Pembrolizumab in MMR-Proficient Metastatic Colorectal Cancer Pharmacologically Primed to Trigger Hypermutation Status

Inclusion Criteria

Entry criteria for SCREENING Phase

• Histologically confirmed diagnosis of metastatic colorectal cancer.
• Documented RAS extended mutations in the archival sample (cohort P only).
• ECOG performance status 0-1.
• SCREENING phase informed consent signed.
• Understanding and accepting the need for undergoing two tumor biopsies if eligible for PRIMING Phase.
• Age ≥ 18 years.
• Availability of all diagnostic FFPE blocks (primary tumor and or metastases), or at least 20 slides (primary tumor and/or metastases). Formalin-fixed, paraffin embedded (FFPE) tissue blocks are preferred to slides.
• Normal organ functions.

Entry Criteria for PRIMING Phase

• Fulfilment of all the SCREENING inclusion criteria;
• PRIMING informed consent signed;
• Confirming the willingness to undergo two tumor biopsies,
• Acceptance that, if the mutational load determination is unfeasible for technical reasons (not enough tissue, substandard test performance, etc.), access to TRIAL phase will not be possible.
• Imaging documented failure of previous standard CRC therapies including fluoropyrimidine, oxaliplatin, irinotecan plus or minus antiangiogenics agents (Bevacizumab, Aflibercept, Regorafenib, others).
• At least one measurable tumor lesion as per RECIST v1.1. Lesions in previously irradiated areas or those that have received other loco-regional therapies (i.e.

percutaneous ablation) should not be considered measurable unless there is clear documented evidence of progression of the lesion since therapy. Imaging must be performed maximum within 28 days prior to enrolment.

• ECOG performance status 0 or 1;
• Following results in the SCREENING Phase tests:
• Proficient MMR status assessed by IHC or MSI-Low status defined by PCR (Bethesda panel);
• Negative score for the MGMT protein expression IHC test;
• Positive score for the MGMT promoter methylation performed on Tissue.
• Women with childbearing potential should complete a pregnancy test and be willing to use highly effective contraceptive methods.
• Normal organ functions.

Entry Criteria for TRIAL Phase

• 1. Fulfilment of all the SCREENING inclusion criteria and Deficient MMR status (IHC) or MSI-High status (PCR) (cohort D only).
• Fulfilment of all the SCREENING and PRIMING inclusion criteria (cohort P only).
• TRIAL Phase informed consent signed (both cohorts).
• Imaging documented PD to TMZ (cohort P only).
• A mutational load value > 20 mutations/MB at TMZ-ML assay (cohort P only).
• Imaging documented failure of previous standard CRC therapies including fluoropyrimidine, oxaliplatin, irinotecan plus or minus targeted agents (Bevacizumab, Aflibercept, Regorafenib, Cetuximab, Panitumumab, others) (cohort D only).
• At least one measurable tumor lesion as per RECIST v1.1. Lesions in previously irradiated areas or those that have received other loco-regional therapies (i.e. percutaneous ablation) should not be considered measurable unless there is clear documented evidence of progression of the lesion since therapy. Imaging must be performed maximum within 28 days prior to enrolment (both cohorts).
• Woman with childbearing potential should complete a pregnancy test and be willing to use highly effective contraceptive methods (both cohorts).
• Normal organ functions. Blood specimens must be collected within 10 days prior to the start of study treatment (both cohorts).

Exclusion Criteria

• A woman of child bearing potential who has a positive urine pregnancy test within 72 hours prior to allocation (see Appendix 3). If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required.
• Has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or with an agent directed to another stimulatory or co-inhibitory T-cell receptor (eg, CTLA-4, OX-40, CD137).
• Has received prior systemic anti-cancer therapy including investigational agents within 4 weeks [could consider shorter interval fo