Iterative prototyping based on lessons learned from the falloposcope in vivo pilot study experience.

Significance: High grade serous ovarian cancer is the most deadly gynecological cancer, and it is now believed that most cases originate in the fallopian tubes (FTs). Early detection of ovarian cancer could double the 5-year survival rate compared with late-stage diagnosis. Autofluorescence imaging can detect serous-origin precancerous and cancerous lesions in ex vivo FT and ovaries with good sensitivity and specificity. Multispectral fluorescence imaging (MFI) can differentiate healthy, benign, and malignant ovarian and FT tissues. Optical coherence tomography (OCT) reveals subsurface microstructural information and can distinguish normal and cancerous structure in ovaries and FTs. Aim: We developed an FT endoscope, the falloposcope, as a method for detecting ovarian cancer with MFI and OCT. The falloposcope clinical prototype was tested in a pilot study with 12 volunteers to date to evaluate the safety and feasibility of FT imaging prior to standard of care salpingectomy in normal-risk volunteers. In this manuscript, we describe the multiple modifications made to the falloposcope to enhance robustness, usability, and image quality based on lessons learned in the clinical setting. Approach: The ∼0.8 mm diameter falloposcope was introduced via a minimally invasive approach through a commercially available hysteroscope and introducing a catheter. A navigation video, MFI, and OCT of human FTs were obtained. Feedback from stakeholders on image quality and procedural difficulty was obtained. Results: The falloposcope successfully obtained images in vivo. Considerable feedback was obtained, motivating iterative improvements, including accommodating the operating room environment, modifying the hysteroscope accessories, decreasing endoscope fragility and fiber breaks, optimizing software, improving fiber bundle images, decreasing gradient-index lens stray light, optimizing the proximal imaging system, and improving the illumination. Conclusions: The initial clinical prototype falloposcope was able to image the FTs, and iterative prototyping has increased its robustness, functionality, and ease of use for future trials.

Study of fallopian tube anatomy and mechanical properties to determine pressure limits for endoscopic exploration.

Falloposcopy is the endoscopic examination of the fallopian tubes, which are challenging to access due to their deep body location, small opening from the uterus, and lumen filled with plicae. We and others have developed endoscopes that are inserted through the uterus guided by a hysteroscope into the tubal ostium. To better understand how to utilize these endoscopes either as standalone devices or in concert with everting delivery balloons, a preliminary study of anatomy and mechanical behavior was performed ex vivo on porcine and human fallopian tubes. Segments of fallopian tubes from the isthmus, ampulla and infundibulum were inflated with saline either to bursting or held at sub-burst pressures with saline or a saline-filled balloon. Formalin fixed, paraffin embedded tissue sections stained with Masson's trichrome were examined for damage to the mucosa and muscularis. Porcine fallopian tubes tolerated saline pressurization at 15 psi for 1 minute without morphological damage. Balloon inflation to 15 psi caused no apparent damage to the muscle layer or rupture of the fallopian tube, but balloon movement within the tube can denude the mucosal epithelial layer. Human fallopian tubes averaged higher burst pressure values than porcine tubes. Under pressurization, the external tube diameter expanded by minimal to moderate amounts. Human and porcine tissues were similar in histological appearance. These studies suggest that moderate pressurization is acceptable but will not appreciably expand the fallopian tube diameter. The results also indicate that pigs are a reasonable model to study damage from falloscopy as seen in human tissue.

Fluorescence and Multiphoton Imaging for Tissue Characterization of a Model of Postmenopausal Ovarian Cancer.

BACKGROUND AND OBJECTIVES: To determine the efficacy of targeted fluorescent biomarkers and multiphoton imaging to characterize early changes in ovarian tissue with the onset of cancer. STUDY DESIGN/MATERIALS AND METHODS: A transgenic TgMISIIR-TAg mouse was used as an animal model for ovarian cancer. Mice were injected with fluorescent dyes to bind to the folate receptor α, matrix metalloproteinases, and integrins. Half of the mice were treated with 4-vinylcyclohexene diepoxide (VCD) to simulate menopause. Widefield fluorescence imaging (WFI) and multiphoton imaging of the ovaries and oviducts were conducted at 4 and 8 weeks of age. The fluorescence signal magnitude was quantified, and texture features were derived from multiphoton imaging. Linear discriminant analysis was then used to classify mouse groups. RESULTS: Imaging features from both fluorescence imaging and multiphoton imaging show significant changes (P < 0.01) with age, VCD treatment, and genotype. The classification model is able to classify different groups to accuracies of 75.53%, 69.53%, and 86.76%, for age, VCD treatment, and genotype, respectively. Building a classification model using features from multiple modalities shows marked improvement over individual modalities. CONCLUSIONS: This study demonstrates that using WFI with targeted biomarkers, and multiphoton imaging with endogenous contrast shows promise for detecting early changes in ovarian tissue with the onset of cancer. The results indicate that multimodal imaging can provide higher sensitivity for classifying tissue types than using single modalities alone. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.

Evaluation of segmentation algorithms for optical coherence tomography images of ovarian tissue.

Ovarian cancer has the lowest survival rate among all gynecologic cancers predominantly due to late diagnosis. Early detection of ovarian cancer can increase 5-year survival rates from 40% up to 92%, yet no reliable early detection techniques exist. Optical coherence tomography (OCT) is an emerging technique that provides depth-resolved, high-resolution images of biological tissue in real-time and demonstrates great potential for imaging of ovarian tissue. Mouse models are crucial to quantitatively assess the diagnostic potential of OCT for ovarian cancer imaging; however, due to small organ size, the ovaries must first be separated from the image background using the process of segmentation. Manual segmentation is time-intensive, as OCT yields three-dimensional data. Furthermore, speckle noise complicates OCT images, frustrating many processing techniques. While much work has investigated noise-reduction and automated segmentation for retinal OCT imaging, little has considered the application to the ovaries, which exhibit higher variance and inhomogeneity than the retina. To address these challenges, we evaluate a set of algorithms to segment OCT images of mouse ovaries. We examine five preprocessing techniques and seven segmentation algorithms. While all preprocessing methods improve segmentation, Gaussian filtering is most effective, showing an improvement of 32 % ± 1.2 % . Of the segmentation algorithms, active contours performs best, segmenting with an accuracy of 94.8 % ± 1.2 % compared with manual segmentation. Even so, further optimization could lead to maximizing the performance for segmenting OCT images of the ovaries.

Comparison of Reproductive Function in Female TgMISIIR-TAg Transgenic and Wildtype C57BL/6 Mice.

Transgenic TgMISIIR-TAg (TAg) mice express the oncogenic virus SV40 in Mullerian epithelial cells. Female TAg mice spontaneously develop epithelial ovarian carcinoma, the most common type of ovarian cancer in women. Female TAg mice are infertile, but the reason has not been determined. We therefore investigated whether female TAg mice undergo puberty, demonstrate follicular development, maintain regular cycles, and ovulate. Ovarian cancers in women commonly develop after menopause. The occupational chemical 4-vinylcyclohexene diepoxide (VCD) accelerates follicle degeneration in the ovaries of rats and mice, causing early ovarian failure. We therefore used VCD dosing of mice to develop an animal model for menopause. The purpose of this study was to characterize reproductive parameters in female TAg mice and to investigate whether the onset of ovarian failure due VCD dosing differed between female TAg and WT C57BL/6 mice. As in WT female mice, TAg female mice underwent puberty (vaginal opening) and developed cyclicity in patterns that were similar between the groups. Vehicle-only TAg mice had fewer ovulations (numbers of corpora lutea) than WT animals. VCD exposure delayed the onset of puberty (day of first estrus) in TAg as compared with WT mice. Morphologic evaluation of ovaries revealed many more degenerating follicles in TAg mice than WT mice, and more VCD-dosed TAg mice were in ovarian failure than VCD-dosed WT mice. These results suggest that despite showing similar onset of sexual maturation, TAg mice have increased follicular degeneration and fewer ovulations than WT. These features may contribute to the inability of female TAg mice to reproduce.

Three-dimensional texture analysis of optical coherence tomography images of ovarian tissue.

Ovarian cancer has the lowest survival rate among all gynecologic cancers due to predominantly late diagnosis. Optical coherence tomography (OCT) has been applied successfully to experimentally image the ovaries in vivo; however, a robust method for analysis is still required to provide quantitative diagnostic information. Recently, texture analysis has proved to be a useful tool for tissue characterization; unfortunately, existing work in the scope of OCT ovarian imaging is limited to only analyzing 2D sub-regions of the image data, discarding information encoded in the full image area, as well as in the depth dimension. Here we address these challenges by testing three implementations of texture analysis for the ability to classify tissue type. First, we test the traditional case of extracted 2D regions of interest; then we extend this to include the entire image area by segmenting the organ from the background. Finally, we conduct a full volumetric analysis of the image volume using 3D segmented data. For each case, we compute features based on the Grey-Level Co-occurence Matrix and also by introducing a new approach that evaluates the frequency distribution in the image by computing the energy density. We test these methods on a mouse model of ovarian cancer to differentiate between age, genotype, and treatment. The results show that the 3D application of texture analysis is most effective for differentiating tissue types, yielding an average classification accuracy of 78.6%. This is followed by the analysis in 2D with the segmented image volume, yielding an average accuracy of 71.5%. Both of these improve on the traditional approach of extracting square regions of interest, which yield an average classification accuracy of 67.7%. Thus, applying texture analysis in 3D with a fully segmented image volume is the most robust approach to quantitatively characterizing ovarian tissue.

Does Mutated K-RAS Oncogene Attenuate the Effect of Sulindac in Colon Cancer Chemoprevention?

The NSAID sulindac has been successfully used alone or in combination with other agents to suppress colon tumorigenesis in patients with genetic predisposition and also showed its efficacy in prevention of sporadic colon adenomas. At the same time, some experimental and clinical reports suggest that a mutant K-RAS oncogene may negate sulindac antitumor efficacy. To directly assess sulindac activity at suppressing premalignant lesions carrying K-RAS mutation, we utilized a novel mouse model with an inducible colon-specific expression of the mutant K-ras oncogene (K-rasG12D ). Tumor development and treatment effects were monitored by minimally invasive endoscopic Optical coherence tomography. Expression of the mutant K-ras allele accelerated azoxymethane (AOM)-induced colon carcinogenesis in C57BL/6 mice, a strain otherwise resistant to this carcinogen. Sulindac completely prevented AOM-induced tumor formation in K-ras wild-type (K-ras wt) animals. In K-rasG12D -mutant mice, a 38% reduction in tumor number, an 83% reduction in tumor volume (P ≤ 0.01) and an increase in the number of adenoma-free mice (P = 0.04) were observed. The partial response of K-RasG12D animals to sulindac treatment was evident by the decrease in mucosal thickness (P < 0.01) and delay in progression of the precancerous aberrant crypt foci to adenomas. Molecular analyses showed significant induction in cyclooxygenase 2 (COX-2), cleaved caspase-3 (CC3), and Ki-67 expression by AOM, but not sulindac treatment, in all genotypes. Our data underscore the importance of screening for K-RAS mutations in individuals with colon polyps to provide more personalized interventions targeting mutant K-RAS signaling pathways. Cancer Prev Res; 11(1); 16-26. ©2017 AACR.

Multispectral fluorescence imaging of human ovarian and fallopian tube tissue for early-stage cancer detection.

With early detection, 5-year survival rates for ovarian cancer exceed 90%, yet no effective early screening method exists. Emerging consensus suggests over 50% of the most lethal form of the disease originates in the fallopian tube. Twenty-eight women undergoing oophorectomy or debulking surgery provided informed consent for the use of surgical discard tissue samples for multispectral fluorescence imaging. Using multiple ultraviolet and visible excitation wavelengths and emissions bands, 12 fluorescence and 6 reflectance images of 47 ovarian and 31 fallopian tube tissue samples were recorded. After imaging, each sample was fixed, sectioned, and stained for pathological evaluation. Univariate logistic regression showed cancerous tissue samples had significantly lower intensity than noncancerous tissue for 17 image types. The predictive power of multiple image types was evaluated using multivariate logistic regression (MLR) and quadratic discriminant analysis (QDA). Two MLR models each using two image types had receiver operating characteristic curves with area under the curve exceeding 0.9. QDA determined 56 image type combinations with perfect resubstituting using as few as five image types. Adaption of the system for future in vivo fallopian tube and ovary endoscopic imaging is possible, which may enable sensitive detection of ovarian cancer with no exogenous contrast agents.

In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis.

Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SHG]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a long-term survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SHG. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer.

Two-photon excited fluorescence imaging of endogenous contrast in a mouse model of ovarian cancer.

BACKGROUND AND OBJECTIVE: Ovarian cancer has an extremely high mortality rate resulting from poor understanding of the disease. In order to aid understanding of disease etiology and progression, we identify the endogenous fluorophores present in a mouse model of ovarian cancer and describe changes in fluorophore abundance and distribution with age and disease. STUDY DESIGN/MATERIALS AND METHODS: A mouse model of ovarian cancer was created by dosing with 4-vinylcyclohexene diepoxide, which induces follicular apoptosis (simulating menopause), and 7,12-dimethylbenz[a]anthracene, a known carcinogen. Imaging of ovarian tissue was completed ex vivo with a multiphoton microscope using excitation wavelength of 780 nm and emission collection from 405 to 505 nm. Two-photon excited fluorescence images and corresponding histologic sections with selective stains were used to identify endogenous fluorophores. RESULTS: The majority of collected fluorescence emission was attributed to NADH and lipofuscin, with additional contributions from collagen and elastin. Dim cellular fluorescence from NADH did not show observable changes with age. Changes in ovarian morphology with disease development frequently caused increased fluorescence contributions from collagen and adipose tissue-associated NADH. Lipofuscin fluorescence was much brighter than NADH fluorescence and increased as a function of both age and disease. CONCLUSIONS: Our finding of NADH fluorescence patterns similar to that seen previously in human ovary, combined with the observation of lipofuscin accumulation with age and disease also seen in human organs, suggests that the findings from this model may be relevant to human ovarian disease. Increased lipofuscin fluorescence might be used as an indicator of disease in the ovary and this finding warrants further study.

Analysis of second-harmonic-generation microscopy in a mouse model of ovarian carcinoma.

Second-harmonic-generation (SHG) imaging of mouse ovaries ex vivo was used to detect collagen structure changes accompanying ovarian cancer development. Dosing with 4-vinylcyclohexene diepoxide and 7,12-dimethylbenz[a]anthracene resulted in histologically confirmed cases of normal, benign abnormality, dysplasia, and carcinoma. Parameters for each SHG image were calculated using the Fourier transform matrix and gray-level co-occurrence matrix (GLCM). Cancer versus normal and cancer versus all other diagnoses showed the greatest separation using the parameters derived from power in the highest-frequency region and GLCM energy. Mixed effects models showed that these parameters were significantly different between cancer and normal (P<0.008). Images were classified with a support vector machine, using 25% of the data for training and 75% for testing. Utilizing all images with signal greater than the noise level, cancer versus not-cancer specimens were classified with 81.2% sensitivity and 80.0% specificity, and cancer versus normal specimens were classified with 77.8% sensitivity and 79.3% specificity. Utilizing only images with greater than of 75% of the field of view containing signal improved sensitivity and specificity for cancer versus normal to 81.5% and 81.1%. These results suggest that using SHG to visualize collagen structure in ovaries could help with early cancer detection.

In vivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-targeted NIR fluorescent probe in the AOM-treated mouse model.

PURPOSE: Increased vascular endothelial growth factor (VEGF) receptor expression has been found at the sites of angiogenesis, particularly in tumor growth areas, as compared with quiescent vasculature. An increase in VEGF receptor-2 is associated with colon cancer progression. The in vivo detection of VEGF receptor is of interest for the purposes of studying basic mechanisms of carcinogenesis, making clinical diagnoses, and monitoring the efficacy of chemopreventive and therapeutic agents. In this study, a novel single chain (sc)VEGF-based molecular probe is utilized in the azoxymethane (AOM)-treated mouse model of colorectal cancer to study delivery route and specificity for disease. PROCEDURES: The probe was constructed by site-specific conjugation of a near-infrared fluorescent dye, Cy5.5, to scVEGF and detected in vivo with a dual-modality optical coherence tomography/laser-induced fluorescence (OCT/LIF) endoscopic system. A probe inactivated via excessive biotinylation was utilized as a control for nonreceptor-mediated binding. The LIF excitation source was a 633-nm He:Ne laser, and red/near-infrared fluorescence was detected with a spectrometer. OCT was used to obtain two-dimensional longitudinal tomograms at eight rotations in the distal colon. Fluorescence emission levels were correlated with OCT-detected disease in vivo. OCT-detected disease was verified with hematoxylin and eosin stained histology slides ex vivo. RESULTS: High fluorescence emission intensity from the targeted probe was correlated with tumor presence as detected using OCT in vivo and VEGFR-2 immunostaining on histological sections ex vivo. The inactivated probe accumulated preferentially on the surface of tumor lesions and in lymphoid aggregate tissue and was less selective for VEGFR-2. CONCLUSION: The scVEGF/Cy probe delivered via colonic lavage reaches tumor vasculature and selectively accumulates in VEGFR-2-positive areas, resulting in high sensitivity and specificity for tumor detection. The combination of OCT and LIF imaging modalities may allow the simultaneous study of tumor morphology and protein expression for the development of diagnostic and therapeutic methods for colorectal cancer.

Quantitative tool for rapid disease mapping using optical coherence tomography images of azoxymethane-treated mouse colon.

Optical coherence tomography (OCT) can provide new insight into disease progression and therapy by enabling nondestructive, serial imaging of in vivo cancer models. In previous studies, we have shown the utility of endoscopic OCT for identifying adenomas in the azoxymethane-treated mouse model of colorectal cancer and tracking disease progression over time. Because of improved imaging speed made possible through Fourier domain imaging, three-dimensional imaging of the entire mouse colon is possible. Increased amounts of data can facilitate more accurate classification of tissue but require more time on the part of the researcher to sift through and identify relevant data. We present quantitative software for automatically identifying potentially diseased areas that can be used to create a two-dimensional "disease map" from a three-dimensional Fourier domain OCT data set. In addition to sensing inherent changes in tissue that occur during disease development, the algorithm is sensitive to exogeneous highly scattering gold nanoshells that can be targeted to disease biomarkers. The results of the algorithm were compared to histological diagnosis. The algorithm was then used to assess the ability of gold nanoshells targeted to epidermal growth factor receptor in vivo to enable functional OCT imaging.