title: Genomic Analysis of Uterine Lavage Fluid Detects Early Endometrial Cancers and Reveals a Prevalent Landscape of Driver Mutations in Women without Histopathologic Evidence of Cancer: A Prospective Cross-Sectional Study.
abstract: BACKGROUND
  Endometrial 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.
  
  
  RESULTS
  Uterine 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.
  
  
  CONCLUSIONS
  Using 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.
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Navya
authors.identifier: http://orcid.org/0000-0002-5223-4808
authors.initials: N
authors.lastname: Nair
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Olga
authors.identifier: http://orcid.org/0000-0002-5223-4808
authors.initials: O
authors.lastname: Camacho-Vanegas
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Dmitry
authors.identifier: http://orcid.org/0000-0001-8610-6540
authors.initials: D
authors.lastname: Rykunov
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Matthew
authors.identifier: http://orcid.org/0000-0003-4059-4467
authors.initials: M
authors.lastname: Dashkoff
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Sandra Catalina
authors.initials: SC
authors.lastname: Camacho
authors.affiliation: Swift Biosciences, Ann Arbor, Michigan, United States of America.
authors.forename: Cassie A
authors.initials: CA
authors.lastname: Schumacher
authors.affiliation: Swift Biosciences, Ann Arbor, Michigan, United States of America.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Irish
authors.affiliation: Swift Biosciences, Ann Arbor, Michigan, United States of America.
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Harkins
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Elijah
authors.identifier: http://orcid.org/0000-0002-1101-0971
authors.initials: E
authors.lastname: Freeman
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Isaac
authors.identifier: http://orcid.org/0000-0002-3999-495X
authors.initials: I
authors.lastname: Garcia
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Elena
authors.identifier: http://orcid.org/0000-0001-5310-9155
authors.initials: E
authors.lastname: Pereira
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Sviatoslav
authors.identifier: http://orcid.org/0000-0002-5368-5109
authors.initials: S
authors.lastname: Kendall
authors.affiliation: Jefferson School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Belfer
authors.affiliation: Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Tamara
authors.initials: T
authors.lastname: Kalir
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Robert
authors.identifier: http://orcid.org/0000-0001-9267-2426
authors.initials: R
authors.lastname: Sebra
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Boris
authors.initials: B
authors.lastname: Reva
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: Dottino
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.|Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.|Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.|Laboratory for Translational Research, Western Connecticut Health Network, Danbury, Connecticut, United States of America.
authors.forename: John A
authors.initials: JA
authors.lastname: Martignetti
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D003430:Cross-Sectional Studies; D004273:DNA, Neoplasm; D016889:Endometrial Neoplasms / Q000235:genetics* / Q000473:pathology; D005260:Female; D016678:Genome; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D011446:Prospective Studies; D007507:Therapeutic Irrigation; D014599:Uterus / Q000378:metabolism*
chemical_list: D004273:DNA, Neoplasm