Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung,Melanoma, and Gastrointestinal Malignancies

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.In modern oncologic practice, patients with advanced-stage non-small cell lung cancer (NSCLC),^{1, 2} melanoma,^{3, 4} and colorectal adenocarcinoma^{5, 6} are often treated with targeted therapies as standard of care or after enrollment in clinical trials. Molecular mutation analysis is the preferred testing modality to guide therapeutic decision making and/or eligibility for biological studies. Therefore, laboratory-developed mutation assays require robust workflows that produce high-quality sequence information from routine clinical specimens, namely formalin-fixed, paraffin-embedded (FFPE) samples. As molecular testing transitions from an ancillary tool to a seminal requirement for optimal oncologic patient management, multiplex sequencing assays with clearly defined content and bioinformatics workflows are essential for accurate and consistent results, reporting, and patient management.Published guidelines endorse which genes to test in a particular tumor type and provide timeframes for receipt of actionable results, but they also grant individual laboratories autonomy to perform mutation testing using any suitable validated method.² Historically at our institution, single-gene mutation analysis for clinically relevant genes was performed either in-house or at a Clinical Laboratory improvement Amendment–certified reference laboratory. Depending on the result, reflex testing was performed for additional genes per mutation frequency or designated algorithms. Unfortunately, this approach introduced considerable turn-around time delays and unnecessary cost, particularly when send-out testing was required. Therefore, we sought testing modalities that could analyze multiple clinically relevant mutations simultaneously, accurately, and expeditiously.Next-generation sequencing (NGS) technologies have revolutionized genomic medicine by allowing high-throughput, parallel sequencing of the human genome.⁷ Currently, however, a large proportion of clinical NGS endeavors are supported by larger academic institutions with shared access to established genomic and bioinformatics research infrastructures, and routine clinical implementation of NGS is complicated by mitigating factors, such as clinical performance, laboratory expertise, lengthy turn-around times, and cost.⁸ Thus, we investigated affordable methods to detect clinically relevant somatic mutations in NSCLC, melanoma, and gastrointestinal (GI) malignancies that generated high-quality sequencing data from FFPE samples, and offered manageable turn-around times. Targeted amplicon-based library preparation methods combined with parallel sequencing offered a practical solution, and recent studies have demonstrated the utility of this approach.^{9, 10}Reversible terminal dideoxynucleotide sequencing chemistry by Illumina (San Diego, CA) consistently generates accurate and reproducible sequencing data.^{11, 12} To use this chemistry for clinical testing, we purchased the bench-top NGS sequencer, the Illumina MiSeq, and paired it with the MiSeq-compatible Illumina TruSight tumor (TST) 26-target amplicon-based library preparation kit. TST targets 26 genes and 174 amplicons selected from College of American Pathologists/National Comprehensive Cancer Network guidelines, relevant publications, and late-phase pharmaceutical clinical trials (Supplemental Table S1). TST offered several advantages over other commercially available mutation testing kits, such as bidirectional targeting of the positive and negative DNA strands, full-exon coverage as opposed to hotspot analysis, and robust vendor-supplied bioinformatics techniques optimized for somatic variant detection. More important, TST library preparation is optimized for FFPE samples, multiple safeguards exist to detect FFPE variant artifacts, and deep sequencing of TST libraries consistently yields high depths of coverage of targeted regions.Somatic mutation testing for many of the TST genes has clinical utility in a wide variety of solid tumors. For example, testing for CTNNB1 exon 3 is performed clinically for diagnostic and prognostic purposes in pediatric desmoid tumors, select PIK3CA hotspot mutations are positive prognostic factors for breast carcinoma, and multiple exons in PDGFRA and KIT are routinely tested in GI stromal tumors to predict response to targeted therapies. More important for our intended validation purposes, all of the clinically relevant genes and regions mutated in NSCLC, melanoma, and colonic adenocarcinoma that were tested in our routine clinical practice were represented. In addition, we could easily incorporate the TST NGS into a 5 business day workflow model, and a cost-analysis demonstrated a reasonable cost per test.Last, TST NGS data are processed from raw sequence (FASTQ) to called variants with on-board MiSeq Reporter software version 2.3, and variant annotations can be performed with Illumina''s VariantStudio software version 2.1 software using standard desktop and laptop computers. The ease of library preparation, sequencing, and data analysis with tools provided by a single vendor best fit our clinical priorities and the resources available at our academic molecular pathology laboratory.Herein, we present our results from the clinical validation of TST NGS using 80 sequenced samples that were selected from 100 FFPE patient samples (40 NSCLCs, 30 melanomas, and 30 GI malignancies). During our validation, we achieved high depths of coverage for multiple clinically relevant variants when multiplexing 8 to 12 samples on a single MiSeq flow cell. TST NGS consistently demonstrated sensitivities comparable to reference assays, showed 100% concordance with known variants, detected novel variants in many samples, and uncovered variants missed by less-sensitive testing modalities. The TST variant-calling pipeline was robust and showed high concordance when compared with an alternative analysis pipeline, and we used an in-house custom Java program to assess laboratory-defined quality control (QC) metrics and streamline clinical reporting (developed by G.H.S., Emory University, http://github.com/ghsmith/coverageQc). More important, although the results detailed herein represent the experience of a single institution, the data and validation strategies shown herein are broadly applicable to most clinical molecular laboratories interested in offering NGS for NSCLCs, melanomas, and GI malignancies as well as many other solid tumors.     

Prevalence and risk factors associated with tuberculin skin test positivity among household contacts of smear-positive pulnionary tuberculosis cases in Umerkot, Pakistan.

STUDY POPULATION AND SETTING: Household contacts of acid-fast bacilli (AFB) sputum smear-positive tuberculosis patients in the Umerkot Taluka, Sindh, Pakistan. OBJECTIVE: To estimate the prevalence of and identify risk factors associated with tuberculin skin test (TST) positivity among household contacts of acid-fast bacilli (AFB) sputum smear-positive pulmonary tuberculosis cases. DESIGN: A cross-sectional study of household contacts of AFB sputum smear-positive tuberculosis cases, registered at the Umerkot Anti-Tuberculosis Association clinic from August 1999 to September 1999. The contact's Mycobacterium tuberculosis infection status was assessed using TST. On the day of the TST, a pre-designed questionnaire was administered to collect data on putative risk factors for TST positivity among contacts. The data were analysed using a marginal logistic regression model by the method of generalised estimating equations (GEE) to determine risk factors independently associated with TST positivity. RESULTS: The prevalence of TST positivity among household contacts of AFB sputum smear-positive index patients was 49.4%. The final multivariate GEE model showed that contact's age and sleeping site relative to the index case, the intensity of the index case's AFB sputum-smear positivity and the contact's BCG scar status were independent predictors of TST positivity among household contacts of AFB sputum smear-positive index cases. CONCLUSIONS: The results suggest that the household contacts of AFB sputum smear-positive tuberculosis patients in a poor neighbourhood of rural Sindh had a high prevalence of M. tuberculosis infection as determined by TST. Poor housing conditions seem to contribute to the spread of M. tuberculosis infection. Early diagnosis of pulmonary TB through evaluation of TST-positive household contacts, followed by appropriate therapy, may prevent further spread of M. tuberculosis infection. We recommend an awareness programme to prevent household contacts from acquiring M. tuberculosis infection from smear-positive pulmonary TB cases.     

Benign Fibrous Histiocytoma: A Rare Case Report and Literature Review

Fibrous histiocytoma is a benign soft tissue tumour that may present as a fibrous mass anywhere in the human body. The involvement of the oral cavity is extremely rare and very few cases have been reported in literature till date. We here report a case of benign fibrous histiocytoma localized in the oral cavity. The clinical and histological features of the lesion are discussed precisely in the light of a literature review of this pathology in oral cavity.