MolDX: Molecular Biomarker Testing for Risk Stratification of Cutaneous Squamous Cell Carcinoma

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42 CFR §410.32(a) Diagnostic x-ray tests, diagnostic laboratory tests, and other diagnostic tests: Conditions

CMS Internet-Only Manual, Pub. 100-02, Medicare Policy Manual, Chapter 15, §80 Requirements for Diagnostic X-Ray, Diagnostic Laboratory, and Other Diagnostic Tests, §80.1.1 Certification Changes

Current molecular biomarker tests that risk stratify individuals with cutaneous squamous cell carcinoma (cSCC) are non-covered by this contractor.

Clinical Background

Cutaneous squamous cell carcinoma (cSCC) is an epidermal keratinocyte-derived malignancy that is the second most common skin cancer.^1,2 Incidence is increasing and varies with latitude in the United States, with higher incidence in areas of greater sun exposure.³ Each year, over 700,000 new cases are diagnosed, 5600 to 12,600 patients develop nodal metastasis, and there are 4000-8800 deaths.^2,4-7 Incidence is highest in non-Hispanic white populations, greater in males compared to females, and increases with age for a mean age at diagnosis of approximately 71 years.³ Ultraviolet (UV) radiation due to cumulative and chronic sun exposure is the most important environmental risk factor, occurring more frequently in those with less pigmented skin types.^8-10 In individuals with highly pigmented skin, cSCC is more frequently found on non-sun exposed areas and is often associated with chronic inflammation, chronic wounds, or scarring.^4-7 cSCC of this etiology is more difficult to treat and exhibits a higher recurrence risk.^11-13

The overall prognosis is favorable with a 5-year survival of approximately 98%.^2,8,14 While most tumors are easily cured with surgical excision, a subset result in recurrence, metastasis, and disease-specific death.^14,15 The risk of nodal metastasis ranges from 1.2-5.8% in cohort and tumor registry studies and risk of death is approximately 2%.^14,16,17 In a cohort of patients treated with Mohs micrographic surgery (MMS), the rate of local recurrence was 2.5%, metastatic disease 1.9% and of disease-specific death 0.57%,¹⁸ underscoring the importance of adequate surgical treatment.

Approaches to Risk-Stratification and Staging

Significant effort has been placed on identifying tumors likely to result in poor outcomes (high-risk cSCC) through development of risk stratification or staging systems that lead to a more individualized, risk-based treatment approach and follow-up schedule.^19-25 However, this has not been defined consistently and currently available approaches have limited positive predictive value (PPV).^14,16,26 The most utilized staging systems and guidelines include the National Comprehensive Cancer Network (NCCN) Guidelines, the American Joint Committee on Cancer 8th Edition (AJCC 8) staging system for Head and Neck Tumors, and the Brigham and Women’s Hospital staging system (BWH).^16,27 Each will be discussed in brief. 

As of 2021, the NCCN guidelines risk stratify cutaneous squamous cell cancer into low, high and very high-risk groups based on outcomes.⁸ The high-risk group is at increased risk of local recurrence and the very high-risk group is at increased risk of both local recurrence and metastasis.⁸ This stratification was validated by a large retrospective dual-center cohort, comparing outcomes of each group in patients treated with MMS or wide local excision (WLE). The cohort included 10,196 tumors from 8727 patients, which were subsequently stratified by NCCN guidelines into low, high, and very high-risk groups.²⁸

Patients with NCCN very high-risk cSCC tumors experienced worse outcomes compared to patients with high-risk and low-risk tumors in terms of local recurrence (LR) (Adjusted 5-year cumulative incidence of 9.4% vs 1.5% and 0.8%, respectively), nodal metastasis (NM) (7.3% vs 0.5% and 0.1% respectively), distant metastasis (DM) (3.9% vs 0.1% and 0.01%, respectively), disease specific death (DSD) (10.5% vs 0.5% and 0.1%, respectively). ²⁸ The study also demonstrated that compared with cSCCs treated with WLE, those treated with MMS or peripheral and deep en face margin assessment (PDEMA) had lower risk of LR (subhazard ratio (SHR), 0.65), DM (SHR, 0.38), and DSD (SHR, 0.55).²⁸ Therefore, in comparison to the NCCN high risk category, which demonstrated rates of metastasis (<1%), the very high-risk category stands to benefit most from further risk stratification and identification of patients with increased risk for nodal and distant metastasis. NCCN guidelines direct risk assessment after biopsy into either the low, high, or very high-risk groups and refer to staging systems, such as AJCC8, as additional resources.

The AJCC 8 came into clinical use in January 2018 with an updated four T stage (T1-T4) cSCC tumor classification for cases located on the head and neck; it was validated by multiple studies.^16,26,29,30 Tumors are considered high-risk when staged as T3 or T4, and most high-risk tumors are staged as T3 due to relative rarity of T4 cases.³¹ More recently, in order to further reduce the prognostic heterogeneity of AJCC 8, the Salamanca refinement of the T3 classification was proposed, with significant difference in outcomes across three groups within T3.³² The BWH staging system was developed in 2013 to provide superior prognostic value compared to an earlier version of AJCC Staging (AJCC 7).^26,32,33 In AJCC7, higher tumor classes (T3 and T4) required the rare finding of bone invasion, and thus most poor outcomes occurred in stage T2.^26,34 BWH staging consists of four T stages (T1, T2a, T2b, and T3) and categorizes tumors by number of observed high-risk features.^26,35 AJCC 8 and BWH classification systems have been validated in studies conducted at single-center academic institutions^26,30,33 and in population-based studies.^16,29

Ruiz et al²⁶ compared the performance of AJCC 8 and BWH tumor classification systems with respect to distinctiveness, homogeneity, monotonicity, and c-statistic in a single-center academic cohort. The two classification systems showed similar homogeneity (proportion of poor outcomes occurring in low tumor classes) and monotonicity (proportion of poor outcomes occurring in high tumor classes). High tumor classes AJCC8 T3/T4 and BWH T2b/T3 accounted for 18% and 9% of total cSCC cases, respectively, as well as 71% and 70% metastases, and 85% and 92% of deaths. AJCC8 T2 and T3 represented 23% of total cases, with statistically indistinguishable outcomes.²⁶ Sensitivity, Specificity, PPV, and NPV of AJCC8 and BWH higher stages to detect NM/DSD were as follows: Sensitivity: 0.73 vs 0.78, Specificity: 0.93 vs 0.85, PPV: 0.30 vs 0.17, NPV: 0.99 vs 0.99.²⁶ C-statistic for LR demonstrated similar discriminative ability by BWH and AJCC8 for LR with a BWH c-statistic of 0.86 (95% CI, 0.79-0.92) and an AJCC8 c-statistic of 0.81 (95% CI, 0.73-0.88) (p = .09). BWH showed superior discriminative ability for NM and DSD with a BWH c-statistic for NM of 0.91 (95% confidence interval (CI), 0.85-0.96) vs an AJCC8 c-statistic of 0.84 (95% CI, 0.76-0.91) (p = .01). For DSD, the BWH c-statistic was 0.97 (95% CI, 0.94-0.99) and the AJCC8 c-statistic was 0.91; (95% CI, 0.88-0.94) (p= .005).²⁶ 

AJCC8 and BWH stratification performance was recently evaluated in a population-based nested case control study examining 887 metastatic cSCC cases and 887 non-metastatic controls, factoring in the Salamanca T3 sub-classification.²⁹ The highest specificity was demonstrated for BWH, being 92.8% (95% (CI 90.8–94.3%), with a PPV of 13.2 (95% CI 10.6–16.2), NPV of 99.0 (95% CI 98.9–99.1) and c-index of 0.84 (95% CI 0.82–0.86). AJCC8 specificity was 80.5 (95% CI 77.7–83.1), PPV was 6.8 (95% CI 6.0–7.7), NPV was 99.2 (95% CI 99.2-99.3) and c-index was 0.78 (95% CI 0.76–0.80). The AJCC8 T3 Salamanca refinement did not show any improvement in AJCC8 T3 cSCC staging, although the number of T3 tumors was small with 37 eligible cases and 37 controls.²⁹ Of note, the odds ratio (OR) for metastasis for AJCC8 T2 was 3.9 (95% CI 2.6-5.8) and for T3, the OR was 11.6 (95% CI 8.3-16.0). The odds ratio for metastasis of BWH T2a was 6.8 (95% CI 4.6-10.1) and for T2b it was 33.3 (95% CI 20.8-53.2), demonstrating significantly higher odds of metastasis with increased stage.²⁹ These data also suggest that AJCC8 tends to upstage low-risk disease, whereas BWH is superior in identification of low-risk patients.³⁷

Surveillance and Treatment Intensity are Influenced by Risk-Stratification Group

The American Academy of Dermatology currently recommends tumor stratification of localized cSCC using NCCN guidelines to provide practical clinical guidance for management and follow up and demonstrates preference for BWH in terms of the most accurate prognostication tool in localized cSCC.³⁸ NCCN high- and very high-risk cSCC tumors are at a greater risk for developing poor outcomes, including local recurrence, nodal metastasis, distant metastasis, and disease specific death. These tumors benefit from risk reduction through MMS or peripheral and deep en face margin assessment (PDEMA).²⁸ NCCN management guidelines for local cSCC recommend MMS or other forms of PDEMA as a primary treatment option for low-risk and high-risk cSCC, and as the preferred surgical technique for very high-risk tumors with consideration of adjuvant radiation therapy (RT) in cases with poor prognostic features. Recent studies have estimated the outcome benefit of adjuvant RT following resection of cSCC with negative margins to be approximately 50% reduction in local and nodal recurrence risks, despite older inconclusive data.³⁹ Therefore, adjuvant RT can be considered for cSCCs with risk factors such as gross clinical radiologic perineural invasion (PNI), multifocal histologic nerve invasion, ≥6 cm tumor diameter, recurrent tumors, high risk for regional or distant metastasis, close surgical margins where further surgery cannot be performed, and desmoplastic or infiltrative tumors in patients who are chronically immunosuppressed.³⁹ In addition, neoadjuvant therapy with cemiplimab has recently demonstrated benefit in patients with resectable cSCC, with application for very high-risk cases.⁴⁰ Suggested follow-up frequency and intensity (including use of imaging) vary according to risk group for local recurrence and metastasis. Patients with very high-risk cSCC that are recurrent or have multiple risk factors that place them in the very high-risk group warrant consideration of sentinel lymph node biopsy (SLNB) prior to PDEMA. At this time, studies assessing the role of SLNB in cSCC have been heterogeneous and small and while there may be prognostic value,^8,41-51 it is unclear if SLNB results in improved patient outcomes.^8,42,44,46

A recent survey of 156 physicians aimed to assess how physicians across fields of dermatology and other cancer specialists from head and neck surgery/surgical oncology, radiation oncology, and medical oncology define high-risk cSCC and approach patient management.²⁷ Results revealed that most dermatologists (89%) and other cancer specialists (93%) apply staging criteria to cSCC, with dermatologists more often staging only high-risk tumors and other cancer specialists preferring to stage all tumors. 71% of the surveyed dermatologists used the BWH staging system along or in conjunction with AJCC8, whereas 71% of other cancer specialists used only AJCC8. A contributing factor to this preference is that BWH staging has been developed by dermatologists, published in dermatology journals, and discussed at dermatology conferences. There was consensus that AJCC T3 and BWH T2b or higher constitute a high enough risk threshold to merit increased management intensity, including radiologic imaging, SLNB, adjuvant radiation therapy, and increased follow up; as such, they would also fall into the ‘very high-risk’ NCCN category. This is aligned with Clinical Practice Guidelines from the American Society for Radiation Oncology that provide a strong recommendation for postoperative radiation therapy (PORT) in patients with T3 and T4 tumors and for patients with desmoplastic or infiltrative tumors in the setting of chronic immunosuppression.⁵² Of note, the survey found that a number of physicians do not use staging systems to drive decision-making with regard to radiologic imaging, SLNB, adjuvant radiation therapy, adjuvant systemic therapy, or increased follow-up. The authors state that the underutilization of consistent and concrete guidelines signifies a major shortcoming in the care of patients with cSCC and calls for urgency in identification of accurate T-stage risk estimates and evidence-based treatment modalities by disease stage.⁵³ The survey did not query participating physicians regarding use of NCCN guidelines for clinical management, although it did assess risk perception of various clinicopathologic factors. The recently added “very high-risk” category in NCCN guidelines represents an effort at unification of management across staging systems and clinicopathologic risk factors.²⁷

Predictive Biomarkers and Gene Expression Profiling

Tumor biomarkers have been used to improve risk prognostication and clinical decision-making in other cancer types⁵⁴ and several are in development to improve risk stratification of cSCC with the goal of bolstering the information provided by the various staging systems. The AJCC8 and BWH staging systems both stand to benefit from a stronger c-index and from improved PPV, as lower PPV may lead to over-treatment and unduly intense follow up.⁵⁴ Oh et al¹⁵ constructed a nomogram using protein markers, Axin2 and p53, and clinical variables such as tumor size, organ transplantation history, poor differentiation, and invasion into subcutaneous fat to estimate individualized risk for recurrence in a retrospective study with 145 cSCC patients, to reveal a c-index of 0.809. Additional investigational biomarkers demonstrating association with poor outcomes in patients with cSCC include PD-L1,^55-58 inositol polyphosphate 5-phosphatase (INPP5a),^59,60 p300,⁶¹ telomerase reverse transcriptase (TERT) promoter mutations,⁶² CD133,⁶³ long non-coding RNAs,⁶⁴ and epidermal growth factor receptor (EGFR) overexpression.^65,66 Further studies are required to determine clinical validity and utility.

DecisionDx-SCC (40-GEP)

Castle Biosciences devised the DecisionDx-SCC (40-GEP) test which harnesses changes in gene expression of 34 metastasis-associated genes and 6 control genes to identify patients with high-risk of metastasis.^35,67-69 The 40-GEP is intended to be used in patients with localized, invasive disease and the presence of one or more risk factors (i.e., high-risk) to guide treatment plans within established management pathways. Risk factors that confer eligibility for testing include: Tumors ≥ 2cm anywhere on the body, tumors located on head, neck, hands, feet, pretibial, genitalia, tumors with poorly defined borders, rapidly growing tumors, neurological symptoms in tumor region, tumor at site of prior radiation therapy or chronic inflammation, immunosuppression, perineural invasion of large or small caliber nerves, poorly differentiated histology, deep invasion and aggressive histologic subtypes, aligning with NCCN high-risk and very high-risk categories. The test is not intended to be used in patients with localized low-risk cSCC, cSCC with the presence of lymphovascular invasion; OR bone invasion; OR all four of the following risk factors: diameter of at least 2 cm, AND poorly differentiated, AND perineural invasion of at least 0.1 mm, AND invasion beyond the subcutaneous fat, cSCC that has evidence of regional or distant metastasis, or on locally recurrent cSCC. As a result, these criteria exclude patients who are in the NCCN low risk category and many tumors that are staged as AJCC8 T1 and BWH T1. The exclusion criteria also exclude tumors staged as BWH T3 and AJCC8 T4 (and possibly some AJCC8 T3 tumors with minor bone invasion). However, it is important to note that patients (for example patients who are immunosuppressed) may be classified as “high-risk” according to NCCN criteria (making them eligible for 40-GEP testing) though they have a stage T1 tumor by AJCC8 or BWH. A significant proportion of BWH and AJCC8 T1 tumors fall into the “high risk” category, and relatively few are considered “very high risk.”²⁸ In all, patients whose risk is either too low or too high to benefit from the 40-GEP are meant to be excluded from testing. Thereby, the test is focused on improving risk stratification within BWH T2a/2b and AJCC8 stages T2/T3, and T1 patients who have other ‘high-risk’ factors. 

The 40-GEP stratifies patients with one or more risk factors into a low metastatic risk (Class 1), moderate metastatic risk (Class 2A) or high metastatic risk (Class 2B) groups. The test was developed using a discovery and development cohort consisting of 202 archival cSCC cases for gene selection and further validated using archival tissue from a separate cohort of 321 high-risk primary cSCC patients with known 3-year outcomes and an overall metastatic rate of 16.2%.³⁵ cSCC tissue and clinical data were obtained from 23 independent centers and enrollment targeted patients with at least one high-risk feature as defined by NCCN guidelines, or AJCC or BWH stage greater than T1, either at the patient or tumor level.³⁵ The 40-GEP signature demonstrated statistically significant capability of stratifying metastatic risk with different 3-year metastatic rates observed for patients with Class 1 (n=203; 8.9%), Class 2A (n=93; 20.4%) and Class 2B (n=25; 60.0%) results.³⁵ The 40-GEP signature maintained predictive value when analyzed in bivariable models with AJCC and BWH staging. Of note, AJCC and BWH also maintained statistical significance in their respective bivariable models with the 40-GEP. A multivariate Cox regression analysis was conducted for 295 patients with clinical features captured prior to definitive surgery including male sex, tumor diameter assessed as a continuous variable per unit increase, immune deficient status, location on head and neck and GEP Class 2A or 2B, to reveal statistical significance for tumor diameter, head, and neck location, as well as 40-GEP Class 2A or 2B (p<0.05). In a separate multivariate Cox regression analysis with histopathological features captured after definitive surgery for 321 cases, statistical significance was achieved for tumor thickness >6mm, poor histological grade, invasion into fat, and a Class 2B result. Of note, a Class 2A result was not statistically significant in this model that included histopathological features only. Clarks Level IV/V and perineural invasion were not significant predictors of metastasis on univariate analysis and were not included in the multivariate model.³⁵

168 cases were missing clinicopathologic data (mostly tumor thickness) and were staged with assumption of null values for missing data which may have resulted in under-staging by BWH and AJCC.³⁵ All specimens underwent central pathology review and restaging according to contemporary staging criteria with medical records reviewed for additional high-risk features. However, cases excised via MMS had no tissue available for review other than the shave biopsy, therefore there is possibility for underreporting of high-stage features and resultant under-staging.³⁵ This is reflected in the higher percentage of metastasis occurring in low T stages than previously reported in the literature and lower sensitivities of AJCC and BWH staging than reported for other cohorts (39% and 25% vs 78% and 73%, respectively).^26,35 Nevertheless, post hoc sensitivity analyses showed robustness of the primary bivariable analyses despite possible under-staging. Accuracy metrics for risk prediction are found in Table 1 below, adapted from Wysong et al.³⁵ The PPV of a Class 2B result was 60%, whereas the PPV of any Class 2 result was similar to AJCC8 and BWH high stage/low stage categories.

Table 1. Accuracy of risk prediction of the 40-GEP and risk assessment methods (n=321)

Ibrahim et al subsequently reported on data from expansion of the previous cohort to 420 patients with high-risk factors, 63 of whom developed regional and/or distant metastases.⁶⁸ The median time to metastasis was 0.9 years.⁶⁸ Of the 420 cases, 212 were classified as Class 1 with a metastatic rate of 6.6%, 185 were classified as Class 2A with a metastatic rate of 20.0%, and 23 were classified as Class 2B with a metastatic risk of 52.2%. When stratified by NCCN high-risk vs very high-risk criteria, Class 1 was associated with a 4.1% metastatic rate in the high-risk cohort vs 11.9% in the very high-risk cohort; Class 2 was associated with a 15.7% metastatic rate in the high-risk vs 25.3% in the very high-risk cohort, and Class 2B was associated with a 37.5% metastatic rate in the high-risk vs 60% in the very high-risk cohort. The overall metastatic rate in the NCCN high-risk cohort was 9.8% vs 23.0% for the very high-risk cohort.⁶⁸ This is significantly higher than the cumulative incidence for metastasis reported by Stevens et al in the NCCN risk stratification validation cohort consisting of 10,196 tumors from 8727 patients.²⁸ 

In multivariate Cox regression analysis with clinicopathologic risk factors that included 40-GEP results along with poor differentiation, perineural invasion, deep invasion, and tumor diameter, the 40-GEP was statistically significant along with poor differentiation and deep invasion. 40-GEP results were also significant in bivariable analysis along with either NCCN risk group, BWH, and AJCC 8 stage, which also remained significant contributors to each respective bivariable model. Sensitivity of the 40-GEP 2B vs 1/2A result was 19%, specificity 96.9%, PPV was 52.2% and NPV was 87.2%, all of which are similar to that reported in Wysong et al.^35,68

Arron et al⁶⁷ evaluated a subset (n=278, 66%) of this cohort from patients with cSCC of the head and neck with an overall metastatic rate of 19.4%. 126 of these patients had Class 1 results (metastatic rate: 8.7%), 134 had Class 2A results (metastatic rate: 24.6%), and 18 had Class 2B results (metastatic rate: 55.6%). In this subset of patients, the PPV of a 40-GEP Class 2B result was 55.6% compared to 37.0% for high stage AJCC8 and 40.0% for high stage BWH, and the difference between the three was not statistically significant. As in the larger cohort, the 40-GEP remained significant in bivariable analyses with AJCC8 and BWH criteria, which were also significant contributors to each respective model. In multivariate analysis with clinicopathologic risk factors, the HR of Class 2A result was 2.28 (95% CI 1.08-4.81) and the HR of a Class 2B result was 4.05 (95% CI 1.34-12.26). Clinicopathologic factors significant along with 40-GEP results in a multivariate Cox regression analysis included tumor diameter, poor differentiation, deep invasion, and male sex.⁶⁴ Sensitivity, specificity, PPV, and NPV of a 2B vs 1/2A result were similar to that reported in Wysong et al and Ibrahim et al.^35,68 

The clinical utility of the 40-GEP lies in its incorporation into existing risk-assessment frameworks to improve prognostic value and facilitate individualized risk assessment, treatment, and follow up. Castle Biosciences aimed to establish clinical utility of the 40-GEP through publications that demonstrate use in clinical practice, clinician surveys at conferences, and proposed frameworks on use within guidelines, such as those from NCCN.^,70-75 Along these lines, Farberg et al intended to refine risk-directed patient management using the 40-GEP along with NCCN guidelines and T stage criteria by examining 300 patients who met NCCN high-risk criteria from the validation cohort presented in Wysong et al.⁷⁰ Of note, this work was performed prior to introduction of the NCCN very high-risk category in 2021, and thereby only includes the former categorization of “high-risk.” As a result, this is not reflective of the most up-to-date classification. The proposed algorithm, which is not currently part of clinical guidelines, starts with NCCN high-risk cSCC patients who subsequently undergo 40-GEP testing and staging by BWH/AJCC8 with the following proposed management strategy⁷⁰:

Table 2: Proposed Risk-Aligned Management Plans within the NCCN Guidelines Framework

(adapted from ⁶⁷):

Additional publications from Castle Biosciences have proposed ways for the 40-GEP to be used in clinical practice.^71-77 Nevertheless, NCCN Guidelines have not incorporated use of the 40-GEP and recommend follow up for high-risk patients to occur every 3-6 months for two years, then 6-12 months for 3 years, then annually for life. This includes complete skin and regional lymph node exam and imaging can be considered if clinical exam is insufficient for following the disease. Adjuvant radiation and systemic therapy would not be recommended, unless the patient has positive post-surgical margins and further surgery is not feasible.⁸ NCCN recommendations for very high-risk patients include follow up every 3-6 months for 2 years, then every 6 months for 3 years, then every 6-12 months for life, along with imaging if clinical exam is insufficient for following disease. SNLB is recommended for very high-risk patients who have multiple risk factors placing them in the very high-risk group with a normal exam of the draining nodal basin. Systemic therapy with or without radiation therapy is recommended in cases of positive margins after MMS or other PDEMA, residual disease after definitive therapy, and for non-surgical candidates. Adjuvant radiation therapy is recommended for cases with positive-post surgical margins if further surgery is not feasible and with negative margins in the setting of extensive perineural, large, or named nerve involvement or other poor prognostic features.⁸ 

In a Castle Biosciences sponsored study, Arron et al⁷¹ convened a panel of Mohs surgeons, surgical oncologists, and radiation oncologists from academic medical centers and community practices and discussed rationales and scenarios where the 40-GEP test result may have clinical utility. Of note, all authors on the panel had disclosed ties to Castle Biosciences. The panel stated that the 40-GEP should not be used as surrogate for standard of care treatment but as an additional data point when determining individualized management plans for high-risk patients.⁷¹ Possible uses of the GEP are explored in relation to nodal evaluation, adjuvant radiation therapy, and follow-up frequency as presented in Table 3 (adapted from ⁷¹).

Table 3. GEP Test Results That May Impact Decisions on Follow-up and Surveillance Intensity During the First Two Years after Diagnosis (Adapted from Arron et al⁶⁸).

Au et al,⁷² describes two retrospective analyses of NCCN very high-risk cases, one with a Class 1 result and the other with Class 2B result. Case 1 was a 65-year-old male with history of renal and liver transplantation and a 1.3 cm poorly differentiated cSCC on his left temple (BWH T2a and AJCC8 T1, NCCN very high-risk). He underwent MMS with possible residual disease. The patient declined further treatment and was disease free at 4 years. He was retrospectively found to have a 40-GEP Class 1 result. Patient 2 was a 69-year-old male with history of liver transplant and 1.5 cm poorly differentiated cSCC on his left temple (BWH T2a, AJCC8 T1, NCCN very high-risk) with subsequent clearing following MMS. The patient developed metastatic cSCC within three months of MMS and was retrospectively found to have a 40-GEP Class 2B result. Au et al⁷² state that Case 1 highlighted a biologically less aggressive tumor that did not recur despite incomplete surgical clearance whereas Case 2 highlighted a more aggressive tumor in spite of clear surgical margins with MMS. The authors state that adjuvant treatment may have been appropriate for Case 2 earlier in the disease course.⁷²

Castle Biosciences has also investigated the clinical impact of the 40-GEP through presentation of clinical vignettes and clinician surveys at conferences showing that clinicians would alter their decisions to perform a SLNB and recommendations for adjuvant radiation therapy and chemotherapy/immunotherapy given 40-GEP results.^73,74 In a Castle Biosciences sponsored study, Singh et al published an algorithm for incorporation of 40-GEP test results into treatment decisions for high-risk cSCC patients. However, this algorithm does not consistently distinguish between NCCN high and very-high risk groups, which have different underlying rates of metastasis, nor does it systematically incorporate staging.⁷⁵ A recent review of cSCC notes that additional data is needed on how best to integrate gene expression profiling into clinical practice⁷⁸ and outcomes data from prospective studies with documented specific changes in management is forthcoming.⁷⁹

Saleeby et al⁷⁹ report on preliminary results from the prospective Clinical Utility and Health Outcomes Study (UTILISE), which aimed to demonstrate patterns of test utilization, distribution of results across clinicopathologic variables, and impact on clinician recommendations for management in Medicare-eligible high-risk patients. The study was conducted at 5 clinical sites and involved 11 unique clinicians (8 dermatologists and 3 dermatology-based physician assistants). The study consisted of two sequential phases, the Lead-in Phase and Clinical Utility Phase. During the Lead-in Phase, clinicians recorded a treatment plan assessment before receiving 40-GEP results for at least five patients. After completion of treatment plan assessment for five patients, clinicians were able to enroll new patients into the clinical utility phase. The Lead-in Phase included a second (post-test) treatment assessment completed after 40-GEP results receipt.⁷⁹

At time of publication, the Lead-in Phase consisted of 31 patients, with 81% having two or more risk factors. 68% (n=21) received a Class 1 result, 25% (n=~8) received a Class 2A result, and 6% (n=1-2) received a Class 2B result, with a median follow up of 41.7 weeks. At time of analysis, one patient with a Class 2A result experienced regional metastasis. The Clinical Utility Cohort included 59 patients, 60.3% of whom had more than one risk factor. This cohort was comprised of 88% Class 1 (n=52) results, and 12% Class 2A results. There were no Class 2B results and no cases of metastasis with a median follow up of 22.5 weeks. The impact on clinician perception of metastatic risk is depicted in Table 4 adapted from Saleeby et al and the impact of the 40-GEP on intensity of management is depicted in Table 5.⁷⁹ 24% of the 11 clinicians reported that they made management changes based on 40-GEP results, 15.5% reported that the test had no impact on their treatment plan, and 58.6% stated that 40-GEP results increased confidence in their original treatment plan. Thereby, the majority (74.1%) of physicians did not change management because of 40-GEP results. The precise changes to management outside of the broad categories “low, moderate, high intensity” management were not published. Clinical outcomes are not available at time of publication, with exception of the single Class 2A patient who developed metastasis.

Table 4. 40-GEP Impact on Clinician Perception of Metastatic Risk

Table 5. 40-GEP Impact on Intensity of Management

The Class 2B results is rare as shown in the study by Saleeby et al described above. In addition, Hooper et al⁷⁶ reviewed 2455 samples submitted during the first year of clinical testing (August 31, 2020-August 31, 2021) and found that 68.8% (n=1687) had a Class 1 result, 28.3% (n=696) had a Class 2A result, and 2.9% (n=72) had a Class 2B result. The majority of Class 1 results were identified in samples with 1–2 risk factors. Class 2A and 2B results were more likely to be found in samples from patients with a greater number of risk factors when compared with Class 1, demonstrating consistency with the increased risk associated with Class 2A and 2B results.⁷⁹ 

Finally, test performance of the 40-GEP has been reported in a population that is 73.3% male and 99.3% Caucasian,⁶⁸ which is representative of the majority of patients with cSCC. Additional studies inclusive of patients of color, who have been shown to have a higher metastatic rate compared to Caucasians,^80-83 are forthcoming.

Patients with cSCC stand to benefit from improved risk stratification to identify those at highest risk of poor outcomes, such as metastasis. However, to demonstrate clinical utility in risk stratification, a new biomarker must provide additional risk stratification (clinical validity) to currently available risk stratification measures (i.e. the combination of staging and clinical plus pathological factors) within a guideline-based standard of care therapeutic framework (i.e. NCCN) to meaningfully reclassify patients with respect to clinical decision thresholds in a manner that improves patient outcomes. To date, this has not been demonstrated for the 40-GEP or other similar tests.

The intended use population for the 40-GEP consists of cSCC patients with one or more high risk factors, aligning with the NCCN high-risk and very high-risk groups. This population is too broad and includes patients who are unlikely to benefit from 40-GEP results. Metastatic rates from the NCCN validation of 10,196 tumors by Stevens et al are significantly lower than metastatic rates presented in the cohort used to develop and validate the 40-GEP. The relatively low underlying rate of metastasis in the NCCN high risk group (<1%) questions the clinical utility of the 40-GEP in this group of patients, as hazard ratios for metastasis of 2.41 (for Class 2A result) to 6.22 (Class 2B result) likely would not lead to significant clinical management changes in a risk category with a baseline metastatic rate of <1%. The NCCN very high-risk group also demonstrated a lower overall rate of metastasis in the NCCN validation when compared to 40-GEP validation cohorts. The difference between metastatic rates in the 40-GEP validation cohort and the NCCN validation questions the generalizability of the patient population that led to the development of the 40-GEP and likely necessitates PPV adjustment to account for the lower prevalence of metastasis outside of the 40-GEP validation cohort. Of note, the prospective study conducted by Saleeby et al reported one patient with metastasis out of 90 (1.1%) high and very high-risk patients, although the study was not complete at time of publication and follow-up data is expected.

The 40-GEP is intended to be used and evaluated in combination with current risk assessment tools (to see their added improvement). NCCN guidelines direct risk assessment after biopsy into either the low, high, or very high-risk groups and refer to other staging systems, such as AJCC8, as additional resources of reference. The American Academy of Dermatology recommends tumor stratification of localized cSCC using NCCN guidelines to provide practical clinical guidance for management and follow up and demonstrates preference for BWH in terms of the most accurate prognostication tool in localized cSCC. The analyses presented in 40-GEP publications to date have not demonstrated clinical validity and clinical utility in this clinical context, as the multivariate analysis with staging was not conducted within the NCCN high risk and very high risk groups separately, but rather in the entire intended use population which includes both high risk (largely stage 1 tumors with low risk for metastasis and higher risk of local recurrence) and very high risk patients (generally higher stage with greater risk of metastasis) and so the benefit of NCCN risk stratification is lost. Given the low metastatic rate in the NCCN high risk category and questionable clinical utility, the clinical validity and clinical utility of the 40-GEP in addition to staging remains to be demonstrated in the NCCN very high-risk group. Importantly, any added risk stratification (clinical validity) within staging must also demonstrate clinical utility with respect to clinical-decision thresholds within the framework of national guidelines.

Publications from Castle Biosciences have proposed frameworks for clinical use of the 40-GEP. However, these are inconsistent and management intensity classification approaches with respect to follow-up frequency, nodal assessment, and adjuvant therapy for Class 1, 2A, and 2B tumors in combination with current risk assessment tools within the NCCN risk framework have not been validated nor have they been supported by independent experts outside of Castle Biosciences. It is also important to note that across all BWH stages, the majority of metastases per stage occur in tumors with Class 1 and Class 2A 40-GEP results and the Class 2B result accounts for a minority of metastases in each stage, and so management changes must be made with care. While there is potential for the 40-GEP to identify patients who are at highest risk of metastasis and for selection of patients for specific treatment options such as adjuvant RT, this has not been convincingly demonstrated at this time. Furthermore, the NCCN expert panel on cSCC states that molecular tests that offer risk stratification for cutaneous squamous cell carcinoma have not yet shown to have additional prognostic benefit over traditional staging in NCCN "very high-risk" squamous cell carcinoma, the patient group in which additional prognostic stratification would be of potential value. 

Finally, test performance of the 40-GEP has been reported in a population that is predominantly male and essentially all Caucasian, which is representative of the majority of patients with cSCC. Although incidence is highest in non-Hispanic white populations, the risk of metastasis is greater in patients of color. Further studies should include representation of test performance characteristics inclusive of and addressed in patients of color.

In conclusion, the current evaluated tests at the time of drafting this policy have not yet demonstrated definitive additional risk stratification beyond that provided by available clinical, pathological, and staging criteria within the NCCN risk framework. Promising evidence is in development for use in selection of patients for adjuvant RT. This contractor will continue to monitor the evidence and may modify coverage based on new information in the pertinent literature and society recommendations.

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