Arethusa consists of three different phases - SCREENING Phase, PRIMING phase, and TRIAL
- SCREENING PHASE: RAS-mutated metastatic CRC patients who have failed standard therapies
are tested for MMR status. MMR-Deficient (MMR-D) patients proceed directly to TRIAL
Phase and are treated with pembrolizumab. MMR-Proficient (MMR-P) patients are further
tested for O6-methylguanine-DNA methyltransferase (MGMT) expression by
immunohistochemistry and promoter methylation analysis. MGMT negative patients proceed
to PRIMING phase. MGMT positive patients go off-study.
- PRIMING PHASE: MMR-P MGMT-negative patients receive TMZ therapy until progression. At PD
(disease progression) patients are biopsied to determine the tumor mutational burden
(TMB). Patients with TMB < 20 mutations/megabase go off-study. Patients with TMB >20
mutations/megabase, proceed to trial phase.
- TRIAL PHASE: Eligible patients are treated with pembrolizumab until disease progression,
unacceptable toxicity, or up to 24 months in patients without disease progression.
Entry criteria for SCREENING Phase
1. Histological confirmed diagnosis of metastatic colorectal cancer;
2. Documented RAS-extended mutations in the archival sample.
3. ECOG performance status 0-1.
4. SCREENING phase informed consent signed.
5. Understanding and accepting the need for undergoing at least two tumor biopsies if
eligible for PRIMING Phase.
6. Age ≥ 18 years;
7. Imaging documented failure of previous standard CRC therapies including
fluoropyrimidine, oxaliplatin, plus or minus anti-angiogenics agents (bevacizumab,
8. 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 registration;
9. Availability of all diagnostic FFPE block or slides (primary tumor, metastases).
Formalin-fixed, paraffin embedded (FFPE) tissue blocks are preferred to slides.
10. Normal organ functions (see Table 6.1.4 below);
Entry Criteria for PRIMING Phase
1. Fulfillment of all the SCREENING inclusion criteria;
2. PRIMING informed consent signed;
3. Confirming the willingness to undergo at least two tumor biopsies,
4. Acceptance that if the mutational load determination from either one mandatory biopsy
is unfeasible for technical reasons (not enough tissue, substandard test performance,
etc.), access to TRIAL phase will not be possible.
5. ECOG performance status 0 or 1;
6. Following results in the SCREENING Phase tests:
- Proficient MMR status on tissue assessed by the IHC MSI test, (Bethesda panel);
- Negative score for the MGMT protein expression IHC test;
- Positive score for the MGMT promoter methylation Methyl BEAMing test performed on
7. Subjects and their partners must be willing to avoid pregnancy.
8. Normal organ functions as defined in the Table 6.1.4 below.
Entry Criteria for TRIAL Phase
1. Fulfillment of all the SCREENING inclusion criteria and Deficient or Proficient MMR
status on tissue assessed by the IHC MSI test, (Bethesda panel)
2. Fulfillment of all the PRIMING Phase inclusion criteria (only cohort P);
3. TRIAL Phase informed consent signed;
4. Imaging documented PD (Disease Progression) to TMZ (only cohort P).
5. A mutational load value > 20 mutations/MB at B-ML assay (only cohort P) ;
6. Women with childbearing potential and their partners must agree to continue using
highly contraceptive methods (see appendix 3).
7. Normal organ functions as defined in the Table 6.1.4 below. Specimens must be
collected within 10 days prior to the start of study treatment.
1. 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.
2. 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,
3. Has received prior systemic anti-cancer therapy including investigational agents
within 4 weeks [could consider shorter interval for kinase inhibitors or other short
half-life drugs] prior to allocation.
1. Note: Participants must have recovered from all AEs due to previous therapies to
≤Grade 1 or baseline. Participants with ≤Grade 2 neuropathy may be eligible.
2. Note: If participant received major surgery, they must have recovered adequately
from the toxicity and/or complications from the intervention prior to starting
4. Has received prior radiotherapy within 2 weeks of start of study treatment (with
pembrolizumab). Participants must have recovered from all radiation-related
toxicities, not require corticosteroids, and not have had radiation pneumonitis. A
1-week washout is permitted for palliative radiation (≤2 weeks of radiotherapy) to
5. Has received a live vaccine within 30 days prior to the first dose of study drug.
Examples of live vaccines include, but are not limited to, the following: measles,
mumps, rubella, varicella/zoster (chicken pox), yellow fever, rabies, and typhoid
vaccine. Seasonal influenza vaccines for injection are generally killed virus vaccines
and are allowed; however, intranasal influenza vaccines (eg, FluMist®) are live
attenuated vaccines and are not allowed.
6. Is currently participating in or has participated in a study of an investigational
agent or has used an investigational device within 4 weeks prior to the first dose of
a. Note: Participants who have entered the follow-up phase of an investigational study
may participate as long as it has been 4 weeks after the last dose of the previous
7. Has a diagnosis of immunodeficiency or is receiving chronic systemic steroid therapy
(in dosing exceeding 10 mg daily of prednisone equivalent) or any other form of
immunosuppressive therapy within 7 days prior to the first dose of study drug.
8. Has a known additional malignancy that is progressing or has required active treatment
within the past 3 years. Note: Participants with basal cell carcinoma of the skin,
squamous cell carcinoma of the skin, or carcinoma in situ (e.g. breast carcinoma,
cervical cancer in situ) that have undergone potentially curative therapy are not
9. Has known active CNS metastases and/or carcinomatous meningitis. Participants with
previously treated brain metastases may participate provided they are radiological
stable, i.e. without evidence of progression for at least 4 weeks by repeat imaging
(note that the repeat imaging should be performed during study screening), clinically
stable and without requirement of steroid treatment for at least 14 days prior to
first dose of study treatment.
10. Has severe hypersensitivity (≥Grade 3) to pembrolizumab and/or any of its excipients.
11. Has severe hypersensitivity (≥Grade 3) to temozolomide and/or any of its excipients.
12. Has active autoimmune disease that has required systemic treatment in the past 2 years
(i.e. with use of disease modifying agents, corticosteroids or immunosuppressive
drugs). Replacement therapy (eg., thyroxine, insulin, or physiologic corticosteroid
replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a
form of systemic treatment.
13. Has a history of (non-infectious) pneumonitis that required steroids or has current
14. Has an active infection requiring systemic therapy.
15. Has a known history of Human Immunodeficiency Virus (HIV). Note: No HIV testing is
16. Has a known history of Hepatitis B (defined as Hepatitis B surface antigen [HBsAg]
reactive) or known active Hepatitis C virus infection. Note: no testing for Hepatitis
B and Hepatitis C is required unless mandated by local health authority.
17. Has a known history of active TB (Bacillus Tuberculosis).
18. Has a history or current evidence of any condition, therapy, or laboratory abnormality
that might confound the results of the study, interfere with the subject's
participation for the full duration of the study, or is not in the best interest of
the subject to participate, in the opinion of the treating investigator.
19. Has known psychiatric or substance abuse disorders that would interfere with
cooperation with the requirements of the trial
20. Is pregnant or breastfeeding, or expecting to conceive or father children within the
projected duration of the study, starting with the screening visit through 120 days
after the last dose of trial treatment..