3.1 Patients

Overall, 866 patients were included in the former study [17], of whom 10 (1.1%) carried a BRAF mutation and were excluded from further analysis. For 274 patients (31.6%), data about RAS extended analysis were not available and thus these patients were excluded from the present study. Overall, 582 patients were included in the present analysis (Online Resource Table 1), of whom 270 (46.6%) were WT, 212 (36.4%) carried a codon 12 KRAS mutation, 49 (8.4%) carried a codon 13 mutation, and 51 (8.7%) carried other KRAS rare mutations or NRAS mutations. Among codon 12 KRAS mutations, the G12D mutation was the most frequent. Details on detected mutations of our population are summarized in Table 1; patient characteristics are summarized in Table 2 and Table 3. In particular, when considering rego-treated patients (Table 2), characteristics were well-balanced between groups, with the exception of number of previous lines of therapy (>4 lines was less frequent in the G12C and G12D groups) and previous administered drugs, since anti-EGFRs were restricted to WT patients. About 50% of patients in each group received TFP/TPI in addition to rego (before or after, with a well-balanced distribution). Even for TFD/TPI-treated patients, characteristics were well-balanced between groups, with the exception of number of previous lines of therapy (>4 lines was less frequent in the G12C and G12D groups) and previously administered drugs, since anti-EGFRs were restricted to all WT patients. About 50% of patients in each group received rego in addition to FTD/TPI (before or after, with a well-balanced distribution).

Table 1 RAS assessment (582 patients)Table 2 Patient characteristics of regorafenib-treated patients [n = 363]Table 3 Patient characteristics of TFD/TPI-treated patients [n = 408]3.2 Efficacy Outcome According to RAS Extended Analysis in Regorafenib-Treated Patients

OS did not significantly differ in rego-treated patients according to RAS extended analysis (p = 0.69) (Fig. 1), although a trend toward a better median survival in patients carrying G12D mutation (12.0 months), Codon 13 mutation (8.0 months) and Codon 12 mutation (7.0 months) has been observed, when compared with WT patients (6.0 months). Accordingly, TTP did not differ significantly in rego-treated patients according to RAS extended analysis (p = 0.71) (Online Resource Fig. 1). However, a non-statistically significant benefit was observed in patients carrying a Codon 12 mutation (3.6 months), a rare mutation (3.9 months), or a G12C mutation (4.5 months), when compared with WT patients (3.0 months).

Fig. 1

Overall survival of Rego-treated patients according to RAS extended analysis

Patients receiving TFD/TPI (before or after, 184 patients) in addition to rego showed a statistically significant gain in OS compared with those not exposed to TFD/TPI (9 vs. 5 months; p = 0.005) (Online Resource Fig. 2a), suggesting an enrichment for favorable prognostic factors. When focusing on rego-treated patients who were exposed to TFD/TPI, a trend toward statistical significance was observed for WT patients (10 months), Codon 13 (12 months), and G12D (14 months; p = 0.05) (Online Resource Fig. 2b).

3.3 Efficacy Outcome According to RAS Extended Analysis in Trifluridine/Tipiracil-Treated Patients

OS did not significantly differ in TFD/TPI-treated patients according to RAS extended analysis (p = 0.09) (Fig. 2), although trend toward a better median survival was observed in WT patients (9 months) in comparison with the whole population (7.0 months). Accordingly, TTP did not significantly differ in TFD/TPI-treated patients according to RAS extended analysis (p = 0.86) (Online Resource Fig. 3). However, a non-statistically significant trend was observed in favor of WT patients (4.0 months) when compared with the whole population (3.7 months).

Fig. 2

Overall survival of TFD/TPI-treated patients according to RAS extended analysis

Patients receiving rego in addition to TFD/TPI (before or after, 188 patients) showed a statistically significant gain in OS when compared with those not exposed to rego (10 vs. 5 months; p < 0.001) (Online Resource Fig. 4a), suggesting an enrichment for favorable prognostic factors. According to RAS extended analysis, a survival gain was equally distributed among groups, without statistically significant differences (Online Resource Fig. 4b).

3.4 Efficacy Outcome According to Each Subgroup

Since no differences concerning efficacy of rego and TFD/TPI according to RAS extended analysis were documented, we analyzed each subgroup separately. These analyses are to be considered exploratory.

Patient characteristics were well-balanced in each population. Statistically significant differences in previously administered drugs for WT, Codon 12, and G12D patients were registered, with missing data being significantly more frequent for the rego-treated population, likely due to the issue that some patients in the rego group had been treated previously. Details of the comparison of patient characteristics in each group have been reported in Online Resource Table 2.

No differences in terms of the response rate were registered between the rego-treated population and the TFD/TPI-treated population in each RAS subgroup. Details about response rate have been reported in Online Resource Table 3. Moreover, concerning dose intensity, dose reductions were more frequent in rego-treated patients (compared with TFD/TPI patients), with statistically significant differences in WT, Codon 12 and Rare subgroups. Details on dose reductions have been reported in Online Resource Table 4, and details of comparison of patient characteristics according to the treatment sequence in each group have been reported in Online Resource Table 5.

Efficacy outcome in the WT group: In WT patients, median rego-related OS was significantly longer in patients receiving TFD/TPI (overall 79 patients [45.0%]; 46 before, 33 after) in addition to rego (p = 0.003) (Fig. 3a). At univariate analysis, including age, sex, number of previous lines, synchronous disease, PS, and sites of disease, no statistically significant differences in terms of OS were observed, with the exception of patients with PS <2 (p = 0.001) and patients with a synchronous disease (p = 0.002). Multivariate analysis was not performed because of the low sample size.

Fig. 3

Rego-related and TFD/TPI-related OS of all-RAS wt patients

At the same time, median TFD/TPI-related OS was significantly longer in patients receiving rego (overall 80 patients [45.1%]; 32 before, 48 after) in addition to TFD/TPI (p = 0.002) (Fig. 3b). At univariate analysis, including age, sex, number of previous lines, synchronous disease, PS, and sites of disease, no statistically significant differences in terms of OS were observed.

When focusing on the treatment administration sequence (comparing patients who received rego followed by TFD/TPI or the reverse sequence), no statistically significant differences were documented (p = 0.13) (Online Resource Fig. 5).

Efficacy outcome in the Codon 12 group: In Codon 12-mutated patients, median rego-related OS showed no differences between patients receiving TFD/TPI (overall 47 patients [52.8%]; 26 before, 21 after) in addition to rego (p = 0.16) (Fig. 4a). Conversely, median TFD/TPI-related OS was significantly longer in patients receiving rego (overall 49 patients [54.4%]; 19 before, 30 after) in addition to TFD/TPI (p = 0.0004) (Fig. 4b). At univariate analysis, including age, sex, number of previous lines, synchronous disease, PS, and sites of disease, no statistically significant differences in terms of OS were observed.

Fig. 4

Rego-related and TFD/TPI-related OS of Codon 12 patients

When focusing on the treatment administration sequence, patients receiving rego before TFD/TPI displayed a significantly longer OS (p = 0.003) (Fig. 5). At univariate analysis, including age, sex, number of previous lines, synchronous disease, PS, and sites of disease, no statistically significant differences in terms of OS were observed, with the exception of patients with liver involvement alone (p = 0.0002). Multivariate analysis was not performed because of the low sample size.

Fig. 5

OS of Codon 12 patients according to sequence (49 pts)

Efficacy outcome in the Codon 13 group: In Codon 13-mutated patients, median rego-related OS showed no differences between patients receiving TFD/TPI (overall 17 patients [58.6%]; 8 before, 9 after) in addition to rego (p = 0.35) (Online Resource Fig. 6a). Accordingly, median TFD/TPI-related OS showed no differences between patients receiving rego (overall 17 patients [44.7%]; 9 before, 8 after) in addition to TFD/TPI (p = 0.11) (Online Resource Fig. 6b).

When focusing on the treatment administration sequence, no statistically significant differences were documented (p = 0.12) (Online Resource Fig. 7), although a trend toward a better performance in patients receiving rego followed by TFD/TPI was observed.

Efficacy outcome in the rare mutations group: In the Rare group, median rego-related OS showed no differences between patients receiving TFD/TPI (overall 16 patients [53.3%]; 10 before, 6 after) in addition to rego (p = 0.80) (Online Resource Fig. 8a). Conversely, median TFD/TPI-related OS was significantly longer in patients receiving rego (overall 16 patients [43.2%]; 5 before, 11 after) in addition to TFD/TPI (p = 0.02) (Online Resource Fig. 8b). At univariate analysis, including age, sex, number of previous lines, synchronous disease, PS, and sites of disease, no statistically significant differences in terms of OS were observed, with the exception of patients with liver involvement alone (p = 0.0002). Multivariate analysis was not performed because of the low sample size.

When focusing on the treatment administration sequence, a trend toward a better performance in patients receiving rego first was observed (p = 0.09) (Online Resource Fig. 9).

Efficacy outcome in the G12C group: In the G12C group, median rego-related OS showed no differences between patients receiving TFD/TPI (overall 6 patients [60.0%]; 4 before, 2 after) in addition to rego (p = 0.98) (Online Resource Fig. 10a). Accordingly, median TFD/TPI-related OS was not significantly longer in patients receiving rego (overall 7 patients [50.0%]; 2 before, 5 after) in addition to TFD/TPI (p = 0.65) (Online Resource Fig. 10b).

When focusing on the treatment administration sequence, no statistically significant differences were documented (p = 0.73) (Online Resource Fig. 11).

Efficacy outcome in the G12D group. In patients in the G12D group, median rego-related OS showed no differences between patients receiving TFD/TPI (overall 19 patients [61.2%]; 9 before, 10 after) in addition to rego (p = 0.42) (Online Resource Fig. 12a). Conversely, median TFD/TPI-related OS was significantly longer in patients receiving rego (overall 19 patients [36.5%]; 10 before, 9 after) in addition to TFD/TPI (p = 0.03) (Online Resource Fig. 12b). At univariate analysis, including age, sex, number of previous lines, synchronous disease, PS, and sites of disease, no statistically significant differences in terms of OS were observed.

When focusing on the treatment administration sequence, no statistically significant differences were documented (p = 0.73) (Online Resource Fig. 13).