Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma

Abstract

Specific human papillomavirus genotypes are associated with most ano-genital carcinomas and a large subset of oro-pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus-associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus-16 or human papillomavirus-18-associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro-pharynx. As negative controls, 18 serum samples from women with human papillomavirus-16-associated high-grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at -80°C (27 cases) or at -20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus-16 E7 and human papillomavirus-18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at -80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus-associated invasive cancers even at sub-clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus-associated high grade cervical intraepithelial neoplasia.

Keywords: anal carcinoma; carcinoma of the head and neck; cervical carcinoma; circulating HPV DNA; ddPCR.

Figure 1

Detection of circulating HPV16 E7 DNA by ddPCR in serum samples from patients with HPV16‐associated cervical carcinoma. (A) Stage II invasive cervical carcinoma (Case N°25); Positive plots are represented in blue (4530 copies/ml); No positive droplets detected from patients with CIN3 or from the negative control. (B) Positive detection in the serum of patients with tonsil (case N°65) and oropharyngeal (case N°69) carcinomas (215 and 4605 copies/ml, respectively). (C) Positive detection in the serum of two patients (cases N°1 and N°12) with micro‐invasive cervical carcinoma (32 and 249 copies/ml, respectively). Positive DNA controls from HPV16 tumour tissues and non‐DNA controls, replaced by elution buffer in no template control (NTC), are indicated.

Figure 2

Rate of c‐HPV DNA (log scale) detected using ddPCR in patients with cervical neoplasias according to clinical criteria. Statistical analysis using t‐test [with Welch's correction for plots (B) to (F)]. In B only stage I and II were compared.

Figure 3

Positive correlation between c‐HPV DNA level (log scale) and tumour size in patients with cervical cancer (n = 37). Spearman correlation: r = 0.51 (p = 0.001). (○) Stage I and (•) stage II/III/IV tumours.

Figure 4

Comparison of c‐HPV DNA levels (log scale) detected using ddPCR in serum and plasma samples from patients diagnosed with HPV‐associated invasive carcinoma of the uterine cervix (A) or the anal canal or oropharyngeal region (B).