Outcomes and prognostic contributors in patients with KRAS mutated non-small cell pulmonary adenocarcinomas: a single institution experience

BackgroundKRAS is the most frequently encountered driver mutation in advanced non-small cell lung cancer (NSCLC). With targeted therapy for the most common KRAS mutation p.G12C on the horizon, the aim of this study is to retrospectively report outcomes in patients with KRAS mutated NSCLC.MethodsThis was a retrospective chart review of 7 hospitals in Texas with reflex biomarker testing in all lung adenocarcinomas. Patients were included if they had pathologically diagnosed adenocarcinoma of any stage originating in the lung with molecularly confirmed KRAS driver mutation of any genotypic subtype. Twelve-month survival was assessed and compared between KRAS p.G12C and all other detected KRAS mutations. Other outcomes including impact of age, sex, smoking status, and pack years smoked were assessed to determine if they had prognostic significance on mortality in KRAS mutated patients.ResultsThere were 58 patients diagnosed with KRAS mutated NSCLC, 63.8% were at an advanced stage at diagnosis, 55.8% of patients were female, and 82.8% were white. The median age was 72 [52–88] years, and 93.1% were either current or prior smokers. KRAS p.G12C was the most common KRAS mutation (44.8%). At diagnosis, patients with KRAS p.G12C had poorer performance statuses compared to other KRAS mutations. A total of 32 (55.2%) patients died, 26 with advanced disease. In this study, current smoking status (P=0.1652), pack years smoked (P=0.6597), age (P=0.5092), sex (P=0.4309), and underlying KRAS codon mutation controlling for stage (P=0.2287) did not impact survival. However, KRAS p.G12C had a numerically lower 12 months overall survival (OS) compared to all other KRAS mutations in both early stage (56.3% vs. 90.9%) and advanced stage (25.0% vs. 47.6%) disease. Of note, 16 (27.6%) patients had prior, concurrent, or second malignancies, but these did not significantly impact OS (P=0.7696).ConclusionsThis study did not find a prognostic difference with sex, smoking history, age, or p.G12C mutation. The patients in this cohort with KRAS p.G12C had a numerically lower 12-month overall survival in both early and advanced stage disease compared to other mutations, and over one-quarter had a notable history of previous and second primary malignancies.     

Frequency and clinicopathologic correlates of KRAS amplification in non-small cell lung carcinoma

Background

Characterization of the non-small cell lung cancer (NSCLC) genome has suggested that KRAS amplification is one of the commonest molecular abnormalities in NSCLC. However, the prevalence and clinicopathologic significance of KRAS amplification, and its relationship with KRAS activating mutations have not been well-defined. The purpose of this study was to establish the prevalence of KRAS amplification in two separate, large NSCLC cohorts, to define the clinicopathologic features of KRAS-amplified NSCLC in a single uniformly treated cohort, and to investigate the interplay between KRAS amplification and KRAS mutation.

Methods

Fluorescence in situ hybridization was utilized to detect KRAS amplification on tissue microarrays constructed from a Swiss cohort of 538 NSCLCs and a series of 402 patients with NSCLC treated in a single institution in New York. DNA sequencing to detect KRAS codon 12 activating mutations was performed on a subset of tumors. Amplification and mutation status were compared with patient baseline characteristics, tumor characteristics, and overall- and disease-free survival.

Results

The prevalence of KRAS amplification was 13.7% in the Swiss cohort and 15.1% in the New York cohort. Among adenocarcinomas, KRAS amplification was associated with larger (mean size 2.8 ± 1.8 cm vs. 2.1 ± 1.3 cm, p = 0.003), less well-differentiated tumors (18% vs. 42%, p = 0.004) that were more likely to be invasive (95% vs. 77%, p = 0.004) and to exhibit angiolymphatic invasion (24% vs. 12%, p = 0.04). These differences were statistically significant within the subset of adenocarcinomas harboring activating KRAS mutations, suggesting a synergistic relationship between amplification and mutation. No significant association between KRAS amplification and nodal metastasis or survival was seen.

Conclusions

KRAS amplification is a common molecular alteration in NSCLC, characterizing ∼15% of tumors. This alteration is associated with indicators of local aggressiveness, and may act synergistically with KRAS mutations to promote tumor progression.     

EGFR and KRAS mutations in Chinese patients with adenosquamous carcinoma of the lung

Adenosquamous carcinoma (ADSQ) is uncommon in non-small cell lung cancer (NSCLC). The frequency rate of ADSQ was 9.7% of 6990 primary lung cancers resected in our department. Many researches have analyzed genetic and molecular alterations in adenocarcinoma (AD) and squamous cell carcinoma (SQ), but few molecular studies have been conducted on heterogeneous ADSQ. The current study was to investigate gene mutations of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene (KRAS) and their correlation with clinical variables in Chinese patients with ADSQ. Histologic features were reviewed, and immunohistochemical (IHC) and molecular (EGFR and KRAS) studies were done in 55 Chinese patients with ADSQ. Microscopically, all the tumors demonstrated dual differentiation with varying proportions of AD and SQ. Based on morphological diagnosis, a combination of multiple IHC markers is helpful for accurately discriminating two undifferentiated histologic subtypes of ADSQ. EGFR mutations were identified in 21 (38.2%) patients: 11 mutations were in exon 19, 1 in exon 20, 7 in exon 21 and double mutations were found in two patients. We also found two new mutations, namely, L747-E749del K754A within exon 19 and H850R within exon 21. Moreover, 16 (29.1%) silent mutations Q787Q in exon 20 were found in the series, five of which coexisted with other mutations. EGFR mutations were more frequently found in patients with size of the tumors ≥3 cm [19/35 (54.3%); 2/20 (10%); P = 0.001] or coexistent double cancer. However, the EGFR mutation was not associated with gender, age, lymph node status, tumor stage and smoking history. KRAS mutations were present in 2 (3.64%) male patients in codon12 (G12C) and none of them showed EGFR mutation. Moreover, identical EGFR and KRAS mutations in both components of ADSQ were further confirmed by microdissection techniques. The data indicated that the incidence of EGFR and KRAS mutations in Chinese patients with ADSQ were similar to those of Asian patients with AD. Furthermore, EGFR silent mutations accounted for a large proportion in ADSQ. Additional prospective studies are needed in order to define the clinical relevance of new and silent mutation variants.     

Prognostic significance of alterations in KRAS isoforms KRAS‐4A/4B and KRAS mutations in colorectal carcinoma

Somatic KRAS mutation is an early well‐known event in colorectal carcinogenesis but a complete understanding of RAS function and dysfunction in colorectal cancer is still to come. Our aim was to study the incidence of KRAS mutation; KRAS splice variants: KRAS4A and KRAS4B; and their relationships with various clinico‐pathological characteristics in colorectal cancer (CRC).In this study, 285 CRC cases were analysed for KRAS mutation by direct DNA sequencing followed by immunohistochemical analysis after validation with real‐time PCR assay, to study the protein expression of KRAS4A and ‐4B isoforms. KRAS gene mutations were seen in 80/285 CRCs (28.1%) and of the mutated cases, the majority of the mutations were seen in codon 12 (81.2%) as opposed to codon 13 (18.8%). CRCs with KRAS mutations were associated with a poor overall survival (p = 0 . 0009). Furthermore, KRAS mutations at codon 12 were associated with a poor overall survival of 64.4% at 5 years compared with a 5‐year overall survival of 75.8% and 78.2% with codon 13 mutation and absence of KRAS mutations, respectively (p = 0 . 0025). KRAS4A protein expression was predominantly seen in the cytoplasm, while KRAS4B protein was nuclear. KRAS4A overexpression was significantly associated with left colon, histology subtype of adenocarcinoma, p27kip1, and cleaved caspase3 expression. Interestingly, KRAS4A overexpression was associated with a better overall survival (p = 0 . 0053). On the other hand, KRAS4B overexpression (33.2%) was significantly associated with larger tumour size (p = 0 . 0234) and inversely correlated with p27kip1 protein (p = 0 . 0159). Both KRAS mutation and KRAS4A were independent prognostic markers in a multivariate analysis with age, gender, stage, differentiation, and MSI status. Our results highlight the differential role of KRAS isoforms in CRC, their utility as a prognostic biomarker, and underline the importance of KRAS alterations as a potential therapeutic target for CRC. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Increased sensitivity of KRAS mutation detection by high‐resolution melting analysis of COLD‐PCR products

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low‐level tumor specific KRAS mutations. Coamplification at lower denaturation temperature‐PCR (COLD‐PCR) is a new form of PCR that selectively amplifies mutation‐containing templates based on the lower melting temperature of mutant homoduplexes versus wild‐type homoduplexes. We have developed a fast COLD‐PCR and high‐resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD‐PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen^® KRAS mutation kit. The sensitivity was increased by 5‐ to 100‐fold for melting temperature decreasing mutations when using COLD‐PCR compared to standard PCR. Mutations, undetectable by the TheraScreen^® kit in clinical samples, were detected by COLD‐PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD‐PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Mutant allele-specific imbalance modulates prognostic impact of KRAS mutations in colorectal adenocarcinoma and is associated with worse overall survival

The prognostic impact of distinct KRAS mutations in colorectal carcinomas is not fully characterized. We hypothesized that the prognostic impact of KRAS mutations is modulated by KRAS mutant allele-specific imbalance (MASI). KRAS MASI was assessed by sequencing electropherograms in KRAS-mutated colorectal carcinomas (N = 394, prospectively tested). The mechanism of KRAS MASI was studied by fluorescence in situ hybridization (FISH; N = 50). FISH showed that KRAS MASI developed by chromosome 12 hyperploidy (9/18, 50%) or KRAS amplification (1/18, 5.5%). KRAS MASI was more common in tumors with KRAS codon 13 than with codon 12 mutations [24/81, 30% vs. 54/313, 17%; odds ratio (OR), 2.0, 95% confidence interval (CI), 1.2-3.5; p = 0.01]. KRAS MASI was correlated with overall survival (N = 358, median follow-up = 21 months). In a multivariate analysis, KRAS codon 13 MASI was an independent adverse prognostic factor (compared to codon 13 mutants without MASI combined with all codon 12 mutants; adjusted hazard ratio, 2.2, 95% CI: 1.2-3.9; p = 0.01). KRAS MASI arises through chromosome 12 hyperploidy or KRAS amplification and, when affects KRAS codon 13, is associated with worse overall survival.     

EGFR and KRAS mutations and altered c-Met gene copy numbers in primary non-small cell lung cancer and associated stage N2 lymph node-metastasis

This study aimed to detect mutations in EGFR and KRAS and alterations of c-Met gene copy number (GCN) changes in primary and lymph node-metastatic NSCLCs. The data showed the concordant rate of EGFR genotype in primary and stage N2 lymph node-metastatic tumors was 95.45%. c-Met GCN in stage N2 lymph nodes was significantly higher than that of the primary tumors (P = 0.038). The results suggest both primary and lymph-node metastases have relatively consistent EGFR mutations and EGFR mutations are not relevant to changes in c-Met GCN. c-Met GCN was increased significantly in EGFR TKI-naive patients with lymph node-metastatic tumors.     

Critical comments for roles of biomarkers in the diagnosis and treatment of cancer

A biomarker is defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic/pharmacodynamic responses to a therapeutic intervention”. Various assays, including immunohistochemistry, gene constitution such as amplification, mutation, and rearrangement, gene and protein expression analysis such as single gene or protein expression, exhaustive analysis and gene or protein signature and single nucleotide polymorphism have been used to identify biomarkers in recent years. No therapeutic effects have yet been predicted based on the results of such exhaustive gene analysis because of low reproducibility although some correlate with the prognosis of patients. Biomarkers such as HER2 for breast cancer or EGFR mutation for lung cancer and KRAS mutation in colon cancer have contributed to identify a patient population that might show a good and bad treatment response, respectively. On the other hand, other biomarkers such as bcr-abl, c-kit gene mutation and CD20 expression, which are positive for CML, GIST and B cell lymphoma, respectively, have crucial biological significance but have not necessarily been used for practical clinical screening since pathological diagnosis coincide with finding of biomarkers. Hence, much work remains to be done in many areas of biomarker research.     

High let-7a microRNA levels in KRAS-mutated colorectal carcinomas may rescue anti-EGFR therapy effects in patients with chemotherapy-refractory metastatic disease

Preclinical and experimental data in vivo indicate that Lethal-7 (Let-7) microRNA downregulates KRAS with antitumor effects in the presence of activating KRAS mutations. We quantified the Let-7a isoform in KRAS-mutated colorectal carcinomas from patients who received salvage cetuximab plus irinotecan. The study population was retrospectively identified among metastatic colorectal cancer patients who underwent third-line therapy with cetuximab plus irinotecan in a period when only epidermal growth factor receptor (EGFR) expression was required for anti-EGFR therapy. In 59 patients harboring KRAS mutations, Let-7a levels were analyzed for association with overall survival (OS) and progression-free survival (PFS) times. An exploratory subgroup analysis was performed using the rs61764370 (LCS6 T>G) polymorphism that experimentally impairs Let-7 binding to KRAS mRNA. In the whole group, higher Let-7a levels were significantly associated with better survival outcomes. For the primary OS endpoint, the multivariate hazard ratio was 0.82 (95% confidence interval, 0.73-0.91; p = .01). The same findings with an accentuated positive effect of high Let-7a levels on both OS and PFS times were observed in an exploratory analysis of the 45 wild-type LCS6 patients (excluding 14 carriers of the LCS6 G allele variant). All survival associations were confirmed after excluding patients with KRAS codon 13 mutations. Among the clinicopathologic features, high Let-7a levels were associated with grade 2-3 skin toxicity (p = .002). In patients with KRAS mutations, Let-7a analysis may serve to identify subgroups of patients who may still benefit from EGFR inhibition and this may open up new perspectives for alternative treatment strategies.