Name: tubal cytology of the fallopian tube as a promising tool for ovarian cancer early detection
Uploaded: 2017-07-25T13:00:00
Description: Watch this Scientific Journal Video about Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection at JoVE.com

The authors would like to acknowledge the Department of Pathology, University of Arizona, for the financial support. The project is partially supported by the Mark and Jane Gibson endowment fund to WZ.

An Institutional Review Board approval was received from University of Arizona for conducting this study. Informed patient consent forms were obtained.
1. Patient Selection
NOTE: An Institutional Review Board approval was obtained from University of Arizona. A total of 38 patients were recruited. The patients&#39; ages ranged from 32 to 86 years old with a mean of 55 years, of whom 26 patients were post-menopausal and 12 patients were pre-menopausal.
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	<li>Obt...

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Prophylactic bilateral salpingectomy or salpingo-oophorectomy has been shown to decrease the risk of developing HGSC in high risk populations, such as women with BRCA gene mutations and strong familial cancer history. However, the sterility and surgical menopause caused by the surgery are serious consequences and make for a difficult decision, especially for women of childbearing age. The previous study has shown excellent correlation between tubal cytology and histology17. We believe that tubal c...

Ovarian high-grade serous carcinoma (HGSC) is the most common and lethal type of ovarian cancer, and remains a major threat to the public health. In the past decade, researchers have suggested that the vast majority of HGSC cases arise from the fallopian tube instead of the ovary itself 1,2,3,4,5,6. Serous tubal intraepithelial carcinoma (STIC) is widely accepted as the precursor of HGSC 1,7,8,9,10,11. So far, early detection of ovarian cancer remains difficult. The current screening protocol with combined cancer antigen 125 (CA-125) and transvaginal ultrasound has shown little effect on patient mortality12,13. Endometrial sampling for ovarian cancer detection has shown an extremely low sensitivity14. Two pioneer studies15,16 explored the usage of laparoscopic direct sampling of benign fallopian tubes and characterized the cytological features of benign tubal epithelium. We believe that direct sampling from the fallopian tube can also be a practical and straightforward way to detecting STIC or HGSC at an early stage when the tumor is not visualized by imaging.
Although the ultimate goal is the utility of minimally invasive laparoscopic screening in patients with high-risk factors, the immediate aims of current study are: 1) To establish the baseline cytological features of benign tubal epithelia; and 2) To test the sensitivity and specificity of tubal cytology in detecting ovarian cancers and cancer precursors. We therefore developed the following baseline tubal cytology method to test the effectiveness of this protocol in detection of HGSC and its precursor STIC17. In this study, we took advantage of the large volume of regularly received surgical specimens, and performed direct sampling of the surgically excised fallopian tube in addition to the routine grossing procedures.
Our recent study17 showed that the cytological diagnosis of malignant or suspicion for malignancy is highly correlated with the histological diagnosis of HGSC (100%). In addition, the tubal cytological evaluation identified intratubal neoplasia in the only two histologically confirmed STIC cases involved in this study. We believe that tubal cytology has great potential in early detection and will provide valuable information for patient management.

<table>
	<tbody>
		<tr>
			<td>Cytobrush plus GT gentle touch of Medscand sample collection kit</td>
			<td>CooperSurgical</td>
			<td>C0112</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep preservCyt solution</td>
			<td>HOLOGIC</td>
			<td>234005</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep 2000 Processor</td>
			<td>HOLOGIC</td>
			<td>70031-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papanicolaou Stain, EA 65</td>
			<td>sigma aldrich</td>
			<td>HT40432</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>sigma aldrich</td>
			<td>652261</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>sigma aldrich</td>
			<td>493538</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>sigma aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>sigma aldrich</td>
			<td>H9627</td>
			<td></td>
		</tr>
		<tr>
			<td>Sakura Tissue Tek 2000 tissue processor</td>
			<td>SAKURA</td>
			<td>TISSUE-TEK VIP 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo Dissecting Scissors,5.5 straight</td>
			<td>Pilgrim medical equiment</td>
			<td>FA710-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemware Forceps</td>
			<td>United state plastic corp</td>
			<td>76122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades,short</td>
			<td>SAKURA</td>
			<td>4785</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades handle,short</td>
			<td>SAKURA</td>
			<td>4786</td>
			<td></td>
		</tr>
	</tbody>
</table>

tubal cytology of the fallopian tube as a promising tool for ovarian cancer early detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

Our previous study showed that sampling directly from the excised fallopian tube using a gentle touch brush could yield quantitatively and qualitatively adequate specimen for further cytological evaluation. In addition, the combination of automated slide preparation and modified Papanicolaou staining enable the pathologist to better visualize the cytological details to make an accurate diagnosis. An example of a manual quick pap stain of a specimen from a benign fallopian tube is illustra...

Watch this Scientific Journal Video about Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection at JoVE.com

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

The overall goal of this protocol is to direct the sample fallopian tube epithelium from the salpingo oophorectomy specimen. For the identification of tubal cytologic features that allow the differentiation of benign tissue from malignant or pre-malignant conditions. This cytology method will help answer some key questions in the gynecologic oncology field regarding early detection of ovarian and fallopian tube cancer.The advantage of this technique is it requires direct sampling of the fallopian tube cells. Another advantage of this technique is that it is a direct and simple method. In the future, it maybe applied during laparoscopic screening for ovarian or fallopian tube cancers in women who are high risk for those diseases.A visual demonstration of the collection method is important as the collection steps can be difficult to learn. We want to make sure that there is minimal disturbance to the fallopian tube epithelium as the histologic diagnosis of the fallopian tube is important in the end, which will then impact clinical management. Demonstrating this procedure is Dr.Howard Chen, whose currently our fourth year pathology resident at the University of Arizona.Immediately after obtaining the tissue, confirm that the uterus, bilateral fallopian tubes and bilateral ovarian are intact. Using small forceps, carefully insert the cytobrush into the lumen of the fallopian tube through the end and slowly rotate the brush clockwise. Moving the brush into the tube, through the infundibular and ampulla regions.When the brush reaches the isthmus, slowly begin rotating the brush in the counter-clockwise direction while pulling the brush back out of the fallopian tube. Next, rotate and swirl the fallopian cell coated brush in freshly prepared preservative solution 10 times. Then tightly cap the sample vial and label it with the patient's information and the laterality of the specimen.The cells can then be stored at four degree celsius for up to six months. Before staining the cells, load the sample vial into an automated processor for slide preparation. When the slides are ready, dip the samples into 95%ethanol, 10 times for one second per dip.Followed by 10 dips in water for one second per dip. Next, submerge the slides in hematoxylin for 10 to 60 seconds, followed by a second water ethanol wash. After the last ethanol immersion, submerge the slides in eosin azure for one to three minutes.Followed by another water ethanol rinse. Then dip the slides in xylene, until the samples become clear. To obtain cyto spins of the harvested fallopian tube cells, first label three slides for each cell sample with the appropriate patient information.Next, attach a filter and a specimen funnel to each slide, and pellet the fallopian tube cells by bench top centrifugation. Remove about half of the supernatant and re-suspend the cells with gentle pipetting, then load 200 microliters of the single cell suspension into each specimen funnel and load the slide assemblies into the cytocentrifuge. At the end of the centrifugation, carefully detach the slides from the funnels and filters and fix the cytospun cells at 95%ethanol for 30 seconds.For sectioning and analysis of the fimbriated end of the fallopian tube sample, first fix the entire specimen for two to four hours in 10%formalin. Next, use small forceps and scissors to carefully detach the fallopian tubes from the uterus at the isthmus, if an adhesion is present, use the forceps and scissors to carefully dissect the fimbriated end from the ovary surface. Amputate the infundibulum and fimbria segment at the distal one to two centimeter end of the fallopian tube and section the infundibulum and fimbria, longitudinally at two millimeter intervals.Submit all of the infundibulum and fimbria sections in total for histologic examination. Then section the isthmus and ampulli, three to five millimeter intervals, collectively submitting the specimens in their entirety. The brush rotation maneuver results in an adequate sampling of the specimen and the manual quick PAP stain provides a good resolution of the nuclear details and the cytoplasmic transparency.Both of which are essential for an accurate evaluation of the tubule cells. Although similar results can be obtained by cytocentrifugation, the quick PAP stain procedure results in an ultimately cleaner background and sharper resolution between the nuclear details in the cytoplasmic transparency. The arrow pointing to the ciliates of the ciliated epithelium.Histologic analysis of the fimbria segment sections can be used to access the morphology of the small ciliated and large non-ciliated secretary cell populations. In the ampulla region of the same section, finger-like fimbria can be observed, as well as a mild disturbance in the villi structure. The cytological appearance of malignancy can be determined by the presence of three dimensional clusters composed of cells with prominent nucleoli and anisonucleosis.Hygrade serious carcinoma-like cell groups can also be observed, as shown within the circle. Within the angulated sheets of normal epithelia cells, as pointed by arrows. Once mastered, this collection and staining procedure can be completed within two hours if performed properly.While attempting this procedure, it is important to avoid cross-contamination with other tissues which can then confound the results. Following this procedure, molecular analysis or immunohistochemistry can be performed on the fake cells, if the cytologic findings are equivocal. This technique lays a foundation for future application to laparoscopic or minimally invasive surgery for screening of women at high risk for ovarian and fallopian tube cancer.After watching this video, you will have a good understanding of how to collect and process tubule epithelium from resected salpingo-oophorectomy specimens.

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