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neu overexpression in 361 patients with ovarian cancer: a multicenter study
Gynecologic Oncology 95 (2004) 89 – 94 www.elsevier.com/locate/ygyno
The prognostic and predictive value of immunohistochemically detected HER-2/neu overexpression in 361 patients with ovarian cancer: a multicenter study
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Eva-Katrin Riener a,*, Norbert Arnoldb, Friedrich Kommossc, Stefan Lauingerb, Jacobus Pfistererb a
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Department of Obstetrics and Gynecology, University of Freiburg Medical School, Freiburg D-79098, Germany b Department of Obstetrics and Gynecology, University of Kiel Medical School, Kiel, Germany c Department of Pathology, University of Heidelberg Medical School, Heidelberg, Germany Received 23 October 2003 Available online 28 August 2004
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Retraction. The article, "The prognostic and predictive value of immunohistochemically detected HER-2/neu overexpression in 361 patients with ovarian cancer: a multicenter study," published in the October issue of the journal [Riener et al.:Gynecol Oncol 2004;95(1):89–94], has been retracted because the Editors and journal staff were notified after production and publication that not all authors had apparently approved the article in its present form. D 2004 Elsevier Inc. All rights reserved.
Introduction
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Tumor biology research has identified numerous growth factors that are involved in malignant transformation and tumor progression. Overexpression of growth factors and growth factor receptors adds to the proliferative burden of tumor cells via various cellular response pathways [1]. The HER family includes tyrosine kinase (TK) receptors with intrinsic TK activity. The human HER2/c-erbB-2/NEU gene encodes a protein called p185HER or HER-2/neu. HER-2/neu is a protooncogene similar to the epidermal growth factor receptor. Cytoplasmatic and mitochondrial expression of HER-2/neu has been documented in normal cells, whereas membrane expression of HER-2/neu seems to be limited to malignant cells [2]. HER-2/neu primarily acts as a heterodimerization partner of various other HER family members (erbB1-4) and controls the complex erB signaling network, mediating cell growth, and differentiation. HER-2/neu overexpression has been documented
* Corresponding author. Fax: +49 761 270 3190. E-mail address: [email protected] (E.-K. Riener). 0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2004.06.048
in various malignancies, among them breast cancer [3], hepatocellular carcinoma [4], multiple myeloma [5], endometrial cancer [6], and pancreatic cancer [7]. The use of an anti HER-2/neu antibody as adjunct to standard chemotherapy has already been incorporated into clinical practice in women with metastatic breast cancer. Of note, the monoclonal anti-HER-2/neu antibody trastuzumab has been shown to significantly increase the clinical benefit of chemotherapy in terms of time to disease progression, rate of response to chemotherapy, duration of response, survival, and risk of death in a prospective randomized trial of 469 women [3]. In contrast to breast cancer, however, less data on HER2/neu expression patterns and the clinical benefit of HER-2/ neu-related therapy strategies in epithelial ovarian cancer are available to date. Moreover, the available data are inconsistent and are often based on small patient samples using variable HER-2/neu staining techniques and scoring systems. For example, HER-2/neu overexpression has been documented to vary widely between 0% and 30% [8–12]. Several studies document a low level of HER-2/neu overexpression at the time of primary diagnosis of epithelial ovarian cancer, after platinum-based chemotherapy, as well
E.-K. Riener et al. / Gynecologic Oncology 95 (2004) 89–94
Materials and methods Patients
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The present study includes assessments of HER-2/neu overexpression in 361 Caucasian patients with ovarian cancer FIGO stages IIB (n = 10), IIC (n = 16), IIIA (n = 27), IIIB (n = 44), IIIC (n = 207), and IV (n = 57), treated between October 1995 and November 1997 during a multicenter study involving over 70 departments of Gynecology and Obstetrics throughout Germany, as described previously [17]. All women took part in a prospective-randomized trial comparing adjuvant taxol/ carboplatin and taxol/cisplatin (AGO-OVAR-3). Lymph node status was available for 207 patients. Lymph node involvement was noted in 104 (50.2%) of these patients. Median age at the time of diagnosis of ovarian cancer was 57.6 (range, 24–83) years. Histologically, 260 tumors were graded as serous adenocarcinomas, 11 as mucinous adenocarcinomas, 31 as endometrioid adenocarcinomas, 7 as mixed-type carcinomas, 20 as undifferentiated carcinomas, and 32 as others. Eighteen tumors were graded as well differentiated, 134 as moderately differentiated, and 198 as poorly differentiated. The median (range) duration of follow-up was 49.1 (38–63) months. Two hundred and fifty-two patients (69.8%) developed recurrent/progressive disease and 179 patients (49.6%) died of cancer-related death during the observation period. CA 125 serum levels were evaluated before surgery. Patients were treated and followed-up according to the AGO-OVAR-3 protocol [17].
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Clinical staging was performed according to the FIGO classification [18]. All cases were reviewed by an experienced pathologist, blinded to the clinical data. Immunohistochemistry
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Tissue specimens were cut into sections of 4–5 Am and mounted on silanized glass slides. Specimens were stored at room temperature between 1 and 42 days before staining. Immunohistochemical staining for HER-2/neu was performed as described previously [19]. Specifically, slides were dried for 60 min at 50–608C in an oven. Slides were then deparaffinized using xylene, ethanol 95%, ethanol 70%, and distilled water. To regain antigenicity, an epitope retrieval solution was used for 40 min in a 908C water bath. After cooling and rinsing, slides were covered with a peroxidase blocking reagent for 5 min, rinsed, and covered with the primary antibody or the negative control reagent (rabbit immunoglobulin) for 30 min. Visualization reagent (dextrane polymer-conjugated peroxidase and goat antirabbit-immunoglobulin) was applied for 30 min, followed by substrate–chromogen solution (DAB) for 10 min. Slides were then counterstained with hematoxylin, mounted, coverslipped, and scored. All tumor samples were scored according to the manufacturer’s instructions (DAKO score) [20] as well as according to Remmele’s score [21]. The DAKO score defines no membrane staining or membrane staining of less than 10% of the tumor cells as 0, weak and incomplete membrane staining of more than 10% of the tumor cells as 1+, weak or moderate complete membrane staining of more than 10% of the tumor cells as 2+, and strong complete membrane staining of more than 10% of the tumor cells as 3+. Scores of 0 and 1+ were interpreted as no HER-2/neu overexpression, scores of 2+ and 3+ were interpreted as HER-2/neu overexpression. Remmele’s score uses a 12 step scale, resulting from multiplying staining intensity (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining) and area of staining (0 = no staining, 1 = 1–9% stained area, 2 = 10–50% stained area, 3 = 51–80% stained area, 4 = 81–100% stained area). Scores of 0–5 were interpreted as no HER-2/neu overexpression, scores of 6–12 were interpreted as HER-2/neu overexpression. For every 7–14 tumor slides, a control slide with human breast cancer cells and three defined HER-2/neu intensity scores (0, 1+, and 3+) was stained and used as positive control. Two independent readers analyzed all samples. To avoid possible misstainings due to time delays, we performed an internal control experiment with 24 cases, two slides of which were stained immediately after cutting and after a 20-day storage period at room temperature. We did not observe any differences in staining intensity after 20 days. Since tissue slides were stored up to 42 days, however, we cannot rule out that staining delay may impact on the frequency of HER-2/neu overexpression.
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as at the time of relapse with over expression rates between 2% and 11% [8]. Some [13,14], but not others [10,11], found HER-2/neu overexpression to correlate with the duration of overall and disease-free survival. The most recently published studies from Denmark and France found HER-2/neu overexpression in 13% and 16%, respectively, and a positive correlation with disease-free and overall survival [15,16]. In summary, the biological role of HER-2/neu dysregulation in epithelial ovarian cancer is controversial and the clinical value of anti-HER-2/neu-specific therapies has not been established. The two largest studies, however, document a prognostic value of HER-2/neu overexpression [15,16]. The purpose of our study was to document HER2/neu expression patterns and their correlation with clinicopathologic parameters and survival in a large and biologically homogenous Caucasian patient collective. For this purpose, we evaluated the overexpression of HER-2/neu in 361 patients enrolled in a prospective-randomized multicenter clinical trial of advanced stage epithelial ovarian cancer International Federation of Gynecology and Obstetrics (FIGO) IIB–IV.
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Results
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All of the women investigated in this study took part in a randomized trial comparing two adjuvant chemotherapy regimens. Women randomized to the taxol/carboplatin and taxol/cisplatin arms showed equal distributions of tumors with HER-2/neu overexpression (6.6% [12/180] vs. 7.2% [13/181]; P = 0.8). HER-2/neu overexpression was detected in 6.9% (25/ 361) of the tumor samples. We observed a membranebound staining pattern in all specimens. HER-2/neu overexpression was significantly associated with tumor stage (P = 0.03), but showed no significant association with lymph node involvement (P = 0.5), tumor grade (P = 0.3), histological type (P = 0.6), residual tumor (P = 0.4), serum CA-125 before therapy (P = 0.2), and patient age (P = 0.8). HER-2/neu overexpression, specified as 0 or 1+, 2+, or 3+ positivity, broken down by tumor stage, lymph node involvement, tumor grade, and histological type, is shown in Table 1. There was no significant difference with regard to patient age, residual tumor, and serum CA-125 before therapy between patients with and without HER-2/ neu overexpression (median, 56.4 vs. 57.2 years; P = 0.8; 13 vs. 17 mm; P = 0.5; 1497 U/ml vs. 1314 U/ml; P = 0.7, respectively). Two independent readers scored the tumor samples using the DAKO and Remmele’s scores. Concordant and disconcordant scores using the DAKO score are given in Table 2. Using the 12-scale Remmele’s score, the two readers had identical readings in 289/358 (80.7%) cases. In 323/358 (90.2%) cases, the readings were identical within the range of F1 scale point. Of the 25 tumors overexpressing HER-2/neu, we performed a fluorescence in situ hybridization (FISH) analysis in 21 tumors. Fourteen and seven of these tumors showed a 2+ and 3+ HER-2/neu overexpression on immunohistochemistry with 0/14 and 4/7 showing HER-2/ neu amplification on FISH.
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Chi-square test was used where appropriate. Survival probabilities were calculated by the product limit method of Kaplan and Meier. Differences between groups were tested using the log-rank test. The results were analyzed for the endpoints of disease-free and overall survival. Survival times of patients disease-free or still alive at the time of last follow-up were censored with the last follow-up date. Comparisons between unpaired groups were made using the Wilcoxon test. Correlations are described using Spearman’s correlation coefficient and Pearson’s correlation coefficient. P values b0.05 were considered statistically significant. We used the SAS statistical software system (SAS Institute Inc., Cary, NC) to do the calculations.
Figs. 1 and 2 show Kaplan–Meier survival analyses for HER-2/neu overexpression and disease-free and overall survival, respectively. In a univariate logistic regression model, HER-2/neu overexpression was not associated with disease-free and overall survival in patients with ovarian cancer (P = 0.6; odds ratio [OR] 1.2, 95% confidence interval [CI], 0.7–1.9 and P = 0.2; OR 1.4, 95% CI 0.8–2.4, respectively). To assess the influence of tumor characteristics such as FIGO stage, tumor grade, and residual tumor on the relationship between HER-2/neu overexpression and disease-free and overall survival, we performed a matched pair analysis. In this analysis, the impact of HER-2/neu overexpression on overall and disease-free survival was compared between women with matched FIGO stages, tumor grades, and residual tumors. We found no significant differences with respect to overall and disease-free survival comparing HER-2/neu overexpression in women with identical FIGO stage (P = 0.7 and P = 0.5, respectively), tumor grade (P = 0.5 and P = 0.4, respectively), and residual tumor (P = 0.2 and P = 0.3, respectively). To test HER-2/neu overexpression as a predictive marker, we evaluated whether HER-2/neu overexpression was associated with response to adjuvant chemotherapy. In a subset of 73 suboptimally debulked patients, pathologic response to adjuvant chemotherapy was evaluated by second-look laparotomy. Suboptimal debulking was defined
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Statistics
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Table 1 HER-2/neu overexpression and clinicopathologic parameters in 361 women with ovarian cancer Clinicopathologic parameters
HER-2/neu Expression DAKO 0, 1+ (%)
DAKO 2+, 3+ (%)
P valuea
9 14 27 44 193 49
(2.7) (4.2) (8.0) (13.1) (57.4) (14.6)
1 (4.0) 2 (8.0) – – 14 (56.0) 8 (32.0)
0.02
94 (49.5) 96 (50.5)
10 (58.8) 7 (41.2)
0.4
Tumor grade G1 G2 G3
18 (5.5) 125 (38.5) 182 (56.0)
– 98 (36.0) 16 (64.0)
0.4
Histological type Serous papillary Endometrioid Mucinous Mixed type Undifferentiated Others
241 29 10 7 18 31
19 2 1 0 2 1
0.6
FIGO stage IIB IIC IIIA IIIB IIIC IV Lymph node involvement Yes No
a
Chi-square test.
(92.7) (93.5) (90.9) (100) (90) (96.9)
(7.3) (6.5) (9.1) (0) (10) (3.1)
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Table 2 HER-2/neu overexpression by two independent readers using the DAKO score Reader
Missing 0 1+ 2+ 3+
1 258 80 16 6
2 2 272 62 17 8
(71.7) (22.2) (4.4) (1.7)
(75.8) (17.3) (4.7) (2.2)
Fig. 2. Kaplan–Meier analysis of overall survival of 361 patients with primary ovarian cancer with HER-2/neu overexpression (dashed line; n = 25) versus patients without HER-2/neu overexpression (full line; n = 336).
between readers 1 and 2 was 0.82 (Spearman’s correlation coefficient).
Discussion
We present a large multicenter study investigating the prognostic impact of HER-2/neu overexpression, previously shown to correlate with the clinical outcome of patients with ovarian cancer in some [13–16], but not in other studies [10,11]. In our series, we confirm recent results in that HER2/neu overexpression was limited to a fraction of patients, that is, 6.9%. HER-2/neu overexpression was not associated with the patients’ disease-free and overall survival length independent of FIGO stage, tumor grading, and residual tumor. Of note, HER-2/neu overexpression was predictive of the response to first-line chemotherapy in suboptimally debulked patients. Although extensively investigated in ovarian cancer, data in the literature on HER-2/neu overexpression are inconsistent. Thus, the clinical value of immunohistochemically detected HER-2/neu overexpression in ovarian cancer has not been established to date. Various reasons may account for the discrepancies reported in previous studies, among them low numbers of patients, different scoring systems, various HER-2/neu antibodies, as well as heterogeneous patient samples with respect to tumor stage and ethnic background. Therefore, we conducted a multicenter study involving a large number of ethnically homogeneous patients from various gynecologic oncology centers in Germany. All patients had advanced stage disease. We used the DAKO scoring system that has emerged as the scoring system of choice in recent years [22]. It has to be acknowledged, however, that the DAKO score is based on and recommended for breast cancer tissue. It is therefore of note that the use of this score as well as the cut-off points for ovarian cancer tissue is experimental. However, we think that it is reasonable to extrapolate IHC
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as presence of residual tumor N1 cm after primary surgical therapy. Women with response to first-line chemotherapy (complete remission [CR] + partial remission [PR]) and no response to first-line chemotherapy (stable disease [SD] + progressive disease [PD]) showed significantly different rates of HER-2/neu overexpression (0% [0/51] vs. 14% [3/ 22]; P = 0.02). All tumor samples were scored according to the DAKO score as well as according to Remmele’s score. Using the DAKO score and the Remmele’s score, HER-2/neu overexpression was detected in 6.9% (25/361) and 6.4% (23/ 361) of the tumor samples, respectively (P = 0.9). The correlation coefficient between HER-2/neu overexpression scores using the DAKO score and the Remmele’s score was 0.81 (Pearson’s correlation coefficient). The prognostic value did not differ between the two scoring methods (DAKO and Remmele). When calculating the survival analyses for overall and disease-free survival length using the Remmele’s score, including the cases that were differentially scored by the Remmele’s scoring method, we also found no significant difference between the groups with and without HER-2/neu overexpression (data not shown). All tumor samples were scored by two independent readers. Using the DAKO score, HER-2/neu overexpression was detected in 6.1% (22/360; reader 1) and 6.9% (25/361; reader 2) of the tumor samples, respectively (P = 1.0). The correlation coefficient of HER-2/neu overexpression scores
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DAKO score
Fig. 1. Kaplan–Meier analysis of disease-free survival of 361 patients with primary ovarian cancer with HER-2/neu overexpression (dashed line; n = 25) versus patients without HER-2/neu overexpression (full line; n = 336).
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patients with epithelial ovarian cancer is questionable. Bookman et al. [9] found a markedly low rate (7.3%) of objective responses to a combination of chemotherapy and anti-HER-2/neu antibody among patients with HER-2/neu overexpression and recurrent or refractory ovarian cancer. Despite the lack of prognostic information of HER-2/neu overexpression, we found that HER-2/neu overexpression was predictive of the response to first-line chemotherapy in suboptimally debulked patients. HER-2/neu overexpression was significantly more common among women who did not respond to first-line chemotherapy compared to those who had evidence of complete or partial remission. This result of our study, however, has to be interpreted with caution, since it is based on a limited subset of only 73 patients, and the influence of the two chemotherapy regimens used in these patients has not been assessed. It is of note, however, that HER-2/neu overexpression has also been correlated with poor responsiveness to chemotherapy and chemoradiotherapy in other malignomas, for example, breast and esophageal cancer [27,28]. In summary, our data confirm previously published results of a low rate of HER-2/neu overexpression and a lack of prognostic value in patients with advanced stage epithelial ovarian cancer. According to our findings and the findings of others, tumor overexpression of HER-2/neu is rare and provides no prognostic information in addition to that provided by established clinicopathologic parameters. Clinical trials involving HER-2/neu targeted therapy seem to be of limited value. On the other hand, however, our data indicate that HER-2/neu overexpression might be clinically useful as a predictive marker in suboptimally debulked patients.
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expression patterns from experience with breast cancer tissue to ovarian cancer tissue. This has also been done by other groups, for example, Hogdall et al. [15] and Varshney et al. [23], and the DAKO score appears to have become the scoring method of choice for most investigators dealing with HER-2/neu immunohistochemistry in ovarian cancer tissue. Of note, the only tumor characteristic positively associated with HER-2/neu overexpression was FIGO stage. Therefore, the low rate of HER-2/neu overexpression in our series, which was limited to advanced stage disease, is unlikely to be biased by low HER-2/neu gene amplification and overexpression in early stage cancer, as reported in previous series [24,25]. In addition, recent data from Finland confirm a low HER-2/neu overexpression rate of 7% in a series of 401 serous ovarian carcinomas [26]. The technique of immunohistochemistry itself can be challenged due to its semiquantitative nature, differing embedding procedures in different centers, and inter- and intra-observer variability in scoring immunohistochemical slides. Therefore, we addressed these issues and found an excellent correlation between the two independent readers as well as the two different scoring systems used, that is, the DAKO score and Remmele’s score. There are limitations to our study. Since we have only investigated Caucasian women, the results of our study cannot be extended to women of other ethnic groups. Another limitation is that only women with advanced stage disease were enclosed in this study. Thus, no statements on the prognostic value of HER-2/neu overexpression in early stage ovarian cancer can be made based on our data. In addition, the low level of HER-2/neu overexpression may also, at least in part, be caused by our exact consideration and exclusion of artifacts, such as edge artifacts, retraction artifacts, and thermal artifacts. Previous studies may have scored these stainings as HER-2/neu overexpression. Since the identification of artifacts is not standardized, this may compromise the comparability between studies. The results of our study demonstrate a low level of HER-2/ neu overexpression at the time of primary diagnosis of epithelial ovarian cancer, and we could not assess a prognostic impact of HER-2/neu overexpression on the duration of disease-free and overall survival. As mentioned above, recently published studies showed similar results [8,22]. Of note, in these studies, the FDA approved DAKO HercepTest and the DAKO scoring system was also used, whereas older studies often used different antibodies and different scoring systems. These findings have clinical implications, because a low level of HER-2/neu overexpression in a rare disease such as ovarian cancer makes HER-2/ neu an unsuitable target for testing innovative therapy strategies. A large number of patients would be necessary to achieve a meaningful statistical power for the detection of small survival benefits in the adjuvant setting. Moreover, a clinical benefit of anti-HER-2/neu antibody-based therapy in
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The prognostic and predictive value of immunohistochemically detected HER-2/neu overexpression in 361 patients with ovarian cancer: a multicenter study
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Eva-Katrin Riener a,*, Norbert Arnoldb, Friedrich Kommossc, Stefan Lauingerb, Jacobus Pfistererb a
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Department of Obstetrics and Gynecology, University of Freiburg Medical School, Freiburg D-79098, Germany b Department of Obstetrics and Gynecology, University of Kiel Medical School, Kiel, Germany c Department of Pathology, University of Heidelberg Medical School, Heidelberg, Germany Received 23 October 2003 Available online 28 August 2004
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Retraction. The article, "The prognostic and predictive value of immunohistochemically detected HER-2/neu overexpression in 361 patients with ovarian cancer: a multicenter study," published in the October issue of the journal [Riener et al.:Gynecol Oncol 2004;95(1):89–94], has been retracted because the Editors and journal staff were notified after production and publication that not all authors had apparently approved the article in its present form. D 2004 Elsevier Inc. All rights reserved.
Introduction
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Tumor biology research has identified numerous growth factors that are involved in malignant transformation and tumor progression. Overexpression of growth factors and growth factor receptors adds to the proliferative burden of tumor cells via various cellular response pathways [1]. The HER family includes tyrosine kinase (TK) receptors with intrinsic TK activity. The human HER2/c-erbB-2/NEU gene encodes a protein called p185HER or HER-2/neu. HER-2/neu is a protooncogene similar to the epidermal growth factor receptor. Cytoplasmatic and mitochondrial expression of HER-2/neu has been documented in normal cells, whereas membrane expression of HER-2/neu seems to be limited to malignant cells [2]. HER-2/neu primarily acts as a heterodimerization partner of various other HER family members (erbB1-4) and controls the complex erB signaling network, mediating cell growth, and differentiation. HER-2/neu overexpression has been documented
* Corresponding author. Fax: +49 761 270 3190. E-mail address: [email protected] (E.-K. Riener). 0090-8258/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2004.06.048
in various malignancies, among them breast cancer [3], hepatocellular carcinoma [4], multiple myeloma [5], endometrial cancer [6], and pancreatic cancer [7]. The use of an anti HER-2/neu antibody as adjunct to standard chemotherapy has already been incorporated into clinical practice in women with metastatic breast cancer. Of note, the monoclonal anti-HER-2/neu antibody trastuzumab has been shown to significantly increase the clinical benefit of chemotherapy in terms of time to disease progression, rate of response to chemotherapy, duration of response, survival, and risk of death in a prospective randomized trial of 469 women [3]. In contrast to breast cancer, however, less data on HER2/neu expression patterns and the clinical benefit of HER-2/ neu-related therapy strategies in epithelial ovarian cancer are available to date. Moreover, the available data are inconsistent and are often based on small patient samples using variable HER-2/neu staining techniques and scoring systems. For example, HER-2/neu overexpression has been documented to vary widely between 0% and 30% [8–12]. Several studies document a low level of HER-2/neu overexpression at the time of primary diagnosis of epithelial ovarian cancer, after platinum-based chemotherapy, as well
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Materials and methods Patients
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The present study includes assessments of HER-2/neu overexpression in 361 Caucasian patients with ovarian cancer FIGO stages IIB (n = 10), IIC (n = 16), IIIA (n = 27), IIIB (n = 44), IIIC (n = 207), and IV (n = 57), treated between October 1995 and November 1997 during a multicenter study involving over 70 departments of Gynecology and Obstetrics throughout Germany, as described previously [17]. All women took part in a prospective-randomized trial comparing adjuvant taxol/ carboplatin and taxol/cisplatin (AGO-OVAR-3). Lymph node status was available for 207 patients. Lymph node involvement was noted in 104 (50.2%) of these patients. Median age at the time of diagnosis of ovarian cancer was 57.6 (range, 24–83) years. Histologically, 260 tumors were graded as serous adenocarcinomas, 11 as mucinous adenocarcinomas, 31 as endometrioid adenocarcinomas, 7 as mixed-type carcinomas, 20 as undifferentiated carcinomas, and 32 as others. Eighteen tumors were graded as well differentiated, 134 as moderately differentiated, and 198 as poorly differentiated. The median (range) duration of follow-up was 49.1 (38–63) months. Two hundred and fifty-two patients (69.8%) developed recurrent/progressive disease and 179 patients (49.6%) died of cancer-related death during the observation period. CA 125 serum levels were evaluated before surgery. Patients were treated and followed-up according to the AGO-OVAR-3 protocol [17].
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Clinical staging was performed according to the FIGO classification [18]. All cases were reviewed by an experienced pathologist, blinded to the clinical data. Immunohistochemistry
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Tissue specimens were cut into sections of 4–5 Am and mounted on silanized glass slides. Specimens were stored at room temperature between 1 and 42 days before staining. Immunohistochemical staining for HER-2/neu was performed as described previously [19]. Specifically, slides were dried for 60 min at 50–608C in an oven. Slides were then deparaffinized using xylene, ethanol 95%, ethanol 70%, and distilled water. To regain antigenicity, an epitope retrieval solution was used for 40 min in a 908C water bath. After cooling and rinsing, slides were covered with a peroxidase blocking reagent for 5 min, rinsed, and covered with the primary antibody or the negative control reagent (rabbit immunoglobulin) for 30 min. Visualization reagent (dextrane polymer-conjugated peroxidase and goat antirabbit-immunoglobulin) was applied for 30 min, followed by substrate–chromogen solution (DAB) for 10 min. Slides were then counterstained with hematoxylin, mounted, coverslipped, and scored. All tumor samples were scored according to the manufacturer’s instructions (DAKO score) [20] as well as according to Remmele’s score [21]. The DAKO score defines no membrane staining or membrane staining of less than 10% of the tumor cells as 0, weak and incomplete membrane staining of more than 10% of the tumor cells as 1+, weak or moderate complete membrane staining of more than 10% of the tumor cells as 2+, and strong complete membrane staining of more than 10% of the tumor cells as 3+. Scores of 0 and 1+ were interpreted as no HER-2/neu overexpression, scores of 2+ and 3+ were interpreted as HER-2/neu overexpression. Remmele’s score uses a 12 step scale, resulting from multiplying staining intensity (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining) and area of staining (0 = no staining, 1 = 1–9% stained area, 2 = 10–50% stained area, 3 = 51–80% stained area, 4 = 81–100% stained area). Scores of 0–5 were interpreted as no HER-2/neu overexpression, scores of 6–12 were interpreted as HER-2/neu overexpression. For every 7–14 tumor slides, a control slide with human breast cancer cells and three defined HER-2/neu intensity scores (0, 1+, and 3+) was stained and used as positive control. Two independent readers analyzed all samples. To avoid possible misstainings due to time delays, we performed an internal control experiment with 24 cases, two slides of which were stained immediately after cutting and after a 20-day storage period at room temperature. We did not observe any differences in staining intensity after 20 days. Since tissue slides were stored up to 42 days, however, we cannot rule out that staining delay may impact on the frequency of HER-2/neu overexpression.
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as at the time of relapse with over expression rates between 2% and 11% [8]. Some [13,14], but not others [10,11], found HER-2/neu overexpression to correlate with the duration of overall and disease-free survival. The most recently published studies from Denmark and France found HER-2/neu overexpression in 13% and 16%, respectively, and a positive correlation with disease-free and overall survival [15,16]. In summary, the biological role of HER-2/neu dysregulation in epithelial ovarian cancer is controversial and the clinical value of anti-HER-2/neu-specific therapies has not been established. The two largest studies, however, document a prognostic value of HER-2/neu overexpression [15,16]. The purpose of our study was to document HER2/neu expression patterns and their correlation with clinicopathologic parameters and survival in a large and biologically homogenous Caucasian patient collective. For this purpose, we evaluated the overexpression of HER-2/neu in 361 patients enrolled in a prospective-randomized multicenter clinical trial of advanced stage epithelial ovarian cancer International Federation of Gynecology and Obstetrics (FIGO) IIB–IV.
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Results
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All of the women investigated in this study took part in a randomized trial comparing two adjuvant chemotherapy regimens. Women randomized to the taxol/carboplatin and taxol/cisplatin arms showed equal distributions of tumors with HER-2/neu overexpression (6.6% [12/180] vs. 7.2% [13/181]; P = 0.8). HER-2/neu overexpression was detected in 6.9% (25/ 361) of the tumor samples. We observed a membranebound staining pattern in all specimens. HER-2/neu overexpression was significantly associated with tumor stage (P = 0.03), but showed no significant association with lymph node involvement (P = 0.5), tumor grade (P = 0.3), histological type (P = 0.6), residual tumor (P = 0.4), serum CA-125 before therapy (P = 0.2), and patient age (P = 0.8). HER-2/neu overexpression, specified as 0 or 1+, 2+, or 3+ positivity, broken down by tumor stage, lymph node involvement, tumor grade, and histological type, is shown in Table 1. There was no significant difference with regard to patient age, residual tumor, and serum CA-125 before therapy between patients with and without HER-2/ neu overexpression (median, 56.4 vs. 57.2 years; P = 0.8; 13 vs. 17 mm; P = 0.5; 1497 U/ml vs. 1314 U/ml; P = 0.7, respectively). Two independent readers scored the tumor samples using the DAKO and Remmele’s scores. Concordant and disconcordant scores using the DAKO score are given in Table 2. Using the 12-scale Remmele’s score, the two readers had identical readings in 289/358 (80.7%) cases. In 323/358 (90.2%) cases, the readings were identical within the range of F1 scale point. Of the 25 tumors overexpressing HER-2/neu, we performed a fluorescence in situ hybridization (FISH) analysis in 21 tumors. Fourteen and seven of these tumors showed a 2+ and 3+ HER-2/neu overexpression on immunohistochemistry with 0/14 and 4/7 showing HER-2/ neu amplification on FISH.
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Chi-square test was used where appropriate. Survival probabilities were calculated by the product limit method of Kaplan and Meier. Differences between groups were tested using the log-rank test. The results were analyzed for the endpoints of disease-free and overall survival. Survival times of patients disease-free or still alive at the time of last follow-up were censored with the last follow-up date. Comparisons between unpaired groups were made using the Wilcoxon test. Correlations are described using Spearman’s correlation coefficient and Pearson’s correlation coefficient. P values b0.05 were considered statistically significant. We used the SAS statistical software system (SAS Institute Inc., Cary, NC) to do the calculations.
Figs. 1 and 2 show Kaplan–Meier survival analyses for HER-2/neu overexpression and disease-free and overall survival, respectively. In a univariate logistic regression model, HER-2/neu overexpression was not associated with disease-free and overall survival in patients with ovarian cancer (P = 0.6; odds ratio [OR] 1.2, 95% confidence interval [CI], 0.7–1.9 and P = 0.2; OR 1.4, 95% CI 0.8–2.4, respectively). To assess the influence of tumor characteristics such as FIGO stage, tumor grade, and residual tumor on the relationship between HER-2/neu overexpression and disease-free and overall survival, we performed a matched pair analysis. In this analysis, the impact of HER-2/neu overexpression on overall and disease-free survival was compared between women with matched FIGO stages, tumor grades, and residual tumors. We found no significant differences with respect to overall and disease-free survival comparing HER-2/neu overexpression in women with identical FIGO stage (P = 0.7 and P = 0.5, respectively), tumor grade (P = 0.5 and P = 0.4, respectively), and residual tumor (P = 0.2 and P = 0.3, respectively). To test HER-2/neu overexpression as a predictive marker, we evaluated whether HER-2/neu overexpression was associated with response to adjuvant chemotherapy. In a subset of 73 suboptimally debulked patients, pathologic response to adjuvant chemotherapy was evaluated by second-look laparotomy. Suboptimal debulking was defined
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Table 1 HER-2/neu overexpression and clinicopathologic parameters in 361 women with ovarian cancer Clinicopathologic parameters
HER-2/neu Expression DAKO 0, 1+ (%)
DAKO 2+, 3+ (%)
P valuea
9 14 27 44 193 49
(2.7) (4.2) (8.0) (13.1) (57.4) (14.6)
1 (4.0) 2 (8.0) – – 14 (56.0) 8 (32.0)
0.02
94 (49.5) 96 (50.5)
10 (58.8) 7 (41.2)
0.4
Tumor grade G1 G2 G3
18 (5.5) 125 (38.5) 182 (56.0)
– 98 (36.0) 16 (64.0)
0.4
Histological type Serous papillary Endometrioid Mucinous Mixed type Undifferentiated Others
241 29 10 7 18 31
19 2 1 0 2 1
0.6
FIGO stage IIB IIC IIIA IIIB IIIC IV Lymph node involvement Yes No
a
Chi-square test.
(92.7) (93.5) (90.9) (100) (90) (96.9)
(7.3) (6.5) (9.1) (0) (10) (3.1)
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Table 2 HER-2/neu overexpression by two independent readers using the DAKO score Reader
Missing 0 1+ 2+ 3+
1 258 80 16 6
2 2 272 62 17 8
(71.7) (22.2) (4.4) (1.7)
(75.8) (17.3) (4.7) (2.2)
Fig. 2. Kaplan–Meier analysis of overall survival of 361 patients with primary ovarian cancer with HER-2/neu overexpression (dashed line; n = 25) versus patients without HER-2/neu overexpression (full line; n = 336).
between readers 1 and 2 was 0.82 (Spearman’s correlation coefficient).
Discussion
We present a large multicenter study investigating the prognostic impact of HER-2/neu overexpression, previously shown to correlate with the clinical outcome of patients with ovarian cancer in some [13–16], but not in other studies [10,11]. In our series, we confirm recent results in that HER2/neu overexpression was limited to a fraction of patients, that is, 6.9%. HER-2/neu overexpression was not associated with the patients’ disease-free and overall survival length independent of FIGO stage, tumor grading, and residual tumor. Of note, HER-2/neu overexpression was predictive of the response to first-line chemotherapy in suboptimally debulked patients. Although extensively investigated in ovarian cancer, data in the literature on HER-2/neu overexpression are inconsistent. Thus, the clinical value of immunohistochemically detected HER-2/neu overexpression in ovarian cancer has not been established to date. Various reasons may account for the discrepancies reported in previous studies, among them low numbers of patients, different scoring systems, various HER-2/neu antibodies, as well as heterogeneous patient samples with respect to tumor stage and ethnic background. Therefore, we conducted a multicenter study involving a large number of ethnically homogeneous patients from various gynecologic oncology centers in Germany. All patients had advanced stage disease. We used the DAKO scoring system that has emerged as the scoring system of choice in recent years [22]. It has to be acknowledged, however, that the DAKO score is based on and recommended for breast cancer tissue. It is therefore of note that the use of this score as well as the cut-off points for ovarian cancer tissue is experimental. However, we think that it is reasonable to extrapolate IHC
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as presence of residual tumor N1 cm after primary surgical therapy. Women with response to first-line chemotherapy (complete remission [CR] + partial remission [PR]) and no response to first-line chemotherapy (stable disease [SD] + progressive disease [PD]) showed significantly different rates of HER-2/neu overexpression (0% [0/51] vs. 14% [3/ 22]; P = 0.02). All tumor samples were scored according to the DAKO score as well as according to Remmele’s score. Using the DAKO score and the Remmele’s score, HER-2/neu overexpression was detected in 6.9% (25/361) and 6.4% (23/ 361) of the tumor samples, respectively (P = 0.9). The correlation coefficient between HER-2/neu overexpression scores using the DAKO score and the Remmele’s score was 0.81 (Pearson’s correlation coefficient). The prognostic value did not differ between the two scoring methods (DAKO and Remmele). When calculating the survival analyses for overall and disease-free survival length using the Remmele’s score, including the cases that were differentially scored by the Remmele’s scoring method, we also found no significant difference between the groups with and without HER-2/neu overexpression (data not shown). All tumor samples were scored by two independent readers. Using the DAKO score, HER-2/neu overexpression was detected in 6.1% (22/360; reader 1) and 6.9% (25/361; reader 2) of the tumor samples, respectively (P = 1.0). The correlation coefficient of HER-2/neu overexpression scores
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Fig. 1. Kaplan–Meier analysis of disease-free survival of 361 patients with primary ovarian cancer with HER-2/neu overexpression (dashed line; n = 25) versus patients without HER-2/neu overexpression (full line; n = 336).
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patients with epithelial ovarian cancer is questionable. Bookman et al. [9] found a markedly low rate (7.3%) of objective responses to a combination of chemotherapy and anti-HER-2/neu antibody among patients with HER-2/neu overexpression and recurrent or refractory ovarian cancer. Despite the lack of prognostic information of HER-2/neu overexpression, we found that HER-2/neu overexpression was predictive of the response to first-line chemotherapy in suboptimally debulked patients. HER-2/neu overexpression was significantly more common among women who did not respond to first-line chemotherapy compared to those who had evidence of complete or partial remission. This result of our study, however, has to be interpreted with caution, since it is based on a limited subset of only 73 patients, and the influence of the two chemotherapy regimens used in these patients has not been assessed. It is of note, however, that HER-2/neu overexpression has also been correlated with poor responsiveness to chemotherapy and chemoradiotherapy in other malignomas, for example, breast and esophageal cancer [27,28]. In summary, our data confirm previously published results of a low rate of HER-2/neu overexpression and a lack of prognostic value in patients with advanced stage epithelial ovarian cancer. According to our findings and the findings of others, tumor overexpression of HER-2/neu is rare and provides no prognostic information in addition to that provided by established clinicopathologic parameters. Clinical trials involving HER-2/neu targeted therapy seem to be of limited value. On the other hand, however, our data indicate that HER-2/neu overexpression might be clinically useful as a predictive marker in suboptimally debulked patients.
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expression patterns from experience with breast cancer tissue to ovarian cancer tissue. This has also been done by other groups, for example, Hogdall et al. [15] and Varshney et al. [23], and the DAKO score appears to have become the scoring method of choice for most investigators dealing with HER-2/neu immunohistochemistry in ovarian cancer tissue. Of note, the only tumor characteristic positively associated with HER-2/neu overexpression was FIGO stage. Therefore, the low rate of HER-2/neu overexpression in our series, which was limited to advanced stage disease, is unlikely to be biased by low HER-2/neu gene amplification and overexpression in early stage cancer, as reported in previous series [24,25]. In addition, recent data from Finland confirm a low HER-2/neu overexpression rate of 7% in a series of 401 serous ovarian carcinomas [26]. The technique of immunohistochemistry itself can be challenged due to its semiquantitative nature, differing embedding procedures in different centers, and inter- and intra-observer variability in scoring immunohistochemical slides. Therefore, we addressed these issues and found an excellent correlation between the two independent readers as well as the two different scoring systems used, that is, the DAKO score and Remmele’s score. There are limitations to our study. Since we have only investigated Caucasian women, the results of our study cannot be extended to women of other ethnic groups. Another limitation is that only women with advanced stage disease were enclosed in this study. Thus, no statements on the prognostic value of HER-2/neu overexpression in early stage ovarian cancer can be made based on our data. In addition, the low level of HER-2/neu overexpression may also, at least in part, be caused by our exact consideration and exclusion of artifacts, such as edge artifacts, retraction artifacts, and thermal artifacts. Previous studies may have scored these stainings as HER-2/neu overexpression. Since the identification of artifacts is not standardized, this may compromise the comparability between studies. The results of our study demonstrate a low level of HER-2/ neu overexpression at the time of primary diagnosis of epithelial ovarian cancer, and we could not assess a prognostic impact of HER-2/neu overexpression on the duration of disease-free and overall survival. As mentioned above, recently published studies showed similar results [8,22]. Of note, in these studies, the FDA approved DAKO HercepTest and the DAKO scoring system was also used, whereas older studies often used different antibodies and different scoring systems. These findings have clinical implications, because a low level of HER-2/neu overexpression in a rare disease such as ovarian cancer makes HER-2/ neu an unsuitable target for testing innovative therapy strategies. A large number of patients would be necessary to achieve a meaningful statistical power for the detection of small survival benefits in the adjuvant setting. Moreover, a clinical benefit of anti-HER-2/neu antibody-based therapy in
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