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One of the most important scientific discoveries in the past 30 years is the causal link between human papilloma virus (HPV) infection of the cervix, and cervical cancer. This finding resulted from the original seminal findings in 1984 by Harald zur Hausen and his group, that HPV 16 can be detected in cervical cancer tissue. In recognition of his discovery, Hausen was awarded the Nobel Prize in Physiology or Medicine in 2008.
HPV is a small DNA virus with a genome of about 8,000 base pairs. It belongs to the papillomavirus family. HPV cannot bind to live tissue, it infects epithelial tissue when, as a consequence of microabrasions and other trauma, the basement membrane is exposed. HPV lesions arise from proliferation of infected basal keratinocytes. HPV can survive for many months and at low temperature without a host.
There are two categories of HPV: those infections that are passed through skin-to-skin contact and those that are sexually transmitted. Typically, an infection with HPV is transient, and the immune system can clear the infection within two years. Currently, there are more than 100 different known HPV genotypes that have been grouped into low-risk and high-risk categories and designated as causing mucosal or cutaneous infections. The types of HPV passed through skin contact typically cause skin warts, especially in children. The most common sexually transmitted disease caused by HPV is genital warts (condyloma acuminatum), a wart-like growth on the cervical or vulval mucosa in females, or on the glans or prepuce in males. Warts are generally the result of infection by low-risk types of HPV, including genotype 6, 11, 32, 40, 42, 44, 54, 55, 61, 62, 64, 71, 72, 74, 81, 83, 84, 87, 89, and 91. High-risk genotypes of HPV include 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, and IS39. The high-risk strains induce cervical dysplasia and can lead to the development of several types of cancers including cancer of the cervix, vulva, vagina, anus, and penis. The most common of these is HPV-associated cervical cancer.
A COMMON INFECTION
HPV infection is the most common sexually transmitted infection worldwide and most sexually active individuals of both sexes will acquire it at some point in their life. On the basis of a meta-analysis of one million women with normal cervical cytology, around 291 million women worldwide are estimated to have HPV infection of the cervix at a given point, corresponding to an average prevalence of 10.4%, though prevalence is higher in women younger than 25 years (16.9%). HPV types 16 and 18 account for roughly 70% of all cervical cancer. Type 16 has been detected in about 24% of women with HPV infection; type 18 has been detected in about 9%.
Prospective studies have shown that the prevalence of HPV includes a mix of incident and persistent infections that have accumulated over time because of lack of clearance. More than 90% of new HPV infections at any age regress in 6-18 months and more persistent infection is a prerequisite for progression to cervical intraepithelial neoplasia (CIN). CIN 1 is an insensitive histopathological sign of HPV infection. CIN 2 includes a heterogeneous group of lesions that have different potential to progress to cancer, and CIN 3 represents the most clinically relevant lesions and is the best surrogate endpoint for cervical cancer in screening and vaccination trials. The probability of clearance of HPV depends on the duration of infection; longer persistence reduces the probability of clearance. HPV infections detected in women aged older than 30 years persist for longer than those in younger women because they are more likely to be persistent infections of long duration.
HPV genotypes have been classified as either carcinogenic or probably carcinogenic. Worldwide, the most common HPV types in cervical cancer were types 16 (57%), 18 (16%), 58 (5%), 33 (5%), 45 (5%), 31 (4%), 52 (3%), and 35 (2%). Types 16, 18, and 45 accounted for a greater, or equal, proportion of infections in cervical cancer compared with normal cytology (panel 1); the ratio between cervical cancer and normal cytology was 3.1:1 for type 16, 1.9:1 for type 18, and 1.1:1 for type 45. Other high-risk types accounted for substantial proportions of CIN 2 and CIN 3, but their contribution to cervical cancer was low, with ratios ranging from 0.9:1 for type 33 to 0.2:1 for type 51. HPV is one of the most powerful human carcinogens and has been implicated in cancers at several sites. Roughly 610,000 new cancers per year (5% of all cancers) have been attributed to HPV infection, of which more than 80% occurred in developing countries. Besides cervical carcinoma, HPV genotypes have been associated with anal cancer, vaginal cancers, cancer of the penis, vulva, and oropharynx (see figure 1).
HPV infects only epithelial cells and depends on the differentiation pathway of epithelial cells to complete its lifecycle. HPV infects cells in the basal layer of the epithelium, probably via microabrasions in the epithelial surface. Infectious internalisation takes several hours, after which viral DNA is released from the capsid and transported into the nucleus as free genetic material, or extrachromosomal episomes. Early gene expression is tightly controlled in the basal epithelial cells with substantial amplification of viral DNA. Replication occurs only in suprabasal, differentiating cells.
HPV encodes two proteins, E6 and E7, that together promote cellular proliferation, prolong cell-cycle progression, and prevent apoptosis. The cell becomes permissive for viral replication and hundreds or even thousands of HPV genomes are generated within a single cell. The capsid proteins L1 and L2 are expressed in the most superficial layers of the epithelium, where viral assembly takes place, and finally, new infectious viral particles (virions) are shed from the epithelial surface. The papillomavirus lifecycle takes 2-3 weeks, the time necessary for a cervical cell to migrate from the basal to most superficial layers of the epithelium, mature, undergo senescence, and die. To complete the infectious lifecycle of the virus, the cell must undergo terminal differentiation, an essential prerequisite for virion assembly and release. However, for some high-risk HPV, E6 and E7 are so effective at blocking negative regulators of the cell cycle that the infected cells never mature. The cells remain actively involved in cell-cycle progression and cease to die.
E7 contributes to oncogenesis through its interaction with the retinoblastoma family members RB1, RBL1, and RBL2, the so-called pocket proteins. E7 binds these proteins and targets them for degradation. This action results in the release and activation of E2F transcription factors that drive the expression of S-phase genes, including those that encode cyclins A and E, which in turn precipitates cell-cycle entry and promotes DNA synthesis. High-risk E5 works with E6 and E7 to drive cellular proliferation and might be a weak cofactor in development of malignancy. Both episomal and integrated copies of the HPV genome frequently co-occur, often within the same cell. In this case, E6 and E7 expression might not be significantly increased.
Papanicolaou (Pap) staining is the gold standard for detecting abnormal cervical epithelial cells, using microscopic analysis of conventional cervical smears or cell suspensions from liquid cytology medium. In the 1930s, cervical cancer was the No. 1 cause of cancer-related death for women in the United States. By 2000, the rate had been cut by almost 90%, largely because widespread adoption of regular cervical cytology exams, or Pap smears, led to a decline in cervical cancer. Currently cervical examinations and Pap tests remain the screening method of choice for most women. Morphological findings from a cytology analysis determine the level of risk for developing cervical malignancy. Cervical epithelial cells determined to be atypical or abnormal, but not yet defined as neoplastic, are given the term ‘atypical squamous cells of undetermined significance’ (ASCUS) or ‘cannot exclude high-grade lesions’ (ASC-H). Most cases of ASCUS signal the presence of low-grade squamous intraepithelial lesions (LSIL), which are generally associated with transient, self-resolving HPV infections. However, some ASCUS findings are associated with underlying high-grade disease, including cervical intraepithelial neoplasia (CIN). Women over the age of 30, who have a persistent infection with high-risk types of HPV, have the greatest risk of developing cervical cancer. Molecular diagnostic tests for HPV can augment screening for cervical cancer when used in conjunction with the Pap smear.
Due to the inability to culture HPV in a laboratory, molecular techniques are the only method available to detect HPV DNA. The HPV genome is double stranded and circular with approximately 8,000 bp. The HPV virus has eight overlapping reading frames, or genes, categorised as either early or late, depending on when they are expressed. Early genes, E1 and E2, participate in genome replication and transcription while the E4 gene promotes the productive phase of the viral lifecycle. The oncoproteins, E6 and E7, form complexes with tumour suppressors, and thus can lead to host cell transformation and progression to cervical cancer. The L1 and L2 late genes encode viral capsid proteins. Most HPV assays target the L1 region for classification of HPV genotypes, although the E1 gene is also used.
The major diagnostic techniques for HPV detection and genotyping are target amplification, signal amplification, and probe amplification. Target amplification duplicates fragments of DNA from a targeted gene sequence. The most well-known example of this is polymerase chain reaction (PCR). Signal amplification uses branched DNA technology or hybrid capture to increase the DNA-proportional signal to detectable levels. Probe amplification includes technologies such as ligase chain reaction, which amplify the probe.
TARGET AMPLIFICATION TECHNIQUES
Polymerase chain reaction is the most commonly used tool in the detection of HPV DNA. In theory, PCR can take a single double-stranded piece of DNA and amplify it to one billion copies after 30 cycles. Polymerase chain reaction consists of the following three basic steps: denaturation, annealing, and extension. Typically, PCR procedures for HPV detection use primers targeted to the viral capsid L1 gene, which can detect numerous HPV types. For type-specific PCR, several repeats of PCR may be necessary in order to determine the specific sequence existing in the sample.
REVERSE LINE BLOT AND LINEAR ARRAY
Probes for multiple HPV types are fixed on a membrane strip, and the PCR product is hybridised to the strip, followed by visual detection. The assay detects 27 different HPV types and the extended edition adds 11 low-risk types, which include 61, 62, 64, 67, 69 to 72, 81, 82, and 89. The results are read with the unaided eye based on a visible band in specific areas of the hybridisation strip. This subjectivity has the potential to affect the interpretation of results.
This system amplifies target DNA using PCR followed by nucleic acid hybridisation. A 96-microwell plate is used and requires only a small sample of 250 μL for testing. The Amplicor assay will detect HPV but will not identify the specific genotype.
Genotyping with this method is based on PCR amplification of the E1 gene by a group of new E1-specific primers, followed by hybridisation to a DNA chip with immobilised HPV oligoprobes. The PapilloCheck DNA-chip setup allows for testing of 12 samples at a time and helps to eliminate false-positive and false-negative results. A laser scanner, CheckScanner, is used to detect excitation from fluorescently labelled probes that bind to the HPV primers. The reporting software, CheckReport, automatically reads and reports the results.
MULTIPLEX HPV GENOTYPING KIT
The Multiplex test is a PCR-based fluorescent bead array that can detect 24 low- and high-risk HPV types. The PCR products are mixed with the Multiplex HPV Genotyping Kit bead mix. This bead mix has 26 populations of beads attached to 24 HPV probes, one β-globin probe, and one control probe. After the PCR products are hybridised, they are labelled using R-phycoerythrin marked streptavidin and read on the Luminex analyser. The individual signatures of the beads can discern the types.
REAL TIME PCR
Real time PCR is a highly sensitive target amplification technique available for HPV-DNA detection. Real time PCR combines fluorescent probes with PCR primers, allowing for accurate quantification of virus present in a sample. Viral load estimation of HPV is a particular advantage of real time PCR, using the nuclear genome to control for cellular content of the sample.
SIGNAL AMPLIFICATION TECHNIQUES
Hybrid Capture Assay: Hybrid capture is a signal amplification technique, meaning that the chemiluminescent or fluorescent signal is amplified to aid detection, rather than the target DNA being amplified by PCR. Specimens containing HPV DNA are hybridised with a HPV-specific RNA probe, creating a DNA:RNA hybrid molecule. The microplate well is coated with antibodies that bind DNA:RNA hybrids, thus capturing the hybrid molecules to the microplate. Alkaline phosphatase-conjugated antibodies bind the hybrid molecules, and a signal is detected on the addition of a chemiluminescent substrate. Signal amplification occurs because several alkaline phosphatase molecules are conjugated to each antibody, and multiple conjugated antibodies can bind each captured hybrid. These assays are only useful for detection of HPV and not specific genotyping.
CERVISTA HPV HR
The Invader technology of the Cervista test is a unique signal amplification technique using two simultaneous isothermal reactions. A DNA probe including a sequence-specific region binds to the HPV DNA molecule. In addition, the proprietary Invader probe also associates with the target sequence, resulting in a 1-base overlapping structure at the key nucleotide. Proprietary enzymes cleave the probe, releasing a 5’oligo flap. Multiple probes are cleaved for each molecule, causing signal amplification. Meanwhile each flap behaves as an Invader oligo in association with a florescence resonance energy transfer (FRET) probe. The FRET probe includes a fluorophore molecule in close proximity to a quencher molecule. Upon association of the flap with the FRET probe, another cleavage reaction occurs, releasing the fluorophore from its quencher. This results in a detectable fluorescent signal.
RT PCR FOR MRNA
Reverse-transcriptase-PCR incorporates an RT step prior to performing basic endpoint or real time quantitative PCR. Using this technique, the level of ultimate protein production of the gene of interest can be elucidated. The overexpression of the HPV E6 and E7 genes is indicated in HPV-induced carcinogenesis, making these genes a potential measure of virulence. Monitoring the expression levels of these genes may allow for screening and monitoring of cancer progression. Reverse-transcriptase-PCR can detect mRNA at much smaller quantities than other mRNA techniques, such as northern blot.
HPV TESTING IN CERVICAL CANCER SCREENING
Because of its crucial causal role and the need for continued expression to maintain the disease phenotype, HPV can be used as a biomarker of cervical cancer and pre-cancer. Randomised trials have shown that HPV DNA testing provides greater sensitivity than cytology does for detection of CIN. Human papillomavirus testing is more reproducible with less subjective analytical characteristics and users need less training and expertise.
Atypical cytology of unknown significance or borderline cytology is associated with underlying CIN2 or worse, in roughly 10% of cases. In atypical cytology of unknown significance or borderline cytology, about 60% test positive for high-risk HPV, which enables HPV testing to identify those at very low risk (HPV negative), who can be routinely recalled at standard screening intervals, whereas a HPV positive result warrants referral to colposcopy. HPV triage of equivocal cytology enables immediate referral for those at risk and routine recall for those who test negative. This approach avoids the need for repeat testing, which is not only inefficient, risking reduced adherence to recall, but also causes distress by prolonging uncertainty and necessitating repeated examination. HPV testing might also be useful as a test of cure after treatment of high grade CIN.
A major benefit of HPV primary screening is the potential for HPV testing to lengthen screening intervals from every year to every five years.
References available on request ([email protected])