**2.1. Precursor lesions of squamous cell carcinoma**

been questioned and gradually replaced by various approaches trying to face the whole group of HPV-related histopathologic abnormalities. In the field of glandular precursor lesions adenocarcinoma in situ is considered the precursor to most invasive cervical adenocarcinomas,

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

Morphology represents a gold standard for lesion diagnosis, since histologic and/or cytologic examination allows in most cases the recognition of viral cytopathic effects and precancerous epithelial alterations; however it can be hampered by inter- and intra-observer variability. In this context, several biomarkers have been investigated for their potential utility in assisting the histopathologic classification of preinvasive lesions and facilitating their distinction from non-HPV induced alterations [16-21]. Human papillomavirus-related intracellular interac‐ tions formed the basis for the identification of markers that may assist in this distinction, including cellular proteins targeted directly by viral oncoproteins, and markers related to the cell cycle, which is disturbed by multiple actions of the virus. Additionally, it is expected that the correlation of slight cellular alterations with new sensitive methods of HPV-detection might lead to the identification of different groups of lesions of clinical significance, as well as

The application of immunohistochemistry and in situ hybridization techniques in the histo‐ pathologic diagnosis of cervical intraepithelial alterations of both squamous and glandular epithelium will be presented in this chapter. The immunohistochemical markers that are currently in use in several laboratories worldwide, as well as some new promising biomarkers will be included. Scientific background for each of these markers and special indications for their application will be summarized. A synoptic review of the pertinent literature will be presented, in an effort to summarize the existing data and the remaining questions at both the

Precursor lesions of both squamous cell carcinoma and adenocarcinoma are well defined, according to our current understanding of cervical neoplasia. However, years of scientific observations and research preceded the recognition of these lesions. Observations concerning spatial and/or temporal relationship between invasive carcinomas and non-invasive, intrae‐ pithelial alterations of the uterine cervix had been repeatedly reported in the past, as early as the end of the 19th century [24-27]. These observations resulted in the recognition of precan‐ cerous alterations of cervical epithelium, for which different terms and definitions have been

The terms carcinoma in situ and dysplasia have been in use for several decades, in order to describe non-invasive, intraepithelial lesions showing cytologic abnormalities akin to those of invasive carcinoma. In the late 1960's the concept of one disease spectrum was introduced, based on observed similarities between groups of lesions, which were considered as different grades of the same disease process, termed cervical intraepithelial neoplasia / CIN [28,29]. This

while the concept of glandular "dysplasia" is being evaluated [11-15].

to the correct application of current morphologic criteria [21-23].

practical and the theoretical level.

used in the following years.

**2. Precursor lesions of cervical carcinoma**

The terms squamous intraepithelial lesion (SIL) of the uterine cervix or cervical intraepithelial neoplasia (CIN) encompass a group of alterations of squamous epithelium that usually occur in or close to the transformation zone and are related to HPV.

Abnormal proliferation and maturation of squamous cells and nuclear atypia, including enlargement, pleomorphism, irregular nuclear borders, and change in chromatin texture, are the characteristics of these intraepithelial lesions. In one group of lesions the observed cellular alterations reflect mainly viral cytopathic effect, corresponding to koilocytic atypia. This is characterized by an abnormal appearing nucleus surrounded by an irregularly shaped cytoplasmic halo with a sharp edge. In these lesions atypia is more conspicuous in the maturing squamous cells, with mild alterations of the basal-parabasal cell morphology. In other groups of lesions cellular atypia is conspicuous in all cell layers: both middle/upper and lower epithelial layers (Fig.1)

Low-grade squamous intraepithelial lesions (LSILs) exhibit differences in density, size and staining of the maturing squamous cells, often accompanied by binucleated cells, cytoplasmic halos, and/or changes in epithelial thickness (Fig.1a)[14]. High-grade squamous intraepithelial lesions (HSILs) exhibit conspicuous nuclear atypia in all epithelial layers, with nuclear crowding, high nuclear:cytoplasmic ratio, loss of normal polarity, irregular nuclear mem‐ branes, and increased mitoses, which can be atypical [32]. In these lesions koilocytosis may be identified or not. There is significant basal/parabasal atypia, with little or no cytoplasmic maturation in the middle-upper layers of the epithelium, and mitotic activity extends to these epithelial layers (Fig.1c).

The differential diagnosis includes mainly: (a) reactive epithelial changes, (b) immature metaplastic changes, and (c) postmenopausal/atrophic epithelia, which may all mimic 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, which allow for a more precise diagnosis in some of the problematic cases.

Herpesvirus, may occasionally pose problems of differential diagnosis. Ancillary techniques

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

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

121

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

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

can be of help in these cases, as described in the next parts of the present chapter.

(a) (b)

**Figure 2.** a-b). AIS. Continuity with benign cervical epthelium is obvious in [b].

**3. HPV in carcinogenesis**

the next part of the present chapter.

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

cervical cancer, but also a necessary cause [6].

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

#### **2.2. Precursor lesions of cervical adenocarcinoma**

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

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

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

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 Herpesvirus, may occasionally pose problems of differential diagnosis. Ancillary techniques can be of help in these cases, as described in the next parts of the present chapter.

**Figure 2.** a-b). AIS. Continuity with benign cervical epthelium is obvious in [b].
