Efficacy of Adjuvant Chemotherapy in Colon Cancer With Microsatellite Instability: A Large Multicenter AGEO Study

JNCI J Natl Cancer Inst (2016) 108(7): djv438 doi:10.1093/jnci/djv438 First published online February 1, 2016 Article Efficacy of Adjuvant Chemotherapy in Colon Cancer With Microsatellite Instability: A Large Multicenter AGEO Study David Tougeron, Guillaume Mouillet, Isabelle Trouilloud, Thierry Lecomte, Romain Coriat, Thomas Aparicio, Gaetan Des Guetz, Cédric Lécaille, Pascal Artru, Gaelle Sickersen, Estelle Cauchin, David Sefrioui, Tarek Boussaha, Aurélie Ferru, Tamara Matysiak-Budnik, Christine Silvain, Lucie Karayan-Tapon, Jean-Christophe Pagès, Dewi Vernerey, Franck Bonnetain, Pierre Michel, Julien Taïeb, Aziz Zaanan Affiliations of authors: Department of Gastroenterology (DT, GS, CS), Department of Medical Oncology (AF), and Department of Molecular Oncology (LKT), Poitiers University Hospital, Poitiers, France; Laboratoire Inflammation, Tissus Epithéliaux et Cytokines (LITEC) - EA 4331, Poitiers University, Poitiers (DT, CS); Methodology and Quality of Life in Oncology Unit, Besançon University Hospital, Besançon, France (GM); Department of Gastroenterology, Ambroise Paré Hospital, Boulogne- Billancourt, France (IT); Department of Gastroenterology (TL) and Department of Biochemistry (JCP), Tours University Hospital, Tours, France, UMR GICC CNRS 7292, Tours François Rabelais University, Tours (TL); Paris Descartes University, Cochin Hospital, Paris, France (RC); Department of Gastroenterology (TA) and Department of Medical Oncology (CDG), Avicenne Hospital, Bobigny, France; Department of Gastroenterology, Bordeaux Nord Aquitaine Clinic, Bordeaux, France (CL); Department of Gastroenterology, Jean Mermoz Lyon Hospital, Lyon, France (PA); Department of Gastroenterology, Nantes University Hospital, Nantes, France (EC, TMB); Department of Gastroenterology, Rouen University Hospital, Rouen, France (DS, PM); Department of Medical Oncology, Saint-Antoine Hospital, Paris, France (TB); Department of Gastroenterology and Digestive Oncology, Georges Pompidou European Hospital, APHP, Paris, France (JT, AZ); Paris Descartes University, Sorbonne Paris Cité, Paris, France (RC, JT, AZ). Correspondence to: David Tougeron, MD, PhD, Department of Gastroenterology, Poitiers University Hospital, 2 rue de la Milétrie, 86000 Poitiers Cedex, France (e-mail: davidtougeron@hotmail.fr/david.tougeron@chu-poitiers.fr). Abstract Background: Deficient mismatch repair (dmmr) colon cancer (CC) is reportedly resistant to 5-fluorouracil (5FU) adjuvant chemotherapy while preliminary data suggest chemosensitivity to oxaliplatin. We assessed the efficacy of fluoropyrimidine with and without oxaliplatin in a large cohort of dmmr CC patients. Methods: This retrospective multicenter study included all consecutive patients who underwent curative surgical resection for stage II or III dmmr CC between 2000 and 2011. Prognostic factors were analyzed using Cox models, and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated. All statistical tests were two-sided. Results: A total of 433 dmmr CC patients were included (56.8% stage II, 43.2% stage III). Mean follow-up was 47.0 months. The patients received surgery alone (n = 263) or surgery plus adjuvant chemotherapy consisting of fluoropyrimidine with (n = 119) or without (n = 51) oxaliplatin. Adjuvant chemotherapy was administered to 16.7% of stage II and 69.0% of stage III CC patients. As compared with surgery alone, adjuvant oxaliplatin-based chemotherapy improved disease-free survival (DFS) in multivariable analysis (HR = 0.35, 95% CI = 0.19 to 0.65, P <.001), contrary to adjuvant fluoropyrimidine alone (HR = 0.73, 95% CI = 0.36 to 1.49, P =.38). In the subgroup analysis, the DFS benefit of oxaliplatin-based chemotherapy was statistically significant in multivariable analysis only in stage III (HR = 0.41, 95% CI = 0.19 to 0.87, P =.02). Conclusion: This study supports the use of adjuvant chemotherapy with fluoropyrimidine plus oxaliplatin in stage III dmmr CC. Received: May 16, 2015; Revised: September 13, 2015; Accepted: December 21, 2015 The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. 1 of 9 Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

D. Tougeron et al. 2 of 9 Colorectal cancer (CRC) is the third most common cancer worldwide, causing more than 600 000 deaths each year (1). Colorectal carcinogenesis is characterized by three major mechanisms: chromosomal instability (CIN), microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP). MSI occurs in approximately 15% of all CRCs. This phenotype is because of a deficient DNA mismatch repair (MMR) system that results in error accumulation within microsatellite regions. Deficient MMR (dmmr) is due to (i) germline mutation in a MMR gene (MLH1, MSH2, MSH6, or PMS2), a situation called Lynch Syndrome (2), (ii) or more commonly to epigenetic inactivation of MLH1 in sporadic cases (3), often associated with the CIMP phenotype (4). Sporadic CRCs with MSI harbor activating mutations in the BRAF V600E oncogene in about 50% of cases (5). The presence of the BRAF V600E mutation rules out Lynch Syndrome (6). Several studies and a large systematic review have shown that the dmmr phenotype is associated with a more favorable stage-adjusted prognosis (7 10). Adjuvant chemotherapy based on 5-fluorouracil (5FU) was shown in the 1990s to reduce the risk of recurrence in patients with stage III colon cancer (CC) (11). In this setting, the dmmr phenotype has been identified as a predictive biomarker of a lack of benefit from adjuvant 5FU (7,12). More recently, three randomized trials showed that adjuvant oxaliplatin combined with 5FU or capecitabine improved disease-free survival (DFS) in patients with stage III CC (13 15). Preclinical data indicate that MSI tumor cells are sensitive to oxaliplatin despite their resistance to 5FU (16). However, clinical studies evaluating the prognostic and/or predictive impact of MMR phenotype in stage III CC patients receiving an adjuvant oxaliplatin-based chemotherapy are limited. The use of adjuvant therapy in stage II CC is controversial. Several but markers of stage II CC at high risk of recurrence have been identified (13,17,18). Adjuvant chemotherapy based on fluoropyrimidine alone or combined with oxaliplatin may be justified for patients with one or more of these markers although its efficacy in this high-risk group has never been prospectively validated (17). Because dmmr tumors are associated with a low risk of recurrence, some clinicians consider that patients with stage II dmmr CC should not receive adjuvant chemotherapy (7,10,12). However, adjuvant chemotherapy has not been specifically evaluated in patients with high-risk stage II dmmr CC. In this multicenter study, we evaluated the impact of adjuvant chemotherapy with fluoropyrimidine alone or combined with oxaliplatin by comparison with surgery alone on DFS in dmmr CC patients. We also analyzed the results with respect to tumor stage (stage II and III) and the molecular mechanism underlying the MMR deficiency (Lynch Syndrome and sporadic cases). differentiated tumors, fewer than eight excised lymph nodes, tumor perforation, or initial bowel obstruction (13,18). The study was approved by the ethics committee Comité de Protection des Personnes Ouest III. MMR Status Determination MMR status was determined by MSI testing and/or analysis of MMR protein expression by immunohistochemistry (IHC). Deficient MMR status was defined by the presence of either high-level tumor DNA MSI or a loss of MLH1, MSH2, or MSH6 protein expression in the tumor. Tumors with discordant results between DNA MSI testing and MMR protein expression were not included in the study. MSI Testing Genomic DNA was extracted from paraffin-embedded or frozen samples. Microsatellite instability was assessed by comparative analysis of normal colon and tumor DNA using the five consensus nucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24 or BAT-25, BAT-26, D5S346, D2S123, and D17S250), as recommended (19,20). Tumor DNA was also used to determine KRAS mutation (codon 12 and 13) and BRAF mutation (V600E). IHC Analysis of MMR Expression Formalin-fixed, paraffin-embedded tumors were stained for MLH1, MSH2, and MSH6 proteins. Protein expression was considered negative when nuclear staining was completely absent in neoplastic cells. Determination of Germline vs Sporadic dmmr Cases Patients under 60 years old and/or with Bethesda II criteria were selected for MMR tumor protein immunochemistry assay (MLH1, MSH2, and MSH6), followed by germline MMR gene analysis if indicated for Lynch Syndrome screening (21,22). Lynch Syndrome was confirmed by identification of a germline MMR mutation (n = 49). When the germline MMR gene mutation analysis was not available (n = 384), patients were considered to have suspected Lynch Syndrome if they exhibited a loss of MSH6 or MSH2 protein expression in the tumor (n = 51) or a loss of MLH1 without BRAF V600E mutation and were younger than age 60 years (n = 18), or if they met the Amsterdam II criteria in the absence of MMR immunochemistry screening (n = 7) (9). All others patients were considered to have sporadic dmmr CC (n = 274), except the patients with missing data (no MMR IHC results and no BRAF V600E status and no Amsterdam II criteria determination), who were excluded from this determination of molecular mechanism underlying the MMR deficiency (n = 34). Methods Study Population This retrospective study was conducted in 11 French medical centers. All consecutive patients with histologically proven stage II or III CC with a dmmr phenotype, treated between January 2000 and December 2011 with surgery alone or followed by adjuvant chemotherapy, were eligible. Patients with rectal cancer, stage I tumors, incomplete curative resection (R1 or R2 resection), or treatment with irinotecan-based adjuvant chemotherapy or targeted therapies were excluded. High-risk stage II colon cancer was defined by T4 stage, VELIPI criteria (vascular emboli, lymphatic invasion, or perineural invasion), poorly Treatment and Outcome Data were collected on the adjuvant chemotherapy regimens, local and distant disease recurrences, and deaths. Routine follow-up consisted of physical examination, biological tests (including serum carcinoembryonic antigen), and computed tomography scan (or ultrasonography) every three months for the first two years and every six months for the following three years. The data were updated in December 2013. Statistical Analyses The primary endpoint was the relationship between adjuvant chemotherapy and DFS in the overall cohort. As a secondary Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

3 of 9 JNCI J Natl Cancer Inst, 2016, Vol. 108, No. 7 endpoint, the influence of the type of adjuvant chemotherapy on DFS was analyzed according to the tumor stage and the mechanism of MMR deficiency. Baseline clinical and pathological variables were described as means and standard deviation for continuous variables and percentages for qualitative variables. Differences in baseline characteristics according to postoperative management were assessed using the chi-square test or Fisher s exact test. DFS was defined as the time elapsed from surgery to the first recurrence or death. Death from any cause and relapse were considered an event. Second cancers were ignored. Overall survival (OS) was defined as the time elapsed from surgery to death. Survival curves were estimated using the Kaplan-Meier method and described using the median and 95% confidence interval (CI). Follow-up was calculated by reverse Kaplan-Meier estimation. Variables with P values of.10 or less in univariate analysis were eligible for the Cox multivariable regression model. Hazard proportionality was checked by plotting log-minus-log survival curves. Variables included in the Cox multivariable model were selected by the stepwise method except for treatment variable. Once the other covariates were selected, the treatment variable was then added to the Cox model in order to estimate the association of adjuvant chemotherapy with DFS and OS. The calibration and goodness-of-fit of the primary final Cox model were assessed by using the extension of the Hosmer-Lemeshow test, with P values greater than.1 at 42 months considered to indicate good agreement. A bootstrap sample procedure was used to assess the internal validity of the model. The predictive value and discriminatory capacity of the final model were evaluated with Harrell s C-index. One thousand random samples of the population were used to derive the 95% confidence interval for the concordance statistic. The C index (0 C 1) is a probability of concordance between predicted and observed survival, with a C of 0.5 for random predictions and a C of 1 for a perfectly discriminating model. Sensitivity analyses were performed to assess the robustness of our primary analysis. First, we evaluated the influence of center of treatment with Cox model shared frailty, with center introduced as a frailty term. Secondly, we performed propensity score with the inverse probability weighting (IPTW) method to confirm the potential benefit of oxaliplatin-based chemotherapy vs surgery, identify in the primary analysis. The probability to receive adjuvant chemotherapy vs surgery alone was estimated using univariate and multivariable logistic models. Fitted values were then extracted from the model and constituted the propensity score. The predictive value of the propensity score was assessed by the area under the receiver operating characteristic curve (AUC). The impact of adjuvant chemotherapy vs surgery alone on DFS was finally tested in propensity score weighted Cox proportional hazards model. Finally, Lynch Syndrome, BRAF V600E, and KRAS mutations were added to the primary multivariable Cox model to test their association with DFS. A P value of less than.05 was considered statistically significant. All statistical tests were two-sided. All analyses were performed using SAS software version 9.3 (SAS Institute, Cary, NC). was determined by IHC alone in 143 patients, MSI testing alone in 118 patients, and both in 172 patients. Overall, 31.3% of patients were considered to have Lynch Syndrome, proven (germline MMR mutation) in 12.3% of cases (n = 49), and suspected in 19.0% of cases (n = 76). Among patients with stage II disease, 149 (60.6%) were considered to have a high risk of recurrence. Adjuvant Therapy and Outcome in the Overall Population Overall, 39.3% (n = 170) of patients received adjuvant treatment, consisting of adjuvant oxaliplatin-based chemotherapy (27.5%, n = 119) or fluoropyrimidine alone (11.8%, n = 51). Adjuvant chemotherapy was administered to, respectively, 16.7% (n = 41/246) and 69.0% (n = 129/187) of patients with stage II and stage III CC. Median age was 64.3 years in patients treated with adjuvant chemotherapy and 78.6 years in the surgery-alone population. In stage III CC, the median ages of patients who did not receive chemotherapy and those who received adjuvant chemotherapy were 83.4 and 66.9 years, respectively. Mean follow-up was 47.0 months (SD = 38.5 51.6). The disease recurrence rates were 5.7% and 20.9% in patients with stage II (n = 14/246) and III (n = 39/187) tumors, respectively. The recurrence rate was 2.4% (n = 1) in stage II patients who received adjuvant chemotherapy and 6.3% (n = 13) in patients who did not receive adjuvant chemotherapy. The respective recurrence rates in patients with stage III disease were 7.0% (n = 9) and 51.7% (n = 30). Sixty-eight patients died among the overall population. The 3-year DFS and OS rates were 76.2% (95% CI = 71.0 to 80.5) and 85.3% (95% CI = 81.0 to 88.7), respectively. Survival According to Adjuvant Therapy The 3-year DFS rates were 75.2% (95% CI = 68.1 to 80.9) in the surgery-alone group, 66.4% (95% CI = 51.0 to 78.0) in the fluoropyrimidine group, and 84.2% (95% CI = 75.3 90.1) in the fluoropyrimidine plus oxaliplatin adjuvant chemotherapy group (Figure 1). In multivariable analysis, patients treated with fluoropyrimidine plus oxaliplatin had statistically significant longer DFS than patients treated with surgery alone (HR = 0.35, 95% CI = 0.19 to 0.65, P <.001) while no survival benefit was observed with fluoropyrimidine alone (HR = 0.73, 95% CI = 0.36 to 1.49, P =.38) (Table 2). The final multivariable Cox model exhibited good calibration (Hosmer-Lemeshow with quintiles P =.77) and discrimination (C = 0.71, 95% CI = 0.65 to 0.82). Bootstrapping results for the internal validation reflected the robustness of the final model with close hazard ratios (95% CIs) (Table 2). Fluoropyrimidine plus oxaliplatin adjuvant chemotherapy was also associated with longer OS as compared with surgery alone after adjustment for prognostic covariates (HR = 0.17, 95% CI = 0.07 to 0.44, P <.001) (Supplementary Table 1, available online). Subgroup Analyses of Survival According to the tumor Stage and Adjuvant Therapy Results Study Population Four hundred thirty-three patients with dmmr CC were included. Median age was 73.1 years (range = 20.2 101.1 years). Stage II and III tumors represented 56.8% (n = 246) and 43.2% (n = 187) of cases, respectively (Table 1). Deficient MMR status In stage III dmmr CC, DFS was longer in patients receiving adjuvant fluoropyrimidine plus oxaliplatin chemotherapy (n = 89) compared with those treated with surgery alone (n = 58) in multivariable analysis (HR = 0.41, 95% CI = 0.19 to 0.87, P =.02) while fluoropyrimidine alone (n = 40) was not associated with a longer DFS compared with surgery alone (HR = 0.66, 95% CI = 0.29 to 1.50, P =.32) (Figure 2A and Table 3; Supplementary Table 2, available online). Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

D. Tougeron et al. 4 of 9 Table 1. Patients and tumor characteristics Variable Missing values All the population Surgery alone Adjuvant chemotherapy No. (%) No. (%) No. (%) P* Total 433 (100) 263 (100) 170 (100) Age, y <.001 <65 157 (36.3) 70 (26.6) 87 (51.2) 65 276 (63.7) 193 (73.4) 83 (48.8) Sex.03 Male 177 (40.9) 97 (36.9) 80 (47.1) Female 256 (59.1) 166 (63.1) 90 (52.9) Years of diagnosis <.001 2004 370 (85.5) 239 (90.9) 131 (77.1) <2004 63 (14.5) 24 (9.1) 39 (22.9) Lynch Syndrome <.001 No 274 (68.7) 189 (76.8) 85 (55.6) MMR mutation 49 (12.3) 23 (9.4) 26 (17.0) Suspected 76 (19.0) 34 (13.8) 42 (27.4) Missing values 34 Tumor perforation.18 No 396 (95.0) 246 (96.1) 150 (93.2) Yes 21 (5.0) 10 (3.9) 11 (6.8) Missing values 16 Initial tumor obstruction.63 No 367 (88.0) 222 (87.4) 145 (89.0) Yes 50 (12.0) 32 (12.6) 18 (11.0) Missing values 16 Tumor site.11 Right 357 (82.4) 223 (84.8) 134 (78.8) Left 76 (17.6) 40 (15.2) 36 (21.2) Tumor differentiation.03 Well- or moderately differentiated 259 (66.1) 166 (70.3) 93 (59.6) Poorly differentiated 133 (33.9) 70 (29.7) 63 (40.4) Missing values 41 Vascular emboli.12 No 224 (61.4) 150 (64.4) 74 (56.1) Yes 141 (38.6) 83 (35.6) 58 (43.9) Missing values 68 Lymphatic invasion.004 No 251 (73.6) 176 (78.6) 75 (64.1) Yes 90 (26.4) 48 (21.4) 42 (35.9) Missing values 92 Perineural invasion.12 No 262 (74.9) 176 (75.9) 86 (72.9) Yes 88 (25.1) 56 (24.1) 32 (27.1) Missing values 83 T stage <.001 T1-T3 310 (72.4) 208 (79.7) 102 (61.1) T4 118 (27.6) 53 (20.3) 65 (38.9) Missing values 5 Lymph node invasion <.001 N0 246 (57.2) 205 (77.9) 41 (24.4) N1 115 (26.6) 35 (13.3) 80 (47.6) N2 70 (16.2) 23 (8.8) 47 (28.0) Missing values 2 Stage <.001 II 246 (56.8) 105 (64.4) 41 (24.1) III 187 (43.2) 58 (35.6) 129 (75.9) BRAF mutation No 159 (55.6) 96 (50.3) 63 (66.3).01 Yes 127 (44.4) 95 (49.7) 32 (33.7) Missing values 147 Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

5 of 9 JNCI J Natl Cancer Inst, 2016, Vol. 108, No. 7 Table 1. Continued Variable Missing values All the population Surgery alone Adjuvant chemotherapy No. (%) No. (%) No. (%) P* KRAS mutation No 219 (86.6) 155 (90.6) 64 (78.1).006 Yes 34 (13.4) 16 (9.4) 18 (21.9) Missing values 180 Adjuvant management Surgery alone 263 (60.7) 263 (100.0) - - Fluoropyrimidine-based 51 (11.8) - 51 (30.0) Oxaliplatin-based 119 (27.5) - 119 (70.0) * Two-sided chi-square test or Fisher s exact test. MMR = mismatch repair. Figure 1. Disease-free survival (DFS) according adjuvant treatment. Survival curves are represented using the Kaplan-Meier method, and survival rates were compared with the use of the two-sided log-rank test. DFS curves are shown according to the adjuvant chemotherapy regimens vs surgery alone. Numbers of patients at risk in each group at various time points are given in the tables below the graph. Adjuvant chemotherapy was mainly administered to patients with high-risk stage II dmmr CC. Among the 246 stage II CC patients, 41 were treated with adjuvant chemotherapy, including 33 with high-risk factors of recurrence. In this subgroup of high-risk stage II CC patients (n = 149), the multivariable analysis showed a trend towards longer DFS in patients receiving fluoropyrimidine plus oxaliplatin chemotherapy (n = 24) compared with surgery alone (n = 116) (HR = 0.13, 95% CI = 0.02 to 1.05, P =.06) while fluoropyrimidine alone (n = 9) was not associated with a longer DFS compared with surgery alone (HR = 0.85, 95% CI = 0.19 to 3.88, P =.83) (Figure 2B and Table 3; Supplementary Table 3, available online). Specific analysis according to the T stage, which is considered to be probably the worst prognostic factor in stage II CC, showed that 3-year DFS rates were 64.6% (95% CI = 0.46 to 0.78) for T4 tumors and 86.0% (95% CI = 0.79 to 0.91) for T1-3 tumors. The robustness of our results was assessed using sensitivity analyses. Firstly, when adding center as a frailty term, oxaliplatinbased adjuvant chemotherapy remains statistically significantly associated with DFS in the overall population and in stage III CC patients (Table 2; Supplementary Table 2, available online). Secondly, we constructed a propensity score with all variables enrolled in Cox univariate analyses, except histologic factors including lymphatic invasion, perineural invasion, and tumor differentiation because of the relative higher rate of missing data. The multivariable logistic regression exhibited an AUC equal to 0.80. By using the IPTW method with propensity score in a Cox univariate model for treatment effect, similar results were obtained (weighted HR = 0.39, 95% CI = 0.23 to 0.66, P <.001), with a DFS benefit in favor of the adjuvant oxalipatin-based chemotherapy compared with surgery alone. Finally, in a sensitivity analysis taking into account the potential prognostic impact of Lynch Syndrome, KRAS exon 2, and BRAF V600E mutations, the DFS benefit of adjuvant oxaliplatin-based adjuvant chemotherapy, as compared with surgery alone, remained statistically significant in multivariable analysis (HR = 0.40, 95% CI = 0.21 to 0.77, P =.006) (Supplementary Table 4, available online). Subgroup Analyses of Survival According to the Mechanism of MMR Deficiency and Adjuvant Therapy In the Lynch Syndrome group (n = 125), 57 patients were treated with surgery alone, while 20 received fluoropyrimidine alone and 48 fluoropyrimidine plus oxaliplatin adjuvant chemotherapy. In Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

D. Tougeron et al. 6 of 9 Table 2. Univariate and multivariable Cox proportional hazards regression models for disease-free survival Univariate analysis Multivariable analysis 1 (n = 359) Multivariable analysis 2* (n = 359) Variable No. patients No. events HR (95% CI) P HR (95% CI) P HR (95% CI) P Total 433 95 Age, y <65 157 23 1 <.001 1.03 1.03 65 276 72 2.83 (1.74 to 4.59) 1.87 (1.78 to 3.25) 1.95 (1.13 to 3.38) (1.13 to 3.49) Sex Male 256 55 1.91 Female 177 40 1.02 (0.68 to 1.54) Years of diagnosis diagnosis 2004 370 74 1.77 <2004 63 21 0.93 (0.55 to 1.57) Tumoral perforation No 396 84 1.31 Yes 21 8 1.47 (0.70 to 3.08) Initial tumor obstruction No 367 76 1.01 1.007 1.01 Yes 50 16 1.96 (1.14 to 3.37) 2.17 (1.23 to 3.83) 2.17 (1.21 to 3.89) (1.10 to 3.73) Tumor site Right 357 79 1.51 Left 76 16 0.84 (0.49 to 1.43) Tumor differentiation Well- or moderately 259 58 1.10 differentiated Poorly differentiated 133 34 1.42 (0.93 to 2.18) Vascular emboli No 224 34 1 <.001 1 <.001 1 <.001 Yes 141 49 2.76 (1.76 to 4.31) 2.36 (1.48 to 3.78) 2.33 (1.44 to 3.79) (1.43 to 3.84) Lymphatic invasion No 251 47 1.07 Yes 90 28 1.54 (0.96 to 2.46) Perineural invasion No 262 48 1 <.001 Yes 88 31 2.17 (1.38 to 3.42) T stage T1-T3 310 56 1 <.001 1.001 1.001 T4 118 38 2.08 (1.38 to 3.15) 2.18 (1.37 to 3.47) 2.22 (1.38 to 3.58) (1.23 to 3.46) Lymph node invasion No 246 40 1.05 Yes 187 55 1.50 (1.00 to 2.26) Adjuvant management Surgery alone 263 59 1 1 1 Fluoropyrimidinebased 51 19 1.02 (0.60 to 1.73).94 0.73 (0.36 to 1.49) (0.31 to 1.45) Oxaliplatin-based 119 17 0.46 (0.27 to 0.79).005 0.35 (0.19 to 0.65) (0.18 to 0.62).38 0.67 (0.32 to 1.39).28 <.001 0.41 (0.22 to 0.77).006 * Multivariable analysis 2: Cox Model shared frailty with center of treatment introduced as a frailty term. CI = confidence interval; HR = hazard ratio. Wald Chi-Square two-sided. Selected for stepwise selection in multivariable Cox model. Internal validation bootstrap 95% CI. comparison with surgery alone, adjuvant chemotherapy with fluoropyrimidine alone (HR = 2.14, 95% CI = 0.73 to 6.33, P =.17) or combined with oxaliplatin (HR = 0.60, 95% CI = 0.20 to 1.81, P =.36) did not statistically significant improve DFS in multivariable analysis (Supplementary Figure 1A, available online). In patients with sporadic dmmr CC (n = 274), 189 patients were treated with surgery alone while 26 received fluoropyrimidine alone, and 59 fluoropyrimidine plus oxaliplatin chemotherapy. Adjuvant oxaliplatin-based chemotherapy statistically significant improved DFS (HR = 0.45, 95% CI = 0.23 to 0.89, P =.02) Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

7 of 9 JNCI J Natl Cancer Inst, 2016, Vol. 108, No. 7 Table 3. Analysis of disease-free survival according to tumor stage and treatment in univariate and multivariable models Disease-free survival Univariate analysis Multivariable analysis* Variable No. of patients (3-year survival rates, %) HR (95% CI) P HR (95% CI) P All patients Surgery alone 263 (75.2) 1 1 Fluoropyrimidine-based 51 (66.4) 1.02 (0.60 to 1.73).94 0.73 (0.36 to 1.49).38 Oxaliplatin-based 119 (84.2) 0.46 (0.27 to 0.79).005 0.35 (0.19 to 0.65) <.001 Stage III Surgery alone 58 (60.9) 1 1 Fluoropyrimidine-based 40 (59.9) 0.70 (0.37 to 1.34).28 0.66 (0.29 to 1.50).32 Oxaliplatin-based 89 (81.1) 0.32 (0.17 to 0.62).001 0.41 (0.19 to 0.87).02 High-risk stage II Surgery alone 116 (78.0) 1 1 Fluoropyrimidine-based 9 (88.9) 0.54 (0.13 to 2.34).41 0.85 (0.19 to 3.88).83 Oxaliplatin-based 24 (92.3) 0.13 (0.02 to 0.98).05 0.13 (0.02 to 1.05).06 * Multivariable Cox model. CI = confidence interval; HR = hazard ratio. Wald Chi-Square two-sided. in multivariable analysis in comparison with surgery alone while adjuvant chemotherapy with fluoropyrimidine alone did not (HR = 0.60, 95% CI = 0.24 to 1.52, P =.28) (Supplementary Figure 1B, available online). In stage III subgroup analysis, only patients with sporadic dmmr CC (n = 117) have a DFS benefit from adjuvant oxaliplatin-based chemotherapy (n = 47) in comparison with those treated by surgery alone (n = 50) in multivariable analysis (HR = 0.43, 95% CI = 0.21 to 0.93, P =.03). No effect of adjuvant chemotherapy was observed in patients with suspected Lynch Syndrome (n = 55), neither for the fluoropyrimidine-alone group (HR = 1.79, 95% CI = 0.37 to 8.65, P =.47) nor for oxaliplatin-based adjuvant chemotherapy (HR = 0.79, 95% CI = 0.15 to 4.11, P =.78). Discussion We report the first retrospective study to compare adjuvant chemotherapy with fluoropyrimidine alone or combined with oxaliplatin vs surgery alone in stage II and III dmmr CC patients. In this large cohort of patients, a statistically significant improvement in DFS was observed in fluoropyrimidine plus oxaliplatin adjuvant chemotherapy but not in fluoropyrimidine alone, as compared with surgery alone in patients with stage III disease. Some other retrospective studies have suggested that adding oxaliplatin to 5FU can restore the efficacy of adjuvant chemotherapy in stage III dmmr CC (23 27). However, these analyses were limited by the small number of dmmr CC patients and the lack of a control group of patients treated with surgery alone. A pooled analysis of the NSABP-C07 and NSABP-C08 trials showed that the benefit of adding oxaliplatin to adjuvant 5FU was not related to MMR status (n = 207) (28 30). In keeping with retrospective analyses of randomized trials, we observed no survival benefit of adjuvant chemotherapy with fluoropyrimidine alone in dmmr CC (7,9,12,31). Indeed, among 1027 stage II and III CC patients included in five previous adjuvant randomized trials in the 1990s, Sargent et al. found that the dmmr phenotype was predictive of a lack of survival benefit from 5FU (12). Adjuvant chemotherapy in stage II tumors is controversial (17). Some authors do not recommend adjuvant treatment for stage II dmmr CC because this phenotype is associated with a good prognosis and resistance to 5FU chemotherapy (7,12,32). However, no data are currently available on the potential benefit of chemotherapy in high-risk stage II dmmr CC. We observed that patients with high-risk stage II dmmr CC tended to have better outcomes with oxaliplatin-based adjuvant chemotherapy compared with surgery alone. These results need to be interpreted with caution because of the small number of the subgroup of patients and contradictory results in the literature (26). Identification of factors more specific for recurrence in stage II dmmr CC could help to predict the potential benefit of adjuvant chemotherapy for these patients (33). The benefit of adjuvant chemotherapy might differ according to the molecular mechanism underlying MMR deficiency (9). Indeed, we observed a statistically significant DFS benefit of oxaliplatin adjuvant-based chemotherapy as compared with surgery alone in sporadic stage III dmmr CC, which was not the case for the Lynch Syndrome group. Previous data suggested that BRAF mutation and/or CIMP phenotype, which are strongly associated with sporadic dmmr CC, confer reduced sensitivity to adjuvant 5FU (28,34 36). These findings could explain the larger survival benefit from the addition of oxaliplatin to fluoropyrimidine in sporadic dmmr patients compared with Lynch Syndrome patients. The main limitations of our study are its retrospective nature, the unbalanced numbers of patients included in the each treatment group, and the relatively small samples sizes of some subgroups of patients. In this nonrandomized study, the treatment allocation was left to investigators discretion after discussion in a multidisciplinary team meeting, which was mainly based on estimated risk of recurrence, age and the period study. Indeed, in the early 2000s, 5FU adjuvant chemotherapy was the preferred option treatment. Then, after the publication of the MOSAIC trial in 2004, FOLFOX chemotherapy became the preferred adjuvant treatment option (13). As already mentioned, 31% of stage III CC patients did not receive adjuvant chemotherapy, which is mainly because of older age and/or postoperative complications. This observation is in accordance with the literature data, showing that approximately 70% of stage III CC had adequate adjuvant treatment; factors affecting therapeutic adherence mainly included the age of the patient, which is inter-related with comorbidities and performance status (37,38). The major strengths of our study are: 1) the large number of dmmr CC Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

D. Tougeron et al. 8 of 9 Figure 2. Disease-free survival (DFS) according to the adjuvant chemotherapy regimen and tumor stage. Survival curves are represented using the Kaplan-Meier method, and survival rates were compared with the use of the two-sided log-rank test. DFS curves are presented according to adjuvant chemotherapy in (A) stage III and (B) high-risk stage II. Numbers of patients at risk in each group at various time points are given in the tables below each graph. patients included (n = 433), 2) the existence of a control group treated with surgery alone, and 3) the adjustment for several clinical and pathological features in the multivariable analyses model. The results provided by our study may be of great interest to clinicians as they could help with decision-making regarding adjuvant treatment for dmmr CC patients. In conclusion, we report in this largest study of dmmr CC patients a statistically significant improvement in DFS with oxaliplatin-based adjuvant chemotherapy in comparison with surgery alone in stage III patients. The observational nature of the study implies caution should be taken in the interpretation of these results. References 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69 90. 2. Aaltonen LA, Salovaara R, Kristo P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med. 1998;338(21):1481 1487. 3. Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of hmlh1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A. 1998;95(12):6870 6875. 4. Toyota M, Ahuja N, Ohe-Toyota M, et al. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A. 1999;96(15):8681 8686. 5. Domingo E, Niessen RC, Oliveira C, et al. BRAF-V600E is not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2 genes. Oncogene. 2005;24(24):3995 3998. Downloaded from https://academic.oup.com/jnci/-abstract/108/7/djv438/2412480

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