University of Groningen Gynaecological malignancies in Lynch syndrome Woolderink, Jorien Maria

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Gynaecological malignancies in Lynch syndrome

Woolderink, Jorien Maria

DOI:

10.33612/diss.84185340

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Publication date:

2019

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Citation for published version (APA):

Woolderink, J. M. (2019). Gynaecological malignancies in Lynch syndrome: surveillance and cancer

characteristics. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.84185340

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Feasibility of

endometrial sampling

by vaginal tampons in

women with

Lynch syndrome

Woolderink JM

1,5

_{, De Bock GH}

2

_{, van Hemel BM}

3

_{, Geuken E}

4

_,

Hollema H

3

_{, Werner N}

3

_{, Mourits MJ}

5

1_{Department of Gynaecology, Martini Hospital Groningen}

2_{Department of Epidemiology, University of Groningen, University Medical Center Groningen} 3 _{Department of Pathology, University of Groningen University Medical Center Groningen} 4_{Department of Pathology, Martini Hospital Groningen}

5 _{Department of Gynaecologic Oncology, University of Groningen} University Medical Center Groningen

Submitted for publication

4

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ABSTRACT

Objective

Endometrial sampling during surveillance of women with Lynch syndrome (LS) is an invasive and painful procedure. The aim of this study was to evaluate the feasibility of a less invasive procedure of collecting vital cells by vaginal tampons.

Materials and methods

In this prospective study, 25 consecutive asymptomatic women with LS or first-degree relatives (FDR) who underwent annual gynaecological surveillance were included. Women were asked to insert a vaginal tampon two-four hours before visiting the outpatient clinic for annual gynaecological surveillance including endometrial sampling. Feasibility was evaluated by patient acceptance, by pain intensity of both procedures assessed by VAS scores (range 0-10) and by evaluating the presence of vital cells in tampons and endometrial sampling. Two pathologists independently evaluated all samples.

Results

All women accomplished the vaginal tampon procedure, where 23/25 women underwent invasive endometrial sampling thereafter. The median VAS score with tampons was 0 (range 0-10) and with invasive endometrial sampling 5.5 (range 1-10) (p<.001). None of the tampon samples analysed by cytology showed endometrial cells and all contained vital squamous cells and granulocytes. Eighteen (78%) invasive endometrial samplings contained enough endometrial tissue for analysis, in which no endometrial abnormalities were found.

Conclusion

Endometrial surveillance by using tampons was a well-accepted and non-painful procedure. All tampon-samples contained vital cells, however no endometrial cells were found in these asymptomatic LS women without endometrial pathology. More research has to be done to evaluate whether this female friendly surveillance by intravaginal tampons can be applicable for endometrial surveillance in women with LS.

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4 INTRODUCTION

In women with Lynch syndrome (LS) the lifetime risk of developing endometrial cancer is 15-55% depending of the type of gene mutation. (1-5) The mean age to develop endometrial cancer in LS is 50-55 years of age, which is ten years earlier than in women with sporadic endometrial cancer. (3-6) Annual surveillance of women with LS to detect endometrial abnormalities in a premalignant or early malignant stage is effective. (6-10) Whether endometrial surveillance should be performed by transvaginal ultrasound (TVU) accompanied with standard endometrial sampling or by TVU alone and endometrial sampling only when indicated, is still under debate. (6,8-10) The current Dutch guideline for LS advises annual endometrial surveillance by transvaginal ultrasound and standard endometrial sampling at the age of 40-60 years. (11) However, the disadvantage of standard endometrial sampling is, that it is an invasive and painful procedure, irrespective of the indication. (12-13) Alternative strategy to avoid the pain is by hysteroscopy under conscious sedation, possibly with colonoscopy in the same setting. (12,14-15)

We searched for another, non-invasive and less painful method to collect endometrial cells. Obtaining cells for diagnostic purpose by using vaginal tampons has been published before in women with cervical cancer, high-grade serous ovarian cancer and endometrial cancer. (16-22) In 1954, it was first described that malignant cells could be obtained by using tampons in women with cervical and endometrial cancer. (16-17) In 2004, a tampon study was performed analysing methylated DNA in cervico-vaginal secretion from women with and without endometrial cancer obtained by tampons. (20) In women with endometrial cancer a higher level of methylated genes was found in vaginal secretion using tampons compared to women without endometrial cancer who underwent a hysterectomy for benign reasons. (20) Recently Bakkum-Gamez et al. performed a tampon study in 66 women before hysterectomy. Of 66 patients, 38 had endometrial cancer and 28 women underwent a hysterectomy for benign reasons. Yield of the tampons in gene level analysis showed a significantly higher methylation rate observed in women with endometrial cancer compared to women with benign endometrial abnormalities. (22) There are no studies about endometrial surveillance by using tampons in asymptomatic women with Lynch syndrome until now.

The aim of this feasibility study was to analyse if this method to collect endometrial cells with tampons is less painful, compared to the current invasive procedure of endometrial sampling, during annual surveillance in women with LS. The secondary

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aim was to evaluate, whether it is possible to find endometrial cells by using tampons, in asymptomatic women with LS and if so, if these cells are vital enough to be analysed by a pathologist.

Materials and methods

For this feasibility study, 25 consecutive women with LS or first-degree relatives (FDR) at 50% risk of LS who underwent annual gynecological surveillance at the Family Cancer Clinic (FCC) of the University Medical Center Groningen or at the gynecology outpatient clinic of the Martini Hospital Groningen were included. Written informed consent was obtained from all patients in this study. The ethics committee of the University Medical Center in Groningen approved the study. All relevant data were entered into a separate password protected database, and protection of a patient’s identity was guaranteed by assigning study specific unique patient numbers.

Inclusion criteria

All asymptomatic pre- and postmenopausal women with LS (i.e. proven carrier of a pathogenic mutation in either MLH1, MSH2, MSH6 or PMS2) or a first-degree relative at 50% risk of the gene mutation who underwent annual gynecological surveillance were included in this study.

Collection of endometrial cells with tampons and endometrial sampling

All women who met the inclusion criteria received written information about the tampon study at home. In total 32 women received information about the tampon study, seven (22%) women declined to participate and 25 (78%) women accepted and were included. If they were willing to participate and returned the signed informed consent form, a standard size cotton vaginal tampon with a flacon of 10 ml saline (NaCl 0,9%) was send by post. At home the women inserted the tampon vaginally two-four hours before the surveillance visit. This time frame was reported to be the optimal period to obtain vital cells of sufficient quality for analysis. (19,22) Before insertion the women were instructed to wet the tampon with 10 ml NaCl 0,9% to stick more cells at the outside of the tampon and to prevent dehydration of the cells. Women were asked not to take a bath or shower after inserting the tampon.

At the outpatient clinic, before starting the physical examination, the tampon was removed by the patient and handed to the gynecologist. The gynecologist immersed the tampon at least 10 times in cytological fixation fluid and hence sprayed Thinprep preservation cytology solution® along the tampon to collect as many cells as possible.

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4

After removing the tampon the women were asked to report the pain score for the

tampon procedure by the Visual Analogue Scale (VAS score). Women were instructed to report the pain score by using a VAS measuring staff, (0 is no pain, 10 is the most severe pain you can imagine), directly after the procedure. Afterwards all women underwent standard gynecological surveillance consisting of a transvaginal ultrasound and subsequent endometrial sampling and a VAS score (range VAS 0-10) was taken for this procedure as well.

Analysis of the samples

The tampon fluid was send for cytological analysis and the endometrial sample was send independently for histological analysis, without mentioning the tampon study.

Cytological analysis

At the pathology laboratory, from each tampon fluid sample one cytological slide was made on the T5000 (Hologic, Cyntyc Corp, Marlborough, MA, USA) using the ThinPrep system and PAP stained. All cytological samples obtained with the tampons were analysed by an experienced cyto-technician followed by a cyto-pathologist. The findings were described in a report. All tampon secretes were analysed by two cyto-pathologists blindly and the results were compared. The presence and quality of endometrial cells and the presence or absence of atypia or endometrial cancer were analysed in the tampon secretes. No differences in pathological outcome of the tampon fluid between two pathologists were found.

Histological analysis

From the endometrial samples for histological analysis, slides were made and HE stained conform the standard procedure. All histopathologic tissue obtained by standard endometrial sampling was analysed according to the standard procedure. The presence or absence of hyperplasia with or without atypia or endometrial cancer in the endometrial samples was analysed. The findings were described in a report. All endometrial biopsy samples were analysed by two pathologists blindly and the results were compared. All endometrial samplings were stored at the Pathology department. No differences in pathological outcome of the endometrial samplings between two pathologists were found.

Data collection

For each woman, patient characteristics and clinical data including parity, the age at the surveillance visit, type of gene mutation, menopausal status, use and type of

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contraceptives, date of last menstrual period, endometrial thickness at transvaginal ultrasound, cytopathology reports of tampons and histopathology reports of endometrial samplings were recorded. VAS scores (range 0-10) after removal of the tampon and at standard endometrial sampling were collected.

Data analysis

Differences in pain by VAS scores between the tampon procedure and standard endometrial sampling were evaluated by Wilcoxon signed rank testing. Endometrial cell yield of both procedures was assessed. Data analysis was performed with SPSS statistics version 20.

RESULTS

Of 32 women who received information about the tampon study, 25 (78%) of the women were included from January 2017 until August 2017. The median age of these 25 women was 47.0 years (range 73-71 years; see Table 1). A total of 15 (60%) women were premenopausal of which three used oral contraceptives, four had a Mirena IUD® and one used depo-provera. Ten (40%) women were postmenopausal, of which one used hormone replacement therapy. The mean endometrial thickness in 15 premenopausal women was 4.5 mm (range 2-18 mm) and in the 10 postmenopausal women 3.0 mm (range 1-7 mm).

Of these 25 women, the median VAS score reported with the tampon procedure was 0 (range 0-10), the median VAS score reported with the 23 endometrial samplings was 5.5 (range 0-10; see Table 2). The level of pain reported with the tampons compared to the endometrial samplings was significantly lower (p<0.001).

In the premenopausal women, in none of the 15 cytological samples obtained with tampons endometrial cells or endocervical cylindric epithelial cells were found, although all samples contained vital granulocytes and squamous cells (see Table 2). Of these premenopausal women, 13/15 (87%) standard endometrial samplings contained enough endometrial tissue for histopathological analysis and no endometrial abnormalities were found. One endometrial sampling contained insufficient tissue for analysis and one woman denied this procedure because of severe pain during the last procedure.

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4

TABLE 1 Characteristics of women with LS during endometrial surveillance by tampons and subsequent endometrial sampling (n=25)

Overall (n=25) Premenopausal

women (n=15) Postmenopausal women (n=10) Age at study entry, mean (SD)

in years

48.8 (9.9) 42.8 (4.3) 57.9 (9.0) Age at study entry, median

(range) in years 47.0 (73-71) 42.0 (37-50) 56.0 (46-71) Gene mutation MLH1 MSH2 MSH6 PMS2 Lynch

First degree relative LS

3 (12%) 5 (20%) 6 (24%) 5 (20%) 1 ( 4%) 5 (20%) 2 (13%) 3 (20%) 5 (33%) 2 (13%) 3 (20%) 1 (10%) 2 (20%) 1 (10%) 3 (30%) 1 (10%) 2 (20%) Menopausal state Premenopausal

Postmenopausal 15 (60%)10 (40%) 15 (100%)Na* Na*10 (100%) Parity Nulliparous Primi/multiparous Unknown 4 (20%) 16 (80%) 5 2 (13%) 13 (87%) 2 (40%) 3 (60%) 5 Hormonal treatment - OC - Mirena IUD - Progesterone - HRT** - None 3 (12%) 5 (20%) 1 ( 4%) 1 ( 4%) 15 (60%) 3 (20%) 4 (27%) 1 ( 7%) - 7 (47%) 1 (10%) 1 (10%) 8 (80%) Endometrial response (mm), Median (range) - 1-2 - >2-4 - >4-6 - >6-8 - >8-10 - >10 - Unknown 3.8 (1-18) 3 (13%) 9 (38%) 5 (21%) 1 ( 4%) 2 ( 8%) 4 (17%) 1 4.5 (2-18) 1 ( 6%) 6 (40%) 2 (13%) 2 (13%) 4 (27%) -3.0 (1-7) 2 (22%) 3 (33%) 3 (33%) 1 (11%) 1 Last period - < 2 weeks - ≥ 2 weeks Postmenopausal Unknown 3 (12%) 11 (46%) 10 (42%) 1 3 (21%) 11 (79%) 1 -10 (-100%)

-*Na: not applicable

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In the postmenopausal women, none of the 10 cytological samples obtained with tampons contained endometrial cells or endo cervical cylindric epithelial cells, although all secretes contained other vital cells (see Table 2). Five (50%) postmenopausal endometrial samplings contained enough endometrial tissue for histopathological analysis and no endometrial abnormalities were found. Four endometrial samplings had an insufficient yield and in one woman the endometrial sampling could not be performed because of a cervical stenosis (see Table 2). Of the four women with an insufficient sample, three had an endometrial thickness of 2-3 mm, and in one woman the endometrial thickness was 7 mm. Because the absence of any symptoms, no further endometrial tissue analysis was performed in this woman.

TABLE 2 Yield of endometrial cells obtained with tampons and standard endometrial sampling

Tampons

(n=25) (%) Endometrial sampling (n=25)

(%)

Median VAS score (range) 0.0 (0-10) 5.5 (1-10)

Quality of cells in the samples

- Good

- No/To less endometrial cells

- Endometrial sampling not obtained

cervical stenosis fear for pain

25 (100%) 25 (100%) 18 (78%) 5 (22%) 1 1 Suffi cient endometrial samples

Premenopausal women Postmenopausal women Insuffi cient endometrial samples Premenopausal women Postmenopausal women 0 -25 (100%) 15 (60%) 10 (40%) 18 (78%) 13 (72%) 5 (28%) 5 (23%) 1 (20%) 4 (80%)

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4 DISCUSSION

In this feasibility study in an unselected series of 25 asymptomatic pre- and postmenopausal women with LS or FDR, vaginal tampon sampling procedures during annual gynecological surveillance for LS were well accepted and less painful than invasive endometrial sampling. The median reported VAS score of the tampon sampling was significantly lower compared to the standard endometrial sampling (0 versus 5.5). All tampon-samples contained vital cells, however no endometrial cells were found in the tampon samples of these asymptomatic women without endometrial pathology.

Standard endometrial sampling is a painful procedure. In a group of women with LS and FDR who underwent annual surveillance, this procedure was reported painful with a median VAS score of 5.0 which is comparable to the here observed median VAS score of 5.5. (13) In the same group of women 20% decided to refrain from endometrial sampling because of pain and choose for preventive surgery or annual gynecological surveillance by TVU only. (13) Two other studies evaluated combined endometrial sampling and colonoscopy in women with LS under conscious sedation and concluded that this combination is a less painful experience in women with LS than endometrial sampling in an office setting without sedation. (14-15)

Of the standard endometrial samplings in our study, 78% contained enough tissue for adequate analysis. This is comparable with 74%-90% reported in other studies. (23-24) In our study all tampon-samples contained vital cells, however no endometrial cells were found, most probably because there were no endometrial (pre)malignancies and hence no shedding of endometrial cells. Thus far, other studies reporting on the collection of endometrial cells with tampons compared the yield in women with and without endometrial cancer. In those studies, it is found that in women with endometrial cancer, malignant cells and methylated DNA could be found on vaginal tampons. (17-20,22,25-32) Especially DNA methylation in cells from women with endometrial cancer compared to women with benign endometrium was a very sensitive procedure to discriminate between women with and without endometrial cancer. (20,22) Other studies found that endometrial cells, shed in cervical smears, may indicate endometrial pathology especially if vaginal bleeding is present. (33-34) DNA methylation assays may potentially be more sensitive because it can be performed on fragments and intact cells are not required. (22)

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In this study using vaginal tampons to collect cells is a well-accepted procedure, further research on the applicability is recommended to find out, whether vaginal tampons can be used for surveillance in women with an increased risk of endometrial cancer. Because women with LS have a lifetime risk of developing endometrial cancer of 15-55% depending on the type of gene mutation and an annual risk of about 2.5% to develop this disease mostly at postmenopausal age, in a larger and long term study it will be expected to find some (pre)malignancies of the endometrial tissue. (1,3,5,35-37) It would be interesting to evaluate if the tampon method is could replace the invasive, painful endometrial sampling.

This is, to the best of our knowledge, the first study, evaluating the feasibility of collecting endometrial cells with tampons in asymptomatic women. This procedure was also never tested before in women with LS. A limitation of the study is that it is still unclear if endometrial abnormalities will be found in symptomatic women with LS by using tampons. Therefore, further research should focus on this topic.

In conclusion: the tampon procedure is a non-painful and well accepted procedure when compared to invasive endometrial sampling during annual gynecological screening in women with LS. The yield of endometrial cells in asymptomatic women with LS was absent. Further research should focus on the effectivity and the applicability of vaginal tampons in the annual screening of women with LS, to avoid unnecessary painful invasive procedures.

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4 REFERENCES

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M M MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH P LH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpC SH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH CAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 SH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM ML 2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH H1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM H6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH AM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 Ep 2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 S2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 M LH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 SH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM ML CAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MS SH2 MSH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpC SH6 PMS2 EpCAM MLH1 MSH2 MSH6 PMS2 EpCAM ML pCAM MLH1 MSH2 MSH6 PMS2 EpCAM MLH1 MSH2 MS