The clinical significance of adenomatous polyposis coli (APC) and catenin Beta 1 (CTNNB1) genetic aberrations in patients with melanoma.

BACKGROUND: Melanoma-intrinsic activated ß-catenin pathway, the product of the catenin beta 1 (CTNNB1) gene, has been associated with low/absent tumor-infiltrating lymphocytes, accelerated tumor growth, metastases development, and resistance to anti-PD-L1/anti-CTLA-4 agents in mouse melanoma models. Little is known about the association between the adenomatous polyposis coli (APC) and CTNNB1 gene mutations in stage IV melanoma with immunotherapy response and overall survival (OS). METHODS: We examined the prognostic significance of somatic APC/CTNNB1 mutations in the Cancer Genome Atlas Project for Skin Cutaneous Melanoma (TCGA-SKCM) database. We assessed APC/CTNNB1 mutations as predictors of response to immunotherapies in a clinicopathologically annotated metastatic patient cohort from three US melanoma centers. RESULTS: In the TCGA-SKCM patient cohort (n = 434) presence of a somatic APC/CTNNB1 mutation was associated with a worse outcome only in stage IV melanoma (n = 82, median OS of APC/CTNNB1 mutants vs. wild-type was 8.15 vs. 22.8 months; log-rank hazard ratio 4.20, p = 0.011). APC/CTNNB1 mutation did not significantly affect lymphocyte distribution and density. In the 3-melanoma institution cohort, tumor tissues underwent targeted panel sequencing using two standards of care assays. We identified 55 patients with stage IV melanoma and APC/CTNNB1 genetic aberrations (mut) and 169 patients without (wt). At a median follow-up of more than 25 months for both groups, mut compared with wt patients had slightly more frequent (44% vs. 39%) and earlier (66% vs. 45% within six months from original diagnosis of stage IV melanoma) development of brain metastases. Nevertheless, time-to-development of brain metastases was not significantly different between the two groups. Fortunately, mut patients had similar clinical benefits from PD-1 inhibitor-based treatments compared to wt patients (median OS 26.1 months vs. 29.9 months, respectively, log-rank p = 0.23). Less frequent mutations in the NF1, RAC1, and PTEN genes were seen in the mut compared with wt patients from the 3-melanoma institution cohort. Analysis of brain melanoma tumor tissues from a separate craniotomy patient cohort (n = 55) showed that melanoma-specific, activated ß-catenin (i.e., nuclear localization) was infrequent (n = 3, 6%) and not prognostic in established brain metastases. CONCLUSIONS: APC/CTNNB1 mutations are associated with a worse outcome in stage IV melanoma and early brain metastases independent of tumor-infiltrating lymphocyte density. However, PD1 inhibitor-based treatments provide comparable benefits to both mut and wt patients with stage IV melanoma.

Enhancing Next-Generation Sequencing-Guided Cancer Care Through Cognitive Computing.

BACKGROUND: Using next-generation sequencing (NGS) to guide cancer therapy has created challenges in analyzing and reporting large volumes of genomic data to patients and caregivers. Specifically, providing current, accurate information on newly approved therapies and open clinical trials requires considerable manual curation performed mainly by human "molecular tumor boards" (MTBs). The purpose of this study was to determine the utility of cognitive computing as performed by Watson for Genomics (WfG) compared with a human MTB. MATERIALS AND METHODS: One thousand eighteen patient cases that previously underwent targeted exon sequencing at the University of North Carolina (UNC) and subsequent analysis by the UNCseq informatics pipeline and the UNC MTB between November 7, 2011, and May 12, 2015, were analyzed with WfG, a cognitive computing technology for genomic analysis. RESULTS: Using a WfG-curated actionable gene list, we identified additional genomic events of potential significance (not discovered by traditional MTB curation) in 323 (32%) patients. The majority of these additional genomic events were considered actionable based upon their ability to qualify patients for biomarker-selected clinical trials. Indeed, the opening of a relevant clinical trial within 1 month prior to WfG analysis provided the rationale for identification of a new actionable event in nearly a quarter of the 323 patients. This automated analysis took <3 minutes per case. CONCLUSION: These results demonstrate that the interpretation and actionability of somatic NGS results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing could potentially improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of up-to-date availability of clinical trials. IMPLICATIONS FOR PRACTICE: The results of this study demonstrate that the interpretation and actionability of somatic next-generation sequencing results are evolving too rapidly to rely solely on human curation. Molecular tumor boards empowered by cognitive computing can significantly improve patient care by providing a fast, cost-effective, and comprehensive approach for data analysis in the delivery of precision medicine. Patients and physicians who are considering enrollment in clinical trials may benefit from the support of such tools applied to genomic data.

Risk Factors for Blood Transfusion with Neck Dissection.

OBJECTIVES: To identify risk factors of perioperative blood transfusions (PBTs) for neck dissection and identify the association of PBTs with other postoperative outcomes. METHODS: This is a retrospective study of the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. The database was queried for neck dissection procedures performed by otolaryngologists from 2006 to 2014. Multivariable logistic regression was used to determine associations between demographic and preoperative factors, mortality, unplanned reoperation, and unplanned readmission with PBTs. RESULTS: Of the 3090 patients included in our study, 346 (11.2%) received a PBT, 249 patients (72.0%) received blood intraoperatively or on postoperative day (POD) 0, and 97 patients (28.0%) received blood within 5 PODs. American Society of Anesthesiologists (ASA) class ≥3 (odds ratio [OR], 2.1; 95% confidence interval [CI], 1.4-3.2), preoperative weight loss (OR, 2.2; 95% CI, 1.5-3.2), and anemia (OR, 5.5; 95% CI, 4.1-7.6) were independently associated with PBTs. Free flaps were also significantly associated with PBTs. PBTs were significantly associated with unplanned return to the operating room within 30 days (OR, 4.31; 95% CI, 3.01-6.18) but not with 30-day unplanned readmission or 30-day mortality. DISCUSSION: Eleven percent of patients undergoing neck dissection receive a PBT. Identifying associated risk factors may reduce PBT among patients with cancer. Comorbid data, such as weight loss, anemia, and ASA class, may be useful in determining risk for transfusion during these procedures. IMPLICATIONS FOR PRACTICE: Awareness of preoperative risk factors for PBT may lead surgeons to reduce the risk of PBT, anticipate the need for transfusion, and manage these patients carefully to prevent unplanned reoperation.

Characteristics and management of sinonasal paragangliomas: a systematic review.

BACKGROUND: Paragangliomas of the nasal cavity and paranasal sinuses, although exceedingly rare, can exhibit frequent and aggressive recurrences. Our objective was to evaluate tumor characteristics, clinical course, management, and associated complications of sinonasal paragangliomas METHODS: A systematic review of the literature was performed utilizing the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, searching for sinonasal paraganglioma. Studies meeting inclusion criteria were assessed for level of evidence. Patient demographics, tumor characteristics, primary intervention, and other clinical characteristics were evaluated. RESULTS: Forty-five relevant studies encompassing 54 patients with sinonasal paraganglioma were identified. The most common tumor locations were the nasal cavity (66.7%), ethmoid sinuses (22.2%), maxillary sinuses (7.4%), and sphenoid sinuses (3.7%). Common presenting symptoms included recurrent epistaxis (68.5%), nasal obstruction (53.7%), and headache (13.0%). Tumors were malignant in 28.6% of patients. Only 4 cases (7.4%) involved functional tumors. Initial management was always surgical, via either an open (63.0%) or endoscopic (33.3%) approach. Radiotherapy was used as adjunctive treatment in 10 cases (18.5%). Recurrence rate was 21.7% and occurred between 12 to 156 months after initial resection. The overall survival was 87.0% with a metastatic rate of 8.7%. CONCLUSION: Sinonasal paragangliomas are vascular neoplasms manifesting clinically with recurrent epistaxis and nasal obstruction. Management goals are total resection with clear margins and long-term follow-up due to tendency for local recurrence. Radiotherapy has been utilized as adjuvant therapy with variable results. Further randomized controlled studies may be invaluable in elucidating these findings.

Improved Tumor Purity Metrics in Next-generation Sequencing for Clinical Practice: The Integrated Interpretation of Neoplastic Cellularity and Sequencing Results (IINCaSe) Approach.

Neoplastic cellularity contributes to the analytic sensitivity of most present technologies for mutation detection, such that they underperform when stroma and inflammatory cells dilute a cancer specimen's variant fraction. Thus, tumor purity assessment by light microscopy is used to determine sample adequacy before sequencing and to interpret the significance of negative results and mutant allele fraction afterwards. However, pathologist estimates of tumor purity are imprecise and have limited reproducibility. With the advent of massively parallel sequencing, large amounts of molecular data can be analyzed by computational purity algorithms. We retrospectively compared tumor purity of 3 computational algorithms with neoplastic cellularity using hematoxylin and eosin light microscopy to determine which was best for clinical evaluation of molecular profiling. Data were analyzed from 881 cancer patients from a clinical trial cohort, LCCC1108 (UNCseq), whose tumors had targeted massively parallel sequencing. Concordance among algorithms was poor, and the specimens analyzed had high rates of algorithm failure partially due to variable tumor purity. Computational tumor purity estimates did not add value beyond the pathologist's estimate of neoplastic cellularity microscopy. To improve present methods, we propose a semiquantitative, clinically applicable strategy based on mutant allele fraction and copy number changes present within a given specimen, which when combined with the morphologic tumor purity estimate, guide the interpretation of next-generation sequencing results in cancer patients.

Clinicopathologic traits and prognostic factors associated with pediatric sinonasal rhabdomyosarcoma.

BACKGROUND: Pediatric sinonasal rhabdomyosarcoma (RMS) is an aggressive and rare malignancy. This is the first multi-institutional study on the prognostic factors associated with outcomes in this population. METHODS: The National Cancer Database was queried for the period from 2004 to 2013 for all cases of malignant sinonasal RMS in the pediatric population. The impact of patients' demographics, tumor characteristics, and Intergroup Rhabdomyosarcoma Study Group (IRSG) staging on survival was assessed using chi-square test, Fisher's exact test, Kaplan-Meier test, and Cox regression analyses. RESULTS: A total of 157 cases of pediatric sinonasal RMS were identified. Mean age at diagnosis was 9.38 years and male patients comprised 48.4% of the cohort. The nasal cavity (31.8%) and maxillary sinus (30.6%) were the most common primary sites. Alveolar was the most common histology (49.7%), followed by embryonal type (32.5%). The majority of patients received solely chemoradiation (52.9%), followed by surgery with adjuvant chemoradiation (30.6%). Five-year overall survival (OS) was 55.2% (±4.5%). Metastatic disease was associated with a poorer 5-year OS rate (24.4% vs 61.5%; p = 0.010). Maxillary sinus site was associated with an improved survival (71.8% vs 47.6%; p = 0.009). On multivariate analysis, chemoradiation with or without surgery was an additional prognostic factor. Although IRSG clinical stages did not correlate with survival, high-risk patients in the IRSG clinical risk groups were associated with poorer survival on multivariate analysis (hazard ratio [HR], 2.005; 95% confidence interval, 1.007-3.993; p = 0.048). CONCLUSION: To date, this is the largest study on pediatric sinonasal RMS. IRSG clinical risk groups may be useful in stratifying high-risk patients with poor prognosis.

Morbidity and mortality associated with ventral skull base surgery: analysis of the National Surgical Quality Improvement Program.

BACKGROUND: Ventral skull base (VSB) surgery has associated morbidity and mortality that is poorly defined. In this study we aim to identify factors associated with adverse events in VSB surgery. METHODS: We queried the database of the American College of Surgeons National Surgical Quality Improvement Program for cases of VSB surgery during the period 2005-2014. Patients with complications, readmissions, reoperations, or mortality were compared to those without adverse events. RESULTS: Nine hundred patients were included; 253 (28.1%) had complications, underwent reoperation, were readmitted, or died. These patients were older (42.6% vs 32.8, p = 0.032) and had higher rates of congestive heart failure (CHF) (3.2% vs 0.2%, p < 0.0001), disseminated cancer (8.3% vs 4.6%, p = 0.032), and preoperative sepsis (8.7% vs 2.2%, p < 0.0001). Other comorbidities included long-term steroid use (13.4% vs 9.0%, p = 0.046) and higher rates of preoperative transfusion (2.4% vs 0%, p < 0.0001). The most common complication was bleeding (13.7%). Preoperative systemic sepsis (odds ratio [OR], 2.6; 95% confidence interval [CI], 1.0-6.6) and lower hematocrit (OR, 2.1; 95% CI, 1.4-3.4) were more likely to be associated with a complication. Those with disseminated cancer (OR, 12.0; 95% CI, 2.9-50.5) were more likely to experience 30-day mortality. Black patients had lower rates of reoperation (OR, 0.3; 95% CI, 0.1-0.8), whereas patients with CHF (OR, 12.6; 95% CI, 1.7-94.4) and hypertension (OR, 2.1; 95% CI, 1.1-4.0) had higher rates of reoperation. Predictors of extended length of stay were Hispanic ethnicity (OR, 2.2; 95% CI, 1.2-4.1) and lower hematocrit (OR, 2.3; 95% CI, 1.5-3.6). CONCLUSION: VSB surgery can involve significant morbidity and mortality, and thus identifying risk factors allows for better prognostication and delivery of care in these patients.

Identification of Germline Variants in Tumor Genomic Sequencing Analysis.

This Correspondence relates to the article by Li et al (Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and the College of American Pathologists. J Mol Diagn 2017, 19:4-23).

Identification of Clonal Hematopoiesis Mutations in Solid Tumor Patients Undergoing Unpaired Next-Generation Sequencing Assays.

PURPOSE: In this era of precision-based medicine, for optimal patient care, results reported from commercial next-generation sequencing (NGS) assays should adequately reflect the burden of somatic mutations in the tumor being sequenced. Here, we sought to determine the prevalence of clonal hematopoiesis leading to possible misattribution of tumor mutation calls on unpaired Foundation Medicine NGS assays. EXPERIMENTAL DESIGN: This was a retrospective cohort study of individuals undergoing NGS of solid tumors from two large cancer centers. We identified and quantified mutations in genes known to be frequently altered in clonal hematopoiesis (DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2, SF3B1, CBL, JAK2) that were returned to physicians on clinical Foundation Medicine reports. For a subset of patients, we explored the frequency of true clonal hematopoiesis by comparing mutations on Foundation Medicine reports with matched blood sequencing. RESULTS: Mutations in genes that are frequently altered in clonal hematopoiesis were identified in 65% (1,139/1,757) of patients undergoing NGS. When excluding TP53, which is often mutated in solid tumors, these events were still seen in 35% (619/1,757) of patients. Utilizing paired blood specimens, we were able to confirm that 8% (18/226) of mutations reported in these genes were true clonal hematopoiesis events. The majority of DNMT3A mutations (64%, 7/11) and minority of TP53 mutations (4%, 2/50) were clonal hematopoiesis. CONCLUSIONS: Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care.See related commentary by Pollyea, p. 5790.

Exceptional Chemotherapy Response in Metastatic Colorectal Cancer Associated With Hyper-Indel-Hypermutated Cancer Genome and Comutation of POLD1 and MLH1.

PURPOSE: A73-year-old woman with metastatic colon cancer experienced a complete response to chemotherapy with dose-intensified irinotecan that has been durable for 5 years. We sequenced her tumor and germ line DNA and looked for similar patterns in publicly available genomic data from patients with colorectal cancer. PATIENTS AND METHODS: Tumor DNA was obtained from a biopsy before therapy, and germ line DNA was obtained from blood. Tumor and germline DNA were sequenced using a commercial panel with approximately 250 genes. Whole-genome amplification and exome sequencing were performed for POLE and POLD1. A POLD1 mutation was confirmed by Sanger sequencing. The somatic mutation and clinical annotation data files from the colon (n = 461) and rectal (n = 171) adenocarcinoma data sets were downloaded from The Cancer Genome Atlas data portal and analyzed for patterns of mutations and clinical outcomes in patients with POLE- and/or POLD1-mutated tumors. RESULTS: The pattern of alterations included APC biallelic inactivation and microsatellite instability high (MSI-H) phenotype, with somatic inactivation of MLH1 and hypermutation (estimated mutation rate > 200 per megabase). The extremely high mutation rate led us to investigate additional mechanisms for hypermutation, including loss of function of POLE. POLE was unaltered, but a related gene not typically associated with somatic mutation in colon cancer, POLD1, had a somatic mutation c.2171G>A[p.Gly724Glu]. Additionally, we noted that the high mutation rate was largely composed of dinucleotide deletions. A similar pattern of hypermutation (dinucleotide deletions, POLD1 mutations, MSI-H) was found in tumors from The Cancer Genome Atlas. CONCLUSION: POLD1 mutation with associated MSI-H and hyper-indel-hypermutated cancer genome characterizes a previously unrecognized variant of colon cancer that was found in this patient with an exceptional response to chemotherapy.

Comprehensive Molecular Characterization of Urachal Adenocarcinoma Reveals Commonalities With Colorectal Cancer, Including a Hypermutable Phenotype.

PURPOSE: Urachal adenocarcinoma is a rare type of primary bladder adenocarcinoma that comprises less than 1% of all bladder cancers. The low incidence of urachal adenocarcinomas does not allow for an evidence-based approach to therapy. Transcriptome profiling of urachal adenocarcinomas has not been previously reported. We hypothesized that an in-depth molecular understanding of urachal adenocarcinoma would uncover rational therapeutic strategies. PATIENTS AND METHODS: We performed targeted exon sequencing and global transcriptome profiling of 12 urachal tumors to generate a comprehensive molecular portrait of urachal adenocarcinoma. A single patient with an MSH6 mutation was treated with the anti-programmed death-ligand 1 antibody, atezolizumab. RESULTS: Urachal adenocarcinoma closely resembles colorectal cancer at the level of RNA expression, which extends previous observations that urachal tumors harbor genomic alterations that are found in colorectal adenocarcinoma. A subset of tumors was found to have alterations in genes that are associated with microsatellite instability (MSH2 and MSH6) and hypermutation (POLE). A patient with an MSH6 mutation was treated with immune checkpoint blockade, which resulted in stable disease. CONCLUSION: Because clinical trials are next to impossible for patients with rare tumors, precision oncology may be an important adjunct for treatment decisions. Our findings demonstrate that urachal adenocarcinomas molecularly resemble colorectal adenocarcinomas at the level of RNA expression, are the first report, to our knowledge, of MSH2 and MSH6 mutations in this disease, and support the consideration of immune checkpoint blockade as a rational therapeutic treatment of this exceedingly rare tumor.

FNA smears as a potential source of DNA for targeted next-generation sequencing of lung adenocarcinomas.

BACKGROUND: Diff-Quik-stained fine-needle aspiration (FNA) smears and touch preparations from biopsies represent alternative specimens for molecular testing when cell block or biopsy material is insufficient. This study describes the use of these samples for targeted next-generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and reports the DNA quality and success rates of FNA smears versus other specimens from 1 year of clinical use. METHODS: A validation set of 10 slides from 9 patients with prior clinical epidermal growth factor receptor (EGFR) Sanger sequencing and KRAS pyrosequencing (5 KRAS-positive/EGFR-negative and 4 KRAS-negative/EGFR-negative) underwent DNA extraction, quality assessment, and targeted NGS. Subsequently, lung adenocarcinoma specimens submitted for NGS solid tumor mutation panel testing in 1 calendar year (60 biopsies, 57 resections, 33 FNA cell blocks, 12 FNA smears, and 10 body fluid cell blocks) were reviewed for specimen adequacy, sequencing success, and DNA quality. RESULTS: All 10 validation samples met the DNA quality threshold (delta Ct threshold < 8; range, -2.2 to 4.9) and yielded 0.5 to 22 µg of DNA. The KRAS and EGFR mutation status from FNA smears according to NGS was concordant with previous clinical testing for all 10 samples. In the 1-year review, FNA smears were 100% successful, and this suggested a performance equivalent to or better than the performance of established specimen types, including FNA cell blocks. DNA quality according to ΔCt was significantly better with FNA smears versus biopsies, resections, and FNA cell blocks. CONCLUSIONS: FNA smears of lung adenocarcinomas are high-quality alternative specimens for a targeted NGS panel with a high success rate in clinical practice. Cancer Cytopathol 2016;124:406-14. © 2016 American Cancer Society.

Identification of Human Papillomavirus Infection in Cancer Tissue by Targeted Next-generation Sequencing.

Human papillomaviruses (HPV) are oncogenic DNA viruses implicated in squamous cell carcinomas of several anatomic sites, as well as endocervical adenocarcinomas. Identification of HPV is an actionable finding in some carcinomas, potentially influencing tumor classification, prognosis, and management. We incorporated capture probes for oncogenic HPV strains 16 and 18 into a broader next-generation sequencing (NGS) panel designed to identify actionable mutations in solid malignancies. A total of 21 head and neck, genitourinary, and gynecologic squamous cell carcinomas and endocervical adenocarcinomas were sequenced as part of the UNCSeq project. Using p16 immunohistochemical results as the gold standard, we set a cutoff for proportion of aligned HPV reads that maximized performance of our NGS assay (92% sensitive, 100% specific for HPV). These results suggest that sequencing of oncogenic pathogens can be incorporated into targeted NGS panels, extending the clinical utility of genomic assays.

Germline Analysis from Tumor-Germline Sequencing Dyads to Identify Clinically Actionable Secondary Findings.

PURPOSE: To evaluate germline variants in hereditary cancer susceptibility genes among unselected cancer patients undergoing tumor-germline sequencing. EXPERIMENTAL DESIGN: Germline sequence data from 439 individuals undergoing tumor-germline dyad sequencing through the LCCC1108/UNCseq™ (NCT01457196) study were analyzed for genetic variants in 36 hereditary cancer susceptibility genes. These variants were analyzed as an exploratory research study to determine whether pathogenic variants exist within the germline of patients undergoing tumor-germline sequencing. Patients were unselected with respect to indicators of hereditary cancer predisposition. RESULTS: Variants indicative of hereditary cancer predisposition were identified in 19 (4.3%) patients. For about half (10/19), these findings represent new diagnostic information with potentially important implications for the patient and their family. The others were previously identified through clinical genetic evaluation secondary to suspicion of a hereditary cancer predisposition. Genes with pathogenic variants included ATM, BRCA1, BRCA2, CDKN2A, and CHEK2 In contrast, a substantial proportion of patients (178, 40.5%) had Variants of Uncertain Significance (VUS), 24 of which had VUS in genes pertinent to the presenting cancer. Another 143 had VUS in other hereditary cancer genes, and 11 had VUS in both pertinent and nonpertinent genes. CONCLUSIONS: Germline analysis in tumor-germline sequencing dyads will occasionally reveal significant germline findings that were clinically occult, which could be beneficial for patients and their families. However, given the low yield for unexpected germline variation and the large proportion of patients with VUS results, analysis and return of germline results should adhere to guidelines for secondary findings rather than diagnostic hereditary cancer testing. Clin Cancer Res; 22(16); 4087-94. ©2016 AACRSee related commentary by Mandelker, p. 3987.

Multi-Institutional FASTQ File Exchange as a Means of Proficiency Testing for Next-Generation Sequencing Bioinformatics and Variant Interpretation.

Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing.

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC.

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

Metabolic and functional genomic studies identify deoxythymidylate kinase as a target in LKB1-mutant lung cancer.

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling showed that Lkb1-null cells had a striking decrease in multiple nucleotide metabolites as compared with the Lkb1-wild-type cells. Thus, LKB1-mutant lung cancers have deficits in nucleotide metabolism that confer hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.