Lavage of the Uterine Cavity for Molecular Detection of Müllerian Duct Carcinomas: A Proof-of-Concept Study

Abstract

Purpose: Type II ovarian cancer (OC) and endometrial cancer (EC) are generally diagnosed at an advanced stage, translating into a poor survival rate. There is increasing evidence that Müllerian duct cancers may exfoliate cells. We have established an approach for lavage of the uterine cavity to detect shed cancer cells.

Patients and methods: Lavage of the uterine cavity was used to obtain samples from 65 patients, including 30 with OC, five with EC, three with other malignancies, and 27 with benign lesions involving gynecologic organs. These samples, as well as corresponding tumor tissue, were examined for the presence of somatic mutations using massively parallel sequencing (next-generation sequencing) and, in a subset, singleplex analysis.

Results: The lavage technique could be applied successfully, and sufficient amounts of DNA were obtained in all patients. Mutations, mainly in TP53, were identified in 18 (60%) of 30 lavage samples of patients with OC using next-generation sequencing. Singleplex analysis of mutations previously determined in corresponding tumor tissue led to further identification of six patients. Taken together, in 24 (80%) of 30 patients with OC, specific mutations could be identified. This also included one patient with occult OC. All five analyzed lavage specimens from patients with EC harbored mutations. Eight (29.6%) of 27 patients with benign lesions tested positive for mutations, six (75%) as a result of mutations in the KRAS gene.

Conclusion: This study proved that tumor cells from ovarian neoplasms are shed and can be collected via lavage of the uterine cavity. Detection of OC and EC and even clinically occult OC was achieved, making it a potential tool of significant promise for early diagnosis.

Conflict of interest statement

Authors' disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article.

© 2015 by American Society of Clinical Oncology.

Figures

Fig 1.

Scheme of the procedure applied for mutation analysis of lavage samples from patients with malignant diseases. When a mutation was detected in the lavage specimen by next-generation sequencing (NGS), it was confirmed by safe sequencing system (SafeSeqS) and analysis of the corresponding tumor tissue. If no mutation was detected by NGS, singleplex methods (SafeSeqS for all 12 patients and digital droplet polymerase chain reaction for six patients) were used to detect a specific mutation previously determined in tumor tissue of 12 patients. (*) Excluded because of previous tubal ligation, germline mutation, or two or more malignant tumors present. MUT, mutation.

Fig 2.

Mutation analysis of lavage specimens by next-generation sequencing (NGS) or singleplex approaches (safe sequencing system [SafeSeqS] and digital droplet polymerase chain reaction [ddPCR]) depicted as percentage of mutant alleles present. (A) Results obtained from analysis of patients with OC included in final analysis. All samples were analyzed by NGS, 12 initially negative samples were analyzed by SafeSeqS, and six of those were also analyzed by ddPCR. (B) Results obtained from analysis of patients with other malignancies, including low-malignant-potential ovarian tumor, uterine carcinosarcoma, and signet ring carcinoma metastasized to the ovaries, as well as five patients with endometrial carcinoma. Disease type and International Federation of Gynecology and Obstetrics stage of disease, if applicable, are listed on the y-axis. ca., carcinoma; Endom., endometrial; potent, potential.

Fig 3.

Distribution of genes affected by mutations, leading to the identification of different sample types (index mutation). A mutation of the TP53 gene is the most important marker in identifying ovarian cancer (OC), whereas KRAS mutations can also be observed in patients with benign diseases. EC, endometrial cancer.