Mutant Allele Frequency Research Articles

Changing Patterns, and Clinical Correlates, of Mutations in Patients with Myelofibrosis over Treatment with Ruxolitinib or Hydroxyurea

Background. About half of patients (pts) with primary (PMF) and post-polycythemia vera/essential thrombocythemia MF (PPV/PET-MF) lose clinical response over prolonged treatment (3y+) with ruxolitinib. Both driver (JAK2, MPL, CALR) and non-driver mutations lack predictive power for the short-term (1y) response (Guglielmelli P, Blood 2015) although pts with >3 mutations are less likely to respond to ruxolitinib (Patel K, Blood 2016). Despite some reductions of Variant Allele Frequency (VAF) of driver mutations, molecular remissions induced by ruxolitinib are exceptional.Aims. To describe modifications of VAF of driver and non-driver mutations and changing patterns of mutation profile, and to correlate with response, in pts with MF receiving ruxolitinib or hydroxyurea.Methods. Sixty-three pts (38 PMF, 25 PPV/PET-MF) in active follow-up (FU) at CRIMM, Florence, were included; 17pts (15 PMF, 2 PPV/PET-MF) received hydroxyurea (HU) and 46 (23 PMF, 23 PPV/PET-MF) ruxolitinib. All pts were annotated for driver mutations and an additional panel of 24 “myeloid” genes by NGS. Paired samples of blood granulocytes were analyzed before starting HU/ruxolitinib therapy (baseline) and at the last FU available while on therapy. For the analysis, we recorded any modification of VAF, either decreasing or increasing, of at least 20% compared to baseline. Mutations were defined “acquired” in case of de novo detection (>2% VAF) and “loss” in case of disappearance of pre-exiting mutation at baseline.Results. The median follow-up from start of therapy was 2.9y for HU-pts and 3.4y Ruxo-pts. At last FU, among Ruxo-pts, 11 (23.9%%) had discontinued for failure, 1 progressed to AML and 16 (34.8%) died; in the HU group, all pts were still on treatment. JAK2 V617F mutation was found in 49 pts (77.8%; 22% HU, 76% Ruxo), CALR mut in 10 (15.9%; 40% HU, 60% ruxo), MPL mut in 1pt in each group; 3 pts were triple negative (2 HU, 1 Ruxo). The most frequent non-driver mutations were ASXL1 (33%), TET2 (20%), NFE2 (15%), ZRSR2 (12%), SRSF2 (8%).HU-pts: During treatment period, JAK2 V617F VAF increased in 2 pts (18%; +43% and +60% vs baseline) and decreased in 1 pt (-63%). No VAF changes were observed in CALR mut pts while MPL VAF increased by 47%. As regards non-driver mutation, VAF increased in 4 pts (31%; median +112%, range +22% to +168%) and remained stable in 13; acquisition of new mutation occurred in 5 pts (29%; 1 each for TET2, TP53 and NRAS and 2 CBL); no pts displayed mutation loss.Ruxo-pts: During treatment period, JAK2 V617F VAF increased in 6 pts (15%; median +27%, range +20% to +50%) and decreased in 9 (23%; median -38%, -21 to -62%). CALR VAF increased of 32% in 1 pt and was unchanged in 5 pts. In the MPL mut pt VAF decreased from 37% to 14%. As regards non-driver mutation, VAF increased in 26% of cases (median +103%) and decreased in 9 pts (23%; median -31%); in 3 pts, loss of 4 mutations was observed. Acquisition of new mutation was observed in 8 pts (17%; 1 each for EZH2, SF3B1, ASXL1, PTPN11, 2 NRAS and 3 KRAS).Response of symptoms or splenomegaly by the IWG-MRT criteria (Tefferi et al, Blood 2013) was achieved in 86% and 57% of pts after a median of 2.9mo and 13.1mo; of these, 13 (34%) and 12 (48%) pts lost clinical response after a median of 3.3y for symptoms and 2.8y for spleen volume reduction. ASXL1 mut at baseline was associated with a significantly lower likelihood of IWG-MRT-defined spleen response over prolonged treatment (32% vs 63% in the absence of ASXL1 mut; P=.040) . The probability of maintaining a spleen response at 2y in ASXL1 mut pts was 33% compared with 87% of unmutated pts. No mutation individually predicted for symptoms response or therapy-discontinuation. Acquisition of at least one mutation was associated with higher rate of discontinuation (36% vs 11% in those pts without clonal evolution, P=.048). Acquisition of 1 and >2 mutations correlated with higher rate of death (63% vs 29% and 45% vs 7%, respectively, compared to pts without clonal evolution).Conclusions. VAF changes of driver and non-driver mutations, as well as loss/acquisition of new non-driver mutations, occurred at comparable rate and with similar patterns in HU- and ruxolitinib-treated pts. Acquisition of new mutations while on ruxolitinib correlated with higher rate of discontinuation and death. DisclosuresVannucchi:Novartis: Honoraria, Speakers Bureau; Shire: Speakers Bureau. Bosi:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees.

Recurrent STAT3 Mutations in the Lymphocytes of Pure Red Cell Aplasia without T-Cell Large Granular Lymphocytic Leukemia

▪Background: Activating somatic mutations of STAT3 are one of the most frequently recognized genetic alterations in T-cell lymphoma including T-cell large granular lymphocytic leukemia (T-LGLL). T-LGLL is associated with certain proportions of patients (pts) with acquired aplastic anemia (AA) and/or myelodysplastic syndromes (MDS). Pure red cell aplasia (PRCA), especially acquired PRCA, is a syndrome defined by an anemia with marked reduction or absence of erythroid production associated with immunological mechanisms. PRCA is classified into primary and secondary types including T-LGLL. Previous reports have described that T-LGLL with STAT3 mutations was closely associated with PRCA. However, it is yet to be clear whether STAT3-mutated T cells would be involved in the pathogenesis of other cytopenias, such as AA, MDS, primary PRCA or secondary PRCA without (w/o) T-LGLL. In this study, we analyzed the profiles of STAT3 gene mutations in bone marrow failure syndrome (BMFS)s including AA/MDS and PRCA.Methods: Peripheral blood samples were collected from 94 patients with BMFSs, including AA, MDS, AA/PNH, and PRCA with or w/o T-LGLL. When possible, T-cell receptor (TCR) Vβ repertoire of the patients was determined by flow cytometry. DNA was extracted from mononuclear cells (MNC), and STAT3 Y640F and D661Y, two hot spot mutations, were detected with sensitive allele specific-PCR (AsPCR) methods. Then we performed deep amplicon sequencing analysis of STAT3 in PRCA pts. CD8+ T cells were sorted if possible, and also subjected to the analyses.Results: The recruited pts included 38 AA, 20 MDS, 7 AA/PNH, 11 PRCA with T-LGLL, and 18 PRCA w/o T-LGLL. Among PRCA pts w/o apparent T-LGLL, 10 had primary PRCA and 8 had secondary PRCA including 2 with thymoma. The clinical characteristics of PRCA are shown in Table 1. TCR Vβ repertoire analysis were performed in 8 PRCA w/o T-LGLL and 5 PRCA with T-LGLL. All pts with PRCA associated with T-LGLL showed restricted Vβ repertoire. On the other hand, only 1pt showed monoclonal Vβ pattern in PRCA w/o TLGLL. AsPCR did not detect Y604F or D661Y mutations in any of 65 pts with AA, MDS, or AA/PNH. In contrast, 2 pts with PRCA and 4 pts with PRCA-T-LGLL were positive for either of the mutations, suggesting STAT3 mutations may be unique to PRCA pts. Further analysis with deep amplicon analysis in the 29 PRCA pts revealed STAT3 mutations in 11 pts (61%) of PRCA w/o T-LGLL, which included 6 pts with primary PRCA and 5 pts with secondary PRCA associated with autoimmune diseases (n=2), 2 thymoma (n=2) and thymic cancer (n=1). The median variant allele frequency of STAT3 mutations was 2.3% (0.2-48.9). N538Y and I653V were most frequent mutations in PRCA w/o T-LGLL. Eight pts (73%) with T-LGLL-associated PRCA were also positive for STAT3 mutation. Thirty-nine % and 70% of STAT3 mutation were found in SH2 domain in PRCA w/o T-LGLL and PRCA with T-LGL, respectively. There was no difference in clinical features between pts with and w/o STAT3 mutations.Discussion: The high frequency of STAT3 mutations in PRCA pts regardless of etiology and the absence of the mutation in pts with AA/MDS suggest that STAT3 mutations may serve as a molecular marker of PRCA. The discordant results with regard to AA/MDS pts from a previous report which detected STAT3 mutations in 4.9% of AA/MDS patients by AsPCR methods (Jerez et al. BLOOD 2013) may be due to the difference in ethnic backgrounds of the pts studied. Given very low frequencies of STAT3 mutations in AA and MDS that were revealed by several previous studies, JAK/STAT signaling systems in the axis of STAT3 mutations may have unique roles in the pathogenesis of PRCA.Conclusion: STAT3 mutations are frequently detected in pts with PRCA even in pts w/o T-LGLL and the JAK/STAT pathway may therefore be therapeutic targets of PRCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Dynamic Epigenetic Landscape in Genetic and Immunophenotypic Stable Pediatric AML

The majority of pediatric acute myeloid leukemia (AML) patients achieve complete remission. However, after this initial response to therapy, relapse occur in around forty per cent. The failure of therapy has been thought to be due to drug resistance resulting either from accumulating mutations, or already a characteristic of leukemic stem cells present at diagnosis. These mechanisms are shown to be true in part of the patients, however in quite a number of these relapses other mechanisms must play a role. In adult leukemias high epigenetic heterogeneity is associated with worse clinical outcomes. Remarkably, the DNMT3, TET and IDH mutations often found in adults are almost non-existent in children, despite the fact the leukemias are phenotypically very alike. We tested the hypothesis that, apart from genetic heterogeneity, epigenetic heterogeneity contributes to therapy resistance and thereby initiation of relapse.Methods: We performed flow cytometric immunophenotypic analyses, whole exome sequencing and epigenome sequencing (by enhanced reduced representation bisulfite sequencing) on bone marrow (BM) samples taken at time of diagnoses, within two weeks after achievement of CR and at time of relapse of three selected patients (diagnosed between 2007-2014, treated according to the Dutch-Belgian DB-AML-01 protocol). Of each patient, apart from whole diagnosis sample and whole relapse sample (all with a minimum of 90% blast cells), the MRD sample was FACS sorted to select for blast cells.Results: Leukemia associated immunophenotypes phenotype found at time of diagnosis remained the same over time with no emerging new immunophenotypes coinciding with emergence of relapses. We further evaluated whether changes in variant allele frequency (VAF) of mutations between diagnosis and subsequent time points could be linked to the development of relapse. Generally, VAF numbers decreased or remained stable over time, with no new mutations arising that characterized relapse. None of the patients had a mutation in DNTM3A, TET or IDH genes, accounting for any epigenetic dysregulation.In contrast to the findings for immunophenotypes and genetics, extensive epigenetic changes were found at the different time points. In samples taken within two weeks after reaching complete remission, a median of 23,665 (range 19,303-28,808) differentially methylated regions (DMRs) was found compared with the diagnosis samples. When comparing the corresponding relapse sample with this CR/MRD sample, another set of DMRs was found: median 19,625 (range 14,680-30,410). In all three patients, the majority of exons, intergenic, intron and promoter regions are uniquely hyper or hypo methylated at time of relapse (median of 88%, range 81%-95%), with a small proportion already differentially methylated at time of remission (median 11%, range 4%-18%). We hypothesize that these changes in epigenetic status occurring after CR, possibly contributes to relapse. Pathway analysis will probably give clues for understanding the pathophysiology and hopefully new targets for treatment.Conclusion: Our preliminary data suggest that epigenetic regulation may play a vital role in development of relapse. Ultimately, a better understanding of the role of epigenetics could open new therapeutic approaches for pediatric AML. DisclosuresOssenkoppele:Roche: Honoraria; Novartis: Research Funding; Karyopharm: Consultancy, Research Funding; J&J: Consultancy, Honoraria; Celgene: Honoraria, Research Funding. Kaspers:Janssen-Cilag: Research Funding. Cloos:Takeda: Honoraria.

Relative Lymphocytosis Is Associated with Improved Outcome in Myelofibrosis and May Account for the Favorable Prognosis of the Calr mutation

Introduction: Neutrophil-Lymphocyte ratio is prognostic in solid tumors and lymphomas which may serve as an index of anti-tumor immune response. The role of immune surveillance in myeloproliferative neoplasms (MPNs) is being increasingly explored. In this study, we aimed to investigate whether such surrogate markers of immune response offers prognostic significance in MPNs. Myelofibrosis (MF) is characterized by leukoerythoblastosis in the peripheral blood which leads to relative lymphopenia. To account for this, we modified the neutrophil:lymphocyte ratio by instead assessing the prognostic significance of relative lymphocytosis in terms of the total white blood cell (WBC):lymphocyte ratio (WLR). Further, given recent evidence of enhanced immunogenicity of CALR mutant MPN cells, we explored if WLR varies based upon driver mutation.Methods: We retrospectively reviewed MF patients seen at our institution between 1/1/2001 and 5/1/2017. MF was defined by 2016 WHO criteria. Laboratory values were calculated at first presentation and overall survival (OS) was calculated from time of first presentation. WLR defined as WBC/absolute lymphocyte count (ALC). A WLR cutpoint of 7 was based on median WLR value.Results: Of 436 total MF patients, 431 had baseline lab values at time of first presentation, and 319 were informative for somatic gene mutations. Two-hundred forty-nine (78%) were JAK2 mut, 36 (11%) were CALR mut, 17 (5%) were MPL mut and 17 (5%) were triple-negative. On univariate analysis, absolute lymphocyte count (ALC) did not correlate with OS (p = 0.82), and there was no difference in ALC based on driver mutation. WBC:ALC ratio (WLR), however, was associated with inferior OS (p = 0.001) in univariate analysis. High WLR directly correlated with age (p < 0.001), monocytosis (p < 0.001), increased peripheral blasts (p < 0.001), immature myeloid cells (p < 0.001), bone marrow cellularity (p < 0.001), splenomegaly (p = 0.001), constitutional symptoms (p = 0.02), IPSS (p < 0.001) and DIPSS prognostic scores (p =0.01), mutations in JAK2 (p < 0.001), TET2 (p = 0.04) and EZH2 (p < 0.001) and total number of somatic mutations (p = 0.009). Low WLR correlated with presence of CALR (p = 0.04) and DNMT3A mutations (p = 0.04). On multivariate analysis, controlling for DIPSS, albumin, and bone marrow fibrosis, WLR remained a significant prognostic co-variate (p = 0.04).Comparison of WLR according to specific driver mutation showed a trend toward lower WLR in CALR mutant versus JAK2 mutant MF (p =0.07). Low WLR was also associated with improved OS (HR 0.67 [0.51-0.89]; p = 0.005) in the entire MF cohort as well as in JAK2 mut (p = 0.002) and CALR mut subgroups (p = 0.06).Lastly, we assessed the relationship between WLR and variable allele frequency (VAF) of JAK2 and CALR driver mutations as well as the commonly occurring somatic mutations ASXL1 (n = 53) and SRSF2 (n = 29). High WLR was directly associated with VAF of JAK2 (p = 0.01) and CALR (p = 0.04), though the correlation was stronger with CALR than JAK2 (r2 = 0.30 vs 0.05). A trend towards a positive correlation was found between WLR and ASXL1 VAF (p = 0.07) whereas there was no correlation with SRSF2 VAF (p = 0.70).Conclusion: WLR is an independent prognostic co-variate in patients with MF with higher ratios conferring inferior outcomes. Decreased WLR in CALR mut compared with JAK2mut MF patients coupled with the strong correlation with CALR VAF suggests this may underlie the more favorable prognosis in CALR mut patients. Further investigations are warranted to discern the biological responses driving the changes in WLR index. [Display omitted] DisclosuresEpling-Burnette:Incyte: Research Funding; Forma Therapeutics: Research Funding; Celgene: Research Funding. Padron:Incyte: Honoraria, Research Funding. Sallman:Celgene: Research Funding. Lancet:Pfizer: Other: Institutional research funding; Biopath, Biosight, Boehringer Ingelheim, Celator/Jazz, Celgene, Janssen, Karyopharm Therapeutics, and Novartis: Consultancy. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria.

Identification of Progression-Associated Mutations in Chronic Myeloid Leukemia (CML) By Comparative Genomic Analysis

Introduction: CML progression from chronic phase (CP) to blast crisis (BC) has been related to the acquisition of additional mutations, but detailed information on the genes involved is lacking. Here we aimed to identify progression-associated driver mutations and potentially targetable pathways.Patients and methods: Bone marrow samples from 58 patients (14 BC-CML, 2 AP and 42 newly diagnosed CP) were collected from Helsinki University Hospital and National Cancer Institute, Cairo University. For CML-CP patients, CD34+ sorted cells were used to facilitate the detection of low variant allele frequency mutations. Genetic alterations were identified by whole exome sequencing (WES) (8 BC, 1 AP and 15 CP samples) and deep targeted sequencing (Nimblegen gene panel, SeqCap EZ comprehensive cancer Design) (10 BC, 1 AP, and 32 CP diagnostic samples and 15 follow up samples) comprising target regions for 578 driver genes with a documented association with cancer. RNA sequencing was used to identify progression-associated gene expression profiles (8 BC and 5 CP diagnostic CD34+ samples).Results: Analysis of data from all 16 advanced patients and 24 CP-diagnostic samples revealed 80 mutations in 62 driver genes in BC samples compared to 69 mutations in 54 driver genes in CP samples. All variants had high oncogenic potential (score >28) as measured by OncoScore (Piazza, R. et al . Sci. Rep. 2017). The median number of mutations per patient (WES data only) was two-fold higher in BC-CML (10, range: 7-14, n=9) than in CP samples (5, range: 1-10, n=11) (p=0.0027). In BC-CML patients, core-binding factor aberrations (RUNX1 mutations and inv(16)) were the most recurrent (n=6) followed by ABL1 (n=5) and BCOR mutations (n=4). Mutations were also discovered in ASXL1 (n=3), NF1, ALK, SETD2, SF3B2, MSH2 (n=2 each), FLT3-TKD, IKZF1 and IDH2 (n=1 each) . In CP-CML, recurrent mutations were detected in ASXL1, TET2 (n=4 each), NOTCH3 (n=3), TET1, KMT2C and PTPRD (n=2 each). CNV data analysis of BC samples identified progression-associated events including +8, inv(16) (n=2 each), i(17q) with P53 loss, LOH in 21q and IKZF1 deletion (n=1 each). Driver mutations known to be associated with hematological malignancies (RUNX1, FLT3 and BCOR) were almost exclusively limited to BC (RUNX1 mutation in one CP-patient who progressed to BC within a year). Epigenetic modifiers (ASXL1, TET1, TET2, and IDH2) represented a major subset of mutated genes in both phases. Mutations in protein tyrosine phosphatase (PTP) genes (putative negative regulators of JAK-STAT signaling) were common in CP patients (n=5), and they were associated with poor response or progression as none of the patients carrying these mutations achieved major molecular remission within 12 months. In CP patients, higher mutation burden (median mutation number from WES data) was associated with poor treatment responses (failure to achieve MMR at 12 months). The median mutation number was almost 2-fold higher in poor responders (8, range: 3-10, n= 5) than in good responders (4.5, range: 2-5, n=4). Variant allele frequencies of putative driver mutations (ex; RUNX1, MECOM, PTP genes) in poorly responding CP patients were tracked in follow up samples, and were found to correlate with BCR-ABL1 levels confirming their presence in the leukemic clone.Conclusion:Comparative analysis of CP-CML and BC-CML patients revealed a group of genes associated with a particular disease phase (e.g. RUNX1, FLT3, and BCOR) of which some could represent therapeutic targets for future studies. In CP-CML patients, mutations in PTP genes were found to correlate with poor response warranting their screening at the time of diagnosis. DisclosuresHeckman:Orion Pharma: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria.

Mutation Profiling Reveals Novel Epigenetics and Splicing Mutations in Chemorefractory Pediatric AML

Despite substantial improvement of treatment outcome in pediatric acute myeloid leukemia (AML), chemotherapy resistance remains to be a major concern. While molecular basis behind therapeutic failure was intensely interrogated, genomic alteration of chemotherapy resistance remains poorly characterized in pediatric AML. Here, we performed whole exon sequencing (WES) to identify gene mutations associated with primary and secondary chemo-resistance in pediatric AML.Taking advantage of the available WES dataset from pediatric AML patients in two hospital centers, we compared mutation frequency of total 45 pediatric AML cases, including 24 cases of induction remission and 21 cases of chemo-resistance, to that of a cohort of 200 adult AML in TCGA dataset. We observed mutation frequency of signaling molecules were much higher in pediatric AML than in adult AML. The most frequent mutated genes are NRAS (27%, 12/45) and KIT (20%, 9/45) in pediatric AML, which has significant higher mutation frequency than in adult AML (NRAS, P=0.0002; KIT, P=0.0001). In consistent with previous findings, epigenetic regulators such as DNMT3A (0%, 0/45), TET2 (0%, 0/45), IDH1/2 (2%, 1/45) and ASXL1/2 (2%,1/45) are rarely mutated in pediatric AML patients, while they are commonly found mutated in adult AML. It's worth noting that we uncovered several novel epigenetic mutations which were not found in TCGA dataset, such as KDM5C, SIRT6, CHD4, SS18 and SETD8.We assessed whether any mutated genes are associated with primary chemo-resistance group as comparing to primary chemo-sensitive group in studies cohorts. The chemo-sensitive group contained 24 primary induction remission samples, and 3 primary chemo-sensitive diagnosis samples of secondary chemo-resistance patients. Besides high recurrence of FLT3-ITD, we observed none of signaling molecule mutations are significantly enriched in primary chemo-resistance group. It's been reported that KIT mutations are frequently mutated in pediatric AML, and associated with adverse outcome. In our studied cohort, KIT mutations are more frequently found in relapsed AML (57%, 4/7), than primary chemo-resistance (28%, 2/7). Thus, poor clinical outcome of KIT mutations is more likely attributed to disease recurrence but not primary chemo-resistance.Furthermore, epigenetic mutations were enriched at primary chemo-resistance group (7/18 vs 2/27, P=0.009), including histone modified enzyme KDM5C, SIRT6, SETD8, splisomal factors PRPF6, PRPF8 and other epigenetic regulators CHD4, and IDH2. We found high allele frequency of KDM5C point mutation (35%) and SIRT6 frameshift mutation (42%) in one chemorefractory t (8:21) translocated AML patient. In addition, we also identified splicing mutation PRPF6 and PRPF8 in chemorefractory AML patients. In light of recent discovery of epigenetic alteration mediating chemo-resistance in adult AML, we thought these epigenetic mutations found in our cohort may also play a critical role in chemo-refractory pediatric AML.The development of new treatment strategies for pediatric AML must be based on an improved understanding of the molecular mechanisms underlying chemotherapy resistance. Previously, Fiona et al, shown that SETBP1 and ASXL1 and RELN mutations were associated with primary chemo-resistance in adult and pediatric AML patients by utilizing targeted sequencing of 670 genes. We have, for the first time, performed whole exon sequencing into 21 pediatric chemo-resistance AML. We identified novel epigenetic and splicing alterations such as KDM5C, SIRT6, SS18, CHD4 and PRPF8, PRPF6, which may play critical role in leukemogenesis and chemo-resistance in pediatric AML patients. Taking together, our study shed light into genetic lesions and evolution patterns underlying chemotherapy resistance and failure in pediatric AML. Larger cohort of patients and functional studies are needed to further understand molecular spectrum underlying chemo-resistance in pediatric AML. DisclosuresNo relevant conflicts of interest to declare.

Mutation Analysis and Allele Frequencies of HFE Gene C282Y, H63D and S65D in Patients with Hyperferritinemia and Healthy Controls from a City in Northeastern Brazil

Hereditary hemochromatosis (HH) is a genetic disease caused by high iron absorption and deposition in several organs. This accumulation results in clinical disturbances such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. The C282Y mutation is well defined in hemochromatosis etiology. Two other mutations have been described, H63D and S65C, associated with a milder form of the disease. The aims of this study were to identify the H63D, S65C and C282Y genetic mutations in the hemochromatosis gene and assess the frequency of these mutations in the HFE protein gene in patients with hyperferritinemia which are sent to the DNA Center laboratory in Natal, Rio Grande do Norte state, in addition to evaluating the correlation between the H63D, C282Y and S65C mutations in hereditary hemochromatosis and serum ferritin. The biochemical dosages and molecular analyses are carried out using the enzymatic method and PCR with enzymatic restriction, respectively. Of the 300 patients investigated, 48.7% showed no mutation and 51.3% some type of mutation: 4.4 % exhibited C282Y heterozygous mutation; 2.7% C282Y homozygous mutation; 31.9% H63D heterozygous mutation; 8.1% H63D homozygous mutation; and 5.1%, heterozygous mutation in both genes. The S65C mutation was studied in 112 patients, 2.7% with heterozygous mutation (S65D/WT) and 1.8% with double heterozygosity (H63D/S65C). With regard to sex, we observed the highest number of cases with molecular changes in men compared to women in the two mutations evaluated. Since it is considered a public health problem and exhibits low toxicity with treatment, early genetic diagnosis of HH is recommended, especially in patients with elevated serum ferritin, thereby preventing severe clinical manifestations resulting from late diagnosis. Our findings show the importance of genetic studies in patients with suspected hereditary hemochromatosis due to the high incidence of this hereditary disease in our region. DisclosuresNo relevant conflicts of interest to declare.

Neoplastic Monocyte-Derived Osteoclasts Are Highly Proliferative, but Sub-Functional in MPN-Associated Myelofibrosis

Background: Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the proliferation of myeloid progenitors. A defining hallmark of MPN-associated myelofibrosis (MF) is the deposition of fibrous tissue inside the bone marrow (BM) stroma, arising either as a primary process (PMF), or secondary to the essential thromocythemia (post-ET MF) or polycythemia vera. In at least 1/3 of cases, BM fibrosis is accompanied by the osteosclerotic thickening of the bone trabeculae. It was generally held that these changes are a reaction of mesenchymal cells to the stimuli from the malignant clone. However, the recent discovery of neoplastic monocyte-derived fibrocytes capable of directly inducing BM fibrosis in situ and in vivo has led us to hypothesize that other tissue remodeling cells of the myeloid lineage may play similar roles in the pathogenesis of MPN-associated MF. Osteoclasts (OC) are giant multinuclear cells formed by the fusion of multiple monocytic progenitors. Apart from being exclusively bone-resorbing cells, OCs have a critical role in regulating bone formation by the mesenchymal cell-derived osteoblasts (OB) and maintaining the hematopoietic BM microenvironment. The aim of this study was to examine the status of these cells in MF patients and explore the effects that the neoplastic OC progenitors exert on the development of MPN-associated osteosclerosis.Methods: In order to quantitate bone remodeling cells, trephine biopsies were obtained from patients with PMF (n = 32) and post-ET MF (n = 27), as well as healthy controls (n = 3). A tyramide signal amplification-based fluorescence IHC approach was employed using TRAP and cathepsin K as OC-specific and CD56and osterix as OB-specific markers. In addition, CD14+ low-density peripheral blood cells from the representative MF patients and healthy controls were cultured in the presence of M-CSF and RANKL. After 3 weeks mature OCs were visualized using fluorescently labeled F-actin and TRAP with DAPI as the nuclear counterstain, and the JAK2V617F allele frequency was assessed via RT-PCR. For the OC functional assay, cells were plated on a mineralized surface, which was subsequently stained using a modified von Kossa method. To assess the OC fusion capacity, two populations of progenitors labeled with different lipophilic probes were mixed in a 1:1 ratio and the colocalized signal was quantified for each mature cell. All imaging was conducted using an automated multispectral or a dual spinning disk confocal system.Results: Based on the multiplex BM imaging analysis, we observed a significant increase in the number of OCs in MF patients compared to healthy controls (median: 21.8 vs. 7.0 per 100 mm of bone perimeter; p < 0.0001) that was followed by a substantial increase in the total number of OBs and the density of trabecular bone. After inducing differentiation of JAK2+ monocytes from MF patients into mature TRAP+ OCs in vitro, these cells consistently showed a high mutant allele frequency. Compared to OCs cultured from healthy monocytes, MF patients' OCs exhibited a 40.5% decrease in the median number of nuclei per cell (p= 0.024) and a tendency to incompletely develop F-actin rings. Simultaneously, there was no change in the mean TRAP positivity between the two groups. Finally, MF OCs were significantly less apt at resorbing mineral-coated areas in contrast to healthy cells (median: 21.5% vs. 7.3%; p= 0.016), and there was a 3-fold decrease in the proportion of fused mature OCs from MF monocytes compared to healthy ones (p= 0.008).Conclusions: The development of OCs is highly skewed towards proliferation in both primary and secondary MPN-associated MF. We were able to demonstrate that neoplastic monocytic progenitors retain their aberrant genetic constitution even after differentiating into mature OCs. However, fusion of such progenitors seems to be profoundly impaired, and MF OCs are unable to fully acquire the phenotypical features associated with efficient bone resorption, particularly multinucleation and the development of actin-rich structures. At least to a certain extent, this process happens independently of the non-malignant stroma, although it seems to be impacting the OBs as well. Taken together, we show evidence that even though MF OCs are hyperproliferative, their function is intrinsically supressed due to the inherited neoplastic burden, which in turn contributes to the osteosclerotic dysplasia of the MPN-affected BM. DisclosuresVerstovsek:Blueprint Medicines Corp: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Pfizer: Research Funding; Lilly Oncology: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Pfizer: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Gilead: Research Funding; CTI BioPharma Corp: Research Funding; Celgene: Research Funding; Promedior: Research Funding.

Oncological evaluation in the perioperative period using cfDNA with BRAF V600E mutation in patients with colorectal cancer

The detection of circulating cell-free DNA (cfDNA) by liquid biopsy is reported to provide prognostic information in colorectal cancer (CRC). Although the frequency of BRAF V600E mutation in CRC is less than 10%, it is associated with poor responses to conventional chemotherapy. We conducted a prospective study to investigate the relationship between the perioperative mutant allele frequency (MAF) of BRAF V600E and tumor recurrence, and to evaluate the possibility of early detection of recurrence. Among 362 patients who underwent radical resection, cfDNA was extracted from the perioperative blood of 11 CRC patients with BRAF V600E mutation and analyzed using the digital polymerase chain reaction (dPCR) system. The median follow-up time was 22 months, and there were four cases of recurrence. Although there was no correlation between recurrence and the perioperative MAF of BRAF V600E, tumor diameter was correlated with the MAF (p = 0.024), and the MAF increased with time in two patients from whom additional samples were obtained prior to recurrence. In this study, we identified a correlation between the pathological tumor diameter and the MAF, but it was difficult to predict recurrence by measuring cfDNA with BRAF V600E mutation in the perioperative period of radical resection of CRC.

Circulating Tumor DNA as a Marker for Treatment Response in Metastatic Melanoma Patients Using Next-Generation Sequencing-A Prospective Feasibility Study.

Simple SummaryDespite the improvement of the prognosis of metastatic melanoma patients through the implementation of targeted and immunotherapies, there is a need to identify biomarkers to predict and monitor treatment response. Therefore, we performed sequencing of paired melanoma tissue biopsies and circulating tumor DNA (ctDNA) from 149 plasma samples using two custom next-generation sequencing (NGS) AmpliSeq™ HD panels to determine the level of concordance. We aimed to evaluate whether ctDNA analysis with NGS could predict and monitor treatment response in a cohort of metastatic melanoma patients; NGS allowed for a comprehensive analysis of cancer-associated mutations in serial plasma samples with high sensitivity. Although a trend could be seen that mutant allele frequency levels over time correlated with or even preceded radiological response to treatment, this finding was not statistically significant in our cohort. Our study demonstrates that NGS gene panels might be useful for treatment monitoring with ctDNA in melanoma patients.We prospectively performed a longitudinal analysis of circulating tumor DNA (ctDNA) from 149 plasma samples and CT scans in Stage III and IV metastatic melanoma patients (n = 20) treated with targeted agents or immunotherapy using two custom next-generation sequencing (NGS) Ion AmpliSeq™ HD panels including 60 and 81 amplicons in 18 genes, respectively. Concordance of matching cancer-associated mutations in tissue and plasma was 73.3%. Mutant allele frequency (MAF) levels showed a range from 0.04% to 28.7%, well detectable with NGS technologies utilizing single molecule tagging like the AmpliSeq™ HD workflow. Median followup time of the tissue and/or plasma positive cohort (n = 15) was 24.6 months and median progression-free survival (PFS) was 7.8 months. Higher MAF ≥ 1% at baseline was not significantly associated with a risk of progression (Odds Ratio = 0.15; p = 0.155). Although a trend could be seen, MAF levels did not differ significantly over time between patients with and without a PFS event (p = 0.745). Depending on the cell-free DNA amount, NGS achieved a sensitivity down to 0.1% MAF and allowed for parallel analysis of multiple mutations and previously unknown mutations. Our study indicates that NGS gene panels could be useful for monitoring disease burden during therapy with ctDNA in melanoma patients.

Non-hotspot PIK3CA mutations are more frequent in CLOVES than in common or combined lymphatic malformations

BackgroundTheragnostic management, treatment according to precise pathological molecular targets, requests to unravel patients’ genotypes. We used targeted next-generation sequencing (NGS) or digital droplet polymerase chain reaction (ddPCR) to screen for somatic PIK3CA mutations on DNA extracted from resected lesional tissue or lymphatic endothelial cells (LECs) isolated from lesions. Our cohort (n = 143) was composed of unrelated patients suffering from a common lymphatic malformation (LM), a combined lymphatic malformation [lymphatico-venous malformation (LVM), capillaro-lymphatic malformation (CLM), capillaro-lymphatico-venous malformation (CLVM)], or a syndrome [CLVM with hypertrophy (Klippel-Trenaunay-Weber syndrome, KTS), congenital lipomatous overgrowth-vascular malformations-epidermal nevi -syndrome (CLOVES), unclassified PIK3CA-related overgrowth syndrome (PROS) or unclassified vascular (lymphatic) anomaly syndrome (UVA)].ResultsWe identified a somatic PIK3CA mutation in resected lesions of 108 out of 143 patients (75.5%). The frequency of the variant allele ranged from 0.54 to 25.33% in tissues, and up to 47% in isolated endothelial cells. We detected a statistically significant difference in the distribution of mutations between patients with common and combined LM compared to the syndromes, but not with KTS. Moreover, the variant allele frequency was higher in the syndromes.ConclusionsMost patients with an common or combined lymphatic malformation with or without overgrowth harbour a somatic PIK3CA mutation. However, in about a quarter of patients, no such mutation was detected, suggesting the existence of (an)other cause(s). We detected a hotspot mutation more frequently in common and combined LMs compared to syndromic cases (CLOVES and PROS). Diagnostic genotyping should thus not be limited to PIK3CA hotspot mutations. Moreover, the higher mutant allele frequency in syndromes suggests a wider distribution in patients’ tissues, facilitating detection. Clinical trials have demonstrated efficacy of Sirolimus and Alpelisib in treating patients with an LM or PROS. Genotyping might lead to an increase in efficacy, as treatments could be more targeted, and responses could vary depending on presence and type of PIK3CA-mutation.

Longitudinal Dynamics of Circulating Tumor Cells and Circulating Tumor DNA for Treatment Monitoring in Metastatic Breast Cancer.

PURPOSELiquid biopsy–based biomarkers, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), are increasingly important for the characterization of metastatic breast cancer (MBC). The aim of the study was to explore CTCs and ctDNA dynamics to better understand their potentially complementary role in describing MBC.METHODSThe study retrospectively analyzed 107 patients with MBC characterized with paired CTCs and ctDNA assessments and a second prospective cohort, which enrolled 48 patients with MBC. CTCs were immunomagnetically isolated and ctDNA was quantified and then characterized through next-generation sequencing in the retrospective cohort and droplet digital polymerase chain reaction in the prospective cohort. Matched pairs variations at baseline, at evaluation one (EV1), and at progression were tested through the Wilcoxon test. The prognostic role of ctDNA parameters was also investigated.RESULTSMutant allele frequency (MAF) had a significant decrease between baseline and EV1 and a significant increase between EV1 and progression. Number of detected alterations steadily increased across timepoints, CTCs enumeration (nCTCs) significantly increased only between EV1 and progression. MAF dynamics across the main altered genes was then investigated. Plasma DNA yield did not vary across timepoints both in the retrospective cohort and in the prospective cohort, while the short fragments fraction showed a potential role as a prognostic biomarker.CONCLUSIONnCTCs and ctDNA provide complementary information about prognosis and treatment benefit. Although nCTCs appeared to assess tumor biology rather than tumor burden, MAF may be a promising biomarker for the dynamic assessment of treatment response and resistance.

REMARRY and PURSUIT trials: liquid biopsy-guided rechallenge with anti-epidermal growth factor receptor (EGFR) therapy with panitumumab plus irinotecan for patients with plasma RAS wild-type metastatic colorectal cancer

BackgroundPrevious clinical trials have demonstrated the potential efficacy of rechallenge with anti- epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) for patients with RAS/BRAF V600E wild-type metastatic colorectal cancer (mCRC). Moreover, post hoc biomarker analyses of clinical trials has suggested that RAS status in circulating tumor DNA (ctDNA) has a high probability to select patients who could benefit from anti-EGFR mAb rechallenge.MethodsThis trial is composed of 2 phases: a monitoring phase (REMARRY) and a trial phase (PURSUIT). A monitoring phase, the REMARRY study, aims to evaluate the dynamics of plasma RAS status during the subsequent treatments after refractory to anti-EGFR therapy in patients with mCRC with RAS/BRAF V600E wild-type tumors who have progressed after a response to previous anti-EGFR therapy, using a highly sensitive digital polymerase chain reaction OncoBEAM RAS CRC kit in a central laboratory (Sysmex, Japan). A trial phase, the PURSUIT trial, is a multicenter, single-arm phase II trial to assess the efficacy and safety of rechallenge therapy with panitumumab plus irinotecan in patients without RAS mutations in ctDNA (plasma RAS negative) in the REMARRY study. Key eligibility criteria of the PURSUIT trial include RAS/BRAF V600E wild-type mCRC in tumor tissue refractory or intolerant to fluoropyrimidine, oxaliplatin, and irinotecan; progression after complete or partial response to previous anti-EGFR therapy; plasma RAS negative (defined as plasma mutant allele frequencies [MAF] of all RAS ≤ 0.1%) within 28 days prior to enrollment; 4 months or more between the last administration of previous anti-EGFR mAb and the start of protocol treatment; and Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) ≤ 1. The primary endpoint is the confirmed objective response rate (ORR). The target sample size of the PURSUIT trial is 50 patients. Biomarker analyses will be performed in parallel using the OncoBEAM RAS CRC kit and a next-generation sequencing-based ctDNA analysis (Guardant360).DiscussionOur trial aims to confirm the clinical benefit of anti-EGFR mAb rechallenge therapy in patients with plasma RAS negative. Moreover, through biomarker analyses, our trial will shed light on which patients would benefit from rechallenge in addition to being plasma RAS negative.Trial registrationThe REMARRY study: UMIN, UMIN000036424. Registered date: April 5, 2019. The PURSUIT trial: jRCT, jRCTs031190096. Registered date: October 1, 2019.

PCR-RFLP molecular confirmation of color dilution alopecia in dogs in Brazil.

Color dilution alopecia (CDA) is a dermatopathy observed exclusively in animals having a diluted coat color. In dogs, color dilution occurs as a result of a single-nucleotide variation (SNV) c.-22G > A in the melanophilin gene. We standardized a PCR-restriction-fragment length polymorphism (PCR-RFLP) technique to identify this mutation and determine its frequency in dogs in Brazil. The standardized PCR-RFLP technique could efficiently identify the SNV c.-22G > A in the melanophilin gene, with mutated allele frequencies of 0.1, 0.1, and 0.0875 in Dachshund, Miniature Pinscher, and Yorkshire Terrier breeds, respectively, with no statistical difference among the breeds (p = 0.252). The mutation was identified in 2 homozygous Dachshund dogs with alopecia, confirming the clinical characteristic of CDA. The standardization of a simpler and more accessible molecular technique for recognition of the SNV c.-22G > A in the melanophilin gene allows identification of heterozygous (phenotypically normal) dogs that can be excluded from reproduction, to avoid the birth of dogs with diluted coat color and consequently CDA.

Genetic and expression deregulation of immunoregulatory genes in rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases globally, and is an important public health concern, associating with early death and systemic complications. Although key development in RA treatment has already been made still RA affected individuals face comorbidity and disability. Therefore, there is a need to discover new risk factors in helping early diagnosis and treatment of RA. The present study is designed to assess the variations of Vitamin D receptor (VDR) and interleukin -6 (IL-6) in RA patients. Polymorphisms of said genes were calculated in 300 RA patients and 300 controls, using Tetra-ARMS polymerase chain reaction. Secondly, expression levels of selected genes were checked using the quantitative PCR (qPCR) and obtained results were evaluated using a different statistical test. Logistic regression analysis showed that frequency of mutant allele of VDR gene polymorphisms (rs11168268, OR = 4.84; 95% CI = 2.94-7.97; p = 0.0001; rs2248098, OR = 1.65; 95% CI = 1.07-2.54; p = 0.02) and IL-6 gene polymorphisms (rs184229712, OR = 2.47; 95% CI = 1.56-3.92, p = 0.0001; rs36215814, OR = 2.14; 95% CI = 1.30-3.53; p = 0.002) was observed significantly higher in RA patients vs controls. Expression analysis showed the significant upregulation of IL-6 (p < 0.0001) and downregulation of VDR gene (p < 0.0001) in RA cases vs controls. ROC curve analysis showed that downregulation of IL-6 (AUC = 0.86, p < 0.001) and upregulation of VDR (AUC = 0.77, p < 0.001) was act as the good diagnostic marker for detection/diagnosis of arthritis. In conclusions, data from the present study showed the significant involvement of VDR and IL-6 gene variations in RA pathogenesis.

Circulating tumor cells, circulating tumor DNA, and disease characteristics in young women with metastatic breast cancer.

Clinical and genomic data from patients with early-stage breast cancer suggest more aggressive disease in premenopausal women. However, the association between age, disease course, and molecular profile from liquid biopsy in metastatic breast cancer (MBC) is not well characterized. Patients were classified as premenopausal (<45years), perimenopausal (45-55years), or postmenopausal (> 55years). Cohort 1 consisted of patients with MBC who consented for prospective serial evaluation of circulating tumor cells (CTCs) using CellSearch™. Cohort 2 included patients who, as part of routine care, had circulating tumor DNA (ctDNA) sequenced by the Guardant360™ assay. Clinicopathologic data were collected from retrospective review to compare disease features between premenopausal and postmenopausal women. Premenopausal women represented 26% of 138 patients in Cohort 1 and 21% of 253 patients in Cohort 2. In Cohort 1, younger patients had a shorter time to metastases and a higher prevalence of lung and brain metastases. Overall, there were similar rates of ≥ 5 CTCs/7.5mL, HER2 + CTC expression, and CTC clusters between pre- and postmenopausal women. However, for those with triple negative breast cancer, premenopausal women had a higher proportion of ≥ 5 CTCs/7.5mL. In Cohort 2, premenopausal women had a higher incidence of FGFR1 (OR 2.75, p = 0.022) and CCND2 (OR 6.91, p = 0.024) alterations. There was no difference in the ctDNA mutant allele frequency or the number of detected alterations between these age groups. Our data reveal that premenopausal women diagnosed with MBC have unique clinical, pathologic, and molecular features when compared to their postmenopausal counterparts. Our results highlight FGFR1 inhibitors as potential therapeutics of particular interest among premenopausal women.

Detection of EGFR Mutations in Plasma cfDNA and Paired CTCs of NSCLC Patients before and after Osimertinib Therapy Using Crystal Digital PCR.

Simple SummaryLiquid biopsy is a useful tool during longitudinal monitoring of NSCLC patients and requires highly sensitive and reliable technologies for accurate detection of genomic alterations. We evaluated and used crystal digital PCR to detect and quantify EGFR mutations in plasma cfDNA and paired CTCs of NSCLC patients before treatment with osimertinib and at progression of disease.Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutant NSCLC. Circulating tumor cells (CTCs) consist a unique source of information at the cellular level. Digital PCR (dPCR) is a valuable tool for accurate and valid analysis of gene mutations in liquid biopsy analysis. In the present study we detected EGFR mutations in ctDNA and paired CTCs under osimertinib therapy at two time points using crystal dPCR and the naica® system (Stilla Technologies). We quantified mutation allele frequencies (MAF) of EGFR mutations in 91 plasma cfDNA samples of 48 EGFR mutant NSCLC patients and in 64 matched CTC-derived genomic DNA samples, and the FDA-cleared cobas® EGFR mutation test in 80 identical plasma samples. Direct comparison between crystal dPCR and the cobas EGFR assay revealed a high concordance for all EGFR mutations. Our comparison of crystal dPCR results in ctDNA with the corresponding primary tissue has shown a strong correlation. EGFR mutations analysis in paired CTC-derived gDNA revealed a high heterogeneity. Crystal dPCR offers the unique advantages of high analytical sensitivity, precision, and accuracy for detecting and quantifying multiple EGFR mutations in plasma cfDNA and CTCs of NSCLC patients.

Association of CYP2C19*2 and *17 genetic variants with hypertension in Pakistani population

Purpose: To investigate the association of *2 and *17 single nucleotide polymorphisms (SNPs) of CYP2C19 gene with hypertension in Pakistani population.&#x0D; Methods: The study was conducted on 527 hypertensive patients and 530 unrelated healthy controls from selected regions of Pakistan. DNA was extracted from leukocytes and all patients and controls were genotyped for two SNPs (rs4244285 and rs12248560) of CYP2C19 gene by allele specific polymerase chain reaction (AS-PCR).&#x0D; Results: Multi-allelic polymorphism in CYP2C19 identified four distinct phenotypes known as ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM) and poor metabolizer (PM) in hypertensive patients and controls. For CYP2C19*2 polymorphisms, overall wild type and mutant allele frequency were 75 and 25 % in hypertensive patients, and 64.2 and 35.8 % in controls. For CYP2C19*17 polymorphisms, the overall wild type and mutant allele frequency were 66.6 and 33.4 % in hypertensive patients and 75.6 % and 24.4 % in controls. Significant difference in allele frequencies for CYP2C19*2 and *17 was demonstrated between hypertensive and non-hypertensive subjects.&#x0D; Conclusion: To the best of our knowledge, this is the first report on CYP2C19 frequencies in hypertensive Pakistani patients. The finds should help clinicians to determine a suitable optimal dosage of some drugs in order to reduce side effects.

Voltage-Gated Sodium Channel (Vgsc) Mutation-Based Pyrethroid Resistance in Aedes aegypti Populations of Three Endemic Dengue Risk Areas of Sri Lanka.

Background Pyrethroid insecticides are widely used in many countries for chemical-based control of Ae. aegypti. Regardless of their efficacy, the constant use of insecticides has induced insecticide resistance mechanisms, such as knockdown resistance (kdr) in mosquitoes. Sri Lankan Vector Controlling Entities (VCE) have been using a variety of pyrethroid insecticides as the primary approach for dengue control. However, development of any resistance among the Aedes mosquitoes has been limitedly studied in the country. Therefore, the current study was conducted to evaluate the prevalence of F1534C, V1016G, and S989P mutations among Ae. aegypti mosquito populations in three dengue endemic high-risk regions of Sri Lanka. Methodology. Immature (both pupae and larvae) stages of Ae. aegypti mosquitoes were collected from Colombo, Gampaha, and Kandy districts of Sri Lanka from February 2018 to December 2019. Polymerase Chain Reaction- (PCR-) based assay for molecular genotyping of mutations was performed to identify the prevalence of kdr mutations in collected Ae. aegypti populations, separately. The frequencies of the resistant and susceptible kdr alleles were determined by using the Hardy–Weinberg equilibrium. Results The Ae. aegypti populations from Colombo, Gampaha, and Kandy districts showed 46%, 42%, and 22% of F1534C mutation allele frequencies, along with 15%, 12%, and 6% of V1016G mutation allele frequencies, respectively. The mutation allele frequencies of S989 in Colombo, Gampaha, and Kandy districts were 9.5%, 8.5%, and 4.5%, respectively. The wild-type (PP) genotype remained predominant within all the three districts, whereas the homogenous (QQ) mutation genotype occurred only in minority. The abundance of Q allele frequency in Ae. aegypti mosquitoes was relatively higher for all the three mutations in Colombo. Conclusions The findings clearly indicate that long-term insecticide applications and multiple use of pyrethroids have led to the acquisition of kdr mutations, leading to the development of insecticide resistance among local Ae. aegypti populations, especially in the Colombo and Gampaha districts. Therefore, evaluation of the prevalence levels of these kdr mutations highlights the necessity for shifting towards novel vector control strategies.

Allelic Variation of Soybean Maturity Genes E1–E4 in the Huang-Huai-Hai River Valley and the Northwest China

Soybean is planted in a wide span of the world, and flowering and maturity time is an important trait determining soybean yield formation and adaptation. Maturity loci E1, E2, E3 and E4 were frequently reported as the most influential genetic loci for soybean flowering and maturity. To understand the allelic variation and assess the phenological traits of cultivars with different E allelic combinations in natural environments, 251 cultivars of maturity group (MG) I–V were field tested in 42 locations across four sub-regions in the Huang-Huai-Hai and Northwest region of China and genotyped with KASP markers for E1–E4 loci. The results indicated that mutant alleles were only found in the E1 and E2 locus, all of the cultivars carried functional alleles in the E3 and E4 loci in this area, with the frequency of mutant allele to be higher in early maturity groups (MGs) than late MGs. Among nine E allelic combinations in this area, one photoperiodic insensitive mutation in E2 loci (E1/e2-ns/E3-Ha/E4 and E1/e2-ns/E3-Mi/E4) made up the largest proportion (25.10 and 18.33%), while two photoperiodic insensitive mutations in both E1 and E2 loci (e1-as/e2-ns/E3-Ha/E4) (1.20%) occupied the lowest proportion in this panel. The major combinations of E locus for MGI, MGII and MG III in this area were E1/E2-dl/E3-Mi/E4, E1/e2-ns/E3-Mi/E4 and E1/e2-ns/E3-Ha/E4, respectively. Cultivars carrying e1-as/e2-ns/E3-Ha/E4 genotype flowered earliest (34 days) on average, 7.6 days earlier than the latest-flowering E haplotype (E1/e2-ns/E3-Ha/E4). This study provided an opportunity to detect the E allelic combinations in the Huang-Huai-Hai River Valley and the Northwest China, which would facilitate the improvement of soybean adaptation in the future.