The ‘Why and How’ of Cervical Cancers and Genital HPV Infection

INTRODUCTION

Invasive squamous cell carcinoma of the cervix is still the most common malignant tumor of the female genital tract in most countries and the most frequent neoplasm among many of the women.^[1] Widespread use of cervical cytological screening programs has resulted in dramatic decrease in the incidence rates of cervical cancer in countries like United States. No other form of cancer documents better the remarkable effects, of screening, early diagnosis and curative therapy, on mortality rates, than does the cervical cancer. The precancerous lesions of cervix have received a great deal of attention due to the easy accessibility of cervix to visual inspection and tissue sampling for morphological as well as molecular diagnosis, and for therapeutic procedures. The role of human papillomavirus (HPV) as a causative agent in cervical precancerous lesions and cancer has been firmly established both epidemiologically and biologically.^[2] Although HPV infection is essential, it is not sufficient to develop cancer, and many other factors such as multiparity, host immune response, hormonal factors, and cigarette smoking also have some definite contribution. Epidemiological studies performed over the past three decades have proved that invasive cervical cancer is a multistep process involving a precursor pre-invasive stage that is closely linked with HPV infection.^[2,3]

HISTORICAL ASPECTS

An Austrian Gynecologist, Schauenstein, was the first one to mention the histological similarity between the noninvasive and the invasive cervical cancer (1908), but the term precancerous lesions of cervix were suggested first by Schottlander and Kermauner in 1912. Schiller, in 1920, mentioned the detailed analysis of histological patterns of various pre-invasive and invasive cervical cancer forms. Robert Meyer and Hinselmann worked separately for early diagnosis of cervical cancer. The credit of introducing a device for magnified inspection of cervix, that is, Colposcope goes to Hinselmann.^[4] In 1941, an innovative smear technique for vaginal cytology (later known as the “Pap smear”) was introduced. J. Ernst Ayre, while working in Florida, (1951) first described and illustrated squamous epithelial cells with a perinuclear “halo” in the smears of uterine cervix. He described them as halo cells. He believed that some long-standing inflammation might be responsible for these peculiar changes. The term “koilocytic atypia” was introduced by Koss and Durfee for abnormal squamous cells in patients with cervical cancer even before the association between HPV and cervical cancer was known. The first evidence of the link between HPV and cervical cancer was the electron microscopic demonstration of viral particles in genital condylomata acuminata. Zur Hausen and Munoz were awarded a Nobel Prize for their dedicated extensive work on HPV. The last two decades have seen a sudden outburst of studies and publications related to association between HPV and cervical cancer. Various immunohistochemical techniques, Southern blot analysis, in situ hybridization, and PCR amplification techniques proved this association which is now a well-established fact today.^[5]

HUMAN PAPILLOMAVIRUS AND HUMAN DISEASE

HPV is associated with a variety of clinical conditions that range from innocuous lesions to cancer. HPV is implicated not only in the causation of cervical cancer but also more than 10% of all human cancers are said to be associated with it. Till today, more than 150 different types of HPV have been described and new types are regularly added to the list. HPV sometimes referred to as the “wart virus” has been recognized for many years as the cause of ordinary skin warts and condylomas. HPV is epitheliotropic and site specific. It is broadly classified into two types, cutaneous and mucosal or genital.

Skin warts, although very common, are benign self-limiting and in general resolve spontaneously within 1–5 years. They are transmitted by direct contact with an infected tissue or indirectly by contact with virus contaminated objects.

Common sites for mucosal papillomas and warts are oral cavity and respiratory mucosa, especially larynx, trunk, and sometimes conjunctiva.^[6] Anogenital warts are most common amongst sexually active individuals. Anogenital cancers are the most important diseases associated with HPV infection. More than 90% of invasive cervical cancers are also associated with HPV infection. Table 1 enlists various HPV types and their clinical manifestations.

HPV VIROLOGY

HPV is a small circular double-stranded DNA virus of Papovaviridae family and containing approximately 7900 base pairs. Mature viral particles are icosahedral (containing 20 faces) and can be visualized with electron microscopy. HPV genome reveals a well-conserved general organization. The viral genome is functionally divided into two regions,

1. The non-coding upstream regulatory region (URR) and

2. The coding region which is represented as open reading frame or ORF [Figure 1a and b].

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Figure 1:

(a) Schematic representation of the genomic organization of HPV. (b) Linear representation of HPV 16 genome (open reading frame).^[7]

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The non-coding region does not code for proteins but contains elements required for regulation of gene expression, DNA replication, and its packaging into virus particles.^[6,7]

The coding region or the ORF can be divided into early and late region based on the specific time schedule in the course of productive infection. The so-called early genes are expressed shortly after infection and before the onset of DNA replication. Products of these genes mediate functions such as replication and expression of viral DNA and also transformation of the host cell. There are total seven early genes from E1 to E7. They are numbered according to their size; the higher the number, the smaller the corresponding ORF. Expression of the early gene products determines whether the HPV infection is active or latent, or may lead to malignant transformation. The late genes code for structural proteins of viral particles and are activated during the final stages of the viral cycle.^[8] The L1 gene codes for the major capsid protein and L2 gene codes for the minor capsid protein.^[9] Table 2 enlists various HPV genes and their functions.

NATURAL HISTORY OF GENITAL HPV INFECTION

Genital HPV infection is a venereally transmitted infection with a life time risk of about 50–80% in women. Most of the infections (75%) clear spontaneously within 12–36 months with an effective immune response. About 40 different genotypes of HPV are known to cause the genital HPV infection. They are further divided into low-risk and high-risk type depending on their propensity to progress to precursor lesions for cervical cancer. Low-risk types are associated low-grade lesions such as cervical condylomas and CIN I or LSIL (low-grade squamous intraepithelial lesion). Although 15 genotypes of high-risk HPV are known, types 16 and 18 are the most common. HPV, though essential, is not sufficient to cause cervical cancer and different cofactors such as tobacco, multiparity, local host immune response, presence of other sexually transmitted diseases, and hormonal factors have also proved their definite role. HPV virus particles are perfectly adapted to their natural host tissue, the differentiating epithelial cells of the skin or mucosa and exploit the cellular machinery for their own purposes. It cannot infect or enter the mature superficial squamous epithelial cells that line the ectocervix, vulva, and vagina. Establishing infection at this site requires cuts, breaks, and microabrasions to gain the virus access to the basal layer of the epithelium through interaction with receptors like alpha-6 integrins. The cervix with its relatively large area of immature squamous epithelium in the transformation zone is particularly vulnerable to HPV infection as it lacks the typical micro ridges present on mature squamous epithelial cells. Normally, the transitional zone coincides with the external os, but in pregnancy, at puberty, and in some contraceptive users, the position of Os and transformation zone may not coincide. This exposes the tissues in the endocervical canal to vagina. This undergoes metaplasia as a protective response and it is during this metaplastic process the epithelium is most vulnerable to viral entry.^[10] HPVs are undergoing a complete life cycle only in fully differentiated squamous epithelium. Existence of the viral genome in the infected cell is central to the life cycle of papilloma viruses and their associated pathologies. After entry into the basal cell, further steps in viral life cycle depend on whether it is low-risk type of virus or high-risk type.

In low-risk group, the virus remains in the circular form not attached to chromosomes, termed as episomal; whereas in high-risk group, the viral DNA gets incorporated into the host cell DNA hence known as integrated. Both forms can multiply in the nuclei of the host cell.

PATHOGENESIS IN LOW-RISK AND HIGH-RISK TYPE HPV

This group of viruses establishes their small double-stranded DNA genome as a circular extra-chromosomal element or episome in the nucleus-infected cells [Figure 2].

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Figure 2:

Pathogenesis of low-risk and high-risk HPV.^[11]

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Two types of infection can occur.

1. Latent infection and

2. Productive infection.

In normal squamous epithelium, cells in the basal layers are dividing as stem cells or transit amplifying cells. After division, one of the daughter cells migrates upwards and begins to undergo terminal differentiation; while the other remains in the basal layer as a slow cycling self-renewing population. Following the entry of HPV into the suprabasal layer through microabrasions, the viral genome replicates automatically with each division of the cell. The papillomavirus DNA replication is totally dependent on the DNA synthesis machinery of the host cell. The factors necessary for viral replication are – (a) the viral DNA sequence to be multiplied, (b) DNA polymerase enzyme, and (c) replication factors. The viral DNA sequence is provided by the early genes of HPV. The replication factors and the DNA polymerase enzyme are available in the nucleus of the dividing basal cell. Viral copies are formed automatically without disturbing the host cell DNA, when the cell divides. Thus, the viral multiplication is seen in low copy number. This infection is called as latent infection. In this type, no tissue changes are seen on light microscopy.^[12]

Other type of infection is productive or permissive type of infection. In this, the epithelial cells containing low copy number of virus particles differentiate and move upwards in the epithelium. The problem for the virus is that the necessary cellular DNA polymerase and replication factors are only available in dividing cells; however, the virus replicates in non-dividing cells. To solve this problem, HPV encodes proteins that, in context of the viral life cycle, reactivate cellular DNA synthesis in non-cycling cells, inhibit apoptosis, and delay the differentiation program of the infected keratinocyte, creating an environment permissive for viral DNA replication.^[13] Now, the virus can produce large copies of viral DNA. As the cell further moves upwards, late genes are expressed that code for structural proteins making capsid. The viral DNA is packaged with the capsid and they are released automatically when the superficial cells are shed off. These cells containing large number of viral particles are nothing but koilocytes. The perinuclear zone in the koilocytes is the storage space for the virus; hence, they are increasingly seen over the superficial layers. This is productive viral infection seen mostly with types 6 and 11 and clinically appearing condylomas and warts due to proliferation of maturing cells. This infection automatically regresses within 1–2 years and has no malignant potential [Figure 3].

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Figure 3:

The HPV life cycle.^[14]

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High-risk HPV types can be distinguished from low-risk HPV types by the structure and function of the E6 and E7 products. In infection with high-risk type of viruses (16, 18, etc.), viral DNA is integrated into host genome. Viral DNA integration into host genome is found in all cases of cervical carcinoma and their metastasis. This viral DNA integration is in itself a mutation with consequences both on viral and cellular genome. Integration of viral genome disrupts or deletes the E2 region resulting in loss of its expression, hence interfering its function of downregulation of transcription of E6 and E7. This leads to increased expression of E6 and E7 genes. The products of E6, E7 genes deregulate host cell growth cycle by binding and inactivating two important tumor suppressor genes p53 and Rb. The E6 gene product selectively binds to p53 and causes its degradation; as a consequence, the normal activities of p53 which govern cell cycle arrest in G1, apoptosis, and DNA repair are impaired. This allows accumulation of additional mutations. Whereas E7 gene product binds to Rb which disrupts the complex between Rb and E2F-1 and makes the factor E2F1 available for transcription of proteins required for entry of the cell into S phase of the cell cycle [Figure 4]. The outcome is stimulation of cellular DNA synthesis and cell proliferation. Next, the E5 gene products induce an increase in mitogen activated protein kinase activity, thereby enhancing cellular responses to growth and differentiation factors. This results in continuous proliferation and delayed differentiation of the host cell.^[15,16]

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Figure 4:

Mechanism in high-risk viruses.^[17]

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CELLULAR TRANSFORMATION

The inactivation of p53 and Rb gene proteins can give rise to an increased proliferation rate and genomic instability. As a consequence, the host cell makes more and more damaged DNA that cannot be repaired, leading to transformed cancerous cells. In addition to the effects of activated oncogenes and chromosome instability, potential mechanisms contributing to transformation include methylation of viral and cellular DNA, telomerase activation, and hormonal and genital factors.^[18] It is plausible that high-risk HPV infection occurs early in life, may persist and in association with other factors promoting cell transformation, and may lead to a gradual progression to more severe disease. Accumulation of mutations is essential for progression toward malignancy and invasion.

GENETIC PREDISPOSITION

Genetic predisposition has a great contribution in cervical cancer. Heritability could affect many factors contributing to the development of cervical cancer including susceptibility to HPV infection, ability to clear HPV infection, and time required for the development of disease. Certain haplotypes such as DOA1 and DQB1 are commonly seen in cervical cancer patients, which may be associated with less effective triggering of immune response against HPV-infected cell. Thus, HLA haplotypes is an important factor in the initial stage as it influences the point between reversion and progression of HPV-induced lesion.^[19,20]

HORMONAL FACTORS

A role of estrogen in cervical carcinogenesis is significant. There is definite evidence of increased cancer risk in women who are long-term users of oral contraceptives containing synthetic estrogen. High parity is also suggested to increase the risk of squamous cell carcinoma of the cervix among HPV-positive women, with repeated exposure to elevated levels of estrogen during pregnancy. Estrogen is physiologically implicated in eversion of the columnar epithelium onto the ectocervix as well as formation of the transformation zone. It is known that the cervical transformation zone featuring squamous metaplasia beside the squamocolumnar junction is highly susceptible to HPV infection and in fact gives rise to most cervical cancers. Aromatase, an enzyme responsible for conversion of androgen to estrogens, is overexpressed in cervical cancer patients. The tumor suppressor gene BRCA1 is reported to repress estrogen- induced transcriptional activity of the estrogen receptor. Thus, estrogen, aromatase, and estrogen-responsive genes may be potential risk factors for initiation and further development of cervical cancer.

HOST IMMUNE RESPONSE

Host immune response plays an important role in regression or reversion of the lesions. The major line of defense against virus is cell-mediated immunity. Therefore, conditions associated with impaired CMI such as transplant recipients and HIV disease show high risk of acquisition and progression of HPV infection. HPV infections with high-risk viral types, persistence of HPV infection, and the presence of squamous intraepithelial lesions are more common in immunocompromised patients than in immunocompetent women. On entry of the virus through cuts in the epithelium, the antigen presenting cells under the epithelium catch the antigen and cause stimulation of cytotoxic, memory, and helper T cells. In majority of cases of genital HPV infection, the HPV is cleared by effective CMI and complete remission is achieved. The time for clearance ranges from months to years depending on the immune response. Poor genital hygiene impairs natural mucosal barriers hence may result in persistent or progressive infection. HPV has developed several mechanisms for evading the immune surveillance. Majority of these mechanisms are related to evading of innate immunity and delaying the adaptive immune response. Other factors are the characteristics of the viral site of infection and the effects of viral oncoproteins. Most important factor that favors the virus to escape from these immune mechanisms is absence of lytic phase hence no cell injury hence no inflammation. There is no viremia during HPV infection. The immune system recognizes the virus only in presence of capsid which is formed in superficial layers of the epithelium where they escape from APC. Release of viral proteins causes inhibition of interferon thus further favoring the viral progression.

SEXUAL FACTORS

The risk of contracting genital HPV infection and cervical cancer is influenced by sexual activity. Multiple sexual partners, multiparity, and onset of sexual activity at early age have an increased risk of HPV infection. Additional association with other sexually transmitted diseases increases the chances of HPV infection. Coinfections with other viruses such as herpes simplex type II and cytomegalovirus also may play a role in the initiation of cervical cancer.

VIRAL FACTORS

The type of HPV variant and the viral load directly correlates with the severity of the disease. HPV variants differ in biological and chemical properties and pathogenicity. The oncogenicity of specific HPV variants appears to vary geographically and also with the ethnic origin. Infections with multiple variants of viruses can also occur. With multiple infections, at least one is of high-risk type.^[21] Although the previous studies do mention about association of herpes simplex virus infection in cervical cancer, it is not proved by molecular and advanced diagnostic techniques.^[22]

OTHER FACTORS

Cervical cancer risk also seems to be independently influenced by other variables including cigarette smoking, alcohol consumption, and diet. Local immune suppression induced by smoking and the mutagenic activity of cigarette components may contribute to persistence of HPV or to malignant transformation similar to that seen in lung.