Japanese Journal of Clinical Oncology Advance Access originally published online on May 31, 2005
Japanese Journal of Clinical Oncology 2005 35(6):324-331; doi:10.1093/jjco/hyi089
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2005 Foundation for Promotion of Cancer Research
Usefulness and Limitation of Multiple Endoscopic Biopsy Sampling for Epidermal Growth Factor Receptor and c-erbB-2 Testing in Patients with Gastric Adenocarcinoma
Departments of 1 Pathology II and 2 Surgery I, National Defense Medical Tokorozawa, Saitama, Japan
For reprints and all correspondence: Hitoshi Tsuda, Department of Pathology II, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: htsuda{at}ndmc.ac.jp
Received February 15, 2005; accepted April 25, 2005
 |
Abstract |
|---|
 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Background: Our objective was to examine the utility of endoscopic biopsy specimens in judging the status of epidermal growth factor receptor (EGFR) and c-erbB-2 genes and proteins in the entire tumor.
Methods: Endoscopic biopsy specimens and specimens of whole representative cut surfaces of corresponding surgically resected tumors were obtained from 14 patients with gastric carcinoma, and immunohistochemistry and fluorescence in situ hybridization were then performed to determine the protein expression and gene amplification profiles, respectively, of EGFR and c-erbB-2 in these biopsy and surgical specimens.
Results: Among the eight endoscopic biopsy specimens obtained from three gastric carcinomas in which EGFR protein overexpression and gene amplification were judged to be positive in the corresponding surgically resected tissue specimens, EGFR overexpression was detected in three specimens (38%), but EGFR amplification was not detected (0%). Among the 19 endoscopic biopsy specimens obtained from five gastric carcinomas in which c-erbB-2 protein overexpression and gene amplification were judged to be positive in the corresponding surgically resected tissue specimens, c-erbB-2 overexpression and amplification (c-erbB-2/CEP17 ratio) were detected in 14 (74%) and 16 (84%) specimens, respectively. All three cases with EGFR overexpression and all five cases with c-erbB-2 overexpression showed intratumor heterogeneity with regard to their EGFR and c-erbB-2 status, respectively.
Conclusions: The c-erbB-2 status could be adequately assessed not only by examining surgically resected materials, but also by examining multiple endoscopic biopsy specimens. On the other hand, to assess the EGFR status accurately, the use of surgically resected samples appeared to be more reliable than the use of multiple endoscopic biopsy samples.
Key Words: epidermal growth factor receptor • c-erbB-2 (HER2) • endoscopic biopsy specimen • fluorescence in situ hybridization • intratumor heterogeneity
|
INTRODUCTION |
|---|
|
TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Epidermal growth factor receptor (EGFR, also known as c-erbB-1) and c-erbB-2 (also known as HER2/neu) are both products of a family of growth factor receptor oncogenes with tyrosine kinase activity. Trastuzumab, a humanized monoclonal antibody against c-erbB-2 protein, is now widely used for the treatment of patients with c-erbB-2-overexpressing and/or c-erbB-2-amplified metastatic breast cancers. Candidates for trastuzumab therapy are identified by appropriate tests for these c-erbB-2 alterations using a combination of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in surgically resected breast cancer tissue blocks (1–3). Although these tests are, in principle, only performed using surgically resected tissue specimens, a few reports have described tests for these c-erbB-2 alterations that were performed using core-needle biopsy specimens (4,5).
Gefitinib, an inhibitor of EGFR tyrosine phosphorylation, has been approved for use in Japan in patients with non-small cell lung cancer. A combination chemotherapy regimen using humanized monoclonal antibodies against EGFR, such as cetuximab (IMC-C225), has also been examined in phase I and II clinical trials for patients with squamous cell carcinoma of the head and neck, lung cancer or pancreatic cancer (6–8). Cetuximab was recently approved by the US Food and Drug Administration (FDA) for use in patients with metastatic colorectal cancer as part of either a single agent regimen or in combination with irinotecan (9). At the same time, the US FDA also recommended the use of an EGFR pharmDx kit (DakoCytomation Inc.) to examine EGFR protein expression and identify patients with colorectal cancer in whom cetuximab is likely to be effective.
In gastric cancer, which is now the second leading cause of cancer-related deaths in men in Japan, EGFR and c-erbB-2 alterations have been detected in 18–28% (10,11) and 6–30% (12–15) of cases, respectively. New therapeutic regimens targeting these tyrosine kinase receptors in combination with existing chemotherapy strategies may improve the prognosis of patients with gastric cancer. In our previous study, EGFR overexpression was detected in 13% of gastric cancers, and a mean EGFR copy number of 
4 or an EGFR/CEP7 ratio of 1.7 was correlated with the EGFR overexpression (16). With regard to c-erbB-2, multiple studies have shown a clear correlation between c-erbB-2 gene amplification, detected by FISH, and c-erbB-2 protein overexpression, detected by IHC, in gastric cancer (16,17). In these reports, most of the materials were obtained from surgically resected tissues, whereas only a few reports studied the c-erbB-2 status using endoscopic biopsy specimens.
For inoperable cases where surgical materials are not available, endoscopic biopsy specimens may provide useful information regarding the EGFR or c-erbB-2 status. Compared with surgically resected materials, however, materials from biopsy specimens may not be representative of the entire tumor, and the results of EGFR and c-erbB-2 tests using these materials would be influenced to some extent by intratumor heterogeneity in gene and protein status. Little information is available, however, regarding the correlation of the status of EGFR and c-erbB-2 between endoscopic biopsy specimens and corresponding surgically resected specimens from patients with gastric cancer.
In the present study, we examined the amplification of the EGFR and c-erbB-2 genes, detected by FISH, and the overexpression of EGFR and c-erbB-2 proteins, detected by IHC, in both endoscopic biopsy specimens and specimens from whole representative cut surfaces of corresponding surgically resected tumors from 14 patients with gastric carcinoma. By comparing the DNA and protein status of EGFR and c-erbB-2 in the biopsy and surgically resected specimens, we examined whether EGFR and c-erbB-2 testing in endoscopic biopsy specimens was sufficient to judge the status of these genes and proteins in the whole tumor. The influence of intratumor heterogeneity on the evaluation of EGFR and c-erbB-2 status in biopsy specimens is also discussed.
|
PATIENTS AND METHODS |
|---|
|
TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
|---|
PATIENTS
We selected a total of 14 gastric carcinoma patients for the present study, based on the previous results using surgically resected materials (16). Three patients showed both EGFR overexpression and gene amplification, whereas two control subjects showed no EGFR overexpression or gene amplification. For these five cases, 14 endoscopic biopsy specimens were obtained, formalin-fixed, paraffin-embedded and used for histopathological diagnosis to be confirmed to contain cancer tissues. They were then examined for EGFR overexpression and gene amplification.
Five additional patients showed both c-erbB-2 overexpression and gene amplification, whereas four control subjects showed no c-erbB-2 overexpression or amplification. For these nine cases, 30 endoscopic biopsy specimens were obtained, formalin-fixed, paraffin-embedded and used for histopathological diagnosis to be confirmd to contain cancer tissues. They were then examined for c-erbB-2 overexpression and gene amplification.
EXAMINATION OF INTRATUMOR HETEROGENEITY
To examine intratumor heterogeneity immunohistochemically, tissue blocks containing the largest representative cut surfaces were made for eight tumors: three cases with EGFR overexpression and five cases with c-erbB-2 overexpression.
The distribution of EGFR or c-erbB-2 overexpression detected by IHC in the largest cut surface from each tumor was then compared with the IHC results for the corresponding endoscopic biopsy specimens. From the largest cut surface, 2–5 cancer tissue sections per case were examined by IHC.
The endoscopic biopsy specimens and the surgically resected specimens were fixed in 10% neutral buffered formalin at room temperature for 24–48 h, dissected, embedded in paraffin, sliced into 5 µm thick tissue sections and mounted on silane-coated glass slides. These sections were stained with hematoxylin–eosin (HE) for routine histological diagnosis, and additional 5 µm thick serial sections were prepared for the FISH and IHC studies. The sections used to study intratumor heterogeneity were only subjected to IHC.
IMMUNOHISTOCHEMISTRY (IHC)
An EGFR pharmDX kit and a Herceptest kit (DakoCytomation) were used to detect EGFR and c-erbB-2 expression, respectively. According to the manufacturer's protocol, after deparaffinization, the sections for EGFR detection were treated with proteinase K solution for 5 min at room temperature, whereas the sections for c-erbB-2 detection were treated with epitope retrieval solution for 40 min at 95°C. After peroxidase blocking for 5 min, the sections were incubated with primary antibody for 30 min at room temperature, then with a labeled polymer for 30 min at room temperature and reacted with 3,3'-diaminobenzidine tetrahydrochloride (DAB)/hydrogen peroxide.
EGFR or c-erbB-2 expression in the carcinoma cells was scored as 3+ if it was strongly stained, and as 2+ if the entire circumference of the cell membrane was weakly to moderately stained in 10% of the constituent carcinoma cells. A score of 1+ was given if incomplete membrane staining was observed in 10% of the carcinoma cells. A score of 0 was given if membrane staining was visible in <10% of the constituent cells or if no membrane staining was apparent. Scores of 2+ and 3+ were classified as overexpression.
FISH
EGFR SpectrumOrange/CEP7 SpectrumGreen and c-erbB-2 SpectrumOrange/CEP17 SpectrumGreen DNA probes (Vysis, Downers Grove, IL, USA) were utilized. FISH was performed according to the manufacturer's protocol, with minor modifications.
The endoscopic biopsy specimen sections were deparaffinized, rehydrated and dried. After treatment with 0.2 N HCl for 20 min and two subsequent washes in 2x SSC (1x SSC: 0.15 M NaCl–0.015 M sodium citrate), the sections were immersed in 1 M sodium thiocyanate for 30 min at 80°C, then washed once in distilled water and twice in 2x SSC. The sections were then treated either with proteinase K (50 µg/ml, Boehringer-Mannheim, Mannheim, Germany) for 10 min at 37°C for EGFR detection or with a protease solution (Vysis) for 30 min at 37°C for c-erbB-2 detection, washed twice in 2x SSC and dried. Subsequently, the sections were refixed in 10% neutral buffered formalin for 10 min followed by two washes in 2x SSC, and dried.
A 10 µl aliquot of the probe was denatured for 5 min at 80°C and immediately cooled on ice. In parallel, the sections were denatured in 70% formamide/2x SSC at 80°C for 5 min, then dehydrated in 70, 85 and 100% ethanol at 4°C for 1 min each. After air drying, the probe was applied to the sections, and a coverslip was placed on the slide. Hybridization was performed for 14–18 h at 37°C. After hybridization, the sections were immersed in 0.3% NP-40/2x SSC to remove the coverslip gently, washed in 0.3% NP-40/2x SSC at 73°C for 2 min and then in 2x SSC at room temperature for 2 min, and counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Vysis).
A Leica DMR fluorescence microscope (Leica, Cambridge, UK) was used to detect the EGFR and c-erbB-2 signals. The red signals representing EGFR or c-erbB-2 were acquired through a Texas red single-bandpass filter (excitation spectrum, 596 nm; absorption spectrum, 620 nm) and the green signals representing CEP7 or CEP17 were acquired through a fluorescein isothiocyanate (FITC) single-bandpass filter (excitation spectrum, 490 nm; absorption spectrum, 520 nm). A DAPI filter (excitation spectrum, 372 nm; absorption spectrum, 456 nm) was used to detect the cell nuclei. Images of each microscopic field were taken using a COHU CCD camera (Leica) and saved in a Q550CW computer. The images obtained through the Texas red filter, the FITC filter and the DAPI filter for each microscopic field were merged using QFISH software (Leica).
For each specimen, the number of EGFR signals was counted for 60 nuclei, and the mean EGFR copy number was calculated by dividing the total number of EGFR signals by 60. The EGFR/CEP7 ratio was also calculated for each specimen by dividing the total number of EGFR signals by the total number of CEP7 signals. The mean c-erbB-2 copy number and the c-erbB-2/CEP17 ratios were calculated in a similar manner. EGFR amplification was defined as the presence of both a mean EGFR copy number of 4.0 and an EGFR/CEP7 ratio of 1.7, according to our previous data (16). C-erbB-2 amplification was defined as a mean c-erbB-2 copy number of 7.0 and a c-erbB-2/CEP17 ratio of 2.0 (16,18).
|
RESULTS |
|---|
|
TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
|---|
EGFR OVEREXPRESSION AND AMPLIFICATION IN BIOPSY SPECIMENS OF GASTRIC CARCINOMA
The detailed EGFR status of eight specimens obtained from three patients with EGFR overexpression (score 3+, n = 2; score 2+, n = 1) and six specimens obtained from two patients without EGFR overexpression are shown in Tables 1, 2 and 3. In two of the three cases with EGFR overexpression in the corresponding surgically resected specimens, at least one of two or three pre-operative endoscopic biopsy specimens had EGFR overexpression (Table 1). Both of the two specimens (100%) from case 1 were scored as 2+ or 3+ for EGFR overexpression. One of the three specimens (33%) from case 2 was scored as 2+ for EGFR overexpression. On the other hand, none of the three specimens (0%) from case 3 showed overexpression. In total, EGFR overexpression was detected in three (38%) of the eight endoscopic biopsy specimens that were obtained from three EGFR-overexpressing gastric carcinomas.
|
|
|
In contrast, EGFR overexpression was not detected in any of the six endoscopic biopsy specimens that were obtained from the two gastric carcinomas without EGFR overexpression in surgically resected specimens.
Of the five gastric carcinoma tumors obtained by surgical resection, three tumors had an EGFR copy number of 4.0 by FISH, and four tumors had an amplified EGFR/CEP7 ratio of 1.7 (Tables 2 and 3) (16). EGFR amplification was not detected in terms of either the mean EGFR copy number or the EGFR/CEP7 ratio (0%) in all eight endoscopic biopsy specimens obtained from these three EGFR-amplified gastric carcinomas (Tables 2 and 3). None of the six endoscopic biopsy specimens from the two gastric carcinomas without EGFR amplification showed EGFR amplification.
C-ERBB-2 OVEREXPRESSION AND AMPLIFICATION IN BIOPSY SPECIMENS OF GASTRIC CARCINOMAS
Of the five surgically resected gastric carcinomas that were judged to exhibit c-erbB-2 overexpression and amplification, the c-erbB-2 expression score, as determined by IHC, was 2+ in one case and 3+ in four cases (16).
In the endoscopic biopsy specimens from these five patients, at least one of the specimens from each tumor was scored as 2+ or 3+ for c-erbB-2 overexpression (Fig. 1). The detailed c-erbB-2 status in the 19 specimens obtained from five c-erbB-2-overexpressing tumors (cases 6–10) and in the 11 specimens obtained from four c-erbB-2-non-overexpressing tumors (cases 11–14) is shown in Tables 4, 5 and 6. The IHC score of the c-erbB-2 in cases 6, 7 and 8 was always 2+ or 3+, whereas three out of four (75%) and two out of six (33%) biopsy specimens in cases 9 and 10 were judged to show c-erbB-2 overexpression, respectively (Table 4). Overall, c-erbB-2 overexpression was detected in 14 out of the 19 specimens (74%) obtained by endoscopic biopsy. The concordance ratio for c-erbB-2 overexpression (74%) between the endoscopic biopsy specimens and the surgically resected specimens tended to be higher than the ratio for EGFR overexpression between the endoscopic biopsy specimens and the surgically resected specimens (38%) (P = 0.08).
|
|
|
|
In contrast, none of the 11 endoscopic biopsy specimens from gastric carcinomas without c-erbB-2 overexpression showed c-erbB-2 overexpression.
Of the five surgically resected gastric carcinomas that were judged to exhibit c-erbB-2 amplification, at least one endoscopic biopsy specimen from each tumor showed obvious gene amplification (Fig. 2). The mean c-erbB-2 copy number and c-erbB-2/CEP17 ratio for each of the 19 specimens obtained from the five gastric carcinomas with c-erbB-2 amplification and the 11 specimens from the four gastric carcinomas without c-erbB-2 amplification are shown in Tables 5 and 6. A mean c-erbB-2 copy number of 7.0 and a c-erbB-2/CEP17 ratio of 2.0 were detected in all of the specimens from cases 6, 7 and 8. A mean c-erbB-2 copy number of 7.0 was detected in three (75%) out of four specimens from case 9 and in three (50%) out of six specimens from case 10. A c-erbB-2/CEP17 ratio of 2.0 was detected in three (75%) out of four specimens from case 9 and in four (67%) out of six specimens from case 10. Overall, c-erbB-2 amplification, in terms of both the mean c-erbB-2 copy number and the c-erbB-2/CEP17 ratio, was detected in 15 (79%) and 16 (84%), respectively, of the 19 endoscopic biopsy specimens obtained from the five gastric cancers with c-erbB-2 amplification. The concordance ratios (79 and 84%) in c-erbB-2 gene amplification between the endoscopic biopsy specimens and the surgically resected specimens were significantly higher than the concordance ratios in EGFR gene amplification between endoscopic biopsy specimens and surgically resected specimens (0%) (P < 0.01).
|
In contrast, c-erbB-2 amplification was not detected in 11 endoscopic biopsy specimens obtained from four gastric carcinomas that had been judged to exhibit no c-erbB-2 amplification in surgically resected specimens.
INTRATUMOR HETEROGENEITY OF EGFR AND C-ERBB-2 OVEREXPRESSION IN SURGICALLY RESECTED MATERIALS
In the three surgically resected specimens with EGFR overexpression and amplification, EGFR expression was examined more closely in the largest representative cut surface of each tumor. All three cases with EGFR overexpression showed intratumor heterogeneity in their expression status (Table 1). These three cases (cases 1, 2 and 3) showed strong to moderate EGFR staining throughout the entire circumference of the cancer cells in 60, 10 and 80% of the tumor area, respectively, whereas they showed no staining or partial, membrane staining in the remaining area of the tumor (Fig. 4).
In the five surgically resected specimens with c-erbB-2 overexpression and amplification, c-erbB-2 expression was examined more closely in the largest representative cut surface of each tumor. All five cases showed intratumor heterogeneity, more or less, in their c-erbB-2 expression status. Three cases (cases 6, 7 and 8) in which all of the endoscopic biopsy specimens had shown both c-erbB-2 overexpression and gene amplification showed strong to moderate c-erbB-2 staining in the entire circumference of the cancer cells in 80, 90 and 50% of the tumor area, respectively, but no staining or partial membrane staining in the remaining 20, 10 and 50% of the tumor area, respectively (Figs 3B and 5). The other two cases (cases 9 and 10), in which only part of the endoscopic biopsy specimens exhibited c-erbB-2 overexpression and gene amplification, showed strong to moderate c-erbB-2 staining in the entire circumference of the cancer cells in 98% of the tumor area but no staining or only partial membrane staining in the other 2% of the tumor area.
|
|
DISCUSSION |
|---|
|
TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Patients with metastatic breast cancer characterized by c-erbB-2 overexpression and/or amplification are candidates for trastuzumab therapy. The response rates to the first-line trastuzumab monotherapy for breast cancer patients with a c-erbB-2 IHC score of 3+ or FISH-based evidence of c-erbB-2 gene amplification have been reported to be 35 and 34%, respectively (2). In gastic carcinoma, the incidence of c-erbB-2 overexpression and/or amplification has been reported to be 6–30% (14,15,19). In a previous study, the incidences of c-erbB-2 overexpression, as determined by IHC, and of c-erbB-2 amplification, as determined by a c-erbB-2/CEP17 ratio of
2.0 using FISH, were 11 and 15%, respectively (16). Similarly to patients with metastatic breast cancer, trastuzumab therapy might be applicable to patients with gastric cancer characterized by c-erbB-2 overexpression and/or amplification, possibly improving the prognosis of these patients. In the present study, c-erbB-2 overexpression and amplification were detected in at least one endoscopic biopsy specimen from each of five gastric cancers confirmed to exhibit overexpression and amplification in surgical specimens. Therefore, the examination of c-erbB-2 overexpression and/or amplification in multiple endoscopic biopsy specimens was considered to represent the status of c-erbB-2 protein and DNA sufficiently. In a total of 19 endoscopic biopsy specimens from five gastric cancers, the detection rates of c-erbB-2 overexpression by IHC and c-erbB-2 gene amplification by FISH were 74 and 84%, respectively. Therefore, FISH tended to reflect the c-erbB-2 status more sensitively than IHC among these cases.
The major cause of the inconsistency in c-erbB-2 overexpression or amplification between the endoscopic biopsy specimens and the surgically resected materials can probably be attributed to intratumor heterogeneity in the c-erbB-2 status. Of the five c-erbB-2-positive cases, c-erbB-2 overexpression was observed in >50% (50–98%) of the tumor area, but the remaining area (2–50%) exhibited no c-erbB-2 overexpression when the largest representative cut surface of the tumor was examined. Intratumor heterogeneity should therefore be considered when the c-erbB-2 status of endoscopic biopsy specimens is analyzed. As seen in Fig. 5, areas of c-erbB-2 overexpression usually cover all or part of the tumor surface, and the examination of multiple endoscopic biopsy specimens is likely to be effective for detecting c-erbB-2 alterations.
In contrast, the influence of intratumor heterogeneity on the evaluation of EGFR status appeared to be larger than that for c-erbB-2 status. Of the three cases that were shown to exhibit EGFR overexpression and amplification in surgically resected materials, EGFR overexpression was only detected in two cases and EGFR gene amplification was not detected in any of the endoscopic biopsy specimens. The inconsistency in EGFR overexpression or amplification in these cases may also be caused by intratumor heterogeneity. In these three cases, the largest representative cut surface of the tumor revealed EGFR overexpression in 10–80% of the tumor area. Since the area of EGFR overexpression was only partially located on the tumor surface, as in cases A and B in Fig. 4, the endoscopic biopsy specimens might not have contained a sample of the EGFR-overexpressing area. The present results therefore suggest that the examination of surgically resected specimens is better suited for the evaluation of EGFR status than the examination of multiple endoscopic biopsy specimens.
|
|
Another cause of the inconsistency between c-erbB-2 and/or EGFR overexpression or amplification between the endoscopic biopsy specimens and the surgically resected materials might have been the effect of the duration of formalin fixation in a minor portion of the cases. In fact, IHC or FISH analysis has been reported to be influenced by overfixation (20,21). The endoscopic biopsy specimens in this study were fixed for
24 h. This fixation time was within the optimal limits, since for both IHC and FISH it is recommend to use sections that have been formalin fixed for <48 h. However, in routinely processed specimens, the materials may have been fixed for longer than 2 days. In particular, small endoscopic biopsy specimens might have been overfixed if the fixation process included successive holidays. In summary, the status of c-erbB-2 can be adequately assessed not only by examining surgically resected materials, but also by examining multiple endoscopic biopsy materials, when these materials are appropriately fixed using 10% formalin for <48 h. On the other hand, for the accurate assessment of the status of EGFR, tumor heterogeneity should be taken into consideration, and the use of surgically resected samples may be more reliable than the use of multiple endoscopic biopsy samples.
|
Acknowledgments |
|---|
This work was supported in part by a grant-in-aid for cancer research from the Foundation for Promotion of Cancer Research and by grants from Chugai Pharmaceutical Co., Fujisawa Pharmaceutical Co. and Taiho Pharmaceutical Co., Japan.
|
References |
|---|
|
TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
|---|
1 Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–92.
2 Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 2002;20:719–26.
3 Seidman AD, Fornier MN, Esteva FJ, Tan L, Kaptain S, Bach A, et al. Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification. J Clin Oncol 2001;19:2587–95.
4 Mezzelani A, Alasio L, Bartoli C, Bonora MG, Pierotti MA, Rilke F, et al. c-erbB2/neu gene and chromosome 17 analysis in breast cancer by FISH on archival cytological fine-needle aspirates. Br J Cancer 1999;80:519–25.[CrossRef][Medline]
5 Klijanienko J, Couturier J, Galut M, El-Naggar AK, Maciorowski Z, Padoy E, et al. Detection and quantitation by fluorescence in situ hybridization (FISH) and image analysis of HER-2/neu gene amplification in breast cancer fine-needle samples. Cancer 1999;87:312–8.[CrossRef][Web of Science][Medline]
6 Herbst RS, Hong WK. IMC-C225, an anti-epidermal growth factor receptor monoclonal antibody for treatment of head and neck cancer. Semin Oncol 2002;29:18–30.[Medline]
7 Bunn PA Jr, Franklin W. Epidermal growth factor receptor expression, signal pathway, and inhibitors in non-small cell lung cancer. Semin Oncol 2002;29:38–44.[Medline]
8 Xiong HQ, Abbruzzese JL. Epidermal growth factor receptor-targeted therapy for pancreatic cancer. Semin Oncol 2002;29:31–7.[Medline]
9 Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004;351:337–45.
10 Lemoine NR, Jain S, Silvestre F, Lopes C, Hughes CM, McLelland E, et al. Amplification and overexpression of the EGF receptor and c-erbB-2 proto-oncogenes in human stomach cancer. Br J Cancer 1991;64:79–83.[Web of Science][Medline]
11 Yasui W, Sumiyoshi H, Hata J, Kameda T, Ochiai A, Ito H, et al. Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res 1988;48:137–41.
12 Yoshida K, Tsuda T, Matsumura T, Tsujino T, Hattori T, Ito H, et al. Amplification of epidermal growth factor receptor (EGFR) gene and oncogenes in human gastric carcinomas. Virchows Arch B 1989;57:285–90.[Medline]
13 Ishikawa T, Kobayashi M, Mai M, Suzuki T, Ooi A. Amplification of the c-erbB-2 (HER-2/neu) gene in gastric cancer cells. Detection by fluorescence in situ hybridization. Am J Pathol 1997;151:761–8.[Abstract]
14 Shun CT, Wu MS, Lin JT, Chen SY, Wang HP, Lee WJ, et al. Relationship of p53 and c-erbB-2 expression to histopathological features, Helicobacter pylori infection and prognosis in gastric cancer. Hepatogastroenterology 1997;44:604–9.[Medline]
15 Yonemura Y, Ninomiya I, Ohoyama S, Kimura H, Yamaguchi A, Fushida S, et al. Expression of c-erbB-2 oncoprotein in gastric carcinoma. Immunoreactivity for c-erbB-2 protein is an independent indicator of poor short-term prognosis in patients with gastric carcinoma. Cancer 1991;67:2914–8.[CrossRef][Web of Science][Medline]
16 Kimura M, Tsuda H, Morita D, Ichikura T, Ogata S, Aida S, et al. A proposal for diagnostically meaningful criteria to classify increased epidermal growth factor receptor and c-erbB-2 gene copy numbers in gastric carcinoma, based on correlation of fluorescence in situ hybridization and immunohistochemical measurements. Virchows Arch 2004;445:255–262.[CrossRef][Medline]
17 Takehana T, Kunitomo K, Kono K, Kitahara F, Iizuka H, Matsumoto Y, et al. Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked immuno-sorbent assay. Int J Cancer 2002;98:833–7.[CrossRef][Medline]
18 Pauletti G, Dandekar S, Rong H, Ramos L, Peng H, Seshadri R, et al. Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol 2000;18:3651–64.
19 David L, Seruca R, Nesland JM, Soares P, Sansonetty F, Holm R, et al. c-erbB-2 expression in primary gastric carcinomas and their metastases. Mod Pathol 1992;5:384–90.[Web of Science][Medline]
20 Selvarajan S, Bay BH, Choo A, Chuah KL, Sivaswaren CR, Tien SL, et al. Effect of fixation period on HER2/neu gene amplification detected by fluorescence in situ hybridization in invasive breast carcinoma. J Histochem Cytochem 2002;50:1693–6.
21 Mitchell MS, Press MF. The role of immunohistochemistry and fluorescence in situ hybridization for HER2/neu in assessing the prognosis of breast cancer. Semin Oncol 1999;26:108–16.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||