Detection of activating mutations in RAS/RAF/MEK/ERK and JAK/STAT signaling pathways
Authors: Zarubina, K., I; Parovichnikova, E. N.; Surin, V. L.; Pshenichnikova, O. S.; Gavrilina, O. A.; Isinova, G. A.; Troitskaia, V. V.; Sokolov, A. N.; Gal'tseva, I., V; Kapranov, N. M.; Davydova, Iu O.; Obukhova, T. N.; Sudarikov, A. B.; Savchenko, V. G.
Issue. The study of activating mutations (NRAS, KRAS, FLT3, JAK2, CRLF2 genes) of RAS/RAF/MEK/ERK and JAK/STAT signaling pathways in B-cell acute lymphoblastic leukemia (B-ALL) in adult patients which are included in Russian multicenter clinical trials. Materials and methods. Within the multicenter study there were 119 adult patients included with de novo B-ALL. The study was considered as prospective and retrospective. The group with BCR-ABL1-negative B-ALL consisted of up to 93 patients (45 male and 48 female, at the age of 17 to 59, the median age - 31), they were treated according to the protocols ALL-2009, ALL-2016. The median follow-up lasted for 19 months (1-119). The group with BCR-ABL1-positive B-ALL with up to 26 patients (10 male and 16 female, at the age of 23 to 78, the median age 34 years) was included in the study as well. The treatment was carried out according to the protocols ALL-2009 and ALL-2012 in combination with tyrosine kinase inhibitors. The median follow-up lasted for 23 months (4-120). The molecular analysis of activating mutations in NRAS, KRAS genes (RAS/RAF/MEK/ERK signaling pathway) and JAK2, CRLF2 genes (JAK/STAT signaling cascade) was performed via Sanger sequencing. The internal tandem duplications (ITDs) in FLT3 gene were studied by fragment analysis. The evaluation of CRLF2 expression was fulfilled via flow cytometry. Results. Activating mutations in NRAS, KRAS, FLT3 genes were found in 22 (23.6%) patients with BCR-ABL1-negative B-ALL. In total, 23 mutations were revealed in the NRAS (n=9), KRAS (n=12), and FLT3 (n=2) genes, according to statistics that was significantly more frequent than with BCR-ABL1-positive B-ALL, these genes mutations were not identified in patients (p=0.007). The frequency of mutations detection in KRAS and NRAS genes in patients with BCR-ABL1-negative B-ALL was comparable as 12.9% (12 of 93) to 9.7% (9 of 93), respectively (p=0.488). One patient was simultaneously revealed 2 mutations in the KRAS gene (in codons 13 and 61). FLT3-ITD mutations were detected in 3.5% (2 of 57) cases of BCR-ABL1-negative B-ALL. In patients with BCR-ABL1-positive B-ALL FLT3-ITD mutations were not assessed. Violations in the JAK/STAT signaling cascade were detected in 4 (4.3%) patients with BCR-ABL1-negative B-ALL. They were represented by the missense mutations of JAK2 gene (n=3) and the overexpression of CRLF2 (n=2); in one patient were detected the overexpression of CRLF2 and a mutation in JAK2 gene simultaneously. No mutations were found in CRLF2 gene. In patients with BCR-ABL1-positive B-ALL no JAK2 mutations were detected. As long as analyzing demographic and clinical laboratory parameters between groups of patients with and without mutations, there were no statistically significant differences obtained. In the analyzed groups of patients, long-term therapy results did not differentiate according to the mutations presence in NRAS, KRAS, FLT3, JAK2 genes. Also, substantive differences were not shown in the rate of the negative status achievement of the minimum residual disease between patients with and without activating mutations in the control points of the protocol (on the 70th, 133rd and 190th days). Conclusion. NRAS, KRAS, FLT3, JAK2 activating mutations do not affect the long-term results of the therapy and the rate of the negative status achievement of the minimum residual disease in patients with BCR-ABL1-negative B-ALL treated by the Russian multicenter clinical trials.
Epigenetic Alterations Are Associated With Gastric Emptying Disturbances in Diabetes Mellitus
CLINICAL AND TRANSLATIONAL GASTROENTEROLOGY
Authors: Narayanan, Susrutha Puthanmadhom; Lee, Jeong-Heon; Bhagwate, Aditya; Kuwelker, Saatchi; Yan, Huihuang; Ordog, Tamas; Bharucha, Adil E.
INTRODUCTION: Epigenetic modifications have been implicated to mediate several complications of diabetes mellitus (DM), especially nephropathy and retinopathy. Our aim was to ascertain whether epigenetic alterations in whole blood discriminate among patients with DM with normal, delayed, and rapid gastric emptying (GE). METHODS: Using the ChIP-seq (chromatin immunoprecipitation combined with next-generation sequencing) assays, we compared the genome-wide enrichment of 3 histone modifications (i.e., H3K4me3, H3K9ac, and H3K27ac) in buffy coats from 20 diabetic patients with gastrointestinal symptoms and normal (n = 6), delayed (n = 8), or rapid (n = 6) GE. RESULTS: Between patients with DMwith delayed vs normal GE, there were 108 and 54 genes that were differentially bound (false discovery rate < 0.05) with H3K27ac and H3K9ac, respectively; 100 genes were differentially bound with H3K9ac in patients with rapid vs normal GE. The differentially bound genes with H3K27ac were functionally linked to the type 2 immune response, particularly Th2 cell activation and function (e.g., CCR3, CRLF2, CXCR4, IL5RA, and IL1RL1) and glucose homeostasis (FBP-1, PDE4A, and CMKLR1). For H3K9ac, the differentially occupied genes were related to T-cell development and function (e.g., ICOS and CCR3) and innate immunity (RELB, CD300LB, and CLEC2D). Compared with normal GE, rapid GE had differential H3K9ac peaks at the promoter site of diverse immunity-related genes (e.g., TNFRSF25 and CXCR4) and genes related to insulin resistance and glucose metabolism. Motif analysis disclosed enrichment of binding sites for transcription factors relevant to the pathogenesis and complications of DM. DISCUSSION: GE disturbances in DM are associated with epigenetic alterations that pertain to dysimmunity, glucose metabolism, and other complications of DM.