Patients

Eligible patients were male or female (> 18 years old) with histologically or cytologycally proven colorectal cancer, an indication of treatment with FOLFIRI ± bevacizumab or cetuximab or panitumumab, the presence of at least one measurable target according to the RECIST criteria, life expectancy of at least 3 months and adequate biological functions (renal, hepatic and haematological). Patients already treated for metastatic disease, taking anti-epileptics drugs, allergic or intolerant to irinotecan, having a contraindication for 5-FU, and with chronic inflammatory bowel disease or bowel obstruction, were excluded. All patients provided written informed consent prior to enrolment.

Study design

The study was approved by the local ethics committee and the competent authority at national level (Agence Nationale de Sécurité des Médicaments et des produits de santé) and registered online at ClinicalTrial.gov (https://clinicaltrials.gov/ct2/show/NCT01963182, 16/10/2013).

This was a prospective phase-II, multicentre, non-randomized trial aiming to demonstrate the feasibility of an individualized dose of irinotecan on the basis of genetic polymorphisms in first-line treatment for mCRC involving the FOLFIRI regimen.

Genotyping

The UGT1A1 genotype was determined within 7 days before the first cycle of treatment, so as to adapt the dose of irinotecan. This analysis was performed by PCR (FRET) with a LightCycler® 2.0 FastStart DNA Master HybProbe (Roche Diagnostics, Meylan, France) and UGT1A1 Light SniP (TIB MOLBIOL, Berlin, Germany). DNA was extracted using QIAamp® DNA Mini and Blood 50 (QIAGEN, Germany).

Treatment

Patients were treated twice a month with the FOLFIRI regimen until progression. The FOLFIRI treatment comprised: 5FU IV bolus 400 mg/m2 on day 1, 5FU infusion 2400 mg/m2 on days 1 and 2, folinic acid 400 mg/m2 on day 1 and irinotecan at the UGT1A1 genotype-adapted dosage on day 1 (180 mg/m2 for UGT1A1 *28/*28, 310 mg/m2 for *1/*28, and 370 mg/m2 for *1/*1 genotype, on the basis of the results of the phase-I study conducted by Toffoli et al. [3]). Patients could receive 5 mg/kg of bevacizumab, 500 mg/m2 of cetuximab or 6 mg/kg of panitumumab, before the FOLFIRI regimen every 2 weeks. Prophylactic G-CSF administration was not allowed as primary prevention.

Dose reduction and discontinuation rules

In case of grade 4 neutropenia, thrombocytopenia or febrile neutropenia, treatment was delayed for 1-–2 week(s) to allow a return to normal (neutrophil count > 1500/mm3 and platelet count > 100,000/mm3) before readministering irinotecan. If the required counts were not achieved on the day of the theoretical recovery (J7 and J15), the irinotecan dose was reduced by 20%. The same process was repeated, with 20% of reduction in dose if haematological recovery was not achieved at the theoretical moment in the cycles. In case of non-haematological grades 3–4 toxicities associated with irinotecan in the inter-treatment period, we waited until the symptoms disappeared completely before administering the medication and proceeded to a dose reduction of 20% in the next cycle. The reduced doses were maintained for all subsequent cycles.

Concomitant care

Granulopoiesis-stimulating factors were administered as a secondary prevention for FOLFIRI-treated patients with grade 4 neutropenia or febrile neutropenia. All symptomatic treatments required for the comfort of the patients were allowed. Concomitant treatments prescribed during the study were at the investigator’s discretion and were to be reported. Any other anti-cancer treatments were prohibited, including any cytotoxic treatment and any hormonal therapy, apart from oestrogen-progestin contraception.

Study objectives and endpoints

The primary objective was to evaluate the benefit in terms of toxicities and response to the individualization of the irinotecan dose according to UGT1A1 polymorphism. The secondary objectives were the pharmacokinetic study of irinotecan, SN38, SN38-G, and bevacizumab, and the evaluation of treatment efficacy.

The primary endpoint was assessed on the basis of the frequency of severe toxicities (defined as grade 4 neutropenia, grades 3 and 4 febrile neutropenia or grade 4 diarrhoea according to the NCI-CTC criteria v.4) and response rate (via imagery) according to the RECIST criteria v.1.1.

For the secondary outcomes, we investigated blood concentrations and the pharmacokinetic AUC of irinotecan, SN38, SN38-G, and bevacizumab, PFS, and response duration.

PFS was defined as the time between diagnosis and progression or death whichever occurred first. Overall survival (OS) was defined as the time between diagnosis and death. Patients were censored at the time of their last recorded follow-up if they were still alive.

Pharmacokinetics

During the first cycle, the pharmacokinetics of irinotecan SN38, SN38-G, and bevacizumab (if present) were explored. Blood samples to monitor treatment exposure (AUC) were regularly collected at several time points:

T0, T30, T60, T90, T120, T240, T480, T720, and T1440 min for FOLFIRI alone and FOLFIRI + vectibix

T0, T30, T60, T90, T120, T180, T210, T240, T270, T300, T420, T660, T900, and T1620 for FOLFIRI + Erbitux

T0, T30, T60, T120, T150, T180, T210, T330, T570, T810, and T1530 for FOLFIRI + Avastin

The plasma concentrations of these molecules were determined using high-performance liquid chromatography coupled with tandem mass spectrometer (HPLC–MS/MS), composed of a TSQ Quantum Ultra™ mass spectrometer (Thermo Fisher Scientific) coupled with Transcend TLX-1 liquid chromatography (Thermo Fisher Scientific). Both molecules were assayed after simple precipitation and the bio-analytical method was validated following ISO-1589 and EMEA guidelines [10]. The Limits of Quantification (LOQ) were 25 ng/ml and 5 ng/mL for irinotecan and SN38, respectively. These analyses were centralized and performed in the clinical pharmacology and toxicology laboratory of Clermont-Ferrand University Hospital.

Irinotecan, SN38, and SN38-G AUCs were calculated using the trapezoidal rule and then compared according to the patients’ UGT1A1 genotype status (*1/*1, *1/*28 and *28/*28) and the dose administered (370 mg/m2, 310 mg/m2 or 180 mg/m2).

Statistical analysis

Given the trade-off between treatment efficacy and treatment toxicity, a two-stage Bryant and Day design was used to plan the study [11]. Concerning the response rate, for the lower limit for rejection we chose a response rate under 35%, and for the upper limit for acceptation a response rate of over 60%. For toxicity, for the lower limit for rejection we chose a toxicity rate over 40% and for the upper limit for acceptation a toxicity rate of under 20%.

A first positive interim analysis involving 16 patients enabled the study to be continued until 47 patients were included. The sample size for the final analysis was n = 34, as some patients were not assessable. Toxicity and efficacy assessments were performed using one-sided exact binomial tests. Toxicity frequency was computed using the maximum grade method.

All descriptive analyses were performed on the whole assessable population (n = 34), and in each UGT1A1 genotype-defined subgroup. AUC distributions of irinotecan, SN38, and SN38-G were compared between groups using Wilcoxon–Mann–Whitney tests. Survival data were analysed using the Kaplan–Meier method. Significance level was set at 5%. Statistical analyses were performed using R software (version 4.1.0; R Core Team, 2021).