Non-hotspot Mutations Research Articles

Abstract 2640: Clonal hematopoiesis identified by matched-normal blood sequencing of solid tumor patients without hematologic malignancy is common and is associated with decreased overall survival

Abstract Background: Recent genomic studies have identified somatic mutations in leukemia genes in asymptomatic individuals without hematologic malignancy. A subset of patients (pts) with clonal hematopoiesis (CH) subsequently develops hematologic malignancies. However, the incidence of CH in pts with solid tumors has not been extensively studied. We sought to assess for CH and its associated clinical impact in pts with solid tumors who were profiled using paired tumor/blood sequencing. Methods: This study included pts who were consented on protocol NCT01775072. All had tumor and blood genomic profiling using the MSK-IMPACT hybridization capture-based next-generation sequencing assay, encompassing all protein-coding exons of 410 cancer-associated genes. In matched blood, we investigated for hotspot mutations from COSMIC database (v71) and non-hotspot mutations in leukemia-associated genes and genes reported in prior CH studies. Mutations were scored as present if variant allele frequency (VAF) in blood was greater than 2% and at least twice the VAF seen in tumor. For cases where at least one mutation exceeded these thresholds, we reduced the VAF threshold in blood to 1% to detect subsequent events. Mutations with VAF >35% in both blood and tumor were excluded as likely germline events. Results: We analyzed 2,146 pts, including 239 pts (11%) with CH. Most commonly identified mutations were in DNMT3A, TET2, and TP53, seen in 167, 52, and 18 pts, respectively. The mean age at time of testing was 62.6 years in pts with CH and 55.4 years in pts without CH (p< 0.001). When comparing baseline blood parameters, there were no statistically significant differences except for higher MCV in CH vs. non-CH pts (91 vs. 90, p = 0.047). 51% of pts with CH had previous radiation therapy compared to 45% of pts without clonal mutations in their blood (p = 0.057). There was no statistically significant difference in the proportion of pts who had received previous chemotherapy (71% of pts in each group). On prospective follow up of pts with CH, no pts have progressed to develop overt hematologic malignancy, with limited follow up (median 13.1 months). Overall survival (OS) was estimated for pts without CH (n = 1907; 1-yr OS: 0.70), CH with 1 mutation (n = 184; 1-yr OS: 0.64), and CH with >1 mutation (n = 55; 1-yr OS: 0.59). The univariate hazard ratios were 1.16 (p = 0.269) and 1.63 (p = 0.015) for 1 mutation and >1 mutation, respectively, when compared to pts without CH. After adjusting for age and sex, a marginal OS association persisted for >1 mutation compared to no CH (HR: 1.46, p = 0.060) Conclusions: CH is common among solid tumor pts without known hematologic malignancy, occurring in 11% of pts, and appears to be associated with decreased OS. Analyses will be replicated in a larger cohort with longer follow up to determine the clinical impact of these mutations in cancer pts. Citation Format: Catherine Coombs, Ahmet Zehir, Sean Devlin, Sumit Middha, Donavan Cheng, Ashwin Kishtagari, David Hyman, David Solit, Mark Robson, Jose Baselga, Maria Arcila, Martin Tallman, Ross Levine, Michael Berger. Clonal hematopoiesis identified by matched-normal blood sequencing of solid tumor patients without hematologic malignancy is common and is associated with decreased overall survival. [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 2640.

Classical V600E and other non-hotspot BRAF mutations in adult differentiated thyroid cancer.

BackgroundBRAF is the most frequently mutated gene in differentiated thyroid cancer (DTC). Previous studies on DTC have well documented high rates of the BRAFV600E mutation in patients of mixed ages. Previous studies either included a mix of pediatric and adult patients or pediatric patients only. However, the prevalence of hotspot and non-hotspot BRAF mutations and its significance in pure adult DTCs is not yet well determined. In this study we determine the frequency of this classical BRAF mutation and other rare BRAF mutations in pure adult DTCs.MethodsA total of 204 adult DTC samples (Age >18 years) were analyzed for mutations in exon 15 of the BRAF gene by performing polymerase chain reaction (PCR) amplification of tumor genomic DNAs and direct sequencing of amplicons using Sanger sequencing. Obtained results were correlated to clinical and pathological characteristics of DTCs. Statistical analyses were performed using SPSS (The Statistical Package for Social Sciences) version 20 software.ResultsOverall, BRAF mutations were identified in 48.5 % (99/204) of adult DTCs. Three rare non-hotspot mutations (T599I, T599dup and K601E) were detected in four tumor samples (2 %). One (K601E) of these non-hotspot mutations occurred in conventional papillary thyroid cancer (CPTC) and other three (T599I, T599dup and K601E) were found in follicular variant PTC. We found significant association between BRAFV600E mutation and age (P < 0.0001), extrathyroidal invasion (P = 0.017), lymph node metastasis (P = 0.038) and TNM stage III/IV (P = 0.001).ConclusionsOur study is the first to report BRAF mutations in a pure adult sample of DTCs of Saudi Arabian ethnicity. Our results show a high rate and a strong prognostic role of the classical BRAFV600E mutation and also suggest a common occurrence of non-hot spot mutations in adult DTC from this highly inbred population.

Hotspot mutations delineating diverse mutational signatures and biological utilities across cancer types.

BackgroundAn important step towards personalizing cancer treatment is to integrate heterogeneous evidences to catalog mutational hotspots that are biologically and therapeutically relevant and thus represent where targeted therapy would likely be beneficial. However, existing methods do not sufficiently delineate varying functionality of individual mutations within the same genes.ResultsWe observed a large discordancy of mutation rates across different mutation subtypes and tumor types, and nominated 702 hotspot mutations in 549 genes in the Catalog of Somatic Mutations in Cancer (COSMIC) by considering context specific mutation characteristics such as genes, cancer types, mutation rates, mutation subtypes and sequence contexts. We observed that hotspot mutations were highly prevalent in Non CpG-island C/G transition and transversion sequence contexts in 10 tumor types, and specific insertion hotspot mutations were enriched in breast cancer and deletion hotspot mutations in colorectal cancer. We found that the hotspot mutations nominated by our approach were significantly more conserved than non-hotspot mutations in the corresponding cancer genes. We also examined the biological significance and pharmacogenomics properties of these hotspot mutations using data in the Cancer Genome Atlas (TCGA) and the Cancer Cell-Line Encyclopedia (CCLE), and found that 53 hotspot mutations are independently associated with diverse functional evidences in 1) mRNA and protein expression, 2) pathway activity, or 3) drug sensitivity and 82 were highly enriched in specific tumor types. We highlighted the distinct functional indications of hotspot mutations under different contexts and nominated novel hotspot mutations such as MAP3K4 A1199 deletion, NR1H2 Q175 insertion, and GATA3 P409 insertion as potential biomarkers or drug targets.ConclusionWe identified a set of hotspot mutations across 17 tumor types by considering the background mutation rate variations among genes, tumor subtypes, mutation subtypes, and sequence contexts. We illustrated the common and distinct mutational signatures of hotspot mutations among different tumor types and investigated their variable functional relevance under different contexts, which could potentially serve as a resource for explicitly selecting targets for diagnosis, drug development, and patient management.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2727-x) contains supplementary material, which is available to authorized users.

Assessment of the clinical application of detecting EGFR, KRAS, PIK3CA and BRAF mutations in patients with non-small cell lung cancer using next-generation sequencing

Background: Next-generation sequencing (NGS) has been widely applied in clinical research, while its application in routine clinical molecular testing requires careful validation. The aim of our study was to assess the clinical usefulness of the NextDaySeq Lung panel on Ion Torrent™ PGM in mutation detection of actionable genes in lung cancer.Methods: The NextDaySeq assay was evaluated by blinded comparisons to Quantitative Real-Time PCR (qPCR) assays with 188 consecutive samples from Chinese patients with non-small cell lung cancer (NSCLC) to detect mutations in EGFR, KRAS, PIK3CA and BRAF. Discordant variants were further validated by Sanger sequencing and independent qPCR and NGS assays.Results: Our results showed 93.3% concordance of reportable variants mutually covered in both NGS and qPCR assays, with a clinical sensitivity of 89.9%, specificity of 97.5%. Through the comparison, the NGS assays demonstrated its advantages in offering more clinical relevant information, such as detecting non-hotspot mutations and providing mutation allele frequencies (MAF) and accurate mutation sequences. The analytical sensitivity of NGS to detect mutations with low MAF needs further improvement.Conclusions: The NextDaySeq Lung panel exhibited good clinical performance, strongly supporting the implementation of the NGS assay in routine clinical use to facilitate therapeutic decision-making for lung cancer patients.

Mutational characteristics of ANK1 and SPTB genes in hereditary spherocytosis.

The aim of this study was to describe the mutational characteristics in Korean hereditary spherocytosis (HS) patients. Relevant literatures including genetically confirmed cases with well-documented clinical summaries and relevant information were also reviewed to investigate the mutational gene- or domain-specific laboratory and clinical association. Twenty-five HS patients carried one heterozygous mutation of ANK1 (n = 13) or SPTB (n = 12) but not in SPTA1, SLC4A1, or EPB42. Deleterious mutations including frameshift, nonsense, and splice site mutations were identified in 91% (21/23), and non-hotspot mutations were dispersed across multiple exons. Genotype-phenotype correlation was clarified after combined analysis of the cases and the literature review; anemia was most severe in HS patients with mutations on the ANK1 spectrin-binding domain (p < 0.05), and SPTB mutations in HS patients spared the tetramerization domain in which mutations of hereditary elliptocytosis and pyropoikilocytosis are located. Splenectomy (17/75) was more frequent in ANK1 mutant HS (32%) than in HS with SPTB mutation (10%) (p = 0.028). Aplastic crisis occurred in 32.0% of the patients (8/25; 3 ANK1 and 5 SPTB), and parvovirus B19 was detected in 88%. The study clarifies ANK1 or SPTB mutational characteristics in HS Korean patients. The genetic association of laboratory and clinical aspects suggests comprehensive considerations for genetic-based management of HS.

Rare RAS Mutations in Metastatic Colorectal Cancer Detected During Routine RAS Genotyping Using Next Generation Sequencing.

Overall survival of metastatic colorectal cancer (mCRC) patients has been improved with the addition of targeted therapy such as anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs) to standard chemotherapy. Retrospective studies and randomized trials showed that the presence of RAS mutations was linked to the absence of clinical response to anti-EGFR mAbs. Patients harboring KRAS and NRAS mutations on exons 2, 3 or 4 have little or no benefit from anti-EGFR therapies. Polymerase chain reaction (PCR)-based assays are routinely used to assess KRAS and NRAS status, whereas deep sequencing with next generation sequencing (NGS) currently represents an alternative method. The objective of our study was to identify KRAS and NRAS non-hotspot mutations using NGS of mCRC tumor samples. DNA was extracted from 188 consecutive formalin-fixed paraffin embedded samples of histologically proven colorectal cancer tumor tissue from patients with mCRC. Following amplification, DNA was sequenced by ultra-deep pyrosequencing. Non-hotspot mutations identified by NGS (frequency of mutated allele range [1.8-70.6%]) were confirmed by Sanger direct-sequencing when possible. NGS procedure was applicable in 94% of the cases and detected mutations in 62% of the samples. Nine uncommon mutational profiles were found with a frequency of mutated allele > 1%. Silent mutations were found in 3.6% of the samples. Mutations at or near functional domains of RAS proteins, other than defined hotspots, were found in 3.6%. NGS proved to be accurate, sensitive and suitable for routine RAS genotyping. Clinical responses to anti-EGFR mAbs are potentially impaired in the presence of these uncommon RAS mutations.

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.

Abstract 544: Recurrent DICER1 hotspot mutations in endometrial cancer and their impact on microRNA biogenesis

Abstract Alternation in genes associated with microRNA (miRNA) biogenesis pathway may lead to miRNA dysregulation, and is implicated in a variety of human malignancies. Previously our group identified recurrent somatic “hotspot” mutations (E1705, D1709, D1810, E1813) in a critical miRNA-processing gene, DICER1, in rare sex cord-stromal tumors. During miRNA biogenesis, the two RNase III domains of DICER1 form an intramolecular dimer, which leads to the cleavage of the precursor miRNA (pre-miRNA) hairpin and generate mature 5p and 3p miRNAs from 5’ and 3’ arms of the precursor hairpin respectively. Studies have shown that the hotspot mutations in the RNase IIIb metal binding domain could impair DICER1's ability to generate mature 5p miRNAs, leading to global loss of 5p miRNAs. Recently, in collaboration with The Cancer Genome Atlas (TCGA), we identified DICER1 hotspot mutations in a small subset of endometrial cancer from TCGA cohort (6/248) as well as our own tumor bank (6/307), suggesting disruption of DICER1 is implicated in a common malignancy. We also found an additional recurrent mutation G1809R and demonstrated that it has similar detrimental effects on miRNA biogenesis as hotspot mutations through deep sequencing and realtime PCR. Using Illumina Miseq targeted resequencing and Sanger sequencing, we observed biallelic DICER1 mutations in RNase IIIb domain in some but not all cases. miRNA deep sequencing confirmed that 5p miRNAs are decreased in both cell line models and endometrial tumors with hotspot mutations. Bioinformatic analysis of RNA sequencing profiles from TCGA dataset predicted hotspot DICER1 mutations to have greater functional impact than non-hotspot DICER1 mutations on gene expression. The oncogenic properties of DICER1 hotspot mutations are currently under investigation. Citation Format: Jiamin Chen, Yemin Wang, Melissa McConechy, Michael Anglesio, Janine Senz, Winnie Yang, Jamie Rosner, Andy Chu, Grace Cheng, Gregg Morin, David Huntsman. Recurrent DICER1 hotspot mutations in endometrial cancer and their impact on microRNA biogenesis. [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 544. doi:10.1158/1538-7445.AM2014-544

G.P.87: Prospects for single antisense oligonucleotide-induced multiple exon skipping for rare non-hotspot mutations in Duchenne muscular dystrophy

Antisense oligonucleotides (AONs) for Duchenne muscular dystrophy (DMD) are designed to induce skipping of a single exon during pre-mRNA splicing, thus restoring the transcript’s open reading frame and consequently synthesis of the deficient dystrophin protein. Although this approach is mutation-dependent, the majority of mutations cluster in a hotspot region from exon 45 to 53, allowing application of one AON to a subpopulation of patients with grouped mutations. Some mutations however are ultra-rare with only few patients worldwide, and require the skipping of an exon outside the hotspot region. Combining these ultra-rare mutations by applying multiple exon skipping is therefore attractive. The feasibility of multiple exon skipping using a cocktail of AONs has been demonstrated, but a drawback is the increased complexity of the development pathway and manufacturing costs. We therefore studied the possibility of designing a single AON capable of inducing simultaneous skipping of multiple exons. The repetitive nature of the central rod domain in dystrophin implies the occurrence of homologous stretches within the transcript. Indeed, in the exon 10 to 40 region several exon pairs were identified sharing such a homologous stretch, which we explored as a potential dual target for a single AON. In-frame exon combinations that would mimic a Becker-like mutation associated with a relatively mild phenotype and that would apply to a considerable patient subpopulation (up to ∼15%) were selected. In vitro proof-of-concept was obtained for healthy donor as well as for DMD patient-derived muscle cells and resulted in novel dystrophin expression. Results demonstrating the applicability in the mdx mouse model in vitro and in vivo will be presented. The data support further (pre) clinical development of such multiple exon skipping AONs, not only for more rare mutations that are not targeted by current single skip programs but also for mutations in the hotspot region.

Whole-exome sequencing in tumor samples from sequenom-wild-type, ALK negative stage IV lung adenocarcinoma (ADC) patients (p).

8070 Background: The majority of lung ADC tumors are characterized by specific genetic features with KRAS mutations (mut) seen in 20-30%, EGFR mut in 15%, EML4-ALK translocations in 5%, and ERBB2 mut in 2%, among others. Some of these genetic alterations are already being used for selecting targeted therapies. However, identification of additional genomic alterations is required. Methods: In the present ongoing study we perform whole-exome sequencing in paraffin-embedded tumor samples from OncoCarta v1.0 panel wild-type (no mut in hotspots of KRAS, EGFR, ERBB2, AKT1, BRAF, PIK3CA genes) and ALK negative (by FISH) stage IV ADC lung cancer p, and in their matched normal tissue samples. Results: To date, a total of 7 tumors and matched normal tissues have been successfully analyzed. We have detected mut in previously identified ADC genes, such as ERBB2, CTNNB1, TP53, SMAD4 or APC. Interestingly, mut were found in genes belonging to the proposed new cancer hallmark ‘epigenetic and RNA regulation’, such as BRD3, EPC1, PHF1 in almost every p included in our study. Regarding alterations that could be considered relevant for lung tumor pathogenesis/growth or as potential targets for treatment therapies, we were able to identify candidates in 4 of the 7 p. In one p, a case of a transmembrane domain ERBB2mut in exon 20 (p.E770delinsEAYVM) not previously detected by the OncoCarta v1.0 panel (that interrogates L755P, G776S/L/V/C, A775_G776insYVMA, P780_Y781insGSP, S779_P780insVGS mut) was found. In another p, three somatic mut in the BRCA1/2 genes were detected. Additionally, one p had an ALK point mut (p.P336K), for which no functional information is available, together with an APC mutation. In the remaining p, a non-hotspot mutation (although previously detected in a colorectal tumor) was found in CTNNB1. Conclusions: In this limited experience of whole-exome sequencing of a subgroup of stage IV lung ADC p, potentially targetable alterations not formerly detected by other techniques were found. We believe that genomic approaches to detecting alterations may be useful in clinical practice and will hopefully provide assistance in making treatment decisions.

This Month in Gastroenterology

This Month in Gastroenterology

Rare Mutations in the PIK3CA Gene Contribute to Aggressive Endometrial Cancer

The molecular basis of endometrial cancer (EC), a common gynecologic malignancy, often includes mutational activation of the PIK3CA and KRAS genes. We aimed to determine the distribution of mutations in the two genes depending on patient clinocopathological characteristics. We sequenced exon 1 of the KRAS gene and exons 9 and 20 of the PIK3CA gene in 108 consecutive EC tumor samples. PIK3CA mutations were present in 24 of the 108 (22.2%) cases and KRAS mutations in 18 of the 108 (16.7%) cases. PIK3CA mutations occurred more frequently in KRAS-mutated samples (7/18, 38.9%; p = 0.06) than in KRAS wild type (17/90, 18.9%) and showed a very high frequency in metastatic tumors (4/9, 44.4%; p = 0.1) and in samples displaying serous differentiation-serous and mixed endometrioid/serous tumors (6/12, 50.0%; p = 0.021)-where KRAS mutations were rare (11.1% and 16.7%, respectively) and did not exist independently of a PIK3CA mutation. Non-hotspot (i.e., non-E542K, -E545K, and -H1047R) mutations in the PIK3CA gene showed higher frequency in metastatic cases (3/9, 33.3%; p = 0.05). Tumors displaying serous differentiation showed a particular pattern-they harbored exclusively mutations in PIK3CA exon 20 (5/12, 41.7%; p = 0.005) and most of these were non-hotspot (4/12, 33.3%; p = 0.02). In all other comparisons exons 9 and 20 mutation distribution did not differ. These results suggest the need for further exploration of the significance of PIK3CA mutations in respect to aggressive EC.

γ-Radiation-induced mutation spectrum in the episomal lacI gene of Escherichia coli under oxic conditions

In this study we have determined the mutation spectrum in the complete episomal lacI gene of Escherichia coli induced by γ-radiation under oxic conditions. Mutants were generated by 60Co γ-irradiation of an E. coli culture of stationary cells in LB medium, under continuous flushing with oxygen. Oligonucleotide probe analysis showed that 14% of the γ-ray-induced mutations were located at the lacI gene hot spot at position 620–632, which is characterized by a triple repeat of the 5′-TGGC-3′ sequence. Previously it was shown that about 70% of the spontaneous mutations were located at this site due to the loss or the addition of a TGGC sequence. The non-hot spot mutations were further characterized by automated sequence analysis. The results show that base pair (bp) substitutions were the main type of γ-ray-induced mutations. Although all types of bp substitutions were observed, 74% of the bp substitutions involved C G base pairs. C G → T A and C G → A T substitutions were predominant, both accounting for 35% of all bp substitutions, whereas A T → C G substitutions were only seldomly observed (3%). A relatively large amount of −1 bp deletions (15% of all mutations) was detected in the γ-ray-induced mutation spectrum, mainly affecting C G base pairs, and 10% were deletions, ranging in size from 11 to 532 bp. It can be concluded that under oxic conditions γ-radiation induces in E. coli mainly bp substitutions of all types but preferentially at C G base pairs, and that the mutations tend to be randomly distributed within the lacI gene sequence.

Mechanisms of spontaneous mutagenesis: An analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene

We have obtained via DNA sequence analysis a spectrum of 174 spontaneous mutations occurring in the lacI gene of Escherichia coli. The spectrum comprised base substitution, frameshift, deletion, duplication and insertion mutations, of which the relative contributions to spontaneous mutation could be estimated. Two thirds of all lacI mutations occurred in the frameshift hotspot site. An analysis of the local DNA sequence suggested that the intensity of this hotspot may depend on structural features of the DNA that extend beyond those permitted by the repeated tetramer at this site. Deletions comprised the largest non-hotspot class (37%). They could be divided into two subclasses, depending on whether they included the lac operator sequence; the latter was found to be a preferred site for deletion endpoints. Most of the deletions internal to the lacI gene were associated with the presence of directly or invertedly repeated sequences capable of accounting for their endpoints. Base substitutions comprised 34% of the non-hotspot events. Unlike the base substitution spectrum obtained via nonsense mutations, G · C → A · T transitions do not predominate. A new base substitution hotspot was discovered at position +6 in the lac operator; its intensity may reflect specific features of the operator DNA. IS1 insertion mutations contributed 12% of the non-hotspot mutations and occurred dispersed throughout the gene in both orientations. Since the lacI gene is not A + T-rich, the contribution of IS1 insertion to spontaneous mutation in general might be underestimated. Single-base frameshift mutations were found only infrequently. In general, they did not occur in runs of a common base. Instead, their occurrence seemed based on the “perfection” of direct or inverted repeats in the local DNA sequence. Three (tandem) duplication events were recovered. No repeated sequences were found that might have determined their endpoints.