Background: Telomerase reverse transcriptase (TERT) activation has been shown to be an important cancer hallmark; the activation and expression of TERT has been documented in >90% of tumors and TERT activation has been touted as a prognostic marker in many cancers. However, there is currently no simple testing modality to detect TERT mRNA expression in surgical pathology specimens. In this study we aim to evaluate and validate the utility and reliability of the TERT RNAscope (R) in-situ hybridization (ISH) assay for the detection of TERT mRNA expression in formalin-fixed, paraffin embedded tissue. Methods and Materials: RNAscope (R) detection for TERT was performed on a Leica Biosystems BOND III research staining robot using the Hs-TERT-O1 (ACD, 481968) probe. Twenty three samples containing 48 tissue types were assessed. TERT genomic alterations were determined by targeted next generation sequencing (NGS), while TERT mRNA expression was determined by both targeted RNA-sequencing and TERT RNAscope (R) and the results compared. Manual vs automated TERT expression quantification methodologies were evaluated for the ISH assay. The expression levels in normal vs. neoplastic tissues were also compared. Results: The RNAscope (R) assay showed high TERT expression in neoplastic tissues, while most normal tissues have no or very low expression levels (p-value= 0.0001, AUC: 0.99). In addition, there was good correlation of TERT expression between the RNAscope (R) assay and RNA-sequencing. For RNAscope (R) quantification, manual calculation of TERT signal/cell ratio based on a count of 100 cells was superior compared to automated signal detection. Conclusion: TERT RNAscope (R) assay is a simple and reliable tool for the evaluation of TERT mRNA expression. TERT signal/cell ratio based on a count of 100 cells is a reproducible and accurate interpretation approach for evaluation of TERT expression.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

Context.-Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription-polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). Objective.-To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID19) patients. Design.-Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. Results.-Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (kappa = 0.23 and kappa = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (kappa = 0.75). Interobserver reliability was almost perfect for IHC (kappa = 0.91) and fair to moderate for RNAish (kappa = 0.380-0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P =.03, Pearson correlation = -0.65) and were more likely to be positive by RNAish (P =.02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. Conclusions.-Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

The relationship of SARS-CoV-2 lung infection and severity of pulmonary disease is not fully understood. We analyzed autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter- and intra- patient heterogeneity of pulmonary virus infection. There was a spectrum of high and low virus cases that was associated with duration of disease and activation of interferon pathway genes. Using a digital spatial profiling platform, the virus corresponded to distinct spatial expression of interferon response genes and immune checkpoint genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

Objectives Human kidney injury molecule 1 (hKIM-1) is a sensitive and specific marker for detection of clear cell renal cell carcinoma (CRCC), papillary renal cell carcinoma (PRCC), and ovarian clear cell carcinoma (OCCC). Its use was limited to a few surgical pathology laboratories because this specific antibody to hKIM-1 was not commercially available. We investigated the diagnostic utility of RNA in situ hybridization/RNAscope in the detection of hKIM-1 in tumors from various organs. Methods RNAscope for hKIM-1 was performed on 1,252 cases on tissue microarray sections, including CRCC (n = 185), PRCC (n = 59), chromophobe renal cell carcinoma (n = 18), oncocytoma (n = 12), OCCC (n = 27), and metastatic CRCC (n = 46). Results Fifty-nine (100%) of 59 PRCCs, 94 (95%) of 99 low-grade CRCCs, 83 (96%) of 86 high-grade CRCCs, and 24 (89%) of 27 OCCCs, and 44 (96%) of 46 metastatic CRCCs were positive for hKIM-1. In contrast, hKIM-1 expression was not seen in normal renal tubules or in most nonrenal tumors. Low-level expression could be seen in a small percentage of urothelial, hepatocellular, and colon carcinomas. Conclusions hKIM-1 is a sensitive and relatively specific marker (1) for diagnosing PRCC, CRCC, and OCCC when working on a tumor of unknown origin and (2) for differentiating CRCC from chromophobe renal cell carcinoma and oncocytoma.

Abstract Anaplastic lymphoma kinase (ALK) rearrangements are involved in ~4-7% of non-small cell lung cancers (NSCLCs). About 80% of ALK rearrangements generate a fusion gene between EML4 and ALK, resulting in overexpression of the fusion protein and constitutive kinase activity. Due to the sensitivity of tumors harboring ALK rearrangements to crizotinib, the identification of these rearrangements is clinically important. In this study, we developed an RNAscope® RNA ISH assay to specifically detect ALK rearrangement leading to upregulation of the 3' region of the ALK gene encoding the kinase domain in FFPE tissues from NSCLC. Two ALK-specific probes targeting ALK exons 1-18 (Hs-ALK E1-E18) and exons 19-29 (Hs-ALK E19-29) were tested in seven NSCLC FFPE samples. In three FISH-confirmed ALK rearrangement positive NSCLC cases, no signal was detected with the Hs-ALK E1-E18 probe, as expected. However, the Hs-ALK E19-29 probe showed positive signals (score 1 or 2) in all three FISH-confirmed cases of NSCLC. Among four ALK rearrangement negative samples (FISH and IHC confirmed), two samples exhibited no positivity with either probe. The other two cases showed low positivity with both probes in parts of the tumors, presumably representing baseline ALK mRNA expression (see table). Our results demonstrate that RNAscope® is a reliable method for the detection of ALK rearrangements that lead to the upregulation of the exons encoding the ALK kinase domain. The RNAscope® assay is both sensitive and specific for the detection of ALK rearrangement in FFPE tissues. Furthermore, the method provides information on the spatial distribution and morphologic context of cells associated with ALK rearrangements in NSCLC. RNAscope HD 2.5 Red assay result scoreRNAscope HD 2.5 Red assay result scoreRNAscope HD 2.5 Red assay result scoreRNAscope HD 2.5 Red assay result scoresample labelALK-FISHIHC ALK-D5F3probe-PPIB (positive control)probe-DapB (negative control)probe-Hs-ALK E1-E18Probe-Hs-ALK E19-29Pathologic DiagnosisAgeGender (M/F)1+N/A2001 (50%)Highly-moderately differentiated invasive mucinous lung adenocarcinoma77F2+N/A2002 (>90%)Moderately-poorly differentiated lung adenocarcinoma32M3+N/A3002 (>90%)Highly-moderately differentiated lung adenocarcinoma, partly mucinous adenocarcinoma49M4--3000Lepidic predominant lung adenocarcinoma58F5--4000Lepidic predominant lung adenocarcinoma63M6--400 (90%), 2(10%)0 (90%), 2(10%)Invasive lung adenocarcinoma (papillary and alveolar predominant, partly micropapillary)48M7--300 (98%), 2(2%)0 (98%), 2(2%)Invasive lung adenocarcinoma (alveolar and lepidic predominant)52M Citation Format: Na Li, Shafei Wu, Mindy Wang, Hongzhe Sun, Zhifu Zhang, Emily Park, Xiao-Jun Ma, Xuan Zeng, Robert Monroe. RNA in situ detection and characterization of ALK rearrangement in NSCLC FFPE tissues [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 5388.

ObjectivesInconsistent data on detection of albumin expression by ribonucleic acid (RNA) in situ hybridization have been reported. We investigated the utility of RNAscope (Advanced Cell Diagnostics, Hayward, CA) in detection of albumin in hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (ICCs), and carcinomas from various organs using manual and automated staining.MethodsRNAscope for albumin detection was performed on 482 cases on tissue microarray sections and on 22 cases of ICC, including 14 surgical resection and eight core biopsy specimens.ResultsThirty-six of 37 (97%) HCCs had detectable mRNA, whereas all non-HCC and non-ICC cases, except one lung adenocarcinoma, were negative for albumin. Fourteen of 22 ICCs (64%) were positive for albumin.ConclusionsRNAscope for albumin is highly sensitive and specific for identifying HCCs and is highly specific and moderately sensitive for detection of ICCs; however, rare carcinomas (non-HCC, non-ICC, and those with no hepatoid histomorphology) can also have aberrant expression of albumin.

IDH1 and IDH2 mutations have been shown to be early events in gliomagenesis. IDH1 mutations are present in ~33% of gliomas and constitute ~90% of all IDH mutations. Approximately 88% of IDH1 mutations involve the substitution c.395G>A leading to the R132H amino acid change. IDH1 mutations, including R132H, are important to recognize clinically as they are more frequent in younger patients and confer a survival advantage. Patients with IDH1 mutations show lower expression of the prognostic markers p53 and Ki-67 and achieve greater benefit from maximal tumor resection. In addition to their prognostic utility, IDH mutations including IDH1 R132H can be useful as diagnostic markers for low-grade gliomas. In difficult to diagnose glial lesions, the presence of IDH mutations strongly favors a diagnosis of glioma versus reactive gliosis. Although PCR and DNA sequencing-based approaches can detect the IDH1 R132H mutation, these techniques require destruction of tissue for nucleic acid isolation and destroy the morphologic context, highlighting the need for a methodology that can be performed on formalin-fixed, paraffin-embedded (FFPE) tissues. In this study, we developed an assay to specifically detect the IDH1 R132H mutation in FFPE samples in expressed IDH1 mRNA transcripts using the novel BasescopeTM technology. IDH1 wild-type (WT) and IDH1 mutation (MT) probes were tested in 6 astrocytoma and 3 glioma FFPE specimens from unique clinical cases. All samples exhibited positivity with the IDH1 WT probe. Two of six astrocytomas and one of three gliomas showed positivity with the IDH1 R132H mutation probe. Among the three positive samples, one astrocytoma exhibited positive signals for the IDH1 R132H mutation in almost all tumor cells, whereas one astrocytoma and one glioma showed only scattered positive signals with the IDH1 R132H mutation probe in a subset of tumor cells. Our findings indicate that the BasescopeTM assay is a novel RNA in situ hybridization assay to visualize point mutations in a highly specific and sensitive manner within the morphologic tissue context. In this study, we demonstrate that the BasescopeTM IDH1 R132H assay is capable of detecting the IDH1 R132H mutation in routine FFPE clinical specimens for the purpose of assisting in the diagnosis of gliomas and providing prognostic information. The BasescopeTM technology also allows for correlation of the frequency and distribution of IDH1 R132H mutations in glial tumors with patient outcomes and overall survival. Citation Format: Na Li, Mindy Wang, Hongzhe Sun, Zhifu Zhang, Xin Wang, Emily Park, Xiao-Jun Ma, Robert Monroe. Detection of IDH1 R132H mutation in situ in human astrocytoma and glioma FFPE samples [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 3333.

The current standard practice of lung lesion diagnosis often leads to inconclusive results, requiring additional diagnostic follow up procedures that are invasive and often unnecessary due to the high benign rate in such lesions (Chest 143:e78S-e92, 2013). The Percepta bronchial genomic classifier was developed and clinically validated to provide more accurate classification of lung nodules and lesions that are inconclusive by bronchoscopy, using bronchial brushing specimens (N Engl J Med 373:243–51, 2015, BMC Med Genomics 8:18, 2015). The analytical performance of the Percepta test is reported here.

The promise of personalized medicine will require rigorously validated molecular diagnostics developed on minimally invasive, clinically relevant samples. Measurement of DNA mutations is increasingly common in clinical settings but only higher-prevalence mutations are cost-effective. Patients with rare variants are at best ignored or, at worst, misdiagnosed. Mutations result in downstream impacts on transcription, offering the possibility of broader diagnosis for patients with rare variants causing similar downstream changes. Use of such signatures in clinical settings is rare as these algorithms are difficult to validate for commercial use. Validation on a test set (against a clinical gold standard) is necessary but not sufficient: accuracy must be maintained amidst interfering substances, across reagent lots and across operators. Here we report the development, clinical validation, and diagnostic accuracy of a pre-operative molecular test (Afirma BRAF) to identify BRAF V600E mutations using mRNA expression in thyroid fine needle aspirate biopsies (FNABs). FNABs were obtained prospectively from 716 nodules and more than 3,000 features measured using microarrays. BRAF V600E labels for training (n=181) and independent test (n=535) sets were established using a sensitive quantitative PCR (qPCR) assay. The resulting 128-gene linear support vector machine was compared to qPCR in the independent test set. Clinical sensitivity and specificity for malignancy were evaluated in a subset of test set samples (n=213) with expert-derived histopathology. We observed high positive- (PPA, 90.4%) and negative (NPA, 99.0%) percent agreement with qPCR on the test set. Clinical sensitivity for malignancy was 43.8% (consistent with published prevalence of BRAF V600E in this neoplasm) and specificity was 100%, identical to qPCR on the same samples. Classification was accurate in up to 60% blood. A double-mutant still resulting in the V600E amino acid change was negative by qPCR but correctly positive by Afirma BRAF. Non-diagnostic rates were lower (7.6%) for Afirma BRAF than for qPCR (24.5%), a further advantage of using RNA in small sample biopsies. Afirma BRAF accurately determined the presence or absence of the BRAF V600E DNA mutation in FNABs, a collection method directly relevant to solid tumor assessment, with performance equal to that of an established, highly sensitive DNA-based assay and with a lower non-diagnostic rate. This is the first such test in thyroid cancer to undergo sufficient analytical and clinical validation for real-world use in a personalized medicine context to frame individual patient risk and inform surgical choice.