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doi:10.1111/jgh.12505
H E PAT O L O G Y
Clinicopathological and prognostic significances of EGFR, KRAS and BRAF mutations in biliary tract carcinomas in Taiwan Yu-Ting Chang,* Ming-Chu Chang,* Kai-Wen Huang,† Chien-Chih Tung,*,‡ Chiun Hsu§ and Jau-Min Wong* Departments of *Internal Medicine, †Surgery and Hepatitis Research Center and §Oncology, National Taiwan University Hospital, College of Medicine, National Taiwan University, and ‡Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital, Taipei, Taiwan
Key words biliary tract carcinomas, BRAF, EGFR, KRAS, survival. Accepted for publication 5 December 2013. Correspondence Dr Yu-Ting Chang, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, No.7 Chung Shan South Road, Taipei, Taiwan. Email: [email protected] Potential conflicts of interest: The authors disclose no conflicts.
Abstract Background and Aim: Biliary tract carcinomas (BTCs) are difficult to diagnose and treat. Epidermal growth factor receptor (EGFR) represents a therapeutic target for the BTCs. Mutations of the EGFR gene and the activation of its downstream pathways, including KRAS and BRAF, predict the sensitivity to anti-EGFR treatment. The aims of this study were to analyze the EGFR, KRAS and BRAF mutations in BTCs and their association with clinical outcomes. Methods: Paraffin-embedded specimens containing 137 BTCs resected at the National Taiwan University Hospital between 1995 and 2004 were analyzed. The exons 18–21 of EGFR gene, the codon 12, 13 and 61 of KRAS gene, and BRAF V600E mutation were analyzed. We examined the correlation between these mutations and the overall survival, tumor location, stage, and differentiation in BTCs. Results: Thirteen (9.5%) BTC patients had EGFR mutations while 23 (16.8%) patients had KRAS mutations. Only one patient had BRAF mutation. Factors influencing survival on univariate analysis were tumor stage, tumor differentiation, and EGFR mutation. On multivariate analysis, EGFR mutation and tumor stage were independent prognostic factors. A correlation between KRAS or BRAF mutations and prognosis was not observed. Conclusions: EGFR and KRAS mutations are not uncommon in BTCs. BRAF mutation is rare in BTCs. EGFR mutation was an independent prognostic marker in BTCs in addition to tumor stage and differentiation. No simultaneous EGFR and KRAS mutations in extrahepatic cholangiocarcinoma and gallbladder carcinoma were found. EGFR and KRAS mutations should be evaluated when tailoring molecular-targeted therapy to patients with BTCs.
Introduction The biliary tract consists of an interconnected system of intra- and extrahepatic ducts that transport bile secreted from the liver to the digestive tract. Biliary tract carcinomas (BTCs), which include cancers of the gallbladders and intra- and extrahepatic biliary trees—are relatively infrequent but highly lethal diseases that are notoriously difficult to diagnose and treat, and have incidences that are increasing worldwide.1,2 In general, the prognosis for patients with advanced BTCs at any site is dismal, with overall 5-year survival rate around 16–44%.3 Surgical resection is the only chance for cure. Local invasion, extensive regional lymph node metastasis, distant metastases, and vascular invasion often preclude resection, and neither radiation nor conventional chemotherapy significantly improves survival or quality of life for
patients with inoperable cancers. Therefore, novel effective therapeutic strategies are urgently required to improve the prognosis. Understanding the pathogenesis and clinicopathological characteristics of BTCs might help to improve the overall treatment strategy for BTCs. Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase of the ErbB receptor family, plays key roles in the diverse processes that stimulate cell proliferation, differentiation, and survival.4 Enhanced signaling from the receptor due to mutation or overexpression contributes to several types of human cancer, including lung, breast, colorectal, pancreatic, head and neck, and bile duct cancer.5–7 Elevated expression of EGFR and its ligand has been reported to correlate with worse prognosis in a variety of cancers, including head and neck, breast, ovarian, colorectal, pancreatic cancers, and cholangiocarcinoma.8–14 Accordingly, EGFR
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represents a validated therapeutic target for the treatment of human cancer. Anti-EGFR antibodies, such as cetuximab, as well as ATP-competitive tyrosine kinase inhibitors, such as erlotinib or gefitinib, have improved the efficacies of conventional chemotherapy and have been approved for the treatment of colorectal cancer and non-small cell lung cancer.15,16 Previous studies have revealed overexpression of EGFR and mutation of this gene in cholangiocarcinoma.7,17–20 Overexpression of EGFR by immunohistochemistry method and somatic mutations of EGFR gene were reported in 47% of cholangiocarcinomas in Taiwan21 and in 8–32% of cholangiocarcinomas in Western countries and Japan.18,19,22 In addition, inhibition of EGFR signaling by gefitinib effectively attenuates the proliferation of cholangiocarcinoma cells in vitro.23 As EGFR kinase inhibitors effectively attenuated cellular growth, these agents may be therapeutically efficacious in human BTCs. A phase II study of erlotinib in patients with advanced biliary cancer showed therapeutic benefit for EGFR blockade with erlotinib in patients with biliary cancer.24 These studies suggest the possibility that like non-small cell lung cancer, the BTCs harbors the EGFR mutations which might be responsible for the clinical response of anti-EGFR therapy. Mutations in the EGFR gene have been investigated in the non-small cell lung cancer because of a reported correlation between mutations and tumor response to treatment with targeted tyrosine kinase inhibitors such as gefitinib and erlotinib.25,26 These mutations have shown some correlation with demographic and histopathologic categories, such as female, non-smoker, and Asian descent.27,28 These somatic mutations can explain the heterogeneous response to gefitinib in lung cancer patients and provide a basis for selecting patients with a high probability of outstanding response to anti-EGFR therapy. The detection of EGFR mutation is considered valuable both for guidance in therapy and for clinical investigations. However, there was no such study like non-small cell lung cancer in BTCs in Taiwan. In addition, few previous studies have clarified associations between the somatic mutations of EGFR gene and expression of this molecule and the clinicopathological factors or prognosis in patients with BTCs. There are two major pathways activated by EGFR: one is RAS/ RAF/MEK/ERK pathway, and the other is PI3K/PTEN/AKT pathway.29–32 EGFR, KRAS and BRAF genotyping are the three most commonly tested genes in solid tumors as they have been linked to resistance or sensitivity to EGFR pathway blockade. The introduction of KRAS testing as a predictive marker to select patients for EGFR-targeted therapies for metastatic colorectal cancer is widely regarded as a key advance in the field of personalized medicine. Current data suggested that together with KRAS mutation, the evaluation of BRAF alterations could also be useful for selecting patients with reduced chance to benefit from EGFRtargeted therapy. Up until now, each of these markers has been mainly assessed as a single event in Taiwan populations. The aims of this study were to analyze the EGFR, KRAS, and BRAF mutations simultaneously in BTCs as well as associations between these mutations and clinicopathological factors or clinical outcome.
cinomas, 45 extrahepatic cholangiocarcinomas, and 35 gallbladder carcinomas resected at the National Taiwan University Hospital between 1995 and 2004 were used for this study. The clinicopathological features of the patients, including TNM staging, tumor location, degree of tumor differentiation, and overall survival, were recorded. The study was approved by the Institutional Review Board at National Taiwan University Hospital. Isolation of genomic DNA and mutational analysis of EGFR, KRAS, and BRAF genes. Tissues from five serial 10-μM sections of retrieved paraffin-embedded formalinfixed slides containing 60% or more tumors were macrodissected from the slides. Genomic DNAs were extracted from deparaffinized samples with the use of the QIAamp DNA Mini kit (Qiagen, Gaithersburg, MD, USA) following the manufacturer’s instructions. The exons of EGFR, KRAS, and BRAF genes were amplified. The primers for amplification were designed using Primer Express Software 3.0 (Applied Biosystems, Foster City, CA, USA). The kinase domain of EGFR coding sequence, from exons 18–21, were tested by high resolution melting analysis (HRMA) first then confirmed by direct sequencing as methods modified from previous report.33 High-resolution melting analysis. The polymerase chain reaction (PCR) was carried out in 96-wells plate with a reaction volume of 10 μL containing genomic DNA (50 ng), 2x LightCycler 480 High Resolution Melting Master Mix (Roche Applied Science, Mannheim, Germany), 2.5 mM MgCl2, and 20 pmol primers. Thermal cycle conditions were 95°C for 5 min, and 45 cycles of 95°C for 10 s and 60°C for 10 s, and the final extension was 72°C for 7 min. Completed PCR plates were analyzed using the LightCycler 480 nd the LightCycler 480 Software 1.5.0 (Roche Applied Science). Direct sequencing of EGFR, KRAS, and BRAF genes. For direct sequencing, the multiplex reaction contained 1x optimized SapphireAmp Fast PCR Master Mix buffer (contain hot start Taq polymerase, dNTP), 20 pmol each primer pair, 100 ng genomic DNA, and nuclease-free water for a final volume of 25 μL. Reactions were performed in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). Thermal cycling conditions were as follows: the initial heat denaturing step was 5 min treatment at 95°C, the second step was 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 0 s (for EGFR exon 19); 45 cycles at 95°C for 30 s; 51°C for 60 s; 72°C for 60 s (for EGFR exons 20 and 21; KRAS exons 1 and 2); 45 cycles at 95°C for 30 s; 54°C for 60 s; 72°C for 60 s (for BRAF exon 15) and the final extension was 72 °Cfor 7 min. The PCR products were purified with the PCR Clean-Up Kit (Geneaid, Taipei, Taiwan) and sequenced using the BigDye Terminator Cycle sequence following the PE Applied Biosystems strategy and Applied Biosystems ABI PRISM3100 DNA Sequencer (Applied Biosystems). All mutations will be confirmed, performing two independent PCR amplifications.
Methods Patient specimens. Paraffin-embedded surgical specimens containing 137 BTCs, including 57 intrahepatic cholangiocar1120
Statistical analysis. The associations of EGFR, KRAS, and BRAF gene mutations among clinicopathological factors were studied by χ2 analysis of proportions, Fisher’s exact test, and Journal of Gastroenterology and Hepatology 29 (2014) 1119–1125
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Student’s t-test when appropriate. Differences in frequencies between subgroups were analyzed using the Kruskal–Wallis test and the Mann–Whitney U-test. Patient survival was analyzed using Kaplan–Meier method, and log–rank tests were performed for univariate survival analysis and the Cox proportional hazards model for multivariate survival analysis. P < 0.05 was accepted as statistically significant. The variables assessed include patient age at diagnosis, sex, tumor location, tumor stage, degree of tumor differentiation, and survival. The statistical calculations were carried out using SPSS statistical software version 11 (SPSS Inc., Chicago, IL, USA).
Results EGFR, KRAS, and BRAF gene mutations in BTCs. EGFR exons 18–21 were analyzed by HRMA using genomic DNA extracted from tissues in the 137 patients. EGFR exons 18–21 were further sequenced. The clinicopathological features and EGFR and KRAS mutations in patients with BTCs are listed in Table 1. In total, 13 patients (9.5%) were identified with EGFR mutations, including seven cases in exon 20: one
Table 1
intrahepatic cholangiocarcinoma in codon 784 (TCC-TTC; amino acid change: Ser-Phe), five extrahepatic cholangiocarcinoma in codon 783 (ACC-ATC; Thr-Ile), codon 800 (GAC-GGC; AspGly), codon 818 (TGT-CGT; Cys-Arg), codon 819 (GTG-ATG; Val-Met), and codon 820 (CAG-CGG; Glu-Arg), and one gallbladder carcinoma in codon 785 (ACC-ATC; Thr-Ile); and six cases in exon 21: one intrahepatic cholangiocarcinoma in codon 837 (GAC-AAC; Asp-Asn), four extrahepatic cholangiocarcinoma in codon 837, codon 851 (GTC-ATC; Val-Ile), codon 873 (GGAGAA; Gly-Glu), and codon 874 (GGC-GAC; Gly-Asp) (Fig. 1), and one gallbladder carcinoma in codon 837 (Table 2). We did not found any samples harboring double mutations in exons 18–21. EGFR mutations were more common in extrahepatic cholangiocarcinoma (9/45, 20%) than in intrahepatic cholangiocarcinoma (2/57, 3.5%), or in gallbladder carcinoma (2/35, 5.7%) (P = 0.01). Twenty-three patients (23/137, 16.8%), including 10 intrahepatic cholangiocarcinoma (10/57, 17.5%), nine extrahepatic cholangiocarcinoma (9/45, 20%), and four gallbladder carcinoma(4/35, 11.4%), had KRAS mutations. Most of the KRAS mutations were located in codon 12 and only one in codon 13. The frequency of KRAS mutation did not differ statistically significantly among the three groups. Two patients with intrahepatic cholangiocarcinoma
Clinicopathological features and EGFR/KRAS mutations in patients with biliary tract carcinoma
Tumor location
Intrahepatic
Extrahepatic
Gallbladder
Total
Case number Gender (male/female) Mean age(range) Tumor grade Well differentiated Moderately differentiated Poorly differentiated Tumor stage I–II III–IV EGFR mutation KRAS mutation
57 32/25 63.6 (39–88)
45 17/28 62.9 (39–82)
35 22/13 67.3 (41–89)
137 71/66 64.3 (39–89)
14 31 12
17 24 4
11 19 5
42 74 21
31 26 2 10
15 30 9 9
7 28 2 4
53 84 13 23
Figure 1 High resolution melting analysis and direct sequence of EGFR gene in in biliary ; B5: 2, ; C5: 3, tract carcinoma. A5: 1, ; D5: 4, ; E5: 5, ; F5: 6, ; G5: 7, ; H5: 8, .
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Table 2
List of EGFR mutations
EGFR exon number
Mutations
Exon 20
783 784 785 800 818 819 820 837 851 873 874
Exon 21
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ACC-ATC TCC-TTC ACC-ATC GAC-GGC TGT-CGT GTG-ATG CAG-CGG GAC-AAC GTC-ATC GGA-GAA GGC-GAC
Amino acid change
Case number ICC/ECC/GB (57/45/35)
Thr-Ile Ser-Phe Thr-Ile Asp-Gly Cys-Arg Val-Met Glu-Arg Asp-Asn Val-Ile Gly-Glu Gly-Asp
0/1/0 1/0/0 0/0/1 0/1/0 0/1/0 0/1/0 0/1/0 1/1/1 0/1/0 0/1/0 0/1/0
EGFR, Epidermal growth factor receptor; ICC/ECC/GB, intrahepatic cholangiocarcinomas/extrahepatic cholangiocarcinomas/gallbladder carcinomas.
had both EGFR and KRAS mutations. Only one extrahepatic cholangiocarcinoma patient was found to have BRAF V600E mutation in our study. EGFR mutation and survival. We conducted survival analysis. In univariate analysis, advanced tumor grade, advanced stage, T, N and M status in TNM stage, and EGFR mutation are all prognostic factors of poor survival (Table 3). In multivariate analysis, EGFR is the strongest independent poor prognostic factor in overall survival (HR 5.655, 95% CI 2.72–11.74, P < 0.0001). Besides, tumor grade and stage are both prognostic factors in overall survival (Table 4 and Fig. 2).
Discussion Novel approaches for the treatment of BTCs require an individualized assessment of early response to identify those individuals who might benefit most from the therapies. Identification of novel, cancer-specific biomarkers to assess the patient’s prognosis and predict response to treatment is a key area of current research in translational oncology. A better understanding of the molecular pathology of BTCs will facilitate the delineation of molecular phenotypes and, as a consequence, define subgroups of patients that respond to specific therapies. In our previous studies, we have found that CDX2 expression is related to clinical outcomes of patients with BTCs and simultaneous overexpression of Aurora-A and Aurora-B is associated with worse survival in patients with BTCs.34,35 In this study, we demonstrated that EGFR mutation was an independent prognostic marker in patients with BTCs in addition to tumor stage and tumor differentiation. Overexpression of EGFR by immunohistochemistry method was reported in 8.1–47% of cholangiocarcinoma in Taiwan and in Japan.18,21 Somatic mutations of EGFR in bile duct and gallbladder carcinoma were reported to be 15% (6/40) in previous study by Leone et al. with a small sample size.19 They found there is no mutation in exon 18 and most of the mutations were found in exon 21, which was similar to our finding. In our series, the frequency of somatic mutations of EGFR gene in BTCs is similar to the previous findings. However, Andersen et al. and Borger et al. ana1122
Table 3 Univariate analysis of survival (months) in patients with biliary tract carcinoma Parameters
Mean
Gender Male Female Tumor location Intrahepatic Extrahepatic Gallbladder Tumor differentiation Well Moderately Poorly Stage I–II II–IV Stage T 1 2 3 4 Stage N 0 1 2 Stage M 0 1 EGFR mutation No mutation Mutation KRAS mutation No mutation Mutation
Median
95% CI
P value
34 34
14 15
23–44 24–44
0.6391 —
31 43 28
14 18 10
21–41 29–57 14–41
0.1129 — —
42 35 12
16 15 8
29–56 25–45 6–18
0.0157 — —
55 22
32 10
40–70 15–29
< 0.00001 —
43 551 29 8
23 15 13 8
30–55 29–73 19–39 6–11
48 12 6
21 8 4
37–59 7–16 3–8
< 0.00001 — — — < 0.00001 — — —
36 5
16 3
28–45 2–8
< 0.00001 —
37 6
16 6
29–46 4–7
< 0.00001 —
36 28
15 11
27–45 11–43
0.4278 —
P value by log–rank test.
lyzed 69 and 89 BTCs, respectively, and they did not find any EGFR mutations except EGFR overexpression by immunohistochemical analysis in Andersen’s series.36,37 The discrepant results might be related to different detecting methods, patient selection with different ethnic and risk factors, and genetic heterogeneity among BTCs. The distribution of somatic mutations of EGFR in our series were found mainly in exons 20 and 21 with different codon mutations as Leone et al. reported. We found that these mutations are related to the poor prognosis of BTCs. However, the relation of these mutations to resistance of drug treatment needs further study. In addition, we found that EGFR mutations were more common in extrahepatic cholangiocarcinoma than in intrahepatic cholangiocarcinoma or in gallbladder carcinoma. BTC represents a heterogeneous groups of malignancies according to the tumor location, different clinical presentation, and gross appearance. The result implies that extrahepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma might be dissimilar tumors. RAS/RAF/MEK/ERK is one of the major pathways activated by EGFR. KRAS belongs to the RAS family that encode guanosine5′-triphosphate-binding proteins as an effector of ligand-bound EGFR. BRAF encodes a serine-threonine protein kinase that is a
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Table 4 Multivariate-adjusted hazard ration of death in patients with biliary tract carcinoma Parameters
Age ∼< 65 years ≥ 65 years Gender Male Female Tumor location Intrahepatic Extrahepatic Gallbladder Tumor differentiation Well Moderately Poorly Tumor stage I-II III-IV EGFR mutation No mutation Any mutation
Adjusted hazard ratio of death (95%CI)
P
1 0.996 (0.98–1.01)
— 0.667
1 0.944 (0.63–1.42)
— 0.784
1 0.529 (0.31–0.89)* 1.084 (0.63–1.86)
— 0.017 0.722
1 1.130 (0.71–1.81) 2.159 (1.13–4.12)*
— 0.610 0.019
1 2.751 (1.67–4.53)**
— 0.000
1 5.655 (2.72–11.74)**
— 0.000
P for these parameters: *< 0.05, **< 0.005.
Figure 2 Kaplan–Meier survival curves for EGFR mutation in biliary tract carcinoma. —, with EGFR mutation; - - -, no EGFR mutation.
downstream effector of activated KRAS. The identification of KRAS mutational status as a predictive marker of response to antibodies against the EGFR has been one of the most significant and practice-changing advances in cancer research. Somatic KRAS mutations have been associated with resistance to EGFRtargeted agents in lung cancer and metastatic colon cancer.38 Previous studies have reported 13–50% prevalence of KRAS mutation in BTCs.39–42 Borger et al. only found 9.2% (8/87) BTCs having KRAS mutations using the SNaPshot system.37 In our study, the frequency of KRAS mutation in BTCs is similar to the reports of previous studies. As most of previous studies, we failed to observe a correlation between KRAS mutations and prognosis of patients with BTCs.39 However, Andersen et al. analyzed 69 BTCs, including 15 extraheptic (hilar) and 54 intrahepatic cholangiocarcinoma, and found that 24.6% of BTCs have KRAS mutation, which was not an independent prognostic factor. However, they integrated KRAS mutation with 238-gene classifier to group patients and found that patients with KRAS mutation were in the poor prognosis group.36 These results imply that a single mutated gene might not be sufficient to be predictive or prognostic markers in BTCs. BRAF mutations have been reported in about 15% of all human cancer with a mutational hot spot at V600E, accounting for more than 90% of mutations in human cancer.43 Mutated BRAF in colorectal cancer can affect the response to anti-EGFR treatment in patients with wild-type KRAS.44,45 However, BRAF mutation is rare in BTCs in our study, which is in accordance with the previous studies.36,37,46,47 The rarity of BRAF mutation in BTCs implied that BRAF mutation is not a key event in carcinogenesis and not a predictive biomarker for prognosis and treatment in BTCs. Ongoing clinical trials and correlative analyses are essential to definitively identify predictive markers and develop therapeutic strategies for patients who may not derive benefit from anti-EGFR therapy. Direct sequencing is the widely used method for EGFR and KRAS mutations detection. However, direct sequencing has shortcomings of high cost, limited sensitivity, and time-consuming. Rapid, sensitive, and reliable methods for detecting mutations are needed for stratification of patients to receive molecular-targeted agents. Numerous multiplex technologies have recently been used to perform high-throughput genotyping on BTC, including transcriptomics array,36 mass spectrometry,42 and SNaPshot Multiplex systems.37 High-resolution melting analysis is a screening technique that has been shown to be able to detect missense mutations as well as deletions and insertions in tumor DNA isolated from paraffin-embedded tissue sections.48 HRMA has been applied to detect germline and somatic mutations, including EGFR and KRAS mutations, with high sensitivity.49 In our previous study, we have applied the HRMA methodology to detect lipoprotein lipase and apolipoprotein CII gene mutations in acute pancreatitis patients with hypertriglyceridemia.50 In this study, we have demonstrated the potential of using HRMA as the rapid screening platform for EGFR mutations in formalin-fixed and paraffin-embedded samples and confirmed the results by direct sequencing. We have found that around 10% of BTCs harbor EGFR mutations and patients with EGFR mutations had worse prognosis. Our findings might explain why clinically only some patients with BTCs had partial response to anti-EGFR therapy. As in colorectal cancer and lung cancer, it is recommended to perform EGFR and KRS mutation analyses to identify patients in BTCs that may respond to anti-EGFR therapy.
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In summary, EGFR and KRAS mutations are not uncommon in patients with BTCs. BRAF mutation is rare in BTCs. EGFR mutation status was an independent prognostic marker in patients with BTCs in addition to tumor stage and tumor differentiation. No simultaneous EGFR and KRAS mutations in extrahepatic cholangiocarcinoma and gallbladder carcinoma were found in our study. Our study provides the possibility of clinical application of HRMA for screening EGFR and KRAS gene mutations before treating BTCs with anti-EGFR therapy.
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for tyrosine kinase inhibitors. Gastroenterology 2012; 142: 1021–31 e15. Borger DR, Tanabe KK, Fan KC et al. Frequent mutation of isocitrate dehydrogenase (IDH)1 and IDH2 in cholangiocarcinoma identified through broad-based tumor genotyping. Oncologist 2012; 17: 72–9. Linardou H, Dahabreh IJ, Kanaloupiti D et al. Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet Oncol. 2008; 9: 962–72. Tannapfel A, Sommerer F, Benicke M et al. Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma. Gut 2003; 52: 706–12. Roa JC, Anabalon L, Tapia O, Melo A, de Aretxabala X, Roa I. [Frequency of K-ras mutation in biliary and pancreatic tumors]. Rev. Med. Chil. 2005; 133: 1434–40. Mutacion del gen K-ras en tumores pancreatico-biliares. Rashid A, Ueki T, Gao YT et al. K-ras mutation, p53 overexpression, and microsatellite instability in biliary tract cancers: a population-based study in China. Clin. Cancer Res. 2002; 8: 3156–63. Voss JS, Holtegaard LM, Kerr SE et al. Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions. Human Pathol. 2013; 44: 1216–22.
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43 Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell 2004; 6: 313–19. 44 Di Nicolantonio F, Martini M, Molinari F et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J. Clin. Oncol. 2008; 26: 5705–12. 45 Loupakis F, Ruzzo A, Cremolini C et al. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br. J. Cancer 2009; 101: 715–21. 46 Goldenberg D, Rosenbaum E, Argani P et al. The V599E BRAF mutation is uncommon in biliary tract cancers. Mod. Pathol. 2004; 17: 1386–91. 47 Xu RF, Sun JP, Zhang SR et al. KRAS and PIK3CA but not BRAF genes are frequently mutated in Chinese cholangiocarcinoma patients. Biomed. Pharmacother. 2011; 65: 22–6. 48 Borras E, Jurado I, Hernan I et al. Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing. BMC Cancer 2011; 11: 406. 49 Do H, Krypuy M, Mitchell PL, Fox SB, Dobrovic A. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies. BMC Cancer 2008; 8: 142. 50 Chang YT, Chang MC, Su TC et al. Lipoprotein lipase mutation S447X associated with pancreatic calcification and steatorrhea in hyperlipidemic pancreatitis. J. Clin. Gastroenterol. 2009; 43: 591–6.
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Clinicopathological and prognostic significances of EGFR, KRAS and BRAF mutations in biliary tract carcinomas in Taiwan Yu-Ting Chang,* Ming-Chu Chang,* Kai-Wen Huang,† Chien-Chih Tung,*,‡ Chiun Hsu§ and Jau-Min Wong* Departments of *Internal Medicine, †Surgery and Hepatitis Research Center and §Oncology, National Taiwan University Hospital, College of Medicine, National Taiwan University, and ‡Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital, Taipei, Taiwan
Key words biliary tract carcinomas, BRAF, EGFR, KRAS, survival. Accepted for publication 5 December 2013. Correspondence Dr Yu-Ting Chang, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, No.7 Chung Shan South Road, Taipei, Taiwan. Email: [email protected] Potential conflicts of interest: The authors disclose no conflicts.
Abstract Background and Aim: Biliary tract carcinomas (BTCs) are difficult to diagnose and treat. Epidermal growth factor receptor (EGFR) represents a therapeutic target for the BTCs. Mutations of the EGFR gene and the activation of its downstream pathways, including KRAS and BRAF, predict the sensitivity to anti-EGFR treatment. The aims of this study were to analyze the EGFR, KRAS and BRAF mutations in BTCs and their association with clinical outcomes. Methods: Paraffin-embedded specimens containing 137 BTCs resected at the National Taiwan University Hospital between 1995 and 2004 were analyzed. The exons 18–21 of EGFR gene, the codon 12, 13 and 61 of KRAS gene, and BRAF V600E mutation were analyzed. We examined the correlation between these mutations and the overall survival, tumor location, stage, and differentiation in BTCs. Results: Thirteen (9.5%) BTC patients had EGFR mutations while 23 (16.8%) patients had KRAS mutations. Only one patient had BRAF mutation. Factors influencing survival on univariate analysis were tumor stage, tumor differentiation, and EGFR mutation. On multivariate analysis, EGFR mutation and tumor stage were independent prognostic factors. A correlation between KRAS or BRAF mutations and prognosis was not observed. Conclusions: EGFR and KRAS mutations are not uncommon in BTCs. BRAF mutation is rare in BTCs. EGFR mutation was an independent prognostic marker in BTCs in addition to tumor stage and differentiation. No simultaneous EGFR and KRAS mutations in extrahepatic cholangiocarcinoma and gallbladder carcinoma were found. EGFR and KRAS mutations should be evaluated when tailoring molecular-targeted therapy to patients with BTCs.
Introduction The biliary tract consists of an interconnected system of intra- and extrahepatic ducts that transport bile secreted from the liver to the digestive tract. Biliary tract carcinomas (BTCs), which include cancers of the gallbladders and intra- and extrahepatic biliary trees—are relatively infrequent but highly lethal diseases that are notoriously difficult to diagnose and treat, and have incidences that are increasing worldwide.1,2 In general, the prognosis for patients with advanced BTCs at any site is dismal, with overall 5-year survival rate around 16–44%.3 Surgical resection is the only chance for cure. Local invasion, extensive regional lymph node metastasis, distant metastases, and vascular invasion often preclude resection, and neither radiation nor conventional chemotherapy significantly improves survival or quality of life for
patients with inoperable cancers. Therefore, novel effective therapeutic strategies are urgently required to improve the prognosis. Understanding the pathogenesis and clinicopathological characteristics of BTCs might help to improve the overall treatment strategy for BTCs. Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase of the ErbB receptor family, plays key roles in the diverse processes that stimulate cell proliferation, differentiation, and survival.4 Enhanced signaling from the receptor due to mutation or overexpression contributes to several types of human cancer, including lung, breast, colorectal, pancreatic, head and neck, and bile duct cancer.5–7 Elevated expression of EGFR and its ligand has been reported to correlate with worse prognosis in a variety of cancers, including head and neck, breast, ovarian, colorectal, pancreatic cancers, and cholangiocarcinoma.8–14 Accordingly, EGFR
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represents a validated therapeutic target for the treatment of human cancer. Anti-EGFR antibodies, such as cetuximab, as well as ATP-competitive tyrosine kinase inhibitors, such as erlotinib or gefitinib, have improved the efficacies of conventional chemotherapy and have been approved for the treatment of colorectal cancer and non-small cell lung cancer.15,16 Previous studies have revealed overexpression of EGFR and mutation of this gene in cholangiocarcinoma.7,17–20 Overexpression of EGFR by immunohistochemistry method and somatic mutations of EGFR gene were reported in 47% of cholangiocarcinomas in Taiwan21 and in 8–32% of cholangiocarcinomas in Western countries and Japan.18,19,22 In addition, inhibition of EGFR signaling by gefitinib effectively attenuates the proliferation of cholangiocarcinoma cells in vitro.23 As EGFR kinase inhibitors effectively attenuated cellular growth, these agents may be therapeutically efficacious in human BTCs. A phase II study of erlotinib in patients with advanced biliary cancer showed therapeutic benefit for EGFR blockade with erlotinib in patients with biliary cancer.24 These studies suggest the possibility that like non-small cell lung cancer, the BTCs harbors the EGFR mutations which might be responsible for the clinical response of anti-EGFR therapy. Mutations in the EGFR gene have been investigated in the non-small cell lung cancer because of a reported correlation between mutations and tumor response to treatment with targeted tyrosine kinase inhibitors such as gefitinib and erlotinib.25,26 These mutations have shown some correlation with demographic and histopathologic categories, such as female, non-smoker, and Asian descent.27,28 These somatic mutations can explain the heterogeneous response to gefitinib in lung cancer patients and provide a basis for selecting patients with a high probability of outstanding response to anti-EGFR therapy. The detection of EGFR mutation is considered valuable both for guidance in therapy and for clinical investigations. However, there was no such study like non-small cell lung cancer in BTCs in Taiwan. In addition, few previous studies have clarified associations between the somatic mutations of EGFR gene and expression of this molecule and the clinicopathological factors or prognosis in patients with BTCs. There are two major pathways activated by EGFR: one is RAS/ RAF/MEK/ERK pathway, and the other is PI3K/PTEN/AKT pathway.29–32 EGFR, KRAS and BRAF genotyping are the three most commonly tested genes in solid tumors as they have been linked to resistance or sensitivity to EGFR pathway blockade. The introduction of KRAS testing as a predictive marker to select patients for EGFR-targeted therapies for metastatic colorectal cancer is widely regarded as a key advance in the field of personalized medicine. Current data suggested that together with KRAS mutation, the evaluation of BRAF alterations could also be useful for selecting patients with reduced chance to benefit from EGFRtargeted therapy. Up until now, each of these markers has been mainly assessed as a single event in Taiwan populations. The aims of this study were to analyze the EGFR, KRAS, and BRAF mutations simultaneously in BTCs as well as associations between these mutations and clinicopathological factors or clinical outcome.
cinomas, 45 extrahepatic cholangiocarcinomas, and 35 gallbladder carcinomas resected at the National Taiwan University Hospital between 1995 and 2004 were used for this study. The clinicopathological features of the patients, including TNM staging, tumor location, degree of tumor differentiation, and overall survival, were recorded. The study was approved by the Institutional Review Board at National Taiwan University Hospital. Isolation of genomic DNA and mutational analysis of EGFR, KRAS, and BRAF genes. Tissues from five serial 10-μM sections of retrieved paraffin-embedded formalinfixed slides containing 60% or more tumors were macrodissected from the slides. Genomic DNAs were extracted from deparaffinized samples with the use of the QIAamp DNA Mini kit (Qiagen, Gaithersburg, MD, USA) following the manufacturer’s instructions. The exons of EGFR, KRAS, and BRAF genes were amplified. The primers for amplification were designed using Primer Express Software 3.0 (Applied Biosystems, Foster City, CA, USA). The kinase domain of EGFR coding sequence, from exons 18–21, were tested by high resolution melting analysis (HRMA) first then confirmed by direct sequencing as methods modified from previous report.33 High-resolution melting analysis. The polymerase chain reaction (PCR) was carried out in 96-wells plate with a reaction volume of 10 μL containing genomic DNA (50 ng), 2x LightCycler 480 High Resolution Melting Master Mix (Roche Applied Science, Mannheim, Germany), 2.5 mM MgCl2, and 20 pmol primers. Thermal cycle conditions were 95°C for 5 min, and 45 cycles of 95°C for 10 s and 60°C for 10 s, and the final extension was 72°C for 7 min. Completed PCR plates were analyzed using the LightCycler 480 nd the LightCycler 480 Software 1.5.0 (Roche Applied Science). Direct sequencing of EGFR, KRAS, and BRAF genes. For direct sequencing, the multiplex reaction contained 1x optimized SapphireAmp Fast PCR Master Mix buffer (contain hot start Taq polymerase, dNTP), 20 pmol each primer pair, 100 ng genomic DNA, and nuclease-free water for a final volume of 25 μL. Reactions were performed in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). Thermal cycling conditions were as follows: the initial heat denaturing step was 5 min treatment at 95°C, the second step was 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 0 s (for EGFR exon 19); 45 cycles at 95°C for 30 s; 51°C for 60 s; 72°C for 60 s (for EGFR exons 20 and 21; KRAS exons 1 and 2); 45 cycles at 95°C for 30 s; 54°C for 60 s; 72°C for 60 s (for BRAF exon 15) and the final extension was 72 °Cfor 7 min. The PCR products were purified with the PCR Clean-Up Kit (Geneaid, Taipei, Taiwan) and sequenced using the BigDye Terminator Cycle sequence following the PE Applied Biosystems strategy and Applied Biosystems ABI PRISM3100 DNA Sequencer (Applied Biosystems). All mutations will be confirmed, performing two independent PCR amplifications.
Methods Patient specimens. Paraffin-embedded surgical specimens containing 137 BTCs, including 57 intrahepatic cholangiocar1120
Statistical analysis. The associations of EGFR, KRAS, and BRAF gene mutations among clinicopathological factors were studied by χ2 analysis of proportions, Fisher’s exact test, and Journal of Gastroenterology and Hepatology 29 (2014) 1119–1125
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Student’s t-test when appropriate. Differences in frequencies between subgroups were analyzed using the Kruskal–Wallis test and the Mann–Whitney U-test. Patient survival was analyzed using Kaplan–Meier method, and log–rank tests were performed for univariate survival analysis and the Cox proportional hazards model for multivariate survival analysis. P < 0.05 was accepted as statistically significant. The variables assessed include patient age at diagnosis, sex, tumor location, tumor stage, degree of tumor differentiation, and survival. The statistical calculations were carried out using SPSS statistical software version 11 (SPSS Inc., Chicago, IL, USA).
Results EGFR, KRAS, and BRAF gene mutations in BTCs. EGFR exons 18–21 were analyzed by HRMA using genomic DNA extracted from tissues in the 137 patients. EGFR exons 18–21 were further sequenced. The clinicopathological features and EGFR and KRAS mutations in patients with BTCs are listed in Table 1. In total, 13 patients (9.5%) were identified with EGFR mutations, including seven cases in exon 20: one
Table 1
intrahepatic cholangiocarcinoma in codon 784 (TCC-TTC; amino acid change: Ser-Phe), five extrahepatic cholangiocarcinoma in codon 783 (ACC-ATC; Thr-Ile), codon 800 (GAC-GGC; AspGly), codon 818 (TGT-CGT; Cys-Arg), codon 819 (GTG-ATG; Val-Met), and codon 820 (CAG-CGG; Glu-Arg), and one gallbladder carcinoma in codon 785 (ACC-ATC; Thr-Ile); and six cases in exon 21: one intrahepatic cholangiocarcinoma in codon 837 (GAC-AAC; Asp-Asn), four extrahepatic cholangiocarcinoma in codon 837, codon 851 (GTC-ATC; Val-Ile), codon 873 (GGAGAA; Gly-Glu), and codon 874 (GGC-GAC; Gly-Asp) (Fig. 1), and one gallbladder carcinoma in codon 837 (Table 2). We did not found any samples harboring double mutations in exons 18–21. EGFR mutations were more common in extrahepatic cholangiocarcinoma (9/45, 20%) than in intrahepatic cholangiocarcinoma (2/57, 3.5%), or in gallbladder carcinoma (2/35, 5.7%) (P = 0.01). Twenty-three patients (23/137, 16.8%), including 10 intrahepatic cholangiocarcinoma (10/57, 17.5%), nine extrahepatic cholangiocarcinoma (9/45, 20%), and four gallbladder carcinoma(4/35, 11.4%), had KRAS mutations. Most of the KRAS mutations were located in codon 12 and only one in codon 13. The frequency of KRAS mutation did not differ statistically significantly among the three groups. Two patients with intrahepatic cholangiocarcinoma
Clinicopathological features and EGFR/KRAS mutations in patients with biliary tract carcinoma
Tumor location
Intrahepatic
Extrahepatic
Gallbladder
Total
Case number Gender (male/female) Mean age(range) Tumor grade Well differentiated Moderately differentiated Poorly differentiated Tumor stage I–II III–IV EGFR mutation KRAS mutation
57 32/25 63.6 (39–88)
45 17/28 62.9 (39–82)
35 22/13 67.3 (41–89)
137 71/66 64.3 (39–89)
14 31 12
17 24 4
11 19 5
42 74 21
31 26 2 10
15 30 9 9
7 28 2 4
53 84 13 23
Figure 1 High resolution melting analysis and direct sequence of EGFR gene in in biliary ; B5: 2, ; C5: 3, tract carcinoma. A5: 1, ; D5: 4, ; E5: 5, ; F5: 6, ; G5: 7, ; H5: 8, .
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Table 2
List of EGFR mutations
EGFR exon number
Mutations
Exon 20
783 784 785 800 818 819 820 837 851 873 874
Exon 21
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ACC-ATC TCC-TTC ACC-ATC GAC-GGC TGT-CGT GTG-ATG CAG-CGG GAC-AAC GTC-ATC GGA-GAA GGC-GAC
Amino acid change
Case number ICC/ECC/GB (57/45/35)
Thr-Ile Ser-Phe Thr-Ile Asp-Gly Cys-Arg Val-Met Glu-Arg Asp-Asn Val-Ile Gly-Glu Gly-Asp
0/1/0 1/0/0 0/0/1 0/1/0 0/1/0 0/1/0 0/1/0 1/1/1 0/1/0 0/1/0 0/1/0
EGFR, Epidermal growth factor receptor; ICC/ECC/GB, intrahepatic cholangiocarcinomas/extrahepatic cholangiocarcinomas/gallbladder carcinomas.
had both EGFR and KRAS mutations. Only one extrahepatic cholangiocarcinoma patient was found to have BRAF V600E mutation in our study. EGFR mutation and survival. We conducted survival analysis. In univariate analysis, advanced tumor grade, advanced stage, T, N and M status in TNM stage, and EGFR mutation are all prognostic factors of poor survival (Table 3). In multivariate analysis, EGFR is the strongest independent poor prognostic factor in overall survival (HR 5.655, 95% CI 2.72–11.74, P < 0.0001). Besides, tumor grade and stage are both prognostic factors in overall survival (Table 4 and Fig. 2).
Discussion Novel approaches for the treatment of BTCs require an individualized assessment of early response to identify those individuals who might benefit most from the therapies. Identification of novel, cancer-specific biomarkers to assess the patient’s prognosis and predict response to treatment is a key area of current research in translational oncology. A better understanding of the molecular pathology of BTCs will facilitate the delineation of molecular phenotypes and, as a consequence, define subgroups of patients that respond to specific therapies. In our previous studies, we have found that CDX2 expression is related to clinical outcomes of patients with BTCs and simultaneous overexpression of Aurora-A and Aurora-B is associated with worse survival in patients with BTCs.34,35 In this study, we demonstrated that EGFR mutation was an independent prognostic marker in patients with BTCs in addition to tumor stage and tumor differentiation. Overexpression of EGFR by immunohistochemistry method was reported in 8.1–47% of cholangiocarcinoma in Taiwan and in Japan.18,21 Somatic mutations of EGFR in bile duct and gallbladder carcinoma were reported to be 15% (6/40) in previous study by Leone et al. with a small sample size.19 They found there is no mutation in exon 18 and most of the mutations were found in exon 21, which was similar to our finding. In our series, the frequency of somatic mutations of EGFR gene in BTCs is similar to the previous findings. However, Andersen et al. and Borger et al. ana1122
Table 3 Univariate analysis of survival (months) in patients with biliary tract carcinoma Parameters
Mean
Gender Male Female Tumor location Intrahepatic Extrahepatic Gallbladder Tumor differentiation Well Moderately Poorly Stage I–II II–IV Stage T 1 2 3 4 Stage N 0 1 2 Stage M 0 1 EGFR mutation No mutation Mutation KRAS mutation No mutation Mutation
Median
95% CI
P value
34 34
14 15
23–44 24–44
0.6391 —
31 43 28
14 18 10
21–41 29–57 14–41
0.1129 — —
42 35 12
16 15 8
29–56 25–45 6–18
0.0157 — —
55 22
32 10
40–70 15–29
< 0.00001 —
43 551 29 8
23 15 13 8
30–55 29–73 19–39 6–11
48 12 6
21 8 4
37–59 7–16 3–8
< 0.00001 — — — < 0.00001 — — —
36 5
16 3
28–45 2–8
< 0.00001 —
37 6
16 6
29–46 4–7
< 0.00001 —
36 28
15 11
27–45 11–43
0.4278 —
P value by log–rank test.
lyzed 69 and 89 BTCs, respectively, and they did not find any EGFR mutations except EGFR overexpression by immunohistochemical analysis in Andersen’s series.36,37 The discrepant results might be related to different detecting methods, patient selection with different ethnic and risk factors, and genetic heterogeneity among BTCs. The distribution of somatic mutations of EGFR in our series were found mainly in exons 20 and 21 with different codon mutations as Leone et al. reported. We found that these mutations are related to the poor prognosis of BTCs. However, the relation of these mutations to resistance of drug treatment needs further study. In addition, we found that EGFR mutations were more common in extrahepatic cholangiocarcinoma than in intrahepatic cholangiocarcinoma or in gallbladder carcinoma. BTC represents a heterogeneous groups of malignancies according to the tumor location, different clinical presentation, and gross appearance. The result implies that extrahepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma might be dissimilar tumors. RAS/RAF/MEK/ERK is one of the major pathways activated by EGFR. KRAS belongs to the RAS family that encode guanosine5′-triphosphate-binding proteins as an effector of ligand-bound EGFR. BRAF encodes a serine-threonine protein kinase that is a
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Table 4 Multivariate-adjusted hazard ration of death in patients with biliary tract carcinoma Parameters
Age ∼< 65 years ≥ 65 years Gender Male Female Tumor location Intrahepatic Extrahepatic Gallbladder Tumor differentiation Well Moderately Poorly Tumor stage I-II III-IV EGFR mutation No mutation Any mutation
Adjusted hazard ratio of death (95%CI)
P
1 0.996 (0.98–1.01)
— 0.667
1 0.944 (0.63–1.42)
— 0.784
1 0.529 (0.31–0.89)* 1.084 (0.63–1.86)
— 0.017 0.722
1 1.130 (0.71–1.81) 2.159 (1.13–4.12)*
— 0.610 0.019
1 2.751 (1.67–4.53)**
— 0.000
1 5.655 (2.72–11.74)**
— 0.000
P for these parameters: *< 0.05, **< 0.005.
Figure 2 Kaplan–Meier survival curves for EGFR mutation in biliary tract carcinoma. —, with EGFR mutation; - - -, no EGFR mutation.
downstream effector of activated KRAS. The identification of KRAS mutational status as a predictive marker of response to antibodies against the EGFR has been one of the most significant and practice-changing advances in cancer research. Somatic KRAS mutations have been associated with resistance to EGFRtargeted agents in lung cancer and metastatic colon cancer.38 Previous studies have reported 13–50% prevalence of KRAS mutation in BTCs.39–42 Borger et al. only found 9.2% (8/87) BTCs having KRAS mutations using the SNaPshot system.37 In our study, the frequency of KRAS mutation in BTCs is similar to the reports of previous studies. As most of previous studies, we failed to observe a correlation between KRAS mutations and prognosis of patients with BTCs.39 However, Andersen et al. analyzed 69 BTCs, including 15 extraheptic (hilar) and 54 intrahepatic cholangiocarcinoma, and found that 24.6% of BTCs have KRAS mutation, which was not an independent prognostic factor. However, they integrated KRAS mutation with 238-gene classifier to group patients and found that patients with KRAS mutation were in the poor prognosis group.36 These results imply that a single mutated gene might not be sufficient to be predictive or prognostic markers in BTCs. BRAF mutations have been reported in about 15% of all human cancer with a mutational hot spot at V600E, accounting for more than 90% of mutations in human cancer.43 Mutated BRAF in colorectal cancer can affect the response to anti-EGFR treatment in patients with wild-type KRAS.44,45 However, BRAF mutation is rare in BTCs in our study, which is in accordance with the previous studies.36,37,46,47 The rarity of BRAF mutation in BTCs implied that BRAF mutation is not a key event in carcinogenesis and not a predictive biomarker for prognosis and treatment in BTCs. Ongoing clinical trials and correlative analyses are essential to definitively identify predictive markers and develop therapeutic strategies for patients who may not derive benefit from anti-EGFR therapy. Direct sequencing is the widely used method for EGFR and KRAS mutations detection. However, direct sequencing has shortcomings of high cost, limited sensitivity, and time-consuming. Rapid, sensitive, and reliable methods for detecting mutations are needed for stratification of patients to receive molecular-targeted agents. Numerous multiplex technologies have recently been used to perform high-throughput genotyping on BTC, including transcriptomics array,36 mass spectrometry,42 and SNaPshot Multiplex systems.37 High-resolution melting analysis is a screening technique that has been shown to be able to detect missense mutations as well as deletions and insertions in tumor DNA isolated from paraffin-embedded tissue sections.48 HRMA has been applied to detect germline and somatic mutations, including EGFR and KRAS mutations, with high sensitivity.49 In our previous study, we have applied the HRMA methodology to detect lipoprotein lipase and apolipoprotein CII gene mutations in acute pancreatitis patients with hypertriglyceridemia.50 In this study, we have demonstrated the potential of using HRMA as the rapid screening platform for EGFR mutations in formalin-fixed and paraffin-embedded samples and confirmed the results by direct sequencing. We have found that around 10% of BTCs harbor EGFR mutations and patients with EGFR mutations had worse prognosis. Our findings might explain why clinically only some patients with BTCs had partial response to anti-EGFR therapy. As in colorectal cancer and lung cancer, it is recommended to perform EGFR and KRS mutation analyses to identify patients in BTCs that may respond to anti-EGFR therapy.
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In summary, EGFR and KRAS mutations are not uncommon in patients with BTCs. BRAF mutation is rare in BTCs. EGFR mutation status was an independent prognostic marker in patients with BTCs in addition to tumor stage and tumor differentiation. No simultaneous EGFR and KRAS mutations in extrahepatic cholangiocarcinoma and gallbladder carcinoma were found in our study. Our study provides the possibility of clinical application of HRMA for screening EGFR and KRAS gene mutations before treating BTCs with anti-EGFR therapy.
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