High Prevalence Of Mutations Research Articles

Genetic and Functional Analysis of GRIN2A in Tumor Samples.

Ionotropic glutamate receptors (iGluRs) are large integral membrane multi-protein complexes that create ion channels in plasma membranes. Upon binding of receptor specific ligands (e.g., glutamate), increased efflux or influx of mono- or divalent cations (e.g., Ca2+) promotes synaptic transmission, cellular migration, and survival. Three classes of iGluRs were originally defined after their respective agonists: AMPA, kainate, and NMDA receptors (NMDARs). Recently, we examined iGluR families at the genetic level using Next-Generation Sequencing (NGS) (whole-exome sequencing (WES)) and discovered a high prevalence of somatic mutations within the gene for one of the NMDAR subunits, GRIN2A, specifically in malignant melanoma. Following confirmation of the somatic mutations, we focused on functional characterization of a subset of the GRIN2A mutants that demonstrated a loss of NMDAR functionality. We used gene expression and protein biochemistry to examine complex formation between GluN1 subunit (encoded by GRIN1) and GluN2A subunit (encoded by GRIN2A), anchorage-independent growth in soft agar and cellular migration. Furthermore, we used shRNA depletion of endogenous GRIN2A in melanoma cells expressing either wild-type GRIN2A or mutant GRIN2A and measured cellular proliferation compared to negative controls. Our data show that somatic mutation of certain residues in GluN2A results in increased survival and is the first such report to demonstrate the functional importance of GRIN2A mutations in melanoma and the significance ionotropic glutamate receptor signaling plays in malignant melanoma.

P3.02b-026 Association of EGFR Exon 19 Deletion and EGFR-TKI treatment duration with Frequency of T790M Mutation in EGFR-Mutant Lung Cancer Patients: Topic: EGFR Biomarkers

The most common event responsible for resistance of epidermal growth factor receptor (EGFR)-tyrosine kinase Inhibitor (TKI) is acquisition of the T790M mutation, which occurs in approximately 50% of patients who initially respond to EGFR-TKI. Recently, third-generation EGFR-TKIs have been shown to exert a remarkable effect against T790M resistance mutation-positive non-small cell lung cancer (NSCLC). T790M is an important predictive marker for third-generation EGFR-TKIs; therefore, determining the clinicopathologic characteristics of T790M-harboring NSCLC showing relapse after EGFR-TKI therapy is of high clinical relevance. we evaluated whether T790M mutation is related to clinicopathologic or prognostic factors in patients with relapse of EGFR-mutant NSCLC after treatment with EGFR-TKIs. We retrospectively reviewed T790M status and clinical characteristics of advanced-stage or recurrent NSCLC patients who had received EGFR-TKI treatment and rebiopsy at Kurume University Hospital between March 2005 and December 2015. we identified 193 patients with advanced or recurrent EGFR-mutant NSCLC who developed resistance to initial EGFR-TKI treatment. Of these patients, 105 (54%) underwent re-biopsy. Adequate histological specimens were available for 73 of these patients, who were enrolled in the study; 38 (52%) of them had T790M mutation and 35 (48%) did not. T790M mutation was present more frequently in patients with exon 19 deletion mutation (63%, 26 of 41) than in those with L858R mutation (38%, 12 of 32) (p=0.035) and in patients who received EGFR-TKI treatment for at least 10 months in total (71%, 32 of 45) than in those who did not (21%, 6 of 28) (p<0.001). The median PFS after initial EGFR-TKI treatment was longer in the T790M mutation-positive group (13.6 months, 95% CI: 9.2-15.8) than in the negative group (7 months, 95% CI: 3.7-8.5) (p=0.037). The median total duration of EGFR-TKI treatment in patients with T790M mutation was 15.3 months, which was significantly longer than that (8.1 months) in patients without T790M mutation (p<0.001). Multivariate analyze revealed that The type of EGFR mutation (exon 19 deletion mutation versus L858R point mutation, p=0.011, OR=0.21, 95% CI=0.05-0.71) and total duration of EGFR-TKI treatment (≥10 months versus <10 months, p<0.001, OR=0.09, 95% CI=0.02-0.28) were significantly associated with the presence of T790M mutation. The patients with EGFR exon 19 deletion mutation and long-term treatment with EGFR-TKI exposure demonstrated a high prevalence of T790M mutation. These data are potentially important for clinical decision making in the NSCLC patients with EGFR mutation.

Mutational Profile of Metastatic Breast Cancers: A Retrospective Analysis

BackgroundMajor advances have been achieved in the characterization of early breast cancer (eBC) genomic profiles. Metastatic breast cancer (mBC) is associated with poor outcomes, yet limited information is available on the genomic profile of this disease. This study aims to decipher mutational profiles of mBC using next-generation sequencing.Methods and FindingsWhole-exome sequencing was performed on 216 tumor–blood pairs from mBC patients who underwent a biopsy in the context of the SAFIR01, SAFIR02, SHIVA, or Molecular Screening for Cancer Treatment Optimization (MOSCATO) prospective trials. Mutational profiles from 772 primary breast tumors from The Cancer Genome Atlas (TCGA) were used as a reference for comparing primary and mBC mutational profiles. Twelve genes (TP53, PIK3CA, GATA3, ESR1, MAP3K1, CDH1, AKT1, MAP2K4, RB1, PTEN, CBFB, and CDKN2A) were identified as significantly mutated in mBC (false discovery rate [FDR] < 0.1). Eight genes (ESR1, FSIP2, FRAS1, OSBPL3, EDC4, PALB2, IGFN1, and AGRN) were more frequently mutated in mBC as compared to eBC (FDR < 0.01). ESR1 was identified both as a driver and as a metastatic gene (n = 22, odds ratio = 29, 95% CI [9–155], p = 1.2e-12) and also presented with focal amplification (n = 9) for a total of 31 mBCs with either ESR1 mutation or amplification, including 27 hormone receptor positive (HR+) and HER2 negative (HER2−) mBCs (19%). HR+/HER2− mBC presented a high prevalence of mutations on genes located on the mechanistic target of rapamycin (mTOR) pathway (TSC1 and TSC2) as compared to HR+/HER2− eBC (respectively 6% and 0.7%, p = 0.0004). Other actionable genes were more frequently mutated in HR+ mBC, including ERBB4 (n = 8), NOTCH3 (n = 7), and ALK (n = 7). Analysis of mutational signatures revealed a significant increase in APOBEC-mediated mutagenesis in HR+/HER2− metastatic tumors as compared to primary TCGA samples (p < 2e-16). The main limitations of this study include the absence of bone metastases and the size of the cohort, which might not have allowed the identification of rare mutations and their effect on survival.ConclusionsThis work reports the results of the analysis of the first large-scale study on mutation profiles of mBC. This study revealed genomic alterations and mutational signatures involved in the resistance to therapies, including actionable mutations.

FAT1 Mutations Influence Time to First Treatment in Untreated CLL

Introduction:Despite mapping of the mutational landscape in patients (pts) with chronic lymphocytic leukemia (CLL), there remains limited information regarding the functional or clinical impact of less common, recurrently mutated genes. Over the past several years, the influence of high-risk mutations has been reported. With the advent of readily available next generation sequencing (NGS), physicians receive a large amount of genomic data, many times without information regarding the clinical impact of less validated, mutations. Using results from a commercially available NGS platform, we identified a relatively high prevalence of mutations in the gene, FAT1, in treatment naïve (TN) CLL pts.FAT1 encodes a protein in the cadherin superfamily, important in regulating WNT signaling, with a role in tumor suppression. Aberrant mutated FAT1 expression has been associated with acute lymphoblastic leukemias with an intermediate clinical course. Loss of protein, through chromosomal deletions has been implicated in many solid tumors and associated with tumor progression. The majority of mutations arise in the cadherin repeats, previously shown to diminish antagonism of β-catenin, allowing its nuclear localization (Morris Nat Genetics 2013). In CLL, FAT1 mutations have been demonstrated to enrich in fludarabine refractory pts (10.3%) but with a relatively low prevalence in untreated pts (1.1%) (Messina, Blood 2014). Given a high prevalence of FAT1 mutations in our database and evidence suggesting mutated FAT1 contributes to tumor evolution, we investigated the clinical impact of clonal FAT1 mutations in pts with CLL.Methods:Patients were identified for inclusion if mutational analysis was performed prior to, or within 12 months of treatment start date. Only pts treated initially with a signal transduction inhibitor (STI) were included. Treated pts were either on protocol or received their STI as standard of care. All pts underwent whole exome profiling with a lymphoid specific NGS panel, including 75 genes (Genoptix Inc.). Analysis was performed on peripheral blood or bone marrow aspirates. The primary outcome was time to first treatment (TTFT). The log-rank test was used to compare Kaplan-Meir curves. Double-sided P values <0.05 were considered significant. Fischer exact test was used to compare baseline characteristics. All calculations were performed using Prism V6.0.Results:In total we analyzed 172 pts, 118 (69%) remain on observation, 54 (31%), have been treated at data cutoff. Nineteen (11%) pts were found to be FAT1 mutated (mFAT1) and 153 (89%) were FAT1 wild type (wtFAT1). The median time from diagnosis to mutational profiling for the cohort was 35 months (range 0-301). We identified 21 total mutations, 17 unique (82% cadherin domain, 18% EGF like region). Recurrent mutations were identified, each occurring in 2 pts (p.A636T, p.P1614L, p.R1257Q, p.R2041H). One pt had an indel and 1 pt had an inframe deletion, the remaining were missense. We found no significant differences between groups in regards to age, IGVH mutation status or co-occurrence of high-risk mutations in NOTCH1, SF3B1, TP53 or ATM. Only 17p deletions occurred significantly more in mFAT1 pts (24%) vs. wtFAT1 (7%), p=0.04, other cytogenetic abnormalities were balanced. mFAT1 pts had significantly shorter TTFT, 50 versus 143 months respectively, p=0.02. We investigated if mFAT1 status could stratify IGVH mutated pts but found no differences in TTFT. Eleven mFAT1 pts have received treatment (82% ibrutinib, 18% acalabrutinib), 8 remain on observation. All mFAT1 pts have responded.Conclusions:Given the increasing use of rapidly available NGS testing, physicians receive genomic information with limited data regarding clinical impact. We identified a higher prevalence of FAT1 mutations in TN pts, than that previously reported. FAT1 mutations associated with deletion 17p but not other high-risk genetic mutations. Recurrent FAT1 mutations were identified and commonly found in the cadherin domain. mFAT1 pts have a significantly shorter TTFT than wtFAT1 pts. When treated with novel agents, there was no difference in response rates. These findings suggest FAT1 mutations in TN pts may be more common than previously reported and identify an intermediate risk for progression. Additional studies investigating the influence of recurrent FAT1 mutations and association with 17p deletions in TN pts are warranted. DisclosuresAllan:Pharmacyclics: Speakers Bureau.

Low Bax/Bcl-2 Ratio and NOTCH1 Mutations Represent Powerful and Synergistic Adverse Prognostic Factors within Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Trisomy 12 is the third most common cytogenetic abnormality in CLL with several distinguishing features including abormal morphology, high prevalence of NOTCH1 mutations (NOTCH1 M) and increased expression of the alpha-integrins CD11a and CD49d (Balatti, 2012; Zucchetto, 2013). Trisomy 12 marks a disease subset characterized by high rates of cell proliferation, disease progression and Richter syndrome transformation (Rossi, 2013). Noteworthy, NOTCH1 M implicate constitutive activation of NOTCH1 signaling which triggers apoptosis resistance and increased survival of CLL cells (Rosati, 2009). Moreover, bax/bcl-2 ratio (bax/bcl-2), marker of apoptosis, predicts chemoresistance and progressive disease in CLL (Del Principe, 2016). The today availability in clinical use of venetoclax (ABT-199), a potent oral anti-bcl-2 peptidomimetic (Seymour, 2014), prompted us to analyze the real impact of the apoptosis (bax/bcl-2) on trisomy 12 CLL prognosis. The primary aims of our research were: 1) to demonstrate a significant association between NOTCH1 M and bax/bcl-2; 2) to correlate NOTCH1 M and bax/bcl-2 with biological and clinical prognosticators; 3) to determine progression-free survival (PFS) and overall survival (OS) upon NOTCH1 M and bax/bcl-2; 4) to evaluate NOTCH1 M and bax/bcl-2 as independent prognostic factors. Therefore we investigated 653 CLL patients, median age 66 years, 363 males and 290 females. Using the 5% cutoff for the FISH cytogenetic abnormalities, 140 (21.4%) cases were classified as trisomy 12 CLL. Bax/bcl-2 was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.32-5.30). The presence of NOTCH1 M was investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1 exon 34. CD49d was >20% in almost all trisomy 12 pts (116/140; 83%). Forty-five patients were NOTCH1 M (32.1%) and 45 were bax/bcl-2 positive (32.1%). There was a very strong correlation between bax/bcl-2 <1.5 and NOTCH1 M (42/45; p<0.0001), corroborating the close dependence of the lacking apoptosis on NOTCH1 M. Both lower bax/bcl-2 and NOTCH1 M were significantly associated with lymphocyte doubling time <12 months (p=0.0077 and p=0.0006), with beta-2 microglobulin >2.2 mg/dl (p=0.0001 and p=0.0007) and LDH >300 U/L (p=0.004 and p=0.00001), thus confirming their significant association both with a high proliferative rate and a high tumor burden. Strong correlations were found between NOTCH1 M or bax/bcl-2 <1.5 and IGHV unmutated status (p<0.0001 and p=0.0005) or ZAP-70 >30% (p<0.0001 and p=0.00002). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with NOTCH1 M vs NOTCH1 wild type [WT] (0% vs 27% at 10 years, p=0.001 and 26% vs 78% at 14 years, p=0.005) or lower bax/bcl-2 (9% vs 38% at 10 years, p=0.0007 and 45% vs 89% at 14 years, p=0.002). Interestingly, NOTCH1 M and bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 M and bax/bcl-2 <1.5 identified a trisomy 12 CLL subset at worst prognosis with regard to PFS (0% vs 40% at 10 years, p=0.00004, Figure) and OS (20% vs 100% at 14 years, p=0.0001, Figure). Therefore, bax/bcl-2 and NOTCH1 M are powerful prognosticators, showing synergistic clinical effects. In multivariate analysis of OS, bax/bcl-2 (p=0.004) together with age (p=0.0006), CD49d (p=0.01) and IGHV status (p=0.03) was confirmed as an independent prognostic factor. In conclusion, the modern strategies to trigger apoptosis and block proliferation, such as venetoclax, may be very effective in this CLL subset, also taking into account that trisomy 12 CD49d+ CLL has abbreviated lymphocytosis with retention of CLL cells within tissues (Thompson, 2014) and consequent poor reduction of lymphadenopathy during treatment with ibrutinib. [Display omitted] DisclosuresLo Coco:Teva: Consultancy, Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; Novartis: Consultancy; Baxalta: Consultancy; Pfizer: Consultancy.

BRAFV600E Transduced Human CD34+ Cells Establish Aggressive Langerhans Cell Histiocytosis in Humanized Mice

IntroductionLangerhans cell histiocytosis (LCH) is the most common histiocytic disease in humans with a complex pathophysiology and unknown etiology. LCH has been recently reclassified from an inflammatory disease to a neoplastic disorder due to the high prevalence of mutations in the MAP kinase pathway. Recurrent mutation in BRAF kinase (BRAFV600E) was detected in more than 50% of patients. We recently showed that BRAFV600E mutation can be detected in the circulating CD34+ cells in LCH patients and that the expression of the mutation in distinct hematopoietic compartments defines the stage and the clinical risk in LCH disease (Berres et al. J Exp Med 2014). Knowledge on induction and progression of LCH is limited due to appropriate models resembling the human disease. We recently generated so called MISTRG mice where the mouse cytokines M-CSF, IL-3, GM-CSF, and thrombopoietin were replaced by their human variants and that in addition express a human transgene of SIRPa (Rongvaux et al. Nat Biotech 2014). These mice efficiently support human myelopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We here tested if xeno-transplantation of BRAFV600E transduced human CD34+ cells in these mice would result in LCH.Materials and MethodsHuman CD34+ HSPCs obtained from healthy donor cord blood samples were lentivirally transduced using a bidirectional promoter vector harboring BRAFV600E and GFP (as a marker for transduction efficiency) in the presence of supportive growth factors. As control, BRAFV600E construct was substituted by luciferase gene. The In vivo experiments were performed by injection of transduced CD34+ into newborn (n=10, of which 5 control and BRAFV600E respectively) and adult (n=8, of which 4 control and BRAFV600E respectively) MISTRG mice, which were sublethally irradiated prior to transplantation. Mice were sacrificed once symptoms of sickness were observed and analyzed using flow cytometry and immunohistochemistry staining for investigating the development of LCH-like disease. The influence of BRAFV600E mutation on proliferation and lineage commitment of human HSPCs was investigated using colony forming unit (CFU) assay and short term liquid cultures. For CFU assays, the transduced human CD34+ cells were plated in M4230 medium in the presence of supportive growth factors. After 14-16 days the obtained colonies were scored microscopically and measured by flow cytometry for further characterization of the cells. For short term liquid cultures, transduced cells were kept in culture in the presence of supportive growth factors and measured by flow cytometry for expression of human myeloid and lymphoid markers.ResultsMice transplanted with human CD34+ HSPCs expressing BRAFV600E developed anemia and reduced WBC and RBC counts. Analysis of lymphoid and non-lymphoid organs demonstrated the accumulation of ''atypical'' BRAFV600E expressing dendritic cells in granuloma-like lesions. Using immunohistochemistry staining we observed the expression of LCH-associated markers, CD1a and CD207 to a varying extent in different organs (Figure 1). We did not observe development of hairy cell leukemia. The in vitro studies revealed that the expression of BRAFV600E blocks the commitment of erythroid progeny toward mature erythrocytes shown by lower number of BFU-E colony type and reduced CD235ahigh-expressing cells. BRAFV600E seemed to increase monocytosis and generation of classical dendritic cells in short term liquid cultures, without affecting the proliferation potential of human HSPCs.Conclusions and outlookWe demonstrate that expression of BRAFV600E in human HSPCs induces LCH-like disease in MISTRG mice. The involvement of wide range of organs resembles high-risk LCH in humans and confirms our already reported data with aggressive LCH being a HSPC disease. Using this model system will further support basic understanding of LCH patho-biology and testing of targeted therapies in LCH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Population-Based Study of Attitudes toward BRCA Genetic Testing among Orthodox Jewish Women.

Given the high prevalence (1 in 40) of BRCA1 and BRCA2 mutations among Ashkenazi Jews, population-based BRCA genetic testing in this ethnic subgroup may detect more mutation carriers. We conducted a cross-sectional survey among Orthodox Jewish women in New York City to assess breast cancer risk, genetic testing knowledge, self-efficacy, perceived breast cancer risk and worry, religious and cultural factors affecting medical decision-making. We used descriptive statistics and multivariable logistic regression models to identify predictors of genetic testing intention/uptake. Among evaluable respondents (n = 243, 53% response rate), median age was 25 and nearly half (43%) had a family history of breast cancer. Only 49% of the women had adequate genetic testing knowledge and 46% had accurate breast cancer risk perceptions. Five percent had already undergone BRCA genetic testing, 20% stated that they probably/definitely will get tested, 28% stated that they probably/definitely will not get tested, and 46% had not thought about it. High decision self-efficacy, adequate genetic testing knowledge, higher breast cancer risk, and overestimation of risk were associated with genetic testing intention/uptake. Decision support tools that improve knowledge and self-efficacy about genetic testing may facilitate population-based BRCA testing among Orthodox Jews.

Association of EGFR Exon 19 Deletion and EGFR-TKI Treatment Duration with Frequency of T790M Mutation in EGFR-Mutant Lung Cancer Patients.

The most common event responsible for resistance to first- and second-generation (1st and 2nd) epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) is acquisition of T790M mutation. We examined whether T790M is related to clinicopathologic or prognostic factors in patients with relapse of EGFR mutant non-small cell lung cancer (NSCLC) after treatment with 1st or 2nd EGFR-TKIs. We retrospectively reviewed the T790M status and clinical characteristics of 73 patients with advanced or recurrent NSCLC who had been treated with EGFR-TKIs and undergone rebiopsy at Kurume University Hospital between March 2005 and December 2015. T790M mutation was more frequent in patients with EGFR exon 19 deletion mutation (63%, 26/41) than in those with L858R mutation (38%, 12/32) (p = 0.035). The median total duration of 1st or 2nd EGFR-TKI treatment was significantly longer in patients with T790M mutation than in those without (15.3 months vs 8.1 months, p < 0.001). Multivariate analysis revealed that the type of EGFR mutation and the total duration of EGFR-TKI treatment were significantly associated with T790M prevalence. Patients with EGFR exon 19 deletion mutation who receive long-term EGFR-TKI therapy show a high prevalence of T790M mutation. The present data are potentially important for clinical decision-making in NSCLC patients with EGFR mutation.

Antiviral drug resistance and hepatitis B: a continuing public health problem.

The use of selective antiviral therapy has been very successful in controlling HBV replication in individuals, leading to a reduction in disease progression and mortality. However, the use of first-generation therapies, often the only available option in low-resource settings, can result in a high prevalence of drug resistance mutations. Variants selected by antiviral therapies targeting the viral polymerase can also result in variants in the viral envelope. These variants can allow the virus to escape the host immune response. The effect of antiviral selection pressure on viral variants that may contribute to immune escape requires further investigation.

Driver oncogenes in Sub-Saharan African patients with non-small cell lung cancer

Non-small cell lung cancer can exhibit driver oncogenes, including epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), that are possible targets for therapy. The prevalence of these rearranged driver oncogenes is influenced by race, smoking habits, and gender. Most data come from Caucasian and Asian populations. To our knowledge, there is no literature available about the prevalence of driver oncogenes in Sub-Saharan Africa, where the tobacco epidemic is still in the early stage. In this small case series, 6 patients of Sub-Saharan African ethnicity with stage IV lung adenocarcinoma are described. EGFR mutation was present in 3/6 patients and ALK rearrangement in 1/6 patients. This incidence seems high but interestingly, all patients were non-smokers or light smokers. In this series, the high prevalence of driver oncogene was probably related to low smoking habits and these initial data in Sub-Saharan Africans suggest high prevalence of driver mutations for this reason.

Clinicopathological, Immunohistochemical, and Genetic Features of Primary Lung Adenocarcinoma Occurring in the Setting of Usual Interstitial Pneumonia Pattern

IntroductionAn association between usual interstitial pneumonia (UIP) and carcinogenesis has been well established. However, few detailed analyses have investigated the clinicopathological, immunohistochemical, and genetic features of patients with primary lung adenocarcinoma (ADC) with UIP (UIP-ADC). MethodsWe identified 44 patients with ADC in the setting of UIP (the UIP-ADC group) (1.9%) from 2309 patients with primary ADC and compared clinicopathological, immunohistochemical, and genetic features between the UIP-ADC group and patients with ADC without UIP (the non–UIP-ADC group). ResultsClinicopathological features of UIP-ADC included an older age at occurrence; male predominance; smoking history; predilection for the lower lobe; large tumor size; high incidence of lymph vessel invasion, pleural invasion, and lymph node metastasis; and poor survival rate. However, the cause of death of patients with UIP-ADC was largely influenced by respiratory complications. Histologically, patients in the UIP-ADC group could be stratified according to invasive mucinous-predominant subtype. Genetically, patients in the UIP-ADC group had lower EGFR and higher KRAS mutation rates compared with patients in the non–UIP-ADC group. ConclusionsUIP-ADC was associated with a poor prognosis owing to the high frequency of perioperative complications rather than the malignancy of the tumor itself. There was a high prevalence of the invasive mucinous-predominant subtype in cases of UIP-ADC. UIP-ADC also had a low prevalence of EGFR mutations and a high prevalence of KRAS mutations. These findings suggest that UIP-ADC should be distinct from non–UIP-ADC.

High Prevalence of Concomitant Oncogene Mutations in Prospectively Identified Patients with ROS1-Positive Metastatic Lung Cancer

Chromosomal rearrangements involving ROS1 define a rare entity of lung adenocarcinomas with exquisite sensitivity to molecularly targeted therapy. We report clinical outcomes and genomic findings of patients with ROS1-positive lung cancer who were prospectively identified within a multiplex biomarker profiling program at the West German Cancer Center. Standardized immunohistochemical (IHC) analysis, fluorescence in situ hybridization (FISH), and hotspot mutation analyses were performed in 1345 patients with advanced cancer, including 805 patients with metastatic lung adenocarcinoma. Clinical and epidemiological data were retrieved from the institutional database. ROS1 positivity by IHC analysis was detected in 25 patients with lung cancer (4.8% of lung adenocarcinomas), including 13 patients (2.5%) with ROS1 FISH positivity with a cutoff of at least 15% of events. Of the ROS1 IHC analysis-positive cases, 36% presented with concomitant oncogenic driver mutations involving EGFR (six cases, five of which were clinically validated by response to EGFR-targeting agents), KRAS (two cases), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA), and BRAF. Three cases initially classified as ROS1 FISH-negative passed the threshold of 15% positive events when repeat biopsies were analyzed at progression. The median overall survival of the ROS1-positive patients (104 months) was significantly superior to that of the 261 patients with EGFR/anaplastic lymphoma kinase/ROS1-negative lung adenocarcinoma (24.4 months, p= 0.044). Interestingly, the overall survival of the 13 ROS1-positive patients with lung cancer from initiation of pemetrexed-based chemotherapy was significantly prolonged when compared with that of 169 pemetrexed-treated patients with EGFR/anaplastic lymphoma kinase/ROS1-negative adenocarcinoma (p= 0.01). ROS1-positive metastatic lung adenocarcinomas frequently harbor concomitant oncogenic driver mutations. Levels of ROS1 FISH-positive events are variable over time. This heterogeneity provides additional therapeutic options if discovered by multiplex biomarker testing and repeat biopsies.

Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations

AbstractPediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers.

High prevalence and predominance of BRCA1 germline mutations in Pakistani triple-negative breast cancer patients.

BackgroundWomen harboring BRCA1/2 germline mutations have high lifetime risk of developing breast/ovarian cancer. The recommendation to pursue BRCA1/2 testing is based on patient’s family history of breast/ovarian cancer, age of disease-onset and/or pathologic parameters of breast tumors. Here, we investigated if diagnosis of triple-negative breast cancer (TNBC) independently increases risk of carrying a BRCA1/2 mutation in Pakistan.MethodsFive hundred and twenty-three breast cancer patients including 237 diagnosed ≤ 30 years of age and 286 with a family history of breast/ovarian cancer were screened for BRCA1/2 small-range mutations and large genomic rearrangements. Immunohistochemical analyses were performed at one center. Univariate and multiple logistic regression models were used to investigate possible differences in prevalence of BRCA1/2 mutations according to patient and tumor characteristics.ResultsThirty-seven percent of patients presented with TNBC. The prevalence of BRCA1 mutations was higher in patients with TNBC than non-TNBC (37 % vs. 10 %, P < 0.0001). 1 % of TNBC patients were observed to have BRCA2 mutations. Subgroup analyses revealed a larger proportion of BRCA1 mutations in TNBC than non-TNBC among patients 1) diagnosed at early-age with no family history of breast/ovarian cancer (14 % vs. 5 %, P = 0.03), 2) diagnosed at early-age irrespective of family history (28 % vs. 11 %, P = 0.0003), 3) had a family history of breast cancer (49 % vs. 12 %, P < 0.0001), and 4) those with family history of breast and ovarian cancer (81 % vs. 28 %, P = 0.0005). TNBC patients harboring BRCA1 mutations were diagnosed at a later age than non-carriers (median age at diagnosis: 30 years (range 22–53) vs. 28 years (range 18–67), P = 0.002). The association between TNBC status and presence of BRCA1 mutations was independent of the simultaneous consideration of family phenotype, tumor histology and grade in a multiple logistic regression model (Ratio of the probability of carrying BRCA1/2 mutations for TNBC vs. non-TNBC 4.23; 95 % CI 2.50–7.14; P < 0.0001).ConclusionGenetic BRCA1 testing should be considered for Pakistani women diagnosed with TNBC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2698-y) contains supplementary material, which is available to authorized users.

P53, a potential predictor of Helicobacter pylori infection-associated gastric carcinogenesis?

Helicobacter pylori (H. pylori) is an ancient and persistent inhabitant of the human stomach that is closely linked to the development of gastric cancer (GC). . Emerging evidence suggests that H. pylori strain interactions with gastric epithelial cells subvert the best- characterized p53 tumour suppressor pathway. A high prevalence of p53 mutations is related to H. pylori infection. H. pylori also accelerates p53 protein degradation by disturbing the MDM2-P53 feedback loop. Additionally, H. pylori triggers the alteration of other p53 isoforms. Dysregulation of p53 by H. pylori infection contributes to gastric carcinogenesis by mediating cell proliferation and apoptosis. This review focuses on the regulation of p53 in H. pylori infection-associated GC.

High prevalence of TERT promoter mutations in micropapillary urothelial carcinoma.

Somatic activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Little is known, however, about TERT-mutation status in the relatively uncommon but clinically aggressive micropapillary (MPC) variant. We evaluated the presence of TERT promoter mutations in MPC of the bladder and upper urinary tract. A retrospective search of our archives for MPC and UC with micropapillary features (2005-2014) was performed. All slides were reviewed to confirm the histologic diagnosis. Thirty-three specimens from 31 patients had FFPE blocks available for DNA analysis and were included in the study. Intratumoral areas of non-micropapillary histology were also evaluated when present. Samples were analyzed with Safe-SeqS, a sequencing error reduction technology, and sequenced using the Illumina MiSeq platform. TERT promoter mutations were detected in all specimens with pure MPC (18 of 18) and UC with focal micropapillary features (15 of 15). Similar to conventional UC, the predominant mutations identified occurred at positions -124 (C228T) (85%) and -146 (C250T) (12%) bp upstream of the TERT ATG start site. In heterogeneous tumors with focal variant histology, intratumoral concordant mutations were found in variant (MPC and non-MPC) and corresponding conventional UC. We found TERT promoter mutations, commonly found in conventional UC, to be frequently present in MPC. Our finding of concordant intratumoral mutational alterations in cases with focal variant histology lends support to the common oncogenesis origin of UC and its variant histology.

High prevalence of DUOX2 gene mutations among children with congenital hypothyroidism in central China

Congenial hypothyroidism (CH) is the most common congenital endocrine disease and is treatable when recognized early enough. We investigated the genetic variants in 12 children diagnosed with CH by newborn screening in Huangshi area central China. Twelve genes commonly involved in CH development were studied. Genomic DNA from peripheral blood was used to amplify all exons of the selected genes, and the constructed sequencing libraries were subjected to next generation high throughput DNA sequencing (NGS). Analysis of the sequencing results identified rare genetic variants in 11 of the 12 patients (91.7%), and two novel rare variants were found in DUOX2 gene and two in TPO gene. Mutations in DUOX2 gene were identified in 10 patients (83.3%), and all these patients were found to carry bi-allelic, tri-allelic mutations or compound mutations with other genes. Recurrent DUOX2 mutations include K530X, R683L, R1110Q, and L1343F. Truncating, splicing, and proven deleterious DUOX2 missense mutations were detected in 50% of the patients. Mutations in TG gene were identified in four patients, and mutations in TPO, THSR, SLC26A4 genes were identified, one in each patient, respectively. The high prevalence of DUOX2 mutations in this cohort of children with CH appears striking and surprising. The clinical implications were discussed.

A Survey of BRCA1, BRCA2, and PALB2 mutations in women with breast cancer in Trinidad and Tobago.

The mortality rate from breast cancer in the nation of Trinidad and Tobago is among the highest of any country in the Caribbean region. The contribution of inherited gene mutations to the burden of breast cancer in Trinidad and Tobago has not been studied. We examined the prevalence of mutations in three susceptibility genes (BRCA1, BRCA2, and PALB2) in breast cancer patients in Trinidad and Tobago. We studied 268 unselected breast cancer patients from Trinidad and Tobago and looked for mutations across the entire coding sequences of BRCA1, BRCA2, and PALB2. Overall, 28 of 268 patients (10.4%) had a mutation in one of the three genes, including 15 in BRCA1, ten in BRCA2, two in PALB2, and one in both BRCA2 and PALB2. There were 25 different mutations identified; of these, four mutations were seen in two patients each. Given the high prevalence of mutations, it is reasonable to offer genetic testing for these three genes to all breast cancer patients in Trinidad and Tobago.

Abstract LB-353: Mutational profile of metastatic breast cancers using whole-exome sequencing: a retrospective analysis of 216 samples from SAFIR01 / 02 / SHIVA / MOSCATO trials

Abstract Background: Major advances have been achieved in the understanding of breast cancer biology using high throughput technologies at the DNA and RNA levels. Most of these advances were made in early breast cancers (eBC) which are cured in about 80% of the cases. Nevertheless, little is known about metastatic breast cancers (mBC) which remain lethal in most of the cases. Methods: Whole-exome sequencing was performed on 216 tumour-normal pairs from mBC patients who underwent a biopsy in the context of the SAFIR01/SAFIR02/SHIVA or MOSCATO prospective trials. TCGA dataset (n = 772) was used to assess frequency of mutations in eBC. Findings: Twelve genes (TP53, PIK3CA, GATA3, ESR1, MAP3K1, CDH1, AKT1, MAP2K4, RB1, PTEN, CBFB, CDKN2A) were identified as drivers using MutSig algorithm (FDR&amp;lt;0.1). Eight genes (ESR1, FSIP2, FRAS1, OSBPL3, EDC4, PALB2, IGFN1, AGRN) were more frequently mutated in mBC as compared to eBC (TCGA, FDR&amp;lt;0.01). HR+/Her2- mBC presented a high prevalence (6.3%) of mutations on genes located on mTOR pathway (TSC1, TSC2) as compared to HR+/Her2- eBC (0.7%, p = 0.0004). A subset of HR+/Her2- mBC (n = 17, 12%) presented a high mutational load (&amp;gt;150 non-synonymous mutations). This subset was observed in 2% of early breast cancers (TCGA, p = 7.1e?06). The prevalence of this subset increased with the time from diagnosis of metastasis to biopsy. Highly mutated HR+/Her2- mBC (n = 17) presented higher rate of PIK3CA mutations (n = 12, 70%), high number of neoantigens, an APOBEC mutational signature and a poor outcome (multivariate analysis, HR = 4.68, 95%CI: 1.8-12.1, p = 0.001). Interpretation: Whole exome sequencing of metastatic breast cancers identifies a subset of HR+/Her2- mBC who present a high mutational load. RB1, PALB2 and TSC1/2 mutations were found enriched in either mBC or HR+ mBC. Fundings: Breast Cancer Research Foundation, Fondation ARC, Fondation Lombard-Odier “Philanthropia”, Odyssea, Operation Parrains Chercheurs, Dassault Foundation, French NCI: INCa-DGOS-INSERM 6043 Citation Format: Maud Kamal, Celine Lefebvre, Thomas Bachelot, Thomas Filleron, Marion Pedrero, Mario Campone, Jean-Charles Soria, Christophe Massard, Christelle Levy, Monica Arnedos, Julie Garrabey, Yannick Boursin, Marc Deloger, Yu Fu, Frederic Commo, Veronique Scott, Ludovic Lacroix, Emmanuel Martin, Veronique Dieras, Anthony Goncalves, Jean-Marc Ferrero, Gilles Romieu, Laurence Vanlemmens, Marie-Ange Mouret-Reynier, Jean-christophe Thery, Pierre Kerbrat, Severine Guiu, Florence Dalenc, Gilles Clapisson, Hervé Bonnefoi, Martha Jimenez, Christophe Le Tourneau, Fabrice Andre. Mutational profile of metastatic breast cancers using whole-exome sequencing: a retrospective analysis of 216 samples from SAFIR01 / 02 / SHIVA / MOSCATO trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-353.

Different patterns of pfcrt and pfmdr1 polymorphism in Plasmodium falciparum isolates from Tehama region, Yemen.

Introduction. Despite the efforts of the malaria control programme, malaria morbidity is still a common health problem in Yemen, with 60% of the population at risk. Plasmodium falciparum is responsible for 99% of malaria cases. The emergence in Yemen of parasite resistance to chloroquine (CQ) prompted the adoption of artemisinin combination therapy (ACT) in 2009, which involves the use of artesunate plus sulphadoxine-pyrimethamine (AS + SP). However, CQ was retained as the drug of choice for vivax malaria. To assess the impact of the change in the malaria treatment policy five years after its introduction, the present study investigated the mutations in the CQ resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes.Method. A molecular investigation of 10 codons of pfcrt (72–76, 220, 271, 326, 356, and 371) and five codons of pfmdr1 (86, 184, 1034, 1042, and 1246) was conducted on P. falciparum isolates from districts with the highest malaria endemicity in the Hodeidah and Al-Mahwit governorates in Tehama region, Yemen. A total of 86 positive cases of falciparum monoinfection were investigated for the presence of mutations related to CQ and other antimalarials using a PCR-RFLP assay.Results. There was a wide prevalence of pfcrt gene mutations with the pfcrt 76T CQ resistance marker being predominant (97.7%). The prevalence of other pfcrt mutations varied from high (75E: 88%) to moderate (74I: 79.1%, 220S: 69.8%, 271E and 371I: 53.5%) or low (326S: 36%, 72S: 10.5%). Mutated pfcrt 72–76 amino acids haplotypes were highly prevalent (98.8%). Among these, the CVIET classic, old-world African/Southeast Asian haplotype was the most predominant, and was mostly found in the isolates from the Khamis Bani Saad district of Al-Mahwit (93.1%) and the AdDahi district of Hodeidah (88.9%). However, it was only found in 26.3% of the isolates from the Bajil district of Hodeidah. Surprisingly, the SVMNT new-world South American haplotype was exclusively detected in 9.3% of the isolates from the Bajil district of Hodeidah. Mutations at Y184F of pfmdr1 were found in all isolates (100%) from all districts. The mutation for codons 1034C and 86Y were found only in the isolates from the AdDahi and Khamis Bani Saad districts. Overall, the AdDahi and Khamis Bani Saad districts were similar in terms of carrying most of the mutations in the pfcrt and pfmdr1 genes, while there was a lower prevalence of mutation in the isolates from the Bajil district.Conclusion. The high prevalence of mutations in pfcrt 5 years after the official cessation of CQ use against P. falciparum suggests that there is sustained CQ pressure on P. falciparum isolates in the study area. Moreover, the low prevalence of mutations in the pfmdr1 gene could be a good indicator of the high susceptibility of P. falciparum isolates to antimalarials other than CQ. A new strategy to ensure the complete nationwide withdrawal of CQ from the private drug market is recommended.