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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.

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.

Context.— Programmed death ligand-1 (PD-L1) immunohistochemistry companion diagnostic assays play a crucial role as predictive markers in patients being considered for immune checkpoint inhibitor therapy. However, because of a convergence of several factors, including recognition of increased types of cancers susceptible to immunotherapy, increasing numbers of immune checkpoint inhibitors, and release of multiple PD-L1 immunohistochemistry antibodies with differing reporting systems, this complex testing environment has led to significant levels of confusion for pathologists and medical oncologists. Objective.— To identify which processes and procedures have contributed to the current challenges surrounding programmed death receptor-1 (PD-1)/PD-L1 companion diagnostics and to propose potential remedies to this issue. This is based upon input from key industrial stakeholders in conjunction with the College of American Pathologists Personalized Health Care Committee. Design.— A meeting of representatives of pharmaceutical and in vitro diagnostic companies along with the Personalized Health Care Committee reviewed the process of release of the PD-L1 companion diagnostic assays using a modified root cause analysis format. The modified root cause analysis envisioned an ideal circumstance of development and implementation of a companion diagnostic to identify shortcomings in the rollout of the PD-L1 assay and to suggest actions to improve future companion diagnostic assay releases. Results.— The group recommended improvements to key principles in companion diagnostics implementation related to multi-stakeholder communication, increased regulatory flexibility to incorporate postapproval medical knowledge, improved cross-disciplinary information exchange between medical oncology and pathology societies, and enhanced postmarket training programs. Conclusions.— The rapidly changing nature of and increasing complexity associated with companion diagnostics require a fundamental review of processes related to their design, implementation, and oversight.

Context.— The 2017 Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists (CAP) tier classification guideline provides a framework to standardize interpretation and reporting of somatic variants. Objective.— To evaluate the adoption and performance of the 2017 guideline among laboratories performing somatic next-generation sequencing (NGS). Design.— A survey was distributed to laboratories participating in NGS CAP proficiency testing for solid tumors (NGSST) and hematologic malignancies (NGSHM). Results.— Worldwide, 64.4% (152 of 236) of NGSST and 66.4% (87 of 131) of NGSHM participants used tier classification systems, of which the 2017 guideline was used by 84.9% (129 of 152) of NGSST and 73.6% (64 of 87) of NGSHM participants. The 2017 guideline was modified by 24.4% (30 of 123) of NGSST and 21.7% (13 of 60) of NGSHM laboratories. Laboratories implementing the 2017 guideline were satisfied or very satisfied (74.2% [89 of 120] NGSST and 69.5% [41 of 59] NGSHM), and the impression of tier classification reproducibility was high (mean of 3.9 [NGSST] and 3.6 [NGSHM] on a 5-point scale). Of nonusers, 35.2% (38 of 108) of NGSST and 39.4% (26 of 66) of NGSHM laboratories were planning implementation. For future guideline revisions, respondents favored including variants to monitor disease (63.9% [78 of 122] NGSST, 80.0% [48 of 60] NGSHM) and germline variants (55.3% [63 of 114] NGSST, 75.0% [45 of 60] NGSHM). Additional subtiers were not favored by academic laboratories compared to nonacademic laboratories (P < .001 NGSST and P = .02 NGSHM). Conclusions.— The 2017 guideline has been implemented by more than 50.0% of CAP laboratories. While most laboratories using the 2017 guideline report satisfaction, thoughtful guideline modifications may further enhance the quality, reproducibility, and clinical utility of the 2017 guideline for tiered somatic variant classification.

Context.— Therapy targeted at 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) 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 criteria for evaluating HER2 were the American Society of Clinical Oncology/CAP 2018 guidelines for breast carcinoma (69.0%; 194 of 281), whereas only 16.0% (45) used guidelines specific to ESC. For CRC, 20.2% (239 of 1185) 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 from the HERACLES trial, and 16.3% (38) used the American Society for Clinical Oncology/CAP 2018 guidelines 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.

PURPOSE Immune checkpoint inhibition (ICI) therapy represents one of the great advances in the field of oncology, highlighted by the Nobel Prize in 2018. Multiple predictive biomarkers for ICI benefit have been proposed. These include assessment of programmed death ligand-1 expression by immunohistochemistry, and determination of mutational genotype (microsatellite instability or mismatch repair deficiency or tumor mutational burden) as a reflection of neoantigen expression. However, deployment of these assays has been challenging for oncologists and pathologists alike. METHODS To address these issues, ASCO and the College of American Pathologists convened a virtual Predictive Factor Summit from September 14 to 15, 2021. Representatives from the academic community, US Food and Drug Administration, Centers for Medicare and Medicaid Services, National Institutes of Health, health insurance organizations, pharmaceutical companies, in vitro diagnostics manufacturers, and patient advocate organizations presented state-of-the-art predictive factors for ICI, associated problems, and possible solutions. RESULTS The Summit provided an overview of the challenges and opportunities for improvement in assay execution, interpretation, and clinical applications of programmed death ligand-1, microsatellite instability-high or mismatch repair deficient, and tumor mutational burden-high for ICI therapies, as well as issues related to regulation, reimbursement, and next-generation ICI biomarker development. CONCLUSION The Summit concluded with a plan to generate a joint ASCO/College of American Pathologists strategy for consideration of future research in each of these areas to improve tumor biomarker tests for ICI therapy.

Context.— Next-generation sequencing–based assays are increasingly used in clinical molecular laboratories to detect somatic variants in solid tumors and hematologic malignancies and to detect constitutional variants. Proficiency testing data are potential sources of information about challenges in performing these assays. Objective.— To examine the most common sources of unacceptable results from the College of American Pathologists Next-Generation Sequencing Bioinformatics, Hematological Malignancies, Solid Tumor, and Germline surveys and provide recommendations on how to avoid these pitfalls and improve performance. Design.— The College of American Pathologists next-generation sequencing somatic and germline proficiency testing survey results from 2016 to 2019 were analyzed to identify the most common causes of unacceptable results. Results.— On somatic and germline proficiency testing surveys, 95.9% (18 815/19 623) and 97.8% (33 890/34 641) of all variants were correctly identified, respectively. The most common causes of unacceptable results related to sequencing were false-negative errors in genomic regions that were difficult to sequence because of high GC content. False-positive errors occurred in the context of homopolymers and pseudogenes. Recurrent errors in variant annotation were seen for dinucleotide and duplication variants and included unacceptable transcript selection and outdated variant nomenclature. A small percentage of preanalytic or postanalytic errors were attributed to specimen swaps and transcription errors. Conclusions.— Laboratories demonstrate overall excellent performance for detecting variants in both somatic and germline proficiency testing surveys. Proficiency testing survey results highlight infrequent, but recurrent, analytic and nonanalytic challenges in performing next- generation sequencing–based assays and point to remedies to help laboratories improve performance.

Context.— Neurotrophic receptor tyrosine kinase (NTRK) fusion testing has both diagnostic and therapeutic implications for patient care. With 2 tumor-agnostic US Food and Drug Administration–approved tropomyosin receptor kinase (TRK) inhibitors, testing is increasingly used for therapeutic decision making. However, the testing landscape for NTRK fusions is complex and optimal testing depends on the clinicopathologic scenario. Objective.— To compare different NTRK testing methods to help pathologists understand test features and performance characteristics and make appropriate selections for NTRK fusion detection for their laboratory and individual patient specimens. Data Sources.— A literature search for NTRK gene fusions and TRK protein was performed, including papers that discussed treatment, testing methodology, and detection or prevalence of fusion-positive cases. Conclusions.— As standard of care in some tumor types, next-generation sequencing (NGS) panel testing is a cost effective and reliable way to detect a broad range of NTRK fusions. The design of the panel and use of DNA or RNA will affect performance characteristics. Pan-TRK immunohistochemistry may be used as a rapid, less expensive screen in cases that will not undergo routine NGS testing, or on specimens unsuitable for NGS testing. Fluorescence in situ hybridization may be appropriate for low-tumor-content specimens that are unsuitable for NGS testing. Quantitative reverse transcription polymerase chain reaction is best suited for monitoring low-level disease of a specific, previously identified target. This information should help laboratories develop a laboratory-specific NTRK testing algorithm that best suits their practice setting and patients' needs.

Context.— In 2016, the College of American Pathologists (CAP) launched the first next-generation sequencing (NGS) in silico bioinformatics proficiency testing survey to evaluate the performance of clinical laboratory bioinformatics pipelines for the detection of oncology-associated variants at varying allele fractions. This survey focused on 2 commonly used oncology panels, the Illumina TruSeq Amplicon Cancer Panel and the Thermo Fisher Ion AmpliSeq Cancer Hotspot v2 Panel. Objective.— To review the analytical performance of laboratories participating in the CAP NGS bioinformatics (NGSB) surveys, comprising NGSB1 for Illumina users and NGSB2 for Thermo Fisher Ion Torrent users, between 2016 and 2019. Design.— Responses from 78 laboratories were analyzed for accuracy and associated performance characteristics. Results.— The analytical sensitivity was 90.0% (1901 of 2112) for laboratories using the Illumina platform and 94.8% (2153 of 2272) for Thermo Fisher Ion Torrent users. Variant type and variant allele fraction were significantly associated with performance. False-negative results were seen mostly for multi-nucleotide variants and variants engineered at variant allele fractions of less than 25%. Analytical specificity for all participating laboratories was 99.8% (9303 of 9320). There was no statistically significant association between deletion-insertion length and detection rate. Conclusions.— These results demonstrated high analytical sensitivity and specificity, supporting the feasibility and utility of using in silico mutagenized NGS data sets as a supplemental challenge to CAP surveys for oncology-associated variants based on physical samples. This program demonstrates the opportunity and challenges that can guide future surveys inclusive of customized in silico programs.

Context.— The College of American Pathologists (CAP), a laboratory accreditation organization with deemed status under the Clinical Laboratories Improvement Amendments of 1988 administers accreditation checklists. Checklists are used by laboratories to ensure regulatory compliance. Peer-level laboratory professionals audit laboratory records during inspections to assess compliance. Objective.— To identify the most frequently cited deficiencies for molecular oncology laboratories undergoing CAP accreditation inspections and describe laboratory improvement opportunities. Design.— The CAP Molecular Oncology Committee (MOC), which is involved in maintaining the Molecular Pathology checklist, reviewed data and inspector comments associated with the most frequently observed citations related to molecular oncology testing from laboratories inspected by the CAP during a 2-year period (2018–2020). Results.— Of 422 molecular oncology laboratories that underwent accreditation inspections, 159 (37.7%) were not cited for any molecular oncology–related deficiencies. For the All Common (COM) and Molecular Pathology checklists, there were 364 and 305 deficiencies, corresponding to compliance rates of 98.8% and 99.6%, respectively. The most frequently cited deficiencies are described. The COM checklist deficiencies were associated most often with the analytic testing phase; the MOL checklist deficiencies were more evenly distributed across the preanalytic, analytic, and postanalytic phases of testing. Conclusions.— Molecular oncology laboratories demonstrated excellent compliance with practices that support high-quality results for patients and the health care providers who use those test results in patient management. This review includes a critical assessment of opportunities for laboratories to improve compliance and molecular oncology testing quality.

Context.— Neoplastic cellularity assessment has become an essential component of molecular oncology testing; however, there are currently no best practice recommendations or guidelines for this potentially variable step in the testing process. Objective.— To describe the domestic and international practices of neoplastic cellularity assessment and to determine how variations in laboratory practices affect neoplastic cellularity assessment accuracy. Design.— Data were derived from 57 US and international laboratories that participated in the 2019 College of American Pathologists Neoplastic Cellularity Proficiency Testing Survey (NEO-B 2019). NEO-B 2019 included 29 laboratory practice questions and 5 images exhibiting challenging histologic features. Participants assessed the neoplastic cellularity of hematoxylin-eosin–stained digital images, and results were compared to a criterion standard derived from a manual cell count. Results.— The survey responses showed variations in the laboratory practices for the assessment of neoplastic cellularity, including the definition of neoplastic cellularity, assessment methodology, counting practices, and quality assurance practices. In some instances, variation in laboratory practice affected neoplastic cellularity assessment performance. Conclusions.— The results highlight the need for a consensus definition and improved standardization of the assessment of neoplastic cellularity. We put forth an initial set of best practice recommendations to begin the process of standardizing neoplastic cellularity assessment.

Context.—The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories (“home brews”), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee.Objective.—To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes: BRAF, EGFR, and KRAS.Design.—There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences for BRAF, EGFR, and KRAS. Performance was analyzed by test method, kit manufacturer, variants tested, and preanalytic and postanalytic practices.Results.—Of 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses for BRAF; 848 [97.5%] acceptable of 870 total responses for EGFR; 942 [97.0%] acceptable of 971 total responses for KRAS). Next-generation sequencing platforms (summed across all analytes and 2 platforms) demonstrated 99.4% accuracy for these analytes (165 [99.4%] acceptable of 166 total next-generation sequencing responses). Slight differences in performance were noted among select commercial assays, dependent upon the particular design and specificity of the assay. Wide differences were noted in the lower limits of neoplastic cellularity laboratories accepted for testing.Conclusions.—These data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

Context.— There has been a rapid expansion of next-generation sequencing (NGS)–based assays for the detection of somatic variants in solid tumors. However, limited data are available regarding the comparative performance of NGS and non-NGS assays using standardized samples across a large number of laboratories. Objective.— To compare the performance of NGS and non-NGS assays using well-characterized proficiency testing samples provided by the College of American Pathologists (CAP) Molecular Oncology Committee. A secondary goal was to compare the use of preanalytic and postanalytic practices. Design.— A total of 17 343 responses were obtained from participants in the BRAF, EGFR, KRAS, and the Multigene Tumor Panel surveys across 84 different proficiency testing samples interrogating 16 variants and 3 wild-type sequences. Performance and preanalytic/postanalytic practices were analyzed by method. Results.— While both NGS and non-NGS achieved an acceptable response rate of greater than 95%, the overall performance of NGS methods was significantly better than that of non-NGS methods for the identification of variants in BRAF (overall 97.8% versus 95.6% acceptable responses, P = .001) and EGFR (overall 98.5% versus 97.3%, P = .01) and was similar for KRAS (overall 98.8% and 97.6%, P = .10). There were specific variant differences, but in all discrepant cases, NGS methods outperformed non-NGS methods. NGS laboratories also more consistently used preanalytic and postanalytic practices suggested by the CAP checklist requirements than non-NGS laboratories. Conclusions.— The overall analytic performance of both methods was excellent. For specific BRAF and EGFR variants, NGS outperformed non-NGS methods and NGS laboratories report superior adherence to suggested laboratory practices.

Context.— With the increasing integration of molecular alterations into the evaluation of hematologic malignancies (HM), somatic mutation profiling by next-generation sequencing (NGS) has become a common clinical testing strategy. Limited data are available about the characteristics of these assays. Objective.— To describe assay characteristics, specimen requirements, and reporting practices for NGS-based HM testing using College of American Pathologists proficiency testing survey data. Design.— The College of American Pathologists NGS Hematologic Malignancies Survey (NGSHM) results from 78 laboratories were used to determine laboratory practices in NGS-based HM testing. Results.— The majority of laboratories performed tumor-only (88.5% [69 of 78]), targeted sequencing of cancer genes or mutation hotspots (98.7% [77 of 78]); greater than 90% performed testing on fresh bone marrow and peripheral blood. The majority of laboratories reported a 5% lower limit of detection for single-nucleotide variants (73.1% [57 of 78]) and small insertions and deletions (50.6% [39 of 77]). A majority of laboratories used benchtop sequencers and custom enrichment approaches. Conclusions.— This manuscript summarizes the characteristics of clinical NGS-based testing for the detection of somatic variants in HM. These data may be broadly useful to inform laboratory practice and quality management systems, regulation, and oversight of NGS testing, and precision medicine efforts using a data-driven approach.

Context.— As laboratories increasingly turn from single-analyte testing in hematologic malignancies to next-generation sequencing–based panel testing, there is a corresponding need for proficiency testing to ensure adequate performance of these next-generation sequencing assays for optimal patient care. Objective.— To report the performance of laboratories on proficiency testing from the first 4 College of American Pathologists Next-Generation Sequencing Hematologic Malignancy surveys. Design.— College of American Pathologists proficiency testing results for 36 different engineered variants and/or allele fractions as well as a sample with no pathogenic variants were analyzed for accuracy and associated assay performance characteristics. Results.— The overall sensitivity observed for all variants was 93.5% (2190 of 2341) with 99.8% specificity (22 800 of 22 840). The false-negative rate was 6.5% (151 of 2341), and the largest single cause of these errors was difficulty in identifying variants in the sequence of CEBPA that is rich in cytosines and guanines. False-positive results (0.18%; 40 of 22 840) were most likely the result of preanalytic or postanalytic errors. Interestingly, the variant allele fractions were almost uniformly lower than the engineered fraction (as measured by digital polymerase chain reaction). Extensive troubleshooting identified a multifactorial cause for the low variant allele fractions, a result of an interaction between the linearized nature of the plasmid and the Illumina TruSeq chemistry. Conclusions.— Laboratories demonstrated an overall accuracy of 99.2% (24 990 of 25 181) with 99.8% specificity and 93.5% sensitivity when examining 36 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 10% or greater. The data also highlight an issue with artificial linearized plasmids as survey material for next-generation sequencing.

Context.— Next-generation sequencing–based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. Objective.— To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. Design.— CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. Results.— The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. Conclusions.— Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.

Context.— Detection of acquired variants in cancer is a paradigm of precision medicine, yet little has been reported about clinical laboratory practices across a broad range of laboratories. Objective.— To use College of American Pathologists proficiency testing survey results to report on the results from surveys on next-generation sequencing–based oncology testing practices. Design.— College of American Pathologists proficiency testing survey results from more than 250 laboratories currently performing molecular oncology testing were used to determine laboratory trends in next-generation sequencing–based oncology testing. Results.— These presented data provide key information about the number of laboratories that currently offer or are planning to offer next-generation sequencing–based oncology testing. Furthermore, we present data from 60 laboratories performing next-generation sequencing–based oncology testing regarding specimen requirements and assay characteristics. The findings indicate that most laboratories are performing tumor-only targeted sequencing to detect single-nucleotide variants and small insertions and deletions, using desktop sequencers and predesigned commercial kits. Despite these trends, a diversity of approaches to testing exists. Conclusions.— This information should be useful to further inform a variety of topics, including national discussions involving clinical laboratory quality systems, regulation and oversight of next-generation sequencing–based oncology testing, and precision oncology efforts in a data-driven manner.

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 HR-HPV 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.

The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories ("home brews"), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee.To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes:There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences forOf 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses forThese data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

There has been a rapid expansion of next-generation sequencing (NGS)-based assays for the detection of somatic variants in solid tumors. However, limited data are available regarding the comparative performance of NGS and non-NGS assays using standardized samples across a large number of laboratories.To compare the performance of NGS and non-NGS assays using well-characterized proficiency testing samples provided by the College of American Pathologists (CAP) Molecular Oncology Committee. A secondary goal was to compare the use of preanalytic and postanalytic practices.A total of 17 343 responses were obtained from participants in theWhile both NGS and non-NGS achieved an acceptable response rate of greater than 95%, the overall performance of NGS methods was significantly better than that of non-NGS methods for the identification of variants inThe overall analytic performance of both methods was excellent. For specific

e13681 Background: Cell-free tumor DNA or circulating tumor DNA tests are increasingly used in clinical care to detect somatic mutations from solid tumors. However, data on laboratory performance characteristics using standardized samples is limited. Methods: Well-characterized reference materials were used for the College of American Pathologists (CAP) cell-free tumor DNA proficiency testing surveys, which consisted of stabilized DNA fragmented to simulate cell-free DNA in a synthetic plasma matrix. For the 2018A, 2018B, 2019A and 2019B surveys, laboratories tested for hotspot mutations (single and dinucleotide sequence changes) in EGFR, BRAF, KRAS, NRAS, and IDH1 at variant allele fractions ranging from 0.1% - 1.0%. As per CAP proficiency testing standards, results were scored according to the known mutation(s) engineered at designated variant allele fractions in each PT sample. Nine laboratories were excluded from analysis because they provided incomplete results. Statistical significance was calculated using a multivariate logistic regression model. Results: In 2018 and 2019, 101 laboratories submitted survey results for at least one proficiency testing mailing. There were 5088 total proficiency testing responses for EGFR, BRAF, KRAS, NRAS, and IDH1 mutations across 12 different samples. For the 3585 responses submitted for BRAF, KRAS, NRAS, and IDH1, sensitivity ranged from 94.6 – 100%, while specificity exceeded 99%. There were no significant differences in performance between analytical methodologies for BRAF, KRAS, NRAS, and IDH1 mutations. Performance characteristics for EGFR mutations among the 1503 responses showed a combined sensitivity of 87.1% and specificity of 98.7%. For laboratories detecting mutations in EGFR, next-generation sequencing methods exhibited a sensitivity (true positivity) of 95.7% while the sensitivity of non-NGS methods was lower at 81.7% ( P= 0.02). Conclusions: These findings demonstrate high sensitivity and specificity for clinical laboratories performing cell-free tumor DNA tests. For EGFR mutations, NGS outperformed non-NGS methods. These data suggest excellent overall agreement among laboratories performing clinical cell-free tumor DNA tests. Further investigation across variant allele fractions and additional variant types is warranted.

Comprehensive next-generation sequencing (NGS) tests are increasingly used as first-line tests in the evaluation of patients with suspected heritable disease. Despite major technical simplifications, these assays still pose significant challenges for molecular testing laboratories. Existing professional guidelines and recommendations provide a framework for laboratories implementing such tests, but in-depth, concrete guidance is generally not provided. Consequently, there is variability in how laboratories interpret and subsequently implement these regulatory frameworks. To address the need for more detailed guidance, the College of American Pathologists with representation from the Association for Molecular Pathologists assembled a working group to create a practical resource for clinical laboratories. This initial work is focused on variant detection in the setting of inherited disease and provides structured worksheets that guide the user through the entire life cycle of an NGS test, including design, optimization, validation, and quality management with additional guidance for clinical bioinformatics. This resource is designed to be a living document that is publicly available and will be updated with user and expert feedback as the wet bench and bioinformatic landscapes continue to evolve. It is intended to facilitate the standardization of NGS testing across laboratories and therefore to improve patient care.

Context.—Fluorescence in situ hybridization (FISH) and brightfield in situ hybridization (ISH) are 2 clinically approved laboratory methods for detecting ERBB2 (HER2) amplification in breast cancer.Objective.—To compare the performance of FISH and brightfield ISH on proficiency testing administered by the College of American Pathologists Laboratory Accreditation Program.Design.—Retrospective review was performed on 70 tissue core samples in 7 separate proficiency testing surveys conducted between 2009 and 2013.Results.—The samples included 13 consensus-amplified tissue cores, 53 consensus-nonamplified cores, and 4 cores that did not reach consensus for FISH and/or brightfield ISH. There were 2552 individual responses for FISH and 1871 individual responses for brightfield ISH. Consensus response rates were comparable for FISH (2474 of 2524; 98.0%) and brightfield ISH (2135 of 2189; 97.5%). The FISH analysis yielded an average HER2 copy number per cell that was significantly higher (by 2.86; P = .02) compared with brightfield ISH for amplified cores. For nonamplified cores, FISH yielded slightly, but not significantly, higher (by 0.17; P = .10) HER2 copy numbers per cell. There was no significant difference in the average HER2 to control ratio for either consensus-amplified or consensus-nonamplified cores. Participants reported “unable to analyze” more frequently for brightfield ISH (244 of 2453; 9.9%) than they did for FISH (160 of 2684; 6.0%).Conclusions.—Our study indicates a high concordance rate in proficiency testing surveys, with some significant differences noted in the technical performance of these assays. In borderline cases, updated American Society of Clinical Oncology/College of American Pathologists cutoff thresholds that place greater emphasis on HER2 copy number per cell could accentuate those differences between FISH and brightfield ISH.

Purpose.—Clinical use of analytical tests to assess genomic variants in circulating tumor DNA (ctDNA) is increasing. This joint review from the American Society of Clinical Oncology and the College of American Pathologists summarizes current information about clinical ctDNA assays and provides a framework for future research.Methods.—An Expert Panel conducted a literature review on the use of ctDNA assays for solid tumors, including preanalytical variables, analytical validity, interpretation and reporting, and clinical validity and utility.Results.—The literature search identified 1338 references. Of those, 390, plus 31 references supplied by the Expert Panel, were selected for full-text review. There were 77 articles selected for inclusion.Conclusions.—The evidence indicates that testing for ctDNA is optimally performed on plasma collected in cell stabilization or EDTA tubes, with EDTA tubes processed within 6 hours of collection. Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens, and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity or clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial. Given the rapid pace of research, reevaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.

Importance The debate about the role of the Food and Drug Administration (FDA) in the regulation of laboratory-developed tests (LDTs) has focused attention on the analytical performance of all clinical laboratory testing. This study provides data comparing the performance of LDTs and FDA-approved companion diagnostics (FDA-CDs) in proficiency testing (PT) provided by the College of American Pathologists Molecular Oncology Committee. Objective To compare the analytical performance of LDTs and FDA-CDs on well-characterized PT samples and to compare the practice characteristics of laboratories using these assays. Design, Setting, and Participants This comparison of PT responses examines the performance of laboratories participating in the College of American Pathologists PT for 3 oncology analytes for which both FDA-CDs and LDTs are used: BRAF , EGFR , and KRAS . A total of 6897 PT responses were included: BRAF (n = 2524; 14 PT samples), EGFR (n = 2216; 11 PT samples), and KRAS (n = 2157, 10 PT samples). US Food and Drug Administration companion diagnostics and LDTs are compared for both accuracy and preanalytic practices of the laboratories. Main Outcomes and Measures As per the College of American Pathologists PT standards, results were scored and the percentages of acceptable responses for each analyte were compared. These were also broken down by the specific variants tested, by kit manufacturer for laboratories using commercial reagents, and by preanalytic practices. Results From analysis of 6897 PT responses, this study demonstrates that both LDTs and FDA-CDs have excellent performance overall, with both test types exceeding 97% accuracy for all 3 genes ( BRAF , EGFR , and KRAS ) combined. Rare variant-specific differences did not consistently favor LDTs or FDA-CDs. Additionally, more than 60% of participants using an FDA-CD reported adapting their assay from the approved procedure to allow for a greater breadth of sample types, minimum tumor content, and instrumentation, changing the classification of their assay from FDA-CD to LDT. Conclusions This study demonstrates the high degree of accuracy and comparable performance of both LDTs and FDA-CDs for 3 oncology analytes. More significantly, the majority of laboratories using FDA-CDs have modified the scope of their assay to allow for more clinical practice variety, rendering them LDTs. These findings support both the excellent and equivalent performance of both LDTs and FDA-CDs in clinical diagnostic testing.

Context.— Recurrent epidermal growth factor receptor (EGFR) mutations are seen in a subset of pulmonary adenocarcinomas. These mutations are targeted by EGFR inhibitors and are a biomarker for response to EGFR inhibitor therapies. Initial data have indicated an increased frequency of activating EGFR mutations in nonsmoking Asian females. However, there are very few studies of global scope that address the question of mutation distribution across the population of lung cancer. Objective.— To determine the frequency of EGFR mutations in exons 18 through 21 detected in clinical laboratories participating in the College of American Pathologists proficiency testing program for EGFR in calendar year 2013. Design.— We reviewed the surveys from 170 clinical laboratories from 20 countries that participated in the College of American Pathologists EGFR proficiency testing program. The proficiency testing includes questions regarding the total numbers of tests performed at each common mutation site, including both activating and resistance mutations, and their frequency. Countries were grouped into regional groups in order to assess frequency of mutation by type, and to indirectly assess ethnic differences in mutation frequencies. Results.— Among the treatment-sensitive activating mutations, the most common are exon 19 mutations (n = 10 802 of 136 533 cases; 7.9% of total cases tested) and the exon 21 L858R mutation (n = 10 351 of 136 533 cases; 7.6% of total cases tested) and the least common are exon 20 mutations (n = 466 of 136 533 cases; 0.3% of total cases tested). The T790M mutation in exon 20 is the more common resistance mutation (n = 1010 of 136 533 cases; 0.7% of all cases tested). The highest activating mutation frequency is seen in southern Asia (n = 4260 of 9337 cases; 46%) and the lowest activating mutation frequencies are in South and North America (n = 113 of 1439 cases and 7926 of 86 654 cases; 8% and 9%, respectively). Conclusions.— Our data confirm that activating EGFR mutations are more common in southern Asia and that the distribution of activating EGFR mutations varies significantly across the regions. Similarly, the frequency and distribution of resistance mutations also show significant variation when comparing southern Asia with other regions.

Context.—With enormous growth in the field of molecular pathology, the reporting of results gleaned from this testing is essential to guide patient care.Objective.—To examine molecular reports from laboratories participating in proficiency testing for required elements to convey molecular laboratory test results to clinicians and patients.Design.—Molecular laboratories participating in the College of American Pathologists (CAP) proficiency testing program for BRAF mutation analysis were solicited to submit examples of final reports from 2 separate proficiency testing reporting cycles. Reports were reviewed for the presence or absence of relevant components.Results.—A total of 107 evaluable reports were received (57 demonstrating a positive result for the BRAF V600E mutation and 50 negative). Methods for BRAF testing varied, with 95% (102 of 107) of reports adequately describing their assay methods and 87% (93 of 107) of reports adequately describing the target(s) of their assays. Information on the analytic sensitivity of the assay was present in 74% (79 of 107) of reports and 83% (89 of 107) reported at least 1 assay limitation, though only 34% (36 of 107) reported on variants not detected by their assays. Analytic and clinical interpretive comments were included in 99% (106 of 107) and 90% (96 of 107) of reports, respectively. Of participants that perform a laboratory-developed test, 88% (88 of 100) included language addressing the development of the assay.Conclusions.—Laboratories participating in BRAF proficiency testing through the CAP are including most of the required reporting elements to unambiguously convey molecular results. Laboratories should continue to strive to report these results in a concise and comprehensive manner.

The College of American Pathologists surveys are the largest laboratory peer comparison programs in the world. These programs allow laboratories to regularly evaluate their performance and improve the accuracy of the patient test results they provide. Proficiency testing is offered twice a year to laboratories performing microsatellite instability testing. These surveys are designed to emulate clinical practice, and some surveys have more challenging cases to encourage the refinement of laboratory practices.This report summarizes the results and trends in microsatellite instability proficiency testing from participating laboratories from the inception of the program in 2005 through 2012.We compiled and analyzed data for 16 surveys of microsatellite instability proficiency testing during 2005 to 2012.The number of laboratories participating in the microsatellite instability survey has more than doubled from 42 to 104 during the 8 years analyzed. An average of 95.4% of the laboratories correctly classified each of the survey test samples from the 2005A through 2012B proficiency challenges. In the 2011B survey, a lower percentage of laboratories (78.4%) correctly classified the specimen, possibly because of overlooking subtle changes of microsatellite instability and/or failing to enrich the tumor content of the specimen to meet the limit of detection of their assay.In general, laboratories performed well in microsatellite instability testing. This testing will continue to be important in screening patients with colorectal and other cancers for Lynch syndrome and guiding the management of patients with sporadic colorectal cancer.

The fraction of malignant cells in tumor tissue submitted for tests of genetic alterations is a critical variable in testing accuracy. That fraction is currently determined by pathologist visual estimation of the percentage of malignant cells. Inaccuracy could lead to a false-negative test result.To describe a prospective, multi-institutional study to determine pathologist estimation accuracy.Ten ×20 magnification images of hematoxylin-eosin-stained colon tissue specimens were sent as an educational component of the College of American Pathologists KRAS-B 2011 Survey. Data from 194 labs were analyzed and compared to a criterion standard with comprehensive manual nuclear counts.Survey responses indicated low interlaboratory precision of pathologist estimation, but mean estimates were fairly accurate. A total of 5 of the 10 cases assessed showed more than 10% of respondents overestimating in a manner that could lead to false-negative test results.The significance of estimation errors resulting in molecular testing failures with implications for patient care is unknown, but the current study suggests false-negative test results may occur.