Supplementary material

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1 Supplementary material

Supplemental methods ... 2

Table S1 Treatment exposure (safety population)... 3

Table S2 Multivariate analysis of prognostic factors for progression-free survival ... 4

Table S3 Safety summary (safety population) ... 5

Table S4 Treatment-emergent adverse events (safety population) ... 6

Table S5 Second-line and later treatments by treatment group (intention-to-treat population) ... 7

Figure S1 Kaplan-Meier estimates of progression-free survival after excluding patients with extended RAS mutations or unknown RAS status (n=16) ... 8

Figure S2 Kaplan-Meier estimates of progression-free survival and overall survival by treatment group and BRAF and PIK3CA status (intention-to-treat population) ... 9

Figure S3 Kaplan-Meier estimates of progression-free survival (A) and overall survival (B) according to the presence of BRAF and/or PIK3CA mutations (n=44) ... 10

Figure S4 Kaplan-Meier estimates of progression-free survival (A) and overall survival (B) according to tumour sidedness in patients with BRAF/PIK3CA mutations (n=44) ... 11

Figure S5 Kaplan-Meier estimates of progression-free survival (A) and overall survival (B) for

FOLFIRI plus bevacizumab vs FOLFIRI plus cetuximab in patients with BRAF/PIK3CA-wild-type left-

sided tumours (n=162) ... 12

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2

SUPPLEMENTAL METHODS

Sample size calculation

In patients with BRAF- and PIK3CA-wild-type tumours, the minimum median progression-free survival, below which the result was deemed to be unacceptable was 8.5 months [1] and the optimal value was 13.0 months. To attain a 2-year progression-free rate with 95% confidence intervals (CI) of

±10%, 89 evaluable patients were required per group. Assuming a maximum of 9% losses during follow-up before progression, it was necessary to recruit 97 patients to the FOLFIRI plus bevacizumab group and 97 patients to the FOLFIRI plus cetuximab group.

In patients with BRAF- and/or PIK3CA-mutated tumours, the minimum acceptable median progression- free survival was 2.5 months [2] and the optimal value was 6 months. With these assumptions, 21 evaluable patients were needed per group. Assuming a maximum of 9% losses during follow-up before progression, it was necessary to recruit 23 patients into the FOLFIRI plus bevacizumab group and 23 patients into the FOLFIRI plus cetuximab group.

Therefore, a total of 240 patients were to be recruited into the trial divided into 4 arms as follows:

 194 patients with BRAF- and PIK3CA-wild-type tumours to be randomly assigned to: FOLFIRI plus bevacizumab (97 patients) and FOLFIRI plus cetuximab (97 patients).

 46 patients with BRAF- and/or PIK3CA-mutated tumours to be randomly assigned to FOLFIRI plus bevacizumab (23 patients) and FOLFIRI plus cetuximab (23 patients).

REFERENCES

1. Price TJ, Hardingham JE, Lee CK, et al. Impact of KRAS and BRAF gene mutation status on outcomes from the phase III AGITG MAX trial of capecitabine alone or in combination with bevacizumab and mitomycin in advanced colorectal cancer. J Clin Oncol 2011;29:2675-82.

2. Tol J, Koopman M, Miller MC, et al. Circulating tumour cells early predict progression-free and

overall survival in advanced colorectal cancer patients treated with chemotherapy and targeted

agents. Ann Oncol 2010;21:1006-12.

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3 Table S1 Treatment exposure (safety population)

Variable

FOLFIRI plus bevacizumab (n=126)

FOLFIRI plus cetuximab (n=113)

Median (range) duration of treatment (weeks)

28.0 (2.0–166.6) 26.1 (2.0–143.4)

Median (range) number of cycles^a 13 (1–76) 12 (1–70)

Median (range) relative dose intensity^b (%)

5-Fluorouracil 88.4 (48.3–101.3) 87.7 (0–104.0)

Irinotecan 89.2 (49.0–101.5) 88.9 (0–104.0)

Bevacizumab 91.2 (54.7–102.3) –

Cetuximab – 82.8 (0–103.6)

Treatment delay, n (%) 99 (78.6) 81 (71.7)

Dose reduction, n (%) 48 (38.1) 44 (38.9)

Note: For patients undergoing surgery during the treatment period, the treatment information prior to the first surgery has been included. Leucovorin was administered using different protocols and so is not included.

aCycle length = 2 weeks.

bRelative dose intensity defined as the quotient between the administered dose and the theoretical dose expressed as a percentage.

FOLFIRI, 5-fluorouracil, leucovorin, and irinotecan.

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4 Table S2 Multivariate analysis of prognostic factors for progression-free survival

Variable Reference group Hazard

ratio

95% CI p value

Treatment arm FOLFIRI plus bevacizumab 0.94 0.65–1.36 0.760

BRAF and PIK3CA status Wild-type 0.76 0.48–1.20 0.241

ECOG performance status 1 1.59 1.10–2.29 0.014

Tumour location Other sites 1.18 0.78–1.78 0.424

Tumour location Right 1.89 1.19–3.01 0.007

Presentation Metachronous 0.57 0.22–1.49 0.253

Surgery for primary tumour No 1.06 0.71–1.58 0.772

Prior treatment No 0.98 0.36–2.66 0.965

Prior radiotherapy No 0.54 0.23–1.31 0.173

Basal CEA levels ≤5 μg/L 0.66 0.42–1.03 0.065

Age <65 years 1.09 0.74–1.62 0.662

Sex Male 0.89 0.59–1.35 0.594

RAS status Mutated or not definable 0.81 0.40–1.64 0.564

CEA, carcinoembryonic antigen; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group;

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5 Table S3 Safety summary (safety population)

BRAF/PIK3CA-wild-type BRAF- and/or PIK3CA-mutated

FOLFIRI plus cetuximab (n=93)

All patients (n=195)

All patients (n=44)

All-grade TEAE 101 (99.0) 93 (100.0) 194 (99.5) 24 (100.0) 20 (100.0) 44 (100.0)

All-grade TEAE treatment-related 100 (98.0) 91 (97.9) 191 (98.0) 23 (95.8) 20 (100.0) 43 (97.7)

Serious TEAE 27 (26.5) 38 (40.9) 65 (33.3) 9 (37.5) 5 (25.0) 14 (31.8)

Serious TEAE treatment-related 16 (15.7) 18 (19.4) 34 (17.4) 4 (16.7) 1 (5.0) 5 (11.4)

Grade ≥3 TEAE 59 (57.8) 64 (68.8) 123 (63.1) 17 (70.8) 16 (80.0) 33 (75.0)

Grade ≥3 TEAE treatment-related 48 (47.1) 45 (48.4) 93 (47.7) 11 (45.8) 15 (75.0) 26 (59.1)

Grade 5 TEAE 4 (3.9) 1 (1.1) 5 (2.6) 2 (8.3) 1 (5.0) 3 (6.8)

Grade 5 TEAE treatment-related 3 (2.9) 0 (0) 3 (1.5) 0 (0) 0 (0) 0 (0)

Data are n (%).

FOLFIRI, 5-fluorouracil, leucovorin, and irinotecan; TEAE, treatment-emergent adverse event.

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6 Table S4 Treatment-related treatment-emergent adverse events (safety population)

Adverse event

BRAF/PIK3CA-wild-type (n=195) BRAF- and/or PIK3CA-mutated (n=44)

All patients (n=195) FOLFIRI plus bevacizumab (n=24)

All patients (n=44)

Grade 1–2

Grade 3–4

Grade 1–2

Grade 3–4

Grade 1–2

Grade 3–4

Grade 1–2

Grade 3–4

Grade 1–2

Grade 3–4

Grade 1–2

Grade 3–4 Diarrhoea 58 (56.9) 8 (7.8) 44 (47.3) 12 (12.9) 102 (52.3) 20 (10.3) 16 (66.7) 3 (12.5) 7 (35.0) 0 (0) 23 (52.3) 3 (6.8) Asthenia 52 (51.0) 4 (3.9) 38 (40.9) 8 (8.6) 90 (46.2) 12 (6.2) 13 (54.2) 4 (16.7) 6 (30.0) 3 (15.0) 19 (43.2) 7 (15.9) Mucositis 38 (37.3) 3 (2.9) 32 (34.4) 3 (3.2) 70 (35.9) 6 (3.1) 14 (58.3) 0 (0) 3 (15.0) 2 (10.0) 17 (38.6) 2 (4.6) Neutropenia 26 (25.5) 23 (22.6) 28 (30.1) 11 (11.8) 56 (27.7) 34 (17.4) 5 (20.8) 5 (20.8) 2 (10.0) 4 (20.0) 7 (15.9) 9 (20.5) Skin toxicity

dermatitis-like

10 (9.8) 0 (0) 59 (63.4) 16 (17.2) 69 (35.4) 16 (8.2) 1 (4.2) 0 (0) 13 (65.0) 3 (15.0) 14 (31.8) 3 (6.8)

Hand–foot skin reaction

9 (8.8) 0 (0) 10 (10.8) 1 (1.1) 19 (9.7) 1 (0.5) 1 (4.2) 0 (0) 4 (20.0) 1 (5.0) 5 (11.4) 1 (2.3)

Pulmonary embolism 0 (0) 2 (2.0) 0 (0) 4 (4.3) 0 (0) 6 (3.1) 0 (0) 0 (0) 0 (0) 1 (5.0) 0 (0) 1 (2.3) Febrile neutropenia 0 (0) 1 (1.0) 1 (1.1) 5 (5.4) 1 (0.5) 6 (3.1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Data are n (%).

Note: Events listed are those for which grade 3/4 events occurred in at least 3% of the BRAF/PIK3CA-wild-type or BRAF- and/or PIK3CA-mutated cohorts.

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7 Table S5 Second-line and later treatments by treatment group (intention-to-treat population)

FOLFIRI plus bevacizumab (n=126)

FOLFIRI plus cetuximab (n=114)

Chemotherapy 93 (73.8) 84 (73.7)

Fluoropyrimidine^a 88 (69.8) 82 (71.9)

Oxaliplatin 74 (58.7) 71 (62.3)

Irinotecan 62 (49.2) 50 (43.9)

TAS-102 12 (9.5) 15 (13.2)

Mitomycin 4 (3.2) 1 (0.9)

Gemcitabine 4 (3.2) 0 (0)

Raltitrexed 4 (3.2) 3 (2.6)

Anti-EGFR agents 67 (53.2) 39 (34.2)

Cetuximab 40 (31.8) 23 (20.2)

Panitumumab 36 (28.6) 19 (16.7)

Anti-angiogenic agents 45 (35.7) 63 (55.3)

Bevacizumab 36 (28.6) 61 (53.5)

Regorafenib 14 (11.1) 13 (11.4)

Aflibercept 0 (0) 2 (1.8)

Other 8 (6.4) 3 (2.6)

Data are n (%). Patients may have received more than 1 agent in each class.

aIncluded 5-fluorouracil, capecitabine, and tegafur.

EGFR, epidermal growth factor receptor; FOLFIRI, 5-fluorouracil, leucovorin, and irinotecan.

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8 Figure S1 Kaplan-Meier estimates of progression-free survival after excluding patients with

extended RAS mutations or unknown RAS status (n=16).

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9 Figure S2 Kaplan-Meier estimates of progression-free survival (A) and overall survival (B) by treatment group and BRAF/PIK3CA status (intention-to-treat population).

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10 Figure S3 Kaplan-Meier estimates of progression-free survival (A) and overall survival (B)

according to the presence of BRAF and/or PIK3CA mutations (n=44).

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11 Figure S4 Kaplan-Meier estimates of progression-free survival (A) and overall survival (B) according

to tumour sidedness in patients with BRAF- and/or PIK3CA mutations (n=44).

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12 Figure S5 Kaplan-Meier estimates of progression-free survival (A) and overall survival (B) for FOLFIRI plus bevacizumab vs FOLFIRI plus cetuximab in patients with BRAF/PIK3CA-wild-type left- sided tumours (n=162*).

Note: Eight patients had RAS-mutant tumours and 3 RAS patients had not definable.