Temozolomide and Irinotecan in Patients With MGMT Silenced Colorectal Cancer After Adjuvant Chemotherapy

Surgical resection is curative for 75% of stage II and 50% of stage III colon cancer patients. The magnitude of benefit of adjuvant chemotherapy in terms of disease-free (DFS) and overall survival (OS) varies according to TNM stage and microsatellite status. Standard adjuvant chemotherapy includes fluoropyrimidine and oxaliplatin regimens for up to six months.

Circulating tumor DNA (ctDNA) detected after surgical resection reflects the presence of micrometastatic disease and pivotal observational studies addressed the prognostic value of ctDNA in the post-surgical setting. Adjuvant chemotherapy can promote the clearance of ctDNA, and ctDNA clearance after adjuvant chemotherapy is prognostic for better DFS in patients with stage III resected cancers and post-operative positive ctDNA.

ctDNA may be investigated as a potential real-time surrogate biomarker of the efficacy of adjuvant therapy, but suggest that patients with ctDNA persistence after standard chemotherapy might be "molecularly metastatic" and may benefit from additional "consolidation" non-cross resistant strategies aimed at clearing micrometastatic disease.

Temozolomide has modest but non-negligible activity (about 10%) in chemo-refractory patients with MGMT methylated mCRC. The response rate to temozolomide-based therapy in pretreated patients is increased to up to 20% when restricting the focus on those with MGMT IHC-negative/MGMT methylated and MSS cancers Significant activity (ORR 26%) and favorable safety profile were reported by the combination of temozolomide and irinotecan (TEMIRI regimen) in patients with pretreated MGMT methylated/MSS mCRC, thus suggesting that the two agents may have synergist activity in line with preclinical data.

Based on all these considerations, there is a strong rationale for investigating TEMIRI regimen as consolidation non-cross resistant therapy in a liquid-biopsy driven interventional trial.

Eligible patients with MGMT-silenced, MSS, radically resected CRC and detectable ctDNA after standard chemotherapy will be enrolled and will receive 6-month post-adjuvant/consolidation TEMIRI (given for up to 6 monthly cycles).

Surgical resection is curative for 75% of stage II and 50% of stage III colon cancer patients. The magnitude of benefit of adjuvant chemotherapy in terms of disease-free (DFS) and overall survival (OS) varies according to TNM stage and microsatellite status. Specifically, in patients with stage II microsatellite stable (MSS) tumors and high risk clinical features (i.e. pT4, lymphovascular invasion, perineural invasion, bowel obstruction, positive surgical margins and inadequately sampled lymph nodes) adjuvant therapy with fluoropyrimidines conditioned a modest but significant DFS benefit, while oxaliplatin-based therapy may be offered to patients with poor prognosis such as those with pT4 disease. In patients with stage III disease adjuvant therapy with oxaliplatin and fluoropyrimidine combinations significantly improved DFS and OS in phase 3 randomized trials. However, oxaliplatin is burdened by dose-cumulative and potentially long-lasting neurotoxicity. Therefore, three-month duration of oxaliplatin-based chemotherapy was compared to six-month in six randomized trials including patients with resected stage (II)/III colon cancer. In the pooled analysis of such trials (IDEA Collaboration), the non-inferiority for DFS of three months adjuvant oxaliplatin-based chemotherapy was not formally demonstrated. However, absolute DFS loss with 3- month therapy was clearly unsignificant from a clinical point-of-view and 3-month duration of oxaliplatin-based adjuvant chemotherapy is now recommended in patients with low risk disease (pT3N1) and particularly when adopting a capecitabine-based regimen (CAPOX).

Circulating tumor DNA (ctDNA) detected after surgical resection reflects the presence of micrometastatic disease and pivotal observational studies addressed the prognostic value of ctDNA in the post-surgical setting. Detectable ctDNA after surgery is prognostic for DFS in patients with resected colon cancer with high specificity in predicting recurrence (-100%), reinforcing its promising role for guiding trials on post-surgical intensification strategies, but ctDNA is also endowed with suboptimal sensitivity (70%), thus limiting its potential usefulness to guide the complete omission of adjuvant chemotherapy. Regarding the impact of adjuvant chemotherapy on micrometastatic disease, adjuvant chemotherapy was able to clear ctDNA in individual patients with resected stage II tumors. Moreover, ctDNA clearance after adjuvant chemotherapy was prognostic for better DFS in patients with stage III resected cancers and post-operative positive ctDNA. Collectively, these data highlight that ctDNA may be investigated as a potential real-time surrogate biomarker of the efficacy of adjuvant therapy, but suggest that patients with ctDNA persistence after standard chemotherapy might be "molecularly metastatic" and may benefit from additional "consolidation" non-cross resistant strategies aimed at clearing micrometastatic disease.

Temozolomide displayed limited activity (overall response rate \[ORR\] 9%) in patients with heavily pretreated metastatic colorectal cancer (mCRC) with MGMT promoter methylation assessed by means of a qualitative assay - methylation-specific PCR. However, even if MGMT promoter methylation is found in up to 40% of patients with colorectal cancer, in-silico analyses and translational analyses showed that only a subset of these tumors (- 10% of all comers) display lack of MGMT expression and negative MGMT IHC staining. In keeping with findings, correlative studies of phase 2 trials showed that MGMT immunohistochemical negativity and higher MGMT methylation % by quantitative assays are associated with temozolomide activity. Finally, proficiency of the mismatch repair is needed for alkylators activity. Therefore, temozolomide might be considered a tailored chemotherapy in patients with MGMT silenced tumors (i.e. those with MGMT negative expression and MGMT promoter methylation) and microsatellite stable (MSS) tumors. Significant activity (ORR 26%) and favorable safety profile were reported by the combination of temozolomide and irinotecan (TEMIRI regimen) in patients with pretreated MGMT methylated/MSS mCRC, thus suggesting that the two agents may have synergist activity in line with preclinical data.

Moving from this rationale we designed a phase 2 proof-of-concept trial aimed at evaluating the activity in terms of ctDNA clearance or "seroreversion" after TEMIRI regimen as a post-adjuvant strategy in patients with MGMT silenced, MSS colorectal cancer (CRC) with positive ctDNA after oxaliplatin-based adjuvant standard chemotherapy.

Eligible patients with MGMT-silenced, MSS, radically resected CRC and detectable ctDNA after standard chemotherapy will be enrolled and will receive 6-month post-adjuvant/consolidation TEMIRI (given for up to 6 monthly cycles).

Eligibility Criteria:
Inclusion Criteria:

* Have provided written informed consent prior to any study specific procedures.
* Age ≥ 18 years.
* Histologically confirmed diagnosis of stage III or T4N0 stage II colon cancer (located 12 cm from the anal verge by endoscopy and above the peritoneal reflection at surgery) or histologically confirmed diagnosis of locally-advanced resectable rectal cancer (proximal margin located at \< 12 cm from the anal verge).
* Radical surgery for patients with colon cancer or preoperative (chemo)-radiotherapy followed by radical surgery for patients with rectal cancer.
* Completion of at least 3 months of oxaliplatin-based (CAPOX or FOLFOX) adjuvant chemotherapy (or candidate to oxaliplatin-based adjuvant chemotherapy if post-surgery pre-screening).
* Availability of the archival FFPE tumor tissue obtained prior to any treatment.
* Acceptance to undergo all the interventional and exploratory liquid biopsies.
* Absent MGMT expression by IHC, MGMT promoter methylation by pyrosequencing (\> 5%) and MSS by standard assessment.
* Presence of ctDNA in the liquid biopsies collected at 2-6 weeks after the last dose of standard adjuvant chemotherapy
* Eastern Cooperative Oncology Group (ECOG) performance status 0-1.
* Completion of adjuvant chemotherapy for a duration of at least three months.
* Adequate organ function as defined below:
* Hematological function indicated by all of the following:

White Blood Cell (WBC) count ≥ 2 x 109/L Absolute neutrophil count (ANC) ≥ 1.5 x 109/L Platelet count ≥ 100 x 109/L Hemoglobin ≥ 9 g/dL (patients may have transfusions and/or growth factors to attain adequate Hb).

- Liver function indicated by all of the following: Total bilirubin \< 1.5 x upper limit of normal (ULN) Aspartate transaminase (AST) and alanine aminotransferase (ALT) \< 3 x ULN Alkaline phosphatase (ALP) \< 2 x ULN.

- Renal function indicated by all of the following: Serum creatinine \< 1.5 x ULN or calculated creatinine clearance \> 40 ml/min.

- Coagulation indicated by all of the following: INR ≤ 1.5 and aPTT ≤ 1.5 x ULN within 7 days prior to the start of study treatment for patients not receiving anti-coagulation. a. NOTE: The use of full-dose oral or parenteral anticoagulants is permitted as long as the INR or aPTT is within therapeutic limits (according to the medical standard of the enrolling institution) and the patient has been on a stable dose of anticoagulants for at least two weeks prior to the start of study treatment.

* Carcinoembryonic antigen (CEA) level ≤ 10 ng/ml.
* No evidence of distant metastases or loco-regional disease by computed tomography scan or magnetic resonance imaging.
* Male subjects with female partners of childbearing potential must be willing to use adequate contraception as approved by the investigator (barrier contraceptive measure or oral contraception).
* Women of childbearing potential must have a negative blood pregnancy test at the baseline visit and must be willing to use adequate contraception as approved by the investigator (barrier contraceptive measure or oral contraception). For this trial, women of childbearing potential are defined as all women after puberty, unless they are postmenopausal for at least 12 months, are surgically sterile, or are sexually inactive.

Exclusion Criteria:

* History of another neoplastic disease, unless in remission for ≥ 5 years. 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 excluded.
* Had an incomplete diagnostic colonoscopy and/or polyps removal.
* Microscopic or macroscopic evidence of residual tumor (R1 or R2 resections). Patients should never have had any evidence of metastatic disease (including presence of tumor cells in the peritoneal lavage).
* Current or recent treatment with another investigational drug or participation in another investigational study.
* Inability to swallow pills.
* Active infection requiring intravenous antibiotics at the start of study treatment.
* Evidence of any other disease, neurologic or metabolic dysfunction, physical examination finding or laboratory finding giving reasonable suspicion of a disease or condition that contraindicates the use of any of the study medications, puts the patient at higher risk for treatment-related complications or may affect the interpretation of study results.

Patient unable to comply with the study protocol owing to psychological, social or geographical reasons.

* Is pregnant or breastfeeding, or expecting to conceive or father children within the projected duration of the study.
* Pregnant or lactating women. Women of childbearing potential with either a positive or no pregnancy test at baseline. Postmenopausal women must have been amenorrheic for at least 12 months to be considered of non-childbearing potential. Sexually active males and females (of childbearing potential) unwilling to practice contraception (barrier contraceptive measure or oral contraception) during the study and until 6 months after the last trial treatment.
* Clinically significant (i.e. active) cardiovascular disease, for example cerebrovascular accidents ≤ 6 months prior to start of study treatment, myocardial infarction ≤ 6 months prior to study enrolment, unstable angina, New York Heart Association (NYHA) Functional Classification Grade II or greater congestive heart failure, or serious cardiac arrhythmia uncontrolled by medication or potentially interfering with protocol treatment.
* Known presence of one of the following UGT1A1 1(TA)6/UGT1A1 36(TA)5; UGT1A1 28(TA)7/UGT1A1 37(TA)8 (homozygous genotype).
* Known presence of galactose intolerance, total lactase deficiency or glucose-galactose malabsorption