PowerPoint Presentation n= 171 p=0.02 Differentiation uncertainty cohort 0% 0 20% 40% 80% 100% 60% 1 2 3 4 5 Time (years) P ro b a b il it y Class 1 Class 2B Class 2A › For clinicians who follow BWH staging, uncertainty in differentiation status may impact patient management. › The 40-GEP provides objective and reproducible prognostic information, including in situations where the distinction between poorly and moderately differentiated histological grading is challenging. › Within this cohort of high-risk cSCC patients, whose BWH stage would change solely due to an alteration in differentiation status, the 40-GEP was able to significantly stratify risk of metastasis. › Incorporating the personalized 40-GEP test results into clinical cSCC risk assessment could enhance current patient management decisions, therefore improving patient outcomes. Incorporating the 40-gene expression profile (40-GEP) test for poorly differentiated cutaneous squamous cell carcinoma (cSCC) tumors mitigates risk assessment uncertainty from histologic grading Aaron S. Farberg, MD1; Jennifer J. Siegel, PhD2; Sarah J. Kurley, PhD2; Alison L. Fitzgerald, PhD2; Anesh Prasai, PhD2; Matthew S. Goldberg, MD2,3; Sherrif Ibrahim, MD4; Sarah I. Estrada, MD5 1Baylor Scott & White Health System, Dallas, TX; 2Castle Biosciences Inc., Friendswood, TX; 3Icahn School of Medicine, New York City, NY 4University of Rochester, Rochester, NY; 5Affiliated Dermatology, Scottsdale, AZ Background › With 1.8 million new cases diagnosed each year, cutaneous squamous cell carcinoma (cSCC) is the second most prevalent skin cancer in the U.S.1 While >95% of cSCC cases are cured by surgery, an estimated 5% progress to nodal or distant metastasis, where survival rates drop to 50-83% and <40%, respectively.2,3 › The degree of differentiation plays a critical role in the progression of cSCC. Multiple studies have established poorly differentiated histology as an independent predictor of poor outcomes.2,4,5 › For patients with primary cSCC and one or more risk factors, the clinically available 40-GEP test accurately classifies likelihood of regional, nodal, or distant metastasis at 3 years post diagnosis (Class 1=low risk, Class 2A=moderate risk, Class 2B= high risk).1,2 The 40-GEP has also demonstrated independent and additive prognostic value in a multivariate model when compared to commonly utilized high-risk factors or Brigham and Women’s Hospital (BWH) staging system (Table 1).6,7 This study was sponsored by Castle Biosciences, Inc. (CBI), which provided funding to the contributing centers for tissue and clinical data retrieval. JJS, SJK, ALF, AP, and MSG are employees and options holders of CBI. ASF is a consultant for CBI. SIE is an independent dermatopathologist contractor for CBI. Methods References 1. Skincancer.org, 2022 2. Schmults, et al. JAMA Derm 2013 3. NCCN v2.2022 4. Thompson, et al. JAMA Derm 2016 5. Karia, et al. JCO 2014 6. Wysong, et al. JAAD 2021 7. Ibrahim, et al. Future Onc. 2021 8. Patel, et al. Cancer Medicine 2022 9. Jagdeo, et al. JAAD 2007 10. Prezzano, et al. Derm Surg 2021 Results For more information: aaron.farberg@gmail.com Conclusions Disclosures Table 2. Differentiation status was altered for 40% of the cohort, impacting BWH stage and potentially treatment decisionsClinical Issue and Objective There is a lack of widely accepted criteria for grading of cSCC tumor tissue. This has led to subjectivity when determining differentiation status, complicated by different specialties’ usage of different staging criteria.8 Multiple studies have shown concordance for cSCC histologic grading is overall weak, especially when comparing moderately differentiated tumors.9,10 The inconsistency in the assessment of this risk factor can adversely impact its value as a prognostic factor due to its direct impact on clinicopathologic tumor staging.9 The objective of this study was to evaluate the ability of the 40-GEP to risk stratify among a high-risk cSCC “differentiation uncertainty cohort” and its impact on staging, therefore its potential to influence treatment decisions. A) Risk factors comprising the Brigham and Women’s Hospital (BWH) staging system.5 B) Overall, 40% of the high-risk cSCC cohort would change differentiation status, with 77.2% being upstaged and 22.8% being downstaged by BWH criteria. This ‘differentiation uncertainty cohort’ is representative of how inconsistencies in assessment of cSCC tissue grading can directly impact staging and potentially treatment decisions. Within the ‘differentiation uncertainty cohort’ (n=171), Kaplan-Meier survival analysis demonstrated statistically significant 3-year metastasis-free survival between all 40-GEP classes. Assessment of number of samples with metastatic outcomes was also evaluated and arranged by 40-GEP class. Table 1. Independent risk assessment by the 40-GEP complements existing systems7 40-GEP Risk Class 3- year MFS (95% CI) Overall Event Rate Non-metastatic (n) Metastatic (n) Class 1 90.1% (97.3-83.5%) 11.3% 63 8 Class 2A 78.6% (87.9-70.3%) 21.4% 66 18 Class 2B 62.5% (91.4-42.8%) 37.5% 10 6 Without 40-GEP 81.9% (87.9-76.3%) 18.7% 139 32 BWH T-stage T1 T2 T3 0 high risk factors T2a: 1 high risk factors T2b: 2-3 high risk factors 4 high risk factors High-risk factors • Poorly differentiated histology • Tumor diameter ≥2cm • Perineural invasion ≥0.1mm • Deep tumor invasion (beyond subcutaneous fat but excluding bone invasion, which qualifies as T3) Calculate percentage of cases that incur a BWH stage change Kaplan-Meier survival analysis at 3 years post diagnosis Number of patients upstaged BWH T-stage n T1 T2a 76 T2a T2b 55 T2b T3 1 Total 132 Number of patients downstaged T2a T1 22 T2b T2a 17 Total 39 Poorly or moderately differentiated tumors “Differentiation uncertainty cohort” (n=171) Modeled to opposing differentiation status Figure 2. The 40-GEP stratifies risk among a histologically ambiguous high-risk cSCC cohort Figure 1. Development of a ‘differentiation uncertainty cohort‘ High-risk cSCC cohort (n=420) Multivariate Cox Regression Risk Factor n Hazard Ratio p value 40-GEP Result Class 1 212 1.00 --- Class 2A 185 2.33 0.013 Class 2B 23 6.86 <0.001 Clinicopathologic Risk Factors Poor Differentiation 58 2.29 0.011 Perineural Invasion 53 1.22 ns Deep Invasion 72 2.05 0.039 Tumor Diameter N/A 1.07 ns Immunosuppression 103 --- --- 40-GEP Result Class 1 212 1.00 --- Class 2A 185 2.98 <0.001 Class 2B 23 9.42 <0.001 BWH T-Stage T1/T2a 364 1.00 --- T2b/T3 56 2.38 0.002 ➢Class 2A result has similar risk to well- established high-risk factors ➢Class 2B result 3-4x as risky as high-risk clinicopathologic factors or T-stage A) B) › A high-risk cSCC cohort7 (n=420) was divided into moderately and poorly differentiated statuses based on clinical pathology reports and by an independent dermatopathologist review. If differentiation status differed between the report and the independent review, poorly differentiated was chosen as the status. The “differentiation uncertainty cohort” (n=171) was then staged by BWH criteria (Table 2A). To represent the subjectivity and inconsistent evaluation that commonly happens with this risk factor, differentiation status was manually changed to the opposing status (i.e., poorly changed to moderately, moderately changed to poorly). Determination of changes to BWH staging were documented to then note wherein changes of management may occur. Kaplan-Meier analysis was used to determine statistical significance of metastasis free survival (MFS) when incorporating the 40-GEP test. = Staged by BWH criteria Scan here for more info Presented at Winter Clinical Dermatology Conferences: January 13-18, 2023, Kohala Coast, Hawaii and February 17-20, 2023, Miami, FL M e ta s ta s is -F re e S u rv iv a l Originally published in Ibrahim et al. 2021 https://castlebiosciences.com/research-development/publications/ Slide 1