Abstract LB-68: Identification of circulating HPV DNA in urine with a dual sequence capture approach.
Authors: Guerrero-Preston Rafael E. , Jedlicka Anne , Diaz-Montes Teresa , Romaguera Josefina , Roa Juan C. , Sidransky David 
Published: August 2014

Abstract
HPV testing is increasingly used for cervical cancer screening in conjunction with cervical cytology. Privacy, cultural, and infrastructure issues challenge the effective implementation of HPV testing in cervical cancer screening. The detection of circulating HPV DNA from urine can be useful for cervical cancer screening.
We captured and sequenced the HPV genome after isolating HPV Transrenal DNA (HPV-TrDNA) and then developed a qPCR-based HPV-TrDNA assay to quantify high risk HPV TrDNA. HPV TrDNA molecules are relatively short, fragmented, pieces of circulating DNA (less than 250 nucleotides) that get filtered by the kidneys and can be isolated from urine.
Methods: We adsorbed cell-free nucelic acids from urine samples (10mL) of 41 women with 23 LGL and 18 HGL cervical lesions and 31 women with normal cytology on a Q-Sepharose resin followed by a silica-based method.
A custom-designed pool of HPV-specific dual sequence capture probes was used for library amplification and target selection (Roche/NimbleGen SeqCap EZ Choice Library). A pre-capture amplification of the library was performed with ligation-mediated PCR (LM-PCR) using primers complementary to the adaptors, followed by two rounds of hybridization to the HPV-specific SeqCap EZ Choice Library.
Amplified captured DNA from HPV infected cell lines, HeLa (HPV 18), CSCC7 (HPV16), and HPV TrDNA samples from premalignant cervical lesions were processed for multiplexed sequencing on the GS Junior system (Roche). Following completion of the pyrosequencing run, signal processing was performed, followed by detailed analyses. For the SYBR green qPCR assay we amplified an HPV E1 region common to thirteen high-risk HPV types for cervical cancer.
Results: We isolated TrDNA from 10mL of urine samples. The mean concentration, total amount, and 260/280 ratio of isolated TrDNA was 34.2 ng/ul;1ug and 1.7 for premalignant samples and 17.2 ng/ul; 0.5ug; and 1.4 for normal samples.
The total number of Pass Filter sequence reads was 100,428, the total number of bases over 41 million and the read length average was 130.3 bp. Most reads (91.63%) mapped to the reference HPV sequences, with the total number of mapped base pairs over 36 million (88%). The number of fully mapped reads was 14,899 (14.84%), and the number of partially mapped reads was 70,686 (88.17%). The average coverage of reference sequences was 78% for HPV 18 in the CSCC7 DNA, 86% for HPV 16 in HeLa DNA, as well as, 100% for HPV 18 and 96% for HPV 16 in TrDNA samples.
The average Ct value for Pre-Capture LM-PCR DNAs was 35.74. The average Ct value for Post-Capture LM-PCR DNAs was 19.73; therefore the Delta Ct value was 16.01. Based on an estimated efficiency for the assay, the approximate fold enrichment was greater than 12,000.
Conclusions: We demonstrated that the HPV genome can be assembled from HPV TrDNA. We are also the first to show that circulating high risk HPV TrDNA can be quantified using SYBR green qPCR assay after dual sequence capture of the HPV genome.
Citation Format: Rafael E. Guerrero-Preston, Anne Jedlicka, Teresa Diaz-Montes, Josefina Romaguera, Juan C. Roa, David Sidransky. Identification of circulating HPV DNA in urine with a dual sequence capture approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-68. doi:10.1158/1538-7445.AM2013-LB-68