Hameed, Meera

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.

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.