Findings

Background

The anti-Her-2 treatment is widely accepted in treating Her-2 expressing breast carcinoma cases, with a significant implication upon their survival [1]. These successful results triggered the optimistic hypothesis that anti-Her-2 treatment could be an option against any carcinoma expressing Her-2. The last decade epithelial ovarian cancer (EOC) has been reported as potentially Her-2 expressing, with a positivity ranging from 4.9% to 70.4% [2, 3]. However its role in EOC survival is at least controversial. Despite this controversy and due to the significant impact of anti-Her-2 treatment on breast cancer, several groups have studied the role of trastuzumab or pertuzumab in Her-2 expressing EOC patients [4, 5]. Interestingly, Her-2 has been reported as interacting with G-protein coupled receptors (GPCRs), both at a signalling and an extracellular level [6]. Of note is the fact that EOC has been reported as widely expressing follicle stimulating hormone receptor (FSHR) [7]. The FSHR expression has been found as a negative prognosticator in EOC patients [7]. Considering the interaction of Her-2 and FSH as possible, we have studied the effect of Her-2 in survival of EOC patients by stratifying them according to their FSHR status.

Patients and methods

Formalin fixed paraffin embedded (FFPE) tissue of 153 EOC patients (mean age: 58.71 years) who had undergone surgery for EOC from 1990 to 2002 at our department were analysed. Histological characterization (serous: n = 108, mucinous: n = 12, endometrioid: n = 21, clear cell: n = 12) and WHO tumour grading (G1: n = 36, G2: n = 52, G3: n = 53) was performed by an experienced gynaecological pathologist (DM). Data on FIGO stage (I: n = 34, II: n = 9, III: n = 102, IV: n = 3) and survival (median: 3.41 years; deaths: n = 103) were available. Patients staged as FIGO II - IV received carboplatin/paclitaxel as an adjuvant chemotherapy. The study was carried out in compliance with the guidelines of the Helsinki Declaration (1975) and was approved by the Ethics Committee of the Ludwig-Maximilians-University of Munich.

FFPE ovarian cancer samples were stained using an FDA-approved Ventana PATHWAY anti-Her-2 (4B5) Rabbit monoclonal antibody (Roche, Mannheim, Germany) on a VENTANA®-Unit (Roche, Mannheim, Germany) and scored as recommended by the manufacturer as 3+, 2+, 1+ or 0 by two blinded independent observers including a gynaecological pathologist (DM) by consensus. Cases that were scored as 2+ underwent fluorescence in situ hybridisation (FISH) as described previously [8]. Immunohistochemical staining for the FSH receptor was assessed as already described by our group [7]. For survival analysis, samples expressing Her-2 at 1+, 2+ or 3+ on a protein level were scored as positive while median FSHR receptor expression (IRS = 3) was used as a cut off to define FSHR positive vs. negative tumours. Gamma/Spearman coefficients, uni- and multivariate survival analysis were employed for statistical analysis. Statistical significance for all tests was set as p < 0.05.

Results

Immunohistochemistry (Figure 1) revealed 12 EOC samples (7.8%) as being Her-2 positive. A high Her-2 immunoreactivity was observed in just one case (3+, n = 1; 0.7%), while another 11 cases were moderately (2+, n = 2; 1.3%) or weakly (1+, n = 9; 5.9%) stained. None of the moderately (2+) expressing cases showed ERBB2 amplification. Eighty two (53.6%) patients were evaluated as negative (IRS ≤ 3) for FSHR expression. Immunoreactivity of Her-2 and FSHR were positively correlated (rho = 0.21; p = 0.009), while Her-2 was not correlated to grade or FIGO stage.

Figure 1
figure 1

Representative microphotographs of FSHR (A, B) and Her-2 (C, D) immunostaining are shown. Patients were stratified according to their FSHR expression status (FSHR negative (A), FSHR positive (B)). The prognostic significance of Her-2 positivity (D) as compared to Her-2 non expressing (C) cases has been evaluated. Scale bars in (A) equal 100 μm and refer to A-D.

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Kaplan-Meier univariate analysis revealed no different outcome of Her-2 expressing vs. non expressing cases though a trend for shortened overall survival was observed in case of Her-2 expression. However patients who did not express FSHR but were at the same time positive for Her-2 emerged to survive significantly shorter (p = 0.001) than those cases that did not express Her-2. Within the group of FSHR negative patients, Her-2 expression (p = 0.004) and FIGO stage (p = 0.003) turned out to be independent prognosticators (Figure 2).

Figure 2
figure 2

Kaplan-Meier plots and multivariate Cox-Regression analysis in EOC patients. Comparison of overall survival of Her-2 positive vs. negative cases (D) revealed no significant difference. However in FSHR negative cases (A) Her-2 positivity was strongly associated with shortened overall survival (p = 0.001) and turned out to be an independent prognosticator (B) in these patients. No link of Her-2 expression and prognosis could be observed in FSHR positive patients (C).

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Discussion

The role of Her-2 in EOC is still unclear. Several studies support a potential role upon EOC survival [9] while others consider it as not being related to prognosis [8]. This can be mainly attributed to the fact that up to-date there is no consensus about the evaluation protocol applied for Her-2 overexpression in EOC. The situation became even more complex due to the fact that different antibodies are used. It can thus be concluded that regarding Her-2 in EOC there is a far more imperative need at least for a consensus in the way Her-2 overexpression will be evaluated.

The existing reports upon efficacy of anti-Her-2 treatment in ovarian cancer patients are few and not encouraging, mainly because the response rate was rather low [4, 5]. Such finding though can be explained possibly by the hypothesis that other molecular pathways interfere with the EGFR/Her-2 signalling [6, 10], potentially minimizing Her-2 impact on ovarian cancer cell proliferation and ultimately to the Her-2 effect on disease progression and prognosis.

Herein we hypothesized that such interference could involve the FSHR and its downstream signalling. Although FSHR-expressing EOC patients have been shown to be of worse prognosis compared to FSHR non-expressing EOC patients [7], an effective selective anti-FSHR intervention has not been published so far. Several clinical trials using anti-gonadotropin receptor (GnR) agents have failed to demonstrate a significant improvement in EOC patient survival [11, 12], most possibly due to the fact that such treatments target simultaneously the luteinizing hormone receptor (LHR) as well, a receptor being a positive prognosticator for EOC survival. Thus, anti-GnR failure can be attributed to the combined cancelling of a negative and a positive prognosticator.

Despite the failure of anti-GnR treatment, the idea of using FSHR - a negative prognosticator for EOC - as a stratifying factor in order to evaluate Her-2 impact on EOC survival, is still appealing. Most possibly FSHR signalling by interfering with Her-2 could abrogate Her-2 signalling and thus its effect on survival. The results presented herein verify this hypothesis. Only in FSHR negative EOC cases does the Her-2 expression present a significant negative impact on patient survival. Of course it could be argued that the sample size of Her-2 positive cases presented herein is rather small. We would agree on such comment, highlighting at the same time though the fact that Her-2 expression in EOC is also restricted. Additionally, by dissecting the Her-2 expressing cases into FSHR positive and negative, we provide an explanation for the restricted impact of anti-Her-2 treatment in EOC. It can be hypothesized that it is likely for EOC patients with the specific phenotype (FSHR negative/Her-2 positive) to benefit from anti-Her-2 treatment. Since, though such patients consist a very small proportion of EOC patients (in our series 3 out of 153), a multicentre clinical study would be necessary to obtain an adequate sample to prove in a statistically significant way whether our current findings and hypothesis are of clinical importance.

Conclusions

In the current report it is demonstrated that Her-2 can be a negative prognosticator only in FSHR negative EOC cases. This observation could be of clinical importance in terms of selecting the patient that would probably benefit from anti-Her-2 treatment. Hence by stratifying EOC patients according to their FSHR expression status, we introduce a diagnostic protocol to effectively select EOC patients that would most probably respond to anti-Her-2 treatment. This observation could be of clinical importance in terms of selecting the patient that would most likely benefit from anti-Her-2 treatment. However due to the small incidence of the FSHR positive/Her-2 negative phenotype in EOC a multicentre study is necessary to verify our result and - as a second step - to assess anti-Her-2 treatment efficacy.

Authors’ information

Udo Jeschke and Darius Dian share senior authorship.