BACKGROUND: Human papillomavirus has been implicated in the carcinogenesis of squamous cell carcinoma of the anal canal. p16 expression and the presence of human papillomavirus DNA have been used to define human papillomavirus-positive patients, but neither approach has been validated against the standard of human papillomavirus E6/7 mRNA expression at this disease site. OBJECTIVE: This study aimed to evaluate the acceptability of p16 immunohistochemistry as a surrogate to E6/7 mRNA expression in identifying human papillomavirus-mediated squamous cell carcinoma of the anal canal. DESIGN: This was a retrospective analysis of a previously constructed tissue microarray. SETTINGS: This study was conducted at a tertiary academic center. PATIENTS: Biopsies and resection specimens from patients diagnosed with squamous cell carcinoma of the anal canal at the study institution from 2005 to 2015 were reviewed for sample adequacy. MAIN OUTCOME MEASURES: Concordance between p16 positivity by immunohistochemistry and E6/7 mRNA expression by in situ hybridization was evaluated. Sensitivity, specificity, and positive predictive value were assessed. RESULTS: Among the 25 patients evaluated, p16 and E6/7 mRNA results were concordant in 24 of 25 specimens (96%). Of the 24 concordant samples, there were 23 true positives (p16+ and E6/7+) and 1 true negative (p16– and E6/7–). One specimen was discordant (p16– and E6/7+) between p16 and E6/7 mRNA (4%). This resulted in a sensitivity of 96% and a specificity of 100%. Positive predictive value of p16 immunohistochemistry for E6/7 mRNA expression was 100%. LIMITATIONS: This study was limited by its retrospective nature and small sample size. It only assessed diagnostic parameters rather than prognostic implications. CONCLUSIONS: In this study, the clinically prevalent method of p16 immunohistochemistry showed excellent concordance with the standard of E6/7 mRNA expression and demonstrated its potential to serve as a surrogate for identifying human papillomavirus-induced squamous cell carcinoma of the anal canal. See Video Abstract at http://links.lww.com/DCR/B448. EVALUANDO LA CONFIABILIDAD Y EL VALOR PREDICTIVO POSITIVO DE P16, COMO SUSTITUTO DE LA EXPRESIÓN DE ARNM DE E6 / 7, MEDIADA POR EL VIRUS DEL PAPILOMA HUMANO, EN CARCINOMA DE CÉLULAS ESCAMOSAS DEL CANAL ANAL ANTECEDENTES: El virus del papiloma humano se ha relacionado en la carcinogénesis del carcinoma de células escamosas del canal anal. La expresión de p16 y la presencia de ADN del virus del papiloma humano, se han utilizado para definir a los pacientes positivos al virus del papiloma humano. Pero ninguno de estos enfoques, han sido validados frente al estándar de oro de la expresión del ARNm del virus del papiloma humano E6 / 7, en este sitio de la enfermedad. OBJETIVO: El estudio tuvo como objetivo, evaluar la aceptabilidad de la inmunohistoquímica del p16, como sustituto de la expresión de ARNm de E6 / 7, en la identificación del carcinoma de células escamosas del canal anal, mediada por virus del papiloma humano. DISEÑO: Fue un análisis retrospectivo de un microarreglo de tejido previamente construido. AJUSTE: El estudio se realizó en un centro académico terciario. PACIENTES: Se revisaron biopsias y muestras de resección de pacientes diagnosticados con carcinoma de células escamosas del canal anal, en la institución del estudio, entre 2005 y 2015 para determinar la idoneidad de la muestra. PRINCIPALES MEDIDAS DE RESULTADO: Se evaluó la concordancia entre la positividad de p16 por inmunohistoquímica y la expresión de ARNm de E6 / 7 por hibridación in situ. Se evaluaron la sensibilidad, especificidad y valor predictivo positivo. RESULTADOS: Entre los 25 pacientes evaluados, los resultados del ARNm de p16 y E6 / 7 fueron concordantes en 24/25 muestras (96%). De las 24 muestras concordantes, hubo 23 positivos verdaderos (p16 + y E6 / 7 +) y un negativo verdadero (p16- y E6 / 7-). Una muestra fue discordante (p16- y E6 / 7 +) entre p16 y ARNm de E6 / 7 (4%). Esto resultó en una sensibilidad del 96% y una especificidad del 100%. El valor predictivo positivo de la inmunohistoquímica de p16 para la expresión de ARNm de E6 / 7 fue del 100%. LIMITACIONES: El estudio estuvo limitado por su naturaleza retrospectiva y por el tamaño pequeño de la muestra. Solamente evaluó los parámetros de diagnóstico, en lugar de las implicaciones pronosticas. CONCLUSIONES: En este estudio, el método clínico prevalente de inmunohistoquímica p16, mostró una excelente concordancia con el estándar de oro de la expresión de ARNm de E6 / 7 y demostró su potencial para servir, como sustituto para identificar el carcinoma de células escamosas del canal anal, inducido por el virus del papiloma humano. Consulte Video Resumen en http://links.lww.com/DCR/B448.
Background: Invasive mucinous adenocarcinoma of the lung (IMA) is pathologically characterized as lung tumor cells with goblet cell morphology containing abundant intracytoplasmic mucin. IMA has been reported to account for 2-10% of lung adenocarcinomas. Genetically, the majority of IMA harbor KRAS mutations. Recently, by analyzing RNA-seq data using human IMA cases, we reported mucus-related genes specifically expressed in IMA, which includes MUC5AC, MUC5B, MUC3, SPDEF, FOXA3 and HNF4 (Guo, Tomoshige et al., 2017). Notably, immunohistochemical analyses have determined that NKX2-1 (also known as TTF-1) is reduced in IMA (Travis et al., 2011); however, other than NKX2-1, genes reduced in IMA compared to normal lungs have not been determined. Methods: In order to further understand the demographic occurrence of IMA, we surveyed the clinical records at the University of Cincinnati College of Medicine in the U.S. and Nagasaki University of School of Medicine in Japan. Genes reduced in IMA compared to normal lungs were identified using the RNA-seq datasets as we have previously reported (Guo, Tomoshige et al., 2017). Using single-cell and sorted RNA-seq datasets (LGEA; Du et al., 2017), genes were further categorized into groups that define different cell types that exist in normal lungs. Results: IMA occurred in 4.6% (3/65 cases in 2017) and 1.9% (2/105 cases in 2017) of lung adenocarcinomas at the University of Cincinnati College of Medicine and Nagasaki University of School of Medicine, respectively. Importantly, analysis using normal lung cell type specification markers obtained from the single-cell and sorted RNA-seq datasets identified that genes that define alveolar type II epithelial cells (e.g., SFTPC and ABCA3) and endothelial cells (e.g., FOXF1 and SOX17) were significantly reduced in IMA compared to normal lungs. Conclusion: IMA comprises mucus-producing goblet cell lineage populations devoid of alveolar type II epithelial and endothelial cell-lineage populations, suggesting a unique tumor microenvironment distinct from other lung adenocarcinomas. Citation Format: Koichi Tomoshige, Minzhe Guo, Tomoshi Tsuchiya, Iris Fink-Baldauf, Takeshi Nagayasu, Kelsey Dillehay McKillip, Yutaka Maeda. Loss of alveolar type II epithelial and endothelial cell lineages in invasive mucinous adenocarcinoma of the lung (IMA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3675.
Quick and reliable identification of actionable genomic alterations is essential to providing high‐quality cancer care. Herein we describe our assessment of Idylla™, a fully automated, cartridge‐based, PCR system for the qualitative detection of actionable genomic alterations in formalin‐fixed, paraffin‐embedded (FFPE) tissue. Three different Idylla™ assays were evaluated to demonstrate the system's ease of use, concordance with established methodology, and overall clinical utility.Thirty‐six patient samples, previously characterized using a clinically validated method, were assessed for EGFR mutations, BRAF mutations, or microsatellite instability (MSI) using Idylla™. The Idylla™ MSI Mutation Assay, which detects mutations in seven novel, tumor specific MSI loci (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, and SULF2), was used to assess microsatellite stability in twelve cases of colorectal cancer. The Idylla™ EFGR Mutation Assay, which detects 51 mutations in exons 18, 19, 20, and 21 of the EGFR gene, was used to identify mutations in twelve cases of lung cancer. Lastly, the Idylla™ BRAF Mutation Assay, which detects seven mutations in codon 600 of the BRAF oncogene, was used to detect mutations in twelve cases of melanoma.Briefly, one 10μm FFPE tissue section was loaded into the cartridge per the manufacturer's instructions. No manual macrodissection was performed. A representative H&E stained slide was retrospectively reviewed and scored for neoplastic cell content by a Pathologist (K.H.). Idylla™ assay results were compared to previously reported results. Three cases, one per assay, were excluded from the final analysis due to lack of neoplastic cell content (NT = no tumor) or assay incompatibility.Overall, the adjusted concordance between the Idylla™ assays and clinically validated methodologies was high. The Idylla™ MSI Mutation Assay was 100% (11/11) concordant with the immunohistochemical assay for mismatch repair (MMR), which included markers MLH1, MSH2, MSH6, and PMS2. The Idylla™ EGFR and Idylla™ BRAF Mutation assays were 91% (10/11) concordant with Sanger sequencing results. Additional studies utilizing a third methodology are needed to further evaluate discordant results.The Idylla™ assays showed high concordance with previously reported results generated using clinically validated methods. Advantages of the Idylla™ system include minimal hands‐on time and rapid turnaround time. In conclusion, Idylla™ offers fast and reliable analysis for a variety of actionable genomic alterations in FFPE tissue.Support or Funding InformationSupport for this project was provided by the Department of Pathology and Laboratory Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The National Cancer Institute (NCI) recognizes that the lack of standardized, high‐quality biospecimen is a significant roadblock to cancer research. Biorepositories help overcome this roadblock by providing human biological samples in support of basic, translational, and clinical research. As custodians of these invaluable samples, biorepositories are responsible for assuring the quality and consistency of the biospecimen collected, stored, and dispensed for research purposes. The University of Cincinnati Biorepository (UCB) was established in 2010 with the goal of creating a centralized institutional biospecimen resource that provides investigators with a streamlined process for accessing high‐quality human biospecimens.The objective of this study was to develop and implement a procedure for assessing and monitoring the quality of biospecimens collected and stored in the UCB, specifically human tissue samples. Pre‐analytical factors such as cold ischemia, tissue processing procedures, and storage temperature and duration are known to affect tissue quality. Provided that low‐quality tissue can have negative impacts on research, it is imperative that we routinely assess the quality of tissue samples collected for research purposes. This is especially true of tissue samples that predate the establishment of the UCB, for which there is little information available regarding the conditions under which these tissues were collected and stored. Furthermore, tissue quality data is also an important tool for validation of standard operating procedures for collection, processing, and storage of tissue samples.Due to the relative instability of RNA and its propensity for degradation, the quality of a tissue sample can be determined by assessing its RNA integrity. Therefore, the UCB implemented a quality assurance program in which approximately 10% of banked biospecimens are randomly assessed for RNA quality on a quarterly basis. Tissue quality was assessed using frozen tissue samples embedded in optimum cutting temperature (OCT) compound. Frozen tissue blocks were sectioned at 10 μm to create tissue scrolls (average of 5 sections per scroll). Total RNA was extracted from the tissue scrolls using the Qiagen RNeasy Micro kit following the manufacturer's recommended protocol. RNA quality was determined using High Sensitivity RNA ScreenTapes on the Agilent 2200 TapeStation. The RNA Integrity Number (RIN) is representative of the quality of RNA and measured on a scaled of 1 to 10, where 1 represents completely degraded RNA and 10 indicates totally intact RNA. Generally, RNA with a RIN ≥7 is considered high‐quality and suitable for most downstream applications.Data collected as part of our quality assurance program demonstrates that on average, cold ischemia up to 2 hours does not negatively affect tissue quality and that tissue samples are generally stable when stored at −80° over several years. In conclusion, the assessment of RNA quality is a useful tool for monitoring the quality of tissue samples collected and stored in a biorepository.Support or Funding InformationSupport for this project was provided by the Department of Pathology and Laboratory Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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