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Kava

Procedure/Surgery: Lavage of the Cavum uteri and proximal Fallopian tubes, performed in the luteal phase of the female cycle

Speiser-catheter for uterine and tubal lavage

In Phase I the sponsor will systematically test conditions for lavage filtration that increase tumor cell fraction without reducing tumor mutation yield. The Sponsor will also transition all lavages to luteal phase timing, when endometrial shedding is least. In Phase II the Sponsor will examine our data in context of clinical characteristics, particularly age, to develop a multivariate model that determines optimal mutant allele frequency (MAF) diagnostic threshold by patient. Furthermore, the sponsor will explore a highly innovative idea, entailing empirically determining each individual's background mutation load, agnostic of the aging or mutagenic exposures responsible, and using this as a personalized calibrator to determine optimal MAF diagnostic threshold.

Case control study in two patient population (AIM 1 in average risk population, AIM 2 in high risk population)

Early Detection of High Grade Ovarian Cancer Using Uterine Lavage EHUD Study and Duplex Sequencing

Pilot Study of Early Detection of High Grade Ovarian Cancer Using Uterine Lavage and Duplex Sequencing

Preliminary testing with two samples indicated that filtration of UtLs to remove clusters of endometrial cells could increase sensitivity for tumor mutations several fold. This dataset will be expanded by analyzing the filtration effect in 10 ULs from patients with HGSC with known TP53 mutations. Each UtL will be divided in half: the first half will be filtered and the second will remain unfiltered. DNA will be extracted from the filtered, filtrate, and unfiltered fractions and analyzed by TP53KDS (total of 30 samples). For each UL, the DNA yield will be compared in each of the fractions and the MAF of the tumor mutation. It is anticipated that the filtered fraction will contain less DNA then the unfiltered fraction but the tumor mutation will be present at a higher frequency (i.e. more is gained by enrichment than is lost by reduction from less available DNA).

SNP allele fraction (AF) will be compared at different dilutions to determine accuracy (expected AF vs. observed AF), precision (AF variation among replicates), and lowest limit of detection achievable.

To asses the reproducibility in clinical use total mutation frequency will be assayed. The coefficient of variation among replicates will be calculated. Overall mutation frequency across TP53 in UtLs samples will be measured for both replicates of each patient. VAF will be approximated by number of non-reference duplex bases/total duplex bases sequenced. Confidence intervals will be computed using a binomial normal approximation.

A detailed mutation profile will be generated from the DS output files: mutant allele frequency (MAF) for all mutated positions, mutation spectrum, predicted pathogenicity to protein function, relationship to known hotspots, and overall mutation load (number of mutant nucleotides divided by the total number of nucleotides sequenced). Following this, samples will be unblinded for cases-controls status.

TP53 MAF from DNA collected by UtLs will be used as predictor for differentiating between average risk patients (AIM I) with and without HGSC by logistic regression modelling. An analogous analysis will be applied to the group of high risk patients (AIM II) where presence and absence of STIC is defining the outcome variable. Cut off values for mutant allele frequency will be suggested in both cases and specificity and sensitivity will be estimated including appropriate confidence intervals.

The association between TP53 mutation frequencies in uterine lavage and leukocytes will be examined and will determine whether false negatives in Aim 1A correspond to cases with increased BB in leukocytes. In addition, TP53 mutation frequencies in leukocytes will be analyzed as a predictor of case control status, again using the leave out 10% procedure. This innovative calibration method will be compared with the simple ROC metrics achieved with the univariate model using a fixed mutation fraction as well as the adjusted multivariate model developed based on other patient characteristics such as age. As a scientific question unrelated to the present aims, it is worth investigating whether leukocyte TP53 mutation load will serve as an independent predictor of patient age, overall health or history of mutagenic exposures.

Comparison of performance of UtL collection with that of Pap smear collection. It will be investigated if any additional power to discriminate cases from controls can be gained by combining information from Pap smears and ULs.

Aiming to evaluate the potential for defining a DNA-Methylation signature for simple PCR testing, dCT-PCR values from 96-plexed high-throughput MSREqPCR analysis will be analysed by bioinformatics \& biostatistics. See Detailed description og the Aim III above for more details.

Paul Speiser, Prof.MD,

Clinical Professor

Lavage of the Cavum uteri and proximal Fallopian tubes, performed in the luteal phase of the female cycle

Paul Speiser, Univ.Prof.Dr.med.

The current study aims at answering the scientific question, whether exfoliated cells from STICs get transported into the uterine cavity via the fallopian tube, and whether it is possible to detect those cells in the lavage fluid from the uterine cavity and proximal fallopian tubes.

To address this question, the investigators will study 20 lavage samples and their 20 corresponding STIC-positive tissue samples in women who opt for risk-reducing bilateral salpingo-oophorectomy (rrBSO) because of increased risk of high grade serous carcinoma of the pelvis (HGSC) (mostly carrying a BRCA mutation), without a history of tubal occlusion for sterilization. Women who opt to have the fallopian tubes removed but the ovaries preserved are eligible for the study too, as are women who opt for rrBSO plus hysterectomy.

The term "high grade serous carcinoma" (HGSC) describes a group of ovarian, tubal and peritoneal cancers with an aggressive biological behavior. HGSC is the leading cause of death from gynecologic malignancy in western civilized countries. Women affected, usually have advanced stage disease with metastatic spread throughout the abdominal cavity at time of diagnosis. Five-year survival rates are in the range of 10 to 30 percent. The specificity of current diagnostic tools (CA-125 and transvaginal ultrasonography) is low and ineffective at detecting HGSC early enough to improve clinical outcomes. Definitive diagnosis of HGSC mostly relies on surgical confirmation. These findings underline the need for an effective test for early detection of HGSC. In the general population, the lifetime risk is 1.5 percent.

Women with germ line mutations in the BRCA1 and BRCA2 gene or a strong family history of epithelial ovarian cancer carry a high risk for breast cancer and/or HGSC development. Familial or inherited syndromes account for approximately 13 percent of cases of invasive epithelial ovarian and fallopian tube cancer. The lifetime risk of ovarian cancer is 35 to 46 percent in women with BRCA1 gene mutations and 13 to 23 percent in those with BRCA2 mutations. Again, even in this population with high-risk for HGSC, the specificity of CA-125 and transvaginal ultrasonography is still too low and ineffective to improve clinical outcomes.

Over the last years, increasing scientific evidence conglomerated that a large proportion of not only familial HGSC develop primarily in the lining of the fallopian tube, that resembles Müllerian epithelium. These precursor lesions are called "serous tubal intraepithelial carcinomas" (STICs) and are characterized by p53 overexpression on immunohistochemistry and high Ki-67 labelling index indicating a high proliferation index. In over 90 percent, STICs carry mutations in the TP53 tumor suppressor gene.

As for today, risk reducing bilateral salpingo-oophorectomy (rrBSO) is the most effective approach to reducing the risk of HGSC in high risk women. Among women with an increased risk of HGSC (most with BRCA mutations) who underwent rrBSO, 4 to 17 percent are found to have a STIC or even invasive neoplasm, and approximately 80 percent of these neoplasms are in the ampullar part of the fallopian tube.

Recent findings highlighted the malignant potential of STICs. On histopathological specimen, intraluminal shedding of tumor cells from STICs can be frequently demonstrated in the fallopian tube. This shedding of tumor cells from STICs appears to be a risk factor for early transperitoneal metastasis frequently found in HGSC. There is a strong clinical need for screening for STICs, since they are the precursor lesion of HGSC. These facts underline the importance of an effective - non-invasive - test for early detection of STICs.

The ovarian surface, the fallopian tubes, the uterine cavity and the peritoneal cavity all together form a communicating compartment. The physiological function of the ciliated lining of the tubes is to transport the egg into the uterine cavity after ovulation thus making it likely that exfoliated cells from STICs can be found in the uterine cavity.

A promising approach for the detection of STICs has been established by Paul Speiser and Robert Zeillinger (Molecular Oncology Group, Department of General Gynaecology and Gynaecologic Oncology, Medical University of Vienna, Austria). This approach is called the ALPINE technique (Austrian Lavage Procedure for the Detection of tubal Intraepithelial Neoplasms) (manuscript under preparation). To facilitate an quick and easy lavage of the uterine cavity and proximal tubes, a special catheter was developed (MEDICOPLAST, MF 13005, catheter for uterine and tubal lavage). The ALPINE technique includes a lavage of the uterine cavity and proximal fallopian tubes and subsequent analysis of this lavage fluid for the presence of pre-malignant and malignant cells.

For the proof of principle that tumor cells from ovarian cancer are shed and can be found in the lavages of the uterine cavity, uterine lavages were collected before a surgical intervention for suspected ovarian malignancy at our institution and at the Catholic University Leuven, Division Gynaecological Oncology, Belgium. After malignancy was confirmed, genetic changes in the TP53 and KRAS genes were determined in tumor tissue. In a set of 9 epithelial ovarian cancer patients (EOC) and 1 ovarian metastases of a signet ring carcinoma, the presence of these genetic changes was examined in lavage samples, using digital droplet PCR (ddPCR). 10 genetic changes were identified in tumor tissue of these patients and 9/10 (90%) of these changes were detected in the corresponding lavage specimen too.

Furthermore, a filter approach, followed by p53 immunofluorescence staining was established, confirming the presence of tumor cells in the lavage sample of one additional patient.

In a next step, lavage samples of 23 ovarian carcinoma patients, and if applicable corresponding tumor tissue, were analysed through deep sequencing by the group of Bert Vogelstein (Johns Hopkins University, Baltimore, USA). The presence of genetic changes, indicative for ovarian cancer, could be confirmed in 18/23 (78.3%) lavage specimen including both, early and advanced stages.

These results are proof that ovarian cancer cells are shed into the fallopian tubes and uterine cavity, and can be collected through our ALPINE technique. The fact that ovarian cancer cells were detected with high sensitivity in the lavage of the uterine cavity and proximal tubes shows that this approach has potential in early diagnosis. Therefore, the investigators are confident that this method could be applied in detection of premalignant changes in high risk patients, as well.

Aim of the study:

The current study aims at answering the scientific question, whether exfoliated cells from STICs get transported into the uterine cavity via the fallopian tube, and whether it is possible to detect those cells in the lavage fluid from the uterine cavity and proximal fallopian tubes.

Methods:

To address this question, the investigators will study 20 lavage samples and their 20 corresponding STIC-positive tissue samples in women who opt for rrBSO because of increased risk of HGSC (mostly carrying a BRCA mutation), without a history of tubal occlusion for sterilization. Women who opt to have the fallopian tubes removed but the ovaries preserved are eligible for the study too, as are women who opt for rrBSO plus hysterectomy.

Lavage of the Uterine Cavity for the Diagnosis of Serous Tubal Intraepithelial Carcinoma

Pilot Study of the Lavage of the Uterine Cavity for the Diagnosis of Serous Tubal Intraepithelial Carcinoma

Women who opt for rrBSO or opt to have the fallopian tubes removed but the ovaries preserved, and women who opt for rrBSO plus hysterectomy. The lavage can be done either in an outpatient setting or in the operating theatre under general anaesthesia.

Study title	NCT ID	Conditions	Interventions	Eligibility	Organization	Link
Early Detection of High Grade Ovarian Cancer Using Uterine Lavage EHUD Study and Duplex Sequencing	NCT04823871	Ovarian Epithel... Carcinoma in Si... Ovarian Cancer	Speiser-cathete...	18 Years - 80 Years	Medical University of Vienna
Lavage of the Uterine Cavity for the Diagnosis of Serous Tubal Intraepithelial Carcinoma	NCT02039388	Ovarian Epithel... Carcinoma in Si... Ovarian Cancer	Lavage of the C...	18 Years - 80 Years	Medical University of Vienna

Spots Global Cancer Trial Database for serous intraepithelial carcinoma (stic)