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* Context.--Next-generation sequencing-based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays. Objective.--To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B). Design.--CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices. Results.--Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results. Conclusions.--These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics., Fusion oncogenes represent valuable biomarkers that aid in diagnostic classification and prognostication, as well as serving as therapeutic targets. (1-4) Examples of structural alterations/mutations that may lead to chimeric fusions [...]

Context.--Therapy targeted at human epidermal growth factor receptor 2 (HER2; also known as ERBB2) was used initially for breast and gastroesophageal carcinoma and has more recently been adopted for endometrial serous carcinoma (ESC) and colorectal carcinoma (CRC). There is evidence that predictive biomarker testing algorithms for HER2 must be tumor type specific and that an algorithm validated for one tumor type cannot be applied to another. Objective.--To describe current laboratory practices for HER2 assessment in ESC and CRC. Design.--We surveyed laboratories participating in the 2021 College of American Pathologists (CAP) HER2 immunohistochemistry proficiency testing program. Results.--The survey was distributed to 1548 laboratories and returned by 1195, of which 83.5% (998) were in the United States. For ESC, 24.0% (287) of laboratories reported performing in-house testing for HER2 by immunohistochemical staining and/or in situ hybridization; of these, 44.3% (127) performed it reflexively on all cases of ESC. The most common criterion for evaluating HER2 was the American Society of Clinical Oncology/CAP 2018 guideline for breast carcinoma (69.0%; 194 of 281), whereas only 16.0% (45) of laboratories used guidelines specific to ESC. For CRC, 20.2% (239 of 1185) of laboratories performed in-house HER2 testing, and 82.0% of these (196) did the test only at the clinician's request. A plurality (49.4%; 115 of 233) used gastroesophageal cancer guidelines when scoring CRC, 30.0% (70) used the CRC scoring system from the HERACLES trial, and 16.3% (38) used the American Society of Clinical Oncology/CAP 2018 guideline for breast carcinoma. Conclusions.--Laboratories vary in their approach to HER2 testing in ESC and CRC. Most laboratories did not report using tumor type-specific recommendations for HER2 interpretation. The lack of standardization could present a challenge to evidence-based practice when considering targeted therapy for these diseases. doi: 10.5858/arpa.2022-0229-CP, As targeted therapy has become available for an increasing number of tumor types, it has become important to assess predictive biomarkers in the pathology laboratory. For example, the monoclonal antibody [...]

Context.—The diagnosis and clinical management of patients with diffuse gliomas (DGs) have evolved rapidly over the past decade with the emergence of molecular biomarkers that are used to classify, stratify risk, and predict treatment response for optimal clinical care.Objective.—To develop evidence-based recommendations for informing molecular biomarker testing for pediatric and adult patients with DGs and provide guidance for appropriate laboratory test and biomarker selection for optimal diagnosis, risk stratification, and prediction.Design.—The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. A systematic review of literature was conducted to address the overarching question, "What ancillary tests are needed to classify DGs and sufficiently inform the clinical management of patients?" Recommendations were derived from quality of evidence, open comment feedback, and expert panel consensus.Results.—Thirteen recommendations and 3 good practice statements were established to guide pathologists and treating physicians on the most appropriate methods and molecular biomarkers to include in laboratory testing to inform clinical management of patients with DGs.Conclusions.—Evidence-based incorporation of laboratory results from molecular biomarker testing into integrated diagnoses of DGs provides reproducible and clinically meaningful information for patient management.

* Context.--Integration of molecular data into glioma classification supports diagnostic, prognostic, and therapeutic decision-making; however, testing practices for these informative biomarkers in clinical laboratories remain unclear. Objective.--To examine the prevalence of molecular testing for clinically relevant biomarkers in adult and pediatric gliomas through review of a College of American Pathologists proficiency testing survey prior to the release of the 2021 World Health Organization Classification of Central Nervous System Tumors. Design.--College of American Pathologists proficiency testing 2020 survey results from 96 laboratories performing molecular testing for diffuse gliomas were used to determine the use of testing for molecular biomarkers in gliomas. Results.--The data provide perspective into the testing practices for diffuse gliomas from a broad group of clinical laboratories in 2020. More than 98% of participating laboratories perform testing for glioma biomarkers recognized as diagnostic for specific subtypes, including IDH. More than 60% of laboratories also use molecular markers to differentiate between astrocytic and oligodendroglial lineage tumors, with some laboratories providing more comprehensive analyses, including prognostic biomarkers, such as CDKN2A/B homozygous deletions. Almost all laboratories test for MGMT promoter methylation to identify patients with an increased likelihood of responding to temozolomide. Conclusions.--These findings highlight the state of molecular testing in 2020 for the diagnosis and classification of diffuse gliomas at large academic medical centers. The findings show that comprehensive molecular testing is not universal across clinical laboratories and highlight the gaps between laboratory practices in 2020 and the recommendations in the 2021 World Health Organization Classification of Central Nervous System Tumors. doi: 10.5858/arpa.2021-0431-CP, Historically, the classification of diffuse gliomas was based in large part on histologic features, including tumor cell morphology, mitotic index, and the presence of necrosis and/or microvascular proliferation. To delineate [...]

Context.--Clinical testing for tumor cell-free DNA (cfDNA) has evolved rapidly, but no practice guidelines exist. Objective.--To summarize cfDNA laboratory practices based on self-reporting and assess preanalytical, analytical, and postanalytical trends that may influence the quality, accuracy, and consistency of cfDNA testing. Design.--Data were derived from the College of American Pathologists cfDNA proficiency testing program submitted by 101 participating laboratories from 2018 to 2019. Results.--Most laboratories performing clinical circulating tumor DNA testing are commercial/nonhospital (71.2%; 72 of 101) and international (77.2%; 78 of 101) laboratories. Commercial laboratories had higher monthly test volumes than hospital-based laboratories (median, 36 versus 7-8) and tended to have larger gene panels (median, 50 versus 11 genes) when panel-based testing was offered. The main clinical indications include therapy selection and treatment/disease monitoring. Plasma is the most commonly accepted specimen, which is predominantly collected in cell-stabilizing tubes. Equal proportions of laboratories use next-generation sequencing (NGS) and non-NGS methods to assess key genes, including EGFR, BRAF, KRAS, NRAS, and IDH1. Most laboratories reported a lower limit of detection (LLOD) of 0.5%, variant allele frequency or less, which did not differ by method, NGS or non-NGS, except for EGFR. Sixty-five percent (17 of 26) of laboratories using the US Food and Drug Administration (FDA)-approved non-NGS EGFR assay report analytical sensitivities higher than 0.5%, as compared to 15% (16 of 104) of laboratories using an alternative NGS or non-NGS method. There is also a wider range in LLODs obtained for the FDA-approved EGFR assay than nonapproved assays. Conclusions.--These results highlight emerging practice trends and serve as a foundation to initiate future practice recommendations. doi: 10.5858/arpa.2021-0585-CP, Cell-free DNA (cfDNA) was first reported in whole blood in 1948, which consists of short fragments of circulating DNA that are approximately 150 to 200 base pairs in length. (1,2) [...]

Context.--Detection of high-risk human papillomavirus (HR-HPV) in squamous cell carcinoma is important for classification and prognostication. In situ hybridization (ISH) is a commonly used HR-HPV-specific test that targets viral RNA or DNA. The College of American Pathologists (CAP) provides proficiency testing for laboratories performing HR-HPV iSh. Objective.--To compare the analytical performance of RNA- and DNA-based ISH methods on CAP HR-HPV proficiency tests. Design.--Data from the 2016-2018 CAP HPV ISH proficiency testing surveys were reviewed. These surveys consist of well-characterized samples with known status for HR-HPV, including 1 to 2 copies, 50 to 100 copies, 300 to 500 copies, and no copies of HR-HPV per cell. Results.--Ninety-five participants submitted 1268 survey results from 20 cores. Overall, RNA ISH had a significantly higher percentage of correct responses than DNA ISH: 97.4% (450 of 462) versus 80.6% (650 of 806) (P < .001). This disparity appears to be the consequence of a superior sensitivity of rNa ISH compared to DNA ISH for samples with 1 to 2 and with 50 to 100 copies of HRHPV per cell: 95.2% (120 of 126) versus 53.8% (129 of 240), P < .001, respectively, and 100% (89 of 89) versus 76.3% (119 of 156), P < .001, respectively. Conclusions.--An assessment of CAP HR-HPV proficiency test performance indicates that RNA ISH shows significantly higher accuracy than DNA ISH owing to higher analytical sensitivity of RNA ISH in tumors with low (1-2 copies per cell) to intermediate (50-100 copies per cell) HR-HPV viral copy numbers. These data support the use of RNA over DNA ISH in clinical laboratories that perform HR-HPV testing as part of their testing algorithms. (Arch Pathol Lab Med. 2020;144:344-349; doi: 10.5858/arpa.2019-0093-CP), High-risk human papillomavirus (HR-HPV) causes HPV-positive squamous cell carcinomas (SCCs) at multiple anatomic sites including the oropharynx, anus, and urogenital tracts. The detection of HR-HPV in tissue samples containing SCC [...]