The first 3 authors contributed equally to this work.
Impact of epidermal growth factor receptor (EGFR) kinase mutations, EGFR gene amplifications, and KRAS mutations on survival of pancreatic adenocarcinoma
Article first published online: 12 MAR 2007
Copyright © 2007 American Cancer Society
Volume 109, Issue 8, pages 1561–1569, 15 April 2007
How to Cite
Lee, J., Jang, K.-T., Ki, C.-S., Lim, T., Park, Y. S., Lim, H. Y., Choi, D.-W., Kang, W. K., Park, K. and Park, J. O. (2007), Impact of epidermal growth factor receptor (EGFR) kinase mutations, EGFR gene amplifications, and KRAS mutations on survival of pancreatic adenocarcinoma. Cancer, 109: 1561–1569. doi: 10.1002/cncr.22559
- Issue published online: 4 APR 2007
- Article first published online: 12 MAR 2007
- Manuscript Accepted: 2 JAN 2007
- Manuscript Revised: 17 DEC 2006
- Manuscript Received: 31 OCT 2006
- pancreatic adenocarcinoma;
- epidermal growth factor receptor;
Erlotinib, in combination with gemcitabine, has shown clinical benefits in pancreatic adenocarcinoma patients. The presence of EGFR mutations and increased EGFR copy numbers in pancreatic adenocarcinoma was explored.
Sixty-six pancreatic cancer patients were included in the analysis. The EGFR mutation was analyzed by DNA sequencing of exons 18–21 in the tyrosine kinase domain. KRAS mutation was analyzed by sequencing codons 12, 13, and 61. Quantitative real-time polymerase chain reaction was performed to analyze the copy number of EGFR.
In the current study the EGFR mutation was harbored in only 1 (1.5%) of the 66 inoperable or metastatic pancreatic adenocarcinoma patients. Amino acid substitution was detected in exon 20 of the EGFR gene. Increased EGFR copy numbers (≥3.0 per cell) were detected in 26 (41%) patients. There was only 1 patient, who had a highly increased EGFR copy number (≥6.0 per cell), who died, 2.1 months from the date of diagnosis. The EGFR amplification did not significantly influence survival in pancreatic adenocarcinoma patients (P = .935). Thirty-two (49%) of the 65 pancreatic adenocarcinomas examined harbored a point mutation in codons 12 (n = 31) and 61 (n = 1) of the KRAS gene. The presence of a point mutation in codon 12 adversely influenced survival of pancreatic cancer patients (P = .030).
The incidence of somatic mutations in the tyrosine kinase domains of EGFR was very low and the increased gene copy number of EGFR did not significantly influence survival. Cancer 2007. © 2007 American Cancer Society.
The majority of patients with metastatic pancreatic cancer suffer from debilitating symptoms and have an extremely poor prognosis and an efficient therapeutic modality is not yet available. Pancreatic cancer is the tenth commonest malignancy in Korea and its incidence has been increasing in the past decade.1 Systemic chemotherapy has been ineffective,2, 3 although gemcitabine has demonstrated a clinical benefit and has become the standard chemotherapy for advanced pancreatic cancer.4 During the past few years a large number of phase III trials have tested the efficacy of gemcitabine in combination with other drugs, of which none showed superiority of combination therapy over gemcitabine alone. Erlotinib, an oral reversible inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, was the first drug to document a superiority of combination therapy with gemcitabine in terms of survival and progression-free survival in a large randomized trial.5
EGFR is a member of the ErbB receptor family of receptor tyrosine kinases, is overexpressed in human pancreatic tissue, and has been implicated in the carcinogenesis of the disease.6 A few studies found a correlation between the coexpression of EGFR and its ligands and the aggressiveness of pancreatic cancer.7, 8 The importance of EGFR in pancreatic carcinogenesis was further supported by the inhibition of pancreatic cancer cell growth and enhanced sensitivity to cisplatin in the presence of a truncated EGFR.9 Recently, approximately 85% of nonsmall-cell lung cancer (NSCLC) patients, who responded favorably to gefitinib or erlotinib, were shown to have somatic mutations in the EGFR gene.10–14 Mutations were located in the 4 exons (exons 18–21) around the ATP-binding pocket of the tyrosine kinase domain, leading to enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib or erlotinib. These mutations were more frequent in women than in men, in adenocarcinoma than in other histologies, in nonsmokers than in smokers, and in patients from Japan than in patients from the West.10–12, 15–17 Given the positive result from a well-designed NCIC-CTG phase III trial of combination treatment of erlotinib and gemcitabine, the EGFR mutational analysis and its clinicopathologic significance should be explored in pancreatic cancer patients.
Activation of the RAS pathway is important in carcinogenesis of various tumor types. Among human cancers, pancreatic cancer shows the highest frequency of KRAS gene mutations.18 Recent studies have suggested a possible ethnic difference in mutation profiles of KRAS between Asians and Westerns.19–21 RAS signaling pathways are commonly activated in tumors in which growth factor receptor tyrosine kinases are overexpressed and involved in mediating the downstream effects of EGFR activation.22 Thus, genetic alterations of both EGFR and KRAS genes may play a critical role in carcinogenesis of pancreatic cancer, although the relation or the impact of EGFR and KRAS mutations is yet to be defined. In this study we analyzed the frequency of EGFR mutations and increased EGFR copy number in 66 pancreatic adenocarcinoma patients. We also studied the association of EGFR mutations with KRAS mutations because EGFR plays a critical role in the RAS signaling cascade.
MATERIALS AND METHODS
Tissue Samples and Patients
From January 2000 to July 2004, 66 patients were diagnosed of unresectable, metastatic pancreatic adenocarcinoma and received at least 1 cycle of cytotoxic chemotherapy. None of these patients were treated with tyrosine kinase inhibitors or monoclonal antibodies targeting EGFR. A complete set of clinical data including sex, age at diagnosis, smoking history, staging, treatment, and vital status were obtained from the medical records of each patient. All pathologic specimens were cut from formalin-fixed paraffin-embedded tumor blocks, and an H & E-stained section was reviewed by 1 pathologist. DNA was prepared from this sections after dissection of tumor to obtain at least 50% tumor cell content. All patients had ductal adenocarcinoma. The grades of differentiation were categorized as well differentiated, moderately differentiated, poorly differentiated, and undifferentiated. Current study was performed according to the institutional review board regarding human subjects.
DNA Sequencing for EGFR
DNA was extracted from 5 paraffin sections of 10 μm thickness containing a representative portion of each tumor block using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). One hundred nanograms of DNA were amplified in a 20-μl reaction solution containing 2 μL of 10× buffer (Roche, Mannheim, Germany), 1.7 to 2.5 mmol/L of MgCl2, 0.3 μM of each primer pairs (exon 18, F: 5′-tccaaatgagctggcaagtg, R: 5′-tcccaaacactcagtgaaacaaa; exon 19, F: 5′-atgtggcaccatctcacaattgcc, R: 5′-ccacacagcaaagcagaaactcac; exon 20, F: 5′-cattcatgcgtcttcacctg, R: 5′-catatccccatggcaaactc; exon 21, F: 5′-gctcagagcctggcatgaa, R: 5′-catcctcccctgcatgtgt), 250 μM of deoxynucleotide triphosphate, and 2.5 units of DNA polymerase (Roche). Amplifications were performed using a 5-minute initial denaturation at 94°C; followed by 30 cycles of 1 minute at 94°C, 1 minute at 57°C, and 1 minute at 72°C, and a 10-minute final extension at 72°C. Polymerase chain reaction (PCR) products were then 2% gel-purified with a Qiagen gel extraction kit (Qiagen). DNA templates were processed for the DNA sequencing reaction using the ABI-PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster, CA) with both forward and reverse sequence-specific primers. Twenty nanograms of purified PCR products were used in a 20-μL sequencing reaction solution containing 8 μL of BigDye Terminator v3.1 and 0.1 μM of the same PCR primer. Sequencing reactions were performed using 25 cycles of 10 seconds at 96°C, 5 seconds at 50°C, and 4 minutes at 60°C. Sequence data were generated with the ABI PRISM 3100 DNA Analyzer (Applied Biosystems). Sequences were analyzed by Sequencer 3.1.1. software (Applied Biosystems) to compare variations.
EGFR Gene Amplification
Quantitative, real-time, TaqMan duplex PCR was performed to analyze the EGFR copy number using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) as previously described by Takano et al.23 Briefly, the EGFR primers were 5′-GGAGGACCGTCGCTTGGT-3′ and 5′-AACACCGCAGCATGTCAAGA-3′; the probe (5′-CACCGCGACCTGGCAGCCA-3′) was labeled with the reporter dye 6-carboxyfluorescein (FAM). The average EGFR copy number per cell was calculated from the differences in the threshold amplification cycles between EGFR and RNaseP. Decreased, normal, increased EGFR copy numbers were defined as less than 1.5, 1.5 to 3.0, and ≥3.0 copies per cell, respectively.
DNA Sequencing for KRAS
Primers used for KRAS were as follows: codon 12, 13, F: 5′-ttatgtgtgacatgttctaat, R: 5′-agaatggtcctgcaccagtaa; codon 61, F: 5′-TCAAGTCCTTTGCCCATTTT, R: 5′-TGCATGGCATTAGCAAAGAC. Amplifications were performed using a 5-minute initial denaturation at 94°C; followed by 30 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C, and a 10-minute final extension at 72°C. DNA sequencing methods were the same as mentioned above.
The statistical analyses of categoric variables were performed using the Fisher exact test. The median overall survival was calculated using the Kaplan-Meier method. Comparisons between different groups were made using the log-rank tests. P values less than 0.05 were considered significant.
EGFR Mutational Analysis
Patient characteristics are provided in Table 1. Sixty-one (92%) patients had metastatic pancreatic cancer at diagnosis. Forty-five (68%) patients had liver metastases at diagnosis. Twenty (30%) patients had a recurrent pancreatic cancer after curative resection. Sixty-four (94%) patients received gemcitabine alone or gemcitabine combined with uracil/tegafur (UFT), capecitabine, or cisplatin as a first-line chemotherapy. Of the 66 tissues examined mutations in the kinase domain of the EGFR gene were identified in 1 (1.5%) tumor tissue, which harbored a substitution in exon 20 (2453G > A, C818Y) (Fig. 1, Table 2). There were no frameshift-type deletion mutations detected in this series. Synonymous single nucleotide polymorphisms were found in 23 (35%) patients in exons 18–21. The most frequently detected polymorphism occurred in exon 20 of the EGFR gene (n = 18, 2361G > A, E787). Other variants observed were as follows: 2 patients at codon 725 (2175G > A, T725) of exon 18; 1 patient at codon 703 (TCT(Leucine) TTT(Leucine)) of exon 18; 1 patient at codon 760 (2280T > C, D760) at exon 19; 1 at codon 758 (2274C > T, I758) at exon 19; and 1 patient at codon 836 (2508C > T, R836) at exon 21. The presence of the frequently observed polymorphism in the EGFR gene (n = 18, 2361G > A, E787) did not influence survival of the patients (13.3 vs 9.1 months, polymorphism (+) vs (−) group, P = .168).
|Current smoker||24 (36)|
|Never smoker||33 (50)|
|Locally advanced disease||5 (8)|
|Metastatic disease||61 (92)|
|Well differentiated||3 (5)|
|Moderately differentiated||35 (53)|
|Poorly differentiated||20 (30)|
|Location of primary tumor|
|Peritoneal seeding||12 (18)|
|Unresectable, locally advanced||5 (8)|
|Previous pancreatectomy||20 (30)|
|Gemcitabine + UFT||14 (21)|
|Gemcitabine + capecitabine||28 (42)|
|Gemcitabine + cisplatin||1 (2)|
|No.||Sex||Age||Smoking status||EGFR polymorphism||EGFR mutation||KRAS mutation||Survival, mo|
|3||M||70||Previous||2361G > A, E787, Exon 20||No||Yes||10.6|
|4||W||48||Current||2103C > T, L703, Exon18||No||No||3.6|
|5||M||60||Current||No||2453G > A, C818Y, Exon20||No||4.1|
|7||M||61||Current||2361G > A, E787, Exon 20||No||No||4.4|
|8||W||51||Never||2175G > A, T725, Exon 18||No||No||3.5|
|14||W||44||Never||2274C > T, I758, Exon 18||No||Yes||5.8|
|18||M||76||Previous||2361G > A, E787, Exon 20||No||Yes||4.3|
|19||M||63||Current||2361G > A, E787, Exon 20||No||Yes||6.8|
|20||M||58||Current||2361G > A, E787, Exon 20||No||Yes||10.8|
|21||M||62||Never||2361G > A, E787, Exon 20||No||Yes||8.4|
|22||M||49||Current||2508C > T, R836, Exon21||No||Yes||6.8|
|27||M||67||Never||2175G > A, T725, Exon 18||No||No||18.0|
|29||M||45||Previous||2361G > A, E787, Exon 20||No||Yes||11.8|
|32||M||47||Current||2361G > A, E787, Exon 20||No||Yes||2.1|
|35||M||56||Previous||2361G > A, E787, Exon 20||No||No||34.6|
|39||W||45||Never||2361G > A, E787, Exon 20||No||Yes||5.7|
|40||W||50||Never||2361G > A, E787, Exon 20||No||Failed||13.5|
|41||M||56||Previous||2361G > A, E787, Exon 20||No||Yes||6.6|
|46||M||77||Never||2361G > A, E787, Exon 20||No||Yes||15.6|
|48||M||43||Current||2361G > A, E787, Exon 20||No||No||7.5|
|49||M||55||Never||2361G > A, E787, Exon 20||No||No||32.8|
|50||M||65||Never||2361G > A, E787, Exon 20||No||Yes||17.2|
|52||M||64||Previous||2361G > A, E787, Exon 20||No||No||16.6|
|53||M||52||Current||2361G > A, E787, Exon 20||No||Yes||16.3|
|65||M||40||Never||2361G > A, E787, Exon 20||No||No||11.7|
EGFR Copy Number
The EGFR copy number was obtained in 63 (96%) of the 66 patients. Of 63 patients, 26 patients (41%) had increased EGFR copy number (≥3.0 per cell). Because there was only 1 patient with an EGFR mutation, no correlation between the EGFR gene copy number and EGFR mutation could be delineated. There was 1 patient who had a high EGFR copy number (≥6.0 per cell) and died after 2.1 months from the date of diagnosis. The EGFR amplification did not significantly influence survival in pancreatic adenocarcinoma patients (P = .935).
KRAS Mutational Analysis
Thirty-two (49%) of the 65 pancreatic adenocarcinomas examined harbored a point mutation in the KRAS gene. All KRAS mutations occurred at codon 12. The most frequently observed point mutation was GGT-GAT in codon 12 (35G > A, 19 of 32, 59%), followed by GGT-GTT at codon 12 (35G > T, 8 of 32 patients, 25%), GGT-CGT (34G > C, 9%, 3 of 26), and GGT-TGT (34G > T, 1 of 26, 4%) in the order of frequency (Fig. 2). There were no mutations in codon 13 and 1 mutation detected in codon 61. The presence of KRAS point mutations at codon 12 adversely influenced median survival time (9.1 vs 13.4 months, KRAS mutation (+) vs (−), P = .030, Fig. 3).
In this study we found a very low incidence of EGFR mutations in pancreatic adenocarcinoma patients. In addition, the proportion of patients with an increased copy number of EGFR (41%) was not as high as in other solid tumor types. The presence of EGFR mutation or increased gene copy number did not significantly influence the survival of pancreatic cancer patients. The amino acid substitution (C818Y within exon 20), which is located within the tyrosine kinase domain of EGFR, was found, which was different from previously described in-frame deletions and deletion mutations in NSCLC tissues.10, 11
To the best of our knowledge the present study represents the largest series to investigate EGFR mutations and amplifications in pancreatic adenocarcinoma. The frequency of EGFR mutation (1.5%) was very low when compared with those in lung cancer (59%, n = 66)23; colon cancer (12%, n = 33)24; squamous cell carcinoma of the head and neck (7%, n = 41)25; and cholangiocarcinoma (14%, n = 22).26 Lee et al.27 analyzed 537 tissues from 98 colon adenocarcinomas, 185 gastric adenocarcinomas, 93 breast ductal carcinomas, 73 hepatocellular carcinomas, and 88 acute adulthood leukemia by PCR-single strand conformation polymorphism (SSCP) analysis and found only 1 silent mutation in exon 18 in breast ductal carcinoma tissue and none in the others. The EGFR mutational analysis on a limited set of samples by a Japanese study group showed no somatic mutations from exons 18 to 21 in 5 pancreatic cancer cell lines28 and a recent report detected 2 (3.6%) EGFR mutations out of 55 patients, both of which were recurrent delE746-A750 mutations.29 These studies along with our data suggest a very low incidence of EGFR mutation in pancreatic adenocarcinoma.
The patterns of EGFR mutation varied among different solid tumors. All of the mutations were in-frame deletion mutations in exon 19 for SCCHN,25 whereas only deletions were found in exon 19 for cholangiocarcinomas.26 The amino acid substitutions in exons 19 and 20 were predominant in colorectal carcinomas (E749K within exon 19, E762G and A767T within exon 20).24 All of these mutations are different from those found in our series (C818Y within exon 20). At this time, it is not clear whether this single mutation represents a true oncogenic mutation or whether it is an uncommon polymorphic variant. We are currently investigating the functional aspect of this particular mutation in our laboratory.
Although a recent NCIC-CTG phase III trial has demonstrated a definite survival benefit of gemcitabine plus erlotinib as compared with gemcitabine alone,5 molecular characterization including EGFR mutation has not been reported in this trial. The predictive and prognostic values of EGFR mutation for sensitivity to gefitinib or erlotinib are now being increasingly recognized. However, unlike NSCLC, it is still not evident that EGFR mutation is associated with sensitivity to EGFR TKIs in pancreatic cancer. Cetuximab is a monoclonal antibody against the EGFR with promising antitumor activity in combination with gemcitabine in pancreatic cancer patients.30 The correlation between cetuximab sensitivity and EGFR mutational status has not been clearly established. In contrast to gefitinib or erlotinib, a recent study demonstrated no association between EGFR mutation and sensitivity to cetuximab in lung cancer patients.31 Although it is well established that the overexpression of EGFR correlates with poor prognosis in pancreatic cancer,7, 8 the association between EGFR mutation and prognosis needs clarification. Interestingly, the EGFR mutation and 58% of KRAS mutations were found in current or previous smokers. In contrast, EGFR mutation has been commonly found in lung cancers from never smokers,10–12 whereas KRAS mutations were associated with cigarette smoking, suggesting the possibility of different pathways in subsets of lung cancer patients. Thus, the relation between smoking status and EGFR or KRAS mutation need further investigation.
We further analyzed the prognostic value of EGFR gene copy number to predict survival, given the fact that it was an independent predictor of gefitinib sensitivity23 and survival in lung cancer.32 Based on our current data, the EGFR gene copy number had limited prognostic significance for survival in pancreatic cancer patients. However, there are some limitations in our methodology for the DNA copy number analysis. Because pancreatic cancer is a highly aneuploid tumor type, it may render the absolute determinations of copy number not possible. In addition, the possibility of stromal contamination may further complicate the assessment of absolute copy number alterations. This hindrance may potentially be overcome by the addition of 1 other reference in the qPCR or parallel fluorescence in situ hybridization (FISH) for absolute copy number determinations in the tumor cells.
Because the RAS-mediated signaling pathway lies downstream of EGFR, it has recently been postulated that both KRAS and EGFR genes are not needed and should show inverse correlation in lung cancer.33, 34 Several groups have reported the inverse correlation between KRAS and EGFR in lung cancer tissues.17, 35 The patient with the EGFR mutation did not harbor a KRAS mutation, although no conclusion could be drawn from our analysis. Point mutations at codon 12 of KRAS are the common oncogene alterations in pancreatic adenocarcinoma.36 The prevalence of KRAS mutations at codons 12 and 13 seems to vary among different ethnic groups. Our results were similar to those reported by the Japanese group: 90% of KRAS codon 12 mutations with GGT-GAT in 67% of cases.37, 38 In a Chinese population the GGT-GAT mutation at codon 12 was the dominant mutation type in pancreatic cancer.21 In contrast, European studies revealed that the prevalence of KRAS codon 12 mutation of 80% with a similar distribution between GGT-GAT (42%) and GGT-GTT (32%).20 In concordance with other studies, codon 12 mutation had an adverse impact on survival of pancreatic cancer patients.
In conclusion, the incidence of somatic mutations in tyrosine kinase domains of EGFR was extremely low and the increased gene copy number of EGFR did not significantly influence survival. The validity of predictive values of the EGFR mutation and gene copy number on gefitinib or erlotinib sensitivity should be investigated in future clinical trials.
- 1Annual report of the Korea Central Cancer Registry Program 2000. Cancer Res Treat. 2002; 34: 77–83., , .
- 3A multispecialty approach to the diagnosis and management of pancreatic cancer. Am J Gastroenterol. 2000; 95: 17–31., , , , , .Direct Link: