HPV-16 in a distinct subset of oral epithelial dysplasia.

Human papillomavirus (HPV) 16 is the most common high-risk HPV type identified in oropharyngeal and cervical neoplasia. Recently, HPV-associated oral epithelial dysplasia with specific histopathologic features and demographics similar to HPV-oropharyngeal carcinoma has been identified. The objective of this study was to evaluate histopathologically all cases of HPV-oral epithelial dysplasia seen in one center and identify HPV types in a subset of cases. Cases with specific histopathology for HPV-oral epithelial dysplasia that were positive both by immunohistochemical studies for p16 and by in situ hybridization for high-risk types of HPV were further analyzed using QIAamp DNA Tissue Kits (Qiagen, Hilden, Germany). DNA was extracted, amplified, and digested with restriction enzymes and run on a polyacrylamide gel. Digestion patterns were visually compared with a database of known HPV digestion patterns for identification. There were 53 specimens included in the analysis. There were 47 males and six females (7.8:1), with a median age of 55 years (range 41-81). The most common site of involvement was the tongue/floor of mouth (77% of cases). Of the 53 cases, 94% exhibited parakeratosis and/or hyperkeratosis. All the cases featured karyorrhexis, apoptosis, and characteristics of conventional carcinoma in situ. The quantity of DNA extracted was sufficient for analysis in 22 cases. HPV-16 was identified in 20/22 (91%) cases. One case was associated with HPV-33 and one with HPV-58 (5% each). Eight of the 53 cases (15%) were associated with invasive squamous cell carcinomas.

Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing.

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome-associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies.

Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total. Rapid heme panel's average coverage is 1500× with <5% of the amplicons with <50× coverage, and it reproducibly detects single nucleotide variants and small insertions/deletions at allele frequencies of ≥5%. Comparison with a capture-based next-generation sequencing assay showed that there is >95% concordance among a wide array of variants across a range of allele frequencies. Read count analyses that used rapid heme panel showed high concordance with karyotypic results when tumor content was >30%. The average turnaround time was 7 days over a 6-month span with an average volume of ≥40 specimens per week and a low sample fail rate (<1%), demonstrating its suitability for clinical application.

KRAS and NKX2-1 Mutations in Invasive Mucinous Adenocarcinoma of the Lung.

INTRODUCTION: Mucinous differentiation is observed in a subset of lung adenocarcinomas with unique clinical and pathological features, but the biology of these neoplasms is poorly understood. METHODS: We apply targeted next-generation sequencing to characterize the mutational profiles of 21 invasive mucinous adenocarcinomas, mixed mucinous/nonmucinous adenocarcinomas, and adenocarcinomas with mucinous features of the lung and validate key findings on 954 additional lung adenocarcinomas from our institution and 514 lung adenocarcinomas from The Cancer Genome Atlas. RESULTS: Sequencing identifies pathogenic mutations in the oncogenes Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), erb-b2 receptor tyrosine kinase 2 (ERBB2), and anaplastic lymphoma receptor tyrosine kinase (ALK) and recurrent mutations in tumor protein p53 (TP53), serine/threonine kinase 11 (STK11), NK2 homeobox 1 (NKX2-1), and SET domain containing 2 (SETD2). In the combined discovery and validation cohorts, we identify nine neoplasms with distinct molecular and pathological features. All are invasive mucinous adenocarcinomas or mixed mucinous/nonmucinous adenocarcinomas with mutations of KRAS and frameshift or nonsense mutations of NKX2-1. Immunohistochemical analysis shows that these neoplasms are associated with altered differentiation states, including loss of expression of the pulmonary marker thyroid transcription factor 1 (also called Nkx2.1) and expression of gastrointestinal markers. CONCLUSIONS: These findings describe recurrent NKX2-1 mutations in invasive mucinous adenocarcinomas of the lung and support NKX2-1 as a lineage-specific tumor suppressor gene in lung carcinogenesis.

MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression.

PURPOSE: Non-small-cell lung cancers (NSCLCs) harboring mutations in MET exon 14 and its flanking introns may respond to c-Met inhibitors. We sought to describe the clinical, pathologic, and genomic characteristics of patients with cancer with MET exon 14 mutations. PATIENTS AND METHODS: We interrogated next-generation sequencing results from 6,376 cancers to identify those harboring MET exon 14 mutations. Clinical characteristics of MET exon 14 mutated NSCLCs were compared with those of NSCLCs with activating mutations in KRAS and EGFR. Co-occurring genomic mutations and copy number alterations were identified. c-Met immunohistochemistry and real-time polymerase chain reaction to detect exon 14 skipping were performed where sufficient tissue was available. RESULTS: MET exon 14 mutations were identified in 28 of 933 nonsquamous NSCLCs (3.0%) and were not seen in other cancer types in this study. Patients with MET exon 14-mutated NSCLC were significantly older (median age, 72.5 years) than patients with EGFR-mutant (median age, 61 years; P < .001) or KRAS-mutant NSCLC (median age, 65 years; P < .001). Among patients with MET exon 14 mutations, 68% were women, and 36% were never-smokers. Stage IV MET exon 14-mutated NSCLCs were significantly more likely to have concurrent MET genomic amplification (mean ratio of MET to chromosome 7, 4.3) and strong c-Met immunohistochemical expression (mean H score, 253) than stage IA to IIIB MET exon 14-mutated NSCLCs (mean ratio of MET to chromosome 7, 1.4; P = .007; mean H score, 155; P = .002) and stage IV MET exon 14-wild-type NSCLCs (mean ratio of MET to chromosome 7, 1.2; P < .001; mean H score, 142; P < .001). A patient whose lung cancer harbored a MET exon 14 mutation with concurrent genomic amplification of the mutated MET allele experienced a major partial response to the c-Met inhibitor crizotinib. CONCLUSION: MET exon 14 mutations represent a clinically unique molecular subtype of NSCLC. Prospective clinical trials with c-Met inhibitors will be necessary to validate MET exon 14 mutations as an important therapeutic target in NSCLC.

A novel method for interpretation of T-cell receptor gamma gene rearrangement assay by capillary gel electrophoresis based on normal distribution.

T-cell receptor gamma (TRG) gene rearrangement status is useful for the differential diagnosis of T-cell lesions. The BIOMED-2 protocol that uses two sets of Jgamma and four sets of Vgamma primers in a multiplex, two-tube reaction followed by capillary gel electrophoresis is emerging as a standard assay for this application. Here, we report a computer-aided method to evaluate the significance of a peak in this TRG clonality assay. A best-fit normal distribution (ND) curve and the chi(2) error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for Vgamma(1-8) is 1). Eighty clinical samples that have been previously analyzed by a GC-clamped primer polymerase chain reaction/denaturing gradient gel electrophoresis assay were reanalyzed with the BIOMED-2 assay and scored by the ND method and four previously published methods: relative peak height (RPH), relative peak ratio (RPR), height ratio (HR), and peak height ratio (Rn). A greater than 90% concordance rate was observed between RPH and ND analysis, whereas RPR, Rn, and HR had a lower threshold to call a peak positive. The advantage of the ND method is that it is more objective, reproducible, and can be automated.