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1 Supplementary Online Content Venook AP, Niedzwiecki D, Lenz H-J, et al. Effect of first-line chemotherapy combined with cetuximab or bevacizumab on overall survival in patients with KRAS wild-type advanced or metastatic colorectal cancer: a randomized clinical trial. JAMA. doi: /jama efigure 1. Kaplan-Meier Estimates of Progression-Free Survival (PFS) by Treatment efigure 2. Forest plots for comparisons of Overall Survival (OS) by Treatment Regimen Overall and within each level of Sex, Race and. Hazard ratios and 95% Confidence Intervals are provided efigure 3. Progression-Free Survival (PFS) by Treatment among patients determined to be wt by expanded RAS analysis (KRAS: exon 2 codons 12, 13; exon 4, codons 117, 146; exon 3 codons 59, 61 or NRAS: exon 2 codons 12, 13; exon 3 codons 59, 61; exon 4 codons 117, 146) efigure 4. Overall Survival (OS) by Treatment Biologic among patients on the mfolfox6 Regimen (Oxaliplatin 85 mg/m2 over 120 minutes, leucovorin 400 mg/m2 over 2 hours followed by 5FU 400 mg/m2 bolus then 5FU 2400 mg/m2 by hour infusion) efigure 5. Overall Survival (OS) by Treatment Biologic among patients on the FOLFIRI Regimen (Irinotecan 180 mg/m2 over 90 minutes, leucovorin 400 mg/m2 over 2 hours followed by 5FU 400 mg/m2 bolus then 5FU 2400 mg/m2 by hour infusion) etable 1. Summary of Major Scientific Amendments to CALGB/SWOG etable 2. Summary of Treatment Administration on CALGB/SWOG by Agent and Treatment Arm This supplementary material has been provided by the authors to give readers additional information about their work.

2 SUPPLEMENTAL MATERIAL efigure 1. Kaplan-Meier Estimates of Progression-Free Survival (PFS) by Treatment* *Tick marks denote the last known follow-up time for patients with no death date reported. Arm N (Events) PFS Median (Interquartile Range) Hazard Ratio (95% CI) P* + Bevacizumab + Cetuximab 559 (516) 578 (528) 10.6 ( ) ( ) ( ) 0.45 *HR and P-value are adjusted for prior adjuvant therapy, prior radiotherapy, protocol chemotherapy and randomization pre- or post-amendment restricting eligibility to KRAS wt.

3 efigure 2. Forest plots for comparisons of Overall Survival (OS) by Treatment Regimen Overall and within each level of Sex, Race and. Hazard ratios and 95% Confidence Intervals are provided

4 efigure 3. Progression-Free Survival (PFS) by Treatment among patients determined to be wt by expanded RAS analysis (KRAS: exon 2 codons 12, 13; exon 4, codons 117, 146; exon 3 codons 59, 61 or NRAS: exon 2 codons 12, 13; exon 3 codons 59, 61; exon 4 codons 117, 146)*. *Tick marks denote the last known follow-up time for patients with no death date reported. Arm N (Events) PFS Median (Interquartile Range) Hazard Ratio (95% CI) P* + Bevacizumab + Cetuximab 256 (227) 270 (247) 11.0 ( ) ( ) ( ) 0.71 *HR and P-value are adjusted for prior adjuvant therapy, prior radiotherapy, protocol chemotherapy and randomization pre- or post-amendment restricting eligibility to KRAS wt.

5 efigure 4. Overall Survival (OS) by Treatment Biologic among patients on the mfolfox6 Regimen (Oxaliplatin 85 mg/m2 over 120 minutes, leucovorin 400 mg/m2 over 2 hours followed by 5FU 400 mg/m2 bolus then 5FU 2400 mg/m2 by hour infusion)* *Tick marks denote the last known follow-up time for patients with no death date reported. Arm N (Events) OS Median (Interquartile Range) HR (95% CI) P* + Bevacizumab + Cetuximab 409 (329) 426 (317) 27.7 ( ) ( ) ( ) 0.03 *HR and P-value are adjusted for prior adjuvant therapy, prior radiotherapy, protocol chemotherapy and randomization pre- or post-amendment restricting eligibility to KRAS wt.

6 efigure 5. Overall Survival (OS) by Treatment Biologic among patients on the FOLFIRI Regimen (Irinotecan 180 mg/m2 over 90 minutes, leucovorin 400 mg/m2 over 2 hours followed by 5FU 400 mg/m2 bolus then 5FU 2400 mg/m2 by hour infusion)* *Tick marks denote the last known follow-up time for patients with no death date reported. Arm N (Events) OS Median (Interquartile Range) Hazard Ratio* (95% CI) P* + Bevacizumab 150 (111) 32.2 ( ) 1.04 ( ) Cetuximab (117) ( ) *HR and P-value are adjusted for prior adjuvant therapy, prior radiotherapy, protocol chemotherapy and randomization pre- or post-amendment restricting eligibility to KRAS wt. etable 1. Summary of Major Scientific Amendments to CALGB/SWOG 80405

7 Action Date Content Revisions to Statistical Considerations Activation 9/15/2005 Update #3 7/15/2007 Formal monitoring of arterial thrombotic events (ATEs) added Update #4 2/15/2008 Efficacy monitoring amended to allow immediate reporting of interim results on the primary hypothesis, OS, and secondary hypothesis, PFS; amended from 20% of expected events. Suspension 6/15/2008 Evidence that KRAS mut tumors unlikely to benefit from cetuximab Update #5 9/9/2008 Eligibility limited to confirmed KRAS wt on exon 2, codons 12, 13 Study now includes preregistration phase to test all patients for K-ras status Patients enrolled preamendment must be tested retrospectively for K-ras status. Reactivation 11/15/2008 Update #6 9/15/2009 Combination biologic treatment arm dropped Update #8 7/12/2010 Results of local CLIA-certified laboratory KRAS testing accepted for eligibility; results must be confirmed centrally Interim analysis of ATEs was added to Statistical Considerations Statistical Considerations were modified to reflect additional looks at data Statistical Considerations were extensively modified to study 2,016 patients with confirmed KRAS wt tumors on three treatment arms The primary analysis was revised to a single comparison of bevacizumab versus cetuximab (for superiority) among 1,142 patients with confirmed KRAS wt tumors; interim analysis of OS and PFS was again revised to begin when 15% of expected OS events were observed Statistical Considerations were amended to study agreement between local and centralized KRAS test results

8 etable 2. Summary of Treatment Administration on CALGB/SWOG by Agent and Treatment Arm +Bevacizumab (N=559) +Cetuximab (N=578) Total (N=1137) 5-Fluorouracil Bolus Number Of Patients Receiving Treatment 542 (97.0%) 554 (95.8%) 1096 (96.4%) Number of Cycles Administered (1 cycle = 8 weeks) Number of Cycles of Treatment Median (Range) 3.0 ( ) 3.0 ( ) 3.0 ( ) % Targeted Dose Median (Range) ( ) ( ) ( ) Dose Adjustments Number of Patients with Adjustments 425 (76.0%) 432 (74.7%) 857 (75.4%) Total Adjustments Reasons For Adjustment Unknown 106 (4.2%) 115 (3.5%) 221 (3.8%) Adverse Event 1989 (77.9%) 2619 (79.6%) 4608 (78.9%) Hypersensivity 32 (1.3%) 24 (0.7%) 56 (1.0%) Patient Refusal or Noncompliance 258 (10.1%) 178 (5.4%) 436 (7.5%) Scheduling 139 (5.4%) 335 (10.2%) 474 (8.1%) Dosing Error 29 (1.1%) 19 (0.6%) 48 (0.8%) 5-Fluorouracil Infusion Number of Patients Receiving Treatment 542 (97.0%) 554 (95.8%) 1096 (96.4%) Number of Cycles Administered Number of Cycles of Treatment Median (Range) 3.0 ( ) 3.0 ( ) 3.0 ( ) % Targeted Dose Median (Range) 100.0( ) 100.0( ) 100.0( ) Dose Adjustments Number of Patients with Adjustments 425 (76.0%) 433 (74.9%) 858 (75.5%) Total Adjustments

9 etable 2: Summary of Treatment Administration on CALGB/SWOG by Agent and Treatment Arm (continued) +Bevacizumab (N=559) +Cetuximab (N=578) Total (N=1137) Reasons For Adjustment Unknown 106 (4.2%) 135 (4.1%) 241 (4.1%) Adverse Event 1987 (77.9%) 2599 (78.9%) 4586 (78.4%) Hypersensivity 32 (1.3%) 24 (0.7%) 56 (1.0%) Patient Refusal or Noncompliance 261 (10.2%) 196 (6.0%) 457 (7.8%) Scheduling 132 (5.2%) 317 (9.6%) 449 (7.7%) Leucovorin Number of Patients Receiving Treatment 539 (96.4%) 553 (95.7%) 1092 (96.0%) Number of Cycles Administered Number of Cycles of Treatment Median (Range) 3.0 ( ) 3.0 ( ) 3.0 ( ) % Targeted Dose Median (Range) 100.0( ) 100.0( ) 100.0( ) Dose Adjustments Number of Patients with Adjustments 314 (56.2%) 325 (56.2%) 639 (56.2%) Total Adjustments Reasons For Adjustment Unknown 164 (7.6%) 157 (5.5%) 321 (6.4%) Adverse Event 1147 (53.2%) 1476 (52.1%) 2623 (52.6%) Hypersensivity 35 (1.6%) 62 (2.2%) 97 (1.9%) Patient Refusal or Noncompliance 300 (13.9%) 236 (8.3%) 536 (10.7%) Scheduling 334 (15.5%) 664 (23.4%) 998 (20.0%) Dosing Error 172 (8.0%) 240 (8.5%) 412 (8.3%) Oxaliplatin Number of Patients Receiving Treatment 396 (70.8%) 409 (70.8%) 805 (70.8%) Number of Cycles Administered Number of Cycles of Treatment Median (Range) 3.0 ( ) 3.0 ( ) 3.0 ( ) % Targeted Dose Median (Range) 99.9 ( ) 99.8 ( ) 99.9 ( ) Dose Adjustments Number of Patients with Adjustments 338 (60.5%) 346 (59.9%) 684 (60.2%) Total Adjustments

10 etable 2: Summary of Treatment Administration on CALGB/SWOG by Agent and Treatment Arm (continued) +Bevacizumab (N=559) +Cetuximab (N=578) Total (N=1137) Reasons For Adjustment Unknown 113 (5.5%) 154 (6.5%) 267 (6.1%) Adverse Event 1641 (80.4%) 1879 (79.7%) 3520 (80.1%) Hypersensivity 64 (3.1%) 63 (2.7%) 127 (2.9%) Patient Refusal or Noncompliance 152 (7.5%) 134 (5.7%) 286 (6.5%) Scheduling 51 (2.5%) 72 (3.1%) 123 (2.8%) Dosing Error 19 (0.9%) 19 (0.8%) 38 (0.9%) Irinotecan Number of Patients Receiving Treatment 151 (27.0%) 148 (25.6%) 299 (26.3%) Number of Cycles Administered 607 (100.0%) 676 (100.0%) 1283 (100.0%) Number of Cycles of Treatment Median (Range) 3.0( ) 3.0( ) 3.0( ) % Targeted Dose Median (Range) 99.9( ) 99.9( ) 99.9( ) Dose Adjustments Number of Patients with Adjustments 159 (28.4%) 148 (25.6%) 307 (27.0%) Total Adjustments Reasons For Adjustment Unknown 181 (15.7%) 162 (11.2%) 343 (13.2%) Adverse Event 645 (55.9%) 921 (63.6%) 1566 (60.2%) Hypersensivity 31 (2.7%) 17 (1.2%) 48 (1.8%) Patient Refusal or Noncompliance 109 (9.5%) 107 (7.4%) 216 (8.3%) Scheduling 126 (10.9%) 182 (12.6%) 308 (11.8%) Dosing Error 1 (0.1%) 7 (0.5%) 8 (0.3%) Cetuximab Number of Patients Receiving Treatment 3 (0.5%) 556 (96.2%) 559 (49.2%) Number of Cycles Administered Number of Cycles of Treatment Median (Range) 1.0 ( ) 3.0 ( ) 3.0 ( ) % Targeted Dose Median (Range) 92.6 ( ) 92.6 ( ) Dose Adjustments Number of Patients with Adjustments 51 (9.1%) 381 (65.9%) 432 (38.0%) Total Adjustments

11 etable 2: Summary of Treatment Administration on CALGB/SWOG by Agent and Treatment Arm (continued) +Bevacizumab (N=559) +Cetuximab (N=578) +Bevacizumab (N=559) +Cetuximab (N=578) Reasons For Adjustment Unknown 222 (40.2%) 161 (5.4%) 383 (10.8%) Adverse Event 133 (24.1%) 2058 (68.5%) 2191 (61.6%) Hypersensivity 2 (0.4%) 35 (1.2%) 37 (1.0%) Patient Refusal or Noncompliance 49 (8.9%) 260 (8.7%) 309 (8.7%) Scheduling 86 (15.6%) 463 (15.4%) 549 (15.4%) Dosing Error 0 (0.0%) 26 (0.9%) 26 (0.7%) Bevacizumab Number of Patients Receiving Treatment 539 (96.4%) 3 (0.5%) 542 (47.7%) Number of Cycles Administered 1985 (100.0%) 3 (100.0%) 1988 (100.0%) Number of Cycles of Treatment Median (Range) 3.0 ( ) 1.0 ( ) 3.0 ( ) % Targeted Dose Median (Range) ( ) ( ) Dose Adjustments Number of Patients with Adjustments 316 (56.5%) 47 (8.1%) 363 (31.9%) Total Adjustments Reasons For Adjustment Unknown 145 (7.2%) 202 (42.1%) 347 (13.9%) Adverse Event 1243 (61.9%) 143 (29.8%) 1386 (55.7%) Hypersensivity 43 (2.1%) 5 (1.0%) 48 (1.9%) Patient Refusal or Noncompliance 296 (14.7%) 53 (11.0%) 349 (14.0%) Scheduling 262 (13.0%) 24 (5.0%) 286 (11.5%) Dosing Error 13 (0.6%) 0 (0.0%) 13 (0.5%)

12 emethods and Results: KRAS and expanded RAS BEAMing mutational analysis Mutational analysis was performed using BEAMing technology 1, 2 a technique built on emulsion polymerase chain reaction that allows detection of one mutant allele in 10,000 wild-type alleles. 3 Experiments were performed by Inostics GmbH (Hamburg, Germany). For the mutational analysis of tumour tissue DNA, archival tumour tissue specimens were examined by a pathologist and samples with fewer than 5% of tumour cells present were considered unevaluable (unless a mutation was subsequently detected). One to three tissue sections were scraped from glass slides and used for DNA isolation according to the kit manufacturer (Epicentre). For the BEAMing assay of tumour tissue DNA, the lower cutoff limit was set at a relatively conservative 1 0% mutant allele to avoid potential false-positive results caused by interpatient contamination resulting from tissue sectioning. The genes and mutations analyzed by BEAMing are: KRAS exon 2: codons 12, 13 exon 4: codons 117, 146 exon 3: codon 59, 61 NRAS exon 2: codons 12, 13 exon 3: codons 59, 61 exon 4: codons 117, 146

13 All KRAS and NRAS mutations examined have been shown to be oncogenically activating. 4, 5 In line with other techniques which may be used clinically to determine RAS mutation status, a cutoff of 1% mutant to wild-type alleles was used to discriminate patients. Tumors were scored as RAS mutant if mutant alleles were detected at a prevalence of 1% of total amplified sequences, regardless of whether all loci were evaluable. Tumors were scored as RAS wildtype only if all 26 mutation assays were evaluable and prevalence of mutant alleles was <1%. Results From 1137 patients we were able to collect 324 tumor tissues from patients enrolled in the chemotherapy and bevacizumab arm and 346 tumor tissues from patients enrolled in the chemotherapy and cetuximab arm. From these 670 wt KRAS exon 2 tumor samples, 621 were successfully analyzed (49 tumor samples lacked sufficient DNA or analyses were incomplete). We identified 95 (15.3%) mutations in the expanded RAS analysis including 1.3% in exon 2 in the wt kras exon 2 cohort. In addition, 1.8% were exon 3, codons 59, 61; 5.9% were exon 4, codons 117, 146 and NRAS: 2.3% exon2, codons 12, 13; 4.2% exon 3, codons 59, 61. One patient had a mutation at both NRAS exon 1 codon 12 and NRAS exon3 codon 61.

14 The patient characteristics from patients from whom tumor tissues were available similar to those without tissue available. These results were presented at ESMO 2014 by Dr. Lenz. 6

15 References 1. Higgins MJ, Jelovac D, Barnathan E, et al. Detection of tumor PIK3CA status in metastatic breast cancer using peripheral blood. Clin Cancer Res 2012; 18: Diehl F, Li M, He Y, et al. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat Methods 2006; 3: Li M, Diehl F, Dressman D, Vogelstein B, Kinzler KW. BEAMing up for detection and quantification of rare sequence variants. Nat Methods 2006; 3: Janakiraman M, Vakiani E, Zeng Z, et al. Genomic and biological characterization of exon 4 KRAS mutations in human cancer. Cancer Res 2010; 70: Smith G, Bounds R, Wolf H, et al. Activating K-Ras mutations outwith 'hotspot' codons in sporadic colorectal tumours - implications for personalised cancer medicine. Br J Cancer 2010; 102: Lenz H, Niedzwiecki D, Innocenti F, et al. CALGB/SWOG 80405: PHASE III trial of irinotecan/5-fu/leucovorin (FOLFIRI) or oxaliplatin/5-fu/leucovorin (mfolfox6) with bevacizumab (BV) or cetuximab (CET) for patients (pts) with expanded ras analyses untreated metastatic adenocarcinoma of the colon or rectum (MCRC). Annals of Oncol. Abstract 501O, 01 September 2014