Somatic Mutations In Exon Research Articles

Use of SuperARMS EGFR Mutation Detection Kit to Detect EGFR in Plasma Cell-free DNA of Patients With Lung Adenocarcinoma

BackgroundThe SuperARMS EGFR Mutation Detection Kit (SuperARMS) is highly selective and sensitive and able to detect 41 of the most common somatic mutations in exons 18 to 21 of the epidermal growth factor receptor gene (EGFR). It allows for the detection of 0.2% to 0.8% mutant DNA in a background of 99.8% to 99.2% normal DNA. The present study assessed the performance of SuperARMS in detecting EGFR mutations in cell-free DNA (cfDNA) samples derived from plasma in patients with advanced lung adenocarcinoma. Materials and MethodsA total of 180 patients with advanced clinical stage lung adenocarcinoma were retrospectively registered. The concordance between the EGFR mutations detected by SuperARMS and ARMS (AmoyDx EGFR 29 Mutations Detection Kit) was analyzed. ResultsOf the 180 samples, 57 (31.7%) were positive for EGFR mutations using SuperARMS, with 38 (21.1%) positive using ARMS. For the entire cohort, the positive, negative, and overall concordance rates were 97.3% (95% confidence interval [CI], 86.2%-99.5%), 85.3% (95% CI, 78.6%-90.2%), and 87.8% (95% CI, 82.2%-91.8%), respectively. The kappa value was 0.69 (95% CI, 0.57-0.81). For the 61 treatment-naive patients and 119 previously treated patients, the kappa values were 0.59 (95% CI, 0.37-0.79) and 0.74 (95% CI, 0.60-0.87), respectively. SuperARMS identified 9 samples harboring the T790M mutation; of these, only 1 (11.1%) was detected using ARMS. ConclusionSuperARMS is a promising plasma-based assay for EGFR mutations, including T790M. It might be useful in advanced-stage lung adenocarcinoma patients whose tissue biopsy samples are insufficient for a traditional diagnostic EGFR assay or for patients with a poor performance status.

Reduced mutation rate in exons due to differential mismatch repair.

While recent studies have revealed higher than anticipated heterogeneity of mutation rate across genomic regions, mutations in exons and introns are assumed to be generated at the same rate. Here we find fewer somatic mutations in exons than expected based on their sequence content, and demonstrate that this is not due to purifying selection. Moreover, we show that it is caused by higher mismatch repair activity in exonic than in intronic regions. Our findings have important implications for our understanding of mutational and DNA repair processes, our knowledge of the evolution of eukaryotic genes, and practical ramifications for the study of the evolution of both tumors and species.

Uterine Fibroids: Bridging Genomic Defects and Chronic Inflammation.

AbstractUterine fibroids (UF; aka leiomyoma, myomas) are the most common benign tumors of female reproductive tract. They are highly prevalent, with 70 to 80% of women burdened by the end of their reproductive years. Fibroids are a leading cause of pelvic pain, abnormal vaginal bleeding, pelvic bulk symptoms, miscarriage, and infertility. They are the leading indication for hysterectomy, and costs exceed 34 billion dollars annually in the United States alone. Recently, somatic mutations in exons 1 and 2 of Med12 gene emerged as common UF driver mutations. Unfortunately, the detailed etiology of UF is not fully realized. Particularly, very little is known about possible dysregulation of inflammatory and immune processes and their possible contribution to UF pathogenesis. The notion on possible impact of altered estrogen and progesterone signaling in UF on inflammatory responses and DNA repair machinery that can conceivably lead to tumor-specific somatic mutation is indeed an intriguing concept which has some foundation in available observation in other hormonally responsive tissues. This review highlights and summarizes our current knowledge on the convergence of such pathways and their relevance for UF pathogenesis.

Visualization portal for genetic variation (VizGVar): a tool for interactive visualization of SNPs and somatic mutations in exons, genes and protein domains.

VizGVar was designed to meet the growing need of the research community for improved genomic and proteomic data viewers that benefit from better information visualization. We implemented a new information architecture and applied user centered design principles to provide a new improved way of visualizing genetic information and protein data related to human disease. VizGVar connects the entire database of Ensembl protein motifs, domains, genes and exons with annotated SNPs and somatic variations from PharmGKB and COSMIC. VizGVar precisely represents genetic variations and their respective location by colored curves to designate different types of variations. The structured hierarchy of biological data is reflected in aggregated patterns through different levels, integrating several layers of information at once. VizGVar provides a new interactive, web-based JavaScript visualization of somatic mutations and protein variation, enabling fast and easy discovery of clinically relevant variation patterns. VizGVar is accessible at http://vizport.io/vizgvar; http://vizport.io/vizgvar/doc/. asolano@broadinstitute.org or allan.orozcosolano@ucr.ac.cr.

Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer

BackgroundThe role of adenomatous polyposis coli (APC) gene in mitosis might be critical for regulation of genomic stability and chromosome segregation. APC gene mutations have been associated to have a role in colon cancer and since gastric and colon tumors share some common genetic lesions, it is relevant to investigate the role of APC tumor suppressor gene in gastric cancer.MethodsWe investigated for somatic mutations in the Exons 14 and 15 of APC gene from 40 diffuse type gastric cancersamples. Rabbit polyclonal anti-APC antibody was used, which detects the wild-type APC protein and was recommended for detection of the respective protein in human tissues. Cell cycle analysis was done from tumor and adjacent normal tissue.ResultsAPC immunoreactivity showed positive expression of the protein in stages I, II, III and negative expression in Stages III and IV. Two novel deleterious variations (g.127576C > A, g.127583C > T) in exon 14 sequence were found to generate stop codon (Y622* and Q625*)in the tumor samples. Due to the generation of stop codon, the APC protein might be truncated and all the regulatory features could be lost which has led to the down-regulation of protein expression. Our results indicate that aneuploidy might occurdue to the codon 622 and 625 APC-driven gastric tumorigenesis, in agreement with our cell cycle analysis. The APC gene function in mitosis and chromosomal stability might be lost and G1 might be arrested with high quantity of DNA in the S phase. Six missense somatic mutations in tumor samples were detected in exon 15 A-B, twoof which showed pathological and disease causing effects based on SIFT, Polyphen2 and SNPs & GO score and were not previously reported in the literature or the public mutation databases.ConclusionThe two novel pathological somatic mutations (g.127576C > A, g.127583C > T) in exon 14 might be altering the protein expression leading to development of gastric cancer in the study population. Our study showed that mutations in the APC gene alter the protein expression and cell cycle regulation in diffuse type gastric adenocarcinoma.

A female case of pleuropulmonary blastoma type 1 whose pulmonary cystic lesion was followed since neonate

Abstract Pleuropulmonary blastoma (PPB) is a rare malignant mesenchymal tumor. It is classified into 3 subgroups, and PPB type 1 is known to be a cystic lesion with good prognosis. Here, we report a case of PPB type 1 seen in a 1-year-old girl whose pulmonary cystic lesion had been followed-up as a congenital pulmonary airway malformation since neonate. The cyst had been diagnosed as congenital pulmonary airway malformation before surgery but the final diagnosis was type 1 PPB. The tumor had a somatic mutation in exon 25 of the DICER 1 gene whereas no germline mutation was found.

The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated invitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

AbstractEssential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed “triple negative.” We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.

Differential overall survival (OS) results in RECORD-3 study based on three distinct mRCC molecular subgroups classified by BAP1 and/or PBRM1 mutations.

561 Background: PBRM1 and BAP1, both of which encode chromatin modulating proteins, have recently been identified as frequently mutated tumor suppressor genes in RCC. However, their correlation with outcomes of targeted therapies is unknown. We explored correlations between overall survival (OS) and mutations in archival tumor samples from RECORD-3, a randomized phase 2 trial comparing first-line everolimus (EVE) then sunitinib (SUN) to first-line SUN then EVE at progression in 471 treatment-naïve mRCC patients (J Clin Oncol 2014; 32:2764). Methods: Somatic mutations in exons of 341 cancer related genes were identified by a next generation sequencing (NGS) assay (MSK IMPACT). Association between genotypes and OS was assessed by Cox PH models and log-rank tests. Results: DNA of 258 archival tumor and 181 matched germline samples was successfully analyzed (median coverage 530X). Three molecular subgroups Gr1 (BAP1 MT [mutant], PBRM1 WT [wild-type]/MT; 17.4%), Gr2 (PBRM1 MT, BAP1 WT; 38.4%), and Gr3 (PBRM1 WT, BAP1 WT; 44.2%) with different OS outcomes after sequential EVE-SUN or SUN-EVE were identified (Table). In the sequential EVE-SUN arm, median OS (months) was shortest with Gr1 (9.8; 95% CI, 7.8–20.0), longest with Gr2 (39.6; 95% CI, 31.7–not estimable), and intermediate with Gr3 (18.1; 13.7–30.0), whereas in the SUN-EVE arm there was no significant difference among groups. When comparing OS between treatment sequences within molecular groups, a trend suggested differences for EVE-SUN vs SUN-EVE in Gr1 (HR 1.5), Gr2 (0.8), and Gr3 (1.2), although comparisons were not statistically significant. Conclusions: Our results suggest that these genotypes may represent distinct RCC molecular subtypes with potentially different predictive/prognostic values on targeted therapies. Different treatment sequences may be considered depending on the genotypes. Clinical trial information: NCT00903175. [Table: see text]

Whole Exome Sequencing Identifies Novel MPL and JAK2 M utations in Triple Negative Myeloproliferative Neoplasms

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic myeloproliferative neoplasms (MPN) characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. Most of the cases are sporadic and driven by somatic mutations, although familial clustering is observed. The most common mutation affecting 50-60% of the cases is JAK2-V617F, while 25-30% of the patients carry somatic mutations in exon 9 of CALR. MPL exon 10 mutations affect ~5% of the cases. JAK2, CALR and MPL mutations are mutually exclusive and account for >90% of ET and PMF cases. In 12% of ET and 5% of PMF cases the disease drivers remain unknown. These patients are termed as triple negative. The mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR. The aim of this study was to identify disease causing mutation in triple negative cases of ET and PMF.To identify the somatic mutations that are potential disease drivers in triple negative MPN we performed whole exome sequencing (WES) on paired samples from the tumor and control tissue of 4 patients with ET and 4 patients with PMF. We identified somatic mutations in 3/8 analyzed cases. In two PMF cases we identified somatic mutations in genes relevant for MPN- TET2, ASXL1, CBL, SRSF2 and a mutation in MPL-S204P. We did not identify a novel recurrent mutation. In the 5 cases without somatic mutations, we looked for germline mutations in genes relevant for MPN. We identified germline mutations MPL-V285E and JAK2-G571S in one PMF case and one case of ET, respectively. SNP microarray analysis for presence of chromosomal aberrations revealed a uniparental disomy of chromosome 6p in the case with MPL -V285E mutation, suggesting clonal hematopoiesis. To determine the frequency of MPL and JAK2 mutations outside exons 10 and 14 in triple negative MPN, we performed Sanger sequencing of all coding exons of MPL in 62 patients and of JAK2 in 49 patients. We detected variants outside exon 10 of MPL in 6/62 cases (9.7%). MPL-T119I, MPL-S204F, MPL-E230G and MPL-Y591D were somatic mutations, while MPL-R321W was germline. We identified an additional patient with MPL-S204P mutation, however the control tissue was not available. JAK2 variants were found in 4/49 cases (8.1%). JAK2-G335D and JAK2-V625F were germline mutations, while for the patients with JAK2-F556V and JAK2-G571S the control tissue was unavailable. In total, we identified non-canonical MPL mutations in 8/70 (11.4%) and JAK2 mutations in 5/57 (8.8%) triple negative cases of ET and PMF. All mutations were heterozygous. The mutations in MPL and JAK2 were mutually exclusive in our patient cohort.The expression of identified MPL mutants did not induce cytokine independent growth of Ba/F3 cells, but the MPL-Y591D expressing cells showed marked hypersensitivity to TPO compared to the wild type. Using a luciferase reporter assay in JAK2-deficient gamma 2A cells, where we transiently expressed the wild type or mutant MPL cDNAs, JAK2, STAT5, STAT5 reporter Spi-Luc, and pRL-TK for transfection control, we could demonstrate that all identified MPL mutations lead to constitutive activation of JAK/STAT signaling. As the detection of activity required longer times (48h) than for the MPL-W515K (24h), we concluded that the identified mutations have a milder effect of the function of MPL. By Western immunodetection we could demonstrate that expression of JAK2-F556V and JAK2-V625F in Ba/F3-MPL cells, lead to the increased phosphorylation of STAT5 in the absence of cytokines. We also observed increased sensitivity to TPO in the Ba/F3 MPL cell lines expressing JAK2-F556V and JAK2-V625F. JAK2-V625F and JAK2-F556V are mild gain-of-function mutations, while JAK2-G335D and JAK2-G571S do not seem to alter the function on the JAK2 protein.The results of our study suggest that sequencing of all coding exons of MPL and JAK2 is recommended for the diagnostic work-up of the ET and PMF patients who do not carry other more common mutations. The lack of evidence for clonal disease in 50% of the triple negative cases and presence of germline mutations suggests that a proportion of cases are likely to be hereditary MPN-like disorders. Application of whole genome sequencing or RNA sequencing for fusion oncogene detection will likely fill in the gap of the remaining triple negative MPN cases with clonal hematopoiesis in which we did not identify a recurrent driving mutation using WES. DisclosuresGisslinger:AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Consultancy; Geron: Consultancy; Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Profile of Calreticulin (CALR) Somatic Mutations in a Cohort of Pakistani Patients with Myeloproliferative Neoplasms

Introduction:Calreticulin (CALR) is a calcium binding multifunctional protein encoded by the CALR gene, whose job is to bind to mis-folded proteins and prevent them from being exported from the endoplasmic reticulum to the golgi apparatus. Outside the endoplasmic reticulum it is found both intra- and extracellularly on the cell surface and has been implicated in diverse processes including proliferation, apoptosis, phagocytosis and immunogenic cell death.In December 2013, somatic mutations in exon 9 of CALR were identified as the second most prevalent acquired nucleotide changes in JAK2 mutation negative essential thrombocythosis (ET) and primary myelofibrosis (PMF) patients but not in polycythaemia vera (PV) patients. At that time CALR mutations were found mutually exclusive with JAK2 and MPL. Recently there have been some reports on co-occurence of JAK2 and CALR mutations, not only in ET and PMF but also in some PV cases. Indels in CALR have also been reported in a JAK2 mutation negative PV patient. All CALR mutations are insertions or deletions resulting in a frameshift and cluster in exon 9 which is the last exon of the gene. Thus far, more than 50 different types of mutations in CALR have been reported. Two specific mutations, Type I (52bp deletion) and Type II (5bp insertion), are most prevalent. Overall, these two mutation types are found in more than 80% of patients with mutant CALR. One report found absence of Type II mutations and scatter point mutations in PMF patients of India. In the light of currently available literature, reported from North America (USA), Europe (Cyprus/Italy/Spain), Eastern Europe (Poland), and East Asia (Mainland China/Taiwan China), indel mutations in CALR exon 9 occur with a similar frequency in both ET and PMF patients regardless of ethnic diversity. CALR mutations have been shown to have important diagnostic and prognostic significance in ET and PMF patients and will likely be incorporated into the World Health Organization diagnostic criteria for MPN.Methods:Clinical and hematological features were obtained from 51 MPN patients. We studied 10 patients with PV, 17 patients with ET, 12 patients with MF and 9 patients with Undifferentiated MPN. JAK2 V617F mutation was analyzed by DNA tetra-primer amplification refractory mutation system (ARMS-PCR). CALR mutations were identified by bi-directional Sanger sequencing.Results:In our cohort, CALR mutations were detected in 7 patients (13.7%; 7/51) of which one patients was positive for JAK2 V617F as well. All those who showed positive results for CALR mutations were ET patients (41.17%; 7/17). Type I mutation was found in 3 patients (42.8%; 3/7) and among them 2 had two new scattered point mutations (c.1081C>G and c.1086C>G) as well, Type II in 2 patients (28.5%; 2/7), and others in 2 patients (28.5%; 2/7). Among those 2 patients with CALR mutations other than Type I or Type II, one had novel CALR mutation pattern of 10bp deletion (c.1130_1139del) whereas other had reported CALR 12bp deletion (c.1214_1225del) profile.When CALR positive ET patients were statistically compared with JAK2 V617F positive patient, insignificant differences were found in platelet counts (p-value 0.198) and hemoglobin level (p-value 0.380) which might be due to the fact that there was only one JAK2 V617F positive patient in the cohort. No differences were also observed in hemoglobin, white blood cell, platelet counts, hepatomegaly, and splenomegaly between CALR positive and CALR negative patients (p-value >0.05).Conclusion:In our cohort of 51 MPN patients CALR mutations were found associated with ET only that is different from other studies worldwide. Prevalence of both scatter point and Type I and Type II CALR mutations are in agreement with worldwide studies but differ from the study conducted on Indian population which shows the need to conduct studies with large sample size in this region to explore the CALR mutation profile in the patients of MPN here. DisclosuresNo relevant conflicts of interest to declare.

Clinical validation of a multipurpose assay for detection and genotyping of CALR mutations in myeloproliferative neoplasms.

To develop a polymerase chain reaction (PCR)-based approach to detect CALR mutations in myeloproliferative neoplasms (MPNs) in a clinical laboratory. DNA was extracted from bone marrow aspirate samples of 67 JAK2 wild-type MPNs (22 with matched peripheral blood), 54 cases of unclassifiable myelodysplastic syndrome/MPN, and 16 cases of atypical chronic myeloid leukemia. We used genomic DNA to detect somatic mutations in exon 9 of CALR and PCR with fluorescently labeled and M13-tagged primers and subjected the products to capillary electrophoresis (CE) followed by Sanger sequencing. Detailed assay performance characteristics were established. We identified CALR mutations in 19 (28.4%) of 67 JAK2-negative MPNs, including 14 type I (52-base pair [bp] deletion), four type II (5-bp insertions), and one type III (18-bp deletion). All mutations were confirmed by Sanger sequencing. Sensitivity studies showed 2.5% and 5% mutation detection levels by CE and Sanger sequencing, respectively, with high reproducibility. This assay allows for rapid, convenient screening for CALR mutations in MPNs, thereby reducing the number of cases that require assessment by Sanger sequencing, reducing labor and improving turnaround time.

MED12 somatic mutations in fibroadenomas and phyllodes tumours of the breast.

Somatic mutations in exon 2 of the mediator complex subunit 12 (MED12) gene have been identified in 60% of breast fibroadenomas (FAs). The aim of this study was to define whether phyllodes tumours (PTs) would harbour MED12 somatic mutations in a way akin to FAs. A collection of 73 fibroepithelial tumours (including 26 FAs, 25 benign PTs, nine borderline PTs and 13 malignant PTs) from 64 patients was retrieved from the authors' institution. Sections from formalin-fixed paraffin-embedded (FFPE) blocks were microdissected to ensure an enrichment in neoplastic stromal elements of >70%. DNA samples extracted from tumour and matched normal tissues were subjected to Sanger sequencing of exon 2 of the MED12 gene. MED12 exon 2 somatic mutations, including 28 somatic single nucleotide variants and 19 insertions and deletions, were found in 65%, 88%, 78% and 8% of FAs, benign PTs, borderline PTs and malignant PTs, respectively. Malignant PTs harboured MED12 exon 2 somatic mutations significantly less frequently than FAs, benign and borderline PTs. Although MED12 exon 2 somatic mutations probably constitute the driver genetic event of most FAs, benign and borderline PTs, our results suggest that the majority of malignant PTs may be driven by other genetic/epigenetic alterations.

Distribution of MED12 mutations in fibroadenomas and phyllodes tumors of the breast--implications for tumor biology and pathological diagnosis.

Somatic mutations in exon 2 of MED12 have been described in benign and malignant smooth muscle cell tumors suggesting a functional role in these neoplasms. Recently fibroadenomas of the breast were also reported to harbor MED12 mutations. Hence, we explored MED12 mutations in fibroepithelial tumors of the breast, histological subtypes of fibroadenomas and phyllodes tumors, to validate and extend previous efforts. Using conventional Sanger sequencing, we profiled 39 cases of fibroepithelial breast tumors comprising classic histological subtypes of fibroadenomas as well as benign and malignant phyllodes tumors for mutations in exon 2 of MED12. MED12 mutations were detected in 60% of all tumor samples with the majority being missense mutations affecting codon 44. Additionally, we report novel in-frame deletions that have not been described previously. Sixty-two percent of the fibroadenomas harbored mutated MED12 with intracanalicular fibroadenomas being the most frequently mutated histological subtype (82%). Of note, 8/11 of benign phyllodes tumors had MED12 mutations while only 1/5 of malignant phyllodes tumors showed mutations in exon 2 of MED12. In conclusion, we confirm the frequent occurrence of MED12 mutations in fibroadenomas, provide evidence that most intracanalicular fibroadenomas closely resembling benign phyllodes as well as benign phyllodes tumors harbor MED12 mutations, and conclude that MED12 mutations in malignant phyllodes tumors appear to be relatively rare.

Characterization of Somatic Mutations in Air Pollution-Related Lung Cancer

Air pollution has been classified as Group 1 carcinogenic to humans, but the underlying tumorigenesis remains unclear. In Xuanwei City of Yunnan Province, the lung cancer incidence is among the highest in China attributed to severe air pollution generated by combustion of smoky coal, providing a unique opportunity to dissect lung carcinogenesis of air pollution. Here we analyzed the somatic mutations of 164 non-small cell lung cancers (NSCLCs) from Xuanwei and control regions (CR) where smoky coal was not used. Whole genome sequencing revealed a mean of 289 somatic exonic mutations per tumor and the frequent C:G→A:T nucleotide substitutions in Xuanwei NSCLCs. Exome sequencing of 2010 genes showed that Xuanwei and CR NSCLCs had a mean of 68 and 22 mutated genes per tumor, respectively (p<0.0001). We found 167 genes (including TP53, RYR2, KRAS, CACNA1E) which had significantly higher mutation frequencies in Xuanwei than CR patients, and mutations in most genes in Xuanwei NSCLCs differed from those in CR cases. The mutation rates of 70 genes (e.g., RYR2, MYH3, GPR144, CACNA1E) were associated with patients' lifetime benzo(a)pyrene exposure. This study uncovers the mutation spectrum of air pollution-related lung cancers, and provides evidence for pollution exposure–genomic mutation relationship at a large scale.

Deciphering the impact of somatic mutations in exon 20 and exon 9 of PIK3CA gene in breast tumors among Indian women through molecular dynamics approach

We examined 25 breast tumor samples for somatic mutations in exon 20 and exon 9 of PIK3CA gene in South Indian population. Genomic DNA was isolated and amplified for PIK3CA gene, followed by direct sequencing of purified polymerase chain reaction products. We identified PI3K3CA mutations in 5 of 25 (20%), including four of the mutations in p.H1047R and one in p.H1047L. Nucleotide base substitution A to G (c.3140A > G) and A to T (c.3140A > T) results in p.H1047R and p.H1047L mutation in exon 20 of PIK3CA gene. We did not observe any mutation in exon 9 of PIK3CA gene. Furthermore, we investigated the effect of mutations on protein structure and function by the combination of sequence and structure-based in silico prediction methods. This determined the underlying relationship between the mutation and its phenotypic effects. Next step, we complemented by molecular dynamics simulation analysis (30 ns) of native and mutant structures that measured the effect of mutation on protein structure. The obtained results support that the application of computational methods helps predict the biological significance of mutations.

Integrative and comparative genomic analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas.

The genetic differences between human papilloma virus (HPV)-positive and -negative head and neck squamous cell carcinomas (HNSCC) remain largely unknown. To identify differential biology and novel therapeutic targets for both entities, we determined mutations and copy-number aberrations in a large cohort of locoregionally advanced HNSCC. We performed massively parallel sequencing of 617 cancer-associated genes in 120 matched tumor/normal samples (42.5% HPV-positive). Mutations and copy-number aberrations were determined and results validated with a secondary method. The overall mutational burden in HPV-negative and HPV-positive HNSCC was similar with an average of 15.2 versus 14.4 somatic exonic mutations in the targeted cancer-associated genes. HPV-negative tumors showed a mutational spectrum concordant with published lung squamous cell carcinoma analyses with enrichment for mutations in TP53, CDKN2A, MLL2, CUL3, NSD1, PIK3CA, and NOTCH genes. HPV-positive tumors showed unique mutations in DDX3X, FGFR2/3 and aberrations in PIK3CA, KRAS, MLL2/3, and NOTCH1 were enriched in HPV-positive tumors. Currently targetable genomic alterations were identified in FGFR1, DDR2, EGFR, FGFR2/3, EPHA2, and PIK3CA. EGFR, CCND1, and FGFR1 amplifications occurred in HPV-negative tumors, whereas 17.6% of HPV-positive tumors harbored mutations in fibroblast growth factor receptor genes (FGFR2/3), including six recurrent FGFR3 S249C mutations. HPV-positive tumors showed a 5.8% incidence of KRAS mutations, and DNA-repair gene aberrations, including 7.8% BRCA1/2 mutations, were identified. The mutational makeup of HPV-positive and HPV-negative HNSCC differs significantly, including targetable genes. HNSCC harbors multiple therapeutically important genetic aberrations, including frequent aberrations in the FGFR and PI3K pathway genes. See related commentary by Krigsfeld and Chung, p. 495.

Calr Is up-Regulated in Patients with Essential Thrombocythemia Independently from Calr and JAK2 Mutations

Somatic mutations in exon 9 of calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations, and absent in patient with polycythemia vera (PV). Among patients with ET or PMF with un-mutated JAK2 or MPL, CALR mutations were detected in 67% of those with ET and 88% of those with primary PMF. Several types of insertions or deletions were identified and all resulted in a frameshift in exon 9 generating a novel C-terminal peptide in the mutant CALR protein. Over expression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with myeloproliferative neoplasms carrying CALR mutations present with higher platelet counts and lower haemoglobin levels than patients with mutated JAK2. Studies suggest these patients have a lower risk of thrombosis and longer overall survival than patients with mutated JAK2.We analysed by Real Time PCR, CALR expression in peripheral blood (PB) of 38 patients affected by ET, 17 JAK2 mutated (45%), 4 CALR (10.5%) mutated, 1 MPL mutated (3%) and 14 with no apparent molecular abnormalities. These were compared with a cohort of healthy volunteers. We found a significant over expression of CALR (median 5.15; range 1.13-270.08) comparing with controls (median 0.38, range 0.18-1). CALR mRNA expression is independent from the CALR mutational status. No significant difference was found comparing CALR expression in CALR mutated (median 4.9, range 1.51-37.14) and CALR/JAK2 un-mutated patients (4.68, range 1.51-28.71). CALR up-regulation is not mutually exclusive with JAK2 mutations; no difference was seen in CALR mRNA between JAK2 mutated (median 5.09, range 1.13-270) and wild type ET patients (median 5.08, range 1.51-37). There was no significant difference when we correlated CALR expression with PLT counts, spleen size or type of cytoreductive therapy. A larger cohort of patients is required to confirm these preliminary findings. DisclosuresNo relevant conflicts of interest to declare.

Novel MED12 gene somatic mutations in women from the Southern United States with symptomatic uterine fibroids.

Although somatic mutations in exon 2 of the mediator complex subunit 12 (MED12) gene have been reported previously in uterine fibroids in women from Finland, South Africa, and North America, the status of these mutations was not reported in the Southern United States women. The aim of this study is to determine the MED12 somatic mutations in uterine fibroids of women from Southern Unites States, which will help to better understand the contribution of MED12 mutations in fibroid tumor biology. Herein, we determined the frequency of MED12 gene exon 2 somatic mutations in 143 fibroid tumors from a total of 135 women from the Southern United States and in 50 samples of the adjacent myometrium using PCR amplification and Sanger sequencing. We observed that the MED12 gene is mutated in 64.33 % (92/143) of uterine fibroid cases in the exon 2 (including deletion mutations). These mutations include 107T > G (4.3 %), 130G > C (2.8 %), 130G > A (7.0 %), 130G > T (2.8 %), 131G > C (2.1 %), 131G > A (20.2 %), and 131G > T (2.1 %). Interestingly, we identified four novel mutations in these patients: 107 T > C (12.8 %), 105A > T (2.1 %), 122T > A (2.1 %), and 92T > A (2.1 %). As expected, we did not observe any mutations in the normal myometrium. Moreover, we found a higher rate of deletion mutations (17.5 %, 25/143) in the above fibroid tumors. Our results clearly demonstrate that the MED12 gene exon 2 is frequently mutated in human uterine fibroids in Southern United States women. These results highlight the molecular pathogenesis of human uterine fibroids with the central role of MED12 somatic mutations.

Abstract 2625: Increased sensitivity to Wnt/beta-catenin inhibitors in hepatocellular carcinoma cells harboring activating mutation of beta-catenin

Abstract [Background] The WNT/β-catenin signaling pathway is a major oncogenic pathway of human hepatocellular carcinoma (HCC). More than 50% of HCCs have aberrant activation of WNT/β-catenin signaling, which are caused by genetic alterations, ligand activation, or cross-activation by other signaling pathways. We hypothesized that activating mutation of CTNNB1, the coding gene of β-catenin, may confer sensitivity to WNT/β-catenin inhibitors in HCC cells. [Materials and Methods] SNU398 is a human HCC cell line harboring a missense somatic mutation in exon 3 of CTNNB1 gene, resulting in S37C mutation. Huh7, HepG2, PLC5, and Hep3B are HCC cells containing no somatic mutations in exon 3 of CTNNB1 gene. HCC cells were treated with various types of WNT/β-catenin pathway inhibitors, including ICG-001 which down-regulates β-catenin/TCF transcriptional activity by interfering the binding of β-catenin with its transcriptional activation complex, XAV939 which targets tankyrases and facilitates degradation of β-catenin, and LGK974 which inhibits Porcupine, an acyltransferase responsible for palmitoylation and secretion of Wnt ligands, and evaluated for in vitro viability by MTT. [Results] ICG-001 as a single agent showed significantly greater anti-proliferation effect in SNU398 than other HCC cells. The 50%-inhibitory concentrations (IC50s) of ICG-001 were 5 μM and &amp;gt;10 μM for SNU398 and other HCC cells, respectively. ICG-001 also induced a more potent suppression of colony- formation, and a more significant increase of subG1 fraction detected by flow cytometry in SNU398 cell than other HCC cells. XAV939 up to 20 μM induced only modest anti-proliferative effect in all HCC cells. However, SNU398 remained the one showing the greatest sensitivity to the growth suppression induced by XAV939. The anti-proliferative effect of LGK974 was modest and showed no significant difference among different HCC cells. [Conclusion] The activating mutation of β-catenin may confer sensitivity to certain WNT/β-catenin pathway inhibitors, such as ICG-001, in HCC cells. (This study was supported by grants NRPB-100CAP1020-2). Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, LI-Chun Lu, Ta-Wen Hsu, Ann-Lii Cheng, Chih-Hung Hsu. Increased sensitivity to Wnt/beta-catenin inhibitors in hepatocellular carcinoma cells harboring activating mutation of beta-catenin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2625. doi:10.1158/1538-7445.AM2014-2625