinib therapy. 30 samples have the optimal molecular response of CML patients. The other 30 samples that have failed molecular response CML patients, which were compared to 30 samples of healthy volunteers as control by depending on the results of CBC film and PCR. BCR-ABL 210 transcript, which was used in this study, is obtained from the patient's file. ResultsThe results based on the comparison of transcript levels showed that there was a significant difference between the positive response and failed response of CML patients (P ≤ 0.0001). The result of miRNA-96 showed that there was no noteworthy difference within both CML patients (P ≥ 0.5030), while miRNA-150 results exhibited a high significant difference (P ≤ 0.0001). The miRNA-96 and miRNA-150 expression levels in all responses of CML patients demonstrated a highly significant difference with P = 0.0134 and P = 0.0002, respectively. The cut-off value of response against failure response of miRNA-96 and miRNA-150 with P value of 1.691 and 1.784, respectively with high sensitivity is a diagnostic value of the difference between the response and failure response. Conclusionmodulating gene expression with different of miRNAs expression has an impact on drug-gene interactions, with consequences for cell growth and death. The gene expression that shows the different level miRNAs (miRNA- 96 and miRNA-150) among of CML patients of imatinib therapy exhibited high expression in response patients than that of the failure response of patients. The gene expression level of miRNA- 96 and miRNA-150 was differed between through different response CML patients.

Avascular necrosis of femoral head: a rare case as a symptom of the chronic phase in adult chronic myelogenous leukemia patients on nilotinib therapy

Chronic myelogenous leukemia (CML) is a malignant clonal disorder of hematopoietic stem cells characterized by abnormal proliferation and accumulation of immature granulocytes. Avascular necrosis (AVN) is a rare case in CML patients where a systematic review reported only 21 cases from 1984 to 2021 of which 17 cases were AVN of the femoral head (AVNFH) and 3 cases were associated with treatment with tyrosine kinase inhibitors, namely imatinib, desatinib and nilotinib. We report a 51-year-old male patient with complaints of hip joint pain. The patient was diagnosed with CML from bone marrow examination with hypercellular results and PCR examination which detected the presence of BCR-ABL transcription in the form of b2a2 fusion. The patient received nilotinib 200 mg every 12 hours. The diagnosis of AVN was established from clinical symptoms and radiological examination. The patient was planned to undergo a total hip replacement.

Challenges of the Treatment with Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia – Case Presentation

We present the case of a 33-year-old male patient diagnosed with chronic phase chronic myeloid leukemia in June 2008. According to treatment protocols enforced at that time, the patient started oral treatment with imatinib 400 mg/day, reaching a major molecular response (MMR) in 18- month time. The molecular response improved over time, with an undetectable BCR-ABL1 transcript level after 36 -48 months of treatment. However, the profound molecular response did not last, requiring a switch to the second line of therapy after 75 months of imatinib. Taking nilotinib 400 mg x2/day, the patient achieved a major molecular response in 12 months, and after 18 months of treatment, the level of BCR-ABL1 became undetectable. However, two vasooclusive events occurred in 2018 – a transient left parietal ischemic stroke and a left femoral artery occlusion – and by ESMO 2017 recommendations, nilotinib treatment was interrupted. The treatment-free remission decision was made with monthly monitoring of the BCR-ABL1 transcript level, but since the 4th month of monitoring, the deep molecular response disappeared. Administration of dasatinib, the third tyrosine kinase inhibitor, resulted in a deep molecular response which currently lasted until now, during the 58 months of treatment. The presented case represents the evolution of the concept of treatment in this haemato-oncological disease during the last decade.

Pediatric chronic myeloid leukemia in myeloid blast crisis

Chronic myeloid leukemia (CML) accounts for 2-3% of childhood leukemias. About 5% of cases present in a blastic phase of CML which clinically and morphologically mimics more common acute leukemias of childhood. We report a case of a 3-year-old male who presented with gradual onset swelling of the abdomen and extremities along with generalized weakness. Examination revealed massive splenomegaly, pallor, and pedal edema. Initial workup showed anemia, thrombocytopenia, and leukocytosis (120,000/uL) with a blast percentage of 35%. Blasts were positive for CD13, CD33, CD117, CD34 and HLA-DR, and stained negative for Myeloperoxidase and Periodic Acid Schiff. Fluorescence in situ hybridization was positive for b3a2/e14a2 junction BCR-ABL1 transcript and negative for RUNX1-RUNX1T1/t(8;21), clinching the diagnosis of CML in myeloid blast crisis. The patient expired within 17 days of diagnosis and initiation of therapy.

Clinical Study on Flumatinib Mesylate Combined with Chemotherapy Followed by Autologous Hematopoietic Cell Transplantation in Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia

<sec><title>Objective</title> To investigate the clinical efficacy and safety of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph⁺ALL) patients treated with second-generation tyrosine kinase inhibitor (TKI) flumatinib mesylate combined with chemotherapy followed by autologous hematopoietic stem cell transplantation(auto-HSCT). </sec><sec><title>Methods</title> From September 2020 to March 2022, clinical informations of Ph⁺ALL patients who received flumatinib mesylate,chemotherapy and auto-HSCT were collected and analyzed. </sec><sec><title>Results</title> Of the 8 patients who were enrolled, a median age was 43 years old. The BCR-ABL transcript type was P190 in patients with the exception of one P210. It was determined that 2 patients suffered from tuberculosis (TB), and 1 patient developed central nervous system leukemia (CNSL). All patients got clinical remissions after the chemotherapy. A total of 4 patients (50%) received auto-HSCT in major molecular response (MMR). However, 1 patient (12.5%) relapsed following auto-HSCT and was switched to dasatinib therapy. Flumatinib was used during the induction and consolidation chemotherapy except the period of HSCT. Myelosuppression and infection were the most common side effects. At a median follow-up of 19.22 months, the 1-year OS rate was 75%. </sec><sec><title>Conclusion</title> Flumatinib combined with chemotherapy followed by auto-HSCT in Ph⁺ALL achieved a great remission rate and safety. </sec>

Severe persistent marrow aplasia in chronic myeloid leukemia (CML): An unusual complication of imatinib.

Imatinib has changed the treatment of chronic myeloid leukemia (CML) drastically since 15 years. It is usually well tolerated, but severe persistent marrow aplasia is an unusual complication of imatinib while using it in CML. The aim of this study is to describe our experience confronting this rare side effect and review the available data worldwide. It was a retrospective analysis conducted at a center from February 2002 to February 2015. This study was endorsed by our Institutional Review Board (IRB) and written consent was taken from all patients. Patients diagnosed as Philadelphia (Ph) chromosome-positive CML either in chronic phase (CP), accelerated phase (AP), or blastic crisis (BC) were included. There were a total of 1,576 patients with CML treated with imatinib during this period. Karyotyping and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) were done at the time of pancytopenia for all patients. In total, 11 (males = 5, females = 6) patients met our inclusion criteria from 1,576 patients of CML. The median age was 58 years (range 32-76). Out of 11 patients 8, 2, and 1 patients were in CP, AP, and BC phases, respectively. The median time of administration of imatinib was 3.3 months (range 1.5-6). The average time of marrow recovery was 10.4 months (range 5-15). Two patients expired (one from septicemia and the other from intracranial hemorrhage). BCR-ABL transcripts level by RT-PCR revealed the existence of the disease in all patients. Imatinib is a very well-tolerated tyrosine kinase inhibitor (TKI), but is associated with persistent myelosuppression when used in older age, advanced phase of the disease, and treated previously. After confirming persistent marrow aplasia, the treatment is mainly supportive. It is striking that the disease is still persistent, which is confirmed by RT-PCR. There is no consensus regarding recalling imatinib at lower doses or the use of second-generation TKI (nilotinib, dasatinib) in these patients.

P047: Correlation between clinico-biological elements and the type of BCR-ABl transcripts in chronic myeloid leukemia

P047: Correlation between clinico-biological elements and the type of BCR-ABl transcripts in chronic myeloid leukemia