Article | Published:

Targeted next-generation sequencing of endometrial cancer and matched circulating tumor DNA: identification of plasma-based, tumor-associated mutations in early stage patients

Modern Pathology (2018) | Download Citation


There is currently no blood-based marker in routine use for endometrial cancer patients. Such a marker could potentially be used for early detection, but it could also help to track tumor recurrence following hysterectomy. This is important, as extra-vaginal recurrence of endometrial endometrioid adenocarcinoma is usually incurable. This proof-of-principle study was designed to determine if tumor-associated mutations could be detected in cell-free DNA from the peripheral blood of early and late stage endometrial endometrioid carcinoma patients. Approximately 90% of endometrioid carcinomas have at least one mutation in the genes CTNNB1, KRAS, PTEN, or PIK3CA. Using a custom panel targeting 30 hotspot amplicons in these four genes, next-generation sequencing was performed on cell-free DNA extracted from plasma obtained from a peripheral blood draw at the time of hysterectomy and the matching tumor DNA from 48 patients with endometrioid endometrial carcinomas. At least one mutation in the tumor was detected in 45/48 (94%) of patients. Fifteen of 45 patients (33%) had a mutation in the plasma that matched a mutation in the tumor. These same mutations were not detected in the matched negative control buffy coat. Presence of a plasma mutation was significantly associated with advanced stage at hysterectomy, deep myometrial invasion, lymphatic/vascular invasion, and primary tumor size. Detecting a plasma-based mutation was independent of the amount of cell-free DNA isolated from the plasma. Overall, 18% of early stage patients had a mutation detected in the plasma. These results demonstrate that mutations in genes relevant to endometrial cancer can be identified in the peripheral blood of patients at the time of surgery. Future studies can help to determine the post-operative time course of mutation clearance from the peripheral blood and if mutation re-emergence is predictive of recurrence.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Jemal A, Ward EM, Johnson CJ, et al. Annual report to the nation on the status of cancer, 1975-2014, featuring survival. J Natl Cancer Inst. 2017;109:djx030.

  2. 2.

    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68:7–30.

  3. 3.

    Amant F, Moerman P, Neven P, et al. Endometrial cancer. Lancet. 2005;366:491–505.

  4. 4.

    Pietrasz D, Pecuchet N, Garlan F, et al. Plasma circulating tumor DNA in pancreatic cancer patients is a prognostic marker. Clin Cancer Res. 2017;23:116–23.

  5. 5.

    Sausen M, Phallen J, Adleff V, et al. Clinical implications of genomic alterations in the tumour and circulation of pancreatic cancer patients. Nat Commun. 2015;6:7686.

  6. 6.

    Strickler JH, Loree JM, Ahronian LG, et al. Genomic landscape of cell-free DNA in patients with colorectal cancer. Cancer Disc. 2018;8:164–73.

  7. 7.

    Rolfo C, Mack PC, Scagliotti GV, et al. Liquid biopsy for advanced non-small cell lung cancer (NSLC): a statement paper from the IASLC. J Thorac Oncol 2018;13:1248–68.

  8. 8.

    Cicchillitti L, Corrado G, De Angeli M, et al. Circulating cell-free DNA content as blood based biomarker in endometrial cancer. Oncotarget. 2017;8:115230–43.

  9. 9.

    Djordjevic B, Hennessy BT, Li J, et al. Clinical assessment of PTEN loss in endometrial carcinoma: immunohistochemistry outperforms gene sequencing. Mod Pathol. 2012;25:699–708.

  10. 10.

    Cheung LW, Hennessy BT, Li J, et al. High frequency of PIK3R1 and PIK3R2 mutations in endometrial cancer elucidates a novel mechanism of regulation of PTEN protein stability. Cancer Discov. 2011;1:170–85.

  11. 11.

    Kurnit KC, Kim GN, Fellman BM, et al. CTNNB1 (beta-catenin) mutation identifies low grade, early stage endometrial cancer patients at increased risk of recurrence. Mod Pathol. 2017;30:1032–41.

  12. 12.

    Singh RR, Patel KP, Routbort MJ, et al. Clinical validation of a next-generation sequencing screen for mutational hotspots in 46 cancer-related genes. J Mol Diagn. 2013;15:607–22.

  13. 13.

    Mehrotra M, Singh RR, Chen W, et al. Study of preanalytic and analytic variables for clinical next-generation sequencing of circulating cell-free nucleic acid. J Mol Diagn. 2017;19:514–24.

  14. 14.

    Bartley AN, Luthra R, Saraiya D, et al. Identification of cancer patients with Lynch syndrome: clinically significant discordances and problems in tissue-based mismatch repair testing. Cancer Prev Res. 2012;5:320–7.

  15. 15.

    Mehrotra M, Singh RR, Loghavi S, et al. Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors. Oncotarget. 2018;9:10259–71.

  16. 16.

    Richman SD, Chambers P, Seymour MT, et al. Intra-tumoral heterogeneity of KRAS and BRAF mutation status in patients with advanced colorectal cancer (aCRC) and cost-effectiveness of multiple sample testing. Anal Cell Pathol. 2011;34:61–66.

  17. 17.

    Nelson AC, Boone J, Cartwright D, et al. Optimal detection of clinically relevant mutations in colorectal carcinoma: sample pooling overcomes intra-tumoral heterogeneity. Mod Pathol. 2018;31:343–9.

  18. 18.

    Christensen E, Nordentoft I, Vang S, et al. Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients. Sci Rep. 2018;8:1917.

  19. 19.

    Vollbrecht C, Lehmann A, Lenze D, Hummel M. Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients. Oncotarget. 2018;9:18529–39.

  20. 20.

    Newman AM, Lovejoy AF, Klass DM, et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol. 2016;34:547–55.

  21. 21.

    Toro PV, Erlanger B, Beaver JA, et al. Comparison of cell stabilizing blood collection tubes for circulating plasma tumor DNA. Clin Biochem. 2015;48:993–8.

  22. 22.

    El Messaoudi S, Rolet F, Mouliere F, et al. Circulating cell free DNA: preanalytical considerations. Clin Chim Acta. 2013;424:222–30.

  23. 23.

    Fiegl H, Gattringer C, Widschwendter A, et al. Methylated DNA collected by tampons—a new tool to detect endometrial cancer. Cancer Epidemiol Biomark Prev. 2004;13:882–8.

  24. 24.

    Bakkum-Gomez JN, Wentzensen N, Maurer MJ, et al. Detection of endometrial cancer via molecular analysis of DNA collected with vaginal tampons. Gynecol Oncol. 2015;137:14–22.

  25. 25.

    Maritschnegg E, Wang Y, Pecha N, et al. Lavage of the uterine cavity for molecular detection of mullerian duct carcinomas: a proof-of-concept study. J Clin Oncol. 2015;33:4293–301.

  26. 26.

    Nair N, Camacho-Vanegas O, Rykunov D, et al. 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. PLoS Med. 2016;13:e1002206.

  27. 27.

    Ni T, Sun X, Shan B, et al. Detection of circulating tumour cells may add value in endometrial cancer management. Eur J Obstet Gynecol Reprod Biol. 2016;207:1–4.

  28. 28.

    Anglesio MS, Papdopoulos N, Ayhan A, et al. Cancer-associated mutations in endometriosis without cancer. N Engl J Med. 2017;376:1835–48.

  29. 29.

    Hafner C, van Oers JM, Hartmann A, et al. Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern. Proc Natl Acad Sci USA. 2007;104:13450–4.

  30. 30.

    Reme T, Travaglio A, Gueydon E, et al. Mutations of the p53 tumour suppressor gene in erosive rheumatoid synovial tissue. Clin Exp Immunol. 1998;111:353–8.

  31. 31.

    Firestein GS, Echeverri F, Yeo M, et al. Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium. Proc Natl Acad Sci USA. 1997;94:10895–900.

  32. 32.

    Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759–67.

  33. 33.

    Kato S, Lippman SM, Flaherty KT, Kurzrock R. The conundrum of genetic “drivers” in benign conditions. J Natl Cancer Inst. 2016;108:djw036.

Download references


This study was funded by the NIH SPORE in Uterine Cancer (RRB and WZ) NIH P50 CA09825 and The Red and Charline McCombs Institute Center for Global Cancer Early Detection (RRB and WZ).

Author information


  1. School of Health Professions, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    • Ana M. Bolivar
    •  & Peter Hu
  2. Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    • Rajyalakshmi Luthra
    • , Meenakshi Mehrotra
    • , Wei Chen
    •  & Bedia A. Barkoh
  3. Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA

    • Wei Zhang
  4. Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    • Russell R. Broaddus


  1. Search for Ana M. Bolivar in:

  2. Search for Rajyalakshmi Luthra in:

  3. Search for Meenakshi Mehrotra in:

  4. Search for Wei Chen in:

  5. Search for Bedia A. Barkoh in:

  6. Search for Peter Hu in:

  7. Search for Wei Zhang in:

  8. Search for Russell R. Broaddus in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Russell R. Broaddus.

About this article

Publication history