Analyzing Toposimerase II-α and HER-2/neu co-amplification seems to be of limited value in epithelial ovarian cancer

To the Editor

Recurrent chemoresistant ovarian cancer remains a therapeutic challenge; in randomized Phase III trials the best response rates to chemotherapy have been at best 12–13% and even so, short-lived [1], [2]. Among available agents, pegylated liposomal doxorubicin is a noteworthy alternative [3]. Pegylated liposomal doxorubicin is, however, quite expensive and has inconvenient side effects as hand-foot syndrome and stomatitis [2]. Consequently, a situation where at least ten patients need to be treated to get one or two responses is definitely less than optimal.

Topoisomerase IIα (TOP2A) is purported to be the main intracellular target of anthracyclines [4]. In breast cancer, TOP2A gene amplification seems to be associated with HER-2/neu gene amplification [5]. Indeed, there is evidence that coamplification of these genes in breast cancer renders the cancer cells especially sensitive to anthracyclines [4], although also conflicting results have been published [5]. After Mano et al. showed that evaluating amplification of TOP2A and HER-2/neu by fluorescent in situ hybridization (FISH) is feasible even in epithelial ovarian carcinoma [6], we decided 1) to perform a preliminary analysis of the rate of TOP2A and HER-2/neu amplification by chromogenic in situ hybridization (CISH) [7] in epithelial ovarian cancer, and 2) to retrospectively correlate the amplification to the clinical efficacy of pegylated liposomal doxorubicin in patients with recurrent chemoresistant ovarian cancer.

Ten patients with recurrent chemoresistant ovarian cancer gave their informed consent to the study. The median age of the patients was 58.5 years (range 43–79). Of the tumors, seven were serous, two were endometrioid, and one was mucinous. We chose both patients who had responded to pegylated liposomal doxorubicin and patients who had not responded. The study was approved by the local Ethics Committee.

The original paraffin blocks from the primary operation we retrieved from the archives. The digoxigenin-labeled probes for HER-2/neu and TOP2A needed for CISH were obtained from Zymec Inc. (South San Francisco, CA). The hybridization method used has been described in detail previously [7]. Hybridization was evaluated with an Olympus BX50 microscope (Olympus, Tokyo, Japan) using an X40 objective. Amplification of HER-2/neu and TOP2A was defined as the presence of six or more copies in more than 30% of nuclei or as presence of an easily identifiable gene copy cluster, the gene copies of which could not be enumerated. The laboratory was blinded as regards to the clinical outcome of the patients.

Four of the carcinomas had responded to pegylated liposomal doxorubicin, as evaluated by the tumor marker Ca-12-5. Three tumors showed stabilization, while the remaining three progressed in spite of the treatment. HER-2/neu gene was found to be amplified only in one endometrioid carcinoma that was unresponsive to doxorubicin. In no instance could we find any amplification of TOP2A gene.

Immunohistochemical (IHC) methods were originally used to study the over-expression of TOP2A and HER-2/neu in cancer cells. Because of difficulties in standardization of IHC, in situ hybridization techniques developed to detect the actual amplification rate have now replaced the IHC methods [8]. Of the in situ hybridization techniques, the newer CISH provides an accurate and practical alternative to the older FISH [4], [7].

To our knowledge, Mano et al. were the first to compare TOP2A and HER-2/neu amplification and expression in epithelial ovarian carcinoma [6]. They found on one hand that the correlation between the expression and the amplification of the respective genes was poor, and on the other hand that there was an excellent correlation between HER-2/neu and TOP2A amplification. Using more stringent criteria, the amplification rates were 12.5 and 7.8%, respectively. The researches did not compare the amplification rates to in vitro or clinical efficacy of pegylated liposomal doxorubicin.

The article by Mano et al. encouraged us to study the amplification rate of TOP2A and HER-2/neu in epithelial ovarian cancer and, moreover, to attempt to correlate the rates to the therapeutic effect of pegylated liposomal doxorubicin. Unfortunately, no tumor was found to harbor amplification of TOP2A, and even HER-2/neu was amplified only once, in a tumor not responsive to doxorubicin. There are obvious shortcomings in our preliminary retrospective study: The samples studied originated from the primary operation, i.e. were chemotherapy-naïve, and pegylated liposomal doxorubicin was started not until the tumors had developed a taxane-platinum resistance. It is possible that the amplification of TOP2A and/or HER-2/neu is a late phenomenon associated e.g. with the development of a resistance to taxanes and platinum compounds in ovarian cancer. It is, however, difficult to obtain histological samples from recurrent ovarian cancer that is as a rule treated with second-line chemotherapy, rather than re-operated.

In conclusion, the rarity of amplification of HER-2/neu, combined with the non-existence of amplification of TOP2A and with a poor correlation to the clinical outcome as found in the present study, discourages us from continuing our efforts.

Acknowledgements

The financial support given by Schering-Plough Finland is appreciated.