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. 2012 Sep 27;139(2):201–209. doi: 10.1007/s00432-012-1319-7

KRAS p.G13D mutations are associated with sensitivity to anti-EGFR antibody treatment in colorectal cancer cell lines

Isabelle Messner 1, Giuseppe Cadeddu 1, Wolfgang Huckenbeck 2, Helen J Knowles 3, Helmut E Gabbert 1, Stephan E Baldus 1, Karl-Ludwig Schaefer 1,✉
PMCID: PMC11824176  PMID: 23015072

Abstract

Purpose

Targeted therapies using the anti-EGFR antibodies panitumumab (Pmab) or cetuximab (Cmab) are currently restricted to patients with metastatic colorectal adenocarcinoma whose tumours do not show a mutation in KRAS. However, recent retrospective studies indicated that patients with tumours mutated in codon 13 of KRAS may benefit from treatment with Cmab in contrast to patients with tumours mutated in KRAS codon 12.

Methods

To study the functional impact of the subtype of KRAS mutations on the efficiency of EGFR-targeted therapies, we correlated the KRAS mutation status of 15 colorectal carcinoma cell lines with the in vitro sensitivity of these cells to Cmab/Pmab. Mutations in the potential predictive biomarkers BRAF and PIK3CA as well as protein expression of EGFR and PTEN were also determined.

Results

Four out of seven KRAS-mutated cell lines were characterised by the p.G13D mutation. Treatment of these cells using Cmab/Pmab induced a significant growth inhibition in contrast to cell lines showing a KRAS mutation at codon 12 or 61. Out of the eight KRAS wild-type cell lines, five were insensitive to Cmab/Pmab. These cell lines were characterised either by BRAF mutation or by absence of EGFR or PTEN protein expression.

Conclusions

Since KRAS p.G13D-mutated tumour cells may respond to EGFR-targeted therapy, we suggest including subtype analysis of KRAS mutations in prospective clinical trials. In KRAS wild-type tumour cells, BRAF mutations and loss of EGFR or PTEN expression may lead to resistance to EGFR-targeted therapy and should be considered as additional negative predictive biomarkers.

Electronic supplementary material

The online version of this article (doi:10.1007/s00432-012-1319-7) contains supplementary material, which is available to authorized users.

Keywords: KRAS, BRAF, PIK3CA, EGFR, Panitumumab, Cetuximab, Colorectal adenocarcinoma

Introduction

The epidermal growth factor receptor (EGFR) is abundantly expressed in a broad spectrum of carcinomas including colorectal adenocarcinoma. After activation by binding of a specific ligand to the extracellular domain, EGFR stimulates several major signal transduction pathways including the RAS/RAF mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase/Pten/Akt pathway. Because these pathways are able to promote tumour cell proliferation, suppression of apoptosis, and invasion or metastasis, EGFR represents one of most promising molecules for targeted therapy of carcinomas (Siena et al. 2009; Tejpar et al. 2010).

The two therapeutic antibodies cetuximab and panitumumab inhibit signalling of EGFR by blocking the ligand-binding site. Clinical trials involving these antibodies, either in monotherapy or in combination with conventional chemotherapy, could show a significant survival benefit for a subgroup of patients suffering from metastatic colorectal cancer (mCRC) (Amado et al. 2008; Di Fiore et al. 2007; Khambata-Ford et al. 2007; Lievre et al. 2008). Unexpectedly, the expression level of the EGFR protein as detected by immunohistochemistry did not correlate with the sensitivity of the tumour cells to anti-EGFR antibodies (Chung et al. 2005; Lenz et al. 2006).

In order to identify other suitable molecular markers to define the subgroup of patients who are most likely to respond to anti-EGFR antibody therapy, the mutation status of the KRAS oncogene has been investigated. Mutations within this EGFR downstream gene are known to hamper the ability of the protein to switch from the GTP-bound active state to the GDP-bound inactive state leading to uncontrolled cell proliferation, increased cell survival, and a higher rate of tumour cell metastasis (Jancik et al. 2010). Indeed, mutations in the KRAS gene were found to be predictive for lack of response to EGFR-targeted antibodies (Amado et al. 2008; Di Fiore et al. 2007; Khambata-Ford et al. 2007; Lievre et al. 2008). As a consequence, mutation testing of KRAS has become mandatory according to the approval of cetuximab and panitumumab for treatment of mCRC by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Hence, only patients with confirmed KRAS wild-type status are currently eligible for adjuvant-targeted therapies using anti-EGFR antibodies.

However, a critical consideration is that there are also reports indicating a benefit of anti-EGFR antibody treatment at least for a small subset of patients with KRAS-mutated colon tumours (Benvenuti et al. 2007). More importantly, in a recent meta-analysis, De Roock et al. (2010) found that the subtype of KRAS mutation may have a predictive value for the treatment of mCRC with cetuximab. Using a pooled data set of 579 patients with chemotherapy-refractory colon carcinoma, p.G13D mutations were found to be associated with longer progression-free and overall survival of patients treated with a combination of cetuximab plus chemotherapy compared to patients with other KRAS-mutated tumours. On the other hand, in the group of p.G13D-mutated tumours treated with cetuximab monotherapy, patients did not show such a significant survival advantage. Therefore, the impact of the subtype of KRAS mutation still has to be elucidated.

A second critical aspect of EGFR-targeted therapies using anti-EGFR antibodies comes from the observation that even in the KRAS wild-type group, less than 50 % of patients respond to EGFR-targeted therapy, indicating that other genes of the EGFR/KRAS/BRAF and EGFR/PI3K/PTEN/AKT pathways may be involved in resistance to anti-EGFR therapeutic strategies (reviewed in (Bardelli and Siena 2010)).

Therefore, the aim of this study was to analyse the functional impact of the subtype of KRAS mutation, together with the impact of additional aberrations in other EGFR downstream genes, on the efficiency of EGFR-targeted therapies in CRC using experimental models. For this purpose, we determined the KRAS mutation status of 15 colorectal carcinoma cell lines in combination with the mutation/expression status of additional potential predictive biomarkers (BRAF, PIK3CA, EGFR, and PTEN) and correlated these data to the in vitro sensitivity of these cells to Cmab and Pmab.

Materials and methods

Colorectal carcinoma cell lines were obtained from the following resources: German Resource Centre for Biological Material DSMZ (Colo320 and SW403), American Type Culture Collection ATCC (HCT15, HCT116, KM12c and SW48), European Collection of Cell Cultures ECACC (HCA7), and N. Stoecklein, Department of General, Visceral and Pediatric Surgery, University of Duesseldorf (CaCO2, Colo205, Colo206F, DLD1, HT29P, LOVO, and SW480). All cells were grown in RPMI1640 with 10 % foetal calf serum (Gibco), 2 mM l-glutamine, and 1 % penicillin/streptomycin. Cells were maintained at 37 °C and 5 % CO2.

Preparation of DNA

Genomic DNA was isolated using the Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, including proteinase K digestion and purification of DNA using appropriate spin columns.

Cell line identity

The identity of all the CRC cell lines was re-evaluated by DNA fingerprint analysis. Using the genres® MPX-2 and MPX-3 kit systems (serac, Bad Homburg, Germany), 9 and 12 different STR markers were determined and compared to data available from American Type Culture Collection (ATCC) or the Interlab cell line collection (ICLC) (see supporting data Table S1).

Identification of mutation status of EGFR downstream genes

To evaluate the KRAS, BRAF, and PIK3CA mutation status, PCR amplification and sequence analysis was performed as already described (Baldus et al. 2010). In order to sequence the EGFR gene exon 18, 19, and 21, primer pairs were used as described elsewhere (Paez et al. 2004).

Analysis of EGFR gene dosage in colon carcinoma cell lines

To identify the EGFR gene copy number in tumour cell lines, cells were formalin-fixed, paraffin-embedded, and finally arranged onto a tissue microarray as already described (Ottaviano et al. 2010). EGFR gene sequences were detected by FISH using the Zytolight SPEC EGFR/CEN7 Dual Color Probe System according to the manufacturer’s protocol (Zytovision, Bremerhaven, Germany).

Determination of sensitivity of colon carcinoma cell lines to panitumumab and cetuximab

For all cell lines growth inhibition induced by treatment with panitumumab (Amgen Inc. Thousand Oaks, CA, USA) or cetuximab (Merck, Darmstadt, Germany) was measured by MTT test. For this purpose, 7,500 cells/well were seeded in a 96-well plate (six repeats per data point) and treated with 10 μg/ml monoclonal antibody for 48 h. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and absorbance was measured at 570 nm. Cell lines showing significant reduction in growth kinetics in two independent series of experiments for both antibodies (Cmab and Pmab) were defined as “sensitive to anti-EGFR antibody”.

Immunohistochemistry of EGFR in colon carcinoma cell lines

Immunohistochemical analysis of the cell line array was performed as already described (Alldinger et al. 2007). For the detection of EGFR protein expression in colorectal cancer cells, the EGFR pharmDx™system (DAKO, Hamburg, Germany) was employed.

Western blot analysis

For the analysis of protein expression in tumour cell lines, Western blot analysis was performed as already described (Schaefer et al. 2006). In brief, 50 μg of total protein lysate was subjected to standard SDS–polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. EGFR protein was detected by the anti-EGFR antibody D38B1, PTEN protein by using the monoclonal rabbit antibody 138G6 (both Cell Signaling Technology/New England Biolabs, Frankfurt, Germany). Detection of GAPDH protein was used as a loading control (rabbit polyclonal anti-GAPDH antibody, Sigma-Aldrich, St Louis, MO, USA).

Statistics

All statistical tests for significance were computed using Microsoft Office EXCEL 2003.

Results

Correlation between KRAS status and responsiveness to anti-EGFR antibody treatment

We classified a set of 15 colorectal carcinoma (CRC) cell lines according to their KRAS status and their responsiveness to anti-EGFR antibody treatment.

Mutations of the KRAS gene were found in seven cell lines, while eight cell lines could be shown to have wild-type status. Mutations were detected in codon 12 (two cell lines), codon 13 (four cell lines, all p.G13D), or codon 61 (one cell line) (Table 1).

Table 1.

Mutational status of 15 colon carcinoma cell lines analysed for KRAS, BRAF, PIK3CA, and EGFR (WT = wild type)

CaCo2 Colo 205 Colo206F Colo 320 DLD-1 HCA-7 HCT 116 HCT 15 HT 29 P KM12c LOVO SW48 SW403 SW480 SW948
KRAS WT WT WT WT p.G13D WT p.G13D p.G13D WT WT p.G13D WT p.G12V p.G12V p.E61L
BRAF WT p.V600E p.V600E WT WT WT WT WT p.V600E WT WT WT WT WT WT
EGFR WT WT WT WT WT WT WT WT WT WT WT p.G719S WT WT WT
PIK3CA WT WT WT WT p.E545K p.D549N WT p.H1047R WT P449T WT WT WT WT WT p.E542K
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To characterise the 15 cell lines as either “responder” or “non-responder”, cells showing significant (t test) growth inhibition in four independent experiments applying 10 μg/ml antibody (2 × cetuximab, 2 × panitumumab) for 48 h were defined as “responders to anti-EGFR antibody”. According to these criteria, seven cell lines were classified as “responders”, while the remaining eight cell lines were defined as “non-responders” (Fig. 1).

Fig. 1.

Fig. 1

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Sensitivity to anti-EGFR antibody treatment. Cells were treated with panitumumab (yellow columns) or cetuximab (blue columns) (10 μg/ml, 48 h), and the resulting viability of the cells was measured by MTT test. For each antibody, two independent series of experiments were performed. A significant (t test) growth reduction compared to untreated control (green columns) is indicated by an asterisk for each run. Only cell lines showing significant growth reduction in all four treatment experiments were regarded as responders. Cell lines are sorted according to KRAS/BRAF wild type (left), mutation in KRAS (middle), and mutation in BRAF (right)

Comparing the overall KRAS status with the sensitivity to EGFR antibody treatment did not reveal a significant correlation between these two parameters. While three out of the eight wild-type cell lines were classified as responder, five KRASwt cell lines did not respond. On the other hand, four of the seven KRASmut cell lines were sensitive to anti-EGFR treatment, while the remaining three KRASmut cell lines were insensitive (p = 0.45, chi-square test).

However, among the group of KRASmut cell lines we observed a strict association between the subtype of mutation and the sensitivity to panitumumab and cetuximab: The three cell lines carrying codon 12 or codon 61 mutations were resistant to antibody treatment. In contrast, the growth of all four cell lines containing the p.G13D mutation was markedly reduced by anti-EGFR treatment (Fig. 1), revealing a significant difference between KRAS p.G13D-mutated CRC cell lines and cell lines showing other KRAS mutations. (p < 0.01, chi-square test).

Comparison of EGFR, BRAF, and PIK3CA status with responsiveness to anti-EGFR antibody treatment

In order to study whether other aberrations within EGFR signalling pathways affect the sensitivity of KRAS wild-type cells to anti-EGFR antibody treatment, we also evaluated the mutation status of BRAF (codon 600), EGFR (exons 18, 19, and 21), and PIK3CA (exons 9 and 20) (Table 1).

BRAF mutations were detected in three out of the eight KRASwt cell lines but were absent in all KRASmut cell lines. A mutation in the EGFR gene was only detected in one KRASwt cell line (SW48), while FISH analysis of EGFR revealed absence of high-level amplification in all the cell lines (data not shown). Mutations in the PIK3CA gene were observed in one KRASwt cell line (HT29P) and three KRASmut cell lines (Table 1).

It was noted that all three BRAF-mutated cell lines were resistant to anti-EGFR antibody treatment (Fig. 1); however, this parameter failed to reach statistical significance (p = 0.10).

Taken together, in the subgroup of eight KRAS wild-type cell lines, no significant correlation between sensitivity to anti-EGFR antibody therapy and genetic aberrations in EGFR, PIK3CA, or BRAF could be demonstrated, even though BRAF mutations were only found in resistant KRASwt cell lines.

Correlation of EGFR and PTEN protein expression with responsiveness to anti-EGFR antibody treatment

We further analysed whether the expression of key regulatory genes of the EGFR pathway was associated with resistance to anti-EGFR treatment.

Western blot analysis demonstrated expression of EGFR and PTEN protein in almost all of the cell lines. A lack of EGFR was observed only for the cell line Colo320 (Fig. 2a), while the absence of PTEN expression was noticed in the cell line KM12c (Fig. 2b). The absence of EGFR in Colo320 was also confirmed by immunocytochemistry (Fig. 3). Both cell lines have wild-type status for KRAS and BRAF but were classified as non-responders to anti-EGFR treatment.

Fig. 2.

Fig. 2

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a EGFR expression by Western blot analysis. b PTEN expression by Western blot analysis. Basal protein expression of a EGFR and b PTEN in 15 colorectal cancer cell lines using GAPDH as loading control

Fig. 3.

Fig. 3

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EGFR expression by immunohistochemistry. Formalin-fixed and paraffin-embedded cells were stained for EGFR expression using standard IHC protocols. Left: cell line DLD1; Right: Colo320, Magnification: ×400

In summary, for the KRAS wild-type cell lines, we found BRAF mutations and loss of EGFR or PTEN protein expression to be associated with resistance to panitumumab or cetuximab. None of these aberrations were detected in KRASmut cell lines.

Discussion

In the last decade several clinical studies have confirmed that targeting the epidermal growth factor receptor (EGFR) with specific monoclonal antibodies may improve outcome in metastatic colorectal carcinoma (mCRC) at least for a subset of patients (reviewed in (Linardou et al. 2008; Normanno et al. 2009)). Trying to define the group of patients who are most likely to benefit from these targeted therapies has identified mutation of the KRAS oncogene—an important intracellular signalling molecule downstream of EGFR—as a strong negative predictor for response to anti-EGFR-based therapies.

However, in a more recent meta-analysis of 579 CRC patients treated with cetuximab, patients exhibiting the p.G13D mutation showed a significantly longer overall and progression-free survival compared to patients whose tumours were characterised by KRAS mutations in codon 12 (De Roock et al. 2010). These findings raise the question whether the group of KRAS p.G13D-mutated tumours defines a subset of tumour cells that may be successfully treated using anti-EGFR antibodies.

In our in vitro study we correlated the sensitivity of CRC tumour cells to anti-EGFR antibody treatment with their KRAS mutation status. While the growth kinetics of all cell lines with p.G13D mutations could be significantly reduced by treatment with anti-EGFR antibodies, none of the cell lines with mutation either in codon 12 or in codon 61 responded to cetuximab and panitumumab.

In CRC, mutations in the KRAS gene are especially common in codon 12 (26 %) and codon 13 (9 %) (Andreyev et al. 2001), while mutations in codon 61 (1.4–2.7 %) and codon 146 (0.4–4.1 %) (Edkins et al. 2006; Loupakis et al. 2009) occur just in a minority of tumours. Mutations in both codons 12 and 13 affect the intrinsic GTPase activity of the KRAS protein, hampering the switch from the GTP-bound active state to the GDP-bound inactive state. However, protein structure and functional analysis performed in the mid-eighties demonstrated that the glycine residue at position 12 is more critical for correct function of wild-type KRAS than the neighbouring glycine at position 13 (Barbacid 1987). Moreover, transfection of NIH3T3 cells with different KRAS variants resulted in a more aggressive phenotype of cells transformed with KRAS mutated in codon 12 compared to cells gaining the codon 13 mutation (Guerrero et al. 2000). Finally, in a clinical study of 194 consecutive colorectal carcinoma cases, codon 13-mutated tumours were significantly less aggressive regarding their potential to generate local or distant metastasis (Finkelstein et al. 1993). Together with our observation that p.G13D-mutated CRC cells are more sensitive to anti-EGFR treatment than codon 12- or codon 61-mutated cells, p.G13D-mutated CRC cells seem to define a less aggressive phenotype of colorectal cancer compared to CRC cells showing other KRAS mutations.

In our in vitro analysis the fully humanised IgG2 antibody panitumumab and the chimeric mouse–human IgG1 antibody cetuximab showed an almost identical growth inhibitory effect on p.G13D-mutated tumour cells. However, such a homogenous pattern is not observed in the clinical situation. Recently, Peeters et al. (2012) reported that in a retrospective analysis of patients treated with or without panitumumab in three different phase III studies, no significant associations were found between the subtype of tumour KRAS mutations and patient outcome. In contrast, Tejpar et al. (2012) expanded their initial analysis (De Roock et al. 2010) on the clinical impact of KRAS pG13D-mutated tumours to patients treated with first-line chemotherapy with or without cetuximab and again observed a benefit from the addition of cetuximab to chemotherapy regarding progression-free survival and tumour response to patients with pG13D-mutated tumours. Since our in vitro data cannot explain this discrepancy between the clinical studies on panitumumab and cetuximab, we speculate that more complex mechanisms like the interaction between antibody-treated tumour cells and the host immune system may be responsible for the differences between the two anti-EGFR antibodies. For example, an enhanced level of activation of natural killer (NK) cells finally leading to antibody-dependent (tumour-)cell cytotoxicity (ADCC) was only observed for cetuximab but not for panitumumab (Lopez-Albaitero and Ferris 2007).

In the second part of our study we observed a lack of response to anti-EGFR treatment in five of eight KRAS wild-type CRC cell lines. We could demonstrate that all five non-responding cell lines were characterised by either BRAF mutation or absence of PTEN or EGFR protein expression. Correspondingly, none of the sensitive cell lines showed any of these aberrations. According to current concepts of EGFR signalling, mutations in the serine/threonine kinase BRAF are leading to aberrant activation of the EGFR-KRAS-BRAF-MEK-ERK pathway (Ikenoue et al. 2003), while loss of the phosphatase PTEN, which controls activity of the PI3 kinase pathway by dephosphorylating phosphoinositide, causes permanent stimulation of the EGFR-PI3K-PTEN-AKT pathway (Silvestris et al. 2009). Our findings on the impact of mutations in BRAF codon 600 or loss of PTEN expression correlate to several clinical and preclinical studies showing that these molecular markers are predictive for the lack of response to anti-EGFR treatment in CRC (Bardelli and Siena 2010; Jhawer et al. 2008; Tejpar et al. 2010). Including other clinical reports emphasising that BRAF mutations are also associated with reduced progression-free and overall survival regardless of anti-EGFR treatment (Tol et al. 2010), BRAF seems to be of both predictive and prognostic value.

In contrast to BRAF mutations and loss of PTEN, the impact of EGFR expression on the sensitivity of CRC cells to anti-EGFR treatment is far from being well understood. In analogy to the expression of ErbB2/Her2 in trastuzumab-sensitive breast cancer cells, expression of EGFR was initially expected to represent a useful predictor of the responsiveness of CRC cells to anti-EGFR antibodies. This assumption was supported by preclinical experiments showing that the response of different tumour cell lines to panitumumab in the xenograft mouse model directly correlated with EGFR expression (Yang et al. 2001). Therefore, at the beginning of anti-EGFR-targeted therapy in CRC, positive EGFR staining of the tumour cells by immunohistochemistry (IHC) was mandatory for patients to be eligible for this treatment. However, subsequent clinical trials have reported significant tumour response to anti-EGFR antibodies even in the group of patients (3–30 %) whose tumours were negative for EGFR protein expression as determined by IHC (Chung et al. 2005; Lenz et al. 2006). These unexpected results may in part be explained by either pitfalls in EGFR immunohistochemistry, including fixation methodology or storage time of tissue samples (Atkins et al. 2004; Lenz et al. 2006), or inter-observer variability (Chung et al. 2005). Also, a heterogeneity between the primary tumour specimen and the corresponding metastases was observed in several studies (Bibeau et al. 2006; Italiano et al. 2005) showing that the lack of EGFR staining in the primary tumour does not exclude expression of EGFR in the corresponding metastases, which represent the principal target of the anti-EGFR therapy.

Therefore, in a clinical setting the assessment of EGFR expression status by immunohistochemistry in the primary tumour does not represent a reliable method to predict response to anti-EGFR-targeted therapy. However, our findings that the only cell line to show no expression of the EGFR protein was insensitive to anti-EGFR treatment—even if no aberrations in KRAS, BRAF, or PTEN were present—may support the original hypothesis that expression of the target itself is important for functional anti-cancer activity of the anti-EGFR antibodies.

Taken together, we found that even KRAS-mutated colorectal carcinoma cells may respond to anti-EGFR treatment if the mutation affects codon 13 of the KRAS gene. On the other hand, KRAS/BRAF wild-type tumour cells may be insensitive to anti-EGFR treatment if no functional PTEN or EGFR proteins are present.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 15 kb) (16KB, docx)

Acknowledgments

The study was supported by the “Forschungskommission” of the Medical Faculty of the University of Duesseldorf (KLS, SEB, project 9772442). HJK is supported by Arthritis Research UK (MP/19200).

Conflict of interest

None declared.

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