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 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.
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.
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.
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.
3. Tubal Cytology Slide Preparation
4. Modified Papanicolaou Staining Protocol
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.
6. Sectioning and Extensively Examining the Fimbriated End (SEE-FIM) Protocol
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 illustrated in Figure 1. As shown in Figure 1, the brush maneuver results in adequate sampling of the specimen. Moreover, the manual quick Pap stain provides good resolution of nuclear details and cytoplasmic transparency, which are essential for the accurate evaluation of the tubal cells.
The comparison between the manual quick Pap stain of a cytology slide and the H&E staining of a cytospin slide is shown in Figure 2. One of the advantages of the above-mentioned slide preparation compared with conventional smear is that it has a much cleaner background and better resolution of nuclear details and cytoplasmic transparency. One example of SEE-FIM of the Fallopian Tube Protocol is demonstrated in Figure 3. As shown in Figure 1, the presence of two cell populations (ciliated cells and secretory cells) is a feature of benign tubal epithelium. The histology of representative sections of the fimbria and ampulla is shown in Figure 4. The brush maneuver shows mild disturbance of villi structure in about one third of specimen.
In our previous study17, the tubal cytological diagnosis correlated with the histological diagnosis of HGSC very well (100%). The cytological appearance of malignancy shows three-dimensional clusters composed of cells with prominent nucleoli and anisonucleosis (Figure 5a). In contrast, the atypical reactive cytology presented as angulated sheets composed of atypical cells with small nucleoli and moderate anisonucleosis without three-dimensional clustering (Figure 5b). HGSC-like cell groups were noted within the context of angulated sheets of normal epithelial cells (Figure 6).
Figure 1: Example of a Manual Quick Pap Stain of the Benign Tubal Epithelium. (A) Background cells. Background cells include small cell clusters consisting of large secretory cells and small ciliated cells, single large (presumably secretory cells) and small (presumably ciliated cells) cells, scattered large and small bare nuclei. (B) Small cell cluster consisting of small ciliated cells and large secretory cells (arrow). (C) Strip of ciliated epithelium (arrow). (D) Tubal epithelium with mesothelial-like sheets. Noted the clinging ciliated cells at the periphery (arrow). Please click here to view a larger version of this figure.
Figure 2: Comparison Between Manual Quick Pap Stain of a Cytology Slide (A) and H&E staining of a cytospin slide (B). A benign angulated cell cluster is present in the center of both the Pap stain and H&E stain slides. Both methods provide good resolution for cytological evaluation of the specimen. However, the Pap stain of the cytology slide yielded a cleaner background. Please click here to view a larger version of this figure.
Figure 3: Illustration of SEE-FIM of the Fallopian Tube. Please note the longitudinal sectioning of the infundibulum and fimbriae segment and the transverse sectioning of the isthmus and ampulla. Please click here to view a larger version of this figure.
Figure 4: Histology of Benign Tubal Epithelium. (A) Fimbriae segment. The tubal epithelium consists of two distinct cell populations: small ciliated cells (arrows) and large non-ciliated secretory cells (circles). Slight villa disturbance is noted. (B) Ampulla section. Note the finger-like fimbria. (C) Mild disturbance of villi structure (arrow). Please click here to view a larger version of this figure.
Figure 5: Comparison Between Atypical Cluster (a) and Three-dimensional Malignant Cluster (b). Malignant cytology shows three-dimensional clusters composed of cells with prominent nucleoli and anisonucleosis (b). Atypical cytology shows angulated sheets composed of atypical cells with small nucleoli and moderate anisonucleosis. Note: No three-dimensional clustering is present (a). This figure has been adopted from reference17.
Figure 6: Correlation Between Cytological and Histologic Appearance of Intraepithelial Neoplasia. (a) Cytological intratubal neoplasia. Cytological intratubal neoplasia showing acquisition of three-dimensional contour and significant anisonucleosis and large cherry red nucleoli (circle) within the context of angulated sheets of normal epithelial cells (arrow). (b) Corresponding surgical pathology section with tubal intraepithelial carcinoma. Note the highly dysplastic cells (circle) adjacent to the relatively normal appearance epithelial cells (arrow). This figure has been adopted from reference17. Please click here to view a larger version of this figure.
95% Ethanol | 5 min |
H2O | 10 sec |
Hematoxylin | 15 sec – 3 min |
Distilled H2O | 5 min |
95% Ethanol | 30 sec |
EA65 | 2 – 5 min |
95% Ethanol | 30 sec |
95% Ethanol | 30 sec |
100% Ethanol | 30 sec |
100% Ethanol | 30 sec |
Xylene | 30 sec |
Xylene | 5 min |
Table 1: Non-Gyn Stain Steps.
95% Ethanol | 10 dips |
H2O | 10 dips |
Hematoxylin | 10 sec-1 min |
Distilled H2O | 10 dips |
95% Ethanol | 10 dips |
EA65 | 1 – 3 min |
95% Ethanol | 10 dips |
100% Ethanol | 10 dips |
Xylene | Dip until clear |
Table 2: Manual Quick Pap Stain Steps.
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 cytology on laparoscopically collected fallopian tube cells has great potential to become a practical, minimally invasive tool for early cancer detection.
The most critical steps for successful collection of fallopian tube cells are: 1) The time between the clamping of blood supply and collection of cells; and 2) Gentle and correct maneuver of the gentle touch brush.
The time (within 30 minutes) between clamping and collection of cells is crucial for good preservation of the morphology and nuclear details of collected cells. Effective communication between pathologists and the surgeons ensure that the specimen is delivered to the collecting site within this period. The vast majority of cases included in the study were performed in the frozen section room. To successfully collect tubal cells, the person who performs the collection should try as best as possible to avoid letting the brush touch the ovarian serosal surface due to the staging implication if the case is malignant. Sometime, this may be difficult due to the proximity of the two organs involved and the complexity of the specimen anatomy. Separation of fallopian tubes from the entire specimen before the collection may be warranted.
The gentle touch brush used in this study can collect a large number of cells that can then be easily transferred to slides or a preservative solution vial, and has a gentle-touch tip designed to minimize trauma to the canal. Moreover, the entire maneuver needs to be slow and gentle to avoid severely disturbing the villous structures of the tubal mucosa. For some cases, it may be difficult to get the brush pass the entire fallopian tube due to obstruction. Nevertheless, it is important to try as best as possible to collect cells from the fimbriae and infundibular portion, since it is believed that the majority of the HGSC originates from the distal end of the fallopian tube. However, the collection should not proceed if the integrity of specimen can be potentially compromised.
Based on our experience, the specimen can be stored in preservative solution for several months without compromising the morphology of the collected cells. The Papanicolaou stain is a commonly used cytological staining technique in cytopathology. The main advantages of this staining procedure include: 1) Good definition of nuclear detail; and 2) Cytoplasmic transparency. In this study, two modified Papanicolaou stain procedures are used for the cytology slides staining, including the automatized Non-Gyn staining procedure and the Quick Pap stain procedure. Both procedures give excellent and reliable slide staining, which shows good preservation of nuclear and cytoplasmic details, and clean background. Although cytospin slides generally have greater background when compared with cytology slides, their H&E stain demonstrated similar quality of cell staining. The H&E staining of these slides can be a suitable alternative method for tubal cytology analysis if an automatic processor is not available.
The SEE-FIM protocol is used for grossing all the fallopian tubes included in this study, including both benign and malignant cases. The SEE-FIM protocol was initially developed for BRCA-positive prophylactic bilateral salpingo-oophorectomy specimens, which is a standard risk-reducing option for BRCA mutation carriers. This protocol allows the maximal exposure of the tubal plicae, from which the STIC is believed to originate. This is currently taken as the gold standard for detecting HGSC tubal extension and STIC. The correlation between tubal cytological finding with SEE-FIM histological finding is crucial for this study to validate the efficiency of the current protocol.
One concern of tubal cell collection using a gentle touch brush is that the maneuver may disturb the integrity of the tubal epithelium and hinder the later accurate histological interpretation of surgical specimens, especially the staging of tumor, if malignancy is identified. However, based on our experience, about one third of cases show mild disturbance of the villi on histology analysis, which generally does not interfere with the final interpretation of the tubal histology.
Our previous study has shown that tubal cytology is sensitive and specific in detecting HGSC and STIC. It will be of great interest to further test whether the combination of tubal cytology with biomarkers staining such as P53 and IMP3 may increase the sensitivity and specificity of detecting serous cancer of tubal origin. Future study will focus on developing a laparoscopic procedure for fallopian epithelium sampling in vivo.
The authors have nothing to disclose.
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.
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 |