Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans, it exhibits a high tumor mutational burden and is more common in immunocompromised patients, which aimed to explore the impact of immunotherapy in this cancer. CSCC shows good response to anti-PD1 immunotherapy, and cemiplimab is the first FDA-approved and the only EMA-approved treatment for this tumor. However, 50% of patients won't respond to anti-PD1 and to date there is little evidence on the reasons for such a lack of effectiveness. Also, anti-PD1 immunotherapy is very safe, but some patients will develop adverse events, and anticipating severe adverse events might help in patients' management. The NGF-GRACE project aims to find biomarkers of response and toxicity, both in the blood and the tumor, using advanced technologies. The goal is to move towards more personalized treatments, better select patients, predict side effects, and improve our understanding of the immune system in CSCC.

The NGF-GRACE project focuses on identifying biomarkers of response and toxicity in patients with advanced cutaneous squamous cell carcinoma (CSCC) undergoing anti-PD1 treatment, being cemiplimab the unique approved drug in Europe. CSCC, characterized by a high mutational burden and increased risk in immunosuppressed patients, has exhibited significant sensitivity to anti-PD1 immunotherapy. By employing cutting-edge technologies such as Next Generation Flow Cytometry and the BD Rhapsody Single Cell System, the study aims to assess the immunological profile in peripheral blood and in the tumor tissue. The ultimate goal is to advance towards a more individualized therapeutic approach, identifying biomarkers that enhance patient selection, anticipate adverse events, and address knowledge gaps in the CSCC tumor immunology.

Background and Rationale CSCC is a type of skin cancer with one of the highest mutational burdens among solid tumors, making it particularly sensitive to immunotherapy, especially PD-1 blockade. Cemiplimab, an anti-PD-1 antibody, has become the first FDA and EMA-approved treatment for advanced CSCC. However, response rates are variable, with around 50% of patients showing clinical benefit. There is an urgent need to identify predictive biomarkers to guide patient selection, anticipate toxicity, and understand resistance mechanisms.

Study Objectives The main objective is to identify biomarker of response and toxicity to cemiplimab in patients with advanced cutaneous squamous cell carcinoma

Study Design The study will enroll approximately 30 patients with locally advanced or metastatic CSCC who will start cemiplimab treatment. Peripheral blood and tumor samples will be collected. A preliminary phase with 5 patients will define optimal sampling timepoints.

Methods Peripheral blood will be analyzed using Next Generation Flow Cytometry (NGF), applying a custom-designed 40-color antibody panel to characterize innate and adaptive immune cells. Also, soluble biomarkers will be evaluated. Single-cell technologies will be implemented for tumor evaluation.

Endpoints and Data Analysis Clinical response will be measured using RECIST 1.1, and toxicity will follow CTCAE v5.0. Statistical analyses will correlate immune profiles with outcomes using appropriate tests (e.g., chi-square, t-tests, ANOVA, Kaplan-Meier survival analysis).

Ethical Considerations The study complies with the Declaration of Helsinki, GCP, and GDPR. Informed consent is mandatory, and data confidentiality is ensured. Samples and data will only be used for approved research purposes.

Impact The NGF-GRACE project seeks to advance precision immunotherapy in CSCC by identifying predictive biomarkers that can improve patient selection, predict adverse events, and inform new therapeutic strategies. The study's innovative technologies and comprehensive design position it to fill critical knowledge gaps in CSCC immuno-oncology.

- Inclusion criteria At least 18 years old

Hepatic function:

1. Total bilirubin ≤1.5x upper limit of normal (ULN) (or ≤3x ULN, if liver metastases).
2. Patients with Gilbert's Disease and total bilirubin up to 3x ULN may be eligible after communication with and approval from the medical monitor
3. Transaminases ≤3x ULN (or ≤5x ULN, if liver metastases)
4. Alkaline phosphatase (ALP) ≤2.5x ULN (or ≤5x ULN, if liver or bone metastases) Renal function: Serum creatinine ≤2x ULN or estimated creatinine clearance >35 mL/min (according the method of Cockcroft and Gault) Creatine phosphokinase (CPK) (also known as CK [creatine kinase]) elevation ≤ grade 2

Bone marrow function:

a. Hemoglobin ≥9.0 g/dL b. Absolute neutrophil count (ANC) ≥1.5 x 109/L c. Platelet count ≥75 x 109/L Anticipated life expectancy >12 weeks

- Exclusion criteria:

1. - Patient who refuses to participate in the study.

2. Being unsuitable for cemiplimab treatment

3. Ongoing or recent (within 5 years) evidence of significant autoimmune disease that required treatment with systemic immunosuppressive treatments, which may suggest risk for immune-related adverse events (irAEs). The following are not exclusionary: vitiligo, childhood asthma that has resolved, type 1 diabetes, residual hypothyroidism requiring only hormone replacement, or psoriasis that does not require systemic treatment.

4. Untreated brain metastasis(es) that may be considered active.

   a. Note in clarification: Patients with previously treated brain metastases may participate provided that the lesion(s) is (are) stable (without evidence of progression for at least 6 weeks on imaging obtained in the screening period), and there is no evidence of new or enlarging brain metastases, and the patients do not require any immunosuppressive doses of systemic corticosteroids for management of brain metastasis(es) within 28 days of the first dose of REGN2810cemiplimab.

5. Immunosuppressive corticosteroid doses (>10 mg prednisone daily or equivalent) within 4 weeks prior to the first dose of REGN2810cemiplimab

   a. Note in clarification: Patients who require brief courses of steroids (eg, as prophylaxis for imaging studies due to hypersensitivity to contrast agents) are not excluded

6. Active infection requiring therapy, including positive tests for human immunodeficiency virus (HIV)-1 or HIV-2 serum antibody, hepatitis B virus (HBV), or hepatitis C virus (HCV)

7. History of pneumonitis within the last 5 years

8. Any anticancer treatment other than radiation therapy (chemotherapy, targeted systemic therapy, imiquimod, photodynamic therapy), investigational or standard of care, within 30 days of the initial administration of REGN2810 cemiplimab or planned to occur during the study period

9. History of documented allergic reactions or acute hypersensitivity reaction attributed to antibody treatments

10. Patients with allergy or hypersensitivity to REGN2810 cemiplimab or to any of the excipients must be excluded.

11. Patients with a history of solid organ transplant (patients with prior corneal transplants may be allowed to enroll after discussion with and approval from the medical monitor)

12. Breastfeeding

13. Positive serum pregnancy test (a false positive pregnancy test, if demonstrated by serial measurements and negative ultrasound, will not be exclusionary, upon communication with and approval from the medical monitor)

14. Receipt of live vaccines (including attenuated) within 30 days of first study treatment

15. Women of childbearing potential (WOCBP)*, or sexually active men, who are unwilling to practice highly effective contraception prior to the initial dose/start of the first treatment prior to the start of the first treatment, during the study, and for at least 6 months after the last dose.

    Optional criteria, depending on the therapy being investigated:

16. Prior treatment with an agent that blocks the PD-1/PD-L1 pathway (If a study is addressing prior unsuccessful treatment with PD-1/PD-L1 inhibitor, it is suggested to exclude patients who had suffered from ≥grade 3 irAE during previous treatment)

17. Prior treatment with other systemic immune-modulating agents within fewer than 28 days prior to the first dose of REGN2810cemiplimab. Examples of immune-modulating agents include therapeutic vaccines, cytokine treatments, or agents that target cytotoxic T-lymphocyte antigen 4 (CTLA-4), 4-1BB (CD137), or OX-40.

    1. Note in clarification: Prior treatment with imiquimod or other topical or intralesional immune modulators will not be exclusionary

Every patient who is going to be treated with cemiplimab is a potential candidate for this study considering the inclusion and exclusion criteria of the registry study, included in SmPC.

Individual participant data (IPD) that underlie the results reported in future publications (after de-identification) will be made available upon reasonable request. This includes data related to baseline characteristics, treatment administration, toxicity, tumor response, survival outcomes, and quality of life assessments

Data will be available starting 12 months after publication of primary results and for a minimum of 5 years thereafter.

Sharing Access Criteria: Qualified researchers with a methodologically sound proposal, as determined by the study steering committee, will be able to access the data. Requests should be directed to the study PI. A data access agreement will be required.