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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 mortality^12,13. Endometrial sampling for ovarian cancer detection has shown an extremely low sensitivity¹⁴. Two pioneer studies^15,16 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.

Protokół

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' 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.

1. Obtain informed consent from each patient.

2. Fallopian Tube Cells Collection Procedure

NOTE: The patients included in the current study were all scheduled for total hysterectomy and bilateral salpingo-oophorectomy for either prophylactic risk reduction or malignancy. The details of surgery were unknown for pathologist who performed the cytology study.

1. Immediately after surgical excision, examine the fresh tissue specimen including uterus, bilateral ovaries, and bilateral fallopian tubes carefully to identify the bilateral fallopian tube.
2. Collect and place fallopian tube cells into the preservative solution (see Table of Materials) vial within 30 min of clamping the blood supply; the recommended preservative is a methanol-based, buffered preservative solution.
   NOTE: Use a gentle touch brush for the tubal cell collection (upcoming steps). With the exception of rare cases in which the fallopian tube is submitted detached from the uterus, inserting a brush head to collect cells from the tubal fimbria occurs typically when the fallopian tube is attached to the uterus.
   1. With the help of a small forceps, insert the brush (see Table of Materials) into the lumen of the fallopian tube through the fimbriae end. Slowly rotate clockwise and move the brush forward through the infundibular portion of the fallopian tube, then the ampulla until it reaches the isthmus.
   2. Slowly rotate and reverse the brush counter-clockwise to pull out the brush. Ensure that the maximal speed of the brush within the lumen is slower than 1 cm/s.
   3. Rinse the brush in the preservative solution (in a vial) by rotating and swirling the brush 10 times in the solution.
   4. Tighten the vial cap.
   5. Record the patient's information and the laterality of the specimen.
   6. Store the vial in a 4 °C refrigerator (for up to) 6 months.

3. Tubal Cytology Slide Preparation

1. Load the sample vial into the automated processor for slide preparation.
   NOTE: We use the automated processor for slide preparation. The following steps are performed automatically after the sample vial is placed into the processor: (i) The cells and debris are separated and dispersed through the rotation of the test filter within the sample vial. (ii) Tubal cells are collected from the exterior surface of the filter by a gentle vacuum. (iii) The filter is inverted and pressed against the slide to let the cells to adhere to the circular area within the slide. (iv) The slide is further fixed in a cell fixative bath and ready for staining and cytology analysis. Unfortunately, there is no alternative manual method, which can yield results with a similar quality.

4. Modified Papanicolaou Staining Protocol

1. Stain the slides made from step 3.1 by running the automatized non-gyn stain procedure, as illustrated in Table 1.
   NOTE: The staining method represents a modification of the Papanicolaou staining technique that we use routinely for non-gynecologic specimens. We use an automated tissue processor to stain some slides. We chose the Non-Gyn staining procedure over the Gyn staining procedure in this study. The Eosin used is EA-65, instead of the EA-50 used for gynecological specimens (Pap smear specimen). In addition, OG-6 in the Gyn staining procedure, which is mainly for squamous cell staining, is not included in the staining procedure since no squamous cells are expected to be seen in this study. EA65 (Eosin Azure) is a counter stain solution comprised of 3 dyes: Eosin Y, Fast green FCF, and Bismarck brown Y. The procedure is illustrated in Table 1.
2. Perform a manual quick Pap stain procedure as follows.
   1. Dip the slide made in step 3.1 into 95% ethanol, 10 times, with each dip taking about 1 s.
   2. Dip the slide into H₂O, 10 times, with each dip taking about 1 s.
   3. Emerge the slide in hematoxylin for 10 - 60 s.
   4. Dip the slide into H₂O, 10 times, with each dip taking about 1 s.
   5. Dip the slide into 95% ethanol, 10 times, with each dip taking about 1 s.
   6. Emerge the slide in EA65 for 1 - 3 min.
   7. Dip the slide into H₂O, 10 times, with each dip taking about 1 s.
   8. Dip the slide into 95% ethanol, 10 times, with each dip taking about 1 s.
   9. Dip the slide in xylene until the slide becomes clear.
      NOTE: This is a modified Papanicolaou staining procedure. This method is used as a more rapid stain method for cases with limited time or space like on-site fine needle aspiration and STAT specimens. This procedure provides comparable quality staining as the automatized Non-Gyn stain procedure. The procedure is illustrated in Table 2.

5. Spin Cells onto Slides

NOTE: Three cytospin slides are made for each specimen. One slide is H&E stained for the morphological comparison with the cytology slide. Two unstained slides were made for the future IHC staining study. The detailed steps of this section are not the focus of this paper; therefore, we will not discuss them in extended details.

1. Prepare a cytocentrifuge with a labeled slide, specimen funnel and filter for each sample to be examined.
2. Concentrate the cells by centrifuging for 5 min at 200 x g.
3. Remove about half of the supernatant and then re-suspend the cells by gentle pipetting.
4. Add 200 µL of each cell suspension to a specimen funnel.
5. Spin at 250 x g for 5 min. Carefully remove the slide(s) from the cytocentrifuge and transfer them to 95% Ethanol for fixation.
6. Air dry prior to staining and for long term storage.
   NOTE: Depending on the cell density on the final slides, step 5.2 and step 5.3 can be skipped or adjusted. In this study, we require at least 2 cell clusters present per high power view for adequate cellular density.

6. Sectioning and Extensively Examining the Fimbriated End (SEE-FIM) Protocol

1. Fix the entire specimen (including uterus, bilateral fallopian tubes, and ovaries) in 10% formalin prior to grossing to minimize exfoliation.
2. Gently and carefully detach the fallopian tubes from the uterus at the isthmus using small forceps and a scissors. If adhesion is present, carefully dissect the fimbriated end from the ovary surface using small forceps and a scissors; these steps are crucial to minimize tumor cross contamination between ovary and fallopian tube due to their intimate proximity.
3. Amputate the infundibulum and fimbriae segment (the distal 1.0 - 2.0 cm of fallopian tube) from the rest of the specimen.
4. Section the infundibulum and fimbriae longitudinally at 2 mm intervals. Submit all sections in toto for histologic examination.
5. Section the isthmus and ampulla at 2 - 3 mm intervals and submit entirely.

Wyniki

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...

Dyskusje

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...

Materiały

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