The occurrence of KRAS mutation is predictive of nonresponse and shorter survival in patients treated by anti–epidermal growth factor receptor (anti-EGFR) antibody for metastatic colorectal cancer (mCRC), leading the European Medicine Agency to limit its use to patients with wild-type KRAS tumors. However, only half of these patients will benefit from treatment, suggesting the need to identify additional biomarkers for cetuximab-based treatment efficacy.

We retrospectively collected tumors from 173 patients with mCRC. All but one patient received a cetuximab-based regimen as second-line or greater therapy. KRAS and BRAF status were assessed by allelic discrimination. EGFR amplification was assessed by chromogenic in situ hybridization and fluorescent in situ hybridization, and the expression of PTEN was assessed by immunochemistry.

In patients with KRAS wild-type tumors (n = 116), BRAF mutations (n = 5) were weakly associated with lack of response (P = .063) but were strongly associated with shorter progression-free survival (P < .001) and shorter overall survival (OS; P < .001). A high EGFR polysomy or an EGFR amplification was found in 17.7% of the patients and was associated with response (P = .015). PTEN null expression was found in 19.9% of the patients and was associated with shorter OS (P = .013). In multivariate analysis, BRAF mutation and PTEN expression status were associated with OS.

BRAF status, EGFR amplification, and cytoplasmic expression of PTEN were associated with outcome measures in KRAS wild-type patients treated with a cetuximab-based regimen. Subsequent studies in clinical trial cohorts will be required to confirm the clinical utility of these markers.

Cetuximab and panitumumab are monoclonal antibodies that target epidermal growth factor receptor (EGFR) and are used alone or in combination with fluoruracil, irinotecan, or oxaliplatin in metastatic colorectal cancer (mCRC).13 Recently, several studies49 have shown that activating mutations in KRAS protein, which partially transduces the activation signal from EGFR, abrogates the therapeutic effect of anti-EGFR therapy. This effect is seen in colorectal cancer patients regardless of line of treatment or which anti-EGFR antibody is used. These results have changed the way anti-EGFR drugs are prescribed. For example, the European Medicine Agency has restricted drug prescription to the set of patients with wild-type KRAS tumors.

However, selecting patients on the basis of tumor KRAS status is an imperfect process. While the test for nonresponse is highly specific (nearly 95% of the patients with mutations of KRAS fail to respond to anti-EGFR therapy), it is not sensitive. In fact, 40% to 60% of patients with wild-type KRAS fail to respond to the treatment.10 This suggests that there are other important molecular determinants of response that have yet to be identified.

Based on the example of KRAS, these additional determinants may be components of the signaling pathways activated by EGFR. Three candidates are the EGFR itself, the phosphatase and tensin homolog gene (PTEN), and the v-raf murine sarcoma viral oncogene homolog B1 (BRAF).11,12 These genes are the targets of genetic alterations in colon cancer,12,13 and they have been associated with the efficacy of therapies directed against EGFR and/or members of EGFR-activated pathways.1425

These data are compelling but not conclusive. Therefore, we have simultaneously ascertained EGFR amplification, PTEN protein expression, and mutations in KRAS and BRAF in a large patient cohort with mCRC. These molecular markers were then evaluated individually and in combination for their ability to predict efficacy of cetuximab treatment.

We collected retrospective tumor samples from 173 patients with mCRC. Collection has been partially described.9 Briefly, patients with mCRC were sequentially ascertained from six hospitals. Three patients received cetuximab only, 141 received a combination of irinotecan and cetuximab, and 28 received a combination of folfiri and cetuximab. All but two patients exhibited clear clinical evidence of having progressed on irinotecan-based therapies. One progressed on folfox-based chemotherapy and received a combination of folfox and cetuximab; one received cetuximab alone in first-line treatment. All but one patient received cetuximab-based therapy as a second-line or greater treatment for mCRC (13 in second-line, 78 in third-line, 49 in fourth-line, 20 in fifth-line, and 12 in sixth-line or greater). This study was approved by the Ile-de-France ethical committee number 2 (ID-RCB-AO1058-47/2008-135). The outcome measures were response based on Response Evaluation Criteria in Solid Tumors (RECIST) criteria (54 patients were responders), progression-free survival (PFS), and overall survival (OS).

The seven most frequent mutations on codons 12 and 13 of KRAS were assessed as previously described.9 BRAF V600E mutation detection was assessed by allelic discrimination using TaqMan probes following the same protocol as that for KRAS mutation. Probes and protocol are available on request.

EGFR In Situ Hybridization

Chromogenic in situ hybridization (CISH) experiments were performed for all the specimens by using the digoxigenylated ZytoDot SPEC EGFR Probe (CE-Marked), the ZytoDot pretreatment kit, and the ZytoDot CISH polymer detection kit, according to the supplier's protocol (Zytovision, Clinisciences, Montrouge, France). The DNA probe and sections were denatured at 95°C and hybridized at 37°C overnight using a HYBrite instrument (Vysis, Downers Grove, IL). An overlook was made with a ×40 dry objective to assess the heterogeneity of the staining on all tumor cells, and 60 nuclei were scored with a ×60 oil objective in at least four areas by two observers independently (A.C. and F.P.-L.). Only tumors with four or more copies of the EGFR gene (focally or in more than 40% of the cells) or with clusters were controlled by fluorescent in situ hybridization (FISH). All other tumors were considered to be FISH-negative. FISH experiments were performed using an EGFR-specific sequence probe (LSI EGFR) and control chromosome enumeration probe 7 (CEP7), according to the manufacturers' recommended protocol (Vysis-Abbott Molecular Diagnostics, Rungis, France) with some minor modifications. The DNA probes and tissue sections were denatured at 85°C for 5 minutes using a HYBrite instrument. An additional wash in distilled water was added before counterstaining and mounting with a solution of 4≪,6-diamidino-2-phenylindole dihydrochloride. As defined previously by Hirsch et al,15 tumors with four or more copies of the EGFR gene in ≥ 40% of the cells (high polysomy) or tumors with EGFR gene amplification (gene-to-chromosome ratio ≥ 2, or presence of gene cluster, or ≥ 10 gene copies in ≥ 10% of the cells) were considered to be FISH-positive, whereas all other tumors were considered to be FISH-negative.

PTEN Expression

PTEN status was determined by immunohistochemistry (IHC) on 3-μm paraffin sections. Immunostaining was performed with a Benchmark XT immunostainer (Ventana, Illkirch, France). After deparaffinization, antigen retrieval was carried out for 60 minutes in CCl citrate buffer (pH = 8) at 95°C. PTEN antibody (AF847; R&D Systems, Lille, France) was incubated for 32 minutes at 37°C and the revelation was made with the Ultraview detection kit (Ventana). The primary antibody dilution varied between 1/200 to 1/400 according to the lot number. A PTEN IHC control slide (8106; Cell Signaling Technology, Ozyme, Saint Quentin en Yvelines, France) was used to optimized the dilution and run with each slide series. Sections were scored semiquantitatively by light microscopy by two pathologists (A.C. and F.P.-L.). Normal endothelial cells were used as an internal positive control. Cytoplasmic and nuclear staining were specified. For each localization, the percentage of stained tumor cells was multiplied by the intensity (1, 2, or 3) and added to a score between 0 and 300. Tumors were then dichotomized into PTEN-negative (cytoplasmic score = 0) or PTEN-positive (cytoplasmic score = > 0) groups.

Statistical Analysis

Statistical analysis was conducted in S-PLUS 7.0 (Insightful, Seattle, WA) using the Cox proportional hazard model for time-to-event data and logistic regression or Fisher's exact test for binary outcomes. Median times-to-event and CIs were obtained from Kaplan-Meier plots and the log-rank test. All P values are two-sided.

We identified 53 (31.3%) activating mutations in codons 12 or 13 of KRAS gene from 169 patients. Genotyping data were unattainable for four samples. The mutations were tested for association with drug efficacy. In fact, the mutation status of 114 of these patients was previously reported and was shown to correlate with cetuximab response.4 Not surprisingly, in the complete set of 169 patients, KRAS is a strong predictor of drug efficacy. KRAS mutations were associated with absence of response (P < .001), with a shorter period of PFS (P < .001) and shorter OS (P < .001; Table 1).


Table 1. Univariate Analysis With Kaplan-Meier Estimates of Median

Table 1. Univariate Analysis With Kaplan-Meier Estimates of Median

Variable Patients With Mutated KRAS (n = 53)
Patients With Nonmutated KRAS (n = 116)
No. 95% CI No. 95% CI No. 95% CI
Responder 1 52 < .001*
    Median PFS, weeks 10 8.0 to 12 30 25.9 to 34 < .001
    Median OS, months 8.4 6.4 to 10.7 14.4 12.0 to 17.9 < .001
Mutated BRAF (n = 5) Nonmutated BRAF (n = 110)
Responder 0 52 .063*
    Median PFS, weeks 8.0 8 to NA 31.4 28 to 34.4 .001
    Median OS, months 6.5 5.9 to NA 14.8 13.0 to 19.6 .001
PTEN cytoplasmic null expression (n = 22) PTEN cytoplasmic non-null expression (n = 89)
Responder 10 41 1*
    Median PFS, weeks 30.0 19.4 to 43 31.4 26.0 to 36 .275
    Median OS, months 11.8 9.1 to 17.9 16.2 13.9 to 20.7 .013
Nonamplified EGFR (n = 79) Amplified EGFR (n = 17)
Responder 29 12 .015*
    Median PFS, weeks 28 19.9 to 34.0 34 29.0 to 65.6 .280
    Median OS, months 13.9 11.4 to 17.3 19.7 13.4 to NA .180

Abbreviations: PFS, progression-free survival; OS, overall survival; NA, not assessable.

*Fisher's exact test.

†Log-rank test.

The V600E mutation in the BRAF gene was determined in the entire series. Five (3%) of 171 tumors were mutated; as expected, all were found in KRAS wild-type tumors. BRAF mutations were tested for association with treatment outcome. Since KRAS mutations are reproducibly associated with outcome after cetuximab therapy, we focused our analyses on patients with KRAS wild-type tumors. But to be complete, in the subgroup of patients with KRAS mutation, the other markers investigated in this study were not associated with outcome measures (Appendix Table A1, online only). In patients with KRAS wild-type tumors (n = 116), BRAF mutations were associated with lack of response (P = .063; Table 1). They were significantly associated with shorter PFS (P < .001) and shorter OS (P < .001; Table 1 and Fig 1). Interestingly, the PFS survival of these patients was similar to that of patients with KRAS mutations (Appendix Fig A1, online only).

EGFR amplification status was determined in 138 of 173 patients. The high dropout rate was due to a technical problem related to the fixation process and therefore is not expected to create patient ascertainment bias. EGFR high polysomy or amplification (FISH-positive phenotype) was found in 22 patients. Among them, 17 (77.3%) occurred in the 96 KRAS wild-type tumors (17.7%).

Restricting our analysis to KRAS wild-type tumors only, we tested for association between EGFR high polysomy or amplification and treatment outcome. In the KRAS wild-type subgroup, EGFR FISH-positive phenotype was associated with higher response rate (RR; 71%) than that in tumors with normal EGFR copy number (37%). This difference was significant (P = .015; Table 1). In addition, there was a trend toward longer PFS and OS in patients with FISH-positive phenotype, but the difference did not reach the threshold of significance (Table 1).

PTEN status was determined by IHC in 162 samples. The evaluation of PTEN cytoplasmic expression as a continuous trait did not demonstrate any significant association with response or PFS or OS. We defined PTEN status for each patient as a dichotomous trait of either expressing PTEN in the cytoplasmic compartment of the cell (ie, any positive tumor cells) or lacking PTEN expression. Thirty-one tumors (19%) were negative for PTEN expression in the tumor cell cytoplasmic compartment. Among them, 22 (19.8%) occurred in the group of 111 wild-type KRAS patients. In this patient subgroup, the lack of PTEN expression was not associated with response or PFS but was associated with a shorter OS (P = .013; Table 1 and Fig 2).

We performed a multivariate analysis of response and OS to determine the respective roles of EGFR, BRAF, and PTEN in determining treatment outcome for colon cancer patients with KRAS wild-type tumors. The multivariate analysis also included adjustments for other variables such as sex, age, number of previous chemotherapy regimens before the use of anti-EGFR therapy, and type of tumor material used for the determination of biomarker status (ie, primary or metastatic tumor). As a result, we found that EGFR and BRAF were significant predictors of response even after adjustment for all other covariates (Table 2). This association was also significant for EGFR in the subset of patients who were wild-type for both KRAS and BRAF (Table 2). Finally, we found that PTEN and BRAF were joint predictors of overall survival (Table 3).


Table 2. Multivariate Logistic Regression Model for Response

Table 2. Multivariate Logistic Regression Model for Response

Variable Categories OR 95% CI P
KRAS wild-type (n = 96)
    EGFR Nonamplified 1.0
Amplified* 4.8 1.4 to 15.7 .007
    BRAF Nonmutated* 1.0
Mutated Infinity .004
KRAS and BRAF wild type (n = 91)
    EGFR Nonamplified 1.0 .002
Amplified* 7.7 1.9 to 31.2

NOTE. Adjusted for sex, age, tumor location (primary or metastatic), and number of previous chemotherapy regimens prior to use of anti–epidermal growth factor receptor therapy (≤ 2 or > 2).

Abbreviation: OR, odds ratio.

*Correlates with response.


Table 3. Multivariate Cox Proportional Hazard Model for Overall Survival

Table 3. Multivariate Cox Proportional Hazard Model for Overall Survival

Variable Categories HR 95% CI P
KRAS wild-type (n = 110)
    BRAF Nonmutated 1.0
Mutated* 6.6 2.4 to 18.2 < .001
    PTEN Non-null expression 1.0
Null expression* 1.8 1.1 to 3.1 .023
KRAS and BRAF wild-type (n = 105)
    PTEN Non-null expression 1.0
Null expression* 1.9 1.1 to 3.2 .026

NOTE. Adjusted for sex, age, tumor location (primary or metastatic), and number of previous chemotherapy regimens prior to use of anti–epidermal growth factor receptor therapy (≤ 2 or > 2).

Abbreviation: HR, hazard ratio.

*Correlates with shorter survival.

There is ample evidence that activating mutations in KRAS are strong predictors of resistance to cetuximab.47,9 However, KRAS is not a perfect predictor since only 40% to 60% of patients with wild-type KRAS tumors respond to treatment. Here, we have provided evidence that in patients with wild-type KRAS, mutations in BRAF are associated with response, PFS, and OS; EGFR amplification is associated with response; and PTEN protein expression is associated with OS. The RRs and frequencies of all the genetic subclasses defined in this study are summarized in Figure 3. These data are based on a retrospective analysis of a nonclinical trial disease cohort and need to be validated in subsequent studies before they are used to inform clinical care. However, they clearly support the emerging view that a comprehensive assessment of genetic alterations in EGFR signaling pathways will enable an accurate prediction of patient benefit from anti-EGFR treatment.

This study adds to the growing evidence that BRAF status predicts benefit from anti-EGFR therapy in colorectal cancer.24,25 In fact, the odds ratio of nonresponse increases from 42 in patients with mutated KRAS to 50 in patients with either KRAS or BRAF mutations. However, the clinical impact of BRAF gene testing depends on the prevalence of BRAF mutations in CRC. Here, the prevalence of BRAF mutations was 3%, which is lower than expected on the basis of previous population studies of microsatellite-stable mCRC.26,27 Moreover, in a recent study24 of mCRC patients treated with cetuximab, the mutation frequency was 9.7%, significantly higher than in this study (P = .02). To eliminate concerns about potential artifacts, we regenotyped 92 of the 116 KRAS wild-type tumors by Sequenom genotyping technology (Sequenom, San Diego, CA; data not shown). No additional BRAF mutants were found. Perhaps the explanation for the low BRAF mutation frequency comes from the fact that BRAF mutations are thought to be negatively prognostic in microsatellite-stable tumors,26 and unlike the patients in Di Nicolantonio et al24, our patients generally survived through multiple rounds of chemotherapy before receiving cetuximab. For example, 30% of our cohort received two or more lines of therapy before cetuximab compared with 46% in the previous study (P = .011). Therefore, at least some patients with BRAF mutations may not have survived long enough to be recruited into this study. But this potential selection bias does not affect the conclusions of this and other studies24, that BRAF status should be taken into account before considering anti-EGFR therapies for mCRC patients. The frequency of BRAF mutations is expected to be higher, and therefore more clinically relevant, in patients with tumors showing microsatellite instability or (assuming anti-EGFR therapies are eventually approved for adjuvant care) in patients with stage III CRC, in which up to 16% of tumors could carry somatic BRAF mutations.2628

In CRC, the association of EGFR amplification with efficacy of anti-EGFR therapy has been controversial. Initially, it was reported that EGFR copy number as determined by FISH was associated with tumor response to cetuximab.14 This association was confirmed and extended to panitumumab in subsequent studies.8,16,22,29 However, there have been discrepant reports. For example, tumor response to anti-EGFR therapies was observed in colorectal tumors without an increase in EGFR copy number,30 and EGFR copy number as determined by quantitative polymerase chain reaction was not associated with drug response in some recent studies.5,31,32 These inconsistencies are due, in part, to difficulties in determining copy number by polymerase chain reaction in samples containing a mixture of somatic and tumor DNA and are also due to the lack of a standardized system for scoring and interpreting FISH data. These inconsistencies have led to the conclusion that this molecular marker would be difficult to include in clinical practice.14,33 To address these issues, we measured EGFR copy number by CISH and FISH and applied the scoring algorithm of Hirsch (Patients and Methods) that has been demonstrated to be associated with response in lung cancer patients treated by cetuximab or EGFR–tyrosine kinase inhibitor–based chemotherapy.15,34,35 This scoring algorithm is relatively simple to apply in clinical practice and therefore could be used to select patients who may benefit from anti-EGFR therapies. It also addresses potential tumor heterogeneity of EGFR amplifications and counts only true polysomic events by excluding cells that are actively dividing (S or M2 phase).

We compared the classification developed by Hirsh and used in this study to the performance of other previously published EGFR FISH thresholds associated with tumor response to cetuximab (ie, EGFR-to-chromosome probe intensity ratios of 2.47,36 2.83,33 and 2.9235). Applying these published thresholds to our set of KRAS wild-type patients discriminated drug responders and nonresponders with an accuracy of 57.7%, 63.9%, and 64.9%, respectively, compared with 64.9% for the Hirsh scoring algorithm (data not shown). Some of the alternative thresholds were also associated with longer PFS and OS in our patient cohort (Table 4), which suggests that there is a biologic link between high EGFR copy number and response to anti-EGFR targeted therapies. Despite these data, we agree with Personeni et al33 that clinical decisions based on EGFR copy number are not warranted as long as FISH technology and scoring are not standardized. Perhaps the scoring system used here and commonly used in lung cancer will help unify the field. Although EGFR status should not be used to exclude patients from treatment (negative predictive value = 0.62 in patients with KRAS and BRAF wild-type tumors), here we have identified a subgroup of patients (16.5% of the entire cohort) who are EGFR FISH-positive (and wild-type for KRAS and BRAF) with cetuximab RRs of approximately 80% (RR, 0.80; 95% CI, 0.51 to 0.95). If confirmed, this selection could identify patients for whom early and aggressive cetuximab-based therapy is clearly warranted.


Table 4. Performance of Reported EGFR Number of Copies Cutoff Point

Table 4. Performance of Reported EGFR Number of Copies Cutoff Point

Reference EGFR Cutoff Point PFS (weeks)*
P OS (months)*
No. 95% CI No. 95% CI
Sartore-Bianchi et al36 > 2.47 32 28 to 36 < .01 15 13 to 20 .12 (NS)
≤ 2.47 12 8 to 32 10 7 to 14
Personeni et al33 > 2.83 33 29 to 36 < .05 16 13 to 22 < .05
≤ 2.83 22 12 to 30 11 8 to 15
Cappuzzo et al35 > 2.92 32 28 to 36 .23 NS 18 13 to 22 .08 (NS)
≤ 2.92 25 15 to 33 12.5 9 to 15

Abbreviations: PFS, progression-free survival; OS, overall survival; NS, not significant.

*Kaplan-Meier estimates of median for patients with KRAS wild-type tumors.

†Log-rank test.

We have also shown that PTEN expression was associated with OS in a cetuximab-treated cohort. This association does not seem to be related to response, which strongly supports the view that PTEN is prognostic (ie, loss of expression identifies a subset of tumors that are likely to have poor outcomes). Since this was a single-arm study, we cannot know if this effect is enhanced by cetuximab treatment or whether it is simply related to the natural history of PTEN-negative tumors. In any case, it seems clear that patients with KRAS wild-type/PTEN-negative tumors require more effective treatment options. mTOR inhibitors,37 which target the AKT pathway downstream of PTEN, could be useful in this set of patients.

Four studies have provided evidence that PTEN status is associated with response in cetuximab-treated mCRC.2022,38 We did not confirm this result, and the reasons for the result are unclear. There are numerous differences between studies, including PTEN antibodies, IHC scoring algorithms, and whether PTEN expression was measured in primary or metastatic tumor tissue. Clearly, these differences could be significant and could contribute to discrepant results. To the contrary, patients with PTEN-negative tumors have poor outcomes regardless of response. Additional studies will be needed to determine the exact role PTEN status plays in influencing patient outcome.

Accurately predicting drug response is the eventual goal of personalized medicine. Somewhat surprisingly, that goal seems achievable for cetuximab. If key regulatory molecules in the signaling pathways downstream of EGFR are mutated so that these pathways gain EGFR-independent activity, then that patient is unlikely to benefit from cetuximab-based therapies. Unfortunately, the predictive algorithm may never be perfect, because the complete list of relevant molecules may never be known. But as the list of approved therapeutics increases, clinicians will be forced to choose between nearly equally effective treatment regimes. The ability to inform that choice with the biomarkers described in this and similar studies should lead to dramatic improvements in patient care.

© 2009 by American Society of Clinical Oncology

See accompanying editorial on page 5866 and article on page 5931

Terms in blue are defined in the glossary, found at the end of this article and online at

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: Julia Reid, Myriad Genetics (C); Steven Stone, Myriad Genetics (C) Consultant or Advisory Role: Pierre Laurent-Puig, Merck Lipha Santé France (C), Amgen France (C); Philippe Rougier, Pfizer (C), Merck Serono (C), sanofi-aventis (C); Michel Ducreux, Amgen (C); Marc Ychou, Merck Lipha Santé (C), Roche (C), Amgen (C), Bayer Pharmaceuticals (C); Fréderic Bibeau, Amgen (C), Merck Serono (C), PGxHealth (C); Frédérique Penault-Llorca, Merck Lipha Santé (C), Amgen (C) Stock Ownership: Julia Reid, Myriad Genetics; Steven Stone, Myriad Genetics Honoraria: Pierre Laurent-Puig, Merck Lipha Santé France, Amgen France; Philippe Rougier, Merck Serono, Amgen, sanofi-aventis; Astrid Lievre, Merck Lipha Santé, Roche; Bruno Landi, Novartis; Michel Ducreux, Amgen, Merck Serono; Marc Ychou, Merck Lipha Santé, Roche, Amgen, Bayer Pharmaceuticals; Fréderic Bibeau, Merck Serono, Amgen; Olivier Bouché, Merck Lipha Santé, Amgen; Frédérique Penault-Llorca, Merck Lipha Santé, Amgen Research Funding: Pierre Laurent-Puig, Merck Lipha Santé France, Amgen France, Myriad Genetics; Fréderic Bibeau, Merck Serono, Amgen, Roche, Pfizer Expert Testimony: None Other Remuneration: None

Conception and design: Pierre Laurent-Puig, Astrid Lievre, Valérie Boige, Steven Stone, Frédérique Penault-Llorca

Financial support: Pierre Laurent-Puig, Frédérique Penault-Llorca

Administrative support: Pierre Laurent-Puig

Provision of study materials or patients: Pierre Laurent-Puig, Anne Cayre, Emmanuel Buc, Jean-Baptiste Bachet, Thierry Lecomte, Philippe Rougier, Astrid Lievre, Bruno Landi, Valérie Boige, Michel Ducreux, Marc Ychou, Fréderic Bibeau, Olivier Bouché, Frédérique Penault-Llorca

Collection and assembly of data: Pierre Laurent-Puig, Anne Cayre, Gilles Manceau, Emmanuel Buc, Jean-Baptiste Bachet, Thierry Lecomte, Philippe Rougier, Astrid Lievre, Bruno Landi, Valérie Boige, Michel Ducreux, Marc Ychou, Olivier Bouché, Frédérique Penault-Llorca

Data analysis and interpretation: Pierre Laurent-Puig, Anne Cayre, Gilles Manceau, Astrid Lievre, Michel Ducreux, Julia Reid, Steven Stone, Frédérique Penault-Llorca

Manuscript writing: Pierre Laurent-Puig, Anne Cayre, Astrid Lievre, Valérie Boige, Julia Reid, Steven Stone, Frédérique Penault-Llorca

Final approval of manuscript: Pierre Laurent-Puig, Anne Cayre, Gilles Manceau, Emmanuel Buc, Jean-Baptiste Bachet, Thierry Lecomte, Philippe Rougier, Astrid Lievre, Bruno Landi, Valérie Boige, Michel Ducreux, Marc Ychou, Fréderic Bibeau, Olivier Bouché, Julia Reid, Steven Stone, Frédérique Penault-Llorca

Glossary Terms

The gene that encodes K-Ras, a protein that is a member of the small GTPase superfamily, in which a single amino acid substitution results in an activating mutation. Alternative splicing gives rise to variants encoding two isoforms that differ in the C-terminal region.

EGFR (epidermal growth factor receptor):

Also known as HER-1, EGFR belongs to a family of receptors (HER-2, HER-3, HER-4 are other members of the family) and binds to the EGF, TGF-α, and other related proteins, leading to the generation of proliferative and survival signals within the cell. It also belongs to the larger family of tyrosine kinase receptors and is generally overexpressed in several solid tumors of epithelial origin.

PTEN (phosphatase and tensin homolog):

PTEN is a tumor suppressor gene with a gamut of regulatory activities. The gene product is a multifunctional molecule. The predominant activity identified for PTEN is its lipid phosphatase activity that converts inositol trisphosphates into inositol bisphosphates, thus inhibiting survival and proliferative pathways that are activated by inositol trisphosphates. PTEN acts to maintain arrest in the G1 phase of the cell cycle and enable apoptosis through an AKT-dependent mechanism.


BRAF is an isoform of RAF. Raf proteins (Raf-1, A-Raf, B-Raf) are intermediate to Ras and MAPK in the cellular proliferative pathway. Raf proteins are typically activated by Ras via phosphorylation, and activated Raf proteins in turn activate MAPK via phosphorylation. However, Raf proteins may also be independently activated by other kinases.


The V600E is the most common oncogenic mutation of BRAF in cancer. The V600E amino acid change results in constitutive activation of the BRAF kinase and promotes cell transformation.

FISH (fluorescent in situ hybridization):

In situ hybridization is a sensitive method that is generally used to detect specific gene sequences in tissue sections or cell preparations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest. FISH uses a fluorescent probe to increase the sensitivity of in situ hybridization.

CISH (chromogenic in situ hybridization):

CISH is a method to detect gene amplification and chromosomal translocations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest. In contrast to FISH (definition), the DNA probe is detected using a simple enzymatic peroxidase reaction and visualized using a conventional microscope.

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We thank Institut du Cancer (PL06-119); Augustin Godart and Violaine Désiré, Inserm-Transfert; Wendy De Roock, Digestive Oncology, University Hospital Gasthuisberg; and Bart Claes, Vesalius Research Center K.U.Leuven for Sequenom genotyping.


Table A1. P Values for Association With Outcome Measures in Patients With KRAS Mutation (n = 41)

Table A1. P Values for Association With Outcome Measures in Patients With KRAS Mutation (n = 41)

Gene Response PFS OS
PTEN .54 .84 .96
EGFR .61 .79 .33

Abbreviations: PFS, progression-free survival; OS, overall survival.

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DOI: 10.1200/JCO.2008.21.6796 Journal of Clinical Oncology 27, no. 35 (December 10, 2009) 5924-5930.

Published online November 02, 2009.

PMID: 19884556

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