Fluorescence in situ hybridization test for detection of endometrial carcinoma cells by non‐invasive vaginal swab

Abstract Endometrial cancer (EC) is the most common gynaecological malignancy with increasing incidence in developed countries. As gold standard, hysteroscopy confirms only 30% of suspected ECs. The detection of EC cells in the vagina by fluorescence in situ hybridization (FISH) after a smear test could reduce invasive procedures in the future. Using array‐based comparative genome hybridization (aCGH) on 65 endometrial carcinomas, most frequently imbalanced regions of the tumour genome were identified. Bacterial artificial chromosomes were used to generate FISH‐probes homologue to these human regions. The FISH test was hybridized on swabs specimens collected from the vaginal cavity. Samples from six patients without EC were selected as a negative control and on 13 patients with known EC as a positive control. To distinguish between benign and EC cases, the cut‐off value has been defined. A first validation of this EC‐FISH Test was performed with swabs from 41 patients with suspected EC. The most common genomic imbalances in EC are around the CTNNB1, FBXW7 and APC genes. The cut‐off is defined at 32% of analysed cells without diploid signal pattern. This differs significantly between the positive and negative controls (p < 0.001). In a first validation cohort of 41 patients with suspected EC, the EC‐FISH Test distinguishes patients with and without EC with a sensitivity of 91% and a specificity of 83%. The negative predictive value is 96%. This is the first report of a non‐invasive EC‐FISH Test to predict EC in women with suspected EC.


| INTRODUC TI ON
Endometrial cancer (EC) is the most common gynaecological malignancy in the developed countries, and its incidence is increasing. 1 One reason for this is the increasing rate of obesity being the major risk factors over the past decades. In 2020, the European Cancer Information system indicated that the estimated age-standardized incidence rate of EC was 28.9/100,000 in Europe and for Germany 24.8/100,000. EC is the ninth cause of cancer death for women in Europe, with an estimated mortality rate of 6.2/100,000. 2 Approximately 90% of ECs are sporadic, and the remaining 10% are hereditary, that is Lynch syndrome. 3 Until recently, the EC classification by Bokhman et al. was the standard, categorizing EC into two pathogenic types using clinical and histopathological variables. The more common oestrogen-dependent endometrioid type I EC have a better prognosis than non-endometrioid type II EC. The rarer form of type II EC (20%) comprises high-grade tumours, which are clinically more aggressive and tend to metastasize. Normally, the later type is diagnosed in the advanced stages and has unfavourable prognosis. 4 Recently, The Cancer Genome Atlas (TCGA) has provided a comprehensive genomic and transcriptomic analysis of 373 EC cases and described the molecular characteristics of these tumours in more detail, which allowed them to stratify EC into four distinct molecular subtypes, [5][6][7] thus becoming the modern standard classification system. EC is currently diagnosed by invasive procedures requiring general anaesthesia. The American College of Obstetricians and Gynecologists (ACOG) committee states that hysteroscopy-guided diagnosis of intrauterine pathologies is considered the gold standard. 8 Postmenopausal bleeding is a frequent and early marker in EC patients. However, the postmenopausal bleeding can be caused by benign conditions as well. In addition, EC can be found in premenopausal women showing irregular vaginal bleeding. In studies looking at vaginal bleeding disorders, only around 10% of patients were diagnosed with EC but all patients underwent an invasive diagnostic procedure. 9 It would be of great benefit for the patient and for the healthcare providers if a predictive marker could identify affected patients, hereby reducing the diagnostic burden. The goal of FISH detection is to reduce the number of diagnostic hysteroscopies in those cases of suspected endometrial carcinoma whose FISH results do not support suspicion. We, therefore, favour a test with the best possible specificity and a high negative predictive value. The falsepositive FISH test results would be identified as actually negative by the diagnostic gold standard of hysteroscopy.
Genomic imbalances that deviate from the normal diploid state could serve as biomarkers to identify tumour cells. Like most solid tumours, the EC is characterized by an accumulation of different genomic imbalances. [10][11][12] These genomic aberrations occur very heterogeneously. Nevertheless, the EC shows recurring patterns.

| DNA preparation
QIAamp® DNA mini kit was used to extract genomic DNA (gDNA) from fresh tissue by the manufacturer's protocol. Genomic DNA from FFPE tissue was isolated by BIOstic® FFPE tissue DNA isolation kit or QIAGEN® GeneRead DNA FFPE kit following the manufacturer's protocol after microdissection of tumour tissue out of 10 μm tissue cuts. The quantity of extracted double stranded DNA was determined by Qubit® 2.0 fluorimeter. The purity of extracted DNA was also measured by using the spectrophotometer NanoDrop™ 2000.

| Generation of FISH probes
Conspicuous aberrant regions with the most frequent changes in the examined ECs were defined as target regions. Within these target regions, we have identified potential bacterial artificial chromosomes (BACs) that could be considered as possible FISH samples.
We obtained corresponding BAC clones from BACPAC Genomics Inc (Richmond, Ca) in lysogeny broth (LB) agar stab cultures. These

| Preparation of brush cells
Simple swab brushes were used for the smears. After the swab, the breakable brush head is transferred to 4 ml Certipur buffer solution (Merck), which was poured into a 15 ml Falcon tube. This could be stored at 4°C for about 2 days until the cells were prepared. The brush head was carefully rinsed in the buffer solution and then removed. The resuspended cells were fixed by adding 1 ml of cold fixative (One part glacial acetic acid and three parts methanol) four times. The suspension was inverted in between. After the suspension has been centrifuged for 5 min at 450 g and 4°C., the supernatant was carefully removed and the pellet was resuspended with 4 ml of cold fixative. This process was repeated twice. The suspen-

| EC-FISH Test
The slide with the smear cells was incubated for 2 min in 2 × saline sodium citrate (SSC) buffer at 37°C. The slide was then transferred to a 37°C. 0.01 M hydrochloric acid with 0.005% pepsin for 15 min.
The slide was washed in phosphate-buffered saline (PBS) buffer, pH 7.4 at room temperature for 3 min. Then, the cells were fixed in PBS with 1% buffered formaldehyde and 20 mM magnesium chloride, pH 7. The slides were washed again for 3 min in PBS at room temperature. The cells were dried in an ascending series of ethanol (70%, 85% and 100%) for 1 min each. Finally, the slides were air dried. At the next step, DNA of the swab cells and the FISH probe DNA were denatured simultaneously. Ten microliters of the FISH probe was denatured under a rubber sealed 22 × 22mm cover glass for 10 min at 75°C. Then, the slide was hybridized at 37°C. in a humid chamber upside down overnight. After hybridization, the cover slip was carefully removed and the slide was washed at room temperature for 2 min in 2× SSC with 0.1% Igepal. The next washing step was carried out for 2 min in 0.4 × SSC with 0.3% Igepal at 72°C. This is followed by another washing step in 2× SSC with 0.1% Igepal at room temperature for 1 min. The slides were incubated again in an ascending series of ethanol (70%, 90% and 100%) each for 1 min.
Finally, the slides were air-dried and covered with the mounting medium containing DAPI. The hybridization was evaluated and viewed on a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Oberkochen, Germany), which is equipped with optical filter sets for FITC and Cy3. We used ISIS (Metasystems, Altlusheim, Germany) as a recording and documentation software. At least three times, 50 interphase nuclei per patient were counted and the average proportion of conspicuous signal patterns was documented. Three and a half times the standard deviation was added to the mean proportion of the negative controls, resulting in a limit value above which the presence of endometrial carcinoma cells could be detected. All signal constellations within an interphase nucleus that deviated from the normal diploid pattern with two signals per each colour (loci) were rated as conspicuous.  (Table 1; Figure 6). In our validation sample, 11 out of 41 patients were histologically proven to have carcinoma uteri (  Figure 5). The signal distribution of the 10 true positive cases (Table 2)

| DISCUSS ION
To our knowledge, this is the first study on using aCGH as a tool to detect EC in women with suspected EC by transvaginal ultrasound.
Using aCGH the chromosome region, 1q23 was identified as the  28 Cuevas et al. 2020 suspect FBXW7 to be involved in mesenchymal-epidermal transition in endometrial carcinoma. 29 The TA B L E 1 Calculation of the cut-off. The proportions of conspicuous signal patterns in the vaginal smears of the negative controls as well as in those of the positive controls and the tumour smears are compared here. The mean value and the standard deviation are calculated below. The cut-off of this EC-FISH Test is calculated from the sum of the mean value and three and a half times the standard deviation. third most common imbalance in our study was found also in more than 60% of the ECs examined by aCGH in the APC gene in chromosomes 5q22.2. The APC gene is a well-known tumour suppressor gene, associated with CTNNB1, with diverse functions, that is cell migration and adhesion. Ignatov and co-authors associate hyper methylation of the APC gene as an early event with the development of EC. 30 Moreno-Bueno and colleagues could not show a functional equivalent to mutations in APC in EC. 31 The imbalances we detected in these three loci of EC with tumour-relevant genes reinforce the assumption that a gene dose effect could influence tumour development. A directly proven impact of the genes on the molecular genetic classification 6 of EC cannot be derived, but their claim is also different. The loci described in their work represent the most common imbalances in EC in our study. Therefore, these loci should serve as biomarkers for the detection of EC cells. The justification for a simple EC detection test can be found in the currently invasive diagnostic procedure to confirm EC. In nearly half of the suspected EC cases, the diagnosis is not confirmed. 32   The high cut-off of the EC-FISH Test is also due to the high proportion of CNAs in the endometrial carcinomas, as Figure 1 confirms. In connection with the chromosomal heterogeneity of the EC, a ploidy standard close to any centromere as an additional FISH signal cannot bring any gain in knowledge. administration (supporting); supervision (lead); writing -review and editing (lead).

ACK N OWLED G EM ENT
We

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.