ReviewEmerging biomarkers and clinical significance of HPV genotyping in prevention and management of cervical cancer
Introduction
Cervical cancer is one of the principal causes of morbidity and mortality among women caused by Human Papilloma Virus (HPV) affecting at least 50% of men and women during their lifetime [1]. The GLOBOCAN database provides a worldwide estimate of mortality and morbidity of cervical cancer in 2012. According to this report, about 5,28,000 newer cases were diagnosed and more than half of them succumbed to it. The global burden of 87% of this disease was observed in less developed countries [2]. In India, cervical cancer is the second most frequent one amongst females. Annually the number of newly diagnosed cases are 1,22,844 and around 67,477 patients die from it. The occurrence of the disease is reported in women aged between 15 and 44 years in India [3], while there is a peak in the diagnosis of cervical cancer between 55 and 59 years [4]. This late diagnosis and increased mortality rate in developing countries is due to the lack of an effective screening program and a high prevalence of HPV [5]. The cervical cancer screening programs cover only 19% of target population in developing countries in comparison to the 63% in developed countries [6].
Previously, Dr. Rous described the progression of papilloma warts into cancer in 1935, while visualization of HPV particles in human warts for the first time was reported in 1949. Cervical cancer gathered interest in the 1970s when Harold zur Hausen discovered, cloned and demonstrated the causative role of HPV in cervical cancer and was awarded Nobel Prize for his remarkable discovery in 2008 [7].
HPV belongs to the Papovaviridae family, a non-enveloped DNA virus measuring 55 nm. The genetic material is enclosed in an icosahedral capsid which contains L1 and L2 capsid proteins. HPV genome is composed of double stranded DNA containing 8000 base pairs that contains a total of 8 genes including early (E) and late (L) genes [8]. The three functional regions in the genome include the upstream non-coding regulatory region or the long control region (LCR). The early region required in the viral replication and oncogenesis consisting of open reading frames (ORF), E1, E2, E4, E5, E6, E7 and finally the late region which encodes the structural proteins L1 and L2 [9]. Fig. 1 shows circular and linear structure of HPV genome [10,11]. Almost a 200 subtypes of HPV have been discovered with more than 30 infecting the genital tract [9] and 11 of them classified as high risk or the oncogenic strains [12]. Type 16 and 18 of the HPV have been mainly associated with cervical cancers [12]. The low risk group HPV causes genital warts or may produce mild dysplasia on the cervix. The HPV 6 and 11 are considered as low risk types. The HPV infection's oncogenic potential rely on the E6 and E7 viral oncogenic activities [13]. The HPV DNA integrates into the host DNA down regulating the E2 protein resulting in over expression of E6 and E7 proteins. These proteins bind and disrupt the functions of p53 and pRB respectively, leading to an increased proliferation of the cells. Proliferating cells are a fertile ground for mutations resulting in oncogenesis [9].
Different screening methods such as cytology (conventional, liquid based, automated pap), visual based screening and high risk HPV testing are presently accessible. Pap test has helped identify many pre-cancerous and cancerous lesions of cervical epithelium and has brought all those under the surveillance umbrella. However, cytological evaluation of cervical cancers have their own limitations which has led to continued research resulting in emergence of varied sophisticated diagnostic tools for cervical cancer. An increase in identification of potential biomarkers in patient studies have been observed in the past decade. Many newer ones are being added to the already existing list of the markers [14].
Section snippets
HPV DNA integration by FISH
The FISH approach was based on a discrete FISH staining that indicates episomal genomes. The punctuated FISH staining predicts integrated viral genomes [15]. Using FISH technique for human DNA, a viral-human combine structure has been suggested in HPV-positive head and neck carcinomas by observation of a punctuated staining [16]. The viral genome was found to be amplified in huge replication foci in differentiating cervical cancer cell lines retaining HPV episomes [17]. When this amplification
Metagenomics studies of HPV
The study, analysis and interpretation of the association between microbiota and cancer has been transformed in the emerging era of metagenomics. It is a reasonable approach to study culture independent micro-organisms that comprise the structural and functional study and their interactions with the habitat [86]. Different new investigations on the infection caused cancers have reported genomic analysis findings of microorganisms inhabiting in the cancerous tissues [87].
The microbiota of a
Global HPV genotype distribution
Globally, the HPV infection burden that leads to cervical cancer is more in developing countries. The oncogenic properties of the HPV positive genotype is different in every region of the world [103]. Globally, about 5% of all cervical malignancies were found to be comprising high risk HPV45 genotype. It is frequently observed in adenocarcinoma than in squamous cell carcinoma. The 300 samples of pre-determined HPV45 were collected from 36 countries and analyzed. The entire open reading frame of
Genotyping
Different types of primers can be designed which target sequences unique to the HPV and thereby amplifying them resulting in enhanced rate of detection. Type specific primers are used for detection of single genotype, which may be a cumbersome process, therefore, many genotypes may be detected using multiple type-specific reactions [117]. Using consensus primer can prove to be less labor intensive, since they target the group specific regions in the HPV genome like the L1 [118] and E2 [119].
Clinical significance of HPV genotyping
Although, cytology-based screening has resulted in decreasing cervical cancer incidence rates in resource-rich countries [133]; however, requirement of multiple Pap tests for identifying precancerous lesions due to higher rates of false-negative results makes it less cost-effective for masses in resource-poor settings. Therefore, more sensitive techniques than the ones based on cytology are now being utilized for early detection of causative agent of cervical cancer. Randomized clinical trials
Conclusion
Cervical cancer deaths are preventable yet the high rates of mortality in developing countries is reflective of an inaccessibility of health care services and health inequity [2]. Several screening and diagnostic tests are routinely practiced clinically. Disease-specific biomarkers including HPV E6/E7 mRNA, p16ink4a or new methylation analyses may work after HPV DNA test found to be positive as secondary markers. This would differentiate women with pre-cancerous conditions who require urgent
Funding
No funding was received for the study.
Author contributions
Ushma Jaykamal Shah, Mohammad Nasiruddin: Conceptualization, Methodology, Writing-Original draft preparation.
Md. Khurshid Alam Khan, Mohammad Riyaz Akhter, Nidhi Singh, Ali A. Rabaan: Data curation, Writing-Original draft preparation, Visualization.
Sajad Ahmad Dar, Shafiul Haque: Data Curation, Writing- Reviewing and Editing, Supervision.
Declaration of competing interest
None.
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These authors contributed equally.