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Japanese Journal of Clinical Oncology 2004 34(12):727-732; doi:10.1093/jjco/hyh137
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© 2004 Foundation for Promotion of Cancer Research

Prognostic Implications of the Expression of erbB2, Topoisomerase II{alpha} and Thymidylate Synthase in Metastatic Gastric Cancer After Fluorouracil-based Therapy

Jacqueline Ming Liu1, Li-Tzong Chen1,5, Anna F. Y. Li2,4, Chew Wen Wu3,4, Chieh Lan1, Tsai Rong Chung1, Her-Shyong Shiah1, Kuan Der Lee1, Tsang Wu Liu1 and Jacqueline Whang Peng1

1 Division of Cancer Research, National Health Research Institutes, 2 Pathology Department and 3 Surgery Department, Veterans General Hospital-Taipei, 4 National Yang Ming University Medical School and 5 Department of Internal Medicine, Kaohsiung Medical University Hospital, Taipei, Taiwan

For reprints and all correspondence: Jacqueline M. Liu, Division of Cancer Research, National Health Research Institutes, c/o A191 Taipei-Veterans General Hospital, Shipai Rd, Sect 2, no. 201, Taipei, Taiwan. E-mail: jmliu{at}nhri.org.tw

Received July 1, 2004; accepted October 9, 2004


   Abstract
TOP
 Abstract
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Objective: This retrospective study aimed to ascertain the expression of erbB2 in relation to topoisomerase II{alpha} (T2{alpha}) and thymidylate synthase (TS) markers in 30 consecutive metastatic gastric cancer patients with a specimen available for study.

Methods: All patients had been entered on consecutive chemotherapeutic clinical trials that were all 5-fluorouracil based. The specimens were evaluated by fluorescence in situ hybridization to ascertain erbB2 and T2{alpha} gene amplification, and by immunohistochemical staining for T2{alpha} and TS protein expression.

Results: erbB2 amplification was detected in 16.7% of specimens, with co-amplification of the T2{alpha} gene in 40%, and 44% had undetectable TS protein expression. Kaplan–Meier survival curves showed significantly prolonged overall survival in patients with erbB2 and T2{alpha} gene amplification, T2{alpha} protein overexpression and absence of TS protein expression (P = 0.0011, P = 0.0048, P = 0.0061 and P = 0.0267, respectively, by log rank test). There was a positive correlation between erbB2 amplification and T2{alpha} amplification, T2{alpha} protein overexpression, and a trend towards absence of TS expression (P = 0.0001, P = 0.003 and P = 0.066 by Fisher's exact test).

Conclusion: High dose fluorouracil/leucovorin-based chemotherapy may have the potential to reverse the adverse effects resulting from erbB2 gene amplification in gastric cancer.

Key Words: gastric cancer • erbB2 • topoisomerase II{alpha} • survival • thymidylate synthase


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Gastric cancer ranks as the eighth most common cancer with the fourth highest mortality rate in Taiwan, the crude incidence and mortality rate being 15 and 11.7/100 000 population, respectively, with 3339 new cases and 2374 deaths reported in the year 2000 (1). The overall 5-year survival for gastric cancer is only 5–15%, because many patients present in a late stage of disease (2). Prognostic factors in association with adverse survival include a diffuse histological type (3), increased depth of cancer invasion, lymph node involvement, tumor aneuploidy, signet ring features and erbB2 expression/amplification (2). Amplification of the erbB2 gene as well as overexpression of the erbB2 protein has been reported in 10–16.4% of gastric cancers (4–8), with no ethnic differences in its occurrence when comparing Japanese and British patients (9). Amplification or overexpression of the erbB2 oncogene has been demonstrated to be an independent poor prognostic factor in gastric cancer (7), and portends for a shortened disease-free and overall survival in patients monitored after curative surgical resection (4–9), as is the case in node-positive breast cancer (10).

In vitro studies demonstrated that increased erbB2 mRNA expression was associated with increased resistance to doxorubicin, carmustine, cisplatin, melphalan, mitomycin and etoposide in a panel of non-small-cell lung cancer cell lines (11). These observations provide a logical drug resistance rationale as the basis of erbB2's adverse effects on patient survival. Analysis of the CALGB 8541 protocol concluded that erbB2 overexpression identified patients most likely to benefit from high doses of adjuvant doxorubicin, suggesting that adequate dosing could overcome the drug resistance inherent in erbB2 overexpression, preliminarily establishing erbB2 as a predictive marker for chemotherapy (12). Subsequent clinical trial analyses suggest that amplification/overexpression of the erbB2 oncogene was a moderate predictive factor for response to anthracyclines (13–16). Molecular genetic studies in erbB2-amplified primary breast cancers demonstrated that 44% had co-amplification of the topoisomease II{alpha} (T2{alpha}) gene and 42% showed a deletion of the gene, with no change in gene copy number in non-erbB2-amplified tumors; concurrent studies in erbB2-amplified cell lines showed that cell lines demonstrating co-amplification of the T2{alpha} gene showed increased sensitivity to doxorubicin, whereas those displaying T2{alpha} deletion manifested drug resistance (17).

Chemotherapeutic regimens of gastric cancers are diverse and varied. In Taiwan, a weekly 24 h or biweekly 48 h high dose 5-fluorouracil (5-FU)/leucovorin (HDFL) infusion-based regimen is usually given up front for gastric cancer, with response rates ranging from 40 to 80%, and median survival from 8 to 11 months (18–22).

In the study of this group of gastric cancer patients with metastatic disease who have received 5-FU-based chemotherapy, we have attempted to ascertain the status of the erbB2 gene and its related markers, including T2{alpha} gene amplification and protein expression, and another closely related marker, the thymidylate synthase (TS) protein, which is the target enzyme inhibited by 5-FU. We also attempted to evaluate the impact of these markers on survival in these patients after HDFL-based chemotherapy.


   SUBJECTS AND METHODS
TOP
 Abstract
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 References
 
PATIENTS
Thirty consecutive gastric cancer patients with metastatic disease, whose specimens were available for study, and who were enrolled in the chemotherapy clinical trials in our cancer division between 1996/7 and 1997/8, were selected for the study. There were 23 males and 7 females (Table 1). Biopsy alone was performed in 11 patients (including three patients who underwent an unsuccessful exploratory laparotomy); 19 underwent gastrectomies. Tumor pathology was reported as poorly differentiated carcinoma in 26 patients, and moderately differentiated in three erbB2-amplified and one erbB2 normal patient. All patients had signed informed consent for clinical trial registration prior to study enrollment review by the hospital Institutional Review Board. All but one patient had been followed until demise; a patient with peritoneal carcinomatosis is still alive 7 years after the initial diagnosis with a carcinoembryonic antigen (CEA) of 30 ng/dl, and continues to take ufur (tagafur + uracil) on a regular basis. These patients were invariably treated with a weekly or bi-weekly continuous infusion (24–48 h) of an HDFL-based regimen (FU, 1600–2600 mg/m2/leucovorin 150–300 mg/m2); see Table 1. Even after tumor progression, the HDFL backbone was carried over into all subsequent salvage regimens. All patients received therapy with almost no interruption up to 1–2 months before demise, when they were placed under hospice care.


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  		Table 1. Patient demographic data (n = 30), and types of chemotherapeutic agents administered

 
FLUORESCENCE IN SITU HYBRIDIZATION
A fluorescence in situ hybridization (FISH) study for erbB2 and T2{alpha} gene copy number was performed using the LSI topoisomerase II{alpha}/CEP and Pathvysion dual color probes (Vysis, IL) according to the manufacturer's instructions. Hybridization occurred with the orange T2{alpha}/erbB2 probe and green CEP17 (staining chromosome 17 centromere) probe (Fig. 1A). At least 50 cells were counted, and amplification of the erbB2 and/or T2{alpha} gene was defined as an orange:green signal ≥2. For this study, borderline amplification was reported if the ratio of orange to green was >1, but <2. The FISH slides are cross-read by the pathologist with the immunohistochemistry slides to ensure correct tumor identification.



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  		Figure 1. (A) Fluorescent in situ hybridization studies of the erbB2 and T2{alpha} gene. The green dots represent the chromosome 17 centromere, and the orange dots erbB2 or T2{alpha} genes. (a–c) erbB2 FISH; (d–f) T2{alpha} FISH. (a) A single cancer cell with 4.5- (18/4) fold amplification of the erbB2 gene. (b) A collection of cancer cells with normal copies of erbB2 per cell. (c) Two cancer cells with 4:2 and 3:2 orange to green dots, after counting 50 cells; the erbB2 to chromosome 17 centromere ratio was between 1 and 2, and defined as borderline amplification for this study. (d) Six-fold amplification of the T2{alpha} gene. (e) The T2{alpha} to chromosome 17 centromere ratio is exactly 1:1. (f) After counting 50 cells, the T2{alpha} to chromosome 17 centromere ratio was between 1 and 2, termed borderline amplification for this study. (B) Immunohistochemical studies of the T2{alpha} and thymidylate synthase (TS) protein expression. The T2{alpha} antibody stains tumor nuclei a reddish color, whereas the TS antibody predominantly stains the cytoplasm. (a) Between 20 and 30% of nuclei have been stained a reddish brown, indicating overexpression of T2{alpha} protein. (b) No staining detected for T2{alpha} protein. (c) A faint pinkish hue is seen in the cytoplasm of most cells, indicating overexpression of thymidylate synthase protein. (d) Negative staining for TS protein.

 
IMMUNOHISTOCHEMISTRY
Immunohistochemistry was done using a T2{alpha} antibody (a gift from J. L. Huang, Institute of Molecular Biology, Academia Sinica, Taiwan), and the TS Ab-1 (Lab Vision, CA), which recognizes a protein of 36 kDa. Briefly, 4–5 µm thick paraffin sections were fixed, deparaffinized, stained with three consecutive 10 min applications of the primary antibody, the secondary biotinylated antibody, then the streptavidin–biotin enzyme conjugate of the DAKO LSAB-2 system (Dako, CA). T2{alpha} was considered positive if >2% of cells showed a reddish brown coloring of the nucleus; at least 50 cells were counted (Fig. 1Ba). TS overexpression would be reported if there was a faint pink hue in the cytoplasm of the cell (Fig. 1Bc).

STATISTICAL ANALYSIS
For purposes of statistical analysis, overall survival was calculated from confirmation of metastatic disease until death. Response to chemotherapy was not evaluated because all patients received varying therapy. Survival was analyzed according to the Kaplan–Meier method, and assessed for significance stratified for different tumor markers using the log-rank test. Correlations between the various tumor markers were done using {chi}2 and Fisher's exact test.


   RESULTS
TOP
 Abstract
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 References
 
erbB2 amplification was demonstrated in five of 30 (16.7%) specimens, and one of 30 (3.3%) cases showed borderline amplification. Co-amplification of the T2{alpha} gene occurred in two of five (40%) patients with erbB2 gene amplification; borderline amplification was detected in three additional patients (including the case with borderline amplification of the erbB2 gene), with normal copy number in the remaining patient. Of the 24 patients with normal copies of the erbB2 gene, T2{alpha} gene copy number was ubiquitously normal. Overall survival time of our gastric cancer patients stratified according to erbB2 (P = 0.0011) and T2{alpha} (P = 0.0048) gene status showed that patients with amplification or borderline amplification of these two genes had significantly prolonged survival, contrary to what has been reported in the literature (Fig. 2A and B). Therefore, in order to investigate this phenomenon further, and since all patients had received 5-FU-based chemotherapy, it was decided to study T2{alpha} and TS protein expression. However, a second round of specimen retrieval only retrieved sufficient material in 27 patients.



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  		Figure 2. Kaplan–Meier survival curves of gastric cancer patients with metastatic disease after treatment with 5-fluorouracil-based chemotherapy, stratified according to selected markers, and assessed for significance using the log-rank test. Improved overall survival is found in patients with erbB2 gene amplification (A) (P = 0.001), T2{alpha} amplification (B) (P = 0.0048), T2{alpha} protein overexpression (C) (P = 0.0061), and absence of thymidylate synthase protein expression (D) (P = 0.0267).

 
In parallel with T2{alpha} gene amplification, T2{alpha} protein expression in tumor nuclei was detected in all six patients with erbB2 amplification/borderline amplification (including the patient with normal copies of the T2{alpha} gene), but also in two cases without T2{alpha} gene amplification. Cytoplasmic TS was undetectable in five of six erbB2-amplified patients, but overexpressed in seven of 21 patients with normal copies of the erbB2 gene. Overall survival was significantly prolonged in patients with T2{alpha} protein expression (P = 0.0061) and absence of TS protein expression (P = 0.0267) (Fig. 2C and D).

The clinico-pathological characteristics and chemotherapy regimen were similar between patients with or without erbB2 amplification/borderline amplification. Response to therapy is not reported for this study, since many patients had no measurable disease, including five of the six patients with increased erbB2 gene copy numbers who had peritoneal carcinomatosis without measurable disease. However, by following tumor markers such as CEA, four of six erbB2-amplified patients attained >50% reduction in tumor markers following HDFL-based therapy. Survival analysis stratified according to amplification or expression of the various oncogenes/drug targets is shown in Fig. 2. There was strong correlation between co-amplification of the erbB2 and T2{alpha} gene (Fisher's exact test, P = 0.0001), erbB2 gene amplification and T2{alpha} protein expression (Fisher's exact test, P = 0.003), and a trend towards negative correlation with TS expression (Fisher's exact test, P = 0.066), see Table 2.


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  		Table 2. Correlation between erbB2 gene status and selected marker gene amplification or protein expression

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
References
 
Amplification of the erbB2 gene occurred in 16.7% (five out of 30) of our gastric cancer patients, which is concordant with previous reports (4–6); co-amplification of the T2{alpha} gene occurred in 40–80% (counting borderline amplified cases) of those patients, concordant with previous breast cancer studies (17). A trend towards erbB2 amplification and absence of TS expression was noted in spite of the limited number of cases. Amplification of the erbB2 oncogene portends an adverse prognosis in breast, ovarian and also gastric cancer (4–10), but this adverse effect can potentially be reversed and manipulated to the patient's advantage by giving anthracycline in adequate doses (12–16). Evidence gained from cell line studies indicate that co-amplification of the T2{alpha} gene is instrumental in increasing sensitivity to T2{alpha}-targeting drugs (17), both substantiating and re-enforcing the clinical observations.

In a Korean study to evaluate the impact of erbB2 overexpression on treatment outcome in gastric cancer after surgery with curative intent, patients were randomized to adjuvant 5-FU/adriamycin or no further treatment. Follow-up analysis determined that in patients with tumors overexpressing the erbB2 protein, the treated subgroup had a prolonged time to local relapse, whereas those with no further treatment experienced a shortened disease-free and overall survival (23). ErbB2 expression predicts responsiveness to 5-FU/adriamycin, which could be an anthracycline effect, a 5-FU effect, or both. In a clinical trial using a bi-weekly HDFL backbone-based regimen for the treatment of pancreatic cancers, a patient with multiple liver metastases attained complete remission with an overall survival of 26 months; the tumor showed amplification of the erbB2 gene and absence of TS protein expression on immunohistochemistry (24).

Significantly prolonged survival of gastric cancer patients with erbB2 amplification after treatment with an HDFL-based regimen was unexpected, since T2{alpha}-targeting drugs were not incorporated into the regimen. Significant survival benefit was also seen in relation to T2{alpha} gene amplification, T2{alpha} protein overexpression and absence of TS expression. Co-amplification of the erbB2 and T2{alpha} genes results from their close proximity on chromosome 17q12–21 (17). Overexpression of the T2{alpha} protein was noted in all six patients with T2{alpha} gene amplification/borderline amplification, but also in four others without gene amplification; those patients all had polysomy (meaning that there were >2 copies of the T2{alpha} gene in their cells). TS is encoded by a gene located at chromosome 18p11–32 (25). It is the main drug target for 5-FU; undetectable expression of TS was significantly correlated to improved survival in our gastric cancer patients, concordant with previously reported data of increased response and prolonged survival in gastric cancer patients with low TS expression undergoing FL-based therapy (26,27). The rate and duration of TS inhibition by 5-FU was significantly greater when given as a 24 h infusion than as a short bolus injection; the prolonged infusion attained maximal tumor killing (28). It does not seem logical that patients with an amplified erbB2 gene attain prolonged survival after HDFL therapy, unless it is associated with a low or absent TS expression; in this study, a trend towards an inverse correlation to TS expression was found, and this should be explored further for validity in a larger prospective clinical trial. However, the survival benefit from HDFL therapy seems more pronounced in erbB2-amplified patients compared with non-TS-expressing patients, so that negative correlation between erbB2 and TS may not fully explain the survival benefit attained by erbB2-amplified patients after HDFL therapy. Although the patient numbers in this study are small, the survival benefit of patients with erbB2 amplification after HDFL therapy is significant, and may be partly attributed to absent TS expression in some tumors. We have established, nevertheless, that erbB2 is amplified in 16.7% of Chinese gastric cancer patients, with co-amplification of the T2{alpha} gene in 40% of cases, and TS protein levels are undetectable in 44% of tumors; these three factors were associated with prolonged overall survival in metastatic gastric cancer patients after high dose 5-FU-based chemotherapy.


   Acknowledgments
 
We thank our research nurses, Hsiao Wei Wu, Wei Chun Lin and Li Ting Shia, without whose meticulous work on data management this manuscipt would not have been possible. We are grateful to the Chen Shuyi Cancer Foundation for financial support.


   References
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 Abstract
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 References
 
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