Biology of Cervical Squamous Neoplasia

Introduction

In the past half-century, cytologic screening, particularly in high-resource settings, has dramatically decreased the burden of squamous cervical cancers, virtually all of which are caused by tissue-specific, persistent infection with human papillomavirus (HPV). The elucidation of the infectious etiology of this disease led to a Nobel Prize in 2008, and to development of preventative vaccines that became commercially available in 2006. This was a public health milestone; worldwide, chronic infections initiate approximately 20% of human cancers, with HPVs causing more malignancies than any other virus. However, because primary prevention strategies are cumbersome and expensive, cervical cancer is still the third most common cause of cancer death in women. Moreover, the incidence of HPV-associated cancers in other anatomic sites, for which no screening algorithms have been developed, is increasing rapidly.

HPV disease is not restricted to the cervix. A growing body of evidence links persistent HPV infection to squamous cancers of the vagina, vulva, anus, and oropharynx. Almost all vaginal and anal squamous cancers and approximately 20% of squamous cancers of the vulva are caused by HPV. The incidence of preinvasive vulvar disease attributed to HPV has increased by 411% over the past three decades, primarily in women under the age 65. HPVs have been identified in one in four squamous carcinomas of the head and neck; to date, the incidence of HPV-associated oropharyngeal cancers is greater in men than in women. Indeed, in the United States, the incidence of HPV-associated cancers of the oropharynx is approximately equal to that of cervical cancer, and is likely to surpass it soon. Because prophylactic vaccines are not administered to the full target population, cohort-appropriate strategies for preventing and treating HPV disease are needed. A better understanding of the biology of intraepithelial, preinvasive HPV disease will inform strategies for screening, secondary prevention, and treatment.

HPV Infection

Like other viruses that cause human cancers, most HPV infections are asymptomatic, eventually cleared, and do not harm the host ( Figure 9.1 ). HPV is an epitheliotropic virus that is essentially endemic. In unvaccinated cohorts, the lifetime risk of acquiring genital HPV at least once is greater than 80%. Over 40 genotypes of HPV can infect the genital tract, 15 of which have been classified as carcinogenic ( Table 9.1 ). These include HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 72. Together, HPV 16 and HPV 18 are the most common carcinogenic genotypes worldwide ( Figures 9.2 and 9.3 ).

HPV infects cervical epithelium shortly after sexual debut, and can be detected in up to 50% of young women who have initiated sexual intercourse in the preceding 36 months. Most women clear an incident HPV infection within 1–2 years. Several host cofactors that have been associated with progression of HPV infection to carcinoma are shown in Table 9.2 . Older women take longer to clear infections, as do smokers, and women with underlying immunosuppression. In the cervix, persistent infection is the proximate cause of squamous cancers and their precursors, high-grade squamous intraepithelial lesion (HSIL), or cervical intraepithelial neoplasia (CIN) grade 2 or 3 (CIN 2, CIN 3).

HPV infections are limited to the suprabasal compartment of nonsterile barrier epithelia in which the immunologic contributions of the local microbiome are incompletely understood. Viral replication, assembly, and release of virions occur in the context of host cellular maturation and desquamation, without cell lysis, and without systemic viremia. Persistent HPV infections increase susceptibility to malignant transformation by a variety of mechanisms, including enhancing genetic instability and cell proliferation, inducing angiogenesis, interfering with intrinsic and extrinsic apoptotic pathways, downregulating expression of adhesion molecules, abrogating DNA damage responses, and interfering with both innate and adaptive immune responses in the lesion microenvironment. Known virally mediated immune-suppressive mechanisms include interfering with type I interferon responses and Toll-like receptor (TLR) signaling, downregulating cell surface MHC class I expression, enhancing secretion of immunosuppressive cytokines, recruitment of immune cells with suppressive function, and downregulating expression of adhesion molecules in lesional neovascular endothelium. Indeed, as we learn more about the immunobiology of persistent infection it becomes less surprising that immune-based therapeutic strategies to date, which have focused solely on eliciting an effector T-cell response to HPV antigens, have failed.

Low-Grade Squamous Intraepithelial Lesions (CIN1)

HPV infects keratinocytes at the basal epithelial layer of the squamocolumnar junction. A productive infection leads to the production of whole infectious virions, and is limited to epithelia that undergo maturation. As HPV does not encode DNA polymerase, host cellular DNA polymerase must be expressed for viral replication. In normal physiologic conditions in stratified squamous epithelium, DNA is copied only in the basal layer. In contrast, in virally infected epithelium, HPV drives DNA replication and cell division in the upper layers of differentiating epithelium, where HPV DNA is packaged into viral capsids, resulting in infectious virions ( Figure 9.4 ). Infectious virions are released not by cell lysis, but in the course of host cell maturation and desquamation, without eliciting an inflammatory response.

HPV infection produces a distinctive cytopathic effect that is recognizable both cytologically and histologically as a sharply demarcated cytoplasmic halo surrounding an enlarged, irregular, hyperchromatic nucleus with uneven chromatin distribution, termed ‘koilocytic atypia.’ Other findings include multinucleation, parakeratosis, and hyperkeratosis. Histologically, HPV infections can be distinguished by koilocytotic changes in differentiating cells, with increased ratios of nuclear to cytoplasmic area, and mitotically active cells above the basal layer. Clinically, the behavior of cytology interpreted as ‘atypical squamous cells of undetermined significance’ with a concurrent positive test for oncogenic HPV DNA, cytology interpreted as ‘low-grade squamous intraepithelial lesion,’ or a tissue biopsy diagnosis of ‘low-grade squamous intraepithelial lesion’ or ‘cervical intraepithelial neoplasia grade I’ (CIN 1) is essentially the same; most eventually regress without intervention. Most CIN 1 lesions maintain HPV as an episome, sustain a complete viral replication cycle, and can be thought of as HPV infection.

High-Grade Squamous Intraepithelial Lesions (CIN 2/3)

HSILs, or CIN grade 2 or 3 (CIN 2/3), are associated with integration of the viral genome into the host genome, and subsequent constitutive expression of the E6 and E7 viral proteins ( Figure 9.5 ). These proteins are functionally required for initiation and persistence of disease. Viral integration sites, although randomly distributed within the human genome, occur principally at sites where human DNA is prone to breakage (e.g., fragile sites), and appear to affect only the expression of the HPV genome itself. Specifically, during integration, E1 and/or E2 are frequently disrupted, while the E6 and E7 viral oncogenes are retained, resulting in constitutive expression. Morphologically at the cellular level, HSILs are characterized by a high nuclear to cytoplasmic ratio. Histologically, high-grade lesions display disarray of the basal layer itself, full thickness lack of cell maturation, and are mitotically active.

Although all squamous cervical cancers arise from untreated HSIL, not all HSILs progress to invasive cancer. Approximately 35% of HSILs undergo complete regression in a time frame of 4–6 months. Lesions associated with HPV 16 are less likely to regress than lesions associated with other HPV types. Because it is not possible to distinguish HSILs that are likely to regress from those that are not, the standard of care is excision or ablation. Excisional approaches include loop electrocautery excision procedure or cold knife conization. The rate of recurrence is 10%. Iterative excisional procedures are associated with cervical incompetence and subsequent preterm birth.