Prognostic Role Of KRAS Mutations Research Articles

56P The prognostic role of KRAS mutations in patients with early-stage lung adenocarcinoma after robotic lobectomy and systematic lymphadenectomy

KRAS in one of the most frequently altered oncogenes in NSCLC. The prognostic role of the KRAS mutations in early-stage NSCLC is still unclear. In this study we aim to analyze the overall survival (OS) and the disease free survival (DFS) of patients with early stage NSCLC underwent lobectomy and systematic lymphadenectomy.

Mutational profile of Brazilian lung adenocarcinoma unveils association of EGFR mutations with high Asian ancestry and independent prognostic role of KRAS mutations

Lung cancer is the deadliest cancer worldwide. The mutational frequency of EGFR and KRAS genes in lung adenocarcinoma varies worldwide per ethnicity and smoking. The impact of EGFR and KRAS mutations in Brazilian lung cancer remains poorly explored. Thus, we investigated the frequency of EGFR and KRAS mutations in a large Brazilian series of lung adenocarcinoma together with patients’ genetic ancestry, clinicopathological and sociodemographic characteristics. The mutational frequency of EGFR was 22.7% and KRAS was 20.4%. The average ancestry proportions were 73.1% for EUR, 13.1% for AFR, 6.5% for AME and 7.3% for ASN. EGFR mutations were independently associated with never-smokers, high-Asian ancestry, and better performance status. KRAS mutations were independently associated with tobacco exposure and non-Asian ancestry. EGFR-exon 20 mutations were associated with worse outcome. The Cox regression model indicated a worse outcome for patients whose were older at diagnosis (>61 y), solid histological subtype, loss of weight (>10%), worse performance status (≥2), and presence of KRAS mutations and EGFR mutational status in TKi non-treated patients. In conclusion, we assessed the clinicopathological and ethnic impact of EGFR and KRAS mutations in the largest series reported of Brazilian lung adenocarcinomas. These findings can support future clinical strategies for Brazilian lung cancer patients.

Mutational analysis of KRAS and its clinical implications in cervical cancer patients.

ObjectiveThe predictive and prognostic role of KRAS mutations in cervical cancer remains inconclusive. The aim of this study was to explore the clinicopathological and prognostic relevance of KRAS mutations in invasive cervical cancers (ICC).MethodsReverse transcription polymerase chain reaction (PCR) and Sanger sequencing were employed to detect KRAS mutations in 876 ICC patients. Quantitative real-time PCR was used to detect human papillomavirus (HPV) 16 and HPV 18.ResultsNon-synonymous mutations of KRAS were identified in 30 (3.4%) patients. These mutations were more common in non-squamous cell carcinoma than in squamous cell carcinoma (SCC) (8.2% vs. 2.2%, respectively, p<0.001) and were associated with HPV 18 infection (p=0.003). The prevalence of mutations was highest (18.2%) in the uncommon histological subtypes followed by adenocarcinoma (AC, 7.3%) and adenosquamous carcinoma (ASC, 5.8%). During the median follow-up of 55 months, compared to patients with wild-type KRAS, a greater percentage of patients with mutant KRAS relapsed (20.0% vs. 42.9%, respectively, p=0.007). The 3-year relapse-free survival was poorer in patients with mutant KRAS than in patients without KRAS mutations (57.1% vs. 81.9%, respectively, p=0.001). Furthermore, the multivariate analysis showed that the presence of a KRAS mutation was an independent predictor for disease recurrence (hazard ratio [HR]=2.064; 95% confidence interval [CI]=1.125–3.787; p=0.019).ConclusionKRAS mutations were predominant in non-SCCs of the cervix and were associated with HPV 18 infection. A combination of KRAS mutation detection and HPV genotyping would be useful in identifying patient with poor prognosis for further interventions.

Toward the molecular dissection of peritoneal pseudomyxoma

Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not-being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations.

Prognostic role of KRAS mutations in Sardinian patients with colorectal carcinoma

The presence of mutations in the KRAS gene is a predictor of a poor clinical response to EGFR-targeted agents in patients affected by colorectal cancer (CRC), but its significance as a global prognostic factor remains unclear. The aim of the present study was to evaluate the impact of the KRAS mutational status on time to first metastasis (TTM) and overall survival (OS) in a cohort of Sardinian CRC patients. A total of 551 patients with metastatic CRC at the time of enrolment were included. Clinical and pathological features of the disease, including follow-up information, were obtained from medical records and cancer registry data. For mutational analysis formalin-fixed paraffin-embedded tissue samples were processed using a standard protocol. The coding sequence and splice junctions of exons 2 and 3 of the KRAS gene were screened for mutations by direct automated sequencing. Overall, 186 KRAS mutations were detected in 183/551 (33%) patients: 125 (67%) were located in codon 12, 36 (19%) in codon 13, and 18 (10%) in codon 61. The remaining mutations (7; 4%) were detected in uncommonly-affected codons. No significant correlation between KRAS mutations and gender, age, anatomical location and stage of the disease at the time of diagnosis was identified. Furthermore, no prognostic value of KRAS mutations was found considering either TTM or OS. When patients were stratified by KRAS mutational status and gender, males were significantly associated with a longer TTM. The results of the present study indicate that KRAS mutation correlated with a slower metastatic progression in males with CRC from Sardinia, irrespective of the age at diagnosis and the codon of the mutation.

KRAS Mutation as a Biomarker for Survival in Patients with Non-Small Cell Lung Cancer, A Meta-Analysis of 12 Randomized Trials.

Because there is no clear consensus for the prognostic implication of KRAS mutations in patients with non-small cell lung cancer (NSCLC), we conducted a meta-analysis based on 12 randomized trials to draw a more accurate conclusion. A systematic computer search of articles from inception to May 1, 2014 using the PubMed, EMBASE, and Cochrane databases was conducted. The enrollment of articles and extraction of data were independently performed by two authors. Our analysis was based on the endpoints overall survival (OS) and progression-free survival (PFS). Nine records (All for OS, 7 for PFS) comprising 12 randomized trials were identified with 3701 patients who underwent a test for KRAS mutations. In the analysis of the pooled hazard ratios (HRs) for OS (HR: 1.39; 95% confidence interval [CI] 1.23-1.56) and PFS (HR: 1.33; 95% CI 1.17-1.51), we found that KRAS mutations are related to poor survival benefit for NSCLC. According to a subgroup analysis stratified by disease stage and line of therapy, the combined HRs for OS and PFS coincided with the finding that the presence of a KRAS mutation is a dismal prognostic factor. However, the prognostic role of KRAS mutations are not statistically significant in a subgroup analysis of patients treated with chemotherapy in combination with cetuximab based on the endpoints OS (P=0.141) and PFS (P=0.643). Our results indicate that KRAS mutations are associated with inferior survival benefits for NSCLC but not for those treated with chemotherapies integrating cetuximab.

K-ras mutational status and its clinical implications in Korean colorectal cancer patients.

641 Background: The incidence of K-ras mutation of exon 2 was reported between 30-40% in colorectal cancer, and it is well known predictive biomarker for EGFR monoclonal antibody. However, its prognostic role has not been defined so far, especially in Eastern country. Methods: From January 2007 to September 2013, patients who were diagnosed as colorectal cancer and examed K-ras exon 2 mutation in Saint Vincent’s hospital were retrospectively reviewed. Results: Among 436 patients, 343 patients with sufficient clinical information were analyzed. The median follow up period was 13.0 (0.2-94.8) months. The incidence of K-ras mutation was 37.9% (codon 12 mutation: 28.6%, codon 13 mutaiton: 9.0%, both: 0.3%). Codon 12 p.G12D was most frequent (52.3%), and codon 13 p.G13D was the next (22.3%). The presence of K-ras mutation was associated with old age (p=0.038), female gender (p=0.034), lesser nodal involvement (p&lt;0.001), but not related to stage, tumor site, and histology. There is no significant clinical differences between codon 12 and 13 mutations. The recurrence free survival in patients who received curative resection was worse in patients with K-ras mutation (p=0.001). The prognostic role of K-ras mutation was more prominent in colon cancer, while it lost its prognostic significance in rectal cancer. Conclusions: The prognostic role of K-ras mutation was different according to primary tumor sites in colorectal cancer.

BRAF and KRAS mutations in liver-resected metastatic colorectal cancer (mCRC) patients (pts).

476 Background: Up today there are no molecular markers to identify mCRC pts candidate to curative liver surgery at high risk of relapse. There are conflicting results about the prognostic role of KRAS mutation in mCRC pts, while BRAF mutation is a well-known negative prognostic factor characterizing a subgroup of mCRCs with a distinct metastatic spread. We analyzed the impact of BRAF and KRAS mutations on relapse free-survival (RFS) and overall survival (OS) in pts undergoing liver resection with curative intent. Methods: Medical records of pts referred to 3 Italian institutions between 1995 and 2012 were reviewed. 3024 mCRC pts were identified. 401 pts (13.3%) underwent liver resection with curative intent and had adequate follow-up. Mutational status was assessable on 360 samples from primary tumors (n=63), metastases (mts) (n=59) or both (n=238). Primary objective was to evaluate the impact of BRAF mutation on RFS in mCRC pts candidate to liver resection. Secondary objectives were to evaluate the prognostic role of BRAF status on OS, and of KRAS status on RFS and OS in this population. Results: BRAF and KRAS were mutated in 11 (3%) and 116 (32%) out of 360 cases respectively. In 238 cases in which paired samples from primary and metastases were available, no discordance was found in BRAF status while 18 cases (7.6%) showed a discrepancy in KRAS status and were thus excluded from analysis. Pts with BRAF mutation had significantly shorter median RFS compared to pts with BRAF wt tumor (5.7 mos vs 11.7 mos, HR=4.25; 95%CI: 1.52-11.88, p=0.005). OS was significantly reduced in pts with BRAF mutated tumors vs wt (HR=5.39; 95%CI: 1.59-18.27, p=0.007). Mutated KRAS was not prognostic for RFS (HR=1.14; 95%CI: 0.87-1.49, p=0.34), while demonstrated a weak prognostic impact on OS (HR=1.52; 95%CI: 1.05-2.21, p=0.03). In this cohort high-risk Fong score were significantly associated with shorter RFS (p&lt;0.0001) and OS (p&lt;0.0001). Conclusions: BRAF mutation is associated with higher probability of relapse and worse outcome in liver resected mCRC. KRAS mutation was not associated with RFS after liver resection. BRAF mutational status may become a new prognostic marker when planning liver resection in mCRC pts.

Pathologic response, KRAS status, and bevacizumab: The Bermuda triangle for outcome prediction after hepatic colorectal cancer metastases (HCRM) resection.

e14716 Background: Clinicopathologic factors are insufficient to stratify prognosis after HCRM resection. The identification of prognostic biomarkers is an unmet clinical need. Even if the prognostic role of KRAS mutations in the metastatic setting is controversial, recent data hypothesized a correlation to worse outcome after HCRM resections. This analysis evaluated the correlation between KRAS mutation, pathological response and outcome. Methods: Fifty-eight pts with HCRM were resected at National Cancer Institute of Milan from 2007 to 2012 following bevacizumab (Bev) (n=30) or cetuximab (Cmab) (n=28) based neoadjuvant regimens. Tumor regression grade (TRG) was classified from TRG 1 for pathological complete response (pCR), to TRG5 for no response; TRG 1-3 for response; TRG 4-5 for scarce/no response. KRAS exons 2 to 4 were sequenced by direct PCR. Results: KRAS missing in 3 pts (1 Bev/2 Cmab), mutated in 23 (42%) vs wild-type in 32 (58%), mutated in 18 (62%) of Bev group vs 5 (19%) of Cmab. Mean TRG: 2.9 (2.2-4) in Bev vs 3.8 (3-5) in Cmab group (p=0.04 at Kruskal-Wallis test). Pathological response higher for Bev vs Cmab (70% vs 39%; p=0.037 at Chi-square test). Bev also increased pCRs (20% vs 0%, p=0.024 at Fisher’s test). Pathological response higher for KRAS mutant vs wild-type (70% vs 41%; p=0.003 at Chi-square test). At log-rank test, DFS significantly shorter for KRAS mutated vs wild-type (7.6 vs 14.6 months; p=0.05), and for TRG4-5 vs TRG1-3 (8.1 vs 15.3 months; p=0.02), while no difference observed according to treatment. OS data are not mature. Multivariable Cox model for DFS shown in the Table. Conclusions: After Bev-based neoadjuvant treatment, patients with KRAS mutated HCRM and without good pathological response may have the worst outcome after curative resection. [Table: see text]

Role of KRAS mutations in the carcinogenesis of mucinous tumors of appendiceal origin with pseudomyxoma peritonei (PMP).

202 Background: Codon 12 KRAS and BRAF mutations may have a role in the mucinous differentiation pathway in colorectal cancer (Bazan V et al, Ann Oncol 2002; Tran et al, Cancer 2011). Elucidating the molecular mechanisms driving this disease is a priority for PMP, with the goal of identifying new markers and therapeutic targets needed to improve disease management. Due to the fact that neoplastic cells may be deceptively bland and sparse histologically within an abundant mucinous background, the adoption of high-sensitivity gene sequencing techniques may be extremely helpful. Methods: Mucinous tumors from 10 PMP cases of appendiceal origin treated in a multidisciplinary team at the National Cancer Institute of Milan were histologically classified according to Bradley classification. Following microdissection and DNA extraction, mutational analysis of KRAS exon 2-4 and BRAF exon 15 were performed by means of PCR, while KRAS exon 2 status was further confirmed through a specific mutant enriched PCR, shown to be a more sensitive approach in colorectal cancer (Molinari et al, Clin Cancer Res 2011). Results: Histological classification was low-grade in 6 and high-grade in 4 tumors. Codon 12 KRAS mutations were detected in 5/10 cases (50%). In the remaining 5 cases: sequencing of exon 4 lead to identification of a first-described A146V mutation in 1 case; with mutant enriched technique, we identified 3 additional codon 12 mutated cases. Due to use of high sensibility techniques and gene sequencing outside hotspot regions, we were able to identify KRAS mutations in 9/10 (90%) specimens. No BRAF exon 15 mutation was detected. Conclusions: The prognostic role of KRAS mutation in PMP patients is under investigation on a larger data-set of National Cancer Institute of Milan and will be presented at the Meeting. However, these preliminary data suggest a major role of KRAS in the carcinogenesis of mucinous tumors of appendiceal origin with PMP.

KRAS G&gt;A mutation favors poor tumor differentiation but may not be associated with prognosis in patients with curatively resected duodenal adenocarcinoma

KRAS mutations have been found in duodenal adenocarcinomas and may have prognostic significance. The purpose of this study was to classify clinicopathological characteristics, microsatellite instability and KRAS mutations and identify possible prognostic role of KRAS mutations in duodenal adenocarcinomas. Demographics, tumor characteristics and survival were recorded for 78 patients with duodenal adenocarcinomas (Stages I-III). KRAS mutations were detected in 27 (34.6%) cases, of which the majority (74.1%) were G>A transitions. Multivariate logistic regression analysis showed that KRAS G>A mutation was significantly associated with late stage (p = 0.025) and poor tumor differentiation (p = 0.035), when compared with wild-type and other than G>A mutations. KRAS G>A mutation carriers were at increased risk for distant relapse (p = 0.022) and had significantly shorter overall survival (OS; log-rank p = 0.045) and a trend toward shorter relapse-free survival (RFS; log-rank p = 0.062) when compared with those who did not carry the KRAS G>A mutation. In multivariate analyses, there was a significant correlation between ≥ 3 positive lymph nodes and poor OS (p < 0.001) and RFS (p = 0.001) and KRAS G>A mutation carriers demonstrated no effect on clinical outcome. In conclusion, KRAS G>A mutation correlates significantly with late stage and poor tumor differentiation in duodenal adenocarcinoma. Among patients who undergo a curative resection of duodenal adenocarcinoma, KRAS G>A mutation carriers will more likely experience distant relapse but may not exhibit a poor prognosis. The number of positive lymph nodes should be incorporated in future staging systems.

549P - Prognostic Role of Kras Mutations Detected by High-Sensitivity Taqmelt PCR Assay in Metastatic Colorectal Cancer

ABSTRACT Background KRAS mutation is present in approximately 35-40% of patients with metastatic colorectal cancer (mCRC) and has been established as a predictive marker of resistance to anti-EGFR therapy, but its role as prognostic factor is not yet clear. This study is aimed to analyze the prevalence and the impact in prognosis of expanded number of KRAS mutations in caucasian mCRC population and the sensitivity of the TaqMelt PCR assay cobas KRAS Mutation Test. Methods A single institution retrospective cohort of 669 consecutive mCRC patients between 2000-9 with clinical follow-up was studied for frequent 7 mutations in codons 12/13 by ARMS-scorpion real-time PCR (Therascreen, Qiagen) and TaqMelt PCR assay cobas KRAS Mutation Test (Roche), which are designed to detect 19 mutations in KRAS codons 12, 13 and 61. DNA was obtained by cobas DNA preparation kit (Roche) from one single 5um FFPE tissue section and by QIAamp DNA FFPE Tissue Kit (Qiagen) from 50um of tissue. Results KRAS mutation was detected by the cobas KRAS Mutation Test in 41.2% of mCRC patients and was correlated with poor overall survival (OS) (p = 0.013), OS from the metastatic diagnosis for metachronous disease (p = 0.025), disease-free survival for metastatic disease (p = 0.012) and time to progression (p = 0.046). KRAS mutation was significantly associated with tumor grade (p = 0.025) and liver as first site of dissemination (p = 0.047). However, KRAS mutations detected by Therascreen did not impact on prognosis. The median of DNA concentration obtained by cobas and QIAamp was 120ng/ul and 80ng/ul, respectively. The frequency of invalid cases was 5.7% (cobas) and 17.9% (Therascreen). Finally, cobas identified 19.2% additional KRAS mutations not detected by Therascreen. Next Generation Sequencing by 454 GS FLX+ System analysis is ongoing to validate discrepant mutations. Conclusions The cobas KRAS Mutation Test is a robust and reproducible assay that, 1) obtains superior DNA recovery from very small amount of FFPE tissue; 2) detects up to 19.2% of mutations not detected by other methods; and, 3) KRAS mutations detected by cobas correlate with poor outcome in mCRC. Disclosure All authors have declared no conflicts of interest.

Prognostic and Predictive Value of K-RAS Mutations in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is a heterogeneous disease, caused by the presence of different clinically relevant molecular subtypes. Genetic mutations are emerging as potential biomarkers of response and treatment selection in patients with NSCLC. Over the past few years, activating mutations of epidermal growth factor receptor (EGFR) have been recognized as the most important predictor of response to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib and also as a favourable prognostic factor. The RAS genes, including H-RAS, K-RAS and N-RAS, encode a family of proteins regulating cell growth, differentiation and apoptosis. Mutations in the K-RAS gene, mainly in codons 12 and 13, have been found in 20-30% of NSCLC tumor samples and occur most commonly, but not exclusively, in adenocarcinoma histology and in heavy smokers. In NSCLC, the presence of K-RAS mutations has generally been considered to be associated with worse prognosis and resistance to systemic therapy in the adjuvant as well as the metastatic setting. In early stage NSCLC, the prognostic role of K-RAS mutations has been evaluated in several studies without definitive conclusion. On the other hand, in advanced NSCLC, the presence of K-RAS mutations identifies a subgroup of patients who do not respond to EGFR-TKI therapy but, at the same time, a positive survival effect from EGFR-TKIs cannot be excluded in these patients. Similarly, K-RAS mutational status does not predict benefit from the anti-EGFR monoclonal antibody cetuximab, highlighting the biological difference between lung cancer and colorectal cancer. As a result of the lack of conclusive data, K-RAS mutations do not represent a validated biomarker for the negative selection of patients who are candidates for anti-EGFR therapy. The aim of this article is to review and discuss the data on the prognostic and predictive value of K-RAS mutations in NSCLC.

Rôle pronostique des mutations du gène KRAS dans le cancer colorectal

The management of patients with advanced or localized colorectal cancer (CRC) remains to date exclusively based on clinical, radiological and pathologic data. In CCR, KRAS mutations are detected in approximately 30-40% of tumours. In patients with a localized CRC, the prognostic role of KRAS mutation is not yet demonstrated with contradictory results in literature. For patients with metastatic CCR, KRAS mutations are a prognosis value for patients treated by anti-EGFR antibody. Indeed, despite the use of monoclonal antibodies against epidermal growth factor receptor (EGFR) in metastatic CRC, the detection of molecular alterations in tumours is not integrated in the routine practice for patient decision-making. In patients treated with anti-EGFR Abs for a metastatic CRC, the detection of KRAS mutations on tumour DNA is now mandatory since several recent studies have clearly demonstrated that the presence of KRAS mutations confer a resistance to these therapies. Considering the lack of sufficient data, the detection of KRAS mutations is not recommended in other clinical setting.

Prognostic Role of K-ras in Patients with Progressive Colon Cancer Who Received Treatment with Marimastat (BB2516)

We determined the prognostic role of K-ras mutation in tumor tissue of patients with refractory colon cancer who received Marimastat (BB2516). DNA was extracted from paraffin-stored tumor tissue of 27 patients who previously failed 5-fluorouracil and were treated with BB2516. The presence of K-ras mutation was characterized by Polymerase Chain Reaction using ras- and p53-specific primers, ras and p53 oncoprotein expression was analyzed by an automated biotin-avidin immunoproxidase technique. Seventeen patients had a normal K-ras sequence and 10 patients had a K-ras mutation. Median survival of patients with a normal ras sequence was 330 days from the time of BB2516 treatment compared with 160 days for patients with a K-ras mutation (p = 0.0442, Wilcoxon; 0.0130 Log-Rank). No differences in age, sex, cancer stage, surgical treatment, or chemotherapy treatment were observed. Abnormalities involving ras expression did not affect survival. By comparison, median survival for patients with p53 mutation or p53 overexpression was both 158 days after BB2516 treatment. Patients having both K-ras and p53 mutations had the poorest median survival of 113 days (p = 0.035). There is a suggestion by univariate analysis that the presence of a K-ras mutation may predict survival in patients with progressive colon cancer. Further assessment with larger patient numbers and multivariate analysis is indicated.