Arabinoxylan from Argentinian whole wheat flour promote the growth of Lactobacillus reuteri and Bifidobacterium breve

Arabinoxylans are part of dietary fibre and have received attention given their emergent prebiotic character. Four arabinoxylans extracts were obtained from Argentinian soft and hard wheat. In vitro assays were performed to describe the extent to which the extracts from whole wheat flour support selective growth of Bifidobacterium breve and probiotic Lactobacillus reuteri ATCC23272 in a defined media. The prebiotic effect was evaluated by three quantitative scores: relative growth, prebiotic activity score and prebiotic index. For prebiotic index equation the growth of Bacteroides and Clostridium strains was compared to that of bifidobacteria and lactic acid bacteria. All the arabinoxylans extracts supported the growth of Lactobacillus and Bifidobacterium, reaching higher prebiotic activity score values than inulin (0·37 and 0·36 for Lactobacillus and Bifidobacterium respectively). AX2 from soft wheat and AX4 from hard showed similar prebiotic index value to commercial inulin (2·64, 2·52 and 2·22 respectively), and AX3 extract presented higher prebiotic index value (4·09) than the positive control and other prebiotic index reported for arabinoxylans. These extracts could be used as prebiotic, synbiotic compositions or novel food prototypes to treat dysbiosis associated with many diseases. Significance and Impact of the Study: The present work demonstrates that AX extracts from Argentinian soft and hard wheat promote efficiently the growth of probiotic strain L. reuteri ATCC23272 and B. breve 286, validated with three different parameters that consider the growth of representative strains of Bacteria genera found in the gut. The evaluation of AX extracts as a food supplement in a murine model could confirm their ability to modulate the microbiome. Novel food prototypes including AX and probiotics could relieve local symptoms and may act as psychobiotics with a beneficial effect on microbiome-brain axis.Fil: Paesani, Candela. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Salvucci, Emiliano Jesus. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Moiraghi, Malena. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Fernandez Canigia, Liliana Beatriz. Hospital Aleman; ArgentinaFil: Perez, Gabriela Teresa. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentin

Clinical outcome of chronic myeloid leukemia imatinib-resistant patients: Do BCRABL kinase domain mutations affect patient survival? First multicenter Argentinean study

In imatinib-treated patients with chronic myeloid leukemia (CML), BCRABL mutations are the most common mechanism of resistance. Here we report the first multicenter Argentinean study investigating mutations in those patients with CML who fail or lose response to imatinib, with or without previous interferon treatment. Point mutations were detected in 36 of 154 patients by direct sequencing. In our series, the single most common mutations were G250E, E255K/V, and M351T. The presence of mutations correlated significantly with accelerated phase, lack of molecular response, and lower cytogenetic and hematological responses. While overall survival did not differ between patients with or without mutations, the probability of progression was higher in patients with mutations. Cases with non-P-loop mutations showed a significantly better overall survival from diagnosis. Multivariate analysis showed that the most significant variables related to the development of mutations were accelerated phase, duration of imatinib treatment, and time delay to starting imatinib. Our results demonstrated that mutation frequency increased with the progression of disease, and suggest that imatinib treatment should be started early.Fil: Bengió, Raquel M.. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Riva, Maria E.. Hospital San Mart́n; ArgentinaFil: Moiraghi, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Lanari, Emilio. Hospital Jose Ramon Vidal ; Gobierno de la Provincia de Corrientes;Fil: Milone, Jorge. Hospital Italiano; ArgentinaFil: Ventriglia, Veronica. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Bullorsky, Eduardo. Hospital Británico de Buenos Aires; ArgentinaFil: de Tezanos Pinto, Miguel. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Murro, Hector. No especifíca;Fil: Bianchini, Michele. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Larripa, Irene Beatriz. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Evaluation of the primitive fraction by functional in vitro assays at the RNA and DNA level represents a novel tool for complementing molecular monitoring in chronic myeloid leukemia

Quantification of BCR-ABL1 mRNA levels in peripheral blood of chronic myeloidleukemia patients is a strong indicator of response to tyrosine-kinase inhibitors (TKI)treatment. However, additional prognostic markers are needed in order to better classify patients. The hypothesis of leukemic stem cells (LSCs) heterogeneity and persistence, suggests that their functional evaluation could be of clinical interest. In this work, we assessed the primitive and progenitor fractions in patients at diagnosis and during TKI treatment using functional in vitro assays, defining a ?functional leukemic burden? (FLB). We observed that the FLB was reduced in vivo in both fractions upon treatment. However, different FLB levels were observed among patients according to their response to treatment, suggesting that quantification of the FLB could complement early molecular monitoring. Given that FLB assessment is limited by BCR-ABL1 mRNA expression levels, we developed a novel detection method of primitive cells at the DNA level, using patient-specific primers and direct nested PCR in colonies obtained from functional in vitro assays. We believe that this methodcould be useful in the context of discontinuation trials, given that it is unknown whether the persistent leukemic clone represents LSCs, able to resume the leukemia upon TKI removal.Fil: Ruiz, María Sol. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Sanchez, María Belén. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Argenomics; ArgentinaFil: Gutierrez, Leandro German. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Instituto Alexander Fleming, Bs. As.; ArgentinaFil: Koile, Daniel Isaac. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Yankilevich, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Mosqueira, Celeste. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Cranco, Santiago. Fundaleu; ArgentinaFil: Custidiano, María del Rosario. Hospital Italiano de La Plata; ArgentinaFil: Freitas, Josefina. Provincia de Buenos Aires. Hospital Nacional Profesor A. Posadas; ArgentinaFil: Foncuberta, Cecilia. Instituto Alexander Fleming; ArgentinaFil: Moiraghi, Beatriz. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Pavlovsky, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Pérez, Mariel Ana. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Ventriglia, Verónica. Provincia de Buenos Aires. Hospital Nacional Profesor A. Posadas; Argentina; ArgentinaFil: Sánchez Ávalos, Julio César Américo. Instituto Alexander Fleming; ArgentinaFil: Mordoh, Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bianchini, Michele. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentin

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1β and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.Fil: de Luca, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Lev, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Camacho, Maria F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Goette, Nora Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Sackmann, Federico. Fundación Para Combatir la Leucemia; ArgentinaFil: Castro Ríos, Miguel A.. No especifíca;Fil: Moiraghi, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Cortes Guerrieri, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Bendek, Georgina. Hospital Italiano; ArgentinaFil: Carricondo, Emiliano. Universidad Austral. Hospital Universitario Austral; ArgentinaFil: Enrico, Alicia. Hospital Italiano; ArgentinaFil: Vallejo, Veronica. Instituto Cardiovascular de Buenos Aires; ArgentinaFil: Varela, Ana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Khoury, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Gutierrez, Marina. Laboratorio Stamboulian; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Platelet toll-like receptors mediate thromboinflammatory responses in patients with essential thrombocythemia

Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR and MPL, which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting.Fil: Marin Oyarzún, Cecilia Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Goette, Nora Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Lev, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: de Luca, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Baroni Pietto, Maria Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Moiraghi, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Castro Ríos, Miguel A.. Consultorios Hematológicos; ArgentinaFil: Vicente, Angeles. Hospital Alemán; ArgentinaFil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

miRNome profiling of clonal stem cells in Ph+ CML

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.Fil: Ruiz, María Sol. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sánchez, María Belén. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Bonecker, Simone. Instituto Nacional de Câncer; BrasilFil: Furtado, Carolina. Instituto Nacional de Câncer; BrasilFil: Koile, Daniel Isaac. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Yankilevich, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Cranco, Santiago. Instituto Alexander Fleming; ArgentinaFil: Custidiano, María del Rosario. Instituto Alexander Fleming; ArgentinaFil: Freitas, Josefina. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Moiraghi, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pérez, Mariel Ana. Gobierno de la Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal General de Agudos "prof. Dr. Rodolfo Rossi".; ArgentinaFil: Pavlovsky, Carolina. Fundación Para Combatir la Leucemia; ArgentinaFil: Varela, Ana Inés. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Ventriglia, Verónica. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Sánchez Ávalos, Julio César Américo. Instituto Alexander Fleming; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Zalcberg, Ilana. Instituto Nacional de Câncer; BrasilFil: Mordoh, Jose. Fundación Cáncer; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valent, Peter. Medical University of Vienna; AustriaFil: Bianchini, Michele. Fundación Cáncer; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Detection of leukemic stem cell (CD26+) in patients with chronic myeloid leukemia with different molecular response

La leucemia mieloide crónica (LMC) se caracteriza por la t(9;22)(q34;q11), generando el gen de fusión BCR-ABL1 que codifica la oncoproteina P210 con actividad constitutiva de tirosina kinasa. Los pa-cientes que presentan una profunda y sostenida res-puesta molecular a los inhibidores de tirosina kinasa (ITK) pueden interrumpir el tratamiento. Sin em-bargo, aproximadamente el 50% de los casos presen-tan recurrencia molecular, probablemente debido a la persistencia de la stem cell leucémica (SCL) quies-cente (no replicativa, transcripcionalmente silente). Recientes publicaciones han demostrado que la ex-presión de la enzima dipeptidil peptidasa IV (CD26) está restringida a la fracción CD45+/CD34+/CD38- de la SCL en LMC y no se ha detectado en otras SCL mieloides/linfoides ni en medula ósea normal. Por esta razón CD26 es considerado un nuevo y especí-fico bio-marcador de LMC.El objetivo del trabajo fue detectar las SCL CD26+ en pacientes con LMC con diferente respuesta molecular (RM) y determinar si estas células persisten aun en casos con respuesta molecular profunda (RMP).Se analizaron 193 muestras de pacientes con LMC (107 sexo masculino y 86 femenino) para evaluar la SCL mediante citometría de flujo usando el panel de anticuerpos monoclonales: CD45, CD34, CD38, CD26, CD117, CD123, CD3 y HLA-DR. En para-lelo se realizó el estudio de la respuesta molecular mediante qRT-PCR BCR-ABL1 (Método Taqman). Ambos estudios se realizaron en simultáneo en la misma muestra, durante el seguimiento en diferen-tes momentos bajo tratamiento con ITK (imatinib, nilotinib o dasatinib). Los pacientes con una reducción de BCR-ABL1 ≥ a 3 log tenían una significativa menor proporción de casos con SCL CD26+ comparado con aquellos que tenían <3 log de reducción de los transcriptos (p<0.0003, OR: 3.4, 95% CI: 1,7 - 6,8). Consideran-do los 76 casos con RMP (33 RM4.0; 38 RM4.5 y 5 RM5.0), solamente 12/76 (16%) mostraron per-sistencia de la SCL CD26+. La presencia de la SCL CD26+ se redujo acorde aumenta la profundidad de la RM: 21%, 13% y 0% en RM4.0, RM4.5 y RM5.0 respectivamente. Nuestros resultados muestran que los pacientes con buena RM (≥3log), se asociaron con baja propor-ción de casos con SCL CD26+. Cuando la detección de SCL se evaluó exclusivamente en los casos con RMP, se observó que el decrecimiento de la SCL se asoció a mayor profundidad de la RM. La stem cell leucémica es altamente quiescente por lo cual podría estar presente aun en casos con respuesta molecular indetectable. En nuestro estudio la persistencia de SCL fue del 16% en casos con respuesta molecular profunda, indicando que la SCL persiste a pesar de la RM alcanzada. Este nuevo abordaje investigando la SCL podría ser útil en el seguimiento a largo plazo y de gran importancia en la evaluación de la recu-rrencia molecular en los casos incluidos en protoco-los de discontinuación.Chronic Myeloid Leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q21) resulting in the BCR-ABL1 fusion gene encoding the P210 oncoprotein with a constitutive tyrosine kinase (TK) activity. It is known that patients with at least two years in deep and sustained molecular response could stop TK inhibitor (TKI) treatment. However, half of them show molecular recurrence, probably due to the persistence of transcriptionally quiescent leukemic stem cells (LSC). Recent studies show that the expression of the enzyme dipeptidylpeptidase IV (CD26) is mainly restricted to the CD45+/CD34+/ CD38- fraction in CML LSC, and it is not found in other myeloid/lymphoid LSC or in normal bone marrow. For this reason, CD26 is considered a novel specific biomarker in CML. The aim of this study was to detect the CD26+ LSC in CML patients with different molecular responses (MR) and to assess if these cells remain even in deep molecular response (DMR). We have evaluated 193 CML patients (107 males and 86 females) for detection of LSC by flow cytometry using the panel: CD45, CD34, CD38, CD26, CD117, CD123, CD3 and HLA-DR and the BCR-ABL1 quantification by qRT-PCR (Taqman method). Both studies were carried out simultaneously on the same sample, during the follow up at different time points under TKI treatment (Imatinib, Nilotinib, Dasatinib). Patients with ≥ 3 BCR-ABL1 log reduction had a significantly lower percentage of cases with CD26+ LSC compared with those who had < 3 log reduction (p<0.0003, OR: 3.4, 95% CI: 1,7 - 6,8). Out of the 76 patients with DMR (33 in MR4.0, 38 in MR4.5 and 5 in MR5.0) only 12/76 (16%) showed persistence of CD26+ LSC. Furthermore, the presence of CD26+ LSC decreased accordingly to the achieved DMR: 21%, 13% and 0% in MR4.0, MR4.5 and MR5.0 respectively, without significant differences. Our results show that patients with good MR (≥3log) were significantly associated with a lower proportion of cases with LSC presence. When the LSC analysis was performed exclusively in cases with DMR, we observed that the decrease of LSC accompanied the deepness of the molecular response. Since the LSC is highly quiescent, it could be present even in cases with undetectable MR. In our study persistence of LSC in cases with DMR was 16%, indicating that these cells remain despite the MR achieved. This new approach to the study of the LSC could be useful in long-term follow-up and of great importance in the evaluation of molecular recurrence in cases included in discontinuation protocols.Fil: Bengio, R. M.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Peña, M.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Palacios, F.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Moiraghi, B.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Negri Aranguren, P.. Instituto Privado de Hematologia y Hemoterapia; ArgentinaFil: Enrico, A.. Hospital Italiano de La Plata; ArgentinaFil: Mariano, R.. Provincia de Entre Rios. Hospital San Martin; ArgentinaFil: Toloza, Maria Jazmin Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Leucemia mieloide Crónica

La LMC es una enfermedad que afecta a las células madre hematopoyéticas. Se caracteriza por la presencia del cromosoma Ph, que resulta de la translocación recíproca entre los cromosomas 9 y 22 [t(9;22) (q34;q11)], y genera la yuxtaposición de los genes BCR y ABL1 dando origen a una proteína oncogénicacon actividad de tirosina kinasa incrementada, alterando las vías de proliferación y supervivencia. Según el punto de ruptura de los genes BCR y ABL1, se generan distintos rearreglos (b2a2 o b3a2, e1a2 y e19a2 ), dando lugar a proteínas de distintos pesos moleculares (P210, P190, P230). En la mayoría de las LMC, se puede detectar el transcripto de la isoforma P210, pero se han descripto casos con P190, P230 u otras con menor frecuencia. El mejor conocimiento de la biología de la enfermedad y la descripción de los mecanismos de resistencia, permitió el desarrollo de tratamientos blanco-moleculares como ITK, logrando una ventaja significativa en la sobrevida de estos pacientes, dada la gran efectividad en la inactivación de la proteína oncogénica.De esta manera, la introducción del imatinib, generó un cambio en el seguimiento de la LMC. La necesidad de mejorar su eficacia y optimizar el manejo de los pacientes llevó al desarrollo de nuevas formulaciones dentro de los ITKs, dasatinib, nilotinib, ponatinib y bosutinib*. La evolución de las técnicas genéticas y moleculares también permitió mejorar el monitoreo de esta enfermedad. La evaluación de la carga tumoral a través de la cuantificación de transcriptos BCR-ABL1 y su actual posibilidad de detectar hasta 4.5 log de reducción de los mismos, así como la posibilidad de evaluar los mecanismos de resistencia con la detección de mutaciones del gen translocado y la descripción de nuevos potenciales sitios de acción, nos muestran que estamos una vez más en un proceso de constante progreso en el manejo de esta patología. Diversas instituciones y grupos de trabajo en el mundo, como ELN, NCCN, NICE, ESMO, han desarrollado recomendaciones para el manejo de la LMC, logrando generar pautas homogéneas, tanto diagnósticas como terapéuticas y de monitoreo.Fil: Beligoy, Luis. No especifíca;Fil: Bendek del Prete, Georgina Emilia. Instituto Universitario Hospital Italiano de Buenos Aires. Rectorado.; ArgentinaFil: Bengió, Raquel. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Bullorsky, Laura. Hospital Británico de Buenos Aires; ArgentinaFil: Enrico, Alicia. Hospital Italiano de La Plata; ArgentinaFil: Franceschi, Erica. Ministerio de Defensa. Ejército Argentino. Hospital Militar Central Cirujano Mayor "Dr. Cosme Argerich"; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Moiraghi, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Osycka, Victoria. No especifíca;Fil: Riveros, Dardo Alberto. No especifíca;Fil: Rojas, Francisca. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Varela, Ana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Ventriglia, Verónica. No especifíca

In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy

IntroductionTreatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%.MethodsIn this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months.ResultsAt the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p&lt;0.0001).DiscussionThis NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1β and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets