[Allogeneic transplantation as a therapeutic option for atypical chronic myeloid leukemia].

Atypical chronic myeloid leukemia (aCML) is a rare disease classified as a myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Recent advances in gene mutational profiling have clarified the characteristics of aCML as a disease entity relative to other MDS/MPNs. Although some studies suggest the efficacy of DNA demethylating agents and tyrosine kinase inhibitors, data about these agents are limited due to the small number of patients. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is only therapeutic option that can provide durable remission for aCML and other MDS/MPNs. Retrospective studies from Europe and Japan revealed the clinical results of allo-HSCT for aCML. This review summarizes the pathogenesis of aCML and the development of allo-HSCT and other therapeutic options.

Efficacy and safety of olverembatinib in adult BCR::ABL1-positive ALL with T315I mutation or relapsed/refractory disease.

Third-generation tyrosine kinase inhibitors (TKIs) have much potential for the treatment of BCR::ABL1-positive leukaemia, particularly that harbouring the ABL1 T315I mutation. Olverembatinib (HQP1351), a novel third-generation TKI, has favourable efficacy and safety profiles in chronic myeloid leukaemia. Here, we present the clinical findings from 31 BCR::ABL1-positive acute lymphoblastic leukaemia (ALL) patients who received olverembatinib. Among the 14 patients with overt relapsed/refractory (R/R) disease (including 10 with the T315I mutation), 71.4% achieved an overall response. Of the other 17 patients with minimal residual disease (MRD)-positive ALL (including 14 with the T315I mutation), 60.0% and 47.1% achieved MRD flow negativity and complete molecular remission, respectively. With a median follow-up time of 16.3 months, the median event-free survival and overall survival were 3.9 and 8.3 months respectively, in overt R/R patients, and 11.5 and 18.4 months in MRD-positive patients. Allogeneic haematopoietic stem cell transplantation further improved outcomes among responders. The safety profile was generally manageable. This study suggests that olverembatinib-based therapy is another promising option for BCR::ABL1-positive ALL in addition to ponatinib, especially for patients with MRD-positive disease and a single T315I mutation.

Dasatinib-Induced Bilateral Chylothorax: A Case Report.

Dasatinib, a BCR-ABL tyrosine kinase inhibitor, is used in the management of Philadelphia-positive chronic myeloid leukemia (CML). Several adverse complications of this targeted immunotherapy have been reported. This case report focuses on a 79-year-old female who presented with acute dyspnea and generalized chest pressure while undergoing management with this specific tyrosine kinase inhibitor. Bilateral chylothorax was diagnosed with the aid of imaging, laboratory studies, and diagnostic thoracentesis. No other risk factors, including trauma, lung malignancies, or congenital anomalies, were detected in this patient. Since no other etiologies for the development of chylothorax were identified, it was concluded that dasatinib therapy was the inciting factor. Dasatinib was discontinued and bosutinib was initiated. A low-fat diet was prescribed, which the patient was amenable to. Six months later, the patient remained stable with no recurrence of chylothorax. The mechanism of dasatinib-induced chylothorax is currently under investigation. The purpose of this report is to raise awareness about dasatinib-induced chylothorax, aid in identifying predisposing risk factors, and enhance understanding of the proper management of this rare complication.

New practice of BCR::ABL1 standardization system based on p210 and p190 BCR::ABL1 reference materials.

Quantification of BCR::ABL1 monitors minimal residual disease, thus critical for patient stratification. While significant progress has been made in enhancing the accuracy of p210 BCR::ABL1 quantification, no equivalent standardization has been conducted for p190 BCR::ABL1. Therefore, we developed p190 BCR::ABL1 reference materials to calibrate the quantitative process through an innovative plasmid-based calibration strategy. Then, we further explored the use of p190 and p210 reference materials to standardize tests in 159 laboratories across China and assessed their detection capability utilizing quality assessment samples. Results suggested that after calibration, the coefficient of variation of detection results decreased from 50.8 %-57.4 % to 24.9 %-36.4 % for p190, and from 37.6 %-49.0 % to 19.1 %-28.5 % for p210. The percentage of laboratories within ± 2-fold of the target values increased from 77.1 %, 76.4 %, 73.2 %, and 74.5 % to 94.3 %, 95.5 %, 92.4 %, and 91.1 % for p190 samples 2023S21-2023S24, and from 72.3 %, 86.2 %, 79.2 %, and 81.1 % to 98.1 %, 99.4 %, 98.1 %, and 96.2 % for p210 samples 2023S11-2023S14. Overall, our study successfully developed and employed p190 and p210 reference materials to promote accuracy and comparability of BCR::ABL1 quantification among laboratories.

Novel ABL1 mutation in a Moroccan CML patient with Imatinib resistance.

Tyrosine Kinase Inhibitors (TKI), such as Imatinib, are known for their effectiveness in achieving complete remission from Chronic Myeloid Leukemia (CML), a malignancy caused by a reciprocal translocation between the terminal fragments of the long arms of chromosomes 9 and 22 that leads to the famous chimeric BCR::ABL1 gene. Mutations in this fusion gene may induce resistance to TKI treatment, which requires prescribing a second-, or third-generation TKI medication. We report here a case of a Moroccan CML patient with secondary resistance to the frontline TKI treatment (Imatinib), in which, BCR::ABL1 cDNA sequencing reveals the novel mutation p.K375M at the ABL1 Kinase Domain. In-silico prediction tools confirm the pathogenicity of the p.K375M substitution. Homology analysis indicated that the residue is highly conserved and located in a stable region. This potentially pathogenic mutation is likely to disrupt the BCR::ABL1-Imatinib binding, leading to the observed resistance. To overcome the treatment resistance, Imatinib should be substituted with a second-generation TKI medication, such as Dasatinib, Bosutinib, or Nilotinib. The present study further widens the spectrum of TKI resistance mutations and emphasizes particularly the crucial role of molecular investigation in personalizing treatment for CML patients, ensuring efficient follow-up and appropriate healthcare.

In Silico Molecular Modeling of Four New Afatinib Derived Molecules Targeting the Inhibition of the Mutated Form of BCR-ABL T315I.

Four afatinib derivatives were designed and modeled. These derivatives were compared to the known tyrosine-kinase inhibitors in treating Chronic Myeloid Leukemia, i.e., imatinib and ponatinib. The molecules were evaluated through computational methods, including docking studies, the non-covalent interaction index, Electron Localization and Fukui Functions, in silico ADMET analysis, QTAIM, and Heat Map analysis. The AFA(IV) candidate significantly increases the score value compared to afatinib. Furthermore, AFA(IV) was shown to be relatively similar to the ponatinib profile when evaluating a range of molecular descriptors. The addition of a methylpiperazine ring seems to be well distributed in the structure of afatinib when targeting the BCR-ABL enzyme, providing an important hydrogen bond interaction with the Asp381 residue of the DFG-switch of BCR-ABL active site residue and the AFA(IV) new chemical entities. Finally, in silico toxicity predictions show a favorable index, with some molecules presenting the loss of the irritant properties associated with afatinib in theoretical predictions.

Association of PARP1 Expression Levels and Clinical Parameters in Different Leukemic Subtypes With BCR::ABL1 p190+ Translocation.

Background/Aim: Although the reciprocal translocation t(9;22)(q34;q11) is a hallmark of chronic myeloid leukemia (CML), it is also present in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Depending on the gene's breakpoint, it is possible to obtain three isoforms, among which p190 stands out for the poor prognosis it induces whenever it appears. Due to the genomic instability induced by BCR::ABL1, it is proposed to expand the applicability of poly-ADP-ribose polymerase-1 (PARP1) and its inhibitors in hematological neoplasms. Materials and Methods: We measured the expression levels of PARP1 by quantitative real-time PCR (qPCR) using TaqMan®, correlating its expression with BCR::ABL1 p190+, to evaluate its influence in the clinic of adult patients. Results: We found that PARP1 is expressed differently in ALL, AML and CML and that p190 transcripts do not follow a linear pattern in these populations. We also found that PARP1 expression is not correlated with age, white blood cell and the amount of p190 transcripts. Conclusion: Despite the lack of statistical correlation between the variables analyzed, the role of PARP1 in BCR::ABL1 leukemia cannot be ruled out, given the instability profile promoted by this translocation. Finally, further studies involving a larger sample of patients are needed, as well as investigations into other molecular pathways that may impact on the pathogenesis of different BCR::ABL1 leukemic subtypes.

HDAC2-miR183-5p Epigenetic Circuit Contributes to the Growth of Philadelphia Chromosome-Positive B-cell Acute Lymphoblastic Leukemia via PTEN/AKT and c-MYC Signaling Pathway.

Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph(+)B-ALL) is a hematological malignancy with a poor prognosis. Epigenetic abnormalities, especially abnormal histone acetylation and microRNAs (miRNAs) dysregulation, are a group of epigenetic patterns that contribute to leukemia progression. However, their regulatory mechanisms in Ph(+)B-ALL have not been fully elucidated. In this study, we identified that miR-183-5p is significantly downregulated in Ph(+)B-ALL and associated with poor prognosis. Moreover, we found that the BCR-ABL fusion gene is a key target gene of miR-183-5p. MiR-183-5p directly targets BCR-ABL gene and induces cell apoptosis via PTEN/AKT and c-MYC signaling pathways. In addition, histone deacetylase inhibitor (HADCi) could mitigate the suppressive effects of HDAC2 on miR-183-5p by promoting promoter acetylation, thereby enhancing cell apoptosis. In conclusion, our results indicate that miR-183-5p is a potential biomarker and suggest that a novel "HDAC2-miR-183-5p epigenetic circuitry regulation" may be involved in the pathogenesis of Ph(+)B-ALL. Taken together, These findings provide new insights into the design of promising molecular-targeted drugs for Ph(+)B-ALL.

Establishment of genomic RNA reference materials for BCR-ABL1 P210 measurement.

Quantitation of BCR-ABL1 with the quantitative reverse transcriptase polymerase chain reaction (RT-PCR) is very important in monitoring chronic myeloid leukemia (CML), which relies on an RNA reference material. A genomic RNA reference material (RM) containing the BCR-ABL1 P210 fusion mutation was developed, and an absolute quantitative method based on one-step reverse transcription digital PCR (RT-dPCR) was established for characterizing the RM. The proposed dPCR method demonstrates high accuracy and excellent analytical sensitivity, as shown by the linear relationship (0.94 < slope < 1.04, R2≧0.99) between the measured and nominal values of b2a2, b3a2, and ABL1-ref within the dynamic range (104-101 copies/reaction). Homogeneity and stability assessment based on dPCR indicated that the RM was homogeneous and stable for 24 months at -80 °C. The RM was used to evaluate inter-laboratory reproducibility in eight different laboratories, demonstrating that participating laboratories could consistently produce copy concentrations of b3a2 and ABL1-ref, as well as the BCR-ABL1/ABL1 ratio (CV < 2.0%). This work suggests that the RM can be employed in establishing metrological traceability for detecting mutations in the BCR-ABL1 fusion gene, as well as in quality control for testing laboratories.

Structure optimization, synthesis and bioactivity evaluation of novel BCR-ABL tyrosine kinase inhibitor targeting T315I mutation.

Chronic Myeloid Leukemia (CML) is a malignant hematologic tumor caused by BCR-ABL fusion protein that binds with ATP to exert tyrosinase activity and persistently activates downstream phosphorylation pathways. The tyrosine kinase inhibitors (TKIs) represented by Imatinib are the key clinical therapy to the CML. While the mutations on the target lead to the serious drug resistance problems, especially the T315I mutation remains an unresolved challenge, and the cardiotoxicity has limited the clinical application of the third generation TKI Ponatinib despite its favorable efficacy against the T315I mutation. Even though, structural optimization of Ponatinib remains a potential strategy to overcome the resistance imposed by the mutation. Herein, we present a series of novel BCR-ABL/T315I tyrosine kinase inhibitors obtained by virtual screening using ZINC21710815, a BCR-ABL/T315I inhibitor reported earlier by our team, as a lead compound, and structural optimization of lead compounds against the T315I mutation, as well as screening of two novel compounds by activity evaluation and mechanistic studies, W4 and W8. W4 and W8 have better cell death-inducing effects and special selectivity against BaF3/T315I, which are worthy of further in-depth study to obtain more desirable anti-CML drugs as lead compounds.

Oxidative lipid damage by naringenin selectively sensitizes chronic myeloid leukemia cell lines and patient samples to Bcr-Abl tyrosine kinase inhibitors.

Chronic myeloid leukemia (CML) treatment with Bcr-Abl tyrosine kinase inhibitors (TKIs) has significantly improved patient outcomes, yet challenges such as drug resistance and persistence of leukemic stem cells persist. This study explores the potential of naringenin, a natural flavonoid, to enhance the efficacy of Bcr-Abl TKIs in CML therapy. We showed that naringenin reduces viability of a panel of CML cell lines regardless of varying cellular origin and genetic mutations, and acts synergistically with dasatinib and ponatinib. Importantly, naringenin is effective in targeting blast crisis CML CD34+ cells by decreasing their colony formation, self-renewal and viability. Compared to CML, naringenin is significantly less effective against normal bone marrow (NBM) counterparts. In addition, naringenin significantly enhances the inhibitory effects of dasatinib in CML but not NBM CD34+ cells. Mechanism studies showed that naringenin's inhibitory effects were associated with the induction of oxidative stress and lipid damage, as evidenced by increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Notably, naringenin upregulated genes related to mitochondrial biogenesis while downregulating antioxidant defense genes. Pretreatment with α-tocopherol, which inhibits lipid-mediated ROS production, completely abolished the ROS increase and restored cell viability, indicating that lysosomal lipid peroxidation plays a crucial role in naringenin's mechanism of action. In a CML xenograft mouse model, the combination of naringenin and dasatinib resulted in remarkably more tumor growth suppression compared to single drug alone. Importantly, this combination was well-tolerated, with no adverse effects on body weight observed. These findings suggest that naringenin, by inducing oxidative lipid damage, enhances the anti-leukemic effects of Bcr-Abl TKIs, offering a promising therapeutic strategy for CML.

A critical review of management of allogeneic transplant-eligible adults with Ph+ acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) in 20%-30% of adult patients contains the Philadelphia (Ph+) chromosome. Historically, Ph+ ALL denoted a markedly inferior outcome and long-term survival in the absence of an allograft was uncommon. However, the advent of targeted therapy directed against the BCR::ABL1 fusion protein with various tyrosine kinase inhibitors (TKIs) has markedly improved the prognosis, resulting in a number of treatment controversies in allograft-eligible patients. Which is the best TKI to use in induction? What is the clinical relevance of the subdivision of Ph+ ALL into multilineage vs lymphoid types? Do all patients in first morphological complete remission (CR1) after induction and consolidation with chemotherapy/TKI require an allograft? If not, what risk factors predict a poor outcome without an allograft? Can chemotherapy-free approaches, such as blinatumomab in conjunction with more potent TKIs, obviate the need for an allograft in high-risk patients? What is the best strategy to deal with persistent or emerging minimal residual disease both pre- and post-transplant? Is maintenance TKI indicated in all patients post allograft? Can salvage therapy and a subsequent allograft cure patients who relapse after not being transplanted in CR1? This manuscript reviews the latest data influencing contemporary management and discusses these controversies.

Low-Level BCR::ABL1 Transcript at Diagnosis in Childhood Leukemia: A 10-Year Single Institution Study.

INTRODUCTION: Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is a high risk form of ALL associated with dismal outcomes in the pre-tyrosine kinase inhibitor (TKI) era. Addition of a TKI to chemotherapy improves outcomes. Therefore, testing for the presence of the Philadelphia chromosome by at least two methods at the time of diagnosis is critical. Diagnostic testing may include karyotype, fluorescent in situ hybridisation (FISH), and RT-PCR for the BCR::ABL1 transcript. The significance of low-level BCR::ABL1 transcript by RT-PCR in the absence of the Philadelphia chromosome on karyotype or by FISH is unknown. METHODS: This is a retrospective review of children diagnosed with acute leukemia at our institution from 2010 to 2020. Those positive for the BCR::ABL1 transcript by qualitative RT-PCR, and negative for t(9;22) by karyotype or FISH were analyzed for demographics, cytogenetic and molecular features at diagnosis and relapse, treatment and outcomes. The Kaplan-Meier method was used to estimate event-free and overall survival. RESULTS: Forty-seven of 306 (15%) patients with Ph- ALL had low-level BCR::ABL1 detected by RT-PCR. Most (77%) had B-cell ALL. The e1a2 transcript was detected most frequently, in 43 (91%) patients. BCR::ABL1 was quantifiable in 12/43 (28%) patients, with a median of 0.0008% (range 0.0003-0.095%). Seven patients (15%) relapsed. No patient with low-level BCR::ABL1 at diagnosis developed Ph + ALL at relapse. There was no difference in 5-year event-free (77% versus 81%, p = 0.407) or overall survival (86% versus 91%, p = 0.3) between children with low-level BCR::ABL1 (n = 47) and those without (n = 259). CONCLUSION: BCR::ABL1 low-level positivity in children with newly diagnosed Ph- ALL is a relatively common finding and did not adversely affect outcome for patients treated using a contemporary risk-adapted approach.

Judicious use of precise fluorescence in situ hybridisation panels guided by population prevalence may assist pragmatic detection of clinically targetable Philadelphia chromosome-like acute lymphoblastic leukaemia fusions: a systematic review.

Diagnosis of Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) in the real-world remains challenging because of definitional complexities, the diverse diagnostic techniques available and the cost, expertise and time involved. We summarise evidence for diagnosis of clinically important Ph-like ALL related genomic lesions using fluorescence in situ hybridisation (FISH) targeting only clinically important and actionable lesions, an accessible and cost-effective diagnostic technique. Electronic databases were interrogated using broad MeSH terms for articles reporting a detailed FISH strategy for diagnosis of Ph-like ALL published since 2014, yielding 653 full text articles and abstracts. We searched the National Library of Medicine Databases including PubMed, Medline, Embase, Cochrane and relevant abstracts. We included studies with a primary aim of determining the utility of FISH for Ph-like ALL diagnosis and studies with broader aims demonstrating Ph-like ALL diagnostic algorithms which partially involved FISH. Nineteen studies met inclusion criteria. Evidence for FISH to detect CRLF2 rearrangements in Ph-like ALL is strongly established and evidence for FISH to detect non-CRLF2 lesions is evolving rapidly. We documented 1620 cases of non-CRLF2 Ph-like lesions diagnosed by FISH. Confirmatory side-by-side methods were applied in six studies (246 samples), four of which demonstrated 100% concordance of FISH results with alternative methods, while two studies demonstrated over 70% sensitivity and specificity. Additional studies demonstrated wide utilisation of FISH in Ph-like ALL classification across diverse geographies and ethnicities, with contrasting prevalence, implicating a need for targeted FISH strategies. In real-world cohorts, it may be clinically useful to prioritise limited early FISH in B-cell ALL (B-ALL) diagnostic algorithms to identify Ph-like abnormalities that respond to locally available kinase inhibitors to promote and prioritise broad access to effective targeted treatment. Additional studies are required to provide adequately powered validations and verifications of targeted Ph-like FISH panels to confirm sensitivity and specificity against side-by-side gold standard methods, and to define optimal local approaches.

SOHO State of the Art Updates and Next Questions | Approach to BCR::ABL1-Like Acute Lymphoblastic Leukemia.

Philadelphia-like (Ph-like) or BCR::ABL1-like acute lymphoblastic leukemia (ALL) is a common high-risk subtype of B-cell precursor ALL (B-ALL) characterized by a diverse range of genetic alterations that challenge diagnose and converge on distinct kinase and cytokine receptor-activated gene expression profiles, resembling those from BCR::ABL1-positive ALL from which its nomenclature. The presence of kinase-activating genetic drivers has prompted the investigation in preclinical models and clinical settings of the efficacy of tyrosine kinase inhibitor (TKI)-based treatments. This was further supported by an inadequate response to conventional chemotherapy, high rates of induction failure and persistent measurable residual disease (MRD) positivity, which translate in lower survival rates compared to other B-ALL subtypes. Therefore, innovative approaches are underway, including the integration of TKIs with frontline regimens and the early introduction of immunotherapy strategies (monoclonal antibodies, T-cell engagers, drug-conjugates, and CAR-T cells). Allogeneic hematopoietic cell transplantation (HSCT) is currently recommended for adult BCR::ABL1-like ALL patients in first complete remission. However, the incorporation of novel therapies, a more accurate diagnosis and a more sensitive MRD assessment may modify the risk stratification and the indication for transplant in these patients.

Increased mRNA expression for serotonin receptor 1B (HTR1B) is associated with thrombosis in BCR::ABL1-negative myeloproliferative neoplasms.

BCR::ABL1-negative myeloproliferative neoplasms (MPNs) are clonal haematopoietic stem cell disorders characterized by specific driver mutations and an increased risk of both macrothrombosis and microthrombosis. Serotonin receptor type 1B (HTR1B) was found to be expressed by various solid tumours, and also primary bone marrow mononuclear cells from myelodysplastic neoplasm and acute myeloid leukaemia patients, representing a potential therapeutic target. In this study we assessed for the first time the expression levels of HTR1B mRNA in the peripheral blood mononuclear cells (PBMC) of 85 newly diagnosed MPN patients, consisting of 28 polycythemia vera, 25 essential thrombocythemia and 32 primary myelofibrosis cases. Levels of HTR1B expression between MPN subtypes and control group were not significantly different. However, at clinical data examination, it was observed that MPN patients with a recent history of major thrombosis and/or signs of impaired microcirculation exhibited significantly higher HTR1B expression levels compared to non-thrombotic MPNs and control group. Moreover, thrombotic MPN patients had significantly higher HTR1B expression than patients with recent thrombosis and absence of MPN diagnostic criteria. These findings suggest that increased levels of HTR1B expression in PBMC might be associated with thrombosis in MPN patients, but larger studies are needed for confirmation, including testing of the receptor protein expression level.

Familial 46, XY Disorder of Sexual Development identified in a Ph+BCR::ABL1P210+ Acute Lymphoblastic Leukemia septuagenarian female with RCBTB2::LPAR6 fusion gene: a case report.

Background: Familial 46, XY Disorder of Sexual Development (DSD) was discovered in a Ph+, BCR::ABL1P210+ Acute Lymphoblastic Leukemia (ALL) female with RCBTB2::LPAR6 fusion gene. Siblings developing 46, XY DSD are extremely rare. Patients with 46, XY DSD have much higher rates of gonadal cancers. Nevertheless, the incidence of hematologic malignancies in patients with DSDs has received little attention. RCBTB2::LPAR6 is a rarely reported fusion gene in ALL. Case presentation: Herein, we report a rare case of a newly diagnosed Ph+, BCR::ABL1P210+ ALL patient who was 77 years old and female by social sex. Whole Exome Sequencing (WES) and RNA sequencing revealed TET2 and NF1 mutations in addition to a rarely reported RCBTB2::LPAR6 fusion gene and 17 other genes with uncertain clinical significance. The patient was surprisingly found to have a male karyotype. On ultrasound, neither the uterus nor the ovaries were discernible. A detailed family and marital history revealed that the patient had undergone surgery at an early age for an unexplained inguinal mass. She had slow pubertal development, scanty menstruation, and few overtly feminine characteristics. She had three marriages, but none succeeded in getting pregnant. The patient had never sought therapy for infertility due to the inaccessibility of medical treatment and a lack of medical knowledge. Her sister, 73 years old and female by social sex, who had amenorrhea in adolescence and was unable to conceive, had the same experience. To our surprise, she also had a male karyotype. Conclusions: Due to the absence of long-term social attention and follow-up, studies on the incidence of hematologic malignancies in patients with 46, XY DSD are incredibly uncommon. Siblings developing 46, XY DSD is extremely rare. We report the oldest patient diagnosed with 46, XY DSD. There have not yet been any reports of familial 46, XY DSD with a concurrent diagnosis of Ph+BCR::ABL1P210+ ALL with a rarely reported RCBTB2::LPAR6 fusion gene.

Molecular biomarkers of leukemia: convergence-based drug resistance mechanisms in chronic myeloid leukemia and myeloproliferative neoplasms.

Leukemia represents a diverse group of hematopoietic neoplasms that can be classified into different subtypes based on the molecular aberration in the affected cell population. Identification of these molecular classification is required to identify specific targeted therapeutic approaches for each leukemic subtype. In general, targeted therapy approaches achieve good responses in some leukemia subgroups, however, resistance against these targeted therapies is common. In this review, we summarize molecular drug resistance biomarkers in targeted therapies in BCR::ABL1-driven chronic myeloid leukemia (CML) and JAK2-driven myeloproliferative neoplasms (MPNs). While acquisition of secondary mutations in the BCR::ABL1 kinase domain is the a common mechanism associated with TKI resistance in CML, in JAK2-driven MPNs secondary mutations in JAK2 are rare. Due to high prevalence and lack of specific therapy approaches in MPNs compared to CML, identification of crucial pathways leading to inhibitor persistence in MPN model is utterly important. In this review, we focus on different alternative signaling pathways activated in both, BCR::ABL1-mediated CML and JAK2-mediated MPNs, by combining data from in vitro and in vivo-studies that could be used as potential biomarkers of drug resistance. In a nutshell, some common similarities, especially activation of PDGFR, Ras, PI3K/Akt signaling pathways, have been demonstrated in both leukemias. In addition, induction of the nucleoprotein YBX1 was shown to be involved in TKI-resistant JAK2-mediated MPN, as well as TKI-resistant CML highlighting deubiquitinating enzymes as potential biomarkers of TKI resistance. Taken together, whole exome sequencing of cell-based or patients-derived samples are highly beneficial to define specific resistance markers. Additionally, this might be helpful for the development of novel diagnostic tools, e.g., liquid biopsy, and novel therapeutic agents, which could be used to overcome TKI resistance in molecularly distinct leukemia subtypes.

Comparative analysis of BCR::ABL1 p210 mRNA transcript quantification and ratio to ABL1 control gene converted to the International Scale by chip digital PCR and droplet digital PCR for monitoring patients with chronic myeloid leukemia.

OBJECTIVES: Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, leading to the BCR::ABL1 fusion gene and hyper-proliferation of granulocytes. Tyrosine kinase inhibitors (TKIs) are effective, and minimal residual disease (MRD) monitoring is crucial. Digital PCR platforms offer increased precision compared to quantitative PCR but lack comparative studies. METHODS: Eighty CML patient samples were analyzed in parallel using digital droplet PCR (ddPCR) (QXDx™ BCR-ABL %IS Kit) and chip digital PCR (cdPCR) (Dr. PCR™ BCR-ABL1 Major IS Detection Kit). RESULTS: Overall, qualitative and quantitative agreement was good. Sensitivity analysis showed positive percentage agreement and negative percentage agreement were both ≥90 %, and the quadratic weighted kappa index for molecular response (MR) level categorization was 0.94 (95 %CI 0.89, 0.98). MR levels subgroup analysis showed perfect categorical agreement on MR level at MR3 or above, while 35.4 % (17/48) of patient samples with MR4 or below showed discordant categorizations. Overall, Lin's concordance correlation coefficient (CCC) for the ratio of %BCR::ABL1/ABL1 converted to the International Scale (BCR::ABL1 IS) was almost perfect quantitative agreement (Lin's CCC=0.99). By subgroups of MR levels, Lin's CCC showed a quantitative agreement of BCR::ABL1 IS decreased as MR deepened. CONCLUSIONS: Both cdPCR and ddPCR demonstrated comparable performance in detecting BCR::ABL1 transcripts with high concordance in MR3 level or above. Choosing between platforms may depend on cost, workflow, and sensitivity requirements.