Reduced subgenomic RNA expression is a molecular indicator of asymptomatic SARS-CoV-2 infection.

Background: It is estimated that up to 80% of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are asymptomatic and asymptomatic patients can still effectively transmit the virus and cause disease. While much of the effort has been placed on decoding single nucleotide variation in SARS-CoV-2 genomes, considerably less is known about their transcript variation and any correlation with clinical severity in human hosts, as defined here by the presence or absence of symptoms. Methods: To assess viral genomic signatures of disease severity, we conducted a systematic characterization of SARS-CoV-2 transcripts and genetic variants in 81 clinical specimens collected from symptomatic and asymptomatic individuals using multi-scale transcriptomic analyses including amplicon-seq, short-read metatranscriptome and long-read Iso-seq. Results: Here we show a highly coordinated and consistent pattern of sgRNA expression from individuals with robust SARS-CoV-2 symptomatic infection and their expression is significantly repressed in the asymptomatic infections. We also observe widespread inter- and intra-patient variants in viral RNAs, known as quasispecies frequently found in many RNA viruses. We identify unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Moreover, these frequently occurring structural variants in SARS-CoV-2 genomes serve as a mechanism to further induce SARS-CoV-2 proteome complexity. Conclusions: Our results indicate that differential sgRNA expression and structural mutational burden are highly correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

Molecular matching and treatment strategies for advanced stage lung cancer at Dartmouth-Hitchcock Medical Center: A three-year review of a Molecular Tumor Board.

Matching of actionable tumor mutations with targeted therapy increases response rates and prolongs survival in lung cancer patients. Drug development and trials targeting genetic alterations are expanding rapidly. We describe the role of a Molecular Tumor Board (MTB) in the design of molecularly informed treatment strategies in our lung cancer patient population. Tumor DNA was sequenced using a 50-gene targeted next-generation sequencing panel. Cases were evaluated by a multidisciplinary MTB who suggested a course of treatment based on each patient's molecular findings. During a three-year period, 21 lung cancer patients were presented at the MTB. All patients lacked common activating EGFR mutations and ALK rearrangements. One patient had Stage IIIb disease; all others were Stage IV; 18 patients had received ≥1 prior line of therapy (range 0-5). Suggestions for treatment with a targeted therapy were made for 19/21 (90.5%) patients, and four patients (21%) underwent treatment with a targeted agent, two as part of a clinical trial. Identified barriers to treatment with targeted therapy included: ineligibility for clinical trials (n â€‹= â€‹2), lack of interest in study/distance to travel (n â€‹= â€‹2), lack of disease progression (n â€‹= â€‹2), poor performance status (n â€‹= â€‹5), decision to treat next with immunotherapy (n â€‹= â€‹3), and unknown (n â€‹= â€‹1). For the majority of lung cancer patients, the MTB provided recommendations based on tumor genetic profiles. Identified barriers to treatment suggest that presentation to the MTB at earlier stages of disease may increase the number of patients eligible for treatment with a genetically informed targeted agent.

The first pancreatic neuroendocrine tumor in Li-Fraumeni syndrome: a case report.

BACKGROUND: Li-Fraumeni syndrome is a cancer predisposition syndrome caused by germline TP53 tumor suppressor gene mutations, with no previous association with pancreatic neuroendocrine tumors (PNETs). Here we present the first case of PNET associated with Li-Fraumeni syndrome. CASE PRESENTATION: This is a 43-year-old female who underwent laparoscopic distal pancreatectomy at age 39 for a well-differentiated grade 2 cystic PNET. When the patient was 41 years old, her seven-year-old daughter was found to have an astrocytoma and a germline TP53 mutation. While undergoing surveillance with 68Gallium-DOTATATE positron emission tomography/computed tomography for her PNET, the patient was found to have a large choroid plexus papilloma in her right temporal lobe. She underwent genetic counseling and testing that identified a germline pathogenic variant in TP53, leading to the diagnosis of Li-Fraumeni syndrome. Her PNET had a hemizygous pathogenic TP53 mutation with loss of the wild-type alternate allele, consistent with loss of heterozygosity and the two-hit hypothesis. She was enrolled in a Li-Fraumeni syndrome protocol and continues surveillance screening with our service. CONCLUSIONS: This is the first PNET reported in association with Li-Fraumeni syndrome. Pancreatic cancer risk is elevated in this syndrome, and our case highlights the need for vigilance in screening for pancreatic neoplasms in these patients.

Variant allele frequencies do not correlate well with myeloblast counts in a clinically validated gene sequencing panel for routine acute myeloid leukemia workup.

Next generation sequencing (NGS) has introduced new types of data, such as variant allele frequencies (VAFs), into the workup of acute myeloid leukemias (AML). There is interest in using NGS to prognosticate disease behavior and monitor residual disease, but the attribution of sequencing data entirely to the leukemic clone may be confounded by VAF contribution from background non-leukemic populations and undetected copy number aberrations. Sixty-eight patients with AML were evaluated by a clinically validated gene sequencing panel at our institution from 2015 to 2018. No correlation was found with a direct comparison of blast counts and VAFs in both primary- and secondary-AML (R2 = 0.0584 and 0.0235, respectively). Only moderate correlations were attained when evaluating against mutant NPM1 allele fraction alone (R2 = 0.5303) or when variants with allelic frequencies >55% of the blast burden were excluded (R2 = 0.4608). VAFs from regular clinical-use gene sequencing panels show poor unrestricted correlation with leukemic blast proportions in AML.

A novel MAP3K7CL-ERG fusion in a molecularly confirmed case of dermatofibrosarcoma protuberans with fibrosarcomatous transformation.

Dermatofibrosarcoma protuberans (DFSP) is a translocation-associated, low-grade sarcoma with fibroblastic differentiation. It is the most common superficial sarcoma, almost exclusively arising within the dermis. In a minority of cases, there is a transition from the conventional morphology to a fibrosarcomatous pattern, known as a fibrosarcomatous DFSP (FS-DFSP). Although a number of different molecular alterations have been described to account for this transformation, it remains poorly understood. Herein we report the first case of a FS-DFSP with a fusion between ERG, an ETS family transcription factor, and MAP3K7CL, a kinase gene rarely observed in fusion gene events.

Synchronous Pulmonary Adenofibroma and Solitary Fibrous Tumor: Case Report and Review of the Literature.

Pulmonary adenofibroma (PAF) is a rare neoplasm that may be related to solitary fibrous tumor (SFT). A subset of PAFs harbor the NAB2-STAT6 fusion that is typical of SFT, but a significant proportion do not. Their distinction is clinically important as SFTs can potentially have an aggressive clinical course, while there has been no report of a PAF behaving in a malignant fashion. We report a case of a 60-year-old male who developed a SFT and PAF in the same lung. The SFT harbored a NAB2-STAT6 fusion, while the PAF did not have any identifiable fusion. This case represents the first instance of a single patient with both of these tumors occurring simultaneously in the same lung.

Undifferentiated Sarcoma as Intermediate Step in the Progression of Malignant Melanoma to Rhabdomyosarcoma: Histologic, Immunohistochemical, and Molecular Studies of a New Case of Malignant Melanoma With Rhabdomyosarcomatous Differentiation.

Malignant melanoma (MM) may display highly variable phenotypic diversity, sometimes associated with loss of immunohistochemical melanocytic markers and acquisition of nonmelanocytic lineage of differentiation. Primary cutaneous MM with rhabdomyosarcomatous differentiation is extremely rare with only 5 reported cases in the literature. To date, a chronological progression of a MM to rhabdomyosarcoma has not been conclusively documented. A 96-year-old man underwent a re-excision of an "atypical fibroxanthoma" of the forearm, which revealed a small lentigo maligna melanoma associated with a dominant dermal high-grade spindle cell nodule admixed with a population of malignant polygonal epithelioid cells. On immunohistochemical studies, the spindle cells were completely negative for all melanocytic markers, whereas a small population of polygonal neoplastic cells at the periphery was positive for Desmin and Myo-D1, supporting early rhabdomyosarcomatous transformation. Several subsequent re-excisions demonstrated merely nodules of malignant pleomorphic epithelioid cells with rhabdomyosarcomatous differentiation and devoid of melanocytic markers. In addition, both rhabdomyosarcomatous component and original MM displayed identical mutations. Therefore, the histologic, immunohistochemical, and molecular findings documented for the first time a chronological progression from an invasive MM to a pleomorphic rhabdomyosarcoma through an intermediate stage of undifferentiated sarcoma/atypical fibroxanthoma. Interestingly, subsequent recurrences of pure rhabdomyosarcomatous component displayed skip lesions/microsatellitosis, marked tumor-infiltrative lymphocytes, and rare junctional nests of rhabdomyosarcomatous cells in the epidermis, histologic features that were not described in primary cutaneous rhabdomyosarcoma and therefore could serve as morphologic clues to the diagnosis of rhabdomyosarcomatous transformation in an MM.

Non-small cell lung cancers with isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations.

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are important metabolic enzymes that convert isocitrate to α-ketoglutarate. IDH1/2 mutations are associated with the development of multiple malignancies. In this study, we examine the prevalence and features of non-small cell lung cancers (NSCLC) with IDH1/2 mutations. From May 2013 to March 2017, 800 lung cancer samples were successfully sequenced for somatic mutations on the Ion Torrent PGM with the 50-gene AmpliSeq Cancer Hotspot Panel v2 on the Ion Torrent PGM (318 chip). Variants were identified using the Ion Torrent Variant Caller Plugin and reference genome hg19. Golden Helix's SVS software was used for variant annotation and prediction of significance. Nine samples (1.1%) from 8 patients harbored an IDH1 (3 p.R132C and 2 p.R132L) or IDH2 mutation (p.R140W, p.R172S, and p.R172T). All patients' tumors had adenocarcinoma histology, and all patients had a smoking history. Eighty-eight percent of patients' tumors had a coexisting KRAS mutation and 6 of 8 were diagnosed at greater than 70 years of age. Five patients presented with stage IV disease and 3 with stage I. After comparison to a cohort of KRAS-mutated NSCLC with a history of smoking, IDH-mutated cases were significantly older but presented with similar rates of advanced-stage disease and sex distribution. Additional studies are needed to understand the role of IDH1/2 mutations in the development of NSCLC, but such patients who have poor prognostic indicators (KRAS mutation and advanced stage at presentation) may benefit from IDH1/2-directed therapies.

Central xanthoma of the jaw in association with Noonan syndrome.

Xanthomas are histiocytic lesions of the skin, soft tissue, and bone and are generally considered to be reactive in nature. When they arise in the bones of the jaw, they are referred to as central xanthomas. New evidence supports the hypothesis that central xanthomas are a separate and distinct entity from their extragnathic counterparts. Noonan syndrome (NS) is an autosomal dominant disorder that has been associated with giant cell lesions, which also commonly occur in the jaw. We present a case of a 15-year-old boy with NS who presented with a radiolucent lesion of the mandible that on excision was found to be a central xanthoma. Although giant cell lesions have been well described in NS, xanthomas of the jaw have not been reported. We will also discuss the entities that must be excluded before making a diagnosis of central xanthoma, as this can affect both treatment and follow-up.

Frequency of Somatic TP53 Mutations in Combination with Known Pathogenic Mutations in Colon Adenocarcinoma, Non-Small Cell Lung Carcinoma, and Gliomas as Identified by Next-Generation Sequencing.

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer. It encodes p53, a DNA-binding transcription factor that regulates multiple genes involved in DNA repair, metabolism, cell cycle arrest, apoptosis, and senescence. TP53 is associated with human cancer by mutations that lead to a loss of wild-type p53 function as well as mutations that confer alternate oncogenic functions that enable them to promote invasion, metastasis, proliferation, and cell survival. Identifying the discrete TP53 mutations in tumor cells may help direct therapies that are more effective. In this study, we identified the frequency of individual TP53 mutations in patients with colon adenocarcinoma (48%), non-small cell lung carcinoma (NSCLC) (36%), and glioma/glioblastoma (28%) at our institution using next-generation sequencing. We also identified the occurrence of somatic mutations in numerous actionable genes including BRAF, EGFR, KRAS, IDH1, and PIK3CA that occurred concurrently with these TP53 mutations. Of the 480 tumors examined that contained one or more mutations in the TP53 gene, 219 were colon adenocarcinomas, 215 were NSCLCs, and 46 were gliomas/glioblastomas. Among the patients positive for TP53 mutations diagnosed with colon adenocarcinoma, 50% also showed at least one mutation in pathogenic genes of which 14% were BRAF, 33% were KRAS, and 3% were NRAS. Forty-seven percent of NSCLC patients harboring TP53 mutations also had a mutation in at least one actionable pathogenic variant with the following frequencies: BRAF: 4%, EGFR: 10%, KRAS: 28%, and PIK3CA: 4%. Fifty-two percent of patients diagnosed with glioma/glioblastoma with a positive TP53 mutation had at least one concurrent mutation in a known pathogenic gene of which 9% were CDKN2A, 41% were IDH1, and 11% were PIK3CA.

Somatic mutation analysis in melanoma using targeted next generation sequencing.

Advanced stage malignant melanoma often responds poorly to therapy with low survival rates. New therapeutic approaches are based upon a growing understanding of the underlying molecular abnormalities. We demonstrate the feasibility of a next generation sequencing (NGS) assay, which targets hotspots in 50 cancer genes, to assess genotypes that may influence therapeutic selection and response. DNA was extracted from formalin fixed paraffin embedded (FFPE) melanoma specimens to create multiplexed libraries which were sequenced. Of the 121 cases, BRAF mutations were present in 48 cases (40%) and NRAS mutations in 24 cases (20%). We identified other gene variants in 20 BRAF-mutated cases. Additional gene variants were also identified in the 57 BRAF wild-type cases. Four patients harbored different gene mutations at metastatic sites as compared to their primary lesions or metastasis from different sites. Concurrent gene variants may provide additional targets for future therapies and may suggest alternative mechanisms of secondary resistance.

Triple-Negative Breast Cancer: Next-Generation Sequencing for Target Identification.

Presently, the ability to study disease at the most fundamental molecular level has led to a reclassification of human cancers into numerous subtypes that vary in disease progression and response to therapy. Similar to most solid tumors, breast cancer is a heterogeneous disease with considerable variation in histologic and biological features. Triple-negative breast cancer (TNBC) is a subtype of breast cancer in which the estrogen receptor and progesterone receptor are not expressed, and human epidermal growth factor-receptor 2 is not amplified or overexpressed. Data derived from highly complex molecular technologies, such as microarrays and next-generation sequencing, have identified gene expression and somatic mutation profile subsets of TNBC that reflect biological behavior more accurately and may lead to further effective therapeutic targets, better prognosis, and improved outcomes. Herein, we review the genomic findings of TNBC and discuss current efforts in precision medicine as they relate to TNBC.

Variant call concordance between two laboratory-developed, solid tumor targeted genomic profiling assays using distinct workflows and sequencing instruments.

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n=17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples.

Improving Adequacy of Small Biopsy and Fine-Needle Aspiration Specimens for Molecular Testing by Next-Generation Sequencing in Patients With Lung Cancer: A Quality Improvement Study at Dartmouth-Hitchcock Medical Center.

CONTEXT: - At our medical center, cytopathologists perform rapid on-site evaluation for specimen adequacy of fine-needle aspiration and touch imprint of needle core biopsy lung cancer samples. Two years ago the molecular diagnostics laboratory at our institution changed to next-generation sequencing using the Ion Torrent PGM and the 50-gene AmpliSeq Cancer Hotspot Panel v2 for analyzing mutations in a 50-gene cancer hot spot panel. This was associated with a dramatic fall in adequacy rate (68%). OBJECTIVE: - To improve the adequacy rate to at least 90% for molecular testing using next-generation sequencing for all specimens collected by rapid on-site evaluation by the cytology laboratory. DESIGN: - After baseline data on adequacy rate of cytology specimens with rapid on-site evaluation for molecular testing had been collected, 2 changes were implemented. Change 1 concentrated all the material in one block but did not produce desired results; change 2, in addition, faced the block only once with unstained slides cut up front for molecular testing. Data were collected in an Excel spreadsheet and adequacy rate was assessed. RESULTS: - Following process changes 1 and 2 we reached our goal of at least 90% adequacy rate for molecular testing by next-generation sequencing on samples collected by rapid on-site evaluation including computed tomography-guided needle core biopsies (94%; 17 of 18) and fine-needle aspiration samples (94%; 30 of 32). CONCLUSION: - This study focused on factors that are controllable in a pathology department and on maximizing use of scant tissue. Optimizing the adequacy of the specimen available for molecular tests avoids the need for a second procedure to obtain additional tissue.

A Phase II Trial of Dovitinib in BCG-Unresponsive Urothelial Carcinoma with FGFR3 Mutations or Overexpression: Hoosier Cancer Research Network Trial HCRN 12-157.

Purpose: To assess the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non-muscle-invasive urothelial carcinoma of the bladder (NMIUC) population.Experimental Design: A multi-site pilot phase II trial was conducted. Key eligibility criteria included the following: Bacillus Calmette-Guerin (BCG)-unresponsive NMIUC (>2 prior intravesical regimens) with increased phosphorylated FGFR3 (pFGFR3) expression by centrally analyzed immunohistochemistry (IHC+) or FGFR3 mutations (Mut+) assessed in a CLIA-licensed laboratory. Patients received oral dovitinib 500 mg daily (5 days on/2 days off). The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate.Results: Between 11/2013 and 10/2014, 13 patients enrolled (10 IHC+ Mut-, 3 IHC+ Mut+). Accrual ended prematurely due to cessation of dovitinib clinical development. Demographics included the following: median age 70 years; 85% male; carcinoma in situ (CIS; 3 patients), Ta/T1 (8 patients), and Ta/T1 + CIS (2 patients); median prior regimens 3. Toxicity was frequent with all patients experiencing at least one grade 3-4 event. Six-month CR rate was 8% (0% in IHC+ Mut-; 33% in IHC+ Mut+). The primary endpoint was not met. Pharmacodynamically active (94-5,812 nmol/L) dovitinib concentrations in urothelial tissue were observed in all evaluable patients. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment.Conclusions: Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition. These results support systemic administration as a viable approach to clinical trials in patients with NMIUC. Long-term dovitinib administration was not feasible due to frequent toxicity. Absent clinical activity suggests that patient selection by pFGFR3 IHC alone does not enrich for response to FGFR3 kinase inhibitors in urothelial carcinoma. Clin Cancer Res; 23(12); 3003-11. ©2016 AACR.

Use of Biosynthetic Controls as Performance Standards for Next-Generation Sequencing Assays of Somatic Tumors: A Multilaboratory Study.

BACKGROUND: Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results. The SeraseqTM Solid Tumor Mutation Mix AF10 and AF20 control materials consist of 26 biosynthetic DNA constructs in a genomic DNA background, each containing a specific variant or mutation of interest and an internal quality marker at 2 distinct allelic frequencies of 10% and 20%, respectively. The goal of this interlaboratory study was to evaluate the Seraseq AF10 and AF20 control materials by verifying their performance as control materials and by evaluating their ability to measure quality metrics essential to a clinical test. METHODS: Performance characteristics were assessed within and between 6 CLIA-accredited laboratories and 1 research laboratory. RESULTS: Most laboratories detected all 26 mutations of interest; however, some discrepancies involving the internal quality markers were observed. CONCLUSION: This interlaboratory study showed that the Seraseq AF10 and AF20 control materials have high quality, stability, and genomic complexity in variant types that are well suited for assisting in NGS assay analytical validation and monitoring routine clinical applications.

Detection of CALR Mutation in Clonal and Nonclonal Hematologic Diseases Using Fragment Analysis and Next-Generation Sequencing.

OBJECTIVES: To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene. METHODS: Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases. CALR screening was primarily performed using fragment analysis polymerase chain reaction, then next-generation sequencing and Sanger sequencing. RESULTS: Among the 18 patients diagnosed with clonal diseases, one had acute myeloid leukemia (positive for trisomy 8), and 17 had myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV). Patients with CML were positive for the BCR-ABL1 fusion. Ten patients were positive for JAK2 (PMF, n = 1; ET, n = 2; PV, n = 7), and three were CALR positive (ET, n = 1; PMF, n = 2). Patients diagnosed with a nonclonal disease were negative for JAK2, BCR-ABL, and CALR mutations. CONCLUSIONS: Screening for CALR mutations is essential in BCR-ABL-negative MPNs since it not only provides valuable diagnostic and prognostic information but also identifies potential treatment targets. Since this study describes the importance of screening for known and novel biomarkers, we described in detail three methods that could be easily integrated into a clinical laboratory.

Clinical Genotyping of Non-Small Cell Lung Cancers Using Targeted Next-Generation Sequencing: Utility of Identifying Rare and Co-mutations in Oncogenic Driver Genes.

Detection of somatic mutations in non-small cell lung cancers (NSCLCs), especially adenocarcinomas, is important for directing patient care when targeted therapy is available. Here, we present our experience with genotyping NSCLC using the Ion Torrent Personal Genome Machine (PGM) and the AmpliSeq Cancer Hotspot Panel v2. We tested 453 NSCLC samples from 407 individual patients using the 50 gene AmpliSeq Cancer Hotspot Panel v2 from May 2013 to July 2015. Using 10 ng of DNA, up to 11 samples were simultaneously sequenced on the Ion Torrent PGM (316 and 318 chips). We identified variants with the Ion Torrent Variant Caller Plugin, and Golden Helix's SVS software was used for annotation and prediction of the significance of the variants. Three hundred ninety-eight samples were successfully sequenced (12.1% failure rate). In all, 633 variants in 41 genes were detected with a median of 2 (range of 0 to 7) variants per sample. Mutations detected in BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA were considered potentially actionable and were identified in 237 samples, most commonly in KRAS (37.9%), EGFR (11.1%), BRAF (4.8%), and PIK3CA (4.3%). In our patient population, all mutations in EGFR, KRAS, and BRAF were mutually exclusive. The Ion Torrent Ampliseq technology can be utilized on small biopsy and cytology specimens, requires very little input DNA, and can be applied in clinical laboratories for genotyping of NSCLC. This targeted next-generation sequencing approach allows for detection of common and also rare mutations that are clinically actionable in multiple patients simultaneously.

Targeted next-generation sequencing detects a high frequency of potentially actionable mutations in metastatic breast cancers.

BACKGROUND: Metastatic breast cancer is a genetically heterogeneous disease and effective therapies for advanced stage disease are limited. METHODS: In this study, distant metastases of 22 formalin-fixed, paraffin-embedded (FFPE) breast cancer samples were sequenced using the Ion Torrent PGM and the 50 gene AmpliSeq Cancer Hotspot Panel v2 from 10ng of extracted DNA using 318 chips. Data analysis was performed with the Ion Torrent Variant Caller Plugin (hg19) and Golden Helix's SVS software for annotation and prediction of the significance of the variants. RESULTS: All patients were female with a median age of 61years (range 37-85years). Metastatic sites included liver (n=6, 27%), skin (n=5, 23%), brain (n=4, 18%), lymph node (n=3, 14%), lung (n=2, 9%), retroperitoneum (n=1, 4.5%), and colon (n=1, 4.5%). Overall, 28 variants in 11 genes were observed. Five (23%) samples showed no alterations and 17 (77%) showed at least one potentially biologically significant variant (BSV) defined as having FDA-approved drugs or clinical trials evaluating their significance. BSVs included mutations in the following genes: TP53 (n=8), APC (n=4), PIK3CA (n=5), MET (n=2), ERBB2 (n=2), AKT1 (n=1), CDKN2A (n=1), KRAS (n=1), and FGFR3 (n=1). CONCLUSIONS: Potentially actionable mutations were identified in the majority of breast cancer metastases. Evaluating metastatic breast tumors using a NGS approach provides a better understanding of the mechanisms behind tumor progression and evolution and also identifies additional potentially beneficial therapeutic targets for patient management or eligibility for clinical trials.