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

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

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<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

miRNome profiling of LSC-enriched CD34+CD38−CD26+ fraction in Ph+ CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool

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.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; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bonecker, Simone. Instituto Nacional de Cancer; BrasilFil: Furtado, Carolina. Instituto Nacional de Cancer; 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. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; 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 Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Zalcberg, Ilana. Instituto Nacional de Câncer; BrasilFil: Mordoh, Jose. Instituto Alexander Fleming; Argentina. Fundación Cáncer; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Valent, Peter. Universidad de Viena; AustriaFil: Bianchini, Michele. Fundación Cáncer; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Elevated plasma levels of IL-6 and MCP-1 selectively identify CML patients who better sustain molecular remission after TKI withdrawal

Abstract 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

DataSheet_1_In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.docx

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 (pDiscussionThis 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.</p