Abstract
Purpose
Biliary tract cancers (BTC) are uncommon in the United States, but are endemic in parts of South America and Asia. BTCs are aggressive tumors associated with poor survival. Activation of HER-2/neu (erbB2) and/or epidermal growth factor receptor (EGFR) are important in breast, colon, and lung cancers. Tumor specimens from patients from the United States and Chile were examined for expression of HER-2/neu, EGFR, and their activated forms (p-erbB2, p-EGFR).
Materials and Methods
Specimens from 77 gallbladder cancers (GBC), 16 extrahepatic bile duct cancers (EHBDC), 21 intrahepatic bile duct cancers (IHBDC), 11 cases of cholecystitis (CHOLE), and 8 normal gallbladders (NGB) were examined for HER-2/neu, p-erbB2, EGFR, and p-EGFR expression by immunohistochemistry (IHC), with scores of 2+ or 3+ defined as positive. HER-2/neu gene amplification was analyzed by double color HER-2/neu gene/chromosome 17 centromere (CEP17) fluorescence in situ hybridization (FISH) assays
Results
HER-2/neu-positive IHC staining was found in 31.2% of GBC, 31.3%, of EHBDC, and 33.3% of IHBDC; 12.5% of CHOLE specimens showed 2+ staining and the remaining CHOLE and NGB were negative. HER-2/neu gene amplification was detected in 20.9% of GBC, 21.4% of EHBDC, and none of IHBDC. There was a significant correlation between IHC 2+ and 3+ and gene amplification (P =.0001).
Conclusions
HER-2/neu amplification was identified in more than 20% of GB and EHBDC. There was strong correlation between HER-2/neu IHC and FISH positivity. These findings indicate a role for HER-2/neu in some subsets of BTC, and provide a rationale for study of HER-2/neu-directed therapies in this setting.
Cancers of the gallbladder and bile ducts (biliary tract cancer) are invasive adenocarcinomas that arise from the epithelial lining of the gallbladder or the intrahepatic (peripheral) and extrahepatic (hilar and distal common bile duct) bile ducts. There were an estimated 10,000 new cases of these cancers in the United States in 2007.1,2 Between 1975 and 1997, both the incidence and mortality from intrahepatic cholangiocarcinoma markedly increased in the United States, with estimated annual percentage changes of 9.1%, and 9.4%, respectively.1,3 High incidence rates are observed in certain subpopulations, including Native Americans and Hispanic women. The highest incidence rates in the world are reported from India, Pakistan, Korea, Japan, Eastern Europe, and some South American countries. In Chile, gallbladder cancer is the most common malignancy in women and the second most common cancer in men.4–6
Although gallbladder cancer, extrahepatic bile duct cancer, and intrahepatic bile duct cancer are related and have similar metastatic patterns, each has distinct clinical presentations, molecular pathology, and prognoses. This group of tumors is characterized by local invasion, extensive regional lymph node metastases, vascular encasement and distant metastases, all of which preclude resection.7 Complete surgical resection offers the only chance for cure; however, only a small percentage of patients present with early stage disease and are considered surgical candidates.8,9 Among those patients who do undergo “curative” resection, recurrence rates are high; thus the majority of patients with biliary tract cancer receive palliative chemotherapy. These tumors are similar in their overall aggressive course and resistance to chemotherapy; systemic chemotherapy has shown response rates in the 0%–25% range and no survival benefit has been demonstrated.
Although the molecular carcinogenic mechanisms of biliary cancers have been well described in animal models,10–15 little is known regarding molecular carcinogenesis in human gallbladder and biliary tract cancer. The human epidermal growth factor receptor family plays a key role in carcinogenesis and disease progression in a number of cancers. The reported immunohistochemical overexpression of HER-2/neu ranges from 10% to 69.6% and from 0% to 82% in gallbladder cancer and cholangiocarcinoma, respectively.16–22 HER-2/neu gene amplification has been reported in 6.8% of biliary tract cancers22 and 100% of cholangiocarcinomas by fluorescence in situ hybridization (FISH), and in 69.6% of gallbladder cancer by polymerase chain reaction (PCR).17
Varying rates of epidermal growth factor receptor (EGFR, HER-1) expression have been reported, ranging from 12.4%– 100%in gallbladder cancer and 10.7%–81% in cholangiocarcinoma.22–25 One study showed EGFR gene amplification positive on FISH in 5.9% of biliary tract cancers.22 Systematic studies evaluating protein expression and gene amplification of HER-2/neu or EGFR in the same set of cases are lacking. The current study was based on the knowledge that the EGFR family of oncogenes is important in many tumor types and that several agents that target the EGFR family are available clinically.
To establish a basis for testing agents that target the EGFR family in biliary tract cancer and improve therapeutic options, analysis of a large number of patient tumor specimens was conducted using immunohistochemical staining and FISH.
MATERIALS AND METHODS
Tissue Specimens
All human tissue specimens were obtained according to a University of Texas M. D. Anderson Cancer Center Institutional Review Board-approved protocol. All patients provided informed consent for the use of their archived tissue for this study. Archival formalin-fixed, paraffin-embedded biliary tract specimens from 11 normal gallbladders, 8 cases of cholecystitis, 77 cases of gallbladder cancer, 16 cases of extrahepatic bile duct cancer, and 21 cases of intrahepatic bile duct cancer were used in this study. Table 1 lists the demographic characteristics, pathologic staging, and histologic findings according to the AJCC (American Joint Committee on Cancer) criteria for patient specimens.21
Table 1.
Characteristics of patients with biliary tract cancer and noncancerous gallbladders
| Tissue type | GBC | EHBDC | IHBDC | NGB | CHOLE |
|---|---|---|---|---|---|
| Number of cases | 77 | 16 | 21 | 11 | 8 |
| Sources | US, Chile | US | US | Chile | Chile |
| Gender (male/female) | 18/59 | 9/7 | 10/11 | 6/5 | 0/8 |
| Mean age, years (range) | 63 (29–86) | 57 (27–77) | 55 (40–76) | 50 (45–56) | 51 (43–61) |
| Histologic grade | |||||
| Well differentiated | 19 | 5 | 5 | ||
| Moderately differentiated | 14 | 8 | 6 | ||
| Poorly differentiated | 42 | 2 | 10 | ||
| Unknown | 2 | 1 | 0 | ||
| Pathologic stage (AJCC, 6th edition) | |||||
| I | 17 | 6 | 3 | ||
| II | 45 | 5 | 0 | ||
| III | 7 | 1 | 6 | ||
| IV | 8 | 3 | 12 | ||
| Unknown | 0 | 1 | 0 | ||
Abbreviations: GBC = gallbladder cancer; EHBDC = extrahepatic bile duct cancer; IHBDC = intrahepatic bile duct cancer; NGB = normal gallbladder; CHOLE = cholecystitis
The biliary tract cancer specimens of US origin were obtained from the Department of Gastrointestinal Medical Oncology at M. D. Anderson Cancer Center (gallbladder cancer = 28, extrahepatic bile duct cancer = 16, intrahepatic bile duct cancer = 21); gallbladder specimens from Chile were provided by the Catholic University Medical School Hospital Santiago, and the Department of Pathology, Universidad de la Frontera, Temuco (normal gall bladder = 11, cholecystitis = 8, gallbladder cancer = 49).
Immunohistochemical Staining
Archived specimens were preserved in 10% buffered formalin within 24 hours and then embedded in paraffin. Routine hematoxylineosin staining was performed for morphologic studies. Four-μm-thick tissue sections for immunohistochemical analysis were deparaffinized and rehydrated. For immunohistochemical evaluation of HER-2 status, HercepTest (Dako A/S, Glastrup, Denmark) testing was performed following the manufacturer’s instructions. Briefly, after deparaffinization and rehydration, sections were immersed in epitope retrieval solution at 99°C for 40 minutes. Endogenous peroxidase activity was blocked by incubation with the provided peroxidase-blocking reagent for 5 minutes. Then the rabbit anti-human HER-2 protein antibody or negative control reagent for evaluating nonspecific staining was applied for 30 minutes at room temperature. After washing the specimens in wash buffer, the specimens were incubated with visualization reagent for 30 minutes and rinsed with wash buffer, followed by substrate chromogen solution for 10 minutes. After rinsing with distilled water, the slides were counterstained with hematoxylin. The breast cancer cells supplied with this kit (MDA-231, MDA-175, and SK-BR-3) were used as positive and negative control specimens.
For immunohistochemical detection of p-erbB2, EGFR and p-EGFR, tissue sections were immersed for 20 minutes in 0.3% hydrogen peroxide with pure methanol. Antigen retrieval was performed by microwave treatment in 10 mM citrate buffer (pH 6.0) at high power for 10 minutes. After washing with phosphate buffered saline (PBS, pH 7.4), sections were incubated in goat serum for 30 minutes for blocking the nonspecific staining. Antibodies used in immunohistochemical staining were as follows: rabbit anti-p-erbB2 (Tyr 1248)-R (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti-EGFR (Cell Signaling Technology, Inc., Beverly, MA), and rabbit antip- EGFR (Tyr 1173)-R (Novus Biologicals, Inc., Littleton, CO). The sections were incubated overnight at 4°C with the respective primary antibodies at the following optimized dilutions: p-erbB2 (1:100), EGFR (1:50), p-EGFR (1:50). After washing in PBS, the sections were incubated at room temperature with the biotinylated anti-rabbit secondary antibody solution for 30 minutes and washed in PBS, followed by treatment with VECTA-STAIN ABC reagent (Vector Laboratories, Inc., Burlingame, CA) for 30 minutes. After washing in PBS, DAB (3,3′-diaminobenzidine tetrahydrochloride) substrate kit (Zymed Laboratories, Inc., South San Francisco, CA) was applied and the sections were counterstained with hematoxylin. Human lung cancer tissue known to react with EGFR antibody was used as positive control. As negative control, slides were incubated with EGFR antibody, which was preabsorbed with EGFR peptide and with secondary antibody without using primary antibodies for excluding nonspecific staining by the secondary antibody.
Evaluation of Immunohistochemical Staining
Evaluation of sections was performed by two investigators independently with respect to the histopathologic characteristics and specific immunoreactivity. For the scoring of HER-2 protein expression, only the membrane staining intensity and pattern were evaluated according to the Hercep-Test scoring system. Cases in which there was no staining or membrane staining in less than 10% of cancerous or noncancerous cells were scored as 0. A faint/barely perceptible membrane staining in more than or equal to 10% of cancerous or noncancerous cells in which only part of the membrane stained was scored as 1+. A weak to moderate complete membrane staining in more than 10% of the cancerous cells was scored as 2+. A strong complete membrane staining in more than 10% of the cancerous cells was scored as 3+. The evaluation of p-erbB2, EGFR, and p-EGFR immunohistochemical analysis used the same criteria as the HER-2 scoring system; each staining was interpreted as negative (0 and 1+) and positive (2+ and 3+) for each protein overexpression.
FISH
The number of HER-2/neu gene copies was determined by the FISH method using the Vysis Path Vysion HER-2/DNA probe kit (Vysis, Downers Grove, IL). Paraffin embedded tissue sections (4 μm thick) were baked overnight at 56°C. After deparaffinization in xylene and dehydration in 100% ethanol, the slides were air dried. Then they were immersed in Vysis pretreatment solution (1N sodium thiocynate [NaSCN]) at 80°C for 14 minutes. After being washing with distilled water for 2 minutes, the slides were treated for 14 minutes at 37°C in a protease solution (pepsin), washed in distilled water for 2 minutes, and air dried. Five μL of the HER-2/DNA and chromosome 17 premixed probe was applied to the target area on each slide. The coverslip was placed over the probe and sealed with rubber cement. Denaturation of the sample DNA and probe DNA was performed on a hot plate at 73°C for 5 minutes. Incubation was performed overnight in a 37°C incubator. After the coverslips were removed, the slides were rinsed in 2X sodium chloride/sodium citrate (SCC)/0.3% NP40, pH 7.15, at 73°C for 2 minutes to remove excess probe, followed by counterstaining with 4,6-diaminidino- 2-phenyl-indole (DAPI). Positive control consisted of a paraffin section of a breast cancer case known to amplify the HER-2/neu gene by FISH.
Evaluation of HER-2/neu Gene Amplification
The Vysis Path Vysion HER-2/DNA probe kit uses two different probes. One is a locus specific identifier (LSI) HER-2/neu labeled in Spectrum Orange and the other is a chromosome enumerator probe (CEP) 17 labeled in Spectrum Green. Signal enumeration was conducted at 1000X magnification with the appropriate filter. HER- 2/neu gene amplification was defined as an LSI HER-2/CEP 17 ratio of 2.0 or greater. Several fields of at least 20 cells were counted and the results were averaged.
The HercepTest for HER-2/neu immunohistochemical staining and the PathVysion probe for detecting HER-2/neu gene amplification by FISH have been validated in many studies in breast carcinoma.25,26
Statistical Analysis
A two-sided χ2 test or Fisher’s exact test was used for comparison of immunohistochemical data between groups. All statistical analysis was carried out using SPSS software. A P value of < .05 was defined as statistically significant.
RESULTS
Immunohistochemical Analysis
Before staining the tissue specimens, we confirmed the staining in the positive controls of HER-2/neu and EGFR. We also recorded negative staining using the negative control reagent for HER-2/neu and preabsorbed EGFR antibody with EGFR peptide. Nonspecific staining was not revealed in the slides stained without primary antibodies for p-erbB2 and p-EGFR. The representative immunohistochemical staining of HER-2/neu on biliary tract specimens is shown in Figure 1A to 1D.
Figure 1.
Representative photomicrographs showing typical immunohistochemical staining by HercepTest on biliary tract cancer. (A) No staining. (B) Faint/barely staining (1+). (C) Weak to moderate staining (2+). (D) Strong staining (3+). Original magnification X66.
The results of the analysis of HER- 2/neu, p-erbB2, EGFR, and p-EGFR overexpression in carcinomas arising in different sites of the biliary tract and noncancerous control lesions are summarized in Table 2. A total of 114 biliary tract cancer patients were included in the immunohistochemical analysis, but 11 samples were excluded for EGFR and p-EGFR staining due to insufficient archival tissue. HER-2/neu overexpression was similar among biliary tract cancer types (gallbladder cancer = 31.2%, extrahepatic bile duct cancer = 31.3%, intrahepatic bile duct cancer = 33.3%). p-erbB2 overexpression was higher for gallbladder cancer (26.0%) than for extrahepatic bile duct cancer (12.5%) and intrahepatic bile duct cancer (9.5%).
Table 2.
Results of immunohistochemistry in biliary tract cancer and noncancerous gallbladder
| Overexpressioin (2+ or 3+)
|
||||
|---|---|---|---|---|
| HER-2/neu | p-erbB2 | EGFR | p-EGFR | |
| Biliary tract cancer | ||||
| GBC | 24/77 (31.2%) | 20/77 (26.0%) | 12/75 (16.0%) | 7/75 (9.3%) |
| EHBDC | 5/16 (31.3%) | 2/16 (12.5%) | 0/12 (0%) | 0/12 (0%) |
| IHBDC | 7/21 (33.3%) | 2/21 (9.5%) | 0/16 (0%) | 0/16 (0%) |
| Total | 36/114 (31.6%) | 24/114 (21.1%) | 12/103 (11.7%) | 7/103 (6.8%) |
| Noncancerous gallbladder | ||||
| NGB | 0/11 (0%) | 0/11 (0%) | 0/11 (0%) | 0/11 (0%) |
| CHOLE | 1/8 (12.5%) | 0/8 (0%) | 0/8 (0%) | 0/8 (0%) |
Abbreviations: GBC = gallbladder cancer; EHBDC = extrahepatic bile duct cancer; IHBDC = intrahepatic bile duct cancer; NGB = normal gallbladder; CHOLE = cholecystitis
In noncancerous gallbladder, 1 (12.5%) of 8 cholecystitis specimens showed HER- 2/neu overexpression. (Specific data not shown, examples appear in Figure 2.) There was a strong correlation (P = .0001) between overexpression of HER-2/neu and overexpression of p-erbB2 in biliary tract cancer (Figure 2A and 2B). Overexpression of EGFR and p-EGFR was found in 16.0% and 9.3%, respectively, of gallbladder cancers, but there was no overexpression in extrahepatic bile duct cancer, intrahepatic bile duct cancer, normal gall bladder, or cholecystitis (Table 2). There was also a strong correlation (P = .0001) of overexpression of EGFR and overexpression of p-EGFR in biliary tract cancer (Table 3, Figure 2C and 2D). We also investigated the relationship between overexpression of HER-2/neu and EGFR in biliary tract cancer, but there were only 3 cases that overexpressed both HER-2/neu and EGFR (Table 3).
Figure 2.
Concordance of immunohistochemical expression between HercepTest (A) and p-erbB2 (B), and EGFR (C) and p-EGFR (D). (A) 3+ HER-2/neu staining was seen at the cancer cells and at the serial section. (B) 2+ p-erbB2 staining was recognized. (C) EGFR showed 3+ staining at the cancer cells and at the serial section. (D) p-EGFR staining was demonstrated as 2+ intensity. Original magnification X66.
Table 3.
Results of HER-2/neu gene amplification analysis by FISH in biliary tract cancer and noncancerous gallbladder HER-2/neu gene amplification.
| Biliary tract cancer | ||
| GBC (n=67) | 14 (20.9%) | |
| EHBDC (n=14) | 3 (21.4%) | |
| IHBDC (n=14) | 0 (0%) | |
| Total (n=95) | 17 (17.9%) | |
| Noncancerous gallbladder | ||
| NGB (n=7) | 0 (0%) | |
| CHOLE (n=6) | 0 (0%) | |
|
HER-2/neu expression |
||
| − | + | |
| 0 and 1+ | 2+ and 3+ (2+, 3+) | |
| HER-2/neu gene amplification | ||
| FISH-positive (n=17) | 2 | 15 (4, 11) |
| FISH-negative (n=78) | 64 | 14 (11, 3) |
| P=.0001 for correlation | ||
Abbreviations: GBC = gallbladder cancer; EHBDC = extrahepatic bile duct cancer; IHBDC = intrahepatic bile duct cancer; NGB = normal gallbladder; CHOLE = cholecystitis
FISH
Because of inadequate slide material, HER-2/neu FISH was performed on 104 out of 114 biliary tract cancer cases and 13 out of 19 noncancerous gallbladder specimens. FISH was considered successful on 95 of the 104 biliary tract cancer (91.3%) and all noncancerous gallbladder specimens (100%). Pretreatment and hybridization results in 6 cases did not meet the evaluation criteria. The gene amplification of HER-2/neu in carcinomas in different sites and noncancerous lesions of the biliary tract is summarized in Table 3. FISH-positive results were obtained in 20.9% of gallbladder cancers and 21.4% of extrahepatic bile duct cancers and in none of the other samples. There were no correlations between gene amplification results for pathologic stage I vs. stage II to IV disease or for well/moderately differentiated vs. poorly differentiated carcinoma (data not shown). HER-2/neu gene amplification and HER-2/neu immunohistochemical overexpression by HercepTest were highly concordant (Table 3, Figure 3). Fifteen (88.2%) of 17 FISH-positive cases showed HER-2/neu overexpression. Results were FISH positive in 11 (78.6%) of 14 cases with HER-2/neu 3+ overexpression and 4 (26.7%) of 15 with HER- 2/neu 2+ overexpression. FISH-positive results were observed in 2 (3.0%) of cases without HER-2/neu overexpression.
Figure 3.
Representative immunohistochemical staining and FISH in biliary tract cancer. (A) Well-differentiated adenocarcinoma showing 3+ intensity of HER-2/neu staining. Original magnification X66. (B) HER-2/neu gene amplification by FISH in the same specimen. Green signal indicates chromosome 17 (CEP 17) and orange signal indicates HER-2/neu gene. HER-2/neu gene copy number was more than 20; thus, orange signals formed clusters. Original magnification X330.
There were no significant differences between US and Chilean gallbladder cancer samples with regard to HER-2/neu immunohistochemical staining or FISH results. immunohistochemical overexpression was observed in 8 (28.6%) of 28 US samples and 16 (32.7%) of 49 Chilean samples; 3 (14.3%) of 21 US samples and 11 (23.9%) of 46 Chilean samples were FISH positive.
DISCUSSION
The EGFR family of receptor tyrosine kinases is at the apex of a complex signal transduction cascade that modulates cell proliferation, apoptosis, migration, and differentiation.26 Overexpression of HER-2/neu and/or EGFR has been implicated to varying degrees in the carcinogenesis of a number of solid tumors, including breast, lung, stomach, pancreas, and colorectal cancers.27–30 HER-2/neu or EGFR overexpression has been widely reported in biliary tract cancer as well.16,17,21,23,25 There has been considerable variability in findings, likely reflecting use of different analytic techniques. To our knowledge, the current study is the first to show HER-2/neu and EGFR status of biliary tract cancer using standard accepted assays. HER-2/neu overexpression (2+, 3+) was identified in 31.2% of gallbladder cancers, 31.3% of extrahepatic bile duct cancers, and 33.3% of intrahepatic bile duct cancers, and gene amplification was positive in 20.9%, 21.4%, and 0%, respectively.
There are discrepancies between HercepTest and FISH results: none of the HerceptTest 2+ intrahepatic bile duct cancer samples showed HER-2/neu gene amplification, and only 26.7% of 2+ gallbladder cancer samples were FISH positive. Similar findings were reported in a study of breast cancer that showed gene amplification in 25% of 2+ cases (29). Nevertheless, there was significant concordance in assay results; whereas FISH is considered a more accurate method to detect HER-2/neu status, the HercepTest appears to be an acceptable predictor for HER-2/neu FISH positivity.
Overall, HER-2/neu gene amplification in gallbladder cancer and extrahepatic bile duct cancer in this study was comparable to rates seen in breast cancer.29,31 We also found that phosphorylation of HER-2/neu was induced in the HER-2/neu-positive cases. Given the efficacy of trastuzumab, a recombinant humanized monoclonal antibody directed against the HER-2/neu protein, in patients with HER-2-positive metastatic breast cancer,32–34 the data presented here indicate that HER-2/neu specific monoclonal antibodies or tyrosine kinase inhibitors may have a therapeutic role in gallbladder cancer and extrahepatic bile duct cancer, but perhaps not intrahepatic bile duct cancer.
The reported prevalence of EGFR overexpression in biliary tract cancer detected by immunohistochemical analysis has varied widely, perhaps in association with examination of only small numbers of cases and use of different analytic techniques. The current study examined a fairly large number of biliary tract cancer cases and employed the most reliable EGFR antibody available, finding EGFR overexpression in 16% of gallbladder cancers and none of the extrahepatic bile duct cancer and intrahepatic bile duct cancer samples. These data are similar to those from another recent study in biliary tract cancer.22
We expected the rate of EGFR expression to be comparable to the level of HER- 2/neu positivity, because overexpression of HER-2/neu results in HER-2/neu–EGFR heterodimerization, which inhibits the degradation of EGFR following EGFR overexpression. 35 In fact, in the biliary tract epithelium of BK5.erbB2 transgenic mice, the transgene of rat erbB2 leads to erbB2- EGFR heterodimer formation and an increase in activation of the EGFR,15 and chemopreventive or therapeutic use of EGFR or dual EGFR-HER-2/neu receptor tyrosine kinase inhibitors was effective in gallbladder cancer in these mice.36 However, the low frequency of simultaneous overexpression of HER-2/neu and EGFR in the current study suggests that the interaction between HER-2/neu and EGFR is not significant in biliary tract cancer carcinogenesis. The finding of cases of overexpression of HER-2/neu irrespective of EGFR expression suggests that there might be some cross-talk with other members of the EGFR family (such as erbB3) and/or other signal transduction pathways.
Although we found a significant correlation between EGFR overexpression and p-EGFR over expression in biliary tract cancer, we also observed that some samples that were 1+ for p-EGFR expression had no EGFR expression (data not shown). This can be explained by the observation that when p-EGFR is upregulated by the ligand connection, internalization of the complex occurs and EGFR disappears from the plasma membrane of the cancer cells.26,35,37 Thus, p-EGFR and not EGFR levels may be important in predicting response to receptor tyrosine kinase inhibitors.38 It is of interest that two recent publications showed that the presence of EGFR mutations in tumors of patients with non–small-cell lung cancer corresponded with sensitivity to receptor tyrosine kinase inhibitors. This gain of function is linked with increased EGFR and downstream activation after ligand binding even though the EGFR protein is not upregulated. 39,40 One study has reported EGFR gene mutations in 6 of 40 biliary tract cancer cases, but did not assess the relationship between gene mutation status and gene amplification and/or p-EGFR expression.41 The potential role of EGFR in biliary tract cancer carcinogenesis thus remains difficult to ascertain.
The frequency of HER-2/neu gene amplification in gallbladder cancer and extrahepatic bile duct cancer was similar to that reported in breast cancer, suggesting some rationale for studying existing anti-HER-2/neu agents in biliary tract cancer patients. Study of a larger number of biliary tract cancer specimens is needed to determine the significance of HER-2/neu overexpression in intrahepatic bile duct cancer. To ascertain whether a rationale exists for studying EGFR-targeted agents in biliary tract cancer, further work needs to be performed to assess EGFR gene amplification and p-EGFR protein level using more accurate and reliable techniques; additional study in larger numbers of patients is also needed to better understand the frequency of EGFR gene mutations in biliary tract cancer and the association of mutation with cancer cell sensitivity to EGFR tyrosine kinase inhibitors.
Thus, the challenge going forward is to determine the clinical relevance of these findings. Although HER-2/neu amplification has proved to be important in a subset of adenocarcinomas of the breast, there are few malignancies in which a relationship between EGFR expression and clinical efficacy of EGFR-targeting agents has been established.
Footnotes
Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Grant support: American Society of Clinical Oncology (ASCO) Foundation Career Development Award 05-91-0325
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