Histopathological Classification Phenotype and Molecular Pathology of Lung Tumors

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2 Histopathological Classification Phenotype and Molecular Pathology of Lung Tumors Elisabeth Brambilla

Introduction Lung cancer is the most common diagnosed cancer and the major cause of mortality (Greenlee et al., 2001) worldwide. With 169,500 new cases per year and 157,400 cancer deaths (Greenlee et al., 2001) in the United States and 182,000 new cases per year and 190,000 cancer deaths in the European Union in 2001, it is the only cancer where incidence and mortality are quite similar. The main risk factor is tobacco carcinogen. Although lung cancer incidence began to decline in men in the United States beginning in 1980, its rate is increasing in women, as a consequence of an increase in the number of women smoking (Travis et al., 1996). This review focuses on histopathological classification and phenotypical characteristics of lung cancer. Histologic evaluation for lung cancer diagnosis is based on several types of biopsy specimens, including bronchoscopy or fine-needle biopsies and video-assisted thoracoscopic biopsy, as well as wedge resection, lobectomy, or pneumonectomy. Light microscopy is sufficient for most of the diagnosis of lung cancer types and subtypes, rendering the need for histochemical stains or immunohistochemistry to a few histologic types. The international standard for histologic Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 1: Molecular Genetics; Lung and Breast Carcinomas

classification of lung tumors is that proposed by the World Health Organization (WHO) and the International Association for the Study of Lung Cancer (Travis et al., 1999). The four major histologic types of lung cancer are squamous cell carcinoma, adenocarcinoma—the incidence of which is increasing at the expense of squamous cell carcinoma—small cell lung carcinoma (SCLC) and large cell lung carcinoma (LCLC). These major types have been subclassified into subtypes, the clinical significance of which might be extremely important such as the bronchioloalveolar carcinoma (BAC) as a variant of adenocarcinoma (Travis et al., 1999).

Preinvasive Lesions Evidence is accumulating that invasive lung cancer is the end result of a multistep and multifocal process in which molecular changes accompany or even precede histological changes. This fulfills the concept of “field cancerization,” which reflects the fact that the entire epithelium is the target of tobacco carcinogens. Active mutagenesis may randomly affect any anatomical location in the bronchial tree. The genetic changes, because of their exquisite specificity or selectivity, are suited to be markers of premalignancy, providing they

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106 are maintained in malignancy. The pathology of preinvasive lesions for lung cancer has attracted increasing interest in recent years because of the importance of early detection of lung cancer to screen high-risk patients using fluorescence bronchoscopy and low-dose spiral and helical computerized tomodensitometry (CT). Early detection aims should therefore be to identify the field effect, evaluate the severity of the lesions in the field, and devise suitable methods of intervention according to individual potential for progression. Histological diagnosis of preneoplasia, as well as that of neoplasia, is currently the most reliable standard of diagnosis, although molecular pathology markers have value in indicating the severity of the cancerization field and the risk of progression into lung cancer.

Squamous Dysplasia and Carcinoma in situ Morphological transformation of the normal bronchial mucosa occurs through a continuous spectrum of lesions, including basal cell hyperplasia; squamous metaplasia; mild, moderate, and severe dysplasia; and carcinoma in situ (CIS). According to the thickness and severity of cytologic atypia within the bronchial epithelium, squamous dysplasia are subclassified as mild, moderate, and severe for the lower third, two-thirds, or all thickness of the bronchial epithelium involved, respectively (Travis et al., 1999). Carcinoma in situ shows full-thickness involvement of the epithelium and marked cytologic atypia. It differs from severe dysplasia by lack of visible maturation and orientation of the cells from the basal to luminal part of the epithelium. Use of the term “microinvasive squamous cell carcinoma” is not recommended because these microinvasive tumors are true squamous cell carcinoma classified as T1 of the tumor size, node, and metastases (TNM) classification and should be separated from carcinoma in situ with involvement of submucosal glands. Pathologists, however, experience the frequency of invasive carcinoma characterized by disruption of basal lamina at less than 300-μ distance from a CIS on serial sections. Serial sections of these areas are thus recommended.

Atypical Alveolar Hyperplasia This is a millimetric lesion that is considered as a preinvasive state for bronchioloalveolar carcinoma. Atypical alveolar hyperplasia (AAH) is characterized by type II cell proliferation that resembles but falls short of criteria for BAC, nonmucinous type. This is an incidental discovery on histologic examination of a lung cancer resection specimen, the incidence of which varies from 5.7–21.4%, depending on the extent of the

search and the criteria used for this diagnosis as well as the range of ages in reported autopsy studies. Most AAH are less than 5 mm in diameter and are multiple. Histologically, AAH consists of a focal proliferation of slightly atypical cuboidal to low columnar epithelial cells along alveolar walls and respiratory bronchioles. Alveolar septa may present slight thickening and discrete lymphoid infiltration.

Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is thought to represent a precursor lesion for carcinoid tumors because a subset of these patients have one or more carcinoid tumors (Travis et al., 1999). It is a rare condition involving peripheral airways at the level of terminal and respiratory bronchioles characterized by linear neuroendocrine cell hyperplasia and tumorlets.

Squamous Cell Carcinoma Variants of squamous cell carcinoma include: Papillary Clear cell Small cell Basaloid Squamous cell carcinoma accounts for approximately 30% of all lung cancers in the United States and 45% in Europe. Its incidence in Europe is progressively decreasing, whereas that of adenocarcinoma is increasing. Two-thirds of squamous cell carcinomas are present as central tumors with a primary site on main system or lobar, whereas one-third are present as peripheral tumors on segmental bronchi. The morphologic features that characterize squamous differentiation include intercellular bridging and keratinization (or individual cell keratinization or squamous pearl formation). These differentiated features are readily apparent in well-differentiated tumors and difficult to detect in poorly differentiated tumors. However, the degree of differentiation does not correlate with prognosis in lung squamous cell carcinoma. Variants described in the WHO classification include papillary, clear cell, small cell, and basaloid subtypes. This last variant has a dismal prognosis compared with poorly differentiated squamous cell carcinoma (Brambilla et al., 1992; Moro et al., 1994). Papillary squamous cell carcinoma often show a pattern of exophytic endobronchial growth. Most squamous carcinoma express high molecular weight cytokeratins (recognized by antibody AE1/AE3,

2 Histopathological Classification of Lung Tumors KL1, 34βE12, anti-cytokeratine 5-6). Very few cases express cytokeratine 7 (Ck7), whereas thyroid transcription factor 1 (TTF-1) is never expressed.

Adenocarcinoma Usual subtypes of adenocarcinoma include: Acinar Papillary Bronchioloalveolar carcinoma Nonmucinous Mucinous Mixed mucinous and nonmucinous or indeterminate Solid adenocarcinoma Adenocarcinoma with mixed subtypes Variants Well-differentiated fetal adenocarcinoma Mucinous “colloid” adenocarcinoma Mucinous cystadenocarcinoma Signet ring adenocarcinoma Clear cell adenocarcinoma Adenocarcinomas account for about 40% of lung cancer in Europe and the United States. Because most adenocarcinomas are histologically heterogeneous, consisting of two or more of the histologic subtypes, 80% of lung adenocarcinomas diagnosed fall into the category of mixed subtype. The acinar and papillary subtypes are recognized by their architectural pattern of tumor cell growth and invasion. A substantially different definition has been given to bronchioloalveolar carcinoma (BAC subtype), which should be restricted to tumors that grow in a purely lepidic fashion. The solid-type adenocarcinoma is a poorly differentiated carcinoma presenting intracytoplasmic mucins that should be at least five mucin droplets in two different high-power fields. Mucin stains recommended are periodic acid–Schiff (PAS) with diastase digestion and Kreyberg staining with alcian blue. Bronchioloalveolar carcinoma is uncommon and probably restricted to fewer than 3% of all lung malignancies. In the 1999 WHO/International Association for the Study of Lung Cancer (IASLC) classification, BAC received a restrictive definition: it is defined as a tumor showing lepidic growth along respected alveolar septa with intact elastic and basal lamina frames, without invasive growth. The lack of invasive growth is added as an essential criterion (Travis et al., 1999) based on clinico-pathological data indicating that patients with less than 2-cm BAC may be curable by economic surgical resection (Noguchi et al., 1995).

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As a result of the definition, this tumor can be considered as a carcinoma in situ at alveolar site. As a consequence of this revised definition of BAC, the literature dealing with these tumors needs complete reevaluation. Indeed, previous to the last classification, BAC included tumors with obvious invasive growth. It is common to observe central scars in pulmonary adenocarcinoma that contains invasive components and a focal BAC-like pattern at the periphery of the tumor. More than 50% of previously called BACs present focal central desmoplastic scaring tissue or intra-alveolar complex papillary growth, whereas the lepidic growth starts around the edge of the scar. For tumors showing malignant tumor cell nests in a desmoplastic central stromal reaction, the diagnosis is adenocarcinoma mixed subtype and the various subtypes present should be mentioned (such as acinar, papillary, or BAC). These are not considered any longer as BAC. According to the 1999 WHO/IASLC classification, a final diagnosis of BAC can only be achieved at examination of a surgical resection specimen. Small biopsies obtained by bronchoscopy or fine-needle sampling may show a lepidic growth pattern suggesting the possibility of BAC but are not sufficient to exclude the presence of an invasive growth. Several important clinicopathological studies have shown the clinical significance of BAC (Noguchi et al., 1995; Suzuki et al., 2000). Noguchi et al. (1995) reported that in a large series of 236 cases, the patients with less than 2-cm peripheral lung adenocarcinoma achieve 100% 5-year survival. Multiple pathologic factors for prognostic assessment associated with 100% 5-year survival include at least one of the following features: 1) a pattern of lepidic growth of more than 75% 2) central scar measuring 5 mm or less; and 3) lack of destruction of the elastic fiber framework by tumor cells (Yokose et al., 2000). The immunohistochemical characteristics of adenocarcinoma may vary according to their histological subtype. They all express epithelial markers (cytokeratins, epithelial membrane antigen (EMA), carcinoembry antigen (CEA). Among cytokeratins, Ck7 is almost constantly expressed, whereas Ck20 is infrequently expressed except for mucinous type BAC, which are often Ck20 positive and Ck7 negative. TTF-1 is expressed in 85% of adenocarcinomas, the most frequently negative ones being mucinous types. Deregulation of oncogenes and tumor suppressor genes are similar to those observed in other non–small cell lung carcinoma except for Ras mutation, which is quite restricted to adenocarcinoma and more characteristic of peripheral type adenocarcinoma. The differential diagnosis of primary lung adenocarcinoma from metastatic colonic adenocarcinoma cannot rely on Ck20 positivity and CdX2 homeobox gene expression because

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108 both are commonly expressed in these two types of tumors (Rossi et al., 2004). A distinction of pulmonary adenocarcinoma from epithelial malignant mesothelioma as a peripheral tumor rests on phenotypical differences. Although primary pulmonary adenocarcinomas express TTF-1, mesotheliomas do not. In contrast with adenocarcinoma, mesothelioma expresses calretinin and cytokeratin 5-6. A number of general adenocarcinoma markers (CEA, CD15, BerEP-4) also help in this distinction.

and EMA stain 85% of SCLC each, in contrast with a specific set of cytokeratin (CK1, 5, 10, 14) recognized by the antibody 34βE12, which is never expressed in pure SCLC. However, this reactivity with 34βE12 enlights the NSCLC component combined with SCLC in the variant of SCLC called small cell lung carcinoma combined. In 85% of SCLC, TTF-1 is expressed. Antigen Mic2 (CD99), characteristic of primitive neuroectodermal tumors (PNET) and small round cell sarcoma, is also present in a large proportion of SCLC. C-kit (CD117) is expressed in about 40% of SCLC.

Small Cell Lung Carcinoma Small cell lung carcinoma (SCLC) accounts for 25% of all lung cancers in the United States as well as in Europe. Two-thirds of SCLC are proximal and present as a perihilar tumor. The 1999 WHO/IASLC classification presents only two types of SCLC: SCLC (with pure SCLC histology) and combined SCLC (combined with any non–small cell type). SCLC has a distinctive histological appearance. Tumor cells have a small size, not exceeding that of three lymphocytes. They have a round or fusiform shape, scant cytoplasm with a nuclear-to-cytoplasmic ratio of 9 to 10, a finely granular nuclear chromatin (salt-and-pepper appearance), and absent or inconspicuous nucleoli (Travis et al., 1999). Owing to the scarcity of cytoplasm, nuclear molding and smearing of nuclear chromatin is frequent, caused by crush artifact. There is usually an extensive necrosis and mitotic rate exceeding 20 and reaching 100 mitoses per 2 mm2 area. Most often, the growth pattern consists of diffuse sheets, although endocrine differentiations with rosettes, palisading, ribbons, and organoid nesting might be seen. The immunohistochemical features of SCLC are not required for the diagnosis of SCLC. However, crush artifact is common in small biopsy specimens for immunohistochemistry for neuroendocrine differentiations and keratins, and common leucocytes antigens become more useful in marking SCLC versus lymphoid cells, respectively. Moreover, the most important differential diagnosis resides in the distinction between SCLC and non–small cell lung carcinoma (NSCLC) because of different therapeutic implications. The most useful and specific neuroendocrine markers for distinction of SCLC are chromogranin A, synaptophysin, and neural cell adhesion molecule (NCAM) (clones 123C3 and CD56). The vast majority (95%) of SCLC are reactive with NCAM antibodies, with a specific membranous pattern. This is the most specific and sensitive marker to distinguish SCLC from NSCLC (Lantuejoul et al., 1998). Twenty percent of small cell lung carcinoma may lack chromogranin A and/or synaptophysin expression. Keratin (AE1–AE3)

Combined Small Cell Lung Cancer A SCLC associated with at least 10% of another NSCLC component is diagnosed as combined SCLC. Therefore a tumor with at least 10% SCLC component is a SCLC combined. The frequency of combined SCLC depends on the extent of histologic sampling and the extent of the associated component. In a recent study on surgically treated SCLC and using a conservative estimate of 10% of tumors showing NSCLC associated for subclassifying a tumor as a combined variant of SCLC, 28% of the SCLC cases showed a combination with NSCLC, more commonly with large cell lung carcinoma followed by adenocarcinoma and squamous cell carcinoma (Nicholson et al., 2002). When SCLC is associated with spindle cell carcinoma, giant cell carcinoma, or carcinosarcoma, the tumor is diagnosed as SCLC combined. Immunohistochemistry might help to recognize associated components such as cytokeratin antibody cocktails, which tend to stain non–small cell lung carcinoma components, a good example of which is CK1, 5, 10, 14 recognized by 34βE12. However, evidence is lacking that pure small cell lung carcinoma and combined small cell lung carcinoma behave differently in regard to prognosis and response to therapy. Following chemotherapy, a mixture of large cells, squamous cell, adenocarcinoma, or giant cells with SCLC may be seen in 15–45% of the cases (Brambilla et al., 1991; Sehested et al., 1986).

Large Cell Lung Carcinoma Large cell carcinoma is a tumor that shows no differentiation pattern allowing classification into squamous cell carcinoma, adenocarcinoma, or small cell carcinoma. These poorly differentiated tumors most often arise in the lung periphery, although they may be located centrally. They frequently appear at gross examination as large, necrotic tumors. Histologically, these consist of sheets or nests of large polygonal cells with vesicular nuclei and prominent nucleoli. Although they are

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undifferentiated by light microscopy, features of squamous cell or adenocarcinoma might be found with electron microscopy examination.

Large Cell Neuroendocrine Carcinoma Large cell neuroendocrine carcinoma (LCNEC) is a variant of large cell carcinoma (Travis et al., 1999). It is a high-grade non–small cell neuroendocrine carcinoma that differs from atypical carcinoid and small cell carcinoma (Travis et al., 1991, 1998). Histologic criteria include (Table 9): 1) neuroendocrine morphology (organoid, palisading, trabecular, or rosette-like growth patterns (Figure 12A); 2) non–small cell cytologic features (large size, polygonal shape, low nuclear-to-cytoplasmic (N/C) ratio, coarse or vesicular nuclear chromatin, and obvious nucleoli; 3) high mitotic rate (≥ 11 per 2 mm2) with a mean of 60 mitoses per 2 mm2; 4) frequent necrosis; and 5) at least one positive neuroendocrine immunohistochemical specific marker or neuroendocrine granules by electron microscopy (Travis et al., 1991, 1999). It is difficult to diagnose LCNEC based on small biopsy specimens because of the frequent lack of neuroendocrine morphology without a substantial sampling of the tumors.

Combined LCNEC

A

B Figure 12 Large cell neuroendocrine carcinoma. A: Numerous

The term combined LCNEC is used for tumors associated with other histologic types of NSCLC, such as adenocarcinoma or squamous cell carcinoma. Any combination of LCNEC with SCLC is diagnosed as

rosettes give this tumor a neuroendocrine morphologic appearance. Mitotic rate is high (HES 400X). B: NCAM is expressed with a membrane pattern (200X).

Table 9 Light Microscopic Features for Distinguishing Small Cell Carcinoma and Large Cell Neuroendocrine Carcinomaa Histologic Feature

Small Cell Carcinoma

Large Cell Neuroendocrine Carcinoma

Cell Size

Smaller (less than diameter of 3 lymphocytes) Higher Finely granular, uniform Absent or faint

Larger

Nuclear/cytoplasmic (N/C) ratio Nuclear chromatin Nucleoli Nuclear molding Fusiform shape Polygonal shape with ample pink cytoplasm Nuclear smear Basophilic staining of vessels and stroma

Characteristic Common Uncharacteristic

Lower Coarsely granular or vesicular, less uniform Often (not always) present, may be prominent or faint Less prominent Uncommon Characteristic

Frequent Occasional

Uncommon Rare

aFrom Travis W.D. et al., 1991. Neuroendocrine tumors of the lung with proposed criteria for large cell neuroendocrine carcinoma: An ultrastructural, immunohistochemical, and flow cytometric study of 35 cases. Am. J. Surg. Pathol. 15:529–533. With permission.

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110 SCLC combined (Travis et al., 1999). A variety of criteria must be used to separate SCLC from LCNEC (Table 9). There is no specific marker to distinguish large cell carcinoma from squamous cell carcinoma and adenocarcinoma. In contrast, there are specific features of LCNEC that distinguish them as a specific variant among large cell carcinomas. This distinction is supported by a worse survival observed for LCNEC compared with classical large cell carcinoma. Ninety percent of LCNECs express NCAM (clone 123C3, Cd56) (Figure 12B) in addition to the typical morphological neuroendocrinoid pattern, and most of them express in addition one or both chromogranin A and synaptophysin (Lantuejoul et al., 1998). Half of LCNECs express TTF1, whereas CK 1, 5, 10, 14 (34βE12) is never expressed in pure LCNEC but is expressed in combined components.

A

Basaloid Carcinoma Basaloid carcinoma is the most prominent variant of large cell carcinoma after LCNEC (Brambilla et al., 1992; Moro et al., 1994; Travis et al., 1999). Basaloid carcinomas represent 3–4% of NSCLC in Europe and always occurs in males and smokers. Most of these tumors develop in proximal bronchi, where they frequently have an endobronchial component. Two-thirds of these tumors arise from long areas on bronchial mucosa and show prolonged and laterally extended in situ carcinoma. About half of the tumors present with a pure basaloid pattern that belongs to a variant of large cell carcinoma. The remaining cases have minor (< 50%) components of squamous cell carcinoma or, more rarely, adenocarcinoma and are thus classified as squamous cell carcinoma (basaloid variant) or adenocarcinoma, respectively. These tumors consist of lobular, trabecular, or palisading gross pattern of relatively small monomorphic cuboidal to fusiform cells with moderately hyperchromatic nuclei, finely granular chromatin, absent or only focally conspicuous nucleoli, scant cytoplasm but an N/C ratio lower than that of SCLC, and high mitotic rate from 20 to 100 mitoses per 2 mm2 (Figure 13A). Neither intercellular bridges nor individual cell keratinization are present, which allow them to be distinguished from poorly differentiated squamous cell carcinoma. Patients with basaloid carcinoma have a significantly shorter survival than those with poorly differentiated squamous cell carcinoma, which deserves this differential diagnosis (Brambilla et al., 1992; Moro et al., 1994). The differential diagnosis between LCNEC and basaloid carcinoma is somewhat puzzling because both may have peripheral palisades and rosettes can be

B

Figure 13 Basaloid carcinoma. A: This tumor consists of lobules of rather small uniform cells with peripheral palisading, and a high rate of mitosis (HES 400X). Chromatin is moderately dense and the nucleolus unconspicuous (400X). B: Cytokeratins 1, 5, 10, 14 stained by antibody 34βE12 are diffusely expressed.

observed in 30% of basaloid carcinoma. Fortunately, immunhistochemical features are quite distinctive. SCLC never expresses CK1, 5, 10, 14 (34βE12 reactively), whereas the vast majority (100% in our series) of basaloid carcinoma are reactive with 34βE12 (Figure 13B). In a quite opposite figure, TTF-1 is never expressed in basaloid carcinoma but is expressed in the majority of LCNEC (Sturm et al., 2001). Other large cell carcinoma variants include clear cell, rhabdoid type and lymphoepithelioma-like carcinoma. This last one displays Epstein-Barr virus genomic sequences at in situ hybridization (Chan et al., 1995).

Adenosquamous Carcinoma Adenosquamous carcinoma accounts for 0.6–2.3% of all lung cancers and is defined as a lung carcinoma having at least 10% of squamous cell or adenocarcinoma components. Adenosquamous carcinoma should

2 Histopathological Classification of Lung Tumors not be confused with mucoepidermoid carcinoma, a malignant epithelial tumor characterized by the presence of squamoid cells, mucin secreting cells, and cells having intermediate type, identical to the same tumors encountered in the salivary glands. Mucoepidermoid carcinoma of high-grade malignancy is differentiated from adenocarcinoma by a variety of features, including a mixture of mucin-containing cells and squamoid cells, transition areas from classical low-grade mucoepidermoid carcinoma, and lack of keratinization. The different immunohistochemical staining characteristics previously described for squamous cell carcinoma and adenocarcinoma are admixed according to the component in adenosquamous carcinoma.

Carcinomas with Pleomorphic Sarcomatoid or Sarcomatous Elements (Sarcomatoid Carcinoma) The subtypes of these carcinomas include: Carcinomas with spindle and/or giant cells Pleomorphic carcinoma Spindle cell carcinoma Giant cell carcinoma Carcinosarcoma Pulmonary blastoma Other This group of lung carcinomas is poorly differentiated and expresses the features and biological behavior of epithelial cells that adopt epithelial to mesenchymal transition in certain conditions of cultures in vitro. Pleomorphic carcinomas tend to be large peripheral tumors invading bronchial lumens, forming endobronchial growth. They often invade the chest wall and are associated with a poor prognosis (Fishback et al., 1994). Because of the characteristic histologic heterogeneity of this tumor, adequate sampling is required and should consist of at least one section per centimeter of the tumor diameter. To enter in this category, a pleomorphic carcinoma should have at least a 10% component of a spindle or giant cells associated with other histological types, such as adenocarcinoma or squamous cell carcinoma (Travis et al., 1999). Pure giant cell or spindle cell carcinomas are extremely rare but cause an obvious challenge in distinguishing them from sarcoma. Pleomorphic and sarcomatoid carcinomas have a dismal prognosis even at stage I. A recent large review of 78 cases of pleomorphic sarcomatoid carcinoma indicated that immunohistochemical features of these carcinomas reflect their epithelial lung epithelial cell origin with TTF-1 expression in 40%

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of them, as well as CK7 positivity and CK20 negativity (Rossi et al., 2003). This class of tumor also includes carcinosarcoma and pulmonary blastoma.

Carcinoid Tumor Subtypes of carcinoid include Typical carcinoid Atypical carcinoid Carcinoid tumors account for 1–2% of all invasive lung malignancies. Typical and atypical carcinoids are characterized histologically by endocrinoid, organoid growth pattern, and uniform cytologic features, consisting of moderate eosinophilic, finely granular cytoplasm, a nuclear with a finely granular chromatin, and inconspicuous nucleoli that can be discretely more prominent in atypical carcinoid. A variety of histologic patterns may occur in atypical and typical carcinoids, including trabecular, palisading, rosette-like, papillary, sclerosing papillary, glandular, paragangliomatous, spindle cell, and follicular patterns. The most distinguishing feature between typical carcinoid and atypical carcinoid is the rate of mitosis and the presence or absence of necrosis. Typical carcinoids show less than 2 mitoses per 2 mm2 area of viable tumor (10× high power field) and no necrosis. The presence of mitosis between 2 to 10 per 2 mm2 or the presence of necrosis (Travis et al., 1998, 1999) define the diagnosis of atypical carcinoids. The presence of features such as cell pleomorphism, vascular invasion, and increased cellularity are of no help in separating typical carcinoid from atypical carcinoid and in allowing stratification of patients for prediction of survival (Travis et al., 1998). Typical carcinoid may well show focal cytologic pleomorphism as do paraganglioma in head and neck area. The necrosis in atypical carcinoid usually consists of small foci centrally located within organoid nests of tumor cells. Although neuroendocrine markers are not required for the diagnosis of carcinoids, they are positive in every case. Carcinoids do not express TTF-1 in contrast with the high-grade neuroendocrine tumors, LCNEC, and SCLC (Sturm et al., 2002). Protein S100, which is a nonspecific antigen, might be present in the nuclei of any kind of neuroendocrine lung tumor. In carcinoids, they are specifically expressed in perilobular cells that have appearance and phenotype of sustentacular cells, as seen in paraganglioma. The differential diagnosis between carcinoids, meningothelial-like bodies, and paraganglioma resides in expression of cytokeratins in

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112 carcinoids but absence of expression in meningotheliallike bodies and in paraganglioma, and the absence of neuroendocrine markers in meningothelial-like bodies.

Molecular Pathology of Lung Carcinoma Molecular pathology refers here to phenotypical markers used to detect abnormal expression of proteins, in situ overexpression of oncogenes, or loss of protein expression of tumor suppressor genes. Both p53 and retinoblastoma gene (Rb), as well as molecules from their pathways, belong to tumor suppressor genes involved in cell cycle regulation and apoptosis; Bcl2 and Bax as well as the Fragile Histidine Trial (FHIT) protein are members of apoptosis markers that influence the cell susceptibility to death; telomerase reactivation reflects cell immortalization; matrix degrading proteases and vascular endothelial growth factor (VEGF) are proteins reflecting angiogenic potential, migratory capacities, and survival of clonal proliferation. Overall, the clonal expansion (tumor growth) might be regarded as the net result of a gain of a cell numbers by increasing intrinsic proliferation (rate of cell division) and loss of cells by decreasing propensity to cell death (escape from apoptosis). The complex genetic and epigenetic changes that result in lung carcinoma are thought to begin before the occurrence of invasive carcinoma in a process referred to as multistep carcinogenesis. A set of phenotypical traits of lung cancer appears at the level of preinvasive lesions.

DNA Damage Damaged DNA may give rise to allelic loss, a frequent early molecular finding in lung carcinogenesis. Loss of heterozygosity (LOH) refers to allelic loss. Chromosomal loci that normally harbor two different polymorphic alleles are assessed for loss of one (loss of heterozygosity, LOH) or both of these alleles (homozygous deletion). Remanant allelic loss at specific chromosomal loci, as a candidate for the presence of tumor suppressor genes, fosters search for mutation of the second allele. Regions that have received considerable attention are chromosomes 3p14-23 (5 loci), 8p21-23, 9p, 17p (p53 locus), and 13q (Rb locus) because of the high density of loss and the probability that tumor suppressor genes are present. One of the best-studied chromosome 3 genes is FHIT. The FHIT gene is situated in a highly fragile histidine site, where it is particularly prone to partial deletion as a result of direct DNA damage by smoking associated carcinogens (Sozzi et al., 1996). Candidate tumor suppressor genes on chromosome 9 include p15INK4A (9p21) and p16 INK4B (9p21).

p16INK4 inhibits activation of cyclinD/Cdk4, 6 kinases and thus impairs progression of the cell through the mitotic cycle. Loss of p16ink4 would cause more rapid progression through the cell cycle. Allelic loss is observed in more than 70% of NSCLC and p16INK4 is inactivated by homozygous loss in nearly half of smoking-associated lung cancers (Gazzeri et al., 1998). Genomic molecular damage accumulating during lung carcinogenesis eventually results in chromosomal rearrangements and aneusomy involving multiple chromosomes. Abnormalities have been described in every chromosome, but some chromosomes and chromosomal loci are more frequently unbalanced than others. Gains of chromosomes 6, 7, and 8 occur in approximately 50% of the NSCLC.

Ras Mutation Ras mutations occur almost exclusively in adenocarcinoma, most frequently at Ki-Ras codon 12, and are strongly related to tobacco smoke nitrosamines. A singlebase mutation at Ki-Ras codon 12, observed in 30% of adenocarcinoma, is responsible for the lack of intrinsic GTPase functions of the Ras protein, which is endowed with constitutive activity toward proliferations. Ras mutation is a relatively late event in the process of Claratype II cell mutagenesis but precedes invasion since it occurs in a proportion of the preneoplastic lesion atypical alveolar hyperplasia. Adenocarcinoma shares similar molecular abnormalities with squamous cell carcinoma, except for the high frequency of Ki-Ras mutations that are not or extremely rarely found in other long tumor types. The frequency of Ki-Ras mutation is much lower in nonsmokers (5%) than in smokers (30%). The restricted number of potential mutants at Ras condon makes Ras mutation a candidate for molecular early detection of adenocarcinoma from exfoliated cells. There is no reliable change of protein expression related to Ras mutation.

p53 Mutations Mutations of TP53 tumor suppressor gene occurs in about 50% of NSCLC and more than 70% of SCLC. TP53 mutations are the most extensively studied mutation in lung cancer. The database maintained at the International Agency for Research on Cancer (IARC) is a valuable resource to study the role of the gene in lung tumorigenesis (Hainaut et al., 2001). Previous reports demonstrated that TP53 mutational spectra of lung cancer was unique from other cancers. In particular, an excess of G:C to T:A transversions was characteristic of lung cancers and was related to smoke exposure. In those who never smoked, there is a reciprocal increase in G:C to A:T transitions.

2 Histopathological Classification of Lung Tumors Squamous cell carcinoma showed the highest frequency of p53 mutations (about 70%) among all histological types of lung carcinoma. p53 is a tumor suppressor gene with functions in G1 arrest and apoptosis in response to cytotoxic stress (stress includes DNA damage and several genetic abnormalities in the gene sequence) that stabilizes P53 and allows immunohistochemical detection. P53 immunoreactivity is fairly correlated with TP53 missense mutation. However, other types of TP53 mutation accounting for a maximum of 20% lead to an absence of functional P53 protein and are thus called null phenotype mutations, not recognized by immunohistochemistry. P53 protein overexpression and, less commonly, mutations may precede invasion. The preinvasive lesions of squamous cell carcinoma, dysplasia, and carcinoma in situ display P53 accumulation and immunoreactivity in an increasing proportion of mild dysplasia to CIS (20–60%).

Rb Gene Inactivation Inactivation of the Rb pathway is frequent in NSCLC, but mechanisms of Rb functions inactivation is different from that seen in high neuroendocrine tumors. Although loss of Rb protein expression is detected in only 15% of NSCLC, RB is frequently inactivated through deregulation of its phosphorylation pathway. Both CdK inhibitor inactivation (p16INK4) and cyclin D1 overexpression contribute to this indirect Rb inactivation and leading to overphosphorylation and loss of Rb function on G1 arrest. Immunohistochemistry is a straightforward method of detecting p16 INK4 inactivation and cyclin D1 overexpression (both occur in 50% of NSCLC). There is a constant inverse relation between loss of Rb, loss of P16, and overexpression of cyclin D1 consistent with P16 and cyclin D1 being exclusively devoted to Rb phosphorylation pathway. The tumor suppressor genes p53 and Rb are frequently mutated in high-grade neuroendocrine lung tumors, including SCLC and LCNEC. In 50–80% of SCLC and LCNEC, p53 mutation and/or p53 protein accumulation and immunoreactivity are observed. Highgrade neuroendocrine tumors share a high frequency of Rb loss of protein expression (80–100%) detectable by immunohistochemistry, compared with nuclear staining of normal cells. p14ARF, a protein encoded at the p16-9p21 locus, is induced by oncogenic stress and exerts its effect through p53 stabilization and transcriptional activation. However, both p14ARF loss and TP53 mutation have been found in SCLC and LCNEC, enlighting p14ARF functions independent of p53. E2F1, which includes p14ARF is frequently overexpressed in high-grade neuroendocrine tumors.

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Aberrant Methylation Epigenetic silencing of the promoter regions of multiple genes are universal in cancers. The major mechanism studied is methylation, although histone deacetylation plays an important cooperative role and may, in fact, precede the onset of methylation. Most of the silenced genes are known or suspected tumor suppressor genes. The methylation profile varies with the tumor type. Carcinoids, SCLC, squamous cell carcinomas, and adenocarcinomas of the lung have unique profiles of aberrant methylation, and the methylation rates of APC, CDH13, and RARβ were significantly higher in adenocarcinomas than in squamous cell carcinomas (Toyooka et al., 2001).

Neuroendocrine Lung Tumors Phenotype The gradual increase of fractional allelic loss and molecular abnormalities for TP53, Rb, p14ARF, E2F1 along the spectrum of neuroendocrine lung tumors strongly supports the grading concept of typical carcinoid as low-grade, atypical carcinoid as intermediate grade, and LCNEC and SCLC as high-grade neuroendocrine lung tumors. However, a continuous spectrum is challenged. MEN1 gene mutation and loss of heterozygosity at the MEN1 gene locus 11q13 was recently demonstrated in 65% of sporadic atypical carcinoids and was not found in high-grade neuroendocrine tumors; TTF-1 is not expressed in carcinoids, whereas it is expressed in high-grade SCLC and LCNEC; gene expression profiling using cDNA microarrays recently compared profiles of carconoids and SCLC showed similarities between carcinoids and central nervous tumors, whereas SCLC has more closely inherited the phenotype of basal bronchial cells.

CONCLUSIONS Genetic and molecular abnormalities in lung cancer are the result of DNA damage, mostly reflecting tobacco carcinogenesis. Their full knowledge and validated tools for their detection are useful in designing new therapeutic strategies and establishing the risk of progression of any early stage into lung cancer. The spectrum of neuroendocrine tumors has not been described as such in the WHO classification in 1999, with the epidemiological, clinical, and behavior as well as prognostic and response to treatment being extremely different. Although they belong to a morphological and biological spectrum sharing neuroendocrine properties and features, their genetic and molecular profiles have more differences than similarities. Histologic subclassification of lung tumors is essentially based on light microscopy in order to achieve

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114 widest application throughout the world and assume comparability and consistency of data. However, techniques, including immunohistochemistry, electron microscopy, tissue culture, and molecular biology, might provide valuable information on carcinogenesis, histogenesis, and differentiation. It is highly recognized that immunohistochemistry or electron microscopy may detect differentiation, specifically regarding histological heterogeneity of lung cancer, that cannot be seen by routine light microscopy. However, these techniques are occasionally required for precise classification. An example of this is LCNEC and malignant mesothelioma that require appropriate immunohistochemical and/or electron microscopy findings to confirm the diagnosis.

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