Here, we identified several proteins that could potentially serve as a suitable target for detecting breast cancer cells within ovarian autografts. One clear application for these markers is the use of NIRF imaging, a technique that can differentiate malignant tissues from non-malignant tissues without reducing the tissue’s viability [
10,
33‐
35]. Designing a NIRF probe directed against E-cadherin shows particular promise, as E-cadherin was expressed by the majority (94 %) of invasive breast tumor cells and was absent on the surface of normal ovarian cells. However, a combination of tumor-selective probes will likely be needed to detect all tumor cells. Based on our results, a combination of probes against E-cadherin, EMA and Her2/neu seems suitable.
Metastatic spread requires the local invasion of the surrounding host tissue by cells that originated from the primary tumor, followed by intravasation in blood and lymphatic vessels, ultimately leading to the dissemination of tumor cells [
36]. E-cadherin and EpCAM mediate cell–cell adhesion, and the downregulation or loss-of-function of these proteins enables cells to escape from solid tumors [
19]. E-cadherin and/or EpCAM are not necessarily expressed in all tumor cells; therefore, metastatic tumor cells might not be detected in some tissues. Furthermore, the majority of metastatic lobular breast cancer cells, which lack E-cadherin expression, will not be detected using a specific anti-E-cadherin probe, even though lobular breast cancer cells are more likely to invade ovarian tissue compared to cells derived from ductal carcinomas [
37,
38].
As mentioned above, we considered tumor cell membranes positive if they showed immunoreactivity of any intensity. As a result, some tumor cells might be more positive than others for the investigated markers. Yet, for NIRF imaging, the staining intensity is less important as long as a significant tumor-to-background-ratio can be achieved.
None of the premenopausal ovaries in our cohort had positive staining in either the stromal cells or the ovarian surface epithelium. However, all markers (except CEA and uPAR) were expressed on epithelial cells in inclusion cysts. Consequently, conjugating antibodies against these markers to a NIR fluorophore will illuminate invasive breast cancer cells in the ovary, as well as inclusion cysts. Because inclusion cysts might differ from metastatic breast cancer cells with respect to their fluorescent configuration, it might be possible to distinguish between these structures. The same strategy might be used to distinguish granulosa cells in primary follicles from metastatic breast cancer cells that express E-cadherin. In addition, full-field optical coherence tomography (FF-OCT), a non-invasive imaging technique that mimics conventional histopathology, might be very useful. In the field of dermatological oncology, FF-OCT has already been proven capable of visualizing sebaceous glands and adipose tissue surrounding hair follicles as well as small malignant skin tumors [
39]. On high magnification, fine architectural details on the subcellular level can be recognized. Therefore, it is expected that FF-OCT will also be able to distinguish inclusion cysts from metastatic tumor cells in ovarian tissue.
One strength of our study is that we examined the expression of tumor markers in tumor tissues obtained from young breast cancer patients who met the criteria for ovarian tissue cryopreservation. Moreover, only biologically active ovaries were analyzed, giving the results clinical relevance, as ovarian tissue is generally cryopreserved before ovarian failure has occurred.
On the other hand, this study has some limitations that merit discussion. First, a relatively small sample size was examined. However, normal ovarian tissue from premenopausal patients is not readily available. We excluded ovaries that were removed due to the presence of a
BRCA gene mutation, as such samples could contain primary ovarian tumor cells [
40], that express the markers investigated in this study. To be certain, we also excluded ovaries from breast cancer patients with unknown mutation status. At the LUMC, normal ovarian tissue from premenopausal breast cancer patients was exclusively available from
BRCA mutation carriers or women with unknown mutation status. As a consequence, ovaries from breast cancer patients were not included in this study. Malignant cells may also be present in ovaries that were removed due to endometrial carcinoma, uterine sarcoma, cervical squamous cell carcinoma, or contralateral ovarian carcinoma; however, the risk of false-positive results was relatively low in our cohort, as all primary tumors were diagnosed at an early stage and the lymph nodes in these patients were clear. Second, the expression of the markers was evaluated on primary invasive breast tumors, since a substantial cohort consisting of ovarian tissues containing breast cancer metastases is scarce. Finally, we examined the expression of tumor markers in relatively small tumor cores using a TMA approach. Yet, the TMA technique is considered an accurate method for examining protein expression in breast cancer tissues [
41].
Recently, the intraoperative use of tumor-targeted fluorescent imaging yielded a high tumor identification rate and enabled the surgeon to detect metastases that could not be detected by visual observation [
42]. For our purpose, tumor-specific NIRF probes could be administered intravenously prior to oophorectomy, after which the removed ovary is dissected into cortical ovarian strips. Detailed fluorescent images could then be obtained using multiphoton microscopy, which provides an inherent submicron spatial resolution that allows revelation of subcellular details with reduced phototoxicity and photobleaching [
43,
44]. Because the NIRF signal lies beyond the red end of the visible spectrum, the signal has enhanced tissue penetration, enabling the identification of fluorescently labeled tumor cells that are located deep within the tissue. Moreover, the low autofluorescence of the tissue at the emission wavelength of the probe provides a high tumor-to-background ratio [
45]. Because of these features and the fact that cortical ovarian fragments can be imaged from both the upper and lower side, thereby increasing the imaging depth even further, NIRF imaging is a promising technique for detecting tumor cells in cortical ovarian strips up to 2 mm in thickness.
In conclusion, we report the identification of tumor markers that may serve as a target for detecting breast cancer cells in ovarian tissue using robust imaging techniques such as NIRF imaging. Based on our analysis, E-cadherin is likely the most suitable target for designing a tumor-specific probe. Further research will focus on examining the expression of these markers on breast cancer metastases in ovaries, refining methods to distinguish breast cancer cells from ovarian inclusion cysts, and examining the clinical feasibility of applying NIRF imaging to the field of fertility preservation.