Increased frequency of CHD1 deletions in prostate cancers of African American men is associated with rapid disease progression without inducing homologous recombination deficiency.

We analyzed genomic data derived from the prostate cancer of African and European American men in order to identify differences that may contribute to racial disparity of outcome and that could also define novel therapeutic strategies. In addition to analyzing patient derived next generation sequencing data, we performed FISH based confirmatory studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CRISPR edited, CHD1 deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. We found that subclonal deletion of CHD1 is nearly three times as frequent in prostate tumors of African American men than in men of European ancestry and it associates with rapid disease progression. We further showed that CHD1 deletion is not associated with homologous recombination deficiency associated mutational signatures in prostate cancer. In prostate cancer cell line models CHD1 deletion did not induce HR deficiency as detected by RAD51 foci formation assay or mutational signatures, which was consistent with the moderate increase of olaparib sensitivity. CHD1 deficient prostate cancer cells, however, showed higher sensitivity to talazoparib. CHD1 loss may contribute to worse outcome of prostate cancer in African American men. A deeper understanding of the interaction between CHD1 loss and PARP inhibitor sensitivity will be needed to determine the optimal use of targeted agents such as talazoparib in the context of castration resistant prostate cancer.

SPOT/Dx Pilot Reanalysis and College of American Pathologists Proficiency Testing for KRAS and NRAS Demonstrate Excellent Laboratory Performance.

CONTEXT.­: The Sustainable Predictive Oncology Therapeutics and Diagnostics quality assurance pilot study (SPOT/Dx pilot) on molecular oncology next-generation sequencing (NGS) reportedly demonstrated performance limitations of NGS laboratory-developed tests, including discrepancies with a US Food and Drug Administration-approved companion diagnostic. The SPOT/Dx pilot methods differ from those used in proficiency testing (PT) programs. OBJECTIVE.­: To reanalyze SPOT/Dx pilot data using PT program methods and compare to PT program data.Also see p. 136. DESIGN.­: The College of American Pathologists (CAP) Molecular Oncology Committee reanalyzed SPOT/Dx pilot data applying PT program methods, adjusting for confounding conditions, and compared them to CAP NGS PT program performance (2019-2022). RESULTS.­: Overall detection rates of KRAS and NRAS single-nucleotide variants (SNVs) and multinucleotide variants (MNVs) by SPOT/Dx pilot laboratories were 96.8% (716 of 740) and 81.1% (129 of 159), respectively. In CAP PT programs, the overall detection rates for the same SNVs and MNVs were 97.2% (2671 of 2748) and 91.8% (1853 of 2019), respectively. In 2022, the overall detection rate for 5 KRAS and NRAS MNVs in CAP PT programs was 97.3% (1161 of 1193). CONCLUSIONS.­: CAP PT program data demonstrate that laboratories consistently have high detection rates for KRAS and NRAS variants. The SPOT/Dx pilot has multiple design and analytic differences with established PT programs. Reanalyzed pilot data that adjust for confounding conditions demonstrate that laboratories proficiently detect SNVs and less successfully detect rare to never-observed MNVs. The SPOT/Dx pilot results are not generalizable to all molecular oncology testing and should not be used to market products or change policy affecting all molecular oncology testing.

RNA Sequencing for Solid Tumor Fusion Gene Detection: Proficiency Testing Practice and Performance Comparison.

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.

Cancers of Unknown Primary: A Descriptive Study in the U.S. Military Health System.

INTRODUCTION: Cancers of unknown primary (CUP) are defined as histologically confirmed metastatic cancers that do not have an identified primary site of origin despite an appropriate diagnostic workup. Although accessibility to and quality of medical care influence diagnosis of cancer including CUP, previous studies describing CUP have generally been conducted in patients with various accessibilities to care. This study aimed to describe the demographic, histologic, and temporal trend characteristics of CUP patients in the DoD Cancer Registry of the Military Health System (MHS), which provides universal health care access, reducing the potential effects of accessibility to care on research results. MATERIALS AND METHODS: The data were obtained from the DoD's Automated Central Tumor Registry (ACTUR), which collects cancer data from beneficiaries who were diagnosed or received treatment in the MHS. We described the demographic and histologic distributions in CUP patients aged 18 years or older diagnosed from 1987 to 2013. We calculated the proportion of CUP patients among all metastatic cancers and the most common histologic categories of those tumors. We then evaluated whether the proportion of histologic types changed over time. RESULTS: CUP comprised 13.3% of all metastatic cancers in ACTUR during the study period. The majority of CUP within ACTUR was moderately and well-differentiated adenocarcinoma (51.3%) and poorly differentiated carcinomas (23.2%) followed by squamous cell carcinomas (12.5%). The percentages of CUP among metastasized cancers of the same histologic category ranged 12%-15% for moderately and well-differentiated adenocarcinomas, squamous cell, and poorly differentiated carcinomas, and 41%-46% for malignant neuroendocrine carcinomas and undifferentiated neoplasms. However, the percentages varied by sex, race, and age for certain pathologies. The proportion of CUP patients among all metastatic cancer patients has steadily declined from 22.4% to 8.3% from 1987 to 2013. CONCLUSION: The proportion and trends of CUP in the ACTUR were generally consistent with other descriptive CUP studies. This study provides a description of CUP in a health care system with universal access in the USA and provides a foundation for future studies on CUP.

Clinical Testing for Tumor Cell-Free DNA: College of American Pathologists Proficiency Programs Reveal Practice Trends.

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.

Current Laboratory Testing Practices for Assessment of ERBB2/HER2 in Endometrial Serous Carcinoma and Colorectal Carcinoma.

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.

Cancers of unknown primary: Survival by histologic type, demographic features, and treatment in the U.S. Military Health System.

BACKGROUND: Cancers of unknown primary (CUP), a group of heterogenous metastatic cancers lacking a known primary site, have poor prognosis. This study compared survival of CUP by histologic type, patient characteristics, and treatment in the U.S. Military Health System (MHS), which provides universal care to its members. METHODS: Patients histologically diagnosed with CUP were identified from the U.S. Department of Defense (DoD)'s Automated Central Tumor Registry. Median survival with 95 % confidence intervals was calculated for demographic and treatment variables by histologic type. A multivariable accelerated failure time model estimated time ratios and 95 % confidence intervals. RESULTS: The study included 3358 CUP patients. The most prevalent CUP in this study was well- and moderately-differentiated adenocarcinomas. Median survival varied by histologic type with squamous cell carcinoma having the longest at 25.1 months and poorly-differentiated carcinomas having the shortest at 3.0 months. For each histologic type, survival was generally similar by sex and active-duty status although women with well- and moderately-differentiated adenocarcinoma had longer survival than their male counterparts. Younger patients tended to have longer survival than those aged 65 years or older. Generally, there were no racial differences in survival except poorer survival for Black patients than White patients in the group of other histologic types. Patients with chemotherapy and radiation treatment generally had improved survival whereas patients with squamous cell carcinoma who received chemotherapy had shorter survival than those without. CONCLUSION: Survival generally did not differ between racial groups, which may be related to equal healthcare access despite racial background. Further studies are warranted to better understand how survival in the MHS compares with that in the general U.S.

Clinical Laboratory Testing Practices in Diffuse Gliomas Prior to Publication of 2021 World Health Organization Classification of Central Nervous System Tumors.

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.

Most Frequently Cited Accreditation Inspection Deficiencies for Clinical Molecular Oncology Testing Laboratories and Opportunities for Improvement.

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.

Tiered Somatic Variant Classification Adoption Has Increased Worldwide With Some Practice Differences Based on Location and Institutional Setting.

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.

Neoplastic Cellularity Assessment in Molecular Testing.

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.

Next-Generation Sequencing Somatic and Germline Assay Troubleshooting Guide Derived From Proficiency Testing Data.

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.

Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next Generation Sequencing-Based Hematologic Malignancy Assays With Survey Material-Specific Differences in Variant Frequencies.

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.

Comparative Performance of High-Risk Human Papillomavirus RNA and DNA In Situ Hybridization on College of American Pathologists Proficiency Tests.

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.

Performance Comparison of Different Analytic Methods in Proficiency Testing for Mutations in the BRAF, EGFR, and KRAS Genes: A Study of the College of American Pathologists Molecular Oncology Committee.

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.

Next-Generation Sequencing (NGS) Methods Show Superior or Equivalent Performance to Non-NGS Methods on BRAF, EGFR, and KRAS Proficiency Testing Samples.

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.

Proficiency Testing of Standardized Samples Shows Very High Interlaboratory Agreement for Clinical Next-Generation Sequencing-Based Oncology Assays.

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.

Comparative Performance of Breast Cancer Human Epidermal Growth Factor Receptor 2 Fluorescence In Situ Hybridization and Brightfield In Situ Hybridization on College of American Pathologists Proficiency Tests.

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.

Predicting Prostate Cancer Progression as a Function of ETS-related Gene Status, Race, and Obesity in a Longitudinal Patient Cohort.

BACKGROUND: ETS-related gene (ERG) oncogenic activation is the most common genomic alteration in prostate cancer (CaP) although it occurs less frequently in African American (AA) versus Caucasian (CA) patients, and the potential role of ERG as a prognostic marker has not been confirmed. OBJECTIVE: This study was conducted to confirm strong racial variation in the prevalence of ERG oncoprotein expression and to examine ERG oncoprotein expression, race, and body mass index as independent and joint predictors of CaP biochemical recurrence (BCR) following radical prostatectomy (RP). DESIGN, SETTING, AND PARTICIPANTS: A retrospective cohort study of CA and AA CaP patients enrolled at Walter Reed National Military Medical Center, who donated clinically annotated, whole-mounted, prostatectomy specimens between 1994 and 2014 following RP, was conducted. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Kaplan-Meier (KM) estimation curves and multivariable Cox proportional hazards models were used to examine time to BCR as a function of ERG status, patient race, and obesity. RESULTS AND LIMITATIONS: Among 930 eligible patients (36.1% AA and 63.9% CA), with 155 (16.7%) BCR events and a median follow-up time of 5.1 yr, ERG oncoprotein expression was significantly less prevalent in index tumors of AA versus CA patients (23.2% vs 49.3%; p<0.0001). KM curves showed significantly poorer BCR-free survival for CA patients with ERG-negative index tumors but not for AA patients. Race-stratified multivariable analyses revealed a significant association between ERG-negative index tumors and poorer BCR-free survival among CA patients (hazards ratio=1.67, confidence interval=1.07, 2.61; p=0.024). Less heterogeneity in ERG expression among AA patients may reduce the ability to show its association with BCR. CONCLUSIONS: Striking racial variation in ERG oncoprotein expression was confirmed. A novel observation was the importance of index tumor ERG-negative status in predicting CaP progression for CA patients. PATIENT SUMMARY: ETS-related gene (ERG) typing of tumors may be useful in prognosticating prostate cancer aggressiveness.