Abstract 2398: Precision molecular biomarkers for the surveillance of gynecologic malignancies: Rapid and efficient pipeline for the design and highly sensitive detection of circulating tumor DNA
Authors: Pereira Elena B. , Camacho-Vanegas Olga , Anand Sanya , Singh Chanpreet , Uzilov Andrew , Sebra Robert , Chappell David , Dottino Peter , Martignetti John A. 
Published: August 2015

Gynecologic malignancies affect over 94,000 women in the U.S. and will result in close to 29,000 deaths this year alone. Currently available biomarkers are inadequate for the early detection of disease recurrence. The development of more sensitive and accurate biomarkers represents a critical and immediate necessity towards improving both patient outcome and quality of life. The measurement of circulating tumor DNA (ctDNA), the so-called “liquid biopsy,” represents a powerful emerging technology capable of providing accurate assessment of both tumor behavior and disease burden, and has great potential for revolutionizing our standard approach to tumor surveillance. The ability to translate the various ctDNA detection technologies from the laboratory to the clinic and begin defining clinical utility will in part be determined by the time and cost efficiencies of the pipelines and inherent detection sensitivities.

We have developed a precision-medicine based approach that couples targeted tumor-specific mutation identification for each patient to droplet digital PCR-based (ddPCR) ctDNA detection. Using two rapid-turnaround sequencing panels (targeted full-gene PacBio single-molecule real time (SMRT) long-read sequencing coupled with the Ion AmpliSeq™Cancer Hotspot Panel v2) in lieu of more expensive and time-consuming approaches like whole-exome or whole-genome sequencing, we comprehensively identified tumor mutations for each of our ovarian and endometrial cancer patients by sequencing normal and tumor DNA from each patient. Analysis of an initial cohort of ovarian and endometrial cancer patients resulted in mutation identification frequencies comparable to previously reported data with mutations in TP53, PTEN, and PIK3CA being most frequently represented. Candidate mutations for ctDNA detection were identified for greater than 80% of subjects using this approach. By targeting specific genes, we raise the likelihood that identified tumor mutations are drivers essential to the disease, rather than passenger mutations that may be selected out during disease progression. ctDNA biomarkers specific to each patient's tumor mutations were generated and tested using ddPCR, allowing for detection down to 0.05% mutant allele fraction. ctDNA levels were assessed longitudinally from prospectively collected serums during multiple time points throughout each patient's clinical course and compared to clinical findings and current gold-standard serum and radiologic tests. ctDNA was successfully detected in all subjects; and to our knowledge for the first time in endometrial cancer.

We demonstrate that our molecular approach allows for rapid, highly sensitive and accurate assessment of patient tumor status and, for some patients, is an improved and better predictor of outcome than current gold-standard surveillance strategies.

Citation Format: Elena B. Pereira, Olga Camacho-Vanegas, Sanya Anand, Chanpreet Singh, Andrew Uzilov, Robert Sebra, David Chappell, Peter Dottino, John A. Martignetti. Precision molecular biomarkers for the surveillance of gynecologic malignancies: Rapid and efficient pipeline for the design and highly sensitive detection of circulating tumor DNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2398. doi:10.1158/1538-7445.AM2015-2398