Current Technologies and Recent Developments for Screening of HPV-Associated Cervical and Oropharyngeal Cancers

Abstract

Mucosal infection by the human papillomavirus (HPV) is responsible for a growing number of malignancies, predominantly represented by cervical cancer and oropharyngeal squamous cell carcinoma. Because of the prevalence of the virus, persistence of infection, and long latency period, novel and low-cost methods are needed for effective population level screening and monitoring. We review established methods for screening of cervical and oral cancer as well as commercially-available techniques for detection of HPV DNA. We then describe the ongoing development of microfluidic nucleic acid-based biosensors to evaluate circulating host microRNAs that are produced in response to an oncogenic HPV infection. The goal is to develop an ideal screening platform that is low-cost, portable, and easy to use, with appropriate signal stability, sensitivity and specificity. Advances in technologies for sample lysis, pre-treatment and concentration, and multiplexed nucleic acid detection are provided. Continued development of these devices provides opportunities for cancer screening in low resource settings, for point-of-care diagnostics and self-screening, and for monitoring response to vaccination or surgical treatment.

Keywords: E6/E7 genotypes; HPV DNA; cervical and oropharyngeal cancers; diagnostics; human papillomavirus; integrated platform; microRNA.

Figure 1

(a) Timeline of cervical cancer progression. Human papillomavirus (HPV) infects the basal cells of the cervical epithelium (dark blue) exposed by wounding, leading to cytological abnormalities; (b) Expression of viral oncogenes E6/E7 accompanies malignant transformation; (b) HPV infection pathway. Following viral entry, viral replication is synchronous with host cellular DNA replication. Integrated and episomal viral DNA produce E6 and E7 oncoproteins. E6 targets p53 for degradation and prevents apoptosis while E7 inactivates retinoblastoma (Rb) tumor suppressors, promoting cell cycle progression. The end result of these processes is cellular transformation. HPV infection also alters transcription of host microRNAs; (c) Samples for detection of HPV E6/E7 mRNA or host microRNA. CIN: cervical intraepithelial neoplasia.

Figure 2

Integrated nucleic acid sensing platform. (a) Schematic of an integrated biosensing platform consisting of a pretreatment, preconcentration and sensing units; (b) Picture of the integrated biochip. Adapted with permission from [196]. PBS: phosphate-buffered saline; TAE: Tris-acetate-EDTA; ssDNA: single-strand DNA.

Figure 3

Sensing unit of the integrated platform. (a) Schematic showing the ion-exchange nanomembrane sensor; (b) Current-voltage characteristics (CVC) showing changes in ohmic, limiting and overlimiting regions for bare anion-exchange membrane (AEM; black), membrane functionalized with oligoprobe (red) and hybridization of DNA/RNA with oligoprobe (blue); (c) The specificity of the sensor was challenged by using a non-target sequence differing by only two base pairs compared to the target sequence. Change in CVC was only observed for the target miR sequence indicating the sensor’s capability to distinguish two-base-pair mismatches. Adapted with permission from [195].

Figure 4

Steps involved in the successful extraction, concentration and detection of miR146a biomarker from oral cancer cell lysate solution. Step 1: The sample is introduced in the pretreatment chamber; Step 2: A baseline CVC is measured for the probe on the sensor; Step 3: An electric field is applied to extract nucleic acid molecules from the pretreatment unit; Step 4: The preconcentration unit is used to concentrate the extracted nucleic acids; Step 5: After allowing target hybridization with the probe, a second CVC is measured to analyze the presence or absence of target.