Prognostic value of KRAS genotype in metastatic colorectal cancer (MCRC) patients treated with intensive triplet chemotherapy plus bevacizumab (FIr-B/FOx) according to extension of metastatic disease

Background Bevacizumab (BEV) plus triplet chemotherapy can increase efficacy of first-line treatment of metastatic colorectal cancer (MCRC), particularly integrated with secondary liver surgery in liver-limited (L-L) patients. The prognostic value of the KRAS genotype in L-L and other or multiple metastatic (O/MM) MCRC patients treated with the FIr-B/FOx regimen was retrospectively evaluated. Methods Tumoral and metastatic samples were screened for KRAS codon 12 and 13 and BRAF mutations by SNaPshot and/or direct sequencing. Fit MCRC patients <75 years were consecutively treated with FIr-B/FOx regimen: weekly 12-h timed flat-infusion/5-fluorouracil (TFI 5-FU) 900 mg/m2, days 1, 2, 8, 9, 15, 16, 22 and 23; irinotecan (CPT-11) 160 mg/m2 plus BEV 5 mg/kg, days 1, 15; oxaliplatin (OXP) 80 mg/m2, days 8, 22; every 4 weeks. MCRC patients were classified as L-L and O/MM. Activity and efficacy were evaluated and compared using log-rank test. Results In all, 59 patients were evaluated: 31 KRAS wild-type (53%), 28 KRAS mutant (47%). At 21.5 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wild-type 90%, 14 months, 38 months; KRAS mutant 67%, 11 months, 20 months. PFS and OS were not significantly different. PFS and OS were significantly different in L-L compared to O/MM evaluable patients. In KRAS wild-type patients, clinical outcome of 12 L-L compared to 18 O/MM was significantly different: PFS 21 versus 12 months and OS 47 versus 28 months, respectively. In KRAS mutant patients, the clinical outcome of 13 L-L compared to 14 O/MM was not significantly different: PFS 11 months equivalently and OS 39 versus 19 months, respectively. Conclusions The KRAS genotype wild-type and mutant does not significantly affect different clinical outcomes for MCRC patients treated with the first-line FIr-B/FOx intensive regimen. KRAS wild-type patients with L-L disease may achieve a significantly prolonged clinical outcome due to integration with secondary liver surgery, with respect to KRAS mutant patients.


Background
Triplet regimens consisting of chemotherapeutic drugs, or doublets plus bevacizumab (BEV) (anti-vascular endothelial growth factor monoclonal antibody) or cetuximab (anti-epithelial growth factor receptor (EGFR) monoclonal antibody) in EGFR-overexpressing and KRAS wild-type metastatic colorectal cancer (MCRC), reported overlapping activity and efficacy in phase III trials, ranging between objective response rate (ORR) 39% to 68%, progression-free survival (PFS) 7.2 to 10.6 months, overall survival (OS) 19.9 to 26.1 months [1]. In 'fit' MCRC patients, these first-line options, integrated with secondary resection of liver metastases, significantly increased survival over doublet regimens [1,2]. More intensive medical treatment consisting of triplet chemotherapy plus targeted agents can further increase activity, thus raising resection rate of liver metastases and clinical outcome [1][2][3][4][5]. Phase II studies, by Masi et al. [3], and by our group [4], proposed BEV addition to triplet chemotherapy, according to FOLFOXIRI/BEV or FIr-B/FOx schedules, reaching ORR 77% and 82%, median PFS 13.1 and 12 months, median OS 30.9 and 28 months, as first-line treatment of MCRC patients. Liver metastasectomies were performed in 32% and 26% overall and 40% and 54% liver-only patients, respectively. Thus, MCRC patients with liver-limited (L-L) disease, integrating FIr-B/FOx intensive regimen and secondary liver surgery significantly improved clinical outcome compared to MCRC patients with multiple metastatic disease, up to median PFS 17 months and median OS 44 months [6].
Clinical outcome (PFS, OS) according to wild-type and mutant genotype assesses the prognostic relevance of a specific biomarker, potentially including the predictive role of effectiveness of treatment strategies. In randomized studies, the predictive relevance of wild-type or mutant genotype can also be specifically assessed by comparing experimental and control arms. The reported median OS values of KRAS wild-type and mutant MCRC patients treated with irinotecan, 5-fluorouracil and leucovorin (IFL) plus BEV were 27.7 and 19.9 months, respectively [18,21]. The prognostic relevance of KRAS or BRAF wild-type compared to KRAS or BRAF mutant genotype was not significantly different, even though the hazard ratio (HR) was 0.64 and 0.38, respectively. A significantly better prognosis was reported only when KRAS/BRAF wild-type patients were compared with patients harboring mutations in the KRAS or BRAF genes (HR 0.51) [18]. KRAS wild-type genotype significantly predicts a favorable clinical outcome of anti-EGFR or anti-vascular endothelial growth factor (VEGF) drugs added to doublet chemotherapy [18,[21][22][23].
In the KRAS mutant genotype, BEV addition to IFL significantly prolonged PFS up to 9.3 months, without increasing OS and activity, compared to IFL [18,21].
Here, we report a retrospective exploratory analysis evaluating the prognostic value of the KRAS genotype in MCRC patients enrolled in a previously reported phase II study [4] and in an expanded clinical program proposing FIr-B/FOx intensive regimen as first-line treatment, also verifying recently reported significantly greater effectiveness in L-L compared to other or multiple metastatic (O/MM) patients [6].

Patient eligibility
MCRC patients were enrolled in a previously reported phase II study [4] and in the expanded clinical program proposing FIr-B/FOx association as first-line treatment. Patients were eligible if they had a histologically confirmed diagnosis of measurable MCRC; were age 18 to 75 years; had World Health Organization (WHO) performance status ≤2; had adequate hematological, renal and hepatic functions; and had a life expectancy more than 3 months. The study was approved by the Local Ethical Committee (Comitato Etico, Azienda Sanitaria Locale n.4 L'Aquila, Regione Abruzzo, Italia) and conducted in accordance with the Declaration of Helsinki. All patients provided written, informed consent.

Mutational analysis
KRAS and BRAF genetic analyses were performed on paraffin-embedded tissue blocks from the primary tumor and/or metastatic sites. Genotype status was assessed for KRAS codon 12 and 13 mutations and BRAF c.1799 T>A (V600E) mutation by SNaPshot ® multiplex screening for KRAS mutations and KRAS/BRAF mutations in 36 and 32 samples, respectively [26,27]; direct sequencing was performed for detection of KRAS mutations in 23 samples and to confirm detected mutations. After treatment with xylene thyocyanate and selection of tumoral cell clusters, DNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues (Applied Biosystems, Courtaboeuf, France) according to manufacturer's instructions. When considering the contamination of tumoral samples by non-malignant cells, a KRAS mutation in the tumor was defined as appearance of a mutant peak with a height of at least one-third compared to the wild-type.

SNaPshot and Direct Sequencing assays
SNaPshot multiplex assay was performed as elsewhere reported [26,27]. Briefly, KRAS exon 2 and BRAF exon 15 were simultaneously amplified by polymerase chain reaction (PCR) using specific primers and purified using NucleoSpin ® Extract II kit (Macherey-Nagel EURL, Hoerdt, France). PCR-amplified DNA was analyzed using the ABI PRISM SNaPshot Multiplex kit (Applied Biosystems, Foster City, CA, USA) and five primers including an additional tail at their 5' end allowing their simultaneous detection. Sense primers allowing the extension at nucleotides KRAS c.34G, c.35G, c.37G, c.38G and BRAF c.1799T were used and a multiplex SNaPshot reaction was performed as reported [26]. KRAS exon 2 sequencing was performed from PCR-amplified tumor DNA using the Big Dye V3.1 Terminator Kit (Applied Biosystems, Foster City, CA, USA). Labeled products were separated using an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Data were analyzed using the GeneMapper Analysis Software version 4.0 (Applied Biosystems, Foster City, CA, USA).

Study design
A retrospective analysis was planned to evaluate prognostic relevance of KRAS genotype on clinical outcome of MCRC patients treated with FIr-B/FOx as first-line treatment. Moreover, patients were classified according to involved metastatic sites, L-L and O/MM [6], to evaluate the relevance of metastatic extension in KRAS wild-type and mutant MCRC patients. Patients with L-L metastases were evaluated at baseline and every three cycles of treatment by a multidisciplinary team, consisting of a medical oncologist, liver surgeon and radiologist, to dynamically evaluate resectability defined according to resectability categories previously reported [6]. Resection rate was evaluated in the intent-to-treat population enrolled. Liver metastasectomies were defined as R0, if radical surgery, R1, if radioablation was added. Surgery was recommended >4 weeks after BEV discontinuation. Clinical evaluation of response was made by computed tomography (CT) scan; positron emission tomography (PET) was added based on investigators' assessment.
Clinical criteria of activity and efficacy were ORR, PFS and OS. ORR was evaluated according to Response Evaluation Criteria In Solid Tumors (RECIST) criteria [28]; pathologic complete response was defined as absence of residual cancer cells in surgically resected specimens. The overall activity of integrated medical treatment and secondary liver surgery, consisting of the sum of clinical complete responses (cCR) and liver metastasectomies was also evaluated, as previously reported [6]. PFS and OS were evaluated using the Kaplan-Meier method [29]. PFS and PFS from surgery were defined, respectively, as the length of time from the beginning of treatment or the date of liver metastasectomy and disease progression or death (resulting from any cause) or to the last contact; OS as the length of time between the beginning of treatment and death or to last contact. The Log-rank test was used to compare PFS and OS in KRAS wild-type versus mutant, L-L versus O/MM, and KRAS wild-type L-L versus O/MM, and KRAS mutant L-L versus O/MM MCRC patients [30].
Median PFS and OS values of MCRC patients treated with FIr-B/FOx were different in KRAS wild-type and mutant patients, even if not significantly, while they were equivalent in the FOLFOXIRI plus BEV study [3]. BEV addition to doublet IFL chemotherapy gave median PFS 13.5 and 9.3 months, median OS 27.7 and 19.9 months in KRAS wild-type and mutant patients, respectively [18,21]. Significantly better prognosis was reported in KRAS/BRAF wild-type patients compared with patients harboring mutations in the KRAS or BRAF genes (HR 0.51) [18]. Direct comparison of OS between KRAS wild-type and mutant MCRC patients treated with BEV-containing chemotherapy failed to significantly differentiate prognosis, as in the present study. Thus, intensive regimens adding BEV to triplet chemotherapy can further increase activity and efficacy in KRAS wild-type and mutant patients. Randomized studies would be able to properly evaluate this.
The high activity of triplet chemotherapy plus BEV regimens correlated with increased resection rate of liver metastases and pathologic CR, particularly in L-L MCRC patients [1,3,4,6]. We recently reported that the clinical outcome of L-L compared to multiple metastatic disease was significantly improved up to median PFS 17 months and median OS 44 months [6] due to the effectiveness of integrated FIr-B/FOx intensive treatment and secondary liver surgery. The present analysis confirms the significantly favorable prognosis of L-L compared to MM patients and show that KRAS wild-type L-L patients, accounting for 20% of fit MCRC patients, could gain 100% overall activity with an integrated medical and surgical approach, due to performed liver metastasectomies and long-lasting cCRs; median PFS 21 months and OS 47 months. A significantly favorable prognosis was demonstrated in KRAS wild-type L-L compared to O/MM patients, even if this represents a retrospective, exploratory analysis in a small cohort of MCRC patients. Using neoadjuvant cetuximab with either FOLFOX6 or FOLFIRI for unresectable colorectal liver metastases, metastasectomies were performed in 38% and 30% patients, respectively [36]. Chrono-IFLO/cetuximab reported a 60% R0 resection rate in unresectable colorectal liver metastases, with ORR 79%, median PFS 14 months and median OS 37 months [5]. Further prospective studies will properly address whether intensive medical treatments, such as FIr-B/FOx, and secondary liver surgery could represent the standard multidisciplinary strategy for KRAS wild-type L-L MCRC patients. In KRAS mutant patients, prevalently harboring c.35 G>A transversion (53.5%), integrated medical and surgical treatment failed to significantly increase PFS and OS in L-L compared to O/MM patients: median PFS was equivalent (11 months), in spite of 54% performed liver metastasectomies in L-L patients; median OS was 39 and 19 months, respectively. These data should be further evaluated in a larger cohort of MCRC patients. A proper multidisciplinary treatment strategy for KRAS mutant patients, showing different aggressiveness [37], sensitivity to medical treatment, and worse clinical behavior, is an unmet need.

Conclusions
KRAS wild-type and mutant genotypes do not significantly affect the clinical outcomes of MCRC patients treated with the first-line FIr-B/FOx intensive regimen. KRAS wild-type patients with L-L disease may achieve significantly greater benefit from integration with liver metastasectomies compared to O/MM metastatic extension, with respect to KRAS mutant patients. The present findings should be verified in prospective trials of multidisciplinary strategies comparing clinical outcome according to KRAS genotype in patients with L-L and O/MM disease.