Quantitative next-generation sequencing-based analysis indicates progressive accumulation of microsatellite instability between atypical hyperplasia/endometrial intraepithelial neoplasia and paired endometrioid endometrial carcinoma.

Atypical hyperplasia/endometrial intraepithelial neoplasia is an accepted precursor to endometrioid-type endometrial carcinoma. Mismatch repair-deficient endometrial carcinomas are also known to be a biologically and clinically distinct subset of tumors. However, the development of microsatellite instability in endometrial carcinogenesis has not yet been evaluated by novel next-generation sequencing-based methods. We examined 17 mismatch repair-deficient endometrioid endometrial carcinomas and their paired atypical hyperplasia/endometrial intraepithelial neoplasia precursors using a next-generation sequencing panel with quantitative microsatellite instability detection at 336 loci. Findings were compared to histological features, polymerase chain reaction-based microsatellite instability testing, immunohistochemical expression of mismatch repair proteins, and tumor mutational burden calculations. All 17 endometrial carcinomas and 8/17 atypical hyperplasia/endometrial intraepithelial neoplasia showed microsatellite instability by next-generation sequencing-based testing. Endometrial carcinoma specimens showed significantly more unstable microsatellite loci than paired atypical hyperplasia/endometrial intraepithelial neoplasia (mean: 40.0% vs 19.9 unstable loci, respectively). Out of nine microsatellite-stable atypical hyperplasia/endometrial intraepithelial neoplasia specimens, four showed mismatch repair loss by immunohistochemistry. All atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens with microsatellite instability were also mismatch repair-deficient by immunohistochemistry. Tumor mutational burden was significantly greater in endometrial carcinoma than in paired atypical hyperplasia/endometrial intraepithelial neoplasia specimens, and tumor mutational burden was significantly correlated with percent unstable microsatellite loci. Paired atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens show progressive accumulation of unstable microsatellite loci following loss of mismatch repair protein expression. Comprehensive next-generation sequencing-based testing of endometrial carcinomas offers new insights into endometrial carcinogenesis and opportunities for improved tumor surveillance, diagnosis, and management.

A pipeline for completing bacterial genomes using in silico and wet lab approaches.

BACKGROUND: Despite the large volume of genome sequencing data produced by next-generation sequencing technologies and the highly sophisticated software dedicated to handling these types of data, gaps are commonly found in draft genome assemblies. The existence of gaps compromises our ability to take full advantage of the genome data. This study aims to identify a practical approach for biologists to complete their own genome assemblies using commonly available tools and resources. RESULTS: A pipeline was developed to assemble complete genomes primarily from the next generation sequencing (NGS) data. The input of the pipeline is paired-end Illumina sequence reads, and the output is a high quality complete genome sequence. The pipeline alternates the employment of computational and biological methods in seven steps. It combines the strengths of de novo assembly, reference-based assembly, customized programming, public databases utilization, and wet lab experimentation. The application of the pipeline is demonstrated by the completion of a bacterial genome, Thermotoga sp. strain RQ7, a hydrogen-producing strain. CONCLUSIONS: The developed pipeline provides an example of effective integration of computational and biological principles. It highlights the complementary roles that in silico and wet lab methodologies play in bioinformatical studies. The constituting principles and methods are applicable to similar studies on both prokaryotic and eukaryotic genomes.

Natural transformation of Thermotoga sp. strain RQ7.

BACKGROUND: Thermotoga species are organisms of enormous interest from a biotechnological as well as evolutionary point of view. Genetic modifications of Thermotoga spp. are often desired in order to fully release their multifarious potentials. Effective transformation of recombinant DNA into these bacteria constitutes a critical step of such efforts. This study aims to establish natural competency in Thermotoga spp. and to provide a convenient method to transform these organisms. RESULTS: Foreign DNA was found to be relatively stable in the supernatant of a Thermotoga culture for up to 6 hours. Adding donor DNA to T. sp. strain RQ7 at its early exponential growth phase (OD600 0.18 ~ 0.20) resulted in direct acquisition of the DNA by the cells. Both T. neapolitana chromosomal DNA and Thermotoga-E. coli shuttle vectors effectively transformed T. sp. strain RQ7, rendering the cells resistance to kanamycin. The kan gene carried by the shuttle vector pDH10 was detected by PCR from the plasmid extract of the transformants, and the amplicons were verified by restriction digestions. A procedure for natural transformation of Thermotoga spp. was established and optimized. With the optimized method, T. sp. strain RQ7 sustained a transformation frequency in the order of 10⁻7 with both genomic and plasmid DNA. CONCLUSIONS: T. sp. strain RQ7 cells are naturally transformable during their early exponential phase. They acquire DNA from both closely and distantly related species. Both chromosomal DNA and plasmid DNA serve as suitable substrates for transformation. Our findings lend a convenient technical tool for the genetic engineering of Thermotoga spp.

High Rates of Ultraviolet-Signature Mutations in Squamous Cell Carcinomas of the Parotid Gland and Prognostic Implications.

In the absence of clear pathologic differences, clinical history may differentiate potential primary parotid squamous cell carcinomas (SCC) from metastases. The presence of an ultraviolet (UV) signature can distinguish between tumors of cutaneous and non-cutaneous origin. This study aimed to investigate rates of UV signature mutations in squamous cell carcinomas of the parotid gland as well as differences in clinical features between tumors of cutaneous and non-cutaneous origin. Clinical and pathologic data were collected from 71 patients with SCC involving the parotid gland, of which 48 had cutaneous, 10 had mucosal, and 13 had no history of SCC. In 34 available cases, genomic DNA was isolated from formalin-fixed paraffin-embedded tissue specimens and sequenced using a targeted hybrid capture 1213 gene panel. Tumor mutational burden and COSMIC (Catalogue of Somatic Mutations in Cancer) mutational signatures were calculated. Most (74%) were UV-positive. Patients with UV-positive tumors were significantly older, white, and had higher rates of sun exposure. Patients with UV-negative tumors had a significantly higher mortality rate and shorter time to death: 6 (67%) died of disease with a median time to death of 9 months compared to 5 (20%) UV-positive patients who died of disease with a median time to death of 32 months. Pathologic features did not significantly vary by clinical history or UV status. The presence of a UV-signature combined with clinical history can be used to determine the primary source of SCC involving the parotid gland. UV-positivity may reflect a less aggressive disease course in an older population.

Follicular Thyroid Neoplasms: Comparison of Clinicopathologic and Molecular Features of Atypical Adenomas and Follicular Thyroid Carcinomas.

In follicular thyroid neoplasms without invasion, a diagnosis of atypical adenoma (AA) (follicular tumor of uncertain malignant potential) may be rendered if atypical features (indefinite capsular/vascular invasion, necrosis, solid growth, increased mitoses) are present. This study compares clinical, histologic, and molecular features of patients with AAs (n=31), nonmetastatic follicular thyroid carcinoma (nmFTC) (n=18), and metastatic follicular thyroid carcinoma (mFTC) (n=38). Patients with mFTC were older. Mitotic activity in areas of solid growth was greatest in mFTC (P=0.05). Oncocytic tumors tended to show solid growth (P=0.04). The presence or frequency of capsular and/or vascular invasion was not different between nmFTC and mFTC. TERT promoter mutations were higher in patients with mFTC (50%) than nmFTC (25%) and AA (10%) (P=0.02). TERT promoter mutation was associated with necrosis (P=0.01) and solid growth plus increased mitoses (P=0.03). Necrosis and TERT promoter mutations were identified in all groups, most frequently in mFTC. The combination of solid growth with increased mitoses, necrosis, and TERT promoter mutation was only seen in follicular carcinomas. Poorly differentiated features, vascular invasion, and TERT promoter mutation correlated with metastasis in FTC. Given the low frequency of necrosis and TERT promoter mutation in AAs, close clinical follow-up is recommended in patients with these findings, especially if additional atypical features (such as solid growth plus mitoses) are present.

Clinical Validation and Implementation of a Measurable Residual Disease Assay for NPM1 in Acute Myeloid Leukemia by Error-Corrected Next-Generation Sequencing.

BACKGROUND: Nucleophosmin 1 (NPM1) is one of the most commonly mutated genes in acute myeloid leukemia, with mutations observed in approximately 30% of all adult cases. The persistence of NPM1 mutations following chemotherapy is associated with a greater risk of relapse as well as a lower rate of survival, making NPM1 measurable residual disease (MRD) an informative clinical target. METHODS: Herein, we have developed a straightforward unique molecular identifier (UMI)-based amplicon next-generation sequencing method for the detection of NPM1-mutated MRD that addresses some of the limitations present in other assays. RESULTS: The NPM1 assay allowed for accurate counting of individual mutant and wild-type molecules down to 0.01% variant allelic frequency. In silico contamination experiments highlighted the ability of this UMI methodology to maximize specificity through dramatic reductions in sequencing/demultiplexing bleed-through error. CONCLUSION: Performance and clinical utility of the NPM1 MRD assay are established via both validation experiments and analyses of live performance over 1.5 years of routine clinical service.

Germline BRCA-Associated Endometrial Carcinoma Is a Distinct Clinicopathologic Entity.

PURPOSE: Whether endometrial carcinoma (EC) should be considered part of the gBRCA1/2-associated hereditary breast and ovarian cancer (HBOC) syndrome is topic of debate. We sought to assess whether ECs occurring in gBRCA carriers are enriched for clinicopathologic and molecular characteristics, thereby supporting a causal relationship. EXPERIMENTAL DESIGN: Thirty-eight gBRCA carriers that developed EC were selected from the nationwide cohort study on hereditary breast and ovarian cancer in the Netherlands (HEBON), and these were supplemented with four institutional cases. Tumor tissue was retrieved via PALGA (Dutch Pathology Registry). Nineteen morphologic features were scored and histotype was determined by three expert gynecologic pathologists, blinded for molecular analyses (UCM-OncoPlus Assay including 1213 genes). ECs with LOH of the gBRCA-wild-type allele (gBRCA/LOHpos) were defined "gBRCA-associated," those without LOH (gBRCA/LOHneg) were defined "sporadic." RESULTS: LOH could be assessed for 40 ECs (30 gBRCA1, 10 gBRCA2), of which 60% were gBRCA/LOHpos. gBRCA/LOHpos ECs were more frequently of nonendometrioid (58%, P = 0.001) and grade 3 histology (79%, P < 0.001). All but two were in the TP53-mutated TCGA-subgroup (91.7%, P < 0.001). In contrast, gBRCA/LOHneg ECs were mainly grade 1 endometrioid EC (94%) and showed a more heterogeneous distribution of TCGA-molecular subgroups: POLE-mutated (6.3%), MSI-high (25%), NSMP (62.5%), and TP53-mutated (6.3%). CONCLUSIONS: We provide novel evidence in favor of EC being part of the gBRCA-associated HBOC-syndrome. gBRCA-associated ECs are enriched for EC subtypes associated with unfavorable clinical outcome. These findings have profound therapeutic consequences as these patients may benefit from treatment strategies such as PARP inhibitors. In addition, it should influence counseling and surveillance of gBRCA carriers.

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.

Complete genome sequence of Thermotoga sp. strain RQ7.

Thermotoga sp. strain RQ7 is a member of the family Thermotogaceae in the order Thermotogales. It is a Gram negative, hyperthermophilic, and strictly anaerobic bacterium. It grows on diverse simple and complex carbohydrates and can use protons as the final electron acceptor. Its complete genome is composed of a chromosome of 1,851,618 bp and a plasmid of 846 bp. The chromosome contains 1906 putative genes, including 1853 protein coding genes and 53 RNA genes. The genetic features pertaining to various lateral gene transfer mechanisms are analyzed. The genome carries a complete set of putative competence genes, 8 loci of CRISPRs, and a deletion of a well-conserved Type II R-M system.