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Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34+CD38−CD26+ and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34+CD38−CD26+ and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26+ (BCR-ABL1+) vs. CD26− (BCR-ABL1−) CD34+CD38− fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34+CD38− fractions that distinguishes between CD26+ (BCR-ABL1+) and their CD26− (BCR-ABL1-) counterparts, providing valuable data for future studies.
ObjectivesThe quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed.MethodsThe laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories.ResultsOur field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA.ConclusionsIn conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.
Desmogleins are involved in cell adhesion conferring structural skin integrity. However, their role in inflammation has been barely studied, and whether desmoglein-4 modulates psoriasis lesions is completely unknown. In this study, we assessed the impact of desmoglein-4 deficiency on the severity of imiquimod (IMQ)-induced skin inflammation and psoriasiform lesions. To this end, desmoglein-4−/− Oncins France Colony A (OFA) with Sprague–Dawley (SD) genetic background were used. Additionally, human RNA-Seq datasets from psoriasis (PSO), atopic dermatitis (AD), and a healthy cohort were analyzed to obtain a desmosome gene expression overview. OFA rats displayed an intense skin inflammation while SD showed only mild inflammatory changes after IMQ treatment. We found that IMQ treatment increased CD3+ T cells in skin from both OFA and SD, being higher in desmoglein-4-deficient rats. In-depth transcriptomic analysis determined that PSO displayed twofold less DSG4 expression than healthy samples while both, PSO and AD showed more than three-fold change expression of DSG3 and DSC2 genes. Although underlying mechanisms are still unknown, these results suggest that the lack of desmoglein-4 may contribute to immune-mediated skin disease progression, promoting leukocyte recruitment to skin. Although further research is needed, targeting desmoglein-4 could have a potential impact on designing new biomarkers for skin diseases.
Treatment-free remission (TFR) in chronic myeloid leukemia (CML) is safe under adequate molecular monitoring, but questions remain regarding which factors may be considered predictive for TFR. Argentina Stop Trial (AST) is a multicenter TFR trial showing that 65% of patients sustain molecular remission, and the prior time in deep molecular response (DMR) was associated with successful TFR. Luminex technology was used to characterize cytokines in plasma samples. Using machine learning algorithms, MCP-1 and IL-6 were identified as novel biomarkers and MCP-1low/IL-6low patients showed eightfold higher risk of relapse. These findings support the feasibility of TFR for patients in DMR and MCP-1/IL-6 plasma levels are strong predictive biomarkers.
Introduction: Treatment-free remission (TFR) is an emerging treatment goal for chronic myeloid leukemia (CML) patients in deep molecular response (DMR). Current evidence shows that 40%-60% of patients relapse while in TFR; and nearly all regain response once tyrosine kinase inhibitors (TKIs) treatment are reinitiated. However a robust predictor of prolonged TFR has not been reported yet. Considering real-life setting, 2 key factors may affect TFR outcome if not properly done: Access to serial molecular monitoring at optimal timepoints and quality laboratory terms as accuracy, sensitivity and rapid results. This motivated the creation of the AST study in our region to guarantee adequate molecular monitoring for TFR in Argentina and characterize new prognostic biomarkers helpful to identify more accurately patients who will be able to sustain TFR. We aimed to assess the proportion of patients with sustained major molecular response (MMR) after TKIs discontinuation and define precise conditions for stopping treatment. Methods: This prospective, multicentre Argentina Stop Trial (AST) trial is recruiting chronic phase CML patients under TKI treatment for at least ≥ 4 years, in DMR (≥MR4.0) sustained for ≥ 2 years in standardized laboratory, confirmed typical BCR-ABL1 transcripts b3a2 and/or b2a2 and aged > 18 years. Molecular tests are centralized in 2 harmonized laboratories and performed monthly for the first 6 months, every 2 months until the first year, and every 3 months during the second year. If patients lost MMR, TKI was restarted immediately. Molecular relapse Free Survival was estimated by Kaplan-Meier method. Difference between survival variables was evaluated through log-rank test. Multivariate analysis was performed through Cox proportional hazards model. The cutoffs of the numerical variables were considered according to the log-rank test. Results: Between February 2019 and July 2020, we evaluated 50 CML patients of whom 46 were enrolled from 7 centers in Argentina and 4 were screening failures. Recruitment was interrupted due to COVID-19 pandemic. Patient median age was 57.5 years (range 24-85). Before discontinuation, TKI treatment was as follows: Imatinib 37/46 (80%), Nilotinib 5/46 (11%) and Dasatinib 4/46 (9%), 2G-TKI as 1st line, 11% of the patients received non-branded treatment. Sokal risk score showed to be low in 22 patients (48%), intermediate in 14 (30%) and high in 10 (22%). Median follow-up was 10 months (range 4-17) and the estimated molecular relapse-free survival was 80.2% (95%CI 69-93) at 6 months Fig 1. Longer DMR durations before discontinuation were associated with increased probability of maintaining response at 6 and 12 months: 83.2% for patients who had >54 months in DMR vs 70% with <54 months and 72% vs 23.3% respectively (p=0.0453) Fig 2. Cox multivariate analysis was performed including different variables as age at diagnosis, time in DMR, time in TKI previous to discontinuation and Sokal risk. The only significant variable associated to improved prognosis was time in DMR (HR 2.8 95%CI 1.002-8.07 p=0.0495). Our cohort had a long time on TKI treatment previous to discontinuation, median 10.5 years (4.16-17.5) probably considering it a favorable factor for the high TFR rates described at 6 months. Among the 46 patients included, 15 (33%) lost MMR, all restarted treatment with the same TKI used before discontinuation, 12/15 (80%) regained MMR with a median time of 3 months (range1-8) and 9/15(60%) obtained MR 4.0 with a median time of 3 months (range1-5). Conclusion: This is the first multicenter study of TKI discontinuation in CML patients in Argentina showing that TKI can be safely discontinued in those who achieve and maintain a DMR before discontinuation. We observed high rates of molecular relapse free survival, although longer follow-up is needed. We must continue with this approach for patients participating in TFR trials or TFR programs in order to decrease the risk of relapse and make this goal a fact in our region. This discontinuation study will allow in a near future significant saving of economic resources and might improve patients quality of life specially in those who are currently experiencing treatment adverse events. Disclosures Pavlovsky: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Speakers Bureau; Pfizer: Speakers Bureau; Pint Pharma: Speakers Bureau. Varela:Novartis: Consultancy, Speakers Bureau. Pavlovsky:Janssen: Membership on an entity's Board of Directors or advisory committees, Other: travel grants, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Varifarma: Speakers Bureau. Moiraghi:BMS: Speakers Bureau; Novartis: Speakers Bureau.
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