Exploiting Circulating Tumour DNA to Intensify the Postoperative Treatment Resected Colon Cancer Patients

Inclusion Criteria, Part I, adjuvant phase:

• Written informed consent to study procedures;

• 18 - 70 years of age ECOG Performance Status ≤ 1 or 71-75 years of age with ECOGPerformance Status 0;

• Histologically confirmed stage III or high-risk stage II adenocarcinoma of colonincluding intraperitoneal rectal cancer. Stage II colon cancers are defined at highrisk if at least one major prognostic factor (pT4, less than 12 nodes examined,clinical presentation with bowel perforation) or at least two minor prognosticfactors (grade 3 or 4, clinical presentation with bowel obstruction, histologicalsigns of vascular or lymphatic or perineural invasion, high preoperative CEA levels)are reported;

• Curative surgery performed no less than 4 and no more than 12 weeks prior torandomization (pending results of ct-DNA analysis, up to 2 cycles of FOLFOX/CAPOXare allowed to start the adjuvant treatment within 8-10 weeks after surgery);

• Contrast-enhanced chest and abdominal CT scan (or abdomen MRI and chest CT ifcontrast-enhanced CT scan is contraindicated) performed after the surgery and priorto randomization with no evidence of metastatic disease;

• Availability of formalin-fixed, paraffin-embedded (FFPE) tumor tissue from thesurgical specimen and blood sample for ct-DNA analysis within 28 days priorrandomization;

• Positive ct-DNA after surgery (central assessment);

• Neutrophils ≥1.5 x 109/L, Platelets ≥100 x 109/L, Hgb ≥ 9 g/dl;

• Total bilirubin ≤1.5 fold the upper-normal limits (UNL), ASAT (SGOT) and/or ALAT (SGPT) ≤2.5 x UNL (or <5 x UNL in the case of liver metastases), alkalinephosphatase ≤2.5 x UNL (or <5 x UNL in case of liver metastases);

• Creatinine clearance ≥50 mL/min or serum creatinine ≤1.5 x UNL;

• Women of childbearing potential must have a negative blood pregnancy test at thescreening visit. For this trial, women of childbearing potential are defined as allwomen after puberty, unless the participants are postmenopausal for at least 12months, are surgically sterile, or are sexually inactive. A postmenopausal state isdefined as no menses for 12 months without an alternative medical cause. A highfollicle stimulating hormone (FSH) level in the postmenopausal range may be used toconfirm a post-menopausal state in women not using hormonal contraception orhormonal replacement therapy. However, in the absence of 12 months of amenorrhea, asingle FSH measurement is insufficient;

• Subjects and their partners must be willing to avoid pregnancy during the trial.Male subjects with female partners of childbearing potential and female subjects ofchildbearing potential must, therefore, be willing to use adequate contraception.

Contraception, starting during study screening visit throughout the study period up to 180 days after the last dose of chemotherapy. Note: Abstinence is acceptable if this is the usual lifestyle and preferred contraception for the subject;

• Will and ability to comply with the protocol.

Inclusion Criteria Part 1 and target-driven part 1, adjuvant phase II study

• Written informed consent to study procedures;

• 18 - 70 years of age ECOG PS ≤ 1 or 71-75 years of age with ECOG PS 0;

• Histologically confirmed stage III or high-risk stage II adenocarcinoma of colonincluding intraperitoneal rectal cancer. Stage II colon cancers are defined at highrisk if at least one major prognostic factor (pT4, less than 12 nodes examined,clinical presentation with bowel perforation) or at least two minor prognosticfactors (grade 3 or 4, clinical presentation with bowel obstruction, histologicalsigns of vascular or lymphatic or perineural invasion, high preoperative CEA levels)are reported;

• For target-driven Part 1 only: HER2+ and RAS wt disease as determined by atissue-based assay (central laboratory assessment);

• Curative surgery performed no less than 4 and no more than 12 weeks prior torandomization (pending results of ct-DNA analysis, up to 2 cycles of FOLFOX/CAPOXare allowed to start the adjuvant treatment within 8-10 weeks after surgery);

• Contrast-enhanced chest and abdominal CT scan (or abdomen MRI and chest CT ifcontrast-enhanced CT scan is contraindicated) performed after the surgery and priorto randomization with no evidence of metastatic disease;

• Availability of formalin-fixed, paraffin-embedded (FFPE) tumor tissue from thesurgical specimen and blood sample for ct-DNA analysis within 28 days priorrandomization;

• Positive ct-DNA after surgery (central assessment);

• Neutrophils ≥1.5 x 109/L, Platelets ≥100 x 109/L, Hgb ≥ 9 g/dl;

• Total bilirubin ≤1.5 fold the upper-normal limits (UNL), ASAT (SGOT) and/or ALAT (SGPT) ≤2.5 x UNL, alkaline phosphatase ≤2.5 x UNL;

• Creatinine clearance ≥50 mL/min or serum creatinine ≤1.5 x UNL;

• For target-driven Part 1 only: Left ventricular ejection fraction (LVEF) ≥50% asassessed by echocardiogram (ECHO) or multiple-gated acquisition (MUGA) scan.

• Availability of formalin-fixed, paraffin-embedded (FFPE) tumor tissue from thesurgical specimen for translational analysis (only for patients eligible forprotocol treatment);

• Women of childbearing potential must have a negative blood pregnancy test at thescreening visit. For this trial, women of childbearing potential are defined as allwomen after puberty, unless they are postmenopausal for at least 12 months, aresurgically sterile, or are sexually inactive. A postmenopausal state is defined asno menses for 12 months without an alternative medical cause. A high folliclestimulating hormone (FSH) level in the postmenopausal range may be used to confirm apost-menopausal state in women not using hormonal contraception or hormonalreplacement therapy. However, in the absence of 12 months of amenorrhea, a singleFSH measurement is insufficient;

• Subjects and their partners must be willing to avoid pregnancy during the trial.Male subjects with female partners of childbearing potential and female subjects ofchildbearing potential must, therefore, be willing to use adequate contraception.Contraception, starting during study screening visit throughout the study period upto 180 days after the last dose of chemotherapy. Note: Abstinence is acceptable ifthis is the usual lifestyle and preferred contraception for the subject;

• Will and ability to comply with the protocol.

Inclusion Criteria, Part II, post-adjuvant phase:

• Written informed consent to study procedures;

• ≥ 18 years of age;

• Histologically confirmed stage III or high-risk stage II adenocarcinoma of colonincluding intraperitoneal rectal cancer. Stage II colon cancers are defined at highrisk if at least one major prognostic factor (pT4, less than 12 nodes examined,clinical presentation with bowel perforation) or at least two minor prognosticfactors (grade 3 or 4, clinical presentation with bowel obstruction, histologicalsigns of vascular or lymphatic or perineural invasion, high preoperative CEA levels)are reported;

• Fluoropyrimidine and oxaliplatin-containing adjuvant treatment for at least 3 months (6 cycles of 5-fluorouracil and oxaliplatin-based therapy or 4 cycles ofcapecitabine and oxaliplatin-based-therapy) and no more than 6 months (12 cycles of 5-fluorouracil and oxaliplatin-based therapy or 8 cycles of capecitabine andoxaliplatin-based-therapy);

• Contrast-enhanced chest and abdominal CT scan (or abdomen MRI and chest CT ifcontrast-enhanced CT scan is contraindicated) performed within 4 weeks from the endof adjuvant therapy and 28 days prior to randomization;

• Availability of FFPE tumor tissue from the surgical specimen and blood sample forct-DNA analysis within 28 days prior to randomization;

• Positive ct-DNA after the end of adjuvant treatment (centrally laboratoryassessment);

• ECOG Performance Status ≤ 1;

• Neutrophils ≥1.5 x 109/L, Platelets ≥100 x 109/L, Hgb ≥9 g/dl;

• Total bilirubin ≤1.5 fold the upper-normal limits (UNL), ASAT (SGOT) and/or ALAT (SGPT) ≤2.5 x UNL (or <5 x UNL in the case of liver metastases), alkalinephosphatase ≤2.5 x UNL (or <5 x UNL in case of liver metastases);

• Creatinine clearance ≥ 50 mL/min or serum creatinine ≤1.5 x UNL;

• Women of childbearing potential must have a negative blood pregnancy test at thescreening visit. For this trial, women of childbearing potential are defined as allwomen after puberty, unless the participants are postmenopausal for at least 12months, are surgically sterile, or are sexually inactive. A postmenopausal state isdefined as no menses for 12 months without an alternative medical cause. A highfollicle stimulating hormone (FSH) level in the postmenopausal range may be used toconfirm a post-menopausal state in women not using hormonal contraception orhormonal replacement therapy. However, in the absence of 12 months of amenorrhea, asingle FSH measurement is insufficient;.

• Subjects and their partners must be willing to avoid pregnancy during the trial.Male subjects with female partners of childbearing potential and female subjects ofchildbearing potential must, therefore, be willing to use adequate contraception.

Contraception, starting during study screening visit throughout the study period up to 180 days after the last dose of chemotherapy. Note: Abstinence is acceptable if this is the usual lifestyle and preferred contraception for the subject;

• Will and ability to comply with the protocol.

Exclusion Criteria:

• Part 1, adjuvant phase and Part 2, post-adjuvant phase

• Any evidence of metastatic disease (radiological or pathological metastasis);

• Macroscopic or microscopic evidence of residual tumor (R1 or R2 resections) aftersurgery;

• Other co-existing malignancies or malignancies diagnosed within the last 5 yearswith the exception of localized basal and squamous cell carcinoma or cervical cancerin situ;

• For Part 1 only: patient with complete dihydropyrimidine dehydrogenase (DPYD)deficiency (homozygous of the following DPYD polymorphisms: c1679GG, c1905+1AA,c2846TT);

• History or evidence upon physical examination of CNS disease unless adequatelytreated;

• Clinical signs of malnutrition;

• Active uncontrolled infections or other clinically relevant concomitant illnesscontraindicating chemotherapy administration;

• Evidence of bleeding diathesis or coagulopathy;

• Clinically significant (i.e. active) cardiovascular disease for examplecerebrovascular accidents (≤6 months), myocardial infarction (≤6 months), unstableangina, New York Heart Association (NYHA) grade II or greater congestive heartfailure, serious cardiac arrhythmia requiring medication;

• Significant vascular disease (i.e. aortic aneurysm requiring surgical repair orrecent arterial thrombosis) within 6 months of study enrolment;

• Lack of physical integrity of the upper gastrointestinal tract, malabsorptionsyndrome, or inability to take oral medication;

• Treatment with any investigational drug within 30 days prior to enrolment or 2investigational agent half-lives (whichever is longer);

• Known hypersensitivity to trial drugs or hypersensitivity to any other component ofthe trial drugs;

• Any concomitant drugs contraindicated for use with the trial drugs according to theproduct information of the pharmaceutical companies;

• Pregnant or lactating women. Women of childbearing potential with either a positiveor no pregnancy test at screening. Sexually active males and females (ofchildbearing potential) unwilling to practice contraception (as defined in section 5.5) during the study and until 180 days after the last trial treatment.

• Part 1, target-driven, adjuvant phase:

• Ongoing ≥ Grade 2 diarrhea of any etiology at screening;

• Presence of known chronic liver disease;

• Known to be positive for hepatitis C infection (positive by polymerase chainreaction [PCR]). Subjects who have been treated for hepatitis C infection arepermitted if they have documented sustained virologic response of at least 12 weeks.

• Known to be positive for hepatitis B (HBV) by surface antigen (HBsAg) expression.Subjects who are positive for either hepatitis B surface antibody (HBsAB) orantibodies to the hepatitis B core antigen (HBcAB) should be screened using PCRmeasurement of hepatitis B DNA levels. Subjects with hepatitis B DNA levels by PCRthat require nucleoside analogue therapy are not eligible for the trial. The latestlocal guidelines should be followed regarding the monitoring of hepatitis B DNAlevels by PCR subjects on study treatment.