**3. HPV in carcinogenesis**

squamous intraepithelial lesions. The distinction of these alterations from HPV-related lesions is based on well-defined morphologic criteria; however, in certain lesions the distinction is less straightforward, and ancillary techniques can be of help, leading to a more precise diagnosis and increased diagnostic reproducibility. Regressing LSILs may also cause a diagnostic problem [33]. On the other hand, ruling out invasion can be difficult in certain high-grade lesions. The next parts of the present chapter are going to describe the ancillary techniques,

120 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

**Figure 1.** a-c). The above lesions represent a spectrum of alterations in cervical biopsies, ranging from low-grade to

The precursor lesion of cervical adenocarcinoma, that is adenocarcinoma in situ (AIS), was introduced as a concept in 1953 and is now acknowledged to be the precursor to most invasive cervical adenocarcinomas [34]. AIS is less common than SIL, with a ratio of AIS/HSIL ranging

Adenocarcinoma in situ is characterized by glands with nuclear hyperchromasia and atypia, increased nuclear:cytoplasmic ratio, pseudostratification or stratification, mitoses and apop‐ totic bodies (Fig.2). It may coexist with SIL and can also be multifocal. It may show a variety of cellular differentiation, and several subtypes have been described, including endocervical,

The diagnosis of glandular dysplasia has been used for intraepithelial alterations of glandular epithelium less pronounced than AIS. However, it has low reproducibility, and it has been suggested that this term should no longer be used in the clinical setting [35], especially since glandular epithelium does not support a productive infection by HPV [33]. It has been suggested that problematic endocervical glandular atypias should be evaluated with special studies [34,35]. The term cervical glandular intraepithelial neoplasia (CGIN), with high grade

Cervical endometriosis, tubal and endometrioid metaplasia, and reparative changes have to be distinguished from AIS. Arias-Stella reaction, atypia due to irradiation, atypical forms of microglandular hyperplasia, as well as other viral infections, specifically Cytomegalovirus and

which allow for a more precise diagnosis in some of the problematic cases.

(a) (b) (c)

**2.2. Precursor lesions of cervical adenocarcinoma**

endometrioid, intestinal, tubal, and stratified [34].

CGIN equating to AIS, is being used in several laboratories [20].

in most series between 1:26 and 1:237 [33].

high-grade lesions.

Human papillomavirus is estimated to comprise a causal agent in 5% of human cancers and is associated with more human cancers than any other virus [36]. Among them, it is associated with the vast majority of cervical cancer cases. In contrast to several other infectious agents, which act as indirect carcinogens by inducing immunosuppression or by preventing apoptosis, high-risk HPVs (HR-HPVs) act mainly as direct carcinogenic factors [3]. Persistent infection by HR-HPVs correlates with increased risk of cervical cancer. However, infection by low-risk HPV types (LR-HPVs), carries a negligible risk of malignant progression. Additionally, other factors, related to the host or the environment, contribute to the development of neoplasia.

Several studies have revealed the complex intracellular interactions, which take place among oncoproteins encoded by human papillomaviruses and their cellular target proteins [3,37-39]. Their complexity is reflected in the long interval between infection and invasive carcinoma detection, often spanning a period of 15 to 25 years [3]. These interactions have offered to investigators the opportunity to study important cellular pathways related to the carcinogenic process, while several participating proteins have been studied for their possible use as markers of HPV infection in biopsy or cytology specimens. These biomarkers are presented in the next part of the present chapter.

#### **3.1. HPV in carcinomas of the anogenital tract**

Cervical cancer represents today a relatively well-studied prototype of a human tumor related to a viral infection, as well as a model for multi-step carcinogenesis. The revealed strong association led to the suggestion that human papillomavirus is not only the main cause of cervical cancer, but also a necessary cause [6].

In addition to cervical cancers, a significant percentage of vaginal, vulvar, penile, anal and perianal carcinomas are HPV-positive [7,40-42], while a fraction of carcinomas in other sites of the human body has also been linked to high-risk [HR] HPV infections. Percentages of HPV positivity observed in carcinomas of the anogenital area are presented in Table 1.

and transformation. High-risk HPV E6 proteins target p53 for proteasomal degradation through association with the cellular ubiquitin ligase E6AP [48,52]. Low-risk HPV E6 proteins can also associate with E6AP; however, high-risk HPV proteins target p53 for ubiquitination. Activation of telomerase is another important facet of E6 functions, which is augmented by

Ancillary Techniques in the Histopathologic Diagnosis of Squamous and Glandular Intraepithelial Lesions…

http://dx.doi.org/10.5772/55897

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HPV E7 proteins interact with the retinoblastoma tumor suppressor protein, pRB, which controls S-phase entry through association with E2F transcription factor family members. They also interact with the related pocket proteins, p107 and p130. High-risk HPV E7 targets pRB for proteasomal degradation, while low-risk HPV E7 binds pRB with lower efficiency (ap‐ proximately 10-fold lower) than the former [47,53]. E7 proteins cause aberrant activation of cdk2 (cyclin-dependent kinase 2), which is associated with cyclins E and A, as well as cdk inhibitors, mainly p21CIP1 and p27KIP1. E7 expression results in dysregulated expression of cyclins E and A [48,54]. Through its multiple interactions, E7 can uncouple keratinocyte differentiation from cell cycle progression and retain differentiating keratinocytes in a DNA

The above interactions form the basis for the application of some important biomarkers used nowadays in many laboratories worldwide. These include p16, a cyclin-dependent kinase inhibitor, often exhibiting increased expression in HPV-related intraepithelial lesions, as well as cyclin E, as will be discussed in the following. Furthermore, proliferation markers, like Ki67, show increased/altered expression, in comparison to non-HPV-infected epithelium of the

In addition, HR-HPV E6 and E7 proteins cooperate to generate mitotic defects and aneuploidy through induction of supernumerary centrosomes and multipolar mitoses in epithelial cells [55], while genomic instability results in the addition of molecular alterations. The detection of abnormal mitoses is a useful morphologic indicator of high-risk HPV-associated lesions [32]. Finally, integration of HPV genome into host chromosomes is an important event in cervical carcinogenesis [56,57]. Integration occurs frequently during malignant progression and may result in dysregulation of E6/E7 expression due to disruption of E2, with associated loss of the

Except for the above interactions, several other factors contribute to the development of neoplasia, and these are related to the host or the environment. Smoking, the use of oral contraceptives, high parity and Chlamydia are associated with a relative risk of 2 to 4 [7,9,58-60]. Immunity plays an important role, and this is reflected in data concerning cervical

**4. Immunohistochemical stains in the diagnosis of cervical intraepithelial**

The role of the pathologist examining a cervical biopsy suspicious for an intraepithelial lesion consists of: a) confirming or excluding the presence of a lesion, b) excluding other entities

lesions in HIV-infected individuals and in transplant-recipients.

E7-induced interactions.

synthesis competent state.

uterine cervix.

inhibitory E2 action.

**lesions**


**Table 1.** Percentage of HPV detection in carcinomas of the anogenital region other than cervical carcinoma [4,7].

The distinction of HPV types into low- and high-risk is based on their association with carcinomas, and this distinction is sometimes challenging, especially in the case of rare/weakly carcinogenic viral types. The most common HPV types detected in cervical carcinomas include HPV 16, 18, 45, 31, 33, 52, 58, and 35, belonging to the high-risk group [43-45]. Low-risk viral types may confer risk reflecting an "at risk behavior" [14].

The fraction of cervical squamous cell carcinomas attributable to HPV16 and HPV18, which comprise the two most common high-risk viral types, is estimated at about 70%, while the respective fraction of cervical adenocarcinomas is 86%. As expected, in low-grade squamous intraepithelial alterations the respective percentages differ, since a significant number of these lesions contain HPVs which do not belong to the most common high-risk types.

#### **3.2. Human papillomavirus oncoproteins and their main interactions with cellular pathways**

HPVs are epitheliotropic double-stranded DNA viruses, whose replication is dependent on the terminally differentiating epithelial tissue. Their circular genome includes several open reading frames (ORFs) encoding for proteins which control early (E) and late (L) viral functions (E1, E2, E4, E5, E6, E7, L1, and L2) [46].

High-risk mucosal HPVs encode three transforming proteins: E5, E6 and E7, which exhibit multiple biological activities. These have been extensively studied in the last few decades; however, several aspects remain to be elucidated [47-48].

HPV E5 is able to transform mouse fibroblasts and keratinocytes in culture [49]. It is believed to contribute to early stages of carcinogenesis and works in concert with E6 and E7 [50-51]. These latter proteins, which often act synergistically, are necessary for the induction and maintenance of the transformed phenotype. They inhibit the function of tumor suppressors p53 and pRb, respectively, whereas their expression enables cells to bypass normal cell cycle checkpoints.

E6 and E7 are required for both the development of precursor lesions of cervical carcinoma, and for maintaining the malignant phenotype of cervical cancer cells [3]. E6 and E7 proteins play critical roles, being able to immortalize human keratinocytes and induce cell proliferation and transformation. High-risk HPV E6 proteins target p53 for proteasomal degradation through association with the cellular ubiquitin ligase E6AP [48,52]. Low-risk HPV E6 proteins can also associate with E6AP; however, high-risk HPV proteins target p53 for ubiquitination. Activation of telomerase is another important facet of E6 functions, which is augmented by E7-induced interactions.

In addition to cervical cancers, a significant percentage of vaginal, vulvar, penile, anal and perianal carcinomas are HPV-positive [7,40-42], while a fraction of carcinomas in other sites of the human body has also been linked to high-risk [HR] HPV infections. Percentages of HPV

positivity observed in carcinomas of the anogenital area are presented in Table 1.

122 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

**Table 1.** Percentage of HPV detection in carcinomas of the anogenital region other than cervical carcinoma [4,7].

The distinction of HPV types into low- and high-risk is based on their association with carcinomas, and this distinction is sometimes challenging, especially in the case of rare/weakly carcinogenic viral types. The most common HPV types detected in cervical carcinomas include HPV 16, 18, 45, 31, 33, 52, 58, and 35, belonging to the high-risk group [43-45]. Low-risk viral

The fraction of cervical squamous cell carcinomas attributable to HPV16 and HPV18, which comprise the two most common high-risk viral types, is estimated at about 70%, while the respective fraction of cervical adenocarcinomas is 86%. As expected, in low-grade squamous intraepithelial alterations the respective percentages differ, since a significant number of these

**3.2. Human papillomavirus oncoproteins and their main interactions with cellular pathways** HPVs are epitheliotropic double-stranded DNA viruses, whose replication is dependent on the terminally differentiating epithelial tissue. Their circular genome includes several open reading frames (ORFs) encoding for proteins which control early (E) and late (L) viral functions

High-risk mucosal HPVs encode three transforming proteins: E5, E6 and E7, which exhibit multiple biological activities. These have been extensively studied in the last few decades;

HPV E5 is able to transform mouse fibroblasts and keratinocytes in culture [49]. It is believed to contribute to early stages of carcinogenesis and works in concert with E6 and E7 [50-51]. These latter proteins, which often act synergistically, are necessary for the induction and maintenance of the transformed phenotype. They inhibit the function of tumor suppressors p53 and pRb, respectively, whereas their expression enables cells to bypass normal cell cycle

E6 and E7 are required for both the development of precursor lesions of cervical carcinoma, and for maintaining the malignant phenotype of cervical cancer cells [3]. E6 and E7 proteins play critical roles, being able to immortalize human keratinocytes and induce cell proliferation

lesions contain HPVs which do not belong to the most common high-risk types.

Vaginal carcinomas 60-91% Vulvar carcinomas 50% Penile carcinomas 30-50% Anal and perianal carcinomas 60-94%

types may confer risk reflecting an "at risk behavior" [14].

however, several aspects remain to be elucidated [47-48].

(E1, E2, E4, E5, E6, E7, L1, and L2) [46].

checkpoints.

HPV E7 proteins interact with the retinoblastoma tumor suppressor protein, pRB, which controls S-phase entry through association with E2F transcription factor family members. They also interact with the related pocket proteins, p107 and p130. High-risk HPV E7 targets pRB for proteasomal degradation, while low-risk HPV E7 binds pRB with lower efficiency (ap‐ proximately 10-fold lower) than the former [47,53]. E7 proteins cause aberrant activation of cdk2 (cyclin-dependent kinase 2), which is associated with cyclins E and A, as well as cdk inhibitors, mainly p21CIP1 and p27KIP1. E7 expression results in dysregulated expression of cyclins E and A [48,54]. Through its multiple interactions, E7 can uncouple keratinocyte differentiation from cell cycle progression and retain differentiating keratinocytes in a DNA synthesis competent state.

The above interactions form the basis for the application of some important biomarkers used nowadays in many laboratories worldwide. These include p16, a cyclin-dependent kinase inhibitor, often exhibiting increased expression in HPV-related intraepithelial lesions, as well as cyclin E, as will be discussed in the following. Furthermore, proliferation markers, like Ki67, show increased/altered expression, in comparison to non-HPV-infected epithelium of the uterine cervix.

In addition, HR-HPV E6 and E7 proteins cooperate to generate mitotic defects and aneuploidy through induction of supernumerary centrosomes and multipolar mitoses in epithelial cells [55], while genomic instability results in the addition of molecular alterations. The detection of abnormal mitoses is a useful morphologic indicator of high-risk HPV-associated lesions [32].

Finally, integration of HPV genome into host chromosomes is an important event in cervical carcinogenesis [56,57]. Integration occurs frequently during malignant progression and may result in dysregulation of E6/E7 expression due to disruption of E2, with associated loss of the inhibitory E2 action.

Except for the above interactions, several other factors contribute to the development of neoplasia, and these are related to the host or the environment. Smoking, the use of oral contraceptives, high parity and Chlamydia are associated with a relative risk of 2 to 4 [7,9,58-60]. Immunity plays an important role, and this is reflected in data concerning cervical lesions in HIV-infected individuals and in transplant-recipients.
