Prognostic relevance of KRAS genotype in metastatic colorectal cancer patients unfit for FIr-B/FOx intensive regimen

First-line triplet chemotherapy plus bevacizumab (FIr-B/FOx) can improve efficacy of metastatic colorectal cancer (MCRC), KRAS wild-type and mutant. Prognostic relevance of KRAS genotype was evaluated in patients unfit for FIr-B/FOx, treated with conventional medical treatments. Consecutive MCRC patients not eligible for FIr-B/FOx regimen due to age (≥75 years) and/or comorbidities were treated with tailored conventional first-line treatments. KRAS codon 12/13 mutations were screened by direct sequencing. Activity and efficacy were evaluated and compared according to medical treatments, age (non-elderly and elderly ≥65 years), comorbidity stage (Cumulative Illness Rating Scale), metastatic extension (liver-limited and other/multiple metastatic), and KRAS genotype, using log-rank. Selected first line treatments were medical in 37 patients (92.5%), and surgical in 3 patients (7.5%). Medical treatment regimens: triplet, 18 (45%); doublet, 15 (37.5%); mono-therapy, 4 (10%). At median follow-up of 8 months, objective response rate (ORR) was 37%, median progression-free survival (PFS) 7 months, liver metastasectomies 8% (liver-limited disease 37.5%), median overall survival (OS) 13 months. Triplet regimens failed to significantly affect clinical outcome, compared to doublet. According to KRAS genotype, ORR, PFS and OS were, respectively: wild-type 50%, 8 months, 13 months; mutant 25%, 6 months, 9 months. KRAS genotype wild-type compared to mutant significantly affected PFS, while not OS. KRAS c.35 G>A mutation (G12D) significantly affected worse PFS and OS compared to wild-type and/or other mutations. KRAS genotype, specifically the c.35 G>A KRAS mutation, may indicate poor prognosis in MCRC patients unfit for intensive medical treatments.

In clinical practice, a decision-making process including functional, nutritional, and co-morbidity status is required to tailor first line medical treatment (10). Elderly status (age >65 years), PS >2, and/or comorbidities represent major features, to limit toxicities and maintain quality of life (QoL). Elderly MCRC patients are prevalent, and a clinical challenge is to select between intensive or tailored medical treatments, by properly weighing expected safety and efficacy, and according to prognostic factors. Retrospective studies showed that elderly patients benefit from 5-fluorouracil (5-FU) (11)(12)(13), irinotecan (CPT-11)-containing therapy (14,15), FOLFOX (16) to the same extent as younger (17)(18)(19). In the OPTIMOX1 trial, ORR 59%, PFS 9.0 months and OS 20.7 months were comparable between old-elderly and younger patients treated with FOLFOX (20). Treatment efficacy was also comparable with BEV associated to 5-FU/CPT-11 (21). In elderly patients, addition of BEV to 5-FU based chemotherapy significantly prolonged PFS (9.2-9.3 months) and OS (17.4-19.3 months) (22,23). In BRiTE and BEAT studies, no different PFS was observed in elderly patients; median OS decreased with age (24,25). In the randomized phase III trial comparing FOLFIRI with FOLFOXIRI, age was not significantly related to activity and efficacy, with OS 16.9 and 19.9 months, respectively (26,27). ORR was significantly lower in older patients treated with FOLFOXIRI (27). Patients underwent metastasectomies without increased morbidity or mortality, irrespective of age. Patients with PS 2 presented a significantly lower OS and PFS, irrespectively of FOLFIRI or FOLFOXIRI chemotherapy regimen (27). Age and/or comorbidities did not affect efficacy in patients treated with cetuximab added to FOLFOX or FOLFIRI (28). In elderly and PS 2 patients, PFS was not increased by addition of panitumumab to FOLFOX (29). A meta-analysis showed that PS 1 compared to PS 2 significantly affect prognosis, regardless of treatment, with ORR 43.8 vs 32%, PFS 7.6 vs 4.9 months, OS 17.3 and 8.5 months, respectively (30). The FOCUS2 randomized trial prospectively evaluated first line chemotherapy options consisting of 80% dose 5-FU or capecitabine, with or without oxaliplatin (OXP), in old-elderly and/or frail patients, and showed that addition of OXP significantly improved ORR (35 vs 13%), a trend of PFS (5.8 vs. 4.5 months, hazard ratio 0.84, p=0.07), but not OS (31), without significantly increasing toxicity, with a negative impact on QoL.
Clinical outcome in wild-type and mutant patients assesses the prognostic relevance of KRAS genotype, depending on differential tumor biological aggressiveness (4), including the predictive effectiveness of treatment strategies. Median OS of patients treated with BEV added to CPT-11/5-FU or triplet chemotherapy was different in KRAS wild-type and mutant patients, but not significantly (4,8,36,37); KRAS wild-type L-L patients may achieve a significantly greater benefit from integration with liver metastasectomies, with respect to mutant patients (4). We recently reported that the prevalent KRAS c.35 G>A (G12D) mutant genotype may significantly affect worse OS of MCRC patients treated with FIr-B/FOx, compared to wild-type or different other mutations (40). Here, we report a retrospective exploratory analysis evaluating tailored first line treatments, the prognostic value of KRAS genotype, and of the c.35 G>A mutation, in consecutive MCRC patients not eligible for intensive first line FIr-B/FOx expanded clinical program, due to age and/or comorbidities.

Materials and methods
Patient eligibility. Consecutive MCRC patients not eligible, due to comorbidities and/or age, for expanded clinical program or ongoing phase II trial proposing intensive regimens consisting of triplet chemotherapy plus targeted agent, were treated in clinical practice with first line medical and/ or surgical treatments, chosen among those in indication for MCRC treatment and approved by Agenzia Italiana del Farmaco (AIFA) for administration in label in Italian public hospitals, and published in Gazzetta Ufficiale Repubblica Italiana ('Elenco dei Medicinali erogabili a totale carico del Servizio sanitaria nazionale', Gazzetta Ufficiale Repubblica Italiana N.1, 2 Gennaio 2009). Thus, it was not a clinical trial and approval by ethics committee and institutional review board was not necessary, because patients were treated with conventional treatments without any additional medical intervention out of the best common clinical practice. Patients had histological confirmed diagnosis of MCRC, age ≥18 years, PS ≤2. Criteria to define patients unfit, or not eligible for intensive regimens were: age ≥75 years; uncontrolled severe diseases; cardiovascular disease (uncontrolled hypertension, uncontrolled arrhythmia, ischemic cardiac diseases in the last year); thromboembolic disease, coagulopathy, preexisting bleeding diatheses; proteinuria >1 g/24 h. Patients were classified according to Cumulative Illness Rating Scale (CIRS) (10). Treatment options were tailored according to age (< or ≥75 years), patient's fitness (PS, CIRS), KRAS genotype. Patients with PS 3 were not treated. All patients provided written, informed consent to the proposed in label treatment option. Other triplet, doublet and mono-regimens were administered according to previously reported schedules (7,41,42). Targeted agents were: BEV (Avastin; Roche, Welwyn Garden City, UK), 5 mg/kg, days 1 and 15; cetuximab (Erbitux; Merck, Darmstadt, Germany), 400 mg/m 2 initial dose, then 250 mg/m 2 /week.

Mutational analysis.
Genetic analyses were performed on paraffin-embedded tissue blocks from primary tumor and/ or metastatic sites, as previously reported (4). Genotype status was assessed for KRAS codon 12 and 13 mutations by direct sequencing. KRAS exon 2 sequence was performed from PCR-amplified tumor DNA using the Big Dye V3.1 Terminator kit, electrophoresis in ABI PRISM 3130xl Genetic Analyzer, and analysis using the GeneMapper Analysis software version 4.0 (Applied Biosystems, Foster City, CA, USA).
Study design. Activity, efficacy, and prognostic relevance of first line treatments, and KRAS genotype on clinical outcomes were evaluated. Patients were classified according to: metastatic extension, L-L and O/MM (3,4); age, non-elderly (<65 years), young-elderly (≥65 <75 years), old-elderly (≥75 years); CIRS stage primary, intermediate, secondary. Clinical evaluation of response was made by CT scan; PET was added based on investigators' assessment. Follow-up was scheduled every two-three months up to disease progression or death. L-L patients were evaluated at baseline and every two-three cycles of treatment by a multidisciplinary team, to evaluate resectability defined according to reported categories (3). Liver metastasectomies were defined as R0, if radical surgery, R1, if radioablation was added. Surgery was recommended >4 weeks after BEV discontinuation.
Clinical criteria of activity and efficacy were ORR, resection rate of metastases, PFS and OS: ORR, evaluated according to RECIST criteria (43); pathologic complete response, defined as no residual cancer cells in surgical specimens; PFS and OS, evaluated using the Kaplan-Meier method (44). PFS was defined as the length of time from the beginning of treatment 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. Log-rank test was used to compare PFS and OS according to medical treatment, KRAS genotype, metastatic extension, age and comorbidity stage (45).
Young-elderly patients eligible for FIr-B/FOx intensive regimen, prevalently characterised by PS 0 (89%) and intermediate CIRS stage (93%), reported ORR 79%, PFS 11 months, OS 21 months, equivalent to overall patients (7,47). A complex decision-making process discriminating patients' fitness, and tailoring a personalized medical treatment, is challenging: patients unfit for FIr-B/FOx can be treated with a two-drug first line combination regimen (31), but showed worse clinical outcome. No increased morbidity, nor mortality was reported in unfit patients who underwent secondary liver metastasectomies, reported as significantly higher in elderly patients (8%) (48).
Overall in MCRC patients treated with BEV added to CPT-11/5-FU, or with more intensive regimens (FIr-B/FOx, FOLFOXIRI/BEV), PFS and OS were not significantly different in KRAS wild-type and mutant (4,6,8,37) as well as in young-elderly patients (47). Recently, KRAS genotype was reported as significantly affecting PFS and OS in patients treated with XelOx/BEV (49). We recently reported in MCRC patients treated with FIr-B/FOx, that the prevalent KRAS c.35 G>A (G12D) mutant genotype may significantly affect worse OS, compared to wild-type or other mutations (40). Present data reported for the first time that in patients unfit for FIr-B/FOx, KRAS wild-type compared to mutant patients showed a significantly different PFS, and not OS. Furthermore, KRAS c.35 G>A mutant genotype may affect significantly worse PFS and OS, compared to wild-type and/ or other mutant, confirming that KRAS genotype, particularly c.35 G>A mutant, confers different biological aggressiveness (35), less effectively overcome by conventional triplet and doublet regimens. The prognostic relevance of KRAS genotype, particularly c.35 G>A mutant (4,40), and the predictive relevance of different medical treatments according to patients' fitness for intensive regimens, should be prospectively evaluated.
In conclusion, in MCRC patients unfit for first line intensive FIr-B/FOx regimen, tailored doublet and triplet medical treatments showed similar activity and efficacy, also according to age and comorbidities. KRAS genotype may indicate different PFS, and c.35 G>A KRAS mutant a significantly worse PFS and OS, compared to wild-type and other mutations. Present findings warrant prospective trials comparing clinical outcome in unfit patients, according to KRAS genotype.