Abstract P2-04-01: Human papillomavirus (HPV) DNA detection in breast cancer by liquid biopsy: Something new on the horizon?
Authors: Bravaccini S , Ravaioli S , Rocca A , Maltoni R , Cristalli C , Marasco E , De Carolis S , Cricca M , Bonafè M 
Published: May 2019

Abstract
Background The presence of human papillomavirus (HPV) DNA in breast cancer (BC) tissues has been widely investigated in recent years. HPV DNA has been detected in ductal lavage fluids and in serum-derived extracellular vesicles of patients with benign or in situ breast lesions. However, there are no data attesting to its presence in liquid biopsies of different BC subtypes or to its impact on prognosis.
Methods We analyzed a total of 72 serum samples for the presence of circulating HPV DNA, of which 20 were from luminal A BC (5 relapsed, 15 non relapsed), 17 from luminal B BC (5 relapsed, 12 non relapsed), 15 from triple-negative BC (6 relapsed and 9 non relapsed), 12 from HER2-positive BC (3 relapsed, 9 non relapsed) and 8 from healthy subjects. Circulating DNA was extracted and purified from 500 μl of serum by Qiamp DNA minikit (Qiagen, Milan, Italy) according to the manufacturer's instructions. HPV DNA was assessed by a high-throughput MALDI-TOF mass spectrometry-based method (Mass Array Platform, Agena Bioscience, Hamburg, Germany). The DNA target sequence was amplified by a multiplex PCR with HPV E6 or E7 gene-specific primers. A primer for primer extension annealing to the amplified product was extended at its 3′ terminal base for each HPV type.
Results HPV DNA was detected in 5 BC patients but in none of the healthy controls. None of the serum samples analyzed showed HPV DNA types 16 or 18. Four of the 5 BC cases had high-risk HPV DNA (type 39,45,52,59) and one had low-risk HPV DNA (type 73). The 4 patients with high risk HPV DNA had low-grade cervical intraepithelial neoplasia (CIN 1) detected by Pap smear prior to the diagnosis of BC. No relation was found between HPV infection and tumor subtype or prognosis. Our in vitro studies also revealed the active release of HPV DNA into the extracellular vesicle compartment of cervical cancer cells.
Table 1.Features of BC patients positive for circulating HPV DNA.Sample IDAge (years)GradingSubtypeDeathRelapseHPV type, riskCervical lesion17471Luminal A-No59, highCIN 139783Luminal B20/03/2015No45, highCIN 145462Luminal B-No39, highCIN 160583TN03/01/2011Yes73, lowHyperplasia63523HER2-positive-Yes52, highCIN 1TN, triple negative; CIN 1:low-grade cervical intraepithelial neoplasia
Conclusions Our findings support the feasibility of HPV DNA evaluation by liquid biopsy in BC. They also suggest that circulating HPV DNA in BC patients might be of cervical tissue origin and that the presence of HPV DNA in BC may be a consequence of its spreading from virus-infected tissue such as that of the uterine cervix.
Citation Format: Bravaccini S, Ravaioli S, Rocca A, Maltoni R, Cristalli C, Marasco E, De Carolis S, Cricca M, Bonafè M. Human papillomavirus (HPV) DNA detection in breast cancer by liquid biopsy: Something new on the horizon? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-04-01.