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Coraglia A.,Institute Investigaciones Hematologicas IIHEMA | Galassi N.,CONICET | Felippo M.,CONICET | Bracco M.M.E.D.,Institute Investigaciones Hematologicas IIHEMA | Bracco M.M.E.D.,CONICET
Journal of Immunology Research | Year: 2016

CD4+ T follicular helper cells (T F H) were assessed in adult patients with common variable immune deficiency (CVID) classified according to the presence of granulomatous disease (GD), autoimmunity (AI), or both GD and AI (Group I) or the absence of AI and GD (Group II). T F H lymphocytes were characterized by expression of CXCR5 and PD-1. T F H were higher (in both absolute number and percentage) in Group I than in Group II CVID patients and normal controls (N). Within CXCR5+CD4+ T cells, the percentage of PD-1 (+) was higher and that of CCR7 (+) was lower in Group I than in Group II and N. The percentages of Treg and T F H reg were similar in both CVID groups and in N. T F H responded to stimulation increasing the expression of the costimulatory molecules CD40L and ICOS as did N. After submitogenic PHA+IL-2 stimulation, intracellular expression of T F H cytokines (IL-10, IL-21) was higher than N in Group I, and IL-4 was higher than N in Group II. These results suggest that T F H are functional in CVID and highlight the association of increased circulating T F H with AI and GD manifestations. © 2016 Ana Coraglia et al. Source

BCR-ABL1 point mutations are the most common cause of resistance in patients with chronic myeloid leukemia (CML) who fail or lose response to tyrosine kinase inhibitors. We have developed a rapid method to screen BCR-ABL1 mutations by high resolution melting (HRM). We designed a strategy based on amplification refractory mutational system-quantitative polymerase chain reaction (ARMS-qPCR) to identify and quantify several clinically relevant mutations. From 856 patients with CML studied during 2 years in our laboratory, we selected 32 who showed persistent levels of BCR-ABL1 transcripts (>0.1%) in at least two consecutive studies. Using our strategy, we identified mutations in 11/32 cases (34.4%), while only two of them (6.2%) were detectable by sequencing. Furthermore, we were able to estimate the timing and dynamics of mutated clones, evaluating retrospective samples from the same patient. In cases with lack or loss of molecular response this analysis might be useful for designing early therapeutic strategies. © 2013 Informa UK, Ltd. Source

Ferri C.,CONICET | Bianchini M.,CONICET | Bengio R.,Institute Investigaciones Hematologicas IIHEMA | Larripa I.,CONICET | Larripa I.,Institute Investigaciones Hematologicas IIHEMA
Blood Cells, Molecules, and Diseases | Year: 2014

Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are the current treatment of chronic myeloid leukemia (CML). BCR-ABL1 point mutations are the principal cause of resistance to treatment; however other mechanisms could be involved in failure to TKI therapy. LYN is a src kinase protein that regulates survival and responsiveness of tumor cells by a BCR-ABL1 independent mechanism. PTEN tumor suppressor gene is downregulated by BCR-ABL1 in CML stem cells and its deletion is associated with acceleration of disease. In this study we evaluated the expression of LYN, PTEN and the ratio of both genes in 40 healthy donors (HD) and in 139 CML patients; 88 of them resistant to TKI in different phases of disease and 51 in chronic phase classified as optimal responders (OR) to TKI [40 treated with imatinib or nilotinib (OR-IN) and 11 treated with dasatinib (OR-D) therapy]. When we analyzed the gene expression values of LYN, an increase was observed only in advanced stages of the disease, however, when we analyzed the ratio between LYN and PTEN genes, the group of resistant patients in chronic phase in imatinib or nilotinib treatment (CP-IN) also showed a significant increase. Resistant patients treated with dasatinib, a src kinase inhibitor, presented a similar ratio to the observed in HD. In addition, the LYN/. PTEN ratio and the LYN expression showed a direct significant correlation with BCR-ABL1 transcript levels in unmutated resistant patients treated with non- src kinase inhibitors. We were able to identify 8/35 (23%) of cases in CP-IN and 4/12 (33%) in accelerated phase and blast phase (AP/BC-IN), in which resistance could be associated with an increase in the ratio of the LYN/. PTEN. Our data suggest that the LYN/. PTEN expression ratio may be a sensitive monitor of disease progression in unmutated CML patients under imatinib or nilotinib treatment. This ratio could detect cases when resistance is related to altered LYN expression, suggesting that the treatment change to a src kinase inhibitor would be most suitable to overcome resistance. © 2013 Elsevier Inc. Source

Gonzalez M.S.,CONICET | De Brasi C.D.,CONICET | De Brasi C.D.,Institute Investigaciones Hematologicas IIHEMA | Bianchini M.,Instituto Alexander Fleming | And 5 more authors.
PLoS ONE | Year: 2014

Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2V617F, is highly desirable. In this study, we present an approach to assess the JAK2V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2V617F spaced from one template for JAK2Wild Type were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2 V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53±4.2% and 51.46±4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2V617F from heterozygosity to homozygosity. © 2014 Gonzalez et al. Source

Ferri C.A.,CONICET | Bianchini M.,CONICET | Bengio R.M.,Institute Investigaciones Hematologicas IIHEMA | Moiraghi E.B.,Servicio de Hematologia del Hospital Ramos Mejia | And 4 more authors.
European Journal of Haematology | Year: 2015

Background: Chronic myeloid leukemia (CML) is a hematological disorder that in rare cases, mainly in CML neutrophilic, presents the e19a2 rearrangement. The encoded product is a 230-KDa protein. Despite the remarkable responses to treatment of most patients, a small but significant fraction of them develop clinical resistance to the tyrosine kinase inhibitors (TKIs). The most common mechanism of resistance is point mutations in the ABL1 kinase domain. The recently approved third-generation TKI ponatinib demonstrated remarkable activity in patients with multi-TKI-resistant disease. Particularly impressive was its efficacy in patients with T315I mutation that is resistant to all other TKIs. Methods: Qualitative PCR was carried out by multiplex approach. Relative transcripts quantification was performed by one-step real-time PCR, with a specific Taqman probe and primers for the e19a2 rearrangement. We carried out a mutational screening by high-resolution melting, and the mutation was identified by Sanger method. The mutation burden was quantified by quantitative PCR using allele-specific primers. Results: In a patient with CML, we identified a PCR product corresponding to e19a2 rearrangement harboring T315I mutation. At the time of mutational analysis, during dasatinib treatment, the T315I clone was 100% and the quantification of BCR-ABL1 was 18%. After ponatinib therapy, the T315I mutation burden decreased down to undetectable levels and the BCR-ABL1 transcripts showed a very low value (0.011%). Conclusions: Here, we report the hematological, cytogenetic, and molecular response of a patient with refractory CML in chronic phase with e19a2 transcripts, carrying T315I mutation that was successfully treated with ponatinib. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Source

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