An upfront-intensified treatment combining all the three active cytotoxic agents in metastatic colorectal cancer (mCRC) including fluoropyrimidines, oxaliplatin, irinotecan (FOLFOXIRI) plus antiangiogenic blockade with bevacizumab significantly improved survival. No biomarkers are available for predicting sensitivity/resistance to single chemotherapeutic drugs, the simultaneous delivery of all active chemotherapeutic agents might overcome resistance to single drugs.

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. Clinical and preclinical synergy has been reported for combination of temozolomide with irinotecan and fluoropyrimidines. Temozolomide could be regarded as a "targeted" chemotherapy for patients with MSS and MGMT silenced tumors. In this subgroup of patients, an intensified triplet upfront regimen including temozolomide, fluoropyrimidines, irinotecan, associated with bevacizumab, could be a novel combination in molecularly super-selected mCRC patients.

Moving from this, the investigators designed this open-label, monocentric, phase 1b study evaluating the safety of the combination regimen 5-fluorouracil, leucovorin, irinotecan, temozolomide and bevacizumab in patients with MGMT silenced and MSS mCRC.

The study will consist in a dose-escalation assessment of the safety of the treatment in subjects with previously untreated MGMT silenced, MSS mCRC. A 3 + 3 design will be used to assess the maximum tolerated dose (MTD) or maximum tested dose of the combination FLIRT-bevacizumab. Upon completion of the phase 1b part, the phase 2 part of the study will start.

An upfront-intensified treatment combining all the three active cytotoxic agents in mCRC including fluoropyrimidines, oxaliplatin, irinotecan (FOLFOXIRI) plus antiangiogenic blockade with bevacizumab significantly improved progression-free survival (PFS), overall survival (OS) and objective response rate (ORR) compared to standard FOLFIRI and bevacizumab irrespective to RAS/BRAF status, at price of higher rate of specific toxicities. Advantages of an intensified regimen include: 1) exposure to all active available drugs, since more than 10-15% of patients would not receive any second-line therapy due to early performance status deterioration; 2) the chance of achieving a high rate (around 36%) of secondary R0/R1 resection of metastases in patients with liver-limited and initially unresectable liver metastases.

Furthermore, results from the phase 3 TRIBE2 study showed that the intensified upfront regimen FOLFOXIRI-bevacizumab followed by the pre-planned reintroduction of the same agents after progressive disease provided a statistically significant and clinically relevant survival benefit when compared with the pre-planned sequential administration of FOLFOX-bevacizumab and FOLFIRI-bevacizumab in unresectable patients with mCRC. Therefore, FOLFOXIRI-bevacizumab regimen is recommended by all major guidelines as one of the possible upfront treatment options for mCRC, and is used in the clinical practice mainly for patients with highly aggressive disease (such as those with right sided and/or RAS or BRAF mutated). Notably, since no biomarkers are available for predicting sensitivity/resistance to single chemotherapeutic drugs, the simultaneous delivery of all active chemotherapeutic agents might overcome resistance to single drugs.

MGMT promoter methylation is found in about 40% of colorectal tumors. MGMT deficiency impairs DNA repair following administration of several alkylating agents, including temozolomide. Temozolomide has limited single-agent activity (around 10%) in patients with pretreated MGMT methylated mCRC. Promising activity has been reported for temozolomide in combination with the potentially synergic drug irinotecan (TEMIRI regimen) in clinically and molecularly selected patients. In a recent phase 2 randomized trial, capecitabine in combination with temozolomide (CAPTEM regimen) displayed similar activity and efficacy with respect to standard FOLFIRI as second-line therapy for MGMT methylated RAS mutated mCRC.

Heterogeneity of MGMT promoter methylation and residual MGMT protein expression might account for lack of activity of temozolomide in patients with MGMT promoter methylation assessed by means of a qualitative-only assay, i.e. methylation-specific PCR (MSP), which has been used as selection assay for patients' enrollment in published trials. Exploratory analyses have consistently shown the role of quantitative assessment of MGMT promoter methylation by means of digital PCR (methylBEAMing) and MGMT protein expression by immunohistochemistry (IHC) as potential predictive factors in mCRC patients treated with temozolomide. In the randomized phase 2 CAPTEM versus FOLFIRI second-line trial, patients with retained MGMT positivity by IHC had poorer outcomes in terms of PFS, OS and disease control rate (DCR: interaction test with arm: P=0.028). Any residual MGMT protein expression has been associated with lack of response to temozolomide across different trials, further supporting the restriction of temozolomide-based therapies for patients with MGMT IHC negativity coupled with gene methylation (MGMT silencing).

Mismatch repair deficiency/microsatellite instability (MSI) has been linked to innate resistance to several alkylating chemotherapeutic agents, including temozolomide, since cytotoxicity of these agents strictly relies on functional mismatch repair. Therefore, patients with MSI-high mCRC are excluded from temozolomide-based therapy.

Temozolomide could be regarded as a "targeted" chemotherapy for patients with MSS and MGMT silenced tumors. In this subgroup of patients, an intensified triplet upfront regimen including temozolomide, fluoropyrimidines, irinotecan, associated with bevacizumab, could be a novel combination in molecularly hyperselected mCRC patients.

Moving from this rationale the investigators designed this phase 1b trial assessing safety, recommended dose and preliminary activity of 5-fluoruracil, irinotecan, temozolomide and bevacizumab (FLIRT-bevacizumab) as a biomarker-guided initial therapy for patients with MGMT silenced and MSS mCRC.

The study will consist in a dose-escalation assessment of the safety of the treatment in subjects with previously untreated MGMT silenced, MSS mCRC.

A 3 + 3 design will be used to assess the maximum tolerated dose (MTD) or maximum tested dose of the combination FLIRT-bevacizumab.

The MTD will be defined as the dose level at which ≥2/3 or ≥2/6 subjects experience a dose-limiting toxicity (DLT). When the MTD or maximum tested dose has been determined or reached, the RP2D to be tested in a future phase II trial will be one dose level below the MTD or the maximum tested dose if MTD will not be reached. At least 6 patients should be treated at the RD during the dose escalation.

The treatment will consist of an induction period of four 28-day cycles of FLIRT- bevacizumab followed by maintenance regimen of 5-FU/LV-bevacizumab administered every 14 days in combination with per os temozolomide according to dose level over days 1-5 every 28 days in patients without progressive disease at the end of the induction period. Patients will undergo tumor assessment at baseline and every 8 ± 1 weeks until confirmed disease progression, unacceptable toxicity, withdrawal of consent, death, whichever occurs first. The treatment will continue until progressive disease, unacceptable toxicities, or consent withdrawal.

The phase 1b part of the study has been completed and the RP2D of temozolomide is 150 mg/sqm on days 1-5 every 28 days. The phase 2 part is ongoing.

(applies to phase 2 part)

Inclusion criteria

1. Histologically confirmed metastatic adenocarcinoma of the colon and/or rectum.
2. Confirmed MGMT promoter methylation by PSQ (> 5%) and absent MGMT expression by immunohistochemistry.
3. Locally assessed pMMR or MSS status.
4. Written informed consent obtained prior to any study procedures.
5. Availability of archival tumor tissue (primary tumor and metastases or at least one of the two) for confirmation of MGMT, MMR/MSI status and biomarker analyses.
6. Availability of blood sample for biomarker analysis.
7. Metastatic colorectal cancer not previously treated with chemotherapy for metastatic disease.
8. At least one measurable lesion according to RECIST 1.1.
9. Age ≥ 18 and less or equal than 75 years.
10. ECOG PS ≤ 1 if patient < 70 years old; ECOG PS 0 if patient 70-75 years old.
11. Life expectancy of at least 12 weeks.
12. Previous (neo)adjuvant fluoropyrimidine or fluoropyrimidine plus oxaliplatin chemotherapy allowed only if more than 6 months elapsed between the end of (neo)adjuvant therapy and first evidence of disease relapse.
13. Neutrophils ≥1.5 x 109/L, Platelets ≥100 x 109/L, Hemoglobin ≥ 9 g/dl.
14. Total bilirubin ≤1.5 fold the upper-normal limits (UNL), AST (SGOT) and/or ALT (SGPT) ≤ 2.5 x UNL (or <5 x UNL in the case of liver metastases), alkaline phosphatase ≤ 2.5 x UNL (or <5 x UNL in case of liver metastases).
15. Creatinine clearance ≥ 50 mL/min or serum creatinine ≤1.5 x UNL.
16. Women of childbearing potential must have a negative blood pregnancy test at the baseline visit. 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. A postmenopausal state is defined as no menses for 12 months without an alternative medical cause. A high follicle stimulating hormone (FSH) level in the postmenopausal range may be used to confirm a post-menopausal state in women not using hormonal contraception or hormonal replacement therapy. However, in the absence of amenorrhea, a single FSH measurement is insufficient.
17. Male subjects with female partners of childbearing potential and female subjects of childbearing potential must be willing to use adequate contraception as approved be the Investigator (barrier contraceptive measure or oral contraception) as outlined in Section 7.6, starting with the screening visit and through 6 months after the last treatment dose. Note: abstinence is acceptable if this is the usual lifestyle and preferred contraception for the subject.
18. Will and ability to comply with the protocol.
19. Is willing and able to provide an adequate archival tumor sample (FFPE) available for molecular screening and exploratory analyses. If the tumor block is not available, a minimum of 25 3-micron unstained sections on charged slides of tumor will be required.

Exclusion criteria

1. Requirement for treatment with any medicinal product that contraindicates the use of any of the study medications, may interfere with the planned treatment, affects patient compliance or puts the patient at high risk for treatment-related complications.
2. Metastatic disease deemed R0 resectable upfront or after induction therapy by means of multidisciplinary team assessment.
3. Radiotherapy to any site within 4 weeks before the study.
4. Presence of one of the following: DPYD2a (c.1905+1G>A); DPYD13 (c.1679 T>G); DPYD D949V (c.2846 A>T); DPYD IVS10 (c.1129-5923 C>G).
5. Presence of one of the following UGT1A1 1(TA)6/UGT1A1 36(TA)5; UGT1A1 28(TA)7/UGT1A1 37(TA)8 (homozygous genotype).
6. In the dose escalation phase, untreated brain metastases or spinal cord compression or primary brain tumors; in the dose expansion phase, known history of brain metastases.
7. History or evidence upon physical examination of central nervous system disease unless adequately treated.8. Active uncontrolled infections or other clinically relevant concomitant illness contraindicating chemotherapy administration.

9. Evidence of bleeding diathesis or coagulopathy. 10. Uncontrolled hypertension and prior history of hypertensive crisis or hypertensive encephalopathy.

11. Clinically significant (i.e. active) cardiovascular disease for example cerebrovascular accidents (≤6 months), myocardial infarction (≤6 months), unstable angina, New York Heart Association (NYHA) grade II or greater congestive heart failure, serious cardiac arrhythmia requiring medication.

12. Significant vascular disease (e.g. aortic aneurysm requiring surgical repair or recent arterial thrombosis) within 6 months of study enrolment.

13. Any previous venous thromboembolism ≥ NCI CTCAE Grade 4. 14. History of abdominal fistula, gastrointestinal perforation, intra-abdominal abscess or active gastrointestinal bleeding within 6 months prior to the first study treatment.

15. Treatment with any investigational drug within 30 days prior to enrollment or 2 investigational agent half-lives (whichever is longer).

16. Other co-existing malignancies or malignancies diagnosed within the last 3 years with the exception of localized basal and squamous cell carcinoma or cervical cancer in situ.

17. Lack of physical integrity of the upper gastrointestinal tract, malabsorption syndrome, or inability to take oral medication.

18. Known hypersensitivity to trial drugs or hypersensitivity to any other component of the trial drugs.

19. Any concomitant drugs contraindicated for use with the trial drugs according to the product information of the pharmaceutical companies.

20. Pregnant or lactating women.