Multi-Institutional Implementation of Clinical Decision Support for APOL1, NAT2, and YEATS4 Genotyping in Antihypertensive Management.

(1) Background: Clinical decision support (CDS) is a vitally important adjunct to the implementation of pharmacogenomic-guided prescribing in clinical practice. A novel CDS was sought for the APOL1, NAT2, and YEATS4 genes to guide optimal selection of antihypertensive medications among the African American population cared for at multiple participating institutions in a clinical trial. (2) Methods: The CDS committee, made up of clinical content and CDS experts, developed a framework and contributed to the creation of the CDS using the following guiding principles: 1. medical algorithm consensus; 2. actionability; 3. context-sensitive triggers; 4. workflow integration; 5. feasibility; 6. interpretability; 7. portability; and 8. discrete reporting of lab results. (3) Results: Utilizing the principle of discrete patient laboratory and vital information, a novel CDS for APOL1, NAT2, and YEATS4 was created for use in a multi-institutional trial based on a medical algorithm consensus. The alerts are actionable and easily interpretable, clearly displaying the purpose and recommendations with pertinent laboratory results, vitals and links to ordersets with suggested antihypertensive dosages. Alerts were either triggered immediately once a provider starts to order relevant antihypertensive agents or strategically placed in workflow-appropriate general CDS sections in the electronic health record (EHR). Detailed implementation instructions were shared across institutions to achieve maximum portability. (4) Conclusions: Using sound principles, the created genetic algorithms were applied across multiple institutions. The framework outlined in this study should apply to other disease-gene and pharmacogenomic projects employing CDS.

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.

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.

Prevalence of nonatheromatous lesions in peripheral arterial disease.

OBJECTIVE: The histopathology of peripheral arterial disease and the accompanying calcification are poorly defined, and it is not known whether this varies according to different risk factors. APPROACH AND RESULTS: Sections from 176 upper and lower leg arteries were examined histologically in specimens from amputations of 60 patients with peripheral arterial disease, of whom 58% had diabetes mellitus, 35% had end-stage renal disease, and 48% had a history of smoking. The most common findings were calcification of the media (72% of arteries) and intimal thickening without lipid (68% of arteries), with the presence of atheromas in only 23% of arteries. Intimal calcification occurred in 43% and was generally much less extensive than medial calcification. Nonatheromatous intimal thickening was frequently severe, resulting in complete occlusion in some vessels. The absence of lipid and macrophages was confirmed by staining with oil red O and staining for CD68. Other than a greater prevalence and severity of medial calcification in end-stage renal disease, the findings did not differ between diabetics, patients with end-stage renal disease, or smokers. CONCLUSIONS: The results indicate that the majority of arteries in patients with peripheral arterial disease have a vascular lesion that is distinct from atherosclerosis, suggesting a different pathogenesis. This pattern does not differ substantially between patients with different risk factors for peripheral arterial disease. The bulk of vascular calcification in the lower extremities is medial rather than intimal.

ERG expression in intraductal carcinoma of the prostate: comparison with adjacent invasive prostatic adenocarcinoma.

Intraductal carcinoma of the prostate is a growth pattern of prostatic adenocarcinoma that has not been well characterized from the molecular standpoint. It remains debatable whether intraductal carcinoma of the prostate represents colonization of benign glands by pre-existing conventional prostatic adenocarcinoma, or progression of high-grade prostatic intraepithelial neoplasia. TMPRSS2-ERG is the most common gene fusion in conventional prostatic adenocarcinoma, identified in about 40-70% of cases. In this study, we compared the expression of ERG in intraductal carcinoma of the prostate and adjacent conventional prostatic adenocarcinoma. Thirty-one confirmed cases of intraductal carcinoma of the prostate, with adjacent conventional prostatic adenocarcinoma and available tissue blocks, were identified at our institution. Immunohistochemical stains were performed for ERG using a rabbit anti-ERG monoclonal antibody. The ERG expression in the intraductal carcinoma of the prostate component was compared with that in the adjacent conventional prostatic adenocarcinoma. Mean patient age was 65 years (range: 48-79 years). Positive ERG expression was identified in 11/31 (35%) cases of intraductal carcinoma of the prostate. In all 11/11 (100%) cases with positive ERG expression in the intraductal carcinoma of the prostate component, ERG expression was also positive in the adjacent conventional prostatic adenocarcinoma. In the 20/31 cases with negative ERG expression in the intraductal carcinoma of the prostate component, ERG was also negative in the adjacent conventional prostatic adenocarcinoma. It is highly conceivable that based on the identical ERG expression (positive or negative) in intraductal carcinoma of the prostate and the adjacent conventional prostatic adenocarcinoma, intraductal carcinoma of the prostate most likely represents colonization of benign glands by adjacent pre-existing conventional prostatic adenocarcinoma.

Extraction force and cortical tissue reaction of silicon microelectrode arrays implanted in the rat brain.

Micromotion of implanted silicon multielectrode arrays (Si MEAs) is thought to influence the inflammatory response they elicit. The degree of strain that micromotion imparts on surrounding tissue is related to the extent of mechanical integration of the implanted electrodes with the brain. In this study, we quantified the force of extraction of implanted four shank Michigan electrodes in adult rat brains and investigated potential cellular and extracellular matrix contributors to tissue-electrode adhesion using immunohistochemical markers for microglia, astrocytes and extracellular matrix deposition in the immediate vicinity of the electrodes. Our results suggest that the peak extraction force of the implanted electrodes increases significantly from the day of implantation (day 0) to the day of extraction (day 7 and day 28 postimplantation) (1.68 +/- 0.54 g, 3.99 +/- 1.31 g, and 4.86 +/- 1.49 g, respectively; mean +/- SD; n = 4). For an additional group of four shank electrode implants with a closer intershank spacing we observed a significant increase in peak extraction force on day 28 postimplantation compared to day 0 and day 7 postimplantation (5.56 +/- 0.76 g, 0.37 +/- 0.12 g and 1.87 +/- 0.88 g, respectively; n = 4). Significantly, only glial fibrillary acidic protein (GFAP) expression was correlated with peak extraction force in both electrode designs of all the markers of astroglial scar studied. For studies that try to model micromotion-induced strain, our data implies that adhesion between tissue and electrode increases after implantation and sheds light on the nature of implanted electrode-elicited brain tissue reaction.