Research Article
Open Access

Genotyping KRAS and EGFR Mutations in Greek Patients With Non-small-cell Lung Cancer: Incidence, Significance and Implications for Treatment

HELENA LINARDOU, VASSILIKI KOTOULA, GEORGE KOUVATSEAS, GIANNIS MOUNTZIOS, VASILIOS KARAVASILIS, EPAMINONDAS SAMANTAS, ANNA KALOGERA-FOUNTZILA, DESPINA TELEVANTOU, KYRIAKI PAPADOPOULOU, XANTHIPI MAVROPOULOU, EMILY DASKALAKI, THOMAS ZARAMBOUKAS, IOANNIS EFSTRATIOU, SOFIA LAMPAKI, GRIGORIOS RALLIS, ELENI RES, KONSTANTINOS N. SYRIGOS, PARIS A. KOSMIDIS, DIMITRIOS PECTASIDES and GEORGE FOUNTZILAS
Cancer Genomics & Proteomics November 2019, 16 (6) 531-541; DOI: https://doi.org/10.21873/cgp.20155

Abstract

Background/Aim: KRAS mutations are reported in 20-25% of non-small cell lung cancer (NSCLC) and their prognostic role is unclear. We studied KRAS and EGFR genotyping in Greek NSCLC patients. Patients and Methods: KRAS and EGFR genotypes were centrally evaluated in 421 NSCLC patients (diagnosed September 1998 -June 2013) and associated with clinicopathological parameters. Outcome comparisons were performed in 288 patients receiving first line treatment. Results: Most patients were male (78.6%), >60 years old (63.9%), current smokers (51.1%), with adenocarcinoma histology (63.9%). EGFR and KRAS mutations were found in 10.7% and 16.6% of all histologies, respectively, and in 14.9% and 21.9% of adenocarcinomas. At 4.5 years median follow-up, KRAS status was an independent negative prognostic factor for overall survival (OS, p=0.016). KRAS mutations conferred 80% increased risk of death in patients receiving first-line treatment (p=0.002). Conclusion: The presence of KRAS mutations is an independent negative prognosticator among Greek NSCLC patients and an independent response predictor to first line treatment.

Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related mortality worldwide, with more than one million deaths per year (1). With our increasing understanding of lung cancer biology, we are now able to recognise many molecular subtypes of NSCLC, based on the identification of diverse molecular events, which play a central role in lung cancer growth and metastasis. These genetic alterations include driver mutations in several genes, such as EGFR, KRAS, BRAF, HER2, or translocations, such as in ALK or ROS-1. Pivotal studies and comprehensive reviews have summarised data on the importance of EGFR mutations and other molecular alterations and their respective inhibitors that have changed the natural history of oncogene-driven NSCLC (2-4). Although the predictive role of activating EGFR mutations on treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) is well established, evidence is still inconclusive on their prognostic value (5-7).

In contrast to EGFR mutations that have been the paradigm shift in lung cancer management, the proto-oncogene Kirsten Rat Sarcoma virus (KRAS) mutations are the ‘waterloo’ of molecular targeting, a continuous story of fruitless attempts in identifying effective therapies, for what is the commonest known oncogene mutated in NSCLC. KRAS mutations are reported in approximately 20-25% of adenocarcinomas, and in a substantially lower number of squamous-cell carcinomas (5-8%) and are thought to be involved in many phases of cancer-cell transformation (8). The most common oncogenic mutations of the KRAS proto-oncogene are point mutations in codons 12 and 13, with the commonest types including G12C, G12V and G12D (9). Preclinical evidence has suggested a differential biological behaviour and chemosensitivity among different types of mutations, resulting in clinical attempts to identify a possible differential prognostic and predictive role (10, 11). KRAS mutations are generally mutually exclusive with EGFR and other oncogenic mutations in NSCLC; however, there are reports of co-existence of these diverse molecular events (12, 13).

Many clinicopathological features, such as gender, age, histology and smoking history have been correlated with EGFR and KRAS mutations. The latter are found more commonly in smokers; nevertheless, recently KRAS mutations have been reported with an incidence of up to 15% in never smokers with NSCLC (14), while there are reports of different mutation types associated with smoking status (15). Interestingly, although for EGFR it has been well established that mutation frequency is ethnicity dependent, very little is known about KRAS mutations frequency among different ethnic groups (16).

In view of the unclear picture of the role of KRAS in advanced NSCLC, and given that ethnicity may play a role on the mutational profiling of tumors, we report here on the first genotype mapping of NSCLC in Greek patients, aiming to investigate the incidence and prognostic significance of KRAS and EGFR mutational status.

Patients and Methods

Study population. In this retrospective analysis, performed by the Hellenic Co-operative Oncology Group (HeCOG), samples from patients with histologically confirmed NSCLC, who had been treated within HeCOG-affiliated centres from September 1998 through June 2013, were centrally evaluated for the presence of KRAS and EGFR mutations. All patients had available clinicopathological data at diagnosis. The following information was collected from the HeCOG clinical database: age at diagnosis, gender, smoking status, stage at diagnosis, histology, and details on treatments received (surgery, first line chemotherapy, platinum compounds, EGFR TKIs), best response achieved, as well as clinical outcomes of first line treatments (ORR, PFS, OS), and EGFR and KRAS mutation status at diagnosis. The study and all treatments were conducted in accordance with the Good Clinical Practice (GCP) guidelines, and the Helsinki Declaration and were approved by the Scientific Committee of HeCOG. The translational protocol was approved by the Bioethics Committee of the Aristotle University of Thessaloniki School of Health Sciences, Faculty of Medicine (4.34/4-6-2010; A13064/16-7-2010). All patients had signed informed consent for the use of their biological material for translational research purposes.

Tissue processing. Formalin-fixed, paraffin-embedded tissue blocks were retrospectively retrieved from the HeCOG tumor repository. Cytologic material was prospectively submitted for genotyping in more recent years (2010-2013). All tumors were initially evaluated at local pathology laboratories. Subsequently, paraffin-embedded tumor blocks and cytologic material in CytoLyt containers (Hologic, Manchester, UK) were sent to the Laboratory of Molecular Oncology of HeCOG for central KRAS and EGFR testing, which was implemented following central histology review. Out of 441 submitted materials, 8 biopsy samples were excluded upfront due to absent or inadequate (<200 per sample) tumor cells on the provided paraffin block. Consequently, biological material was processed for 433 patients. Tumors were centrally reviewed for histology and tumor cell content (TCC%). Manual macrodissection was applied for enrichment in TCC wherever possible. DNA was extracted with a standard protocol using the QIAamp DNA mini kit (Qiagen, Hilden, Germany), measured in an Eppendorf Biophotometer, and normalized at 50 ng/μl.

KRAS and EGFR genotyping. All 433 samples were submitted for KRAS genotyping with a routinely used qPCR Taqman-MGB allelic discrimination assay targeting the 7 most common mutations in codons 12 and 13 (17). All samples were also analysed with dd-sequencing on nested PCR products with M13-coupled, intron-spanning primers for KRAS exon 2 (coordinates according to GRCh38 for KRAS on chr12: 25245453-25245233). Mutations in the ATP-binding pocket of the EGFR kinase domain were assessed with dd-sequencing as above for the following GRCh37 coordinates on chr7: exon 18 (55241512-55241795); exon 19 (55242380-55242570); exon 20 (55248954-55249194); and, exon 21 (55259354-55259591). Samples were sequenced in both directions with the BigDye Terminator v1.1 Cycle Sequencing Kit and analysed in an ABI3130XL system (Applied Biosystems/Life Technologies). Samples were considered as non-informative (a) with qPCR if the cycle threshold [CT; crossing point (CP)] was ≥36 for the control wild type allele contained in each assay, and (b) with dd-sequencing, for failed sense and antisense capillary electrophoresis for all targets in both genes. By using these criteria, informative sequencing data were obtained for 424 tumors (96% of all submitted tumors; 98% of analyzed samples). The 10 underperforming samples corresponded to 7 biopsies, 1 surgical specimen and 2 fine needle aspirates from the tumor.

Figure 1.
Figure 1.

REMARK diagram.

Statistical methods. Associations between KRAS, EGFR mutations (mutated vs. wild-type) and clinicopathological parameters were performed in the entire cohort as well as in the first-line treated subgroup of patients and were evaluated with the chi-square or Fisher's exact test (where appropriate). Progression-free survival (PFS) was measured from the date of initiation of first line treatment until the first verified disease progression, death from any cause or date of last contact, whichever occurred first, while overall survival (OS) from the date of initial diagnosis until death from any cause or last contact. PFS was assessed in the subgroup of patients with available treatment and follow-up data, following first line treatment, while OS was examined in the entire cohort. Surviving patients (for OS and PFS) and patients without relapse (for PFS) were censored at the date of last contact. The Kaplan–Meier method was used for estimating time-to-event distributions, while log-rank tests were used for comparison of survival curves among groups. The associations between the examined factors and relapse/mortality rates were evaluated with hazard ratios estimated with Cox proportional hazards models.

Statistical significance was set at 5% (two-sided). The statistical analysis complied with the reporting recommendations for tumor marker prognostic studies (18) and was performed using the SAS software (SAS for Windows, version 9.3, SAS Institute Inc., Cary, NC, USA).

Results

Patients and genotype characteristics. KRAS and EGFR genotypes were evaluated in a total of 421 NSCLC patients. Among these, 288 patients had received first line treatment for advanced, inoperable disease (Figure 1); 18 patients had not received chemotherapy, while treatment status was not available for 17.6% of the patients, probably due to the retrospective nature of the analysis. Clinicopathological characteristics are detailed in Table I. More patients were males (78.6%), older than 60 years (63.9%), current smokers (51.1%) and had tumors with adenocarcinoma histology (63.9%). With respect to histologic type, because all tumors and genotyping methods were applied before the end of 2013, bronchioloalveolar carcinoma (BAC) was distinguished from adenocarcinomas; the term has been discontinued in the currently valid 2015 WHO Classification of Lung Tumors (19) and this tumor type is herein discussed as adenocarcinoma.

EGFR mutations were found in 45 tumors and KRAS mutations in 70 tumors, corresponding to 10.7% and 16.6% of all histological types, respectively, and to 14.9% and 21.9% of adenocarcinomas. Mutation details can be found in the following link (https://www.hecog.gr/images/stories/pdf/PAPERS_ONLINE/EGFR_and_KRAS_mutation_details_for_all_421_NSCLC_patients.pdf). Most EGFR mutations were of the classical type (72.6%), with an almost equal representation of the exon 19 p.E746_A750delELREA and the exon 21 p.L858R point mutation (Figure 2A); 41 of them were registered in the COSMIC database. The T790M resistance mutation was found in one tumor. The most common KRAS mutations were p.G12C, p.G12D and p.G12V (Figure 2B). EGFR and KRAS mutations were mutually exclusive except for one tumor that had both EGFR and KRAS mutations. Finally, it should be noted that 23 out of the 45 patients with an EGFR activating mutation received first line treatment with an EGFR TKI, probably since these drugs were not the standard of care for EGFR mutant NSCLC until 2011.

Associations. EGFR mutations were significantly associated with female gender, adenocarcinoma histology and non-smoking status, as previously described (Tables II and III). KRAS mutations were associated with adenocarcinoma histology in the population with available first line data. In our study, the presence of KRAS mutations was not associated with smoking status either in the whole study population or among patients with advanced disease receiving first-line treatment (Tables IV and V).

Effect of KRAS/EGFR mutational status on overall survival in the whole study population. Regarding overall survival (OS) in the whole patient population with available follow-up data (N=377), independent favourable prognostic factors were the best response to treatment, (CR or PR vs. other, Wald's p=0.017), platinum vs. non-platinum treatment p=0.001, earlier disease stage (I-II) vs. advanced disease stage (III-IV) (p<0.001), and surgical removal of the primary tumour vs. no surgery (p<0.001). At a median follow-up of 4.5 years, in the univariate analysis for the entire population, KRAS mutant status was an adverse prognostic factor for overall survival (p=0.016), while EGFR status did not show any significant associations with OS (p=0.45), most likely due to the small sample size. The effect of KRAS mutation was strong, with a median difference in OS of approximately 10 months between KRAS wt and mut patients (Figure 3).

View this table:
Table I.

Clinicopathological patient characteristics.

Effect of KRAS/EGFR mutational status on outcomes in patients with advanced NSCLC receiving first-line treatment. In the subpopulation of patients receiving first line treatment for advanced disease (N=288), the presence of KRAS mutations, was also an independent adverse prognostic factor for survival with an 80% increase in the risk of death (HR=1.80, 95%CI=1.25-2.60, p=0.002, Figure 4), while EGFR status did not show any significant associations (p=0.53). In the same population, it seemed that patients receiving platinum-based chemotherapy had improved OS vs. those receiving non-platinum-based chemotherapy, irrespective of EGFR status (HR=0.45, 95%CI=0.14-1.54, p=0.21 and HR=0.86, 95%CI=0.64-1.15, p=0.31 for the EGFR mut and wt subgroups respectively, test for interaction p=0.31), even though significance was not reached. When we combined the effect of the presence of any mutation (either EGFR or KRAS) on OS in patients treated with first-line platinum-based chemotherapy, a marginally non-significant difference was observed, with patients harbouring either EGFRmut or KRASmut tumors bearing worse prognosis as compared to any wt (HR=1.52, 95%CI=0.97-2.38, p=0.066). Furthermore, KRAS mutational status showed shorter OS, as compared to either EGFR mut or wt status on both genes (Log-rank p=0.015) for all patients receiving first line treatment regardless of platinum or non-platinum regimen (Figure 5). Detailed analyses according to type of response (complete or partial response vs. other) did not show any significant differences based on EGFR or KRAS status, most likely due to smaller sample sizes.

The effect of different mutation types on outcome. No significant associations were observed in analyses per EGFR mutation type (EGFR classic vs. non-classic), most likely due to the small patient numbers. With respect to KRAS mutation type, analysis has shown that patients with the most common mutations (p.G12C/D/V) vs. other less common, had similar outcomes [median OS 12.6 months (95%CI=8.6-20.6) vs. 11.8 months (95%CI=0.2-16.4)] and again these fared significantly worse than KRAS wt patients (median OS 22.9 months (95%CI=19.2-28.4), log-rank p=0.009).

Discussion

The genomic diversity of NSCLC across different geographical areas and ethnic groups necessitates analyses in ethnically homogenous populations in order to elucidate epidemiological differences and to capture genomic alterations with clinical relevance. To our knowledge, this is the first publication of data on central EGFR and KRAS genotyping in the Greek population with NSCLC. We found that the incidence of EGFR mutations in Greek patients was 10.7% across all histological types and 14.9% in adenocarcinomas. A recent large meta-analysis of 456 published studies on the prevalence of EGFR mutations (30,466 patients with an EGFR mutation reported among 115,815 NSCLC patients), showed an overall pooled EGFR mutation prevalence of 32.3%, which varied by geographical area, with Asia exhibiting the higher prevalence (38.4%), followed by North and South America (24.4%) and Europe showing the lowest prevalence (14.1%) (20). Results varied similarly in ethnic groups, with a prevalence of 17.4% in Caucasians rising to 19.2% in patients with adenocarcinoma (20). Furthermore, very few studies have reported on intra-ethnic differences across Europe. From the published data, the EGFR mutation incidence in Europe ranges between 6% in Switzerland, 11% in France to 37.5% in Germany, and is dependent on ethnicity and on clinicopathological characteristics (21, 22). Different incidences have been previously reported for Greek patient populations, from as low as 8.2% to as high as 15.83% (23). In these studies, however, analyses were performed in local laboratories and included patients with diverse characteristics, i.e. in the study with the higher prevalence, 82.5% of the patients had adenocarcinomas (23). In our population, all tumor samples were centrally genotyped and 63.9% of the patients had adenocarcinoma, which is representative for the Greek population; the reported incidence is within the expected range for a Caucasian population in Europe, and is consistent with the incidence reported in 2016 in a large cohort of approximately 1,500 Greek patients included in a National program for early access to EGFR-TKis (10.04%) (24). Furthermore, in agreement with previous knowledge, the presence of EGFR mutations in our study was significantly associated with female gender, adenocarcinoma histology and non-smoking status (25, 26).

Figure 2.
Figure 2.

Types of mutations detected. A: EGFR mutations, B: KRAS mutations.

With respect to KRAS mutations, the corresponding prevalence was 16.6%, rising to 21.9% among adenocarcinomas. In various studies in the European population, the reported incidence varies from 15-25%, depending on the geographical area, ethnicity and clinicopathological characteristics (9, 27). Given the high prevalence of KRAS mutations among smokers and the fact that Greece is one of the European countries with the highest percentage of smokers (51%) (28), one could expect a higher incidence than the one observed. Since, however, this is a centrally genotyped population, these results are valid and the low incidence might be attributed to epidemiological differences and genomic diversity. As previously described, KRAS mutations were associated with adenocarcinoma histology and younger age (<60 years), while the frequencies of different KRAS mutation types were also within the previously reported ranges in European studies (23, 29). Smoking status is known to correlate with KRAS mutations and there are recent reports associating different smoking habits with different mutation types (14, 15).

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Table II.

Associations of EGFR mutation status with clinicopathological parameters in all patients.

One of the main aims of our study was to investigate the prognostic and predictive role of KRAS and EGFR mutations in NSCLC patients, both in the whole study population and in the subgroup receiving first line chemotherapy. We have found that at a median follow-up of 4.5 years, in the univariate analysis for the entire population, KRAS status was prognostic for worse overall survival (p=0.016). This effect was consistent, with a 10-month difference in OS between KRAS mutated and wild-type patients. When the effect of KRAS mutations on OS of patients treated with platinum-based therapy, was compared among different mutational groups (EGFR mut vs. KRAS mut vs. EGFR+KRAS wt), it was shown that patients with KRAS mutations retained the worst outcome regardless of the type of first line treatment (platinum or non-platinum). On the other hand, EGFR status did not show any significant associations with OS, most likely due to the small sample size in our study. These results come to add to the existing evidence on the prognostic role of KRAS mutations in advanced NSCLC, which remains largely controversial. In the early disease setting, a meta-analysis of four large adjuvant trials reported that KRAS mutational status was not prognostic for neither PFS nor OS (15). On the other hand, data in the metastatic setting are more persuasive on the negative prognostic impact of KRAS mutations, although contradictory studies have been published (9, 29-34). A recently published meta-analysis in the advanced disease setting suggested a detrimental effect of KRAS mutations on survival in patients with adenocarcinoma histology (9); more recent publications, focusing on the detection of KRAS mutations in cell-free DNA (cfDNA) in blood samples, attempted to provide stronger supportive evidence on the adverse prognostic role of KRAS in advanced NSCLC (31, 35, 36). Finally, recently published data have suggested that KRAS-mutation incidence and prognostic value are metastatic site-specific in lung adenocarcinoma, implicating KRAS mutations as independent predictors for the development of brain and bone metastases (37, 38).

View this table:
Table III.

Associations of EGFR mutation status with clinicopathological parameters in patients treated with first line chemotherapy.

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Table IV.

Associations of KRAS mutation status with clinicopathological parameters in all patients.

Figure 3.
Figure 3.

Effect of KRAS mutational status on overall survival (OS) in the whole study population.

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Table V.

Associations of KRAS mutation status with clinicopathological parameters in patients with available first line clinical data.

Figure 4.
Figure 4.

Effect of KRAS mutational status on overall survival (OS) in the subgroup of first line treated patients.

Another open issue with respect to KRAS status is its predictive value for response and outcome to first line therapies in NSCLC. In colorectal cancer it is well established that KRAS mutational status adversely affects response to EGFR-targeted agents (27); however, in NSCLC, a similar view is not generally accepted for response prediction to EGFR TKIs (39), although a few studies have proposed KRAS mutations as a predictive factor of poor response to first line chemotherapy (40-42). Of note, among patients with advanced NSCLC treated with immune check-point inhibitors, the presence of KRAS mutations was predictive for response to immunotherapy (43), probably due to the higher incidence of KRAS mutations in smokers, who usually carry a larger tumor mutation load (9). On the other hand, in patients with tumors harbouring activating EGFR mutations and treated with EGFR-TKIs, the presence of KRAS mutations was not associated with prognosis (44).

Previous reports have indicated that different point mutations might confer different biological behaviour, resulting in differential prognostic and possibly predictive effect for the various mutations. Although preclinical data are highly suggestive of this idea of a diverse molecular entity (10), the clinical evidence is not yet there (9, 25). In our study, when analyses were performed according to KRAS mutation type, it was shown that patients with the most common mutations (p.G12C/D/V) vs. other less common had similar outcomes (median OS 12.6 months vs. 11.8 months) and again these fared significantly worse than KRAS wt patients (median OS 22.9 months). Similarly, we were not able to observe significant associations or differences when analyses were performed per EGFR mutation type (EGFR classic vs. non-classic), most likely in both cases due to small patient numbers.

In the present study, EGFR and KRAS genotype incidences and prognostic significance are presented for Greek patients with metastatic NSCLC. Our results confirm the adverse prognostic significance of KRAS mutations for survival, both in the whole study population and importantly among patients receiving platinum-based first line treatment, while we were not able to detect prognostic differences among diverse types of mutations. These results add to the existing body of evidence for the role of KRAS mutations and support the negative prognostic value of this biomarker in advanced NSCLC. Furthermore, our results showed that the presence of KRAS mutations can strongly predict the response to first line platinum-based therapy, as patients with KRAS mutant tumours faired significantly worse than those with KRAS wt tumours, with first line platinum-based treatment. These results further suggest that the presence of KRAS mutations could indicate the need for alternative or additional therapies to platinum-based chemotherapy, strengthening the evidence that KRAS mutations represent a marker of resistance to platinum-based therapy.

Figure 5.
Figure 5.

Overall survival according to mutational status for all patients receiving first-line treatment.

Acknowledgements

Part of this work has been presented at the 15th World Conference on Lung Cancer, Sydney, Oct 27-30, 2013 and at the ESMO 2018 Congress, Oct 19-23, Munich.

The Authors are indebted to patients and their families for their trust and generous provision of biological material for research purposes.

The Authors wish to thank Helen Sgouramalli, MSc for block sectioning, Emily Daskalaki for excellent technical assistance with NGS, Maria Moschoni for data coordination and Stella Dallidou, MBA for secretarial assistance.

Footnotes

  • Authors' Contributions

    HL and VK designed the study, GK provided statistical analysis, HL, VK and GM contributed primarily in the writing of the manuscript, while all other authors contributed in the collection of data, completion of study, and reviewing the writing of the manuscript. All Authors approved the final manuscript.

  • This article is freely accessible online.

  • Conflicts of Interest

    The Authors declare no conflicts of interest regarding this study.

  • Received August 1, 2019.
  • Revision received September 15, 2019.
  • Accepted September 18, 2019.

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Genotyping KRAS and EGFR Mutations in Greek Patients With Non-small-cell Lung Cancer: Incidence, Significance and Implications for Treatment
HELENA LINARDOU, VASSILIKI KOTOULA, GEORGE KOUVATSEAS, GIANNIS MOUNTZIOS, VASILIOS KARAVASILIS, EPAMINONDAS SAMANTAS, ANNA KALOGERA-FOUNTZILA, DESPINA TELEVANTOU, KYRIAKI PAPADOPOULOU, XANTHIPI MAVROPOULOU, EMILY DASKALAKI, THOMAS ZARAMBOUKAS, IOANNIS EFSTRATIOU, SOFIA LAMPAKI, GRIGORIOS RALLIS, ELENI RES, KONSTANTINOS N. SYRIGOS, PARIS A. KOSMIDIS, DIMITRIOS PECTASIDES, GEORGE FOUNTZILAS
Cancer Genomics & Proteomics Nov 2019, 16 (6) 531-541; DOI: 10.21873/cgp.20155
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