Mesonephric-like adenocarcinoma of the ovary with squamoid morular metaplasia, aberrant ß-catenin expression, and concurrent FGFR2 and CTNNB1 mutations: a case report.

In general, endometrioid-defining features such as squamoid morular metaplasia are not thought to be associated with mesonephric adenocarcinoma (MA) and mesonephric-like adenocarcinoma (MLA). Here, we report a case of FGFR2-mutated ovarian MLA with squamoid morular metaplasia accompanied by aberrant nuclear and cytoplasmic ß-catenin expression and CTNNB1 mutation. Histologically, the tumor showed classical MLA histology, including well-formed glands with intraluminal eosinophilic secretions and cells with papillary thyroid carcinoma-like nuclei. Squamoid morular metaplasia was intimately associated with the tumor. Glandular epithelial elements, including those immediately associated with the squamoid morules, were negative for ER, but positive for both GATA3 and PAX8; aberrant ß-catenin expression was limited to the squamoid morules. This case illustrates the ability of mesonephric neoplasia to exhibit histological features previously thought to be restricted to an endometrioid phenotype.

A simplified molecular method to detect high-risk HPV using the Aptima HPV assay on head and neck FNA smears.

BACKGROUND: Fine-needle aspiration is used as a diagnostic tool in head and neck oropharyngeal squamous cell carcinoma and its metastases. Prognosis and treatment rely on the presence or absence of the human papilloma virus. The purpose of this study was to validate the performance of the Aptima HPV assay using Hema-Diff stained fine-needle aspiration smears in the diagnosis of human papilloma virus-related oropharyngeal squamous cell carcinoma using a simplified method to obtain tumor cells for testing. METHODS: Patients with a diagnosis of squamous cell carcinoma and positive p16 immunohistochemical staining were identified. Aptima Specimen Transport Media was used to remove tumor cells from the Hema-Diff stained slides using a moistened swab. The selected cells were tested for high risk-human papilloma virus using the Aptima HPV assay and Aptima HPV 16 18/45 genotype assay. The results were compared with the p16 immunohistochemical staining of the related cell block and surgical specimens. RESULTS: Twenty-one of the 21 (100%) p16-positive cases were found to be positive for high risk-human papilloma virus, whereas 20 of 21 (95%) negative cases were found to be negative for high risk-human papilloma virus using the Aptima HPV assay. CONCLUSION: The Aptima HPV assay can be used to detect high-risk human papilloma virus in Hema-Diff stained fine-needle aspiration smears of oropharyngeal squamous cell carcinoma with a sensitivity of 100% and a specificity of 95%. This provides a valuable alternative to p16 immunohistochemical staining of cell block sections that often lack appropriate numbers of tumor cells.

Development of a rapid, simple, and sensitive point-of-care technology platform utilizing ternary NanoLuc.

Point-of-care tests are highly valuable in providing fast results for medical decisions for greater flexibility in patient care. Many diagnostic tests, such as ELISAs, that are commonly used within clinical laboratory settings require trained technicians, laborious workflows, and complex instrumentation hindering their translation into point-of-care applications. Herein, we demonstrate the use of a homogeneous, bioluminescent-based, split reporter platform that enables a simple, sensitive, and rapid method for analyte detection in clinical samples. We developed this point-of-care application using an optimized ternary, split-NanoLuc luciferase reporter system that consists of two small reporter peptides added as appendages to analyte-specific affinity reagents. A bright, stable bioluminescent signal is generated as the affinity reagents bind to the analyte, allowing for proximity-induced complementation between the two reporter peptides and the polypeptide protein, in addition to the furimazine substrate. Through lyophilization of the stabilized reporter system with the formulated substrate, we demonstrate a shelf-stable, all-in-one, add-and-read analyte-detection system for use in complex sample matrices at the point-of-care. We highlight the modularity of this platform using two distinct SARS-CoV-2 model systems: SARS-CoV-2 N-antigen detection for active infections and anti-SARS-CoV-2 antibodies for immunity status detection using chemically conjugated or genetically fused affinity reagents, respectively. This technology provides a simple and standardized method to develop rapid, robust, and sensitive analyte-detection assays with flexible assay formatting making this an ideal platform for research, clinical laboratory, as well as point-of-care applications utilizing a simple handheld luminometer.

A "Null" Pattern of p16 Immunostaining in Endometrial Serous Carcinoma: An Under-recognized and Important Aberrant Staining Pattern.

The ability to distinguish endometrial serous carcinoma (SC) from high-grade endometrioid adenocarcinoma is of great importance given their differences in prognosis and management. In practice, this distinction typically relies upon the use of a focused immunohistochemical panel including p53, p16, and mismatch repair proteins. The expression of p16 is characteristically strong and diffuse in SCs, and weak and/or patchy in many high-grade endometrioid adenocarcinomas. Here, we report a subset of SCs that are entirely negative for p16 immunostaining, a pattern we refer to as "p16 null." This pattern was identified in 2 of 63 cases of SC diagnosed at our institution-1 with histologically classic features and 1 with ambiguous high-grade histologic features. These tumors otherwise showed a SC signature by immunohistochemical and demonstrated an SC pattern of genetic mutations. No mutation in the gene for p16, cyclin-dependent kinase inhibitor 2A (CDKN2A), was identified in either case. However, molecular correlates for the absent p16 expression were present, including homozygous deletion of CDKN2A in one case and hemizygous deletion of CDKN2A with promotor hypermethylation of the remaining allele in the other case. To our knowledge, this constitutes the first report conclusively demonstrating the existence of a small subset of SCs that are completely negative by p16 immunohistochemistry, and the molecular lesions responsible for this pattern. In the context of an otherwise clinically and histologically classic example of SC, we endorse this "null" p16 staining pattern as an alternative aberrant staining pattern that should not deter one from committing to this diagnosis.

Oil immersed lossless total analysis system for integrated RNA extraction and detection of SARS-CoV-2.

The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.

Revealing fine-scale spatiotemporal differences in SARS-CoV-2 introduction and spread.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties following the statewide "Safer at Home" order, which went into effect 25 March 2020. Our results suggest patterns of SARS-CoV-2 transmission may vary substantially even in nearby communities. Understanding these local patterns will enable better targeting of public health interventions.

An Unusual Case of Actinomucor elegans: A Challenging Diagnosis.

BACKGROUND Actinomucor elegans is an unusual cause of mucormycosis and can be difficult to identify by conventional methods. Mucormycosis has a very high mortality rate, especially among immunocompromised individuals. Due to the morbid and progressive nature of opportunistic fungal infections, early diagnosis is paramount for effective disease management. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI) and Sanger sequencing are useful methods for rapid diagnosis of unusual fungal pathogens. CASE REPORT We report a fatal case of mucormycosis caused by A. elegans in an immunocompromised man. The pathogen was isolated from a large nasal septal black eschar that developed rapidly during tooth extraction in a patient with myelodysplastic syndrome and diabetes mellitus. After unsuccessful identification by conventional methods, A. elegans was identified using MALDI and Sanger sequencing. CONCLUSIONS Diagnosing fungal organisms poses many difficulties, but amidst the technological evolution in pathogen identification, there are useful methods for rapid identification, including MALDI and sequencing. With these powerful tools, earlier diagnosis will give health professionals an advantage against potentially fatal fungal infections.

Fatal Myocarditis Following Treatment with the PD-1 Inhibitor Nivolumab.

Therapeutic antibodies targeting the programmed cell death protein 1 (PD-1) pathway function as immune checkpoint inhibitors, allowing the immune system to recognize tumors which otherwise escape immune surveillance. However, these agents can also elicit an autoimmune response by inhibiting the ability of non-neoplastic tissues and regulatory cells to suppress the immune system. Here we present a fatal case of active myocarditis in a 55-year-old man with non-small-cell lung cancer which occurred following monotherapy with the PD-1 inhibitor nivolumab (Opdivo). He presented with acute right-sided heart failure and died 1 day after admission. Postmortem examination revealed multiple gelatinous lesions in the myocardium of the interventricular septum and the bilateral atria and ventricles which had microscopic features diagnostic of myocarditis. Subsequent studies failed to identify an infectious cause. Immune checkpoint inhibitors are an increasingly common addition to anticancer regimens and they should be considered in the evaluation of acute myocarditis.

FNA smears of pancreatic ductal adenocarcinoma are superior to formalin-fixed paraffin-embedded tissue as a source of DNA: Comparison of targeted KRAS amplification and genotyping in matched preresection and postresection samples.

BACKGROUND: The current study was conducted to compare DNA yield, including normalization to nuclear area, DNA amplification functionality, and detection of KRAS mutations between matched fine-needle aspiration (FNA) specimens and pancreatic resections diagnostic of pancreatic ductal adenocarcinoma. METHODS: A retrospective sample of 30 matched single FNA smears and macrodissected formalin-fixed, paraffin-embedded (FFPE) curls (2 5-µm curls) were compared by measuring the following: nuclear area (via digital image analysis), DNA yield (via NanoDrop spectrophotometry and Quantus fluorometry), and polymerase chain reaction threshold cycles for KRAS amplifications. Variants in KRAS codons 12/13 and 61 were detected by fluorescent melt curve analyses, followed by Sanger DNA sequencing. RESULTS: Despite a similar nuclear area, FNA smears yielded greater DNA per nuclear area via 2 DNA quantification methods. KRAS codon 12 mutations were detected in 23 of 30 FNA specimens (77%) compared with 17 of 30 matched FFPE specimens (57%), for a concordance rate of 74%. No KRAS codon 13 or 61 mutations were detected. CONCLUSIONS: FNA specimens are a more optimal source of DNA, and represent an important resource in the preresection and postresection molecular analysis of pancreatic ductal adenocarcinoma. Cancer Cytopathol 2017;125:838-47. © 2017 American Cancer Society.

KRAS and GNAS Co-Mutation in Metastatic Low-Grade Appendiceal Mucinous Neoplasm (LAMN) to the Ovaries: A Practical Role for Next-Generation Sequencing.

BACKGROUND Low-grade appendiceal mucinous neoplasms (LAMNs) are cytologically low-grade tumors of the appendix and are a frequent cause of pseudomyxoma peritonei. They can become a diagnostic challenge when they metastasize to the ovaries, where they may mimic primary ovarian mucinous tumors. CASE REPORT We report the case of a patient with very large bilateral ovarian mucinous tumors and a concurrent minute LAMN incidentally discovered in a grossly normal appendix. A primary ovarian tumor was suspected, but histological analysis of the ovaries suggested an appendiceal origin. Immunohistochemical studies were not informative and a consensus regarding the source of the ovarian tumors could not be reached within our department. Subsequent next-generation sequencing of tumors from the right ovary, left ovary, appendix, and matched normal tissue demonstrated identical somatic point mutations in KRAS and GNAS present in all tumors. The patient was diagnosed with metastatic LAMN and did not receive further treatment. She remains disease-free after 15 months of close observation. CONCLUSIONS Determining the tissue of origin in low-grade mucinous tumors of the ovaries can be challenging when a concurrent LAMN is identified in the appendix. In cases where histology and immunohistochemistry are insufficient to render a diagnosis, the presence of concurrent KRAS and GNAS mutations in both tumors strongly favors a diagnosis of metastatic LAMN. We emphasize the utility of targeted next-generation sequencing to establish tissue of origin in challenging cases when LAMN is suspected as the source of mucinous ovarian tumors.

Implementation and Clinical Utility of an Integrated Academic-Community Regional Molecular Tumor Board.

PURPOSE: Precision oncology develops and implements evidence-based personalized therapies that are based on specific genetic targets within each tumor. However, a major challenge that remains is the provision of a standardized, up-to-date, and evidenced-based precision medicine initiative across a geographic region. MATERIALS AND METHODS: We developed a statewide molecular tumor board that integrates academic and community oncology practices. The Precision Medicine Molecular Tumor Board (PMMTB) has three components: a biweekly Web-based teleconference tumor board meeting provided as a free clinical service, an observational research registry, and a monthly journal club to establish and revise evidence-based guidelines for off-label therapies. The PMMTB allows for flexible and rapid implementation of treatment, uniformity in practice, and the ability to track outcomes. RESULTS: We describe the implementation of the PMMTB and its first year of activity. Seventy-seven patient cases were presented, 48 were enrolled in a registry, and 38 had recommendations and clinical follow-up. The 38 subjects had diverse solid tumors (lung, 45%; GI, 21%; breast, 13%; other, 21%). Of these subjects, targeted therapy was recommended for 32 (84%). Clinical trials were identified for 24 subjects (63%), and nontrial targeted medicines for 16 (42%). Nine subjects (28%) received recommended therapy with a response rate of 17% (one of six) and a clinical benefit rate (partial response + stable disease) of 38% (three of eight). Although clinical trials often were identified, patients rarely enrolled. CONCLUSION: The PMMTB provides a model for a regional molecular tumor board with clinical utility. This work highlights the need for outcome registries and improved access to clinical trials to pragmatically implement precision oncology.

INSM1: A Novel Immunohistochemical and Molecular Marker for Neuroendocrine and Neuroepithelial Neoplasms.

OBJECTIVES: Neuroendocrine neoplasms (NENs) are heterogeneous neoplasms, which are sometimes malignant, although predicting metastasis is difficult. INSM1 is a transcription factor expressed transiently in embryonic neuroendocrine (NE) tissue, thought to coordinate termination of cell division with differentiation of NE and neuroepithelial cells. In adult tissues, INSM1 has been identified in multiple tumors of NE or neuroepithelial origin but has not been thoroughly investigated as a potential neoplastic marker. METHODS: We evaluated INSM1 as a semiquantitative immunohistochemical (IHC) marker for NE and neuroepithelial neoplasms and as a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) marker for gastrointestinal NENs (GI-NENs). RESULTS: Using IHC, we found in normal adult tissue that INSM1 expression was highly restricted to nuclei of NE cells and tissues. INSM1 was not detected in any adult nonneoplastic, non-NE tissue. In neoplastic tissue, INSM1 was detectable by IHC in 88.3% of 129 NEN specimens. In contrast, INSM1 was detected by IHC in only one of 27 neoplasms without a neuroepithelial or NE component. Using qRT-PCR, we evaluated INSM1 gene expression in 113 GI-NEN specimens. CONCLUSIONS: INSM1 expression was significantly increased in neoplastic vs nonneoplastic tissue. Furthermore, among midgut GI-NENs, neoplasms with known metastases showed significantly higher expression than those that had not yet metastasized.

High-risk human papillomavirus DNA detected in primary squamous cell carcinoma of urinary bladder.

CONTEXT: We reported previously that more than one-third (37%) of primary bladder squamous cell carcinomas (SCCs) demonstrate diffuse p16 immunoreactivity independent of gender. This observation made us question whether p16 overexpression in bladder carcinoma is due to human papillomavirus (HPV)-dependent mechanisms. OBJECTIVES: To determine whether the presence of high-risk HPV (HR-HPV) DNA could be detected in these tumor cells. DESIGN: Fourteen cases of primary bladder SCC, which were positive for p16 by immunohistochemistry, were probed for the detection of HR-HPV by in situ hybridization and the signal amplification Invader assay. Samples positive for detection of HR-HPV by Invader assay were amplified by using HR-HPV type-specific primers, and amplification products were DNA sequenced. RESULTS: Detection of HR-HPV by the in situ hybridization method was negative in all cases (0 of 14). However, in 3 of 14 cases (21.4%), the presence of HR-HPV DNA was detected with the Cervista HPV HR Invader assay, which was followed by identification of genotype. All positive cases were confirmed by using HR-HPV type-specific amplification followed by DNA sequencing. Identified HR-HPV genotypes included HPV 16 (2 cases) and HPV 35 (1 case). CONCLUSIONS: High-risk HPV DNA is detectable in a subset of primary bladder SCCs. Based on the well-documented carcinogenic potential of HR-HPV, there is a necessity for additional studies to investigate the role of HR-HPV in bladder carcinogenesis.

Structural profiles of TP53 gene mutations predict clinical outcome in diffuse large B-cell lymphoma: an international collaborative study.

The purpose of this study is to correlate the presence of TP53 gene mutations with the clinical outcome of a cohort of patients with diffuse large B-cell lymphoma (DLBCL) assembled from 12 medical centers. TP53 mutations were identified in 102 of 477 patients, and the overall survival (OS) of patients with TP53 mutations was significantly worse than those with wild-type TP53 (P < .001). However, subsets of TP53 mutations were found to have different effects on OS. Mutations in the TP53 DNA-binding domains were the strongest predictors of poor OS (P < .001). Mutations in the Loop-Sheet-Helix and Loop-L3 were associated with significantly decreased OS (P = .002), but OS was not significantly affected by mutations in Loop-L2. A subset of missense mutations (His158, His175, Ser245, Gln248, His273, Arg280, and Arg282) in the DNA-binding domains had the worst prognosis. Multivariate analysis confirmed that the International Prognostic Index and mutations in the DNA-binding domains were independent predictors of OS. TP53 mutations also stratified patients with germinal center B cell-like DLBCL, but not nongerminal center B cell-like DLBCL, into molecularly distinct subsets with different survivals. This study shows the prognostic importance of mutations in the TP53 DNA-binding domains in patients with DLBCL.