The study was terminated on the basis on an evaluation of the efficacy and safety of the 4 included and treated patients.

The trial is designed as an investigator initiated prospective phase 2 study in patients with metastatic pMMR colorectal cancer (CRC) to determine the safety and efficacy of calcium electroporation (CaEP) performed concurrently with irreversible electroporation (IRE) followed by a PD-1 inhibitor (pembrolizumab).

The investigators hypothesize that Ca-EP targeting the primary CRC tumor combined with IRE targeting a metastasis will be a promising, safe two target approach to ensure sufficient immune response both locally and systemic to potentiate the efficacy of immunotherapy in patients with pMMR metastatic CRC.

Inclusion Criteria:

1. Signed informed consent

2. Age ≥ 18 years of age

3. Histologically confirmed stage IV, non-resectable pMMR colorectal cancer

4. The primary malignant tumor is left sided (cancer of the splenic flexure and cancer in regions distal to the splenic flexure, including the rectum)

5. The primary tumor is described as reachable at index endoscopy

6. At least two metastatic tumors must be present. One metastatic tumor, that in the opinion of the investigators is amenable to IRE, and at least one additional metastatic tumor that will not undergo IRE. Both lesions must be accessible for biopsy

7. Previous chemotherapy a), or b):

   1. Patients refractory to, intolerable of, or refusing standard chemotherapy options including 5-FU, irinotecan, oxaliplatin, bevacizumab and EGFR-inhibitors e.g. panitumumab/cetuximab (if RAS/RAF wild type)
   2. Patients with favourable biological disease, characterized by

   i. Non-progressive disease ≥ 6 months after last administration of prior 1st line chemotherapy or ≥ 18 months since diagnosis of metastatic disease

1. Patients in this category must have been exposed to an EGFR-inhibitor if RAS/RAF wild type

8. Life expectancy greater than 3 months

9. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1

10. Adequate bone marrow function:

a. Hemoglobin ≥ 5.6 mmol/L or ≥ 9 g/dL, b. Absolute neutrophil count (ANC) ≥ 1.5 × 109/L c. Platelet count ≥ 75 × 109/L

11. Adequate kidney function:

a. Estimated glomerular filtration rate (eGFR) ≥ 60 mL/min or creatinine ≤1.5 X upper limit of normal (ULN)

12. Adequate liver function:

a. Total bilirubin ≤ 1.5 × ULN b. Alanine aminotransferase (ALT): ≤2.5 X ULN or ≤5 X ULN for subjects with liver metastases c. Aspartate aminotransferase (AST): ≤2.5 X ULN or ≤5 X ULN for subjects with liver metastases d. Albumin: >25 g/L

13. Adequate coagulation function:

a. International Normalized Ratio (INR) ≤1.5 X ULN unless subject is receiving anticoagulant therapy as long as prothrombin Time (PT) or partial prothrombin time (PTT) is within therapeutic range of intended use of anticoagulants b. Activated Partial Thromboplastin Time (aPTT) ≤1.5 X ULN unless subject is receiving anticoagulant therapy as long as PT or PTT is within therapeutic range of intended use of anticoagulants

14. Follow the conditions regarding fertility, pregnancy, or lactation:

1. Female and male participants of reproductive potential (for definition refer to appendix 16.1) must agree to avoid becoming pregnant or impregnating a partner, respectively, while receiving pembrolizumab and for 120 days after the last dose
2. Participants of reproductive potential must use (or have their partner use) an acceptable method of contraception, as outlined in appendix 16.1, during heterosexual activity, while receiving pembrolizumab and for 120 days after the last dose
3. Women of reproductive potential (WORP) must have a negative serum or urine pregnancy test (minimum sensitivity 25 IU/L or equivalent units of HCG) within 72 hours prior to receiving the first dose of pembrolizumab.
4. Women must not be breastfeeding.

Exclusion Criteria:

1. Prior treatment with an immune checkpoint inhibitor (e.g., anti-PD-1, anti-PD-L1, anti-PD-L2 agent, or anti-CTLA-4 agent)

2. Concurrent treatment with an investigational medicinal product

3. Radiotherapy or major surgery within the last two weeks prior to entering the study

4. Any serious or uncontrolled medical disorder that, in the opinion of the investigator, may increase the risk associated with study participation or study drug administration, impair the ability of the subject to receive protocol therapy, or interfere with the interpretation of study results E.g

   1. Uncorrectable coagulation disorder.
   2. Highly inflamed gastrointestinal tissue which is ulcerated and bleeding
   3. Known history of, or any evidence of interstitial lung disease or active, non-infectious pneumonitis
   4. Has known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the study

5. Patients should be excluded if they have an active, known or suspected autoimmune disease (except thyroiditis with replacement therapy and type I diabetes mellitus).

6. Patients should be excluded if they have a known history of human immunodeficiency virus (HIV) (HIV 1/2 antibodies), active chronic, acute hepatitis B (e.g., HBsAg reactive), or hepatitis C (e.g., HCV RNA is detected).

7. Patients should be excluded if they have a condition requiring systemic treatment with either corticosteroids (> 10 mg daily prednisone equivalents) or other immunosuppressive medications within 14 days of study drug administration. Inhaled or topical steroids and adrenal replacement doses > 10 mg daily prednisone equivalents are permitted in the absence of active autoimmune disease.

8. Allergies and Adverse Drug Reaction:

   i. History of allergy to study drug components ii. History of severe hypersensitivity reaction to any monoclonal antibody

9. Patients are excluded if they have active brain metastases or leptomeningeal metastases. Subjects with brain metastases are eligible if metastases have been treated and there is no magnetic resonance imaging (MRI) evidence of progression for [lowest minimum is four weeks or more] after treatment is complete and within 28 days prior to the first dose of nivolumab administration. There must also be no requirement for immunosuppressive doses of systemic corticosteroids (> 10 mg/day prednisone equivalents) for at least two weeks prior to study drug administration

10. Absolute contraindications for IRE:

    1. Implanted pacemaker or ICD unit.
    2. History of epilepsy
    3. History of cardiac (ventricular) arrhythmia
    4. Recent myocardial infarction
    5. Congestive heart failure (>NYHA class 2)
    6. Uncontrollable hypertension

11. Relative contraindications for IRE:

    1. Atrial fibrillation
    2. Combined severe stenosis of the common hepatic artery and main portal vein branch