Pilot Study of Early Detection of High Grade Ovarian Cancer Using Uterine Lavage and Duplex Sequencing
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 发起方
- 入组人数
- 406
- 试验地点
- 10
- 主要终点
- Variant allele frequencies (VAF) for variants identified in both unfiltered and filtrate uterine lavage from the same patient.
研究概览
简要总结
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.
详细描述
APPROACH PHASE I The University of Washington has demonstrated diagnostic proof of principle; however, further optimization and validation is needed for industrial scale deployment. In Phase I the UtL filtration method will be optimized (Aim 1), analytical performance on the commercial workflow validated (Aim 2) and a sample collection milestone met (Aim 3). Aim 1. Determine utility of uterine lavage filtration to maximize enrichment for tumor derived DNA. Preliminary testing with two samples indicated that filtration of UtLs to remove clusters of endometrial cells could increase sensitivity for tumor mutations several folds. This data set will be expanded by analysing the filtration effect in 10 UtLs 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 analysed by TP53 DS (total of 30 samples). For each UtL, the DNA yield in each of the fractions and the MAF of the tumor mutation will be compared. 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. If sensitivity is improved (i.e. more is gained by enrichment than is lost by reduction from less available DNA), filtration will be used on all future samples collected. Aim 2. Validation of optimized assay: accuracy, precision, limit of detection, and reproducibility. TwinStrand has developed a streamlined DS workflow using improved adapters, ligation chemistry and a high throughput 96 well plate-based format amenable to liquid handling robots and compatible with CLIA laboratory standards.
TwinStrand has also developed an optimized cloud-based analysis pipeline that supports automated parallel processing of multiple samples. The optimized process for the detection of TP53 mutations in UtL samples will be validated. To assess accuracy, technical precision, and lower limit of detection, DNA samples from two individuals that differ in genotype at >5 SNP sites in or near TP53 will be mixed, in ratios from 1:100 1:5,000, two UtL. The mixtures will be sequenced at ~10,000x molecular depth in two independent experiments. 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 test assay reproducibility in its intended clinical use, sequencing will be repeated on the 30 samples used Aim 1, which will encompass a wide range of tumor MAFs. The Sponsor will calculate the coefficient of variation among replicates. Based on pilot studies The Sponsor anticipate excellent reproducibility (CV<5%). Aim 3. Sample collection. Collection of samples proposed in Phase II will be started under appropriate IRB/Ethic Committee approval at each participating institution.
After collection, samples are shipped to the Institute for Cancer Research at the Medical University of Vienna who will perform UtL filtration and DNA extraction.
Paired DNA from Pap smears and peripheral leukocytes will also be extracted. Isolated DNAs will be assigned a de identification number and shipped to TwinStrand for Duplex Sequencing.
Phase I Milestones:
研究设计
- 研究类型
- Interventional
- 分配方式
- Non Randomized
- 干预模型
- Parallel
- 主要目的
- Diagnostic
- 盲法
- None
入排标准
- 年龄范围
- 18 Years 至 80 Years（Adult, Older Adult）
- 性别
- Female
- 接受健康志愿者
- 否
入选标准
- •Written informed consent obtained
- •Age ≥ 18 years and ≤ 80 years
- •Women undergoing primary surgery for suspected High grade serous carcinoma (HGSC) If premenopausal in luteal phase (minimum 14 days after last day of menstrual period) or:
- •Women with high risk for breast or ovarian cancer (HBOC) undergoing prophylactic resection of tubes with or without ovaries. If premenopausal in luteal phase except amenorrhea under hormonal contraception (incl. levonorgestrel -IUD)
排除标准
- •Incapacitated women
- •Pregnant women
- •Prior hysterectomy
- •Prior bilateral salpingectomy
- •Prior tubal ligation
- •First half of menstrual cycle
- •Interval debulking
- •Current cytotoxic chemotherapy
结局指标
主要结局
Variant allele frequencies (VAF) for variants identified in both unfiltered and filtrate uterine lavage from the same patient.
时间窗: Day 1
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).
Variant allele frequencies (VAF) for variants identified in uterine lavage ans pap smear from the same patient.
时间窗: Day 1
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.
Total mutation frequency (UtLs)
时间窗: Day 1
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.
TP53 mutation frequencies in leukocytes
时间窗: Day 1
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.
SNP allele fraction (AF) comparison at different dilutions: expected AF vs. observed AF, AF variation among replicates (UtLs).
时间窗: Day 1
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.
dCT-PCR values from 96-plexed high-throughput MSREqPCR analysis
时间窗: Day 1
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.
Mutant allele frequency (MAF) for all mutated positions, mutation spectrum (UtLs),
时间窗: Day 1
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.
次要结局
未报告次要终点
研究者
Paul Speiser, Prof.MD,
Clinical Professor
Medical University of Vienna