1Laboratory of Cancer Biology, Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere; Departments of 2 Oncology and 5Pathology, Tampere University Hospital, Tampere; 3Medicity Research Laboratories, and Department of Medical Biochemistry and Molecular Biology, University of Turku; 4Turku Graduate School of Biomedical Sciences, University of Turku, Turku; 9Department of Oncology, Turku University Hospital, Turku, Finland; 6Department of Pathology, University of Oulu and Oulu University Hospital, Oulu; Departments of 7 Cardiothoracic Surgery and 8 Oncology, Helsinki University Central Hospital, Helsinki, Finland
* Correspondence to: Professor J. Isola, Laboratory of Cancer Biology, Institute of Medical Technology, University of Tampere & Tampere University Hospital, P.O. Box 607, FIN-33101 Tampere, Finland. Tel: +358-3-2156729; Fax: +358-3-2158923; Email: jorma.isola{at}uta.fi
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: The frequency of HER-2/neu and Topoisomerase II gene amplification was studied in adenocarcinomas of the stomach (n=131) and the gastroesophageal junction (n=100) by chromogenic in situ hybridization (CISH). Sensitivity of a gastric cancer cell line N87 with HER-2/neu amplification to trastuzumab was studied by a cell viability assay and compared with that of a HER-2 amplified breast cancer cell line SKBR-3. Growth inhibition of N87 cells was also verified in vivo in N87 xenograft tumors.
Results: HER-2/neu amplification was present in 16 (12.2%) of the 131 gastric and in 24 (24.0%) of the 100 gastroesophageal adenocarcinomas. Co-amplification of Topoisomerase II was present in the majority of gastric (63%) and esophagogastric junction cancers (68%) with HER-2/neu amplification. HER-2/neu amplification was more common in the intestinal histologic type of gastric cancer (21.5%) than in the diffuse (2%) or the mixed/anaplastic type (5%, P=0.0051), but it was not associated with gender, age at diagnosis or clinical stage. Presence of HER-2/neu amplification was associated with poor carcinoma-specific survival (P=0.0089). HER-2/neu targeting antibody trastuzumab inhibited the growth of a p185HER-2/neu overexpressing gastric and breast carcinoma cell lines (N87 and SKBR-3) with equal efficacy.
Conclusions: HER-2/neu amplification is common in the intestinal type of gastric carcinoma, and it is associated with a poor outcome. HER-2 might be a useful target in this disease, and this hypothesis deserves to be investigated in clinical trials.
Key words: CISH, gastric carcinoma, HER-2, Lauren's classification, trastuzumab
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Breast cancer patients with HER-2/neu positive tumors often benefit from topoisomerase II inhibitor-based chemotherapy (commonly doxorubicin or epirubicin; reviewed in [4]). This finding may be explained by co-amplification of HER-2/neu and Topoisomerase II
genes, the latter being located adjacent to HER-2/neu at the chromosome band 17q12-q21 [5
, 6
]. Studies carried out in vitro indicate that Topoisomerase II
gene amplification leads to overexpression of topoisomerase II
protein, which, in turn, makes the cells more sensitive to doxorubicin by providing an increased number of targets for the drug [6
].
In this study, we examined amplification of HER-2/neu and Topoisomerase II in a series consisting of 131 adenocarcinomas of the stomach, and 100 adenocarcinomas of the cardia and the distal esophagus using chromogenic in situ hybridization (CISH). To explore gastric carcinoma as a potential target for anti-HER-2 therapies, we studied the sensitivity of a gastric cancer cell line with HER-2/neu amplification to trastuzumab and compared its sensitivity with that of a breast cancer cell line with HER-2/neu amplification in vitro and in vivo.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines
A gastric carcinoma cell line N87 and the breast carcinoma cell lines SKBR-3 and T47D were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were maintained in RPMI medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin and 50 mg/ml streptomycin. All cell lines were initially tested for the presence of HER-2/neu gene amplification by FISH as previously described [5, 6
].
Chromogenic in situ hybridization
The slides were de-paraffinized and incubated in 0.1 M TrisHCl (pH 7.3) in a temperature-controlled microwave oven [at 92°C for 10 min, followed by cooling for 20 min at room temperature (RT)]. After a wash with PBS, enzymic digestion was carried out by applying 100 µl of digestion enzyme to the slides for 1015 min at RT (Digest-All III solution, Zymed Inc., South San Francisco, CA). The slides were then washed with PBS and dehydrated with graded ethanols. Ten microliters of the ready-to-use digoxigenin-labeled HER-2/neu or the Topoisomerase II DNA probe (Zymed) was applied to the slides, which were covered with coverslips and sealed with rubber cement. The sections were denatured on a thermal plate (94°C for 3 min) and hybridization was carried out overnight at 37°C. After hybridization, the slides were washed with 0.5x SSC (standard saline citrate; 5 min at 75°C), followed by three washes in PBS (at RT). The probes were detected by means of sequential incubations with mouse anti-digoxigenin (diluted 1:300; Roche Biochemicals, Mannheim, Germany), anti-mouse-peroxidase polymer (Powervision+, Immunovision Inc., Daly City, CA) and diaminobenzidine chromogen. The tissue sections were lightly counterstained with hematoxylin and embedded. A positive and a negative control sample (tumors with and without HER-2 amplification in FISH) was included in every hybridization batch.
Quantitative RT-PCR
Total RNA was extracted from cell cultures using RNAzol B reagent (Tel-Test Inc., Gainesville, FL) according to the manufacturer's protocol. After treatment with DNAse I (Life Technologies Inc., Rockville, MD), 1 µg of total RNA was subjected to cDNA synthesis using random hexamer (Promega, Madison, WI) primers and M-MLV RNase H minus reverse transcriptase (Promega) in a total volume of 25 µl according to the manufacturer's protocol. To control for false positive amplification resulting from contaminating chromosomal DNA, duplicate reactions were performed in the absence of the reverse transcriptase enzyme. TaqManTM quantitative RT-PCR analysis was employed to determine the expression levels of each HER receptor mRNA. The design and evaluation of specificity of the oligonucleotide sets are described in detail elsewhere [9]. PCR was carried out using 300 nM of primers, 200 nM of probes, 12.5 µl of TaqManTM universal PCR Master Mix (PE Biosystems, Foster City, CA) and 0.5 µl of reverse transcribed template cDNA in a total volume of 25 µl. Thermal cycling (ABI PRISM 7700 Sequence Detector; PE Biosystems, Foster City, CA) was started with initial steps of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. CT values (the cycle number at which the signal exceeds a fixed threshold value) were determined and normalized against an internal control (ß-actin mRNA expression) from parallel samples. Expression of each HER transcript was presented as the percentage of HER mRNA expression relative to the expression of the control ß-actin mRNA.
Western blot analysis
HER protein expression was analyzed by western blotting as described elsewhere [10]. Primary antibodies EGFR (1005), NEU (C-18), ErbB-3 (C-17) and ErbB-4 (C-18) (all from Santa-Cruz Biotechnology Inc., Santa Cruz, CA) were used in 1:1000 dilution for detection or HER1, p185HER2/neu, HER3 and HER4, respectively. Peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA) was used as a secondary antibody in 1:10 000 dilution, and visualized by ECL (Amersham Biosciences, Piscataway, NJ) and autoradiography.
Trastuzumab sensitivity assays
Cellular responses of the cell lines N87 and SKBR-3 to trastuzumab (Herceptin®, Roche, Basel, Switzerland) were estimated by counting cell numbers by a MTS color assay (Promega, Madison, WI) and using a hemocytometer. For the MTS assays, cells were seeded on 96-well plates (1 x 104 cells/well) in serum-free RPMI supplemented with or without trastuzumab and maintained in the same medium for up to 6 days. MTS color solution (Promega; 20 µl/well) was added for the last 3 h at each time point, and the intensity of staining, reflecting the number of viable cells, was quantified spectrophotometrically according to the manufacturer's instructions. To count the cell numbers with a hemocytometer, cells were seeded on six-well plates (1 x 105 cells/well) in serum-free RPMI supplemented with or without trastuzumab, and cultured for up to 6 days.
N87 cells were also tested for trastuzumab sensitivity when grown as xenograft tumors in female nu/nmri nude mice (Harlan Netherland, Horst, The Netherlands). The tests were carried out with the approval of the ethics committee of the University of Tampere. Trastuzumab was given intraperitoneally at a dose of 5 mg/kg and compared with the same dose of a similar humanized antibody rituximab, which is directed against CD20 differentiation antigen not expressed in carcinoma cells. Tumor growth was measured using a caliper and tumor volume was calculated using the formula Tvol=/6x larger diameter x (smaller diameter)2.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Sensitivity to trastuzumab
Gastric cancer cell line N87 and the breast carcinoma cell line SKBR-3 showed high level HER-2/neu gene amplification by initial FISH testing, whereas T47D showed normal copy numbers (data not shown). In vitro doseresponse curves to trastuzumab were determined using the cell viability test and the actual cell counts were calculated using a hemocytometer. These experiments indicated that trastuzumab inhibited the growth of N87 and SKBR-3 at about equal efficacy (Figure 3A and data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The observed prevalence of HER-2/neu gene amplification (12.2%) in gastric adenocarcinomas was within the same range as found in a FISH-based study recently reported by Takehana and others, who found HER-2 to be amplified in about 8% of the cases [12]. Our results show that HER-2/neu amplification is strongly associated with the intestinal histologic type as defined according to Lauren's classification (21.5% versus 2.2% in the diffuse type, P=0.0051). In three studies [13
15
], immunohistochemical p185HER-2/neu overexpression has been more common in the intestinal than in the diffuse type of gastric adenocarcinoma, but because of the wide variation in the prevalence of p185HER-2/neu expression, it is difficult to evaluate the clinical importance of these findings [13
15
]. The present results also pinpoint the usefulness of Lauren's classification, since this groups gastric adenocarcinomas into different pathobiologically relevant subgroups. As in breast carcinomas, where HER-2/neu gene amplification is a common feature of invasive ductal carcinomas and an uncommon feature in lobular carcinomas, the intestinal type of gastric cancer shows high prevalence of HER-2/neu amplification in contrast to diffuse gastric carcinoma. According to our results, HER-2/neu gene amplification associates similarly, but inversely into different histological subtypes (according to Lauren's classification) as E-cadherin mutations [16
]. They are typical for diffuse gastric and lobular invasive breast carcinomas, but rare in intestinal gastric and ductal invasive breast carcinomas [16
]. HER-2/neu amplification was found in 24% of gastroesophageal adenocarcinoma cases, the proportion of which is almost the same as we found in the intestinal type of gastric adenocarcinoma. These cancer types are histologically indistinguishable.
The majority of published studies have found HER-2 expression to be of prognostic value in gastric adenocarcinoma [11]. In our study, amplification of HER-2/neu was strongly associated with poor carcinoma-specific survival in gastric carcinoma (P=0.0089). This was particularly evident in the subgroup of intestinal type of cancers (P=0.0019), which is usually considered to associate with more favorable prognosis than the diffuse type of gastric adenocarcinoma [17
]. HER-2/neu gene amplification was not associated with the clinical stage, indicating that the poor outcome is likely to be explained by aggressive tumor biology of cancers with HER-2/neu amplification. The prognostic significance of HER-2/neu amplification is controversial in gastroesophageal carcinoma [11
]. In this study, there was a trend towards poor survival among patients with cancer with HER-2/neu amplification, but this was not statistically significant, and could reflect the overall poor prognosis of this tumor entity with a median survival of only 9 months in the present series.
The present drug sensitivity assays suggest that the presence of HER-2/neu amplification may turn out to be a more important feature in therapeutic prediction than in prognostication in gastric and esophageal adenocarcinomas. Analysis of a HER-2/neu amplified gastric carcinoma cell line N87 indicated that it was as sensitive to trastuzumab as the HER-2/neu amplified breast carcinoma cell line SKBR-3, which is a widely used reference in trastuzumab sensitivity studies [1820
]. To our knowledge, this is the first published demonstration of efficacy of trastuzumab in gastric cancer, and gives the theoretical background for testing this drug clinically in gastric carcinoma patients with amplified HER-2/neu either as a single agent or in combination with conventional chemotherapy. Interestingly, HER-2/neu amplification was associated with overexpression of HER-1 (EGFR), which might turn out to be important because of the potential synergic effect between trastuzumab and some EGFR inhibiting drugs such as Iressa® [20
].
We have previously shown that simultaneous amplification of Topoisomerase II is a common feature of HER-2/neu amplified breast tumors [6
]. There is accumulating evidence that this co-amplification might in part explain the spectrum of chemosensitivity associated with HER-2/neu amplified breast tumors [4
]. As in breast cancer, Topoisomerase II
was co-amplified with HER-2/neu in gastric adenocarcinomas, although the frequency of co-amplification may be even higher than in breast cancer. Two-thirds of the gastroesophageal and gastric adenocarcinomas with HER-2/neu gene amplification showed co-amplification of Topoisomerase II
, whereas in breast cancer, 3040% of tumors have co-amplification of these genes. This suggests that a majority of HER-2/neu amplified gastric and gastroesophageal adenocarcinomas might be sensitive to topo II inhibiting drugs. This is in line with clinical findings, and topo II inhibitors such as etoposide and doxorubicin are widely used cytotoxic drugs in the treatment of gastric cancer.
![]() |
Acknowledgements |
---|
Received for publication August 17, 2004. Revision received October 21, 2004. Accepted for publication October 22, 2004.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Menard S, Casalini P, Campiglio M et al. HER2 overexpression in various tumor types, focusing on its relationship to the development of invasive breast cancer. Ann Oncol 2001; 12: S15S19.[ISI]
3. Scholl S, Beuzeboc P, Pouillart P. Targeting HER2 in other tumor types. Ann Oncol 2001; 12: S81S87.[Medline]
4. Yarden Y, Sliwkowski MX. Untangling the ErbB signaling network. Nat Rev Mol Cell Biol 2001; 2: 127137.[CrossRef][ISI][Medline]
5. Jarvinen TA, Tanner M, Barlund M et al. Characterization of topoisomerase II alpha gene amplification and deletion in breast cancer. Genes Chromosomes Cancer 1999; 26: 142150.[CrossRef][ISI][Medline]
6. Jarvinen TA, Tanner M, Rantanen V et al. Amplification and deletion of topoisomerase II alpha associate with ErbB-2 amplification and affect sensitivity to topoisomerase II inhibitor doxorubicin in breast cancer. Am J Pathol 1999; 156: 839847.[ISI]
7. Sihvo EI, Salminen JT, Salo JA. The epidemiology of oesophageal adenocarcinoma: has the cancer of gastric cardia an influence on the rising incidence of oesophageal adenocarcinoma? Scand J Gastroenterol 2000; 35: 10821086.[CrossRef][ISI][Medline]
8. Watanabe H, Jass JR, Sobin LH. Histological typing of oesophageal and gastric tumours. World Health Organization International Histological Classification of Tumours, 2nd edition. Berlin: Springer Verlag 1990.
9. Junttila TT, Laato M, Vahlberg T et al. Identification of patients with transitional cell carcinoma of the bladder overexpressing ErbB2, ErbB3, or specific ErbB4 isoforms: real-time reverse transcription-PCR analysis in estimation of ErbB receptor status from cancer patients. Clin Cancer Res 2003; 9: 53465357.
10. Kainulainen V, Sundvall M, Määttä JA et al. A natural ErbB4 isoform that does not activate phosphoinositide 3-kinase mediates proliferation but not survival or chemotaxis. J Biol Chem 2000; 275: 86418649.
11. Ross JS, McKenna B. The HER-2/neu oncogene in tumors of the gastrointestinal tract. Cancer Invest 2001; 19: 554568.[CrossRef][ISI][Medline]
12. Takehana T, Kunimoto K, Kono K 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: 833837.[CrossRef][ISI][Medline]
13. Lin JT, Wu MS, Shun CT et al. Occurrence of microsatellite instability in gastric carcinoma is associated with enhanced expression of erbB-2 oncoprotein. Cancer Res 1995; 55: 14281430.[Abstract]
14. Wu MS, Shun CT, Wang HP et al. Genetic alterations in gastric cancer: relation to histological subtypes, tumor stage, and Helicobacter pylori infection. Gastroenterology 1997; 112: 14571465.[ISI][Medline]
15. Polkowski W, van Sandick JW, Offerhaus GL et al. Prognostic value of Lauren's classification and c-erbB-2 oncogene overexpression in adenocarcinoma of the esophagus and gastroesophageal junction. Ann Surg Oncol 1999; 6: 290297.
16. Berx G, Becker KF, Hofler H, van Roy F. Mutations of the human E-cadherin (CDH1) gene. Hum Mutat 1998; 12: 226237.[CrossRef][ISI][Medline]
17. Hochwald SN, Kim S, Klimstra DS et al. Analysis of 154 actual five-year survivors of gastric cancer. J Gastrointest Surg 2000; 4: 520525.[CrossRef][ISI][Medline]
18. Pegram MD, Finn RS, Arzoo K et al. The effect of Her-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells. Oncogene 1997; 15: 537547.[CrossRef][ISI][Medline]
19. Pegram M, Hsu S, Lewis G et al. Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers. Oncogene 1999; 18: 22412251.[CrossRef][ISI][Medline]
20. Normanno N, Campiglio M, De LA et al. Cooperative inhibitory effect of ZD1839 (Iressa) in combination with trastuzumab (Herceptin) on human breast cancer cell growth. Ann Oncol 2002; 13: 89.