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

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

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

Neoplasias mieloproliferativas crónicas BCR-ABL negativas, manifestación clínica y evolución. Experiencia en el Hospital Italiano de Buenos Aires

BCR-ABL negative myeloproliferative neoplasms(MPNs) are a heterogeneous group of clonal hematopoietic disorders characterized by the overproduction of differentiated hematopoietic cells, includingpolycythemia vera (PV), essential thrombocythemia(ET), primary myelofibrosis (PMF) and prefibroticprimary myelofibrosis (prePMF).The WHO classification was revised in 2016 adding specific molecular findings with an impact on diagnosis and treatment. This integrates clinical, molecular and pathological criteria and provides a central role to morphological examination of the bone marrow (BM). It also discriminates between essential thrombocythemia (ET) and the prefibrotic phase of primary myelofibrosis (pre PMF).In order to better understand this entity and to have its own population data, a retrospective cohort study was carried out at the Hospital Italiano de Buenos Aires, which included 264 patients with a diagnosis of Philadelphia-negative chronic myeloproliferative neoplasms between January 2004 and December 2017 and a pathological review of 64 patients with a diagnosis of essential thrombocythemia prior to 2016 (using the criteria of the new WHO classification).The analysis showed that the characteristics of our population are similar to those reported in the literature, with rates of global survival and transformation similar to the global population. Regarding the review of bone marrow pathology, the diagnosis of ET was confirmed in 56% of the patients. In patients who were reclassified to PMF prefibrotic stage (44%), a higher percentage of medullary fibrosis and splenomegaly was found at diagnosis. Las neoplasias mieloproliferativas BCR-ABL negativas son un grupo heterogéneo de trastornos hematopoyéticos clonales caracterizados por la sobreproducción de células hematopoyéticas diferenciadas.Incluye policitemia vera (PV), trombocitemia esencial (TE), mielofibrosis primaria (MFP) y mielofibrosis primaria prefibrótica (MFPpre).La clasificación de la OMS 2001-2008 fue revisada en el año 2016 agregando hallazgos moleculares específicos con un impacto en el diagnóstico y el tratamiento. Integra criterios clínicos, moleculares y patológicos y mantiene un papel central para el examen morfológico fino de la médula ósea.Con la finalidad de comprender mejor esta entidad y de contar con datos poblacionales propios, se realizó un estudio de cohorte retrospectiva en el Hospital Italiano de Buenos Aires que incluye 264 pacientes con diagnóstico de neoplasia mieloproliferativa crónica Phi negativa entre enero 2004 y diciembre 2017y se realizó la revisión anatomopatológica de 64 pacientes con diagnóstico de trombocitemia esencialprevio al 2016 (utilizando los criterios de la nueva clasificación de la OMS).El análisis demostró que las características de nuestra población son similares a las reportadas en la bibliografía, con tasas de sobrevida global y de transformación similares en la población global.En cuanto a la revisión de la patología de la médula ósea, el diagnóstico de TE fue confirmado en el 56%de los pacientes. En los pacientes que recategorizaron a mielofibrosis primaria en estadio prefibrótico(44%) se encontró un porcentaje mayor de fibrosis medular y de esplenomegalia al diagnóstico

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