• Predicting deep molecular response to imatinib treatment for Chronic Myeloid Leukemia. • Leveraging machine learning methods. • Utilizing data from a developing country (Brazil) where cost considerations affect treatment.

Click to increase image sizeClick to decrease image size AcknowledgmentsThe authors acknowledge with gratitude Dr. Marcelo Land, Dr. Renata Binato, and Moisés Rocha. We thank Drs. Soheil Meshinchi and Michael Loken for their invaluable suggestions and Alice Theophilo Teixeira de Matos Bch. for editing the figures. This work was dedicated to the Brazilian indigenist Bruno Pereira in memoriam.Authors’ contributionsCapela de Matos RR, Silva MLM, and Ribeiro RC participated in the design of the study and manuscript writing; Rouxinol M performed the clinical history of the patient; Capela de Matos RR and Silva MLM conducted the conventional cytogenetic analyses and performed FISH; Costa ES and Mello FV performed and revised the immunophenotypic studies; Ferreira GM and Zalcberg I performed the NGS studies; Bonecker S performed the RT-PCR, RT-qPCR analyses, and literature review; Capela de Matos RR, and Ferreira GM participated in the manuscript editing; Silva MLM, Zalcberg I, Abdelhay E, and Ribeiro RC revised the manuscript critically for important intellectual content.Disclosure statementThe authors report there are no competing interests to declare.Additional informationFundingThis work was supported by FAPERJ (project no. E-26/210.419/2019, 248798), Brazil; the Ministry of Health (MS), Brazil; the National Cancer Control Program INCA, Brazil.

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.

CAR-T-cell therapy has shown considerable advance in recent years, being approved by regulatory agencies in US, Europe, and Japan for the treatment of refractory patients with CD19+ B-cell leukemia or diffuse large B-cell lymphoma. Current methods for CAR-T-cell production use viral vectors for T-cell genetic modification and can take up to 15 days to generate the infusion product. The development of simple and less costly manufacturing protocols is needed in order to meet the increasing demand for this therapy. In this present work, we generated 19BBz CAR-T cells in 8 days using a protocol based on the non-viral transposon-based vector Sleeping Beauty. The expanded cells display mostly a central memory phenotype, expressing higher levels of inhibitory receptors when compared with mock cells. In addition, CAR-T cells were cytotoxic against CD19+ leukemia cells in vitro and improved overall survival rates of mice xenografted with human RS4;11 or Nalm-6 B-cell leukemias. Infused CAR-T cells persisted for up to 28 days, showing that they are capable of long-term persistence and antitumor response. Altogether, these results demonstrate the effectiveness of our protocol and pave the way for a broader application of CAR-T-cell therapy.

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 CML LSC, normal hematopoietic stem cells (HSC) obtained from the same 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 and HSC from CML 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 patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. In silico analysis revealed an association between validated microRNAs and multiple metabolic pathways, suggesting that these molecules may be mediators of the previously reported dysregulation of LSC metabolism. This is the first report of the LSC miRNome that distinguishes between BCR-ABL1 + LSC and their BCR-ABL1 - counterparts, providing valuable data for future studies. * aUPD : acquired uniparental disomy BFU-E : burst-forming unit-erythroid BM : bone marrow CFU-GEMM : colony-forming unit-granulocyte erythroid macrophage megakaryocyte CFU-GM : colony-forming unit-granulocyte macrophage CML : chronic myeloid leukemia FACS : fluorescent-activated cell sorting GO : Gene Ontology HD : healthy donor HSC : hematopoietic stem cells KEGG : Kyoto Encyclopedia of Genes and Genomes LSC : leukemic stem cells LT-HSC : (murine) long-term repopulating hematopoietic stem cell NGS : next-generation sequencing PB : peripheral blood MNC : mononuclear cells Ph : Philadelphia chromosome Pre-L-NSC : pre-leukemic neoplastic stem cells qPCR : quantitative PCR RT : reverse transcription snoRNAs : small nucleolar RNAs snRNAs : small nuclear RNAs TKI : tyrosine kinase inhibitors tRNAs : transfer RNAs

Despite acceptable results of imatinib in the treatment of chronic myeloid leukemia (CML), some patients fail to acquire a complete cytogenetic response (CCyR), which may be caused by polymorphisms in the pharmacogenetic genes. The study aimed to evaluate the association of two polymorphisms in the ABCB1 and ABCG2 genes with cytogenetic response to imatinib and the risk of CML development.We genotyped ABCB1 (c .2677G/T/A) and ABCG2 (c .421C/A) polymorphisms by PCR-RFLP, T-ARMS-PCR methods in 111 patients with CML and 102 sex- and age-matched healthy subjects. CCyR was determined by standard chromosome banding analysis (CBA).Analysis of polymorphisms showed significant association of ABCG2 c.421CA genotype (p < 0.0001; OR = 0. 17), and ABCG2c.421A allele (p < 0.0001; OR = 0.31) with decreased risk of CML. Moreover, ABCB1c.2677GT- ABCG2c.421CC combined genotype (p = 0.017; OR = 4.20) was associated with increased risk of CML. Analysis of the joint effect of SNP-smoking combination showed that smoker subjects with the ABCB1c.2677GG/GT (p = 0.001; OR = 15.96, p = 0.001; OR = 8.13, respectively) or ABCG2c.421CC genotypes (p = 0.001; OR = 5.82) had the increased risk of CML, while the risk of the CML in non-smokers carrying the ABCG2c.421CA (p < 0.0001; OR = 0. 18) genotype was strongly decreased compared with reference group. Regarding drug response, ABCG2c.421 CC/CA genotypes in the smoker patients were associated with an increased risk of resistance to imatinib (p < 0.0001; OR = 7.02, p = 0.018; OR = 4.67, respectively).Our results suggest the impact of ABCG2c .421C/A polymorphism on CML development, and smoking may have a synergistic role in the risk of CML and resistance to imatinib.

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Data show 40% of chronic myeloid leukemia (CML) patients maintain complete molecular remission (CMR), i.e. failure to detect BCR-ABL1, consenting to termination of Imatinib mesylate (IM) therapy, following undetectable disease for ≥2 years by quantitative PCR (Q-PCR). These findings suggest majority of the patients experience molecular relapse. Furthermore, majority relapse in the first 6 months, implying Q-PCR assay sensitivity is suboptimum, to confidently identify patients for discontinuation of IM. Droplet digital PCR (ddPCR) is suggested to have sensitivity that is one log greater than the Taqman (Q-PCR) assay. If verified, ddPCR would enhance safe withdrawal of IM therapy from CML patients. Here we present data comparing ddPCR with Q-PCR.

We report an extremely rare case of a female child who presented the onset of primary myelofibrosis (PMF) harboring JAK2 (Janus Kinase 2 gene) mutation (JAK2V617F) when she was 15 months old. She was monitored over 25 years, a period in which she was treated with spleen radiotherapy and recombinant interferon α. She also underwent splenectomy when she was 13 years old, due to massive splenomegaly, anemia and various infection disease episodes. The longstanding evolution of the patient enabled us to verify that there were no complications related to post-splenectomy events and/or blast transformation. To the best of our knowledge, this is the first reported case of severe PMF with JAK2 mutation in a child. We provide evidence that a better quality of life and long survival in pediatric PMF may be provided by splenectomy.

Musashi 2 (also known as MSI2), a mRNA binding protein is reported to control critical stem cell fate decisions by binding to the 3’untranslated region of target mRNAs, thereby inhibiting translation. MSI2 is preferentially expressed in hematopoietic tissue, in particular early myeloid progenitors. Moreover, investigators suggest upregulated MSI2 disrupts regulatory pathway/s leading to hematopoietic stem cell proliferation, impaired myeloid differentiation and worse clinical prognosis in CML and AML (Kharas et al. Nat Med. 2010; 16:903; Ito et al. Nature. 2010; 466:765). Indeed we have confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in chronic phase (CP), irrespective of lymphoid or myeloid transformation. Furthermore, we have shown MSI2 and BCR-ABL1 expressions correlate. Here we report data implying MSI2 functions viaTGFβ1 signalling pathway.

Despite the unprecedented success of tyrosine kinase inhibitors (TKIs), the clinical management of 20–30% of patients with chronic myeloid leukemia (CML) experiencing primary or secondary resistanc...