PurposeType 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 MethodsLavage 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.ResultsThe 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.ConclusionThis 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.

Highlights d Ovarian cancer can be detected by ultra-accurate sequencing of uterine lavage DNA d However, low-frequency TP53 mutations also exist in normal tissue of healthy women d TP53 mutations are increasingly selected for with age, revealing somatic evolution d Age-associated, cancer-like mutations challenge specificity for cancer detection

RT-qPCR is a highly sensitive approach to detect rare transcripts, as derived from circulating tumor cells (CTCs) in the blood of cancer patients. However, the presence of unwanted leukocytes often leads to false positive results. Here, we evaluated whether the micro-fluidic Parsortix™ technology is appropriate to remove these leukocytes and thereby finally to improve the overall approach.In this study, we established a workflow including the micro-fluidic Parsortix™ technology for the molecular detection of CTC related transcripts. Background levels of EpCAM, PPIC, TUSC3, and MAL2 were efficiently removed due to an up to 106-fold depletion of leukocytes. The presence of these gene markers was observed in Parsortix™-enriched blood samples from patients with primary and recurrent gynecological cancer (32% and 14%), as well as in 86% of the metastatic breast cancer samples, at a very high specificity. In the ovarian cancer samples, PPIC was the most prominent gene marker, contributing to all positive cases and at least to 70% of the positive cases after pre-amplification of the respective target genes. Expanding the analytical panel up to 29 gene markers further increased the positivity rate (primary gynecological cancer: 95%, recurrent gynecological cancer: 100%, metastatic breast cancer: 92%).The established workflow strongly improved the overall molecular analysis of the target cells by the efficient removal of contaminating cells, and, thereby offers great promise for the molecular characterization of CTCs.