Continuous Global Improvement of Human Papillomavirus (HPV) Genotyping Services: The 2022 and 2023 HPV LabNet International Proficiency Studies.

The International Human Papillomavirus (HPV) Reference Center launches annual global HPV genotyping proficiency panels to enhance the precision and international standardization of HPV genotyping services. This study aims to assess the proficiency levels achieved in the global HPV genotyping proficiency panels conducted in 2022 and 2023, and to evaluate trends in performance over time. The proficiency panels comprised 44 blinded samples each, including 40 samples containing various purified plasmids corresponding to HPV types combined with human DNA, plus four control samples (one negative control and three extraction controls). Proficiency required a sensitivity of 50 International Units (IU)/5 µL for HPV 16 and HPV 18 500 IU/5 µL for HPVs 6, 11, 31, 33, 45, 52, and 58 and 500 genome equivalents (GE)/5 µL for other HPV types in both single and multiple infections, while avoiding false positivity. In 2022, 78 laboratories submitted a total of 154 data sets, and in 2023, 81 laboratories contributed 141 data sets. Most data sets (87%, 258/295) utilized commercially available HPV assays. Proficiency was common, with 77% of data sets meeting the proficiency criteria in 2022 and 79% in 2023. False positive results significantly decreased from 22% in 2022 to 13% in 2023. The high proficiency and increasing specificity in HPV genotyping services indicates progress toward more reliable HPV testing. High accuracy is crucial for supporting global efforts in HPV and cervical cancer elimination.

Challenges and improvements in HER2 scoring and histologic evaluation: insights from a national proficiency testing scheme for breast cancer diagnosis in China.

BACKGROUND: In 2022, our team launched the pioneering national proficiency testing (PT) scheme for the pathological diagnosis of breast cancer, rapidly establishing its credibility throughout China. Aiming to continuously monitor and improve the proficiency of Chinese pathologists in breast pathology, the second round of the PT scheme was initiated in 2023, which will expand the number of participating institutions, and will conduct a nationwide investigation into the interpretation of HER2 0, 1+, and 2+/FISH- categories in China. METHODS: The methodology employed in the current round of PT scheme closely mirrors that of the preceding cycle in 2022, which is designed and implemented according to the "Conformity assessment-General requirements for proficiency testing"(GB/T27043-2012/ISO/IEC 17043:2010). More importantly, we utilized a statistics-based method to generate assigned values to enhance their robustness and credibility. RESULTS: The final PT results, published on the website of the National Quality Control Center for Cancer ( http://117.133.40.88:3927 ), showed that all participants passed the testing. However, a few institutions demonstrated systemic biases in scoring HER2 0, 1+, and 2+/FISH- with accuracy levels below 59%, considered unsatisfactory. Especially, the concordance rate for HER2 0 cases was only 78.1%, indicating challenges in distinguishing HER2 0 from low HER2 expression. Meanwhile, areas for histologic type and grade interpretation improvement were also noted. CONCLUSIONS: Our PT scheme demonstrated high proficiency in diagnosing breast cancer in China. But it also identified systemic biases in scoring HER2 0, 1+, and 2+/FISH- at some institutions. More importantly, our study highlighted challenges in the evaluation at the extreme lower end of the HER2 staining spectrum, a crucial area for further research. Meanwhile, it also revealed the need for improvements in interpreting histologic types and grades. These findings strengthened the importance of robust quality assurance mechanisms, like the nationwide PT scheme conducted in this study, to maintain high diagnostic standards and identify areas requiring further training and enhancement.

High success rate of first proficiency testing for RET fusions and RET mutations in lung and thyroid cancer samples by various methods.

This study describes the external quality assessment (EQA) scheme for molecular testing of RET alterations in non-small cell lung cancer (NSCLC), medullary thyroid carcinomas (MTC), and non-MTC. The lead panel institute and Quality Assurance Initiative in Pathology (Qualitätssicherungs-Initiative Pathologie [QuIP] GmbH) selected formalin-fixed paraffin-embedded (FFPE) tissue from MTC for RET mutation testing by next-generation sequencing (NGS) methods and FFPE tissue from NSCLC and non-MTC for RET gene fusion testing using either in situ hybridisation (ISH) or NGS methods, forming 3 sub-schemes of the EQA scheme. Tissue material underwent an internal validation phase followed by an external testing phase. The internal validation phase served as a cross-validation step conducted by panel institutes. In the external testing phase, the number of participating institutes in the RET point mutation sub-scheme, RET fusion (ISH) sub-scheme, and RET fusion (NGS) sub-scheme was 32, 24, and 38, respectively. The reported success rates for external testing were 96.0%, 89.5%, and 93.5% for the RET point mutation, the ISH RET fusion, and the NGS RET fusion EQA sub-schemes, respectively. These findings confirm the reliability of the NGS method in detecting RET alterations and align with current screening recommendations. Overall, 31 institutes were certified for RET point mutation testing by NGS methods, 22 institutes were certified for RET fusion testing by ISH, and 36 institutes were certified for RET fusion testing by NGS methods. Results can be employed to inform real-world diagnostic decisions in Germany, Austria, and Switzerland.

Multinational proficiency tests for EGFR exon 20 insertions reveal that the assay design matters.

Insertion mutations in exon 20 of the epidermal growth factor receptor gene (EGFR exon20ins) are rare, heterogeneous alterations observed in non-small cell lung cancer (NSCLC). With a few exceptions, they are associated with primary resistance to established EGFR tyrosine kinase inhibitors (TKIs). As patients carrying EGFR exon20ins may be eligible for treatment with novel therapeutics-the bispecific antibody amivantamab, the TKI mobocertinib, or potential future innovations-they need to be identified reliably in clinical practice for which quality-based routine genetic testing is crucial. Spearheaded by the German Quality Assurance Initiative Pathology two international proficiency tests were run, assessing the performance of 104 participating institutes detecting EGFR exon20ins in tissue and/or plasma samples. EGFR exon20ins were most reliably identified using next-generation sequencing (NGS). Interestingly, success rates of institutes using commercially available mutation-/allele-specific quantitative (q)PCR were below 30% for tissue samples and 0% for plasma samples. Most of these mutation-/allele-specific (q)PCR assays are not designed to detect the whole spectrum of EGFR exon20ins mutations leading to false negative results. These data suggest that NGS is a suitable method to detect EGFR exon20ins in various types of patient samples and is superior to the detection spectrum of commercially available assays.

Proficiency testing of diagnosis in histopathology and immunohistochemistry of breast pathology in China: results from a pilot work of National Single Disease Quality Control Program for breast cancer.

AIM: Pathologists are currently supposed to be aware of both domestic and international guidelines for breast cancer diagnosis, but it is unclear how successfully these guidelines have been integrated into routine clinical practice in China. Thus, this national proficiency testing (PT) scheme for breast pathology was set up to conduct a baseline assessment of the diagnostic capability of pathologists in China. METHODS: This national PT plan is designed and implemented according to the "Conformity assessment-General requirements for proficiency testing" (GB/T27043-2012/ISO/IEC 17043:2010). Five cases of breast cancer with six key items, including histologic type, grade, ER, PR, HER2, and Ki67, were selected for testing among 96 participants. The final PT results were published on the website of the National Quality Control Center for Cancer ( http://117.133.40.88:3927/cn/col22/362 ). RESULTS: Our study demonstrated that the median PT score was 89.5 (54-100). Two institutions with scores < 67 were deemed unacceptable. The accuracy of histologic type, ER, PR, HER2, and Ki67 was satisfactory (all > 86%). However, the histologic grade showed low accuracy (74.0%). The unacceptable results mainly included incorrect evaluation of histologic grade (36.7%), inaccurate evaluation of ER/PR/HER2/Ki67 (28.2%), incorrect identification of C-AD as IBC-NST (15.7%), inappropriate use of 1+/2+/3+ rather than staining percentage for ER/PR (6.1%), misclassification of ER/PR < 1% weak expression as positive staining (1.4%), and no evaluation of histologic grade in ILC, MC, and IMC (5.8%). CONCLUSIONS: our nationwide PT program exhibited a satisfactory baseline assessment of the diagnostic capability of pathologists in China. More importantly, we identify some areas for further improvement.

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.

Busulfan Interlaboratory Proficiency Testing Program Revealed Worldwide Errors in Drug Quantitation and Dose Recommendations.

BACKGROUND: The clinical outcomes of busulfan-based conditioning regimens for hematopoietic cell transplantation (HCT) have been improved by personalizing the doses to target narrow busulfan plasma exposure. An interlaboratory proficiency test program for the quantitation, pharmacokinetic modeling, and busulfan dosing in plasma was developed. Previous proficiency rounds (ie, the first 2) found that 67%-85% and 71%-88% of the dose recommendations were inaccurate, respectively. METHODS: A proficiency test scheme was developed by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML) and consisted of 2 rounds per year, with each round containing 2 busulfan samples. In this study, 5 subsequent proficiency tests were evaluated. In each round, the participating laboratories reported their results for 2 proficiency samples (ie, low and high busulfan concentrations) and a theoretical case assessing their pharmacokinetic modeling and dose recommendations. Descriptive statistics were performed, with ±15% for busulfan concentrations and ±10% for busulfan plasma exposure. The dose recommendations were deemed accurate. RESULTS: Since January 2020, 41 laboratories have participated in at least 1 round of this proficiency test. Over the 5 rounds, an average of 78% of the busulfan concentrations were accurate. Area under the concentration-time curve calculations were accurate in 75%-80% of the cases, whereas only 60%-69% of the dose recommendations were accurate. Compared with the first 2 proficiency test rounds (PMID 33675302, October, 2021), the busulfan quantitation results were similar, but the dose recommendations worsened. Some laboratories repeatedly submit results that deviated by more than 15% from the reference values. CONCLUSIONS: The proficiency test showed persistent inaccuracies in busulfan quantitation, pharmacokinetic modeling, and dose recommendations. Additional educational efforts have yet to be implemented; regulatory efforts seem to be needed. The use of specialized busulfan pharmacokinetic laboratories or a sufficient performance in busulfan proficiency tests should be required for HCT centers that prescribe busulfan.

Nicotine and Tobacco Alkaloid Testing and Challenges: Lessons Learned From a College of American Pathologists Proficiency Testing Survey.

CONTEXT.­: Consequences related to nicotine (NIC) use remain a major health concern, leading to demand for testing to detect NIC, metabolites such as cotinine (COT), and related tobacco alkaloids, including anabasine (ANAB). NIC-related testing is not standardized among laboratories, nor are there clinical or regulatory guidelines to inform decisions such as appropriate screening cutoffs or limits of quantitation. OBJECTIVE.­: To evaluate analytical performance and reporting practices of laboratories that perform NIC-related testing by reviewing participant responses to the Nicotine and Tobacco Alkaloid (NTA) Proficiency Testing Survey. DESIGN.­: NTA results were retrieved from 2017 (the first year of the survey) through 2020. Survey participants, methodologies, and results were evaluated for all analytes, and simulated grading was performed for COT. Additional data, including limits of quantitation, qualitative cutoffs, and reasons for testing, were reviewed. RESULTS.­: Participant growth was steady for qualitative COT testing. Participation was stable for NIC, ANAB, and quantitative COT testing. Overall, participants performed well on survey challenges. However, reporting thresholds were widely divergent, ranging from 10 to 3000 ng/mL and 0.5 to 300 ng/mL, respectively, for qualitative and quantitative COT testing. Screening cutoffs were as high as 100 ng/mL for ANAB and 1000 ng/mL for NIC. CONCLUSIONS.­: Although participating laboratories performed well on the NTA Survey, the wide diversity of qualitative and quantitative reporting thresholds creates substantial risk for misinterpretation of results, and could lead to analytical concerns such as excessively high false-negative or false-positive rates. NIC-related testing would benefit from evidence-based guidelines to drive standardization of reporting.

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.

Four-Year Laboratory Performance of the First College of American Pathologists In Silico Next-Generation Sequencing Bioinformatics Proficiency Testing Surveys.

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.

Lessons Learned, Challenges Taken, and Actions Made for "Precision" Immunohistochemistry. Analysis and Perspectives From the NordiQC Proficiency Testing Program.

Immunohistochemistry (IHC) has for decades been an integrated method within pathology applied to gain diagnostic, prognostic, and predictive information. However, the multimodality of the analytical phase of IHC is a challenge to ensure the reproducibility of IHC, which has been documented by external quality assessment (EQA) programs for many biomarkers. More than 600 laboratories participate in the Nordic immunohistochemical Quality Control EQA program for IHC. In the period, 2017-2021, 65 different biomarkers were assessed and a total of 31,967 results were evaluated. An overall pass rate of 79% was obtained being an improvement compared with 71% for the period, 2003-2015. The pass rates for established predictive biomarkers (estrogen receptor, progesterone receptor, and HER2) for breast carcinoma were most successful showing mean pass rates of 89% to 92%. Diagnostic IHC biomarkers as PAX8, SOX10, and different cytokeratins showed a wide spectrum of pass rates ranging from 37% to 95%, mean level of 75%, and attributed to central parameters as access to sensitive and specific antibodies but also related to purpose of the IHC test and validation performed accordingly to this. Seven new diagnostic biomarkers were introduced, and all showed inferior pass rates compared with the average level for diagnostic biomarkers emphasizing the challenge to optimize, validate, and implement new IHC biomarkers. Nordic immunohistochemical Quality Control operates by "Fit-For-Purpose" EQA principles and for programmed death-ligand 1, 2 segments are offered aligned to the "3-dimensional" approach-bridging diagnostic tests, drugs to be offered, and diseases addressed. Mean pass rates of 65% and 79% was obtained in the 2 segments for programmed death-ligand 1.

Resultados del ejercicio de calidad de la Sociedad Latinoamericana de Genética Forense, 2023 / Results of the quality exercise of the Latin American Society of Forensic Genetics, 2023

Introducción: La Sociedad Latinoamericana de Genética Forense, desde el año 2003 organiza ejercicios colaborativos de comparación interlaboratorios a fin de apoyar el fortalecimiento de los laboratorios de Genética Forense de Latinoamérica, siendo el ejercicio de calidad de SLAGF el único que incluye muestras óseas. Objetivo: Presentar los resultados del análisis del ejercicio de calidad de SLAGF correspondientes al año 2023. Metodología: Se diseñó un ejercicio práctico con cinco muestras: dos hisopados bucales (M1 y M2), una muestra de sangre en FTA (M3), una muestra mezcla 1:1 de sangre- sangre en FTA (M4) y un resto óseo (M5), Se envió a los laboratorios un ejercicio teórico con cinco casos; dos de los cuales eran opcionales. Al igual que cinco ejercicios teóricos para los peritos independientes participantes. Por primera vez se incluyó un ejercicio de IVD opcional, los ejercicios están disponibles en: http://slagf.org/resultados-control- slagf-2023/x Resultados: Participaron 30 laboratorios y 12 peritos independientes. En el ejercicio práctico, la muestra cadavérica de restos óseos presentó los mayores desafíos, en la parte teórica fue el ejercicio de IVD. Conclusión: Los desafíos que enfrentan los laboratorios de Genética Forense Latinoamericanos, reflejados en el ejercicio de calidad de SLAGF 2023, son similares a los encontrados por otros grupos que realizan ejercicios de control de calidad en Genética Forense...(AU)

[Interlaboratory comparison for determination of cadmium in drinking water].

OBJECTIVE: To analyze and evaluate the testing capability of cadmium in drinking water in the laboratories of the provincial and municipal centers for disease control and prevention across the country by implementing the interlaboratory comparison project. METHODS: The preparation method of the secondary standard materials were used as the reference for the sample preparation in the interlaboratory comparison project. The homogeneity and stability of the samples and short-term stability for simulated transportation were tested by single factor analysis of variance(ANOVA) and linear regression and mean consistency test(t test). On top of using the kernel density estimation to test the distribution of laboratory test result, we adopted precision statistical method to analyze the laboratory test result and used Z-score to evaluate the testing ability of each participating laboratory. RESULTS: A total of 409 laboratories throughout the country participated in the proficiency testing program.383 laboratories(93.6%) of participating laboratories, obtained satisfactory result. Results provided by 4 laboratories(1.0%) of total participating laboratories, were found suspicious in their capacities. Finally, there were 22 laboratories(5.4%) of total participating laboratories, with result found to be outliers. CONCLUSION: The statistical result of the interlaboratory comparison project show that the testing capability of cadmium in drinking water has been ranked as satisfactory in the laboratories of the provincial and municipal centers for disease control and prevention across the country, and the testing capability of a small number of laboratories requires further improvement.

Continual Improvement of the Reliability of Next-Generation Sequencing-Based ctDNA Analysis: A Long-Term Comparison of ctDNA Detection in China.

BACKGROUND: Since circulating tumor DNA (ctDNA) sequencing is increasingly being applied in clinical management of patients with cancer, its testing accuracy has become a matter of serious concern. To address this issue, a long-term ctDNA analysis proficiency testing (PT) scheme for next-generation sequencing (NGS) was launched in China in 2018, serving as an educational tool for assessing and improving the testing quality of NGS-based ctDNA detection. METHODS: Feedback from participating laboratories across 23 different PT samples containing different variants with varying variant allele frequency was collected between 2018 and 2021. To further show the landscape of changing conditions in accuracy and reliability of NGS-based ctDNA testing, performance was analyzed by evaluating the cfDNA extraction kits, testing panels, target enrichment strategies, and sequencing platforms. RESULTS: During the 4 years, 2745 results reported from 504 laboratories were evaluated. Only 66.3% of results from laboratories were entirely in concordance with the expected results. Nonetheless, along with an increasing number of participating laboratories, the number of errors occurring in laboratories, and the proportion of laboratories that experienced errors both showed a significant downward trend. No obvious differences in the error rates were found regarding the kit manufacturers or sequencing platform. Moreover, the individual performances of the laboratories improved when they participated in more PT scheme rounds. CONCLUSIONS: These data demonstrated that the performance of individual Chinese laboratories for NGS-based ctDNA analysis continuously improved over time with participation in PT schemes. However, further care must also be taken in standardized operations and validations.

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.

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.

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.