Date Published: December 27, 2016
Publisher: Public Library of Science
Author(s): Navya Nair, Olga Camacho-Vanegas, Dmitry Rykunov, Matthew Dashkoff, Sandra Catalina Camacho, Cassie A. Schumacher, Jonathan C. Irish, Timothy T. Harkins, Elijah Freeman, Isaac Garcia, Elena Pereira, Sviatoslav Kendall, Rachel Belfer, Tamara Kalir, Robert Sebra, Boris Reva, Peter Dottino, John A. Martignetti, Elaine Rene Mardis
Abstract: BackgroundEndometrial cancer is the most common gynecologic malignancy, and its incidence and associated mortality are increasing. Despite the immediate need to detect these cancers at an earlier stage, there is no effective screening methodology or protocol for endometrial cancer. The comprehensive, genomics-based analysis of endometrial cancer by The Cancer Genome Atlas (TCGA) revealed many of the molecular defects that define this cancer. Based on these cancer genome results, and in a prospective study, we hypothesized that the use of ultra-deep, targeted gene sequencing could detect somatic mutations in uterine lavage fluid obtained from women undergoing hysteroscopy as a means of molecular screening and diagnosis.Methods and FindingsUterine lavage and paired blood samples were collected and analyzed from 107 consecutive patients who were undergoing hysteroscopy and curettage for diagnostic evaluation from this single-institution study. The lavage fluid was separated into cellular and acellular fractions by centrifugation. Cellular and cell-free DNA (cfDNA) were isolated from each lavage. Two targeted next-generation sequencing (NGS) gene panels, one composed of 56 genes and the other of 12 genes, were used for ultra-deep sequencing. To rule out potential NGS-based errors, orthogonal mutation validation was performed using digital PCR and Sanger sequencing.Seven patients were diagnosed with endometrial cancer based on classic histopathologic analysis. Six of these patients had stage IA cancer, and one of these cancers was only detectable as a microscopic focus within a polyp. All seven patients were found to have significant cancer-associated gene mutations in both cell pellet and cfDNA fractions. In the four patients in whom adequate tumor sample was available, all tumor mutations above a specific allele fraction were present in the uterine lavage DNA samples. Mutations originally only detected in lavage fluid fractions were later confirmed to be present in tumor but at allele fractions significantly less than 1%. Of the remaining 95 patients diagnosed with benign or non-cancer pathology, 44 had no significant cancer mutations detected. Intriguingly, 51 patients without histopathologic evidence of cancer had relatively high allele fraction (1.0%–30.4%), cancer-associated mutations. Participants with detected driver and potential driver mutations were significantly older (mean age mutated = 57.96, 95% confidence interval [CI]: 3.30–∞, mean age no mutations = 50.35; p-value = 0.002; Benjamini-Hochberg [BH] adjusted p-value = 0.015) and more likely to be post-menopausal (p-value = 0.004; BH-adjusted p-value = 0.015) than those without these mutations. No associations were detected between mutation status and race/ethnicity, body mass index, diabetes, parity, and smoking status. Long-term follow-up was not presently available in this prospective study for those women without histopathologic evidence of cancer.ConclusionsUsing ultra-deep NGS, we identified somatic mutations in DNA extracted both from cell pellets and a never previously reported cfDNA fraction from the uterine lavage. Using our targeted sequencing approach, endometrial driver mutations were identified in all seven women who received a cancer diagnosis based on classic histopathology of tissue curettage obtained at the time of hysteroscopy. In addition, relatively high allele fraction driver mutations were identified in the lavage fluid of approximately half of the women without a cancer diagnosis. Increasing age and post-menopausal status were associated with the presence of these cancer-associated mutations, suggesting the prevalent existence of a premalignant landscape in women without clinical evidence of cancer. Given that a uterine lavage can be easily and quickly performed even outside of the operating room and in a physician’s office-based setting, our findings suggest the future possibility of this approach for screening women for the earliest stages of endometrial cancer. However, our findings suggest that further insight into development of cancer or its interruption are needed before translation to the clinic.
Partial Text: Endometrial cancer is the most common gynecologic malignancy in the United States, with 60,000 incident cases and greater than 10,000 deaths estimated for 2016. Alarmingly, both the incidence and associated mortality are rising . By 2030, endometrial cancer is projected to surpass colorectal cancer to become the third most common cancer among women in the United States . Despite its already high prevalence and increasing morbidity and mortality, no effective screening exists for endometrial cancer. Specifically, no screening methods can effectively detect either pre-malignant lesions (primary prevention) or early-stage cancers (secondary prevention). The lack of screening is particularly significant because when detected early, endometrial cancer survival rates are dramatically improved. The 5-year survival for localized disease is 95%, whereas it is <20% for disease that has metastasized . The study was conducted from September 2015 to November 2016. Patient samples were collected during the months of September 2015 to April 2016, with DNA extraction being performed concurrently with sample collection. NGS, Sanger sequencing, and digital droplet PCR were performed on these samples and validation sets from February 2016 to October 2016. Data analysis was performed once all samples were sequenced and histopathology results confirmed. This prospective study on women undergoing hysteroscopy and dilation and curettage as a diagnostic procedure demonstrated the ability of ultra-deep targeted sequencing of uterine lavage sampling to identify not only early-stage endometrial cancers but also a surprisingly high burden of driver mutations in a majority of women in our cohort who were without histopathologic evidence of cancer. As such, we believe these molecular findings simultaneously offer both the future promise of screening women for endometrial cancer while also providing a rare opportunity to explore the processes that are associated with the early steps of defining cancer development and its abrogation. Source: http://doi.org/10.1371/journal.pmed.1002206