PowerPoint Presentation A comprehensive diagnostic offering workflow increases the rate of actionable results of the 23- and 35-gene expression profile tests for use as ancillary diagnostic tools for difficult-to-diagnose melanocytic lesions Background Methods › Diagnostic discordance in suspicious cutaneous melanocytic lesions is well documented and particularly prevalent among difficult-to-diagnose cases, for which histopathology may be insufficient for a definitive diagnosis.1-4 › The 23-gene expression profile (GEP; myPath Melanoma) and 35-GEP (DiffDx-Melanoma) tests are clinically available objective ancillary tools that facilitate diagnosis of melanocytic lesions with ambiguous histopathology. The tests use proprietary algorithms to produce results of likely benign, intermediate, or malignant.5-7 › The 23-GEP has shown accuracy metrics of over 90% sensitivity in multiple clinical studies that included patient outcomes.8-10 However, the 23-GEP historically has resulted in ~23% of cases receiving either a technical failure or an intermediate result, which can be perceived as nonactionable.6,11-13 The 35-GEP test can address this shortcoming and showed both an increased sensitivity in the first validation cohort and a decreased nonactionable rate of 8.5%.7 › Clinical utility has been demonstrated with benign and malignant GEP test results;11,14 therefore, those test results are defined as actionable. Acknowledgments & Disclosures References › This study was sponsored by Castle Biosciences, Inc. › All authors are employees and shareholders of Castle Biosciences, Inc. 1. Shoo, B. A. et al. J Am Acad Dermatol 2010. 62 (5) 751–756. 2. Gerami, P. et al. Am J Surg Pathol 2010. 34 (6) 816–821. 3. Haws, B. et al. J Cutan Pathol 2012. 39 (9) 844–849. 4. Elmore, J. G. et al. BMJ 2017. 357 (1) j2813. 5. Clarke, L. E. et al. J Cutan Pathol 2015. 42 (4) 244–252. 6. Clarke, L. E. et al. Cancer 2017. 123 (4) 617–628. 7. Estrada, S. et al. SKIN 2020. 4 (6) 506–522. 8. Ko, J. S. et al. Cancer Epidem Biomar Prev 2017. 26 (7) 1107–1113. 9. Ko, J. S. et al. Human Pathology 2019. 86 213–221. 10. Clarke, L. E. et al. Personalized Medicine 2020. 17 (5) 361–371. 11. Cockerell, C. J. et al. Medicine 2016. 95 (40) e4887. 12. Minca, E. C. et al. Mod Pathol 2016. 29 (8) 832–843. 13. Castillo, S. A. et al. Am J Dermatopathol 2020. 42 (12) 939-947. 14. Farberg, A. et al. SKIN 2020. 4 (6) 523–533. Results Matthew S Goldberg, MD1,2, Jennifer J Siegel, PhD1, Brooke H Russell, PhD1, Jason H Rogers, MSc1, Kyle R Covington, PhD1, Kristen M Oelschlager, RN1, Trisha M Poteet1, Jeffrey K Wilkinson, PhD1, Michael D Berg, PhD1, Katherine Falkowski, PhD1, Sarah J Kurley, PhD1, and Armineh Kajoian, MD1 1Castle Biosciences, Inc., Friendswood, TX 2Icahn School of Medicine at Mount Sinai, New York, NY For more information: mgoldberg@castlebiosciences.com Objective › Today, both the 23- and 35-GEP are offered from a single laboratory as part of a comprehensive diagnostic offering (CDO) workflow. Unless preferred otherwise by the ordering clinician, clinical samples are processed first through the 23-GEP test, and if a technical failure or intermediate result is received, processed to the 35-GEP (Figure 1). › Here, we report test result metrics from archival research cases and from this clinical workflow. › Combining the 23-GEP and 35-GEP tests into one workflow leverages the strengths of both assays. › The CDO workflow demonstrated a high rate of accuracy in research cases, with 94.7% sensitivity and 89.5% specificity. › The CDO workflow for ambiguous melanocytic lesions has substantially improved reporting of clinically actionable results from a historic rate of ~77% for the 23-GEP alone to over 98%. › Eligible cases with a malignant result from the CDO can also be subsequently run on the 31-GEP prognostic test (Decision Dx-Melanoma), without requiring extra tissue, to predict the likelihood of recurrence and sentinel lymph node biopsy positivity. Conclusions Figure 1. Clinical workflow of the comprehensive diagnostic offering *Does not generate a test report. Cases with Intermediate or Technical Fail results from the 23-GEP undergo testing with the 35-GEP. GEP, gene expression profile. › Melanocytic lesions and associated de-identified clinical data from patients ≥18 years of age were included in this study. Samples were acquired under an IRB-approved protocol or were previously submitted for clinical testing for the 31-GEP. Research samples were independently reviewed by at least 2 dermatopathologists for diagnostic adjudication, were blinded to the original diagnosis, and included in the study if they received at least 2 out of 3 diagnostic concordance (Table 1). The study also included clinical cases submitted to Castle Biosciences for CDO testing with results reported since implementation of the CDO workflow between June 3 and August 31, 2021 (Table 2). › All cases not receiving a benign or malignant result from the 23-GEP were run on the 35-GEP, except for pediatric cases (<18 years), which were only run on the 23-GEP and excluded from this analysis. Technical fail included samples with insufficient quantity of RNA and/or control or discriminant gene amplification failure based on the requirements for each test. Scan or click here for more info › Clinical test results were analyzed over a 3-month period. › The 23-GEP test gave an actionable result of benign or malignant in 77.8% of cases, which is comparable to past reporting in ambiguous cases for this test6,11 (Table 2). › Nonactionable classifications of the 23-GEP test were 22.2% (12.9% intermediate and 9.4% technical failure). These cases then underwent testing with the 35-GEP test, and an additional 20.9% of originally submitted cases received an actionable result. Only 1.1% of cases received a final intermediate test result (i.e., from both tests); the technical failure rate for the CDO was 0.2% (Table 2). › This clinical workflow increased the rate of an actionable report from 77.8% to 98.7% when compared with 23-GEP testing alone (Table 2). › The clinical workflow results were 59.5% benign, 39.1% malignant, 1.1% intermediate, and 0.2% technical failure. Melanocytic lesion of uncertain malignant potential 23-GEP Benign Intermediate or Technical Fail* 35-GEP Benign Intermediate Technical Fail* Malignant Malignant Table 2. Clinical test results of the comprehensive diagnostic offering Cases with Intermediate or Technical Fail results from the 23-GEP undergo testing with the 35-GEP. GEP, gene expression profile. Research Cohort, n=738 CDO 95% CI Sensitivity 94.7% 92.4-96.8 Specificity 89.5% 86.3-92.7 PPV 90.1% 87.7-93.4 NPV 94.1% 91.4-96.4 Intermediate 0.8% n=6 Clinical CDO Testing Actionable (%) Nonactionable (%) 23-GEP alone 77.8% 22.2% Subsequent 35-GEP 20.8% 1.3% Overall 98.7% 1.3% › The Research Cohort was comprised of 738 FFPE archival biopsy samples from adults ≥18 years of age with cutaneous melanocytic lesions with a consensus diagnosis reviewed by at least three independent dermatopathologists who were blinded to the original diagnosis. All samples were run on the 23-GEP, and any intermediate or technical fail samples were subsequently run on the 35-GEP per the current clinical CDO workflow (Figure 1). › Accuracy metrics demonstrate high performance of the CDO workflow (Table 1). Table 1. Accuracy metrics in research cases from the comprehensive diagnostic offering NPV, negative predictive value; PPV, positive predictive value; CI, confidence interval. https://castletestinfo.com/