System for Informatics in the Molecular Pathology Laboratory: An Open-Source End-to-End Solution for Next-Generation Sequencing Clinical Data Management.

Next-generation sequencing (NGS) diagnostic assays increasingly are becoming the standard of care in oncology practice. As the scale of an NGS laboratory grows, management of these assays requires organizing large amounts of information, including patient data, laboratory processes, genomic data, as well as variant interpretation and reporting. Although several Laboratory Information Systems and/or Laboratory Information Management Systems are commercially available, they may not meet all of the needs of a given laboratory, in addition to being frequently cost-prohibitive. Herein, we present the System for Informatics in the Molecular Pathology Laboratory (SIMPL), a free and open-source Laboratory Information System/Laboratory Information Management System for academic and nonprofit molecular pathology NGS laboratories, developed at the Genomic and Molecular Pathology Division at the University of Chicago Medicine. SIMPL was designed as a modular end-to-end information system to handle all stages of the NGS laboratory workload from test order to reporting. We describe the features of SIMPL, its clinical validation at University of Chicago Medicine, and its installation and testing within a different academic center laboratory (University of Colorado), and we propose a platform for future community co-development and interlaboratory data sharing.

Noninvasive Follicular Thyroid Neoplasms With Papillary-like Nuclear Features Are Genetically and Biologically Similar to Adenomatous Nodules and Distinct From Papillary Thyroid Carcinomas With Extensive Follicular Growth.

CONTEXT: - Proposed noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTPs), formerly noninvasive encapsulated papillary carcinoma, follicular variant (PTC-FV), is an indolent tumor with follicular growth and frequent RAS mutations. OBJECTIVE: - To detect histologic and molecular differences separating NIFTP from follicular adenomas (FAs) and invasive carcinomas, particularly papillary carcinomas with extensive follicular growth (PTC-EFGs) and invasive encapsulated PTC-FV (IE-PTC-FV). DESIGN: - Sixty-one tumors were reviewed histologically and reclassified into 32 NIFTPs (52%), 4 IE-PTC-FVs (7%), 14 PTC-EFGs (23%), and 11 FAs (18%). Next-generation sequencing for mutations in 50 genes was performed. Clinical outcomes were recorded. RESULTS: - The NIFTPs and FAs were well circumscribed and unencapsulated. The FAs had bland nuclei, whereas the NIFTPs showed at least 2 of 3 (67%; sufficient) nuclear features (enlargement, irregular contours, chromatin clearing). The IE-PTC-FVs had follicular growth, sufficient nuclear features, and extensive capsular invasion. The PTC-EFGs had a median of 5% papillae with intrathyroidal invasion (broad-based, sclerotic, or small follicle growth patterns); intranuclear pseudoinclusions were present only in PTC-EFGs (9 of 14; 64%). Mutations included RAS in 20 of the 32 NIFTPs (62%), 4 of the 11 FAs (36%), and 3 of the 4 IE-PTC-FVs (75%); BRAF K601E in 1 NIFTP (3%); BRAF V600E in 5 PTC-EFGs (36%). No NIFTPs or FAs recurred or metastasized. All 4 IE-PTC-FVs (100%) had hematogenous metastasis. Two PTC-EFGs (14%) had lymphatic metastasis. CONCLUSIONS: - The morphologic similarity and RAS mutations in FAs, NIFTPs, and IE-PTC-FVs supports the genetic similarity of those follicular neoplasms in contrast to the unique presence of BRAF V600E mutations in PTC-EFGs. Using strict diagnostic criteria supported by molecular testing, tumors with extensive follicular growth can be classified into follicular type or RAS-like (FA, NIFTP, IE-PTC-FV) versus papillary type or BRAF V600E-like (PTC-EFG).

Risk factors, prevalence, and site concordance of human papillomavirus in high-risk Greek men.

The aim of this study was to determine the risk factors, genotype-specific prevalence, and concordance of human papillomavirus (HPV) infections at three anatomical sites in a cohort of high-risk Greek men. Patients were recruited from sexually transmitted infection and HIV clinics in Athens. Samples were obtained from oral, penile, and anal sites of 294 study participants and HPV testing was performed on 882 samples using next-generation sequencing. Patients also completed a questionnaire assessing risk factors for infection. The mean age of the participants was 33.1, 30% identified as men who have sex with men (MSM), and 21% were HIV positive. The prevalence of HPV was 49%; it was the highest at anal sites (33%) compared with 23% at penile sites (P=0.008) and 4% at oral sites (P<0.001). The most common HPV types in order of frequency were 6, 44, 16, 53, and 89. The genotype concordance rate was the highest between the penile and anal sites (7%), followed by 2% for anal-oral concordance. Identifying as MSM [adjusted odds ratios (aOR)=6.75, P<0.001] and being HIV positive (aOR=2.89, P=0.026) were significant risk factors for anal HPV infection, whereas alcohol use (aOR=0.45, P=0.002) was associated negatively with infection. The only significant risk factor for oral infection was an older age of sexual debut (aOR=1.32, P=0.038). Nearly half of our study participants tested positive in at least one of three anatomical sites. Using next-generation sequencing, we could identify high-risk types that are not covered by the current vaccine and would be missed by traditional HPV testing kits.

High prevalence of MiTF staining in undifferentiated pleomorphic sarcoma: caution in the use of melanocytic markers in sarcoma.

AIMS: The diagnosis of undifferentiated pleomorphic sarcoma (UPS) may be challenging, as other lesions with undifferentiated spindle cell morphology must be excluded, including melanoma. Microphthalmia-associated transcription factor (MiTF) stains naevi and epithelioid melanomas, as well as some mesenchymal neoplasms. The aim of this study was to evaluate the prevalence of MiTF and melanocytic markers in UPS and a subset of atypical fibroxanthoma (AFX). METHODS AND RESULTS: MiTF, SOX10, Melan-A, HMB45 and S100 immunostaining was performed on resection specimens from 19 UPSs and five AFXs. Next-generation sequencing of 50 genes was performed in UPSs to exclude dedifferentiated melanoma. In 17 of 19 UPSs (89%), tumour cells showed nuclear positivity for MiTF that was not eliminated by casein block. Three showed focal nuclear staining for HMB45, which was eliminated by casein block. One showed focal nuclear vacuole staining for S100 with red but not brown chromogen. None expressed SOX10 or Melan-A. Mutational analysis of 15 UPSs with adequate DNA showed no mutations within hotspot regions of BRAF, KIT, or NRAS. Four of five AFXs (80%) stained with MiTF; other markers were negative. CONCLUSION: There is a high prevalence of nuclear MiTF expression in UPSs (89%) and AFXs (80%). Rare UPSs showed non-specific nuclear HMB45 or S100 staining. These findings argue against using MiTF in isolation to differentiate between UPS or AFX and melanoma, and caution in interpreting focal staining for a single additional melanocytic marker. Casein block may eliminate non-specific staining. MiTF should be used to support a diagnosis of melanoma only if multiple melanocytic markers are positive.

Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays.

Next-generation sequencing (NGS) genomic oncology profiling assays have emerged as key drivers of personalized cancer care and translational research. However, validation of these assays to meet strict clinical standards has been historically problematic because of both significant assay complexity and a scarcity of optimal validation samples. Herein, we present the clinical validation of 76 genes from a novel 1212-gene large-scale hybrid capture cancer sequencing assay (University of Chicago Medicine OncoPlus) using full-data comparisons against multiple clinical NGS amplicon-based assays to yield dramatic increases in per-sample data comparison efficiency compared with previously published validations. Using a sample set of 104 normal, solid tumor, and hematopoietic malignancy specimens, head-to-head NGS data analyses allowed for 6.8 million individual clinical base call comparisons, including 2729 previously confirmed variants, with 100% sensitivity and specificity. University of Chicago Medicine OncoPlus showed excellent performance for detection of single-nucleotide variants, insertions/deletions up to 52 bp, and FLT3 internal tandem duplications of up to 102 bp or larger. Highly concordant copy number variant and ALK/RET/ROS1 gene fusion detection were also observed. In addition to underlining the efficiency of NGS validation via full-data benchmarking against existing clinical NGS assays, this study also highlights the degree of performance similarity between hybrid capture and amplicon assays that is attainable with the application of strict quality control parameters and optimized computational analytics.

Polyglucosan bodies in intramuscular nerve branches are a poor predictor of GBE1 mutation and adult polyglucosan body disease.

INTRODUCTION: Adult polyglucosan body disease (APBD) is associated with formation of polyglucosan bodies in peripheral nerve branches. Some muscle biopsies show these inclusions in intramuscular nerve branches. It has not been established whether the presence of multiple polyglucosan bodies in intramuscular peripheral nerve branches could or should suggest testing for APBD. METHODS: Fifteen muscle biopsies from adults between the ages of 36 and 84 years, all showing polyglucosan bodies in intramuscular peripheral nerve twigs, were tested by sequencing of the GBE1 gene. RESULTS: In 4 patients, testing identified heterozygous missense mutations not previously described. No homozygous or compound heterozygous mutations were identified. CONCLUSIONS: The presence of polyglucosan bodies in intramuscular nerve twigs by itself, even if they are multiple, is not an indication of APBD. Further testing may only be indicated in patients with clinical disease manifestations.

Amplicon Indel Hunter Is a Novel Bioinformatics Tool to Detect Large Somatic Insertion/Deletion Mutations in Amplicon-Based Next-Generation Sequencing Data.

Amplicon-based targeted next-generation sequencing assays are used widely to test for clinically relevant somatic mutations in cancer. However, accurate detection of large insertions and deletions (indels) via these assays remains challenging. Sequencing reads that cover these anomalies are, by definition, different from the reference sequence, and lead to variable performance of alignment algorithms. Reads with large indels may be aligned incorrectly, causing incorrect calls, or may remain unmapped and essentially ignored by downstream informatics pipeline sub-processes. Herein, we describe Amplicon Indel Hunter (AIH), a novel large (>5-bp) indel detection method that is reference genome independent and highly sensitive for the identification of somatic indels in amplicon-based, paired-end, next-generation sequencing data. We validated the algorithm for sensitivity and specificity using a set of clinical cancer samples with Clinical Laboratory Improvement Amendment-confirmed indels as well as in silico mutated data sets. The AIH detected 100% of tested large indels with relatively higher mutant allele frequencies compared with a variety of traditional aligners, which showed variably reduced sensitivity and specificity by comparison. The AIH especially outperformed alignment-based methods for detection of all tested FLT3 internal tandem duplications up to 102 bp. Because AIH runs in parallel to traditional alignment-based informatics pathways, it can be readily incorporated into nearly any analysis pipeline for somatic mutation detection in paired-end amplicon-based data.

Quantitative trait locus analysis identifies Gabra3 as a regulator of behavioral despair in mice.

The Tail Suspension Test (TST), which measures behavioral despair, is widely used as an animal model of human depressive disorders and antidepressant efficacy. In order to identify novel genes involved in the regulation of TST performance, we crossed an inbred strain exhibiting low immobility in the TST (RIIIS/J) with two high-immobility strains (C57BL/6J and NZB/BlNJ) to create two distinct F2 hybrid populations. All F2 offspring (n = 655) were genotyped at high density with a panel of SNP markers. Whole-genome interval mapping of the F2 populations identified statistically significant quantitative trait loci (QTLs) on mouse chromosomes (MMU) 4, 6, and X. Microarray analysis of hippocampal gene expression in the three parental strains was used to identify potential candidate genes within the MMUX QTLs identified in the NZB/BlNJ x RIIIS/J cross. Expression of Gabra3, which encodes the GABA(A) receptor alpha3 subunit, was robust in the hippocampus of B6 and RIIIS mice but absent from NZB hippocampal tissue. To verify the role of Gabra3 in regulating TST behavior in vivo, mice were treated with SB-205384, a positive modulator of the alpha3 subunit. SB-205384 significantly reduced TST immobility in B6 mice without affecting general activity, but it had no effect on behavior in NZB mice. This work suggests that GABRA3 regulates a behavioral endophenotype of depression and establishes this gene as a viable new target for the study and treatment of human depression.

Adaptation and limitations of established hemagglutination inhibition assays for the detection of porcine anti-swine influenza virus H1N2 antibodies.

Hemagglutination inhibition (HI) has been a reliable method for determining porcine antibody levels to the well-characterized swine influenza virus (SIV) H1N1 and H3N2 subtypes. However, the recent emergence of the novel H1N2 serotype of SIV and the persistence of 2 other serotypes (H1N1 and H3N2) in the United States swine population represents a significant challenge to diagnostics. Both standardized and modified HI protocols were used in a blinded study to examine a collection of 50 control sera representing a total of 12 swine that were experimentally inoculated with one of the 3 SIV subtypes. Using these control sera data, a statistical basis for analysis was established in an attempt to classify 30 field sera with known case histories or seroprevalance into SIV serotype categories. By this approach 57% of the field sera could be classified into specific categories. The remaining samples that could not be classified reliably were most likely composed of heterogeneous anti-SIV antibody populations. These results indicate that although serological differentiation might be possible in a controlled environment, applications of these methods to field samples are currently problematic. Approaches other than HI will be required to fulfill the current need for SIV diagnostics and surveillance when specific serotype identification is required.