Genetic markers for toxicity of adjuvant oxaliplatin and fluoropyrimidines in the phase III TOSCA trial in high-risk colon cancer patients

We investigated 17 polymorphisms in 11 genes (TS, MTHFR, ERCC1, XRCC1, XRCC3, XPD, GSTT1, GSTP1, GSTM1, ABCC1, ABCC2) for their association with the toxicity of fluoropyrimidines and oxaliplatin in colorectal cancer patients enrolled in a prospective randomized trial of adjuvant chemotherapy. The TOSCA Italian adjuvant trial was conducted in high-risk stage II–III colorectal cancer patients treated with 6 or 3 months of either FOLFOX-4 or XELOX adjuvant chemotherapy. In the concomitant ancillary pharmacogenetic study, the primary endpoint was the association of polymorphisms with grade 3–4 CTCAE toxicity events (grade 2–4 for neurotoxicity). In 517 analyzed patients, grade ≥ 3 neutropenia and grade ≥ 2 neurotoxicity events occurred in 150 (29%) and in 132 patients (24.8%), respectively. Diarrhea grade ≥ 3 events occurred in 34 (6.5%) patients. None of the studied polymorphisms showed clinically relevant association with toxicity. Hopefully, genome-wide association studies will identify new and more promising genetic variants to be tested in future studies.

pharmacokinetic and pharmacodynamics of anti-cancer drugs and this may contribute to the differences in toxicity among patients 3 .
In the last decades, some genetic variants involved in the oxaliplatin and the fluoropyrimidines pathways were identified as potential predictors of toxicity 4,5 . However, the majority of clinical data have been obtained from retrospective analyses including a limited number of patients. In fact, none of the studied polymorphisms showed sufficient evidence for use in clinical practice 4,5 . Prospective analyses from randomized clinical trial represent a unique opportunity for evaluating association between genetic variants and clinical outcomes and are necessary for confirming the predictive role for toxicity of candidate polymorphisms [6][7][8][9] .
TOSCA (Three Or Six Colon Adjuvant) is a large randomized trial addressing the role of a shorter duration of an adjuvant oxaliplatin/ fluoropyrimidines regimen in surgically resected stage III and highrisk stage II colorectal cancer 10 . We adopted this clinical trial for planning a robust pharmacogenetic assessment for toxicity focusing on candidate polymorphisms, which had showed promising associations in previous studies [6][7][8][11][12][13] .

Patients, Materials and Methods
TOSCA trial. Patients included in this study represent a subgroup of the 3.759 patients with surgically-resected, stage III and high-risk stage II colorectal cancer recruited in TOSCA trial between 2007 and 2011 10 . This is an Italian intergroup, multicentre, randomized, non-inferiority phase III study in high-risk stage II and stage III colon cancer patients treated with 3 or 6 months of either FOLFOX-4 or XELOX adjuvant chemotherapy, sponsored by GISCAD (Italian Group For The Study Of Gastrointestinal Cancer) and supported by Italian Medicines Agency (AIFA) 10 . Patients eligible for the TOSCA study were asked to give further and specific written informed consent for the pharmacogenetic study. All experiments were performed in accordance with relevant guidelines and regulations and the Local Ethics Committee of each institution approved the Study.
Assessment and management of chemotherapy toxicity. Selected hematologic and non-hematologic toxicities (anemia, leukopenia, neutropenia, thrombocytopenia, asthenia, diarrhea, mucositis stomatitis, vomiting, nausea, hepatic toxicity, skin toxicity, neurotoxicity) were assessed at the start of each cycle using Common Toxicity Criteria for Adverse Events (CTCAE) version 2.0. All adverse events at any time were monitored and reported. Toxicity was managed as follows: in case of grade $ 3 hematologic toxicity or persistent grade 2 the dose of all drugs was reduced by 25%. In case of grade $ 3 non-hematologic toxicity the dose of the related drugs was reduced by 50%. In case of grade $ 3 or persistent grade 2 neurotoxicity, oxaliplatin dose was reduced by 20%. Oxaliplatin was definitely stopped if grade $ 2 neurosensory symptoms persisted between cycles.
Molecular assessments. This prospective study was planned as a confirmatory analysis of genetic variants, which had shown some putative predictive effect for toxicity in previous studies [6][7][8][11][12][13] . Seventeen polymorphisms in eleven genes involved in DNA repair and drug metabolism and as drug targets, were selected from various reports as being potentially predictive of 5-fluorouracil (5-FU) or oxaliplatin toxicity (Table 1) in colorectal cancer patients. For each polymorphism, patients were considered in three groups: homozygous wild type (p2); heterozygous (pq); homozygous variant (q2). We also considered the model with merged heterozygous and homoxygous risk variant carriers.
Analysis plan, sample size, and statistics. According to the planned management of toxicity in TOSCA trial, we chose outcome measures and endpoints, which reflects clinically relevant degrees of both hematologic and non-hematologic toxicity. Primary outcome was defined as the occurrence of a grade 3-4 toxicity (grade 2, 3, 4 for neurotoxicity) considering in each patient the maximum grade of toxicity (MGT) reported during treatment. Secondary outcome was the time to toxicity (TTT), defined as the time from date of randomization in TOSCA trial to the date of first grade $ 2 event for neurotoxicity and $3 event for other toxicities. Subjects without such a toxicity event at the time of analysis were censored at the date they were last known to be event-free while on treatment. The treatment compliance was described in terms of treatment interruption and dose intensity, defined as the dose given in mg/m 2 per week. Logistic regression and Cox proportional hazard models were used to assess the effects of genotypes on MGT and TTT, respectively, adjusting for treatment duration (6 or 3 months). For each polymorphism, toxicity analysis was performed across the three group genotypes (p 2 , pq, q 2 ) and after grouping carriers of the heterozygous and homozygous risk genotypes.
Sample size calculation was based on an expected prevalence of at higher risk allele of at least 30% and assuming a 25% risk of toxicity. Accordingly, 440 patients (105 events) would allow the detection of an odds ratio (OR) of at least 2.0 associated to the group with unfavorable genotypes with a power of 90% and a I type error of 5%, for a bilateral test. Deviation from the Hardy-Weinberg equilibrium was assessed using the Pearson x 2 test. Analyses were performed with SAS 9.2 (SAS Institute, Cary, NC). All reported p values are two-sided, and confidence intervals (CIs) are at the 95% level. A p value , 0.05 was considered statistically significant.    This figure represents 81% of patients randomized in the same period and by the same centers in the main TOSCA trial study. Seventeen patients were not assessable; five patients who were never treated, two patients because of unavailability of treatment data, and ten due to technical problems about blood sampling. Therefore, the analysis was conducted on 517 patients. Characteristics of the 517 patients are shown in Table 2. Patients' baseline characteristics were consistent with those of the whole trial population (data not reported). Most patients were randomized to FOLFOX-4 because option for XELOX regimen was introduced only during the late phase of accrual of this ancillary study. Toxicity caused by adjuvant chemotherapy is reported in Table 3. Again, the spectrum and the frequency of toxicities did not differ from those observed in whole trial population (data on file). The target number of events was reached for neutropenia (150/517 patients, 29%) and neurotoxicity (132/517, 25.5%), only. Dose intensity and treatment interruptions are shown in Table 4 and Table 5, respectively. Dose intensity for patients randomized in 6 months arms is slightly lower than that reported for patients randomized in 3 months arms.
Genetic assessments. Table 1 lists the studied genetic variants and the distribution of genotypes of patients. Consistent with previous observations, genotype frequency did not differ from those observed in Caucasian population. Allele frequencies of all polymorphisms were consistent with the Hardy-Weinberg equilibrium (x 2 ; p . .05) and with values in the published literature.
Pharmacogenetics for neutropenia and neurotoxicity. The results of pharmacogenetic analyses in the 517 patients for neutropenia and neurotoxicity are shown in Table 6 and Table 7, respectively. A weak association was observed between the GST-T1/M1 null/null genotype (presence of homozygous deletion in both genes) and neutropenia according to MGT (OR 1.99, 95%CI 1.06-3.73; p 5 0.03) and TTT (HR 1.70, 95%CI 1.03-2.78; p 5 0.04), when compared with wildtype genotype. As far as the ABCC1 rs2074087 genotype and neurotoxicity is concerned, the planned statistical analyses could not be performed due to low model convergence. Regarding neurotoxicity no evidence of association was found between polymorphisms and MGT and TTT.
Other toxicities. Despite the number of events was less than required, therefore decreasing the power of the tests, the analyses showed some statistically significant association. In presence of the

Discussion
This study assessed 17 polymorphisms in 11 genes thought to be associated with toxicity of fluoropyrimidines or oxaliplatin. To the best of our knowledge this is the first and the largest prospective pharmacogenetic analysis in a randomized trial of adjuvant chemotherapy in colorectal cancer. Candidate polymorphisms were selected on the basis of previous promising data from retrospective or single arm studies. The prospective accrual of patients achieved the required number of events for neutropenia and neurotoxicity, however only GST-T1/M1 was statistically associated to neutropenia and the strength of this association was very low. Therefore, no polymorphism showed a clinically relevant association with neurotoxicity and neutropenia. The results on the other toxicities should be looked at with caution because of the low number of events.
To date, five randomized clinical trials in colorectal cancer have incorporated pharmacogenetic analysis [6][7][8][9] , but only one study in the adjuvant setting 14 . In the US Intergroup N9741 pharmacogenetic analysis there were 114 patients treated with IFL chemotherapy, 299 patients treated with FOLFOX-4 regimen and 107 patients who received IrOX chemotherapy 6 . Therefore, despite the 520 initial patients assessed for pharmacogenetic analyses, this remarkable study population was diluted among three treatments arms, with a small number of patients assessable for an oxaliplatin-based regimen. In this study, $grade 3 neutropenia, neurotoxicity and diarrhea occurred in the 27%, 13% and 13% of patients respectively. In the FOLFOX-4 regimen analysis, the GST-P1 TT genotype carriers were more likely to suffer from febrile neutropenia and to discontinue the treatment because of neurotoxicity, carriers of the GST-M1 null genotype were at increased risk of neutropenia 6 . In the Fédération Francophone de Cancérologie Digestive 2000-05 trial, metastatic colorectal cancer patients were randomized to receive 5-FU plus leucovorin followed by FOLFOX-6, followed by FOLFIRI (arm A), or FOLFOX-6 followed by FOLFIRI (arm B). The pharmacogenetic analysis included 346 patients who received more regimens in a different sequence 7 . There was a remarkable frequency of $grade 2 neurotoxicity (about half of the patients) and $grade 3 myelotoxicity in about one-third of the patients. The XPD C allele (rs13181) was significantly associated with an increased risk of FOLFOX-induced hematologic toxicity (p 5 0.01). In the pharmacogenetic analysis associated with the randomized FOCUS UK trial, 1.188 patients were assessed 8 . In this study, metastatic colorectal cancer patients were randomized to receive three treatment strategies according to a different sequence of the following regimens: 5-FU alone, irinotecan alone, 5-FU with irinotecan and 5-FU with oxaliplatin. Only 280 patients were assessable for first-or second-line oxaliplatin-based chemotherapy. No significant pharmacogenetic association was found in this study 8 . The most recently published analysis in metastatic colorectal cancer patients depicts the results of a large panel of genetic variants in a robust sample of more than 2,000 patients enrolled in the COIN trials in UK 9 . Again, this study ruled out clinically relevant associations between pharmacogenetics and clinical outcomes of patients treated with fluoropyrimidine/oxaliplatin with or without cetuximab 9 .
As far as the adjuvant setting is concerned, the recently published pharmacogenetic study from the QUASAR2 trial has investigated the role of fluoropyrimidine-related polymorphisms in 927 patients who were randomized between capecitabine and capecitabine with bev- acizumab. Of the 36 assessed polymorphisms only four TS and DPYD genetic variants were associated with grade $ 3 global toxicity, but with modest predictive power 14 .
Considering the characteristics of the above mentioned studies, we would emphasize the remarkable sample size in the adjuvant setting of our oxaliplatin-based study population, as well as the quality of pharmacogenetic analyses in a prospective and controlled collection of clinical data 10 . It seems that we recorded a lower frequency of grade $ 2 neurotoxicity and grade . 3 neutropenia than previously reported in the literature 2 . Generally, we observed a global lower incidence of toxicity events than expected. This finding is likely related to the accuracy of physicians in the monitoring of patients with early detection of signs of side-effects and consequently, their conservative attitudes towards treatment delays and dosereductions.
However, this did not jeopardize the study plan of the ancillary pharmacogenetic study and a sufficient number of events for neurotoxicity and neutropenia was observed. Unfortunately, given the low rate of other severe toxicities, we cannot rule out the risk of observing false-negative associations in these cases.
A number of drug-and host-related variables contribute to pharmacodynamic and pharmacokinetic changes of chemotherapy drugs. Therefore, because of the moderate functional effects of polymorphism in the enzyme/target activity, their clinical impact may be masked according study populations and clinical settings. This may also explain the heterogeneity of results across pharmacogenetic studies. On the whole, we highlight the necessity for large-scale validation trials before pharmacogenetic findings from small studies are incorporated into clinical practice [12][13][14][15] . In fact, our findings, together with the results of the analyses in metastatic colorectal cancer 6-9 and other malignancies 3 , mitigate the positive expectations for the routine use of pharmacogenetics. It is a matter of fact that in spite of the growing burden of small, retrospective published studies on the predictive/prognostic role of polymorphisms in colorectal cancer patients, only UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) and dihydrophyrimidine dehydrogenase (DPYD) genetic variants have shown a promising level of evidence for clinical practice 16 . However, we did not study the UGT1A1*28 genotype analysis since it is typically associated with Irinotecan pharmacokinetic and toxicity 16 . As far as the DPYD IVS14 1 1G . A splice mutation is concerned, we did not include this variant for 5-fluorouracil toxicity analysis because of its very low frequency 16 . In fact, there were 2 heterozygous carriers in the 346 patients (0.5%) of the French trial 6 , 4 heterozygous carriers in the 520 patients (0.7%) of US trial 7 and 12 heterozygous carriers in the 1088 patients (1.1%) of FOCUS trial 8 .
Pharmacogenetics may still offer a unique opportunity for tailoring the administration of chemotherapy and novel biologic agents to cancer patients. Hopefully, new sophisticated techniques such as SNP arrays and genome-wide association studies (GWAS) will identify new and more promising genetic variants to be tested in future studies 17,18 .