Adenocarcinoma

Adenocarcinomas are tumors that produce malignant-appearing glands with tubular, acinar, or papillary differentiation and whose cells may demonstrate mucin production and secretion (Box 65-1). On gross examination, the tumors may manifest peripherally with pleural retraction (puckering), as central or endobronchial masses, as diffuse pleural involvement resembling a malignant mesothelioma, arising or associated with a scar, or as a diffuse parenchymal pneumonic-like tumor. The histologic pattern and organization may show a well-differentiated acinar pattern with intracytoplasmic vacuoles or more poorly differentiated features with solid malignant growth pattern and minimal mucin expression, as demonstrated by histochemical and/or immunohistochemical staining assays. In some cases, the intracytoplasmic vacuole distends the cytoplasm and compressively deforms the nucleus to the cell margin, forming a “signet-ring” cell. The tumors may arise at various levels of the airway with malignant changes in respiratory, bronchiolar, and alveolar epithelium. Malignant transformation of these various epithelia may be associated with protean histologic and cytologic characteristics and growth patterns of invasion.

The nature of tumor growth and progression depends on whether the cell of origin is a bronchial gland, ciliated columnar cell, goblet cell, nonciliated bronchiolar cell, or type II pneumocyte. The histologic growth pattern may show a mixture of various types including acinar, papillary, solid, and lepidic (growth along alveolar interstitium), and the cytologic features may include mucinous and nonmucinous differentiation (Figures 65-2 and 65-3). The grading of adenocarcinomas is based on the histopathologic assessment of the extent of glandular differentiation and architectural pattern, cytologic expression and pleomorphism, nuclear characteristics and stratification, and mitotic activity and tumor necrosis. Most adenocarcinomas tend to be of mixed pattern, are intermediate in differentiation or moderately differentiated, and show a predominant acinar-type glandular arrangement. As expected, predominantly solid adenocarcinomas tend to demonstrate high-grade, poorly differentiated cytologic features with prominent mitotic activity and tumor necrosis. Adenocarcinomas may have clear cell features resulting from the accumulation of intracellular glycogen or mucins, and such tumors must be differentiated from metastatic clear cell renal carcinomas; the presence of intracytoplasmic lipid and glycogen is diagnostic of renal tumors of the clear cell type.

Figure 65-2 Adenocarcinoma, acinar type. A, Adenocarcinoma with acinar growth pattern. (Hematoxylin-eosin stain [H&E], 10×.) B, Cytologic preparation showing intracytoplasmic vacuoles. (Diff-Quik stain, 40×.) C, Adenocarcinoma with tubuloacinar growth pattern. (H&E, 10×.) D, Cytologic preparation showing sheetlike growth and intracytoplasmic vacuoles. (Diff-Quik stain, 40×.)

Figure 65-3 Adenocarcinoma, papillary and lepidic types. A, A papillary growth pattern is evident on this preparation. (Hematoxylin-eosin stain [H&E], 20×.) B, A micropapillary pattern can be seen. (H&E, 20×.) C, Mucinous adenocarcinoma with lepidic growth pattern. (H&E, 4×.)

Histopathologic characteristics that correlate with a relatively poor prognosis in adenocarcinomas of the lung include a predominant solid pattern and high-grade cytologic features with mitotic figures and tumor necrosis, a micropapillary relative to a papillary histologic pattern, extension to and involvement of the visceral pleura, extensive lymphovascular tumor embolic spread, involvement of bronchial and hilar lymph nodes, and tumor-associated secondary obstructive lobar pneumonia. In addition, resection specimens that show intralobar satellite (metastatic) nodules and involvement of the surgical resection margins are associated with disease recurrence.

The histologic subtypes of adenocarcinoma appear to correlate with a history of cigarette smoking. Relative to nonsmokers, smokers more often have adenocarcinoma of the solid histologic type, and the extent of the solid component within the adenocarcinoma shows a dose-response relationship with pack-years of smoking. The solid subtype of adenocarcinoma also demonstrates a higher frequency of lymphovascular invasion and pleural involvement, and these histopathologic features are predictive for recurrence in stage I adenocarcinoma.

Historically, the most common cases of pulmonary adenocarcinoma were of the mixed type, with several histologic subtypes, and often the tumor had a so-called bronchioloalveolar carcinoma (BAC) growth margin. As originally defined, BAC is characterized by a lepidic growth pattern with atypical tumor cells (nonmucinous serous cells or mucinous cells) proliferating along the interstitial framework and not demonstrating stromal invasion or lymphovascular invasion. Tumors that manifest a BAC pattern often exhibit “ground glass” features on radiographs, and mixed-type invasive adenocarcinomas have central solid density with parenchymal distortion.

As a result of improper utilization and ambiguity of these pathologic terms, a group of international societies proposed a new classification that incorporated the histopathologic pattern, the radiographic features, and the clinical presentation and outcome. On the basis of the accumulated clinical experience with cases in which tumors 3 cm or less in diameter, with a pure lepidic growth pattern, were completely excised, followed by excellent survival, the term adenocarcinoma in situ (AIS) was adopted. These preinvasive lesions were judged to be extensions of a precursor lesion, termed atypical adenomatous hyperplasia (AAH), which also showed a lepidic growth pattern but were smaller in size (up to 5 mm in diameter). Those tumors that demonstrated an overall AIS pattern but also contained central minimal stromal invasion (less or equal to 5 mm) were designated minimally invasive adenocarcinoma (MIA). These latter cancers usually are nonmucinous and are associated with an excellent clinical outcome. In order to render a diagnosis of AIS or MIA, the entire lesion must be excised and processed for microscopic evaluation. These tumor types are excluded when histologic features of lymphovascular invasion, pleural involvement, and tumor necrosis are identified. It is recommended that the designation BAC be abandoned and that in its place, the proper histologic classification be applied, which may be AIS, MIA, adenocarcinoma with a predominant or mixed lepidic pattern, or mucinous adenocarcinoma. Tumors that were historically diagnosed as “nonmucinous BAC” may now occur as AIS, MIA, or lepidic-dominant adenocarcinomas. Many of these cases tend to be found in women, with immunohistochemical profiles of CK7 and thyroid transcription factor-1 (TTF-1) immunoreactivity and frequent epidermal growth factor receptor (EGFR) mutational status. By contrast, the historical designation of “mucinous BAC” is recommended to be changed to invasive mucinous adenocarcinoma, with immunohistochemical profiles of CK20 immunoreactivity and nonreactivity for TTF-1. These tumors frequently are associated with KRAS mutation and no EGFR mutation.

Invasive adenocarcinomas, representing a considerable majority (greater than 75% of cases), are those that exhibit significant stromal invasion and possible lymphovascular invasion and infiltration of the visceral pleura. These cancers need to be measured, because the size of the invasive component governs the tumor-node-metastasis (TNM) staging and the potential for tumor dissemination. The classification also recommends that the predominant pattern of the adenocarcinoma be designated and that the term “mixed pattern” be abandoned. The assessment of the components of the adenocarcinoma not only is important for the proper classification but also is useful for the correct identification of multiple pulmonary nodules as either multiple primary tumors or T3 or T4 tumors. Obviously, tumors that differ in histologic pattern and those that have an in situ component represent multiple primary tumors. It also is possible to differentiate whether the separate nodules are multiple T1 tumors by determining the relative distribution of the adenocarcinoma subtypes. For example, two nodules of adenocarcinoma may be regarded as separate T1 tumors if one is predominantly of the acinar subtype while the other nodule lacks the acinar pattern and is composed of predominant solid variant.

The consensus classification also addressed the recommended approach with small biopsy specimens (a common problem with fiberoptic biopsy samples) and cytologic preparations. The pathologist must make every attempt to avoid the diagnosis of “lung cancer not otherwise specified,” because the identification of a characteristic histologic pattern is important for tumor management. In a majority of cases, the distinction between adenocarcinoma and squamous cell carcinomas may be made using routine hematoxylin-eosin–stained (H&E) sections. In those cases of poorly differentiated carcinomas, mucin histochemical methods and a limited panel of immunohistochemical stains may achieve a more definitive result. Adenocarcinomas will show luminal or intracytoplasmic mucin positivity and TTF-1 immunoreactivity. Squamous cell carcinomas will lack mucin staining and will demonstrate reactivity for p63 or CK5/6 but not TTF-1.

Squamous Cell Carcinoma

Squamous cell carcinoma is a malignant tumor arising from bronchial epithelium with a strong etiologic association with cigarette smoking, in contrast with adenocarcinoma. It most commonly is central in location and arises from the main, lobar, or segmental bronchi. Squamous cell carcinomas usually appear as white or gray masses with a variable degree of fibrosis, frequently associated with central necrosis and cavitations. Histologic hallmarks of squamous origin, namely keratinization and intercellular bridges, are seen in relation to the degree of the differentiation; although readily appreciated in well-differentiated tumors, they may be only focally present or even absent in poorly differentiated ones (Figure 65-4). In selected cases, when squamous nature cannot be ascertained by routine light microscopy, immunohistochemistry studies are invaluable. Squamous cell carcinomas usually are immunoreactive for high-molecular-weight keratin, CK5/6, and p63 and generally are nonreactive for TTF-1, CK7, and neuroendocrine markers (CD56, synaptophysin, chromogranin). Although not widely used, electron microscopy also can highlight features of squamous differentiation, such as prominent desmosomes and cytoplasmic aggregates of intermediate keratin filaments.

Figure 65-4 Squamous cell carcinoma. A, In this preparation, note the keratin pearl. (Hematoxylin-eosin stain, 20×.) B, Cytologic preparation showing cytoplasmic keratinization. (Papanicolaou smear, 100×.)

Recognizing histologic variants of squamous cell carcinoma, including papillary, clear cell, small cell, and basaloid types, is useful mainly in excluding the various differential diagnostic considerations. Their behavior is primarily a reflection of the degree of differentiation, with basaloid and small cell variants portending a worse outcome.

Pattern of spread is somewhat different depending on the anatomic location. Central tumors either grow along the epithelium, with or without invasion into subepithelial tissue, or expand as an endobronchial polypoid growth. In advanced cases, these tumors may directly involve hilar mediastinal tissue, including lymph nodes. Peripheral tumors, on the other hand, form a solid nodule and may directly break through the pleura into the chest wall or diaphragm.

Staging is governed by the TNM system. Regardless of the location, squamous cell carcinomas are more likely to be locally aggressive and invade adjacent structures. Distant metastasis, albeit far less common than with other subtypes of lung cancer, has a predilection for brain, liver, adrenals, lower gastrointestinal tract, and distant lymph nodes. In general, squamous cell carcinomas are associated with better prognosis and survival rate, as compared with stage-matched adenocarcinomas.

Small Cell Carcinoma and Neuroendocrine (Carcinoid) Tumors

Neuroendocrine tumors, those that contain dense core neurosecretory granules composed of serotonin, histamine, and other bioactive amines, include small cell and large cell neuroendocrine carcinomas and typical and atypical carcinoid tumors. As a group, these tumors represent about one fifth of primary lung neoplasms, with small cell carcinoma being the most common in this category. These tumor types share a histologic pattern of uniform to pleomorphic cells in an organoid arrangement of tumor nests and trabecula (Figure 65-5). Carcinoid tumors may be considered as well-differentiated neuroendocrine carcinomas with little to no mitotic activity (less than 2 mitotic figures per 2-mm² microscopic field) or tumor necrosis. Carcinoid tumors may occur in nonsmokers, in whom they manifest as a vascular rich endobronchial mass, although some carcinoid tumors may have a peripheral localization and have spindle cellular features. The tumors are low-grade malignancies with approximately 15% nodal metastases at diagnosis; nevertheless, affected patients have a greater than 90% 5-year survival. The atypical carcinoid tumors are neuroendocrine carcinomas of intermediate grade that have an organoid arrangement with more irregular and atypical cytologic pattern, identifiable tumor necrosis, and countable mitotic activity (2 to 10 mitoses per 2-mm² microscopic field). In contrast with typical carcinoid tumors, atypical carcinoid may have nearly 50% lymph node metastases on presentation and a lower 5-year survival rate.

Figure 65-5 Carcinoid tumor. A, Note the nesting arrangement of cells. (Hematoxylin-eosin stain, 40×.) B, Cytologic preparation showing a uniform cellular pattern. (Diff-Quik stain, 40×.)

The high-grade poorly differentiated neuroendocrine tumors consist of the small cell (neuroendocrine) carcinoma and the large cell neuroendocrine carcinoma. The latter two have, respectively, smaller and larger cells with considerable cytologic atypia, prominent mitotic activity (more than 10 mitotic figures per 2-mm² microscopic field), and abundant tumor necrosis. All of the neuroendocrine tumors will be immunohistochemically reactive with broad-spectrum cytokeratins, CD56, and, specifically, immunoreactive for neuroendocrine markers, such as chromogranin and synaptophysin. The high-grade neuroendocrine carcinomas are immunoreactive for TTF-1, more so than are the intermediate-grade and well-differentiated types.

Close attention to the morphologic details in conjunction with ancillary studies is important to differentiate small cell carcinoma not only from non–small cell carcinoma but also from other neuroendocrine tumors of lung, because they differ substantially in their demographics, clinical presentation, and behavior. Small cell carcinoma of lung generally is a disease of elderly men (mean age, 65 years) who are almost always current or former heavy smokers. These demographic findings are in sharp contrast with those in patients with typical carcinoid, who present at a younger age (mean, 45 to 50 years), with no difference in regard to gender or smoking history. The disparity also extends to associated prognosis and clinical behavior. Whereas typical carcinoid tumors are known for their indolent behavior and excellent survival rates, small cell carcinomas, at the other end of the spectrum, are fatal, with many patients presenting with distant metastasis at the time of diagnosis. Small cell carcinomas typically are central in location and frequently appear radiographically as hilar or perihilar masses with mediastinal lymphadenopathy and lobar collapse. Extensive necrosis is almost always present.

The key histopathologic features in the diagnosis of small cell carcinoma are nuclear morphologic features, such as finely granular chromatin, absence of prominent nucleoli, fragility, molding, and extensive crush artifact, as well as indistinct cytoplasmic borders and high mitotic figure count (more than 10 per 10 high-power fields) (Figure 65-6). Large zones of geographic necrosis also are common. Classically, a great deal of emphasis was placed on the small cell size or nuclear size, as suggested by the nomenclature. The usefulness of this criterion is questionable, however, in view of the wide variability in size, with frequent overlap with large cell neuroendocrine carcinomas. Large cell neuroendocrine carcinomas show a low-power neuroendocrine architecture, as seen in carcinoid tumors; however, the cell morphology closely resembles that of non–small cell carcinomas, with vesicular chromatin, prominent nucleoli, and abundant cytoplasm—features completely different from those of small cell carcinoma (Figure 65-7). Additionally, these tumors are highly mitotic (more than 10 mitotic figures per 10 high-power fields) and often show extensive necrosis, similar to that in small cell carcinomas, and in keeping with their high-grade nature.

Figure 65-6 Small cell carcinoma. A, In this preparation, note the partial nesting pattern and tumor necrosis. (Hematoxylin-eosin stain [H&E], 20×.) B, Nuclear molding and mitotic activity are evident in this preparation. (H&E, 40×.) C, Cytologic preparation showing inconspicuous cytoplasm and nuclear fragmentation. (Diff-Quik stain, 20×.)

Figure 65-7 Large cell carcinoma. A, In this preparation, a high-grade pattern with prominent nucleoli is evident. (Hematoxylin-eosin stain, 40×.) B, On this specimen from a large cell neuroendocrine carcinoma, note the trabecular and nesting pattern of a neuroendocrine tumor. (Hematoxylin-eosin stain, 20×.)

Positive immunoreactivity with at least one neuroendocrine marker (synaptophysin, chromogranin) is required for the diagnosis of small cell carcinoma. By contrast, the main role of immunohistochemistry studies in small cell carcinoma is to exclude a wide range of other high-grade round blue cell tumors in the differential diagnosis. Cytokeratin immunoreactivity, especially the characteristic dotlike positivity, aids in excluding a lymphoid process, reactive or neoplastic, in instances of considerable morphologic overlap, particularly in small and poorly preserved samples. However, strong membranous staining with cytokeratins and diffuse staining with 34βE12 are characteristic of non–small cell carcinomas and generally are not seen in small cell carcinoma. The neuroendocrine markers synaptophysin and chromogranin, although strongly expressed in carcinoid, may be very focal in location or entirely absent in small cell carcinoma, and in such cases, CD56 is a more sensitive indicator of neuroendocrine differentiation. TTF-1, a very useful marker in establishing lung origin in adenocarcinoma, is seen in an overwhelming majority of small cell lung carcinomas, but it also may be expressed by extrapulmonary small cell carcinomas such as bladder and prostate cancers and therefore cannot be used to establish the site of origin. Although Ki-67 (MIB1) proliferation index is not an integral part of the current WHO classification of lung neuroendocrine tumors, it is of value, especially in the evaluation of small biopsy samples and cytologic specimens, when tissue is limited and poor preservation or extensive crush artifact during procurement may be inevitable. It has been suggested that Ki-67 (MIB1) percent positivity may be used as a diagnostic adjunct to morphologic examination: for typical carcinoid, less than 2%; for atypical carcinoid, less than 20%; and significantly, greater than 20% for small cell and large cell neuroendocrine carcinomas.

Traditionally, small cell carcinomas have been divided into limited-stage disease (treatable within a single radiation portal) and extensive-stage disease (contralateral or distant metastasis) with regard to prognosis. Recent large-scale analyses have shown the value of TNM staging for all neuroendocrine tumors of the lung, including carcinoid tumors.

Large Cell Carcinoma

By definition, large cell carcinoma of lung is an undifferentiated carcinoma lacking morphologic evidence of small cell carcinoma, squamous cell carcinoma, or adenocarcinoma. In other words, it represents a diagnosis of exclusion. These tumors typically are composed of sheets and nests of large polygonal cells with moderate amount of cytoplasm, vesicular chromatin and prominent nucleoli (Figure 65-7). Although the classification is purely based on morphologic grounds, judicious use of ancillary studies (as discussed previously), especially with small biopsy specimens, is of significant value in separating the poorly differentiated squamous cell carcinomas and adenocarcinomas from the truly undifferentiated large cell carcinoma. These tumors share similar demographic and clinical features with other non–small cell carcinomas of the lung, because they commonly are seen in elderly men with smoking history.

Large cell carcinomas are preferentially seen at the periphery of the lung. Several histologic subtypes are recognized: basaloid, clear cell, and lymphoepithelioma-like carcinomas. Although grouped under large cell carcinomas in the 2004 WHO classification, large cell neuroendocrine carcinoma, as strictly defined in the previous section, is best categorized as a neuroendocrine tumor, closely resembling small cell carcinoma. Nevertheless, the mere focal expression of neuroendocrine markers as detected on immunohistochemistry studies, seen in some large cell carcinomas, should not be interpreted as diagnostic for large cell neuroendocrine carcinoma. In general, large cell carcinomas are high-grade tumors that are similar in prognosis and pattern of spread to stage-matched non–small cell carcinomas, with performance status at the time of diagnosis and disease extension considered to be the most important clinical prognostic parameters. Metastases arise most commonly in the hilar and mediastinal nodes, followed by pleura, liver, bone, brain, abdominal nodes, and pericardium. The basaloid subtype carries a worse prognosis, even in the lower stages, with more common metastasis to the brain. On the other hand, lymphoepithelioma-like carcinomas generally are diagnosed at a later stage of disease but are associated with a better prognosis.

Pleural Malignancies

Diffuse malignant mesothelioma is a pleura-based cancer that may be histopathologically composed of a biphasic growth pattern of combined epithelial and sarcomatoid components. The tumor also may manifest in a monophasic form of either epithelioid cancer resembling a pleural adenocarcinoma or a spindle sarcomatoid form resembling a mesenchymal sarcoma. The diagnosis is usually but not exclusively rendered with a history of asbestos exposure, similar to that for pleural asbestosis or asbestos-related diseases and malignancies, but with a lower threshold of lung asbestos burden. Other proposed causes of diffuse malignant mesothelioma are chest radiation therapy and exposure to other mineral silicates, such as vermiculite and erionite. Erionite originally was identified in Turkey but is now appreciated to have widespread deposits in the western United States (i.e., Dakotas, Nevada, Arizona, and California). The diagnosis is based on appropriate clinical, radiographic, and histopathologic findings of a painful pleura-based tumor with scattered multiple nodules or a coalescent tumor encasing the lung. Malignant mesothelioma may obliterate the pleural cavity or be associated with an exudative pleural effusion. The tumor may invade the underlying lung and typically infiltrates through the diaphragm.

Malignant mesothelioma exhibits characteristic surgical pathology features that must be distinguished from reactive mesothelial proliferation and chronic fibrosing pleuritis. Reactive mesothelial hyperplasia frequently is present in the setting of collagen vascular diseases, drug or inflammatory reactions, and trauma or surgery. Of importance, reactive mesothelial hyperplasia lacks invasion of the stromal connective tissue and fat, does not undergo necrosis, and typically is not immunoreactive for EMA (epithelial membrane antigen) and p53 (tumor suppressor gene product). In the setting of exposure to asbestos, collagenous pleural plaques may be identified and associated with either reactive mesothelial hyperplasia or an incipient diffuse malignant mesothelioma. Separation of chronic pleuritis from malignant mesothelioma also is based on the lack of mesothelial invasion in pleuritis and the presence of an organizing zonal layered fibroinflammatory process. Desmoplastic malignant mesothelioma will, on the other hand, show stromal invasion, haphazard growth pattern, and metastatic spread. Reactive mesothelial cells and those of malignant mesothelioma are immunoreactive for cytokeratin. The histopathologic appearance of reactive mesothelial conditions is organized and layered, in contrast to the haphazard cellularity and invasion in malignant mesothelioma.

The histologic pattern of the epithelioid type of diffuse malignant mesothelioma appears as an invasive tubular and papillary malignancy that mimics a pleura-based adenocarcinoma or a metastatic adenocarcinoma. The monophasic sarcomatoid mesotheliomas resemble spindle cell sarcomas, which may manifest as primary or metastatic cancers. The most common form of malignant mesothelioma is the epithelioid variant and may show a variety of histologic features, such as tubulopapillary, glandular, or solid patterns. Cytologic examination of malignant pleural effusions shows irregular clusters of atypical mesothelial-like cells in pools of hyaluronic acid. Malignant mesotheliomas also may present in the peritoneal cavity and mimic ovarian cancers in women. Biphasic mesotheliomas are less common in the abdomen than in the pleura, and abdominal sarcomatoid mesotheliomas are rare. The distinction between malignant mesothelioma and adenocarcinoma in the pleura and peritoneal cavity is based on histologic features and supported by histochemical and immunohistochemical staining and ultrastructural findings. Mesotheliomas will be positive for Alcian blue, a stain that disappears with pretreatment with hyaluronidase. Most adenocarcinomas are histochemically positive for mucicarmine stains, although a few mesotheliomas also may stain positive. On immunohistochemical assays, malignant mesotheliomas will be immunoreactive for calretinin, keratin 5/6, WT-1 protein, and podoplanin (D2-40), in contrast with adenocarcinomas, which are immunoreactive for CEA (carcinoembryonic antigen), MOC-31, B72.3, Ber-EP4, and TTF-1. Both mesotheliomas and adenocarcinomas will be positive on assay for pancytokeratins, which will demonstrate the viability of the tissue for immunohistochemical assessments. Historically, the best diagnostic discriminator for distinguishing malignant mesothelioma from adenocarcinoma has been electron microscopic examination of the tumor. The ultrastructural appearance of malignant mesothelioma on transmission electron microscopy shows long slender microvillous processes with frequent branching and lacking a glycocalyx, in contrast with short blunt straight microvilli of adenocarcinoma, containing a glycocalyx.

Epidermal Growth Factor Receptor Gene

EGFR is a transmembrane signaling protein that transduces extracellular signals, due to binding of EGFR ligands, into intracellular changes mediated by activating tyrosine kinase phosphorylations. EGFR, also called HER1 (human epidermal growth factor receptor 1), is in the same family as HER2, another membrane receptor that is amplified in approximately 20% of breast cancers. In breast cancer, HER2 is biologically associated with a relatively aggressive type of breast cancer, but it also is a therapeutic target for directed antibodies or small molecule tyrosine kinase inhibitors (TKIs). When EGFR is mutated, predominantly in nonsmokers, it adopts an activated state, as if constantly bound to activating ligands or growth factors. EGFR activation induces downstream signals that inhibit apoptosis and stimulate cell proliferation and migration and motility. Targeted TKIs are effective for tumor regression when non–small cell carcinomas have activating EGFR mutations, as opposed to tumors with wild-type EGFR. EGFR-activating mutations tend to be present in a minority of lung cancers (10% to 20%) and appear most commonly in nonmucinous adenocarcinomas exhibiting a well-differentiated acinar or papillary growth pattern with a lepidic growth margin and presenting demographically in younger Asian women who were never-smokers or light smokers. In this last demographic subgroup, the incidence of EGFR mutation may rise to 40% to 50%. Large cell carcinomas also have shown EGFR mutation; however, the rate of such mutations in squamous cell carcinoma is only a few percent, and this low frequency is not sufficient for routine analysis. Immunohistochemical identification of EGFR in tumor tissue has no correlation with clinical tumor response to TKIs. The EGFR mutation is the molecular marker, more significant than EGFR amplification, for targeted therapy and is a predictive factor for a therapeutic response. Fluorescence in situ hybridization (FISH) assays have been attempted to identify EGFR amplification and show some concordance with therapeutic response, because EGFR mutations correlate with amplification. Adenocarcinomas with nonmutated but amplified EGFR gene have been recognized, and these tumors show a partial response to tyrosine kinase inhibition.

The standard tests for EGFR mutations may be performed on formalin-fixed paraffin-embedded tissue and consist of direct DNA sequencing or mutation-specific assays investigating mutations that usually are found in exons 19 and 21. In instances of tumor recurrence due to resistance to TKIs, the sequencing analysis should be expanded to include exons 18 and 20. The acquired resistance tends to occur in about a year after TKI therapy is started and may be related to a new mutation in the active site; other causes for TKI resistance are due to MET gene amplification in 10% to 15% of cases. EGFR genetic testing shows concordant results with the presence of an activating mutation between the primary tumor and its metastasis in a majority of cases; however, an average discordance of 25%, as a result of tumor heterogeneity and progression, has been noted.

KRAS Oncogene

KRAS is an oncogene found on the short arm of chromosome 12 (12p) and shows mutations in approximately 20% of non–small cell lung cancers and amplification of the gene in a smaller set of cancers. The p21 protein (i.e., K-RAS), encoded by KRAS, couples with a cytoplasmic GTPase to convert guanosine triphosphate (GTP) to guanosine diphosphate (GDP) in order to modify transductive signals from the cytoplasm to the nucleus, thereby controlling cellular growth and survival pathways. When KRAS is mutated, GTP is maintained, and the signal is persistently in the “active” state. Activating mutations of KRAS are present in a variety of epithelial tumors, including colon and pancreatic cancers, and lead to activating phosphorylation of mitogen-activated protein kinase (MAPK), inducing nuclear signals of cellular proliferation and tumor progression. KRAS mutations and amplifications demonstrate clinicopathologic correlations with older age combined with male gender, history of heavy cigarette smoking, and advanced disease at presentation, with relatively aggressive tumors and poor clinical outcome. Histologically, KRAS mutations tend to be identified in poorly differentiated adenocarcinomas with solid growth pattern and lacking a lepidic growth pattern. Of interest, the mucinous lepidic pattern is associated with KRAS mutations. These high-grade adenocarcinomas also show lymphovascular invasion, mitotic activity, and tumor necrosis. K-RAS is downstream from EGFR effects and when activated is independent of EGFR status, thereby conveying resistance to tyrosine kinase inhibitors of EGFR function.

EML4/ALK

A new addition to molecular markers that predict a clinically favorable response to targeted therapy is the EML4–ALK translocation. The proper name of the fusion gene product is echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase. The short arm of chromosome 2 (2p) may contain multiple small inversions and or translocations whose permutations generate in-frame fusions of the 5′ portion of the EML4 gene with the 3′ end of the ALK gene, creating a chimeric tyrosine kinase with an intracellular kinase domain. Breakpoints of the EML4 gene occur at several possible exon sites, although the breakpoint of the ALK gene tends to be specific for the kinase domain at exon 20. This fusion activation of the ALK gene is infrequent (occurring in less than 10% of non–small cell lung cancers), but the incidence may be enriched in lung cancers present in younger persons without significant smoking history in whom EGFR mutations and KRAS mutations are lacking. EML4–ALK translocations are histologically correlated with high-grade adenocarcinomas exhibiting acinar and solid growth patterns that contain mucinous and signet ring differentiation. From early studies, it appears that tyrosine kinase–targeted therapy for EGFR mutations is not effective treatment for EML4–ALK mutations. EML4–ALK fusion also appears to be selective for adenocarcinomas, because it is lacking in the other types of bronchogenic carcinomas.

The test for the translocation is based on reverse transcriptase–polymerase chain reaction (RT-PCR) assay of tumor tissue RNA, which amplifies and detects certain of the fusion constructs. The assay limitation is due to the identification of only the translocations of EML4 exons 6 and 13 with ALK exon 20. The fusion gene also may be assayed by FISH techniques, and recent studies suggest that immunohistochemical methods also may yield accurate results.