The availability of a reliable model for ovarian neoplasms may provide methods to discriminate between benign and malignant ovaries using non-destructive tools with adequate resolution to resolve fine structural features of ovarian tissue. Optical coherence tomography (OCT) and laser-induced fluorescence (LIF) spectroscopy are complementary imaging modalities, providing high resolution structural imaging and spectral signatures derived from the biochemical composition of tissue specimens, respectively. We utilize combined OCT-LIF to image the ovaries of a post-menopausal rat model of ovarian carcinogenesis. Comparison of OCT images and histopathology demonstrated that OCT could provide information on the microstructural features of cyclic, acyclic and malignant ovary consistent with features described in previous studies of both rat35
and human ovary.15–19
LIF was able to characterize spectral differences in fluorescence emission attributed to collagen, NADH/FAD and hemoglobin absorption among cyclic, acyclic and neoplastic ovaries. These findings support the potential application of OCT-LIF to distinguish a benign from a malignant ovary.
The visualization of precursor lesions and early neoplastic foci is key to improving understanding of ovarian pathogenesis through the development of animal models and new diagnostic tools to differentiate benign ovarian changes from cancer. Specifically, the ability to identify a small solid tumor focus developing within a benign cyst ( and D
) is promising. In this study and in a prior in vivo study of human ovary,15
OCT identified epithelial invaginations and inclusions, which are thought to be precursor lesions to aggressive ovarian carcinomas.37
The high resolution and subsurface imaging capabilities of OCT and LIF allow visualization of these lesions where they would likely be unidentifiable by any method of surface evaluation. OCT and LIF are non-destructive imaging modalities which can be packaged into a small diameter dual modality device.36,38
As these characteristics provide the means for in vivo imaging, future work should aim to evaluate in vivo ovarian imaging of animal models serially over time to observe tumor progression from early neoplasms or preneoplastic lesions to better characterize these lesions and further enhance understanding of ovarian tumor carcinogenesis.
Laser induced fluorescence spectroscopy was able to successfully elucidate the biochemical compositions of normal cyclic, acyclic and SCST to provide preliminary spectral criteria for differentiating these categories. In comparisons of normal cycling ovaries and SCST, the mean emission intensities were statistically significantly different at 390, 420 and 450 nm in both solid and cystic tissues. As the OCT criteria for solid SCST and CL are quite similar, the ability to distinguish these two entities both with signal attenuation comparisons and fluorescence spectroscopy supports the use of dual modality imaging. In comparisons of 390 nm/450 nm fluorescence emission among solid regions of normal cycling ovaries (containing the largest numbers of CL) and solid SCST, the application of a set cut-off provided a sensitivity of 88% to SCST and a specificity of 60% to normal cycling ovaries. Additionally, the ability to potentially distinguish normal follicles from cystic SCST with fluorescence spectroscopy both complements and strengthens the OCT criteria developed to distinguish these two tissue types.
The LIF spectral findings in this study are consistent with previously reported LIF spectral characterization of human ovarian tissue and neoplasms,16,34
but are inconsistent with the results found in a prior study of fluorescence spectroscopy in a VCD/DMBA treated rat ovarian carcinogenesis model35
and previous studies of fluorescence spectroscopy in other types of neoplastic tissues, including cervix, colon and bladder,27
both of which report neoplastic tissues as generally characterized by a relative increase in 450 nm emission due to increased metabolic activity and a relative decrease in 390 nm emission from collagen. Sex cord-stromal tumors have a thin collagenous capsule surrounding the neoplastic mass, which may be the source for the increased 390 nm emission. Second harmonic generation studies of the ovary show an increase in collagen as well as a change in collagen crosslinking which gives an increased signal from collagen and may also be found here.39
These tumors are characterized as slow growing neoplasms, which may result in decreased metabolic activity and a relative decrease in 450 nm emission from NADH, specifically when compared with metabolically active cycling ovaries.
Differentiating normal follicles, epithelial inclusions and cystic SCST will also be crucial. Given the small size of most epithelial inclusions and the large spot size of the fluorescence excitation beam, we are unable to evaluate if these entities can be distinguished by fluorescence spectroscopy. Future studies will utilize focused-beam fluorescence spectroscopy to evaluate if epithelial inclusions can be distinguished from follicles and cystic neoplasms.
In previous studies of fluorescence spectroscopy in VCD/DMBA treated rat models, DMBA exposure was accomplished by introducing a DMBA soaked suture into the ovary. This resulted in a significant inflammatory reaction which may have resulted in a higher 450 nm emission peak from the metabolic activity of the inflammatory cells. Additionally, the number of tumors in the previous fluorescence spectroscopy study of the VCD/DMBA ovarian carcinogenesis model were small (n = 3) and were a mix of epithelial and stromal tumors, which may also account for the discrepancy in fluorescence signatures.35
Future studies will be aimed at further characterizing the fluorescence signature of larger numbers of a variety of ovarian neoplasms.
The vast majority of human ovarian malignancies are epithelial in origin.1
The animal model used in this study expressed stromal ovarian neoplasms, specifically SCST which represent <0.5% of human ovarian malignancies.40
There is a very large difference between the two forms of ovarian malignancy in stage at time of diagnosis; greater than 75% of epithelial ovarian neoplasms are stage III or IV at the time of diagnosis, but approximately 70% of stromal ovarian neoplasms are at stage I at the time of diagnosis due to more specific symptomology from neoplasm hormone production.1,40
Although this is a relatively rare cancer, the ability to distinguish an ovarian cancer from benign ovary would have significant clinical utility, particularly if it was confined to the ovary. To date no reliable rodent model of human ovarian cancer exists, so the availability of a rat model capable of providing reliable stromal tumors may be useful in characterizing the pathogenesis of these tumor types. The successful application of OCT-LIF to a rodent model of ovarian cancer providing preliminary imaging criteria to differentiate cyclic ovaries mimicking pre-menopausal human females, acyclic ovaries mimicking postmenopausal human females, and stromal neoplasms is encouraging and should also be applicable to other types of ovarian tumors as it has been evaluated in previous imaging of human epithelial ovarian carcinoma.2,16
Future studies will evaluate additional animal models with the intent to develop a model for epithelial ovarian cancer. Additionally, as this was a pilot study, the bilateral ovaries of a single animal were included in a given treatment group despite the fact that only the right ovary was treated with saline or DMBA, unless the ovary was found to be neoplastic, in which case it was assigned to the neoplasm group. Histological evaluation and OCT imaging showed no structural differences between the bilateral ovaries of a single rat, indicating that although the treatment was directly applied to the right ovary, the effects of the treatment appear to be systemic. However, in future studies we will treat the bilateral ovaries as dependent but separate in analysis to determine if direct treatment of one ovary has equal effects on the bilateral, untreated ovary.
An additional limitation to this study was the large spot size of the fluorescence incident light (1.25 mm) compared to the OCT lateral resolution (18 µm). Due to this discrepancy, the data are not directly comparable as the fluorescence data encompasses a much larger tissue volume than is depicted in the OCT images. Future studies will be aimed at developing focused OCT-LIF probes such that direct comparisons between OCT and LIF data can be reliably performed.
In this study, we present the successful implementation of combined OCT-LIF to image a post-menopausal rat model of ovarian stromal cancer. Comparison of OCT images and corresponding histopathology allowed for the description of (1) preliminary microstructural features of normal cyclic ovary including follicles, CL and CL remnants, (2) features of acyclic ovaries including presence of degenerating follicles, increases in stromal collagen, presence of epithelial invaginations/inclusions and vascular changes associated with VCD and DMBA treatment and (3) features of both solid and cystic SCSTs. LIF was able to characterize spectral differences in fluorescence emission attributed to collagen, NADH/FAD and hemoglobin absorption among cyclic ovaries, acyclic ovaries and SCSTs. Additionally, future ex vivo and in vivo imaging will evaluate a finer disease spectrum including normal ovary, benign cyst (simple and inclusion), cyst adenoma, borderline tumors and malignant neoplasms of epithelial origin in addition to neoplasms of stromal and germ cell origin to develop more concrete qualitative and quantitative criteria to aid with differentiation of these entities.