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FIGURE 4.21 High-grade keratinizing dysplasia associated with microinvasive carcinoma.

FIGURE 4.23 High-grade, nonkeratinizing dysplasia (CIS).

FIGURE 4.24 High-grade dysplasia. The basal layer is crowded by cells with high nucleus/cytoplasmic ratios, with enlarged pyknotic nuclei. Parabasal layers are occupied by cells with clear cytoplasm and enlarged nuclei. Contrast these features with the bland cytology of contiguous normal squamous epithelium, present in the upper portion of the figure.

FIGURE 4.24 High-grade dysplasia. The basal layer is crowded by cells with high nucleus/cytoplasmic ratios, with enlarged pyknotic nuclei. Parabasal layers are occupied by cells with clear cytoplasm and enlarged nuclei. Contrast these features with the bland cytology of contiguous normal squamous epithelium, present in the upper portion of the figure.

hyperplasia shows an increase in epithelial thickness secondary to an increase in the stratum spinosum, with no cellular atypia in the basal and parabasal layers. In abnormal hyperplasia, basal or parabasal layers are increased, encompassing up to one-half of the mucosal thickness, but their nuclei, although enlarged, lack significant cytological atypia. The occurrence of significant nuclear atypia and dyskeratosis, associated with preservation of the overall epithelial architecture, characterizes atypical hyperplasia. In CIS, cells with the cytological features of malignancy occupy the majority of the epithelium, which has lost its regular stratification and shows very frequent mitoses [13,19,22]. The approximate correlation between the

FIGURE 4.23 High-grade, nonkeratinizing dysplasia (CIS).

FIGURE 4.25 High-grade, nonkeratinizing dysplasia (CIS).

FIGURE 4.22 Dyskeratosis involving a single cell and a cell nest within the epithelium.

laryngeal intraepithelial neoplasia (LIN) [16,17], as an alternative to dysplasia. The proposed advantages of the designation "intraepithelial neoplasia" are manifold. This definition matches the currently used designation for the cervix uteri [cervical intraepithelial neoplasia CIN], which has replaced the old designation of dysplasia and thus allows more standardized reporting of preneoplastic lesions across different anatomical sites. Furthermore, in the authors' intentions, this designation is more clinically oriented and broader than "dysplasia" and allows the inclusion of nonmorphological parameters, i.e., molecular markers, within the grading system. Although biologically sound, this suggestion, however, has not met acceptance in routine clinical practice.

Kambic and Gale proposed a distinction of dysplasia for the larynx into simple, abnormal, atypical hyerplasia, and CIS. In this classification, known as Ljubljana classification, the emphasis is on cytological features [13,19,22]. Simple

TABLE 4.2 Major Classification Schemes of Dysplasia and Approximate Correspondence of Definitions

WHO Mild Moderate Severe dysplasia CIS

Crissman SIN-LINI SIN-LINII SIN-LINIII CIS

Kambic Simple Abnormal Atypical hyperplasia CIS

different types of dysplasia is sketched in Table 4.2. However, the Ljubjana and the WHO systems are not easily reducible one to the other, as demonstrated by large variations in the classification of lesions between the two groups. Thus, of 13 cases diagnosed as mild dysplasia in the WHO system, 3 were reclassified as simple, 8 as abnormal, and 1 as atypical hyperplasia; of 20 cases of moderate dysplasia, 2 were reclassified as simple, 10 as abnormal, 6 as atypical hyperplasia, and 1 as carcinoma in situ-, and of 14 cases of severe dysplasia, 1 was reclassified as simple, 8 as atypical hyperplasia, and 1 as carcinoma in situ [23]. Nine lesions were placed in a group of "large cell hyperplasia," a category not described in the original Ljubjana classification [23]. Like other multi-tiered grading systems, the WHO system, which is used most commonly in pathological practice, has high inter- and intraobserver variability [24]. The morphological distinction between severe dysplasia and CIS is particularly difficult and is not reproducible [10]; however, these lesions have a similar risk of progression into overt cancer [10]. These findings justify a distinction into two tiers: low-grade (including mild and moderate dysplasia) and high-grade (including severe dysplasia and CIS), dysplasia, provided that a mutual understanding of the terminology is instituted among pathologists, surgeons, and oncologists.

C. Molecular Markers of Dysplasia

Since the initial histological characterization of prema-lignant conditions of the UADT, one of the major advances in this field has been identification of the molecular alterations with which they are associated. While biologically justified and crucial in reducing the variability intrinsic in a morphology-based system [24], the inclusion of molecular markers has yet to find a role in the classification of dysplasia. However, understanding the molecular basis of progression for UADT preneoplastic lesions is invaluable in understanding the biology of SCC. Furthermore, it may be clinically relevant if molecular markers of dysplasia are used as screening tools for early detection or as intermediate markers in chemoprevention trials. The prototype model of molecular alterations associated with preneoplastic progression is the colon, where sequential molecular alterations have first been described to occur alongside the morphological progression from normal to cancer [25], A similar accumulation of alterations has been described in the UADT in increasing morphological grades of dysplasia.

Molecular alterations occurring in preneoplastic lesions of the upper aerodigestive tract belong to two main groups, reflecting abnormalities in either cellular differentiation or cell cycle control. Changes of the first group include those affecting the profile of keratin expression and were the first to be reported. In the normal epithelium, low molecular weight keratins (LMW) are expressed in the basal layer and high molecular weight keratins (HMW) are expressed in the stratum spinosum. Abnormal cells express, regardless of their position, LMW keratins, whereas HMW keratins are expressed either in the uppermost, keratinized layer or not at all in dysplasia. Thus, suprabasal expression of LMK, such as CK19, has been proposed to constitute a marker of dysplasia [18,26], altough this finding cannot reliably distinguish hyperplasia from true dysplasia [1],

The foremost alteration in cell cycle regulation occurring in dysplasia is the occurrence of an increased proliferative rate in association with increasing morphological grades. This has been assessed traditionally by morphological evaluation, i.e., counting mitoses. However, more recently, the discovery of PCNA and Ki-67 antigens, expressed exclusively by proliferating cells, has allowed an objective evaluation of the proliferative rate [3,27-30], Using both methods, a continuum of increasing proliferative rates is seen in increasing grades of dysplasia. In contrast, the only population to show infrequent positivity in normal mucosa is the basal cell, compatible with its role as a progenitor cell [3,29,30]. Interestingly, in simple mucosal hyperplasia, only the basal layer shows positive staining for proliferative antigens, setting it aside from true dysplasia, where the expression of this antigen extends to suprabasal cells [1],

Alterations in many molecules controlling the cell cycle are frequent in dysplasia and are likely responsible for its hyperproliferative state. A continuum of increasing positivity is observed in the rate of p53 positivity, as detected by immunohistochemistry in lesions of increasing histological grades [30-32], While negative in normal epithelium, p53 is found in 9.4 to 32% of low-grade and 33 to 50% of highgrade dysplasia [30,32], Suprabasal expression of CK 19 and PCNA is associated with positivity for p53 in the majority of cases of dysplasia [1], highlighting the link existing between abnormal differentiation and cell cycle alterations. Alterations in the distribution of the cyclin kinase inhibitor p21 also occur. Whereas only the intermediate layer of the normal epithelium expresses this marker, the entire dysplas-tic epithelium shows positivity [33].

Increasing percentages of EGFR positivity are also seen, seemingly associated with the dysplastic component [31], Increasing percentages of aneuploid populations are also observed in increasing grades, progressing from 33% of SINI to 78% of SINII and 100% of SINII [21],

Apoptosis increases in parallel to the proliferative rate, as observed morphologically or by DNA in situ-labeling techniques [30,34]. The relevance of alterations in the apoptotic rate is highlighted by data showing a change in expression of the antiapoptotic protein bcl-2 (the target gene deregulated as a consequence of the 14; 18 translocation occurring in most follicular lymphomas) in dysplasia. While normally found only in 37% of normal squamous mucosa, where its expression is restricted to the basal layer, its expression raises to 71 % of dysplastic lesions and 80% of invasive ones in the nasopharynx [35].

Genetic studies of preneoplastic lesions for loss of heterozygosity (LOH) have shown that increasing grades of dysplasia show accumulation of genetic deletions, compatible with the commonly accepted model envisioning cancer as the result of multiple genetic "hits." The earliest and most common changes occur at sites 3p and 9p. The 9p locus harbors genes for the kinase inhibitors pi6 and pi9, which are altered frequently in HNSCC, as well as in malignancies from other sites. The 3pl4 and 3p21 sites harbor several candidate tumor suppressor genes, including the gene deleted in Von Hippel Lindau (VHL) disease, the DNA mismatch repair enzyme hMLHl, affected in hereditary nonpolyposis colon cancer, the retinoic acid receptor [3, and the fragile histidine triad (FHIT) gene [36], While the identification of the specific suppressor gene(s) located at 3pl4 and 3p21, whose loss is responsible for the development of cancer, is still unresolved, the FHIT gene has been proposed to be a specific target of cigarette smoke carcinogens [37-39] and shown to behave as a tumor suppressor gene in vitro [40]. In a retrospective study correlating molecular alterations with progression to overt cancer, losses at 3p and 9p loci were the most common lesions in both nonprogressing and progressing premalignant lesions. However, they were virtually always present in progressing lesions, compatible with a model whereby they are necessary but not sufficient for malignant transformation. The occurrence of additional chromosomal losses was shown to confer a much higher risk of progression, comparable to losses at 3p and 9p alone [41], These included chromosomal sites 4q, 8p, llq, and, notably, 17p [41,42]. Because this latter site is the locus of p53, these data are compatible with histochemical data quoted previously, showing alterations in p53 as an important event in the progression of premalignant lesions. These data are overall compatible with a model envisioning loss of genetic material in chromosome 3p and 9p loci, as involved in initiation [41], and additional genetic losses, including p53, as involved in progression. Data are summarized in Table 4.3.

Changes in the basement membrane have also been described in dysplasia. Normal and hyperplastic mucosa is usually associated with a prominent and continuous basement membrane, as assessed by an immunohistochemical stain with collagen type IV and laminin. The basement membrane is usually prominent and continuous in mild to moderate dysplasia; in contrast, in severe dysplasia/carcinoma in situ it is often thinned and discontinuous. However, some

TABLE 4.3 Incidence of Molecular Alterations in Low- and High-Grade Dysplasia
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