Lavage of the Uterine Cavity for Diagnosis of Ovarian Carcinomas
Phase: NA
Conditions: High Grade Ovarian Serous Adenocarcinoma, Stage III Ovarian Cancer AJCC v8, Stage IIIA Ovarian Cancer AJCC v8, Stage IIIA1 Ovarian Cancer AJCC v8, Stage IIIA2 Ovarian Cancer AJCC v8, Stage IIIB Ovarian Cancer AJCC v8, Stage IIIC Ovarian Cancer AJCC v8, Stage IV Ovarian Cancer AJCC v8, Stage IVA Ovarian Cancer AJCC v8, Stage IVB Ovarian Cancer AJCC v8
Sponsor: University of Washington

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Lavage of the Uterine Cavity for Diagnosis of Ovarian Cancer 
 
NCT03606486 
 
Date: 11/9/23 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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STUDY PROTOCOL 
 
LAVAGE OF THE UTERINE CAVITY FOR DIAGNOSIS OF OVARIAN 
CARCINOMAS 
 
PRINCIPAL INVESTIGATOR: Barbara Norquist, MD 
TITLE: Associate Professor 
INSTITUTION: University of Washington 
ADDRESS: 1959 NE PACIFIC ST, BOX 356460 
      Seattle, WA  98195 
Phone: 206-616-1620 
Email: bnorquis@uw.edu 
 
CO-INVESTIGATOR: Rosa Ana Risques, PhD 
Title: Assistant Professor 
INSTITUTION: University of Washington 
ADDRESS: 1959 NE PACIFIC ST, BOX 356460 
      Seattle, WA  98195 
Email: rrisques@uw.edu 
 
Funding: 
Minnesota Ovarian Cancer Alliance Early Detection Grant 
 
 
SUMMARY OF PROPOSED RESEARCH 
The goal of this project is to develop a minimally invasive test to detect ovarian cancer, by 
searching for mutations from the tumor in samples obtained from the cervix (Pap smears), and 
from the uterus (uterine lavage) in women with ovarian cancer and women with a genetic 
predisposition to malignant neoplasm of the ovary. We plan a pilot study of 60 women with 
ovarian cancer and with genetic predispositions to malignant neoplasm of the ovary. Pap smear 
and uterine lavage samples will be collected while the woman is under anesthesia for planned 
debulking surgery or prophylactic surgery. A novel, highly sensitive and accurate technique, 
Crispr-Duplex sequencing, will be used to detect tumor associated mutations in TP53 (the most 
commonly mutated gene in ovarian cancer) within these samples. These results will be 
compared to sequencing results in the tumor itself for comparison, and Pap and uterine lavage 
will be compared to each other to determine the optimal test.  
 
A. BACKGROUND AND SIGNIFICANCE 
In 2017, more than 22,000 women will be diagnosed with ovarian cancer and more than 14,000 
women will die from the disease, making it the leading cause of death from gynecologic cancer1. 
Ovarian, peritoneal, and fallopian tube carcinoma can clinically be considered one disease, 
referred to collectively as OC. The majority of patients present with advanced stage III or IV 
disease, with 5-year survival rates of 34% and 18%, respectively2. In contrast, patients 
diagnosed with stage I disease have 90% 5-year survival2, demonstrating the critical importance 
of early detection. Efforts at screening for early stage OC using ultrasound and serum 

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biomarkers such as CA-125 have been unsuccessful at reducing mortality from this disease3, 4. 
Women with inherited mutations in BRCA1 and BRCA2 are at increased risk of developing OC, 
with lifetime risks estimated near 50% for BRCA1 and 20% for BRCA25, 6. Therefore, for high-
risk women, national guidelines recommend removal of the ovaries and fallopian tubes after 
completion of childbearing7. Surgical prevention dramatically reduces the incidence of OC in 
BRCA1 and BRCA2 mutation carriers, and improves all-cause mortality8, 9. Risks of surgery, 
premature menopause, and loss of childbearing potential are balanced against these benefits. 
Women at high risk need more sensitive and specific screening options when they need to 
delay surgery. Mathematical models have estimated that women with BRCA1 and BRCA2 
mutations who go on to develop OC spend at least 4 years with pre-invasive or early stage (I 
and II) lesions10, providing a window in which an adequately sensitive screening method could 
lead to early detection. As these lesions are often microscopic or <1cm, they are not detected 
clinically by standard imaging techniques, and many early lesions will have a normal CA-125.  
 
The limitations of CA-125 and ultrasound have led investigators to consider molecular 
approaches to early detection. The first demonstration that OC could be detected using 
minimally invasive sample collection came from Kinde et al, sequencing DNA from Pap 
smears11. Using an error-corrected next generation sequencing (NGS) method called Safe-
SeqS, they were able to detect mutations in tumor DNA in 41% of Pap smears from women with 
OC. To improve the yield of tumor DNA, Speiser and Zeillinger developed a uterine lavage 
technique that allows for collection of samples from closer proximity to the OC. As the ovaries, 
fallopian tubes, and uterine cavity are in communication with each other, they hypothesized that 
uterine lavage could reliably capture OC DNA. They sequenced DNA from uterine lavage 
samples using NGS for a panel of potentially mutant genes, and were able to identify OC 
related mutations in 18/30 (60%) cases, with most being in the gene TP5312. Additional tumor 
mutations were identified in an additional 6 uterine lavage samples that were tested with digital 
droplet PCR (ddPCR), once the tumor specific mutation was determined by sequencing the OC 
itself. While this demonstrates an important proof of principle that the mutation can be detected, 
it is not helpful as a screening test as it would require a biopsy of the OC first. Given these 
studies’ limitations in sensitivity, other sequencing techniques are needed in order to make this 
type of testing feasible.  
 
Next generation sequencing techniques commonly introduce low-level errors that can appear to 
be mutations, masking true tumor-associated mutations that are present at very low 
frequencies, as would be expected for early stage cancers. In order to not miss these true 
mutations, substantial improvements to these methods are needed. Duplex Sequencing (DS), 
developed in the Loeb lab at the University of Washington13, overcomes this problem by 
reducing the error rate to <1 in 10 million. This accuracy is accomplished by independently 
sequencing the two strands of each DNA molecule, as true mutations will be present at the 
same position in both strands, and artifacts or errors are present in only one of the two strands. 
Dr. Risques and colleagues have utilized DS to accurately detect mutations as infrequent as 
4x10-4 in tumor DNA taken from peritoneal fluid samples in women with OC14. They then began 
collaborating with the investigators that developed the uterine lavage technique described 
above, and sequenced TP53 in uterine lavage samples from OC patients with DS, identifying 
mutations in 8/10 (80%) samples (see preliminary data). TP53 is mutated in >96% of high-grade 
serous OCs15, in addition to most serous tubal intraepithelial carcinomas (STICs) 16, making 
TP53 an optimal target for OC early detection. The main difficulty with DS is that efficiency is 

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low (~1%), requiring at least 1μg of DNA to achieve adequate sequencing coverage. Utilizing 
Crispr- Cas9 genome fragmentation, rather than random sonication, increases efficiency and 
requires much less input DNA.  
 
Preliminary data: 
In collaboration between the University of Washington and the Medical University of Vienna, DS 
was applied to 10 uterine lavage samples from women with OC, and 11 controls. Tumor 
associated TP53 mutations were detected with DS in 8/10 (80%) women with OC, and not in 
controls, manuscript in preparation by Risques et al. Interestingly, biological background 
mutations in TP53 were found in all tested subjects, including the healthy controls, a finding that 
was also present in the previous study of DS using peritoneal fluid from women with and without 
OC14. Tumor related TP53 mutations tended to have higher allele frequencies, and in this study 
a cutoff of 0.01 was effective at differentiating between these two findings. Moreover, this 
threshold allowed for discrimination between women with OC from controls with 70% sensitivity 
and 100% specificity, a diagnostic performance superior to any other OC test currently 
available. Preliminary data suggests that filtration of the uterine lavage samples to remove 
clusters of endometrial cells results in an increased allele frequency of tumor associated 
mutations, and ongoing efforts are optimizing this protocol. This study provides evidence that 
uterine lavage combined with DS increases the sensitivity of tumor TP53 mutation detection 
over other sequencing methods.  
 
B. General Description of the Study 
This study aims to collect specimens from 50 women with high-grade serous OC and 10 women 
who are undergoing prophylactic surgery for genetic predisposition to malignant neoplasm of 
the ovary, while they are under anesthesia for planned surgery at a single site, the University of 
Washington. There will be a total of at least 60 participants in the study. 
 
Dr. Norquist will coordinate the collection of samples including a Pap smear, uterine lavage, and 
tumor at the time of planned primary surgery. DNA will be extracted from all samples. Dr. 
Risques will coordinate the TP53 sequencing utilizing the Crispr-DS method on the Pap smear 
and uterine lavage extracted DNA. Primary tumors will be sequenced in order to confirm that the 
mutations identified in the Pap and/or uterine lavage are present in the tumor. We will compare 
the sensitivity for OC detection of Pap smear compared to uterine lavage, in order to optimize 
our assay.  
 
Results will not be released to either patients or clinicians, or used for any treatment decisions.  
 
C. Study Objectives  
 
Objectives: 
1) To collect samples from 50 women with high-grade serous OC and 10 women who are 
undergoing prophylactic surgery for genetic predisposition to malignant neoplasm of the 
ovary, including Pap smears, uterine lavage, and a tumor sample.  
2) To test the DNA from Pap smears and uterine lavage samples for tumor-derived TP53 
mutations, using the new technology of Crispr-Duplex Sequencing. 
3) Determine the sensitivity and specificity of Pap smear or uterine lavage in detection of 
tumor-derived TP53 mutations. 

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D. Study Design 
Study population and recruitment:  
Patients with suspected OC and women who are undergoing prophylactic surgery for genetic 
predisposition to malignant neoplasm of the ovary will be identified and consented pre-
operatively for participation in the study. This process is already in place at the University of 
Washington as nearly all patients with OC or those undergoing prophylactic surgery for genetic 
predisposition to malignant neoplasm of the ovary are consented pre-operatively for our 
Gynecologic Oncology Tissue Bank.  
 
Eligibility: 
Inclusion criteria: 
• 
Women over the age of 18 with suspected ovarian cancer or genetic predisposition to 
malignant neoplasm of the ovary 
• 
Planned surgery 
• 
Have a uterus and no history of tubal occlusion 
 
Exclusion criteria: 
• 
Unable to speak English 
• 
Unable to provide informed consent 
• 
Prior hysterectomy 
 
Sample collection:  
After induction of anesthesia, samples will be collected, starting with a standard liquid-based 
Pap smear. Uterine lavage is then carried out. First the cervix is cleansed with a betadine 
solution as is standard prior to surgery involving the uterus. The three-way endometrial catheter 
is then inserted into the cervical canal and 10ml of sterile saline are used to irrigate the uterine 
cavity, as previously described12.  The three-way uterine lavage catheter developed by Oncolab 
Diagnostics has received FDA designation as a nonsignificant risk (NSR) device. It is 
commercially available, and is currently being utilized in 3 ongoing clinical trials (NCT02039388, 
NCT02062697, and NCT02518256). Finally, samples of the OC itself are collected for those 
with suspected OC during the abdominal surgery for comparison. Uterine lavage samples will 
be filtered to remove clusters of endometrial cells to decrease the amount of non-tumor DNA.  
 
Molecular analysis: 
DNA will be extracted from each of the three samples collected per case (Pap, uterine lavage, 
and tumor). Pap smear and uterine lavage samples will be sequenced for TP53 mutations using 
the Crispr-Duplex Sequencing method. Patients will be excluded from the study if the diagnosis 
of high-grade serous OC is not confirmed by the final pathology results.  
 
Data collection: 
Baseline demographic and clinical variables will be collected on each patient per procedures 
already in place with our gynecologic oncology Tissue Bank.  
 
Data privacy and Safety Monitoring: 

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There will be limited access to authorized users who will use password protection and encrypted 
storage devices. Data will be stored in a locked cabinet on an encrypted flash drive. Access to 
electronic records are password protected. Team members will complete confidentiality and 
security training. No identifiers will be used in any study reports, publications or presentations. 
 
Adverse Events:  
Adverse events are unlikely with this study. Enrolled subjects are women who are already 
planned to undergo surgery for their suspected ovarian cancer, and they will be under 
anesthesia during collection. Collection is designed to occur in parallel with other portions of the 
surgery, so we do not anticipate it will prolong surgical time. Uterine perforation or bleeding, or a 
pelvic infection could occur, although it is unlikely based on the known safety of procedures 
such as endometrial biopsy. If any such adverse events occur, they will be reported.  
 
 
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