Malignant pulmonary NET is a distinct subset of lung neoplasms [51
]. In the recently revised World Health Organization (WHO) classification of lung tumors, this category embraces typical carcinoid, atypical carcinoid, large cell neuroendocrine carcinoma (LCNEC), and SCLC [9
]. In this spectrum of neoplasms, typical carcinoid represents the lowest grade neoplasm, and SCLC and LCNEC represent the highest grade neoplasms. All of these tumors share, to varying degrees, certain histological, ultrastructural, immunohistochemical and molecular characteristics. Microscopically, neuroendocrine architectural characteristics include organoid nesting, palisading, a trabecular pattern and rosette-like structures. Mitoses, necrosis, and pleomorphism are present in varying degrees in this spectrum of neoplasms, as outlined in the WHO classification scheme. Cytomorphologic characteristics such as cell and nuclear size and shape, chromatin pattern, nucleoli, and amount of cytoplasm also vary among these neoplasms.
Neuroendocrine differentiation can be demonstrated by electron microscopy or IHC in virtually all typical and atypical carcinoid tumors, and in a smaller percentage of the higher grade neuroendocrine neoplasms. For this reason, IHC is helpful for confirming neuroendocrine differentiation, but has limited value for separating individual neuroendocrine tumors from each other. A wide range of immunohistochemical markers to detect neuroendocrine differentiation has been studied [52
]. Chromogranin, synaptophysin and neural cell adhesion molecule (NCAM)-CD56 are the most reliable and widely used, offering confident results with high sensitivity and specificity [44
]. Ultrastructurally, synaptophysin is present in microvesicles, whereas chromogranin is present in secretory granules [63
]. These differences suggest that chromogranin and synaptophysin may be complementary generic neuroendocrine markers. CD57 (Leu-7) is also a frequently used neuroendocrine marker. However, CD57 is not restricted in its distribution to neuroendocrine tumors, since its reactivity is also present in non-neuroendocrine tumors including prostate carcinomas, thymomas, and a variety of small round blue cell tumors [64
]. Therefore, the use of CD57 antibody alone is unreliable for specific identification of neuroendocrine tumors. Neuron-specific enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate in the glycolytic pathway, and was widely used in earlier times to identify neuroendocrine differentiation. Because of its broad reactivity in non-neuroendocrine neoplasms, this antibody has been replaced by more specific neuroendocrine markers that are now available. Using TMA analysis [7
], Nitadori and coauthers systematically studied 48 antibodies and the phenotypic differences between LCNEC and SCLC. They found four proteins were significantly over-expressed in LCNEC as compared to SCLC: CK7, 113 vs. 49 (p<0.03); CK18, 171 vs. 120 (p<0.001); E-cadherin, 77 vs. 9 (p<0.001); and beta catenin, 191 vs. 120 (p<0.03). These antibodies may be useful in the routine differential diagnosis between LCNEC and SCLC, but further studies are needed.
Immunohistochemical staining for neuroendocrine substances is also not restricted to neuroendocrine neoplasms. Lung neoplasms that are not classified by histological criteria as neuroendocrine neoplasms may express neuroendocrine markers. Up to 20% of NSCLCs that do not show neuroendocrine morphology by light microscopy demonstrate immunohistochemical and/or ultrastructural evidence of neuroendocrine differentiation [65
]. These tumors are collectively referred to as “NSCLC with neuroendocrine differentiation” (NSCLC-ND). If histological features of a more specific histological type of NSCLC are seen (i.e., squamous differentiation), then the neoplasm should be classified according to its specific histological features (i.e., squamous cell carcinoma) and a comment made regarding neuroendocrine differentiation.
Currently, the diagnosis of LCNEC requires confirmation of neuroendocrine differentiation by either IHC or electron microscopy, in a large cell carcinoma with neuroendocrine architectural characteristics. If the tumor is a large cell carcinoma with neuroendocrine architectural characteristics but no neuroendocrine staining, the term “large cell carcinoma with neuroendocrine architecture” has been used. If the tumor is a large cell carcinoma with neuroendocrine staining but without neuroendocrine architectural features, then it can be classified as a “large cell carcinoma with neuroendocrine differentiation”.
Basaloid carcinoma can occasionally be difficult to distinguish from SCLC and LCNEC, and IHC can offer assistance [48
]. Basaloid carcinoma typically displays a solid, nested or trabecular growth pattern, nuclear palisading at the periphery of the neoplastic lobules, rosette-like structures in one-third of the cases, and commonly comedo-type necrosis. However, in contrast to LCNEC, neoplastic cells are characterized by hyperchromatic nuclei, increased nuclear to cytoplasmic ratio and inconspicuous nucleoli. Basaloid carcinomas usually express low and high molecular weight keratins and only about 10% of basaloid carcinomas focally express a neuroendocrine marker, with only 5% to 20% of tumor cells immunopositive in these cases [49
]. TTF-1 is negative.
Sturm et al
have recently shown the power of a specific cytokeratin antibody CK34βE12 to discriminate basaloid carcinoma from LCNEC [54
]. Monoclonal antibody 34βE12 (CK34βE12) recognizes a subset of high molecular weight cytokeratins identified as 1, 5, 10, and 14 in Moll's catalog [68
]. It is expressed in basaloid carcinomas, but not in LCNECs [54
]. In tumor pathology, Morice and Ferreiro first retrospectively investigated the expression of CK34βE12 in upper aerodigestive tract tumors. 22 of 23 basaloid squamous cell carcinomas showed strong positivity for CK34βE12, whereas none of the 10 small cell carcinomas showed staining [69
]. Similar results were subsequently reported in lung cancers. Lyda and Weiss showed that CK34βE12 was immunoreactive in 97% of squamous cell carcinomas, but only in 1 of 37 (3%) SCLCs and focally positive in 1 of 6 (17%) LCNECs [44
]. Viberti et al
investigated the expression of CK34βE12 in cytological specimens and transbronchial biopsies [70
]. In their study, CK34βE12 was negative in 93% (40/43) of SCLCs and in all six other neuroendocrine tumors (2 typical carcinoids, 2 atypical carcinoids and 2 LCNECs), while strong positivity was observed in virtually all non-neuroendocrine tumors: squamous cell carcinomas (15 of 16), adenocarcinomas (10 of 10) and basaloid carcinoma (4 of 4). All these findings suggest that expression of CK34βE12 is largely restricted to non-neuroendocrine pulmonary carcinomas.
Sturm and colleagues' recent study also confirmed the lack of staining for CK34βE12 in most neuroendocrine lesions [54
]. In their large cohort of neuroendocrine lesions, all cases of neuroendocrine cell hyperplasia (n=15), tumorlet (n=23), typical (n=27) and atypical carcinoid (n=23), as well as some LCNECs were completely negative for CK34βE12. While the majority of high grade NETs lacked CK34βE12 immunoreactivity, combined carcinomas and a proportion of histologically pure cases demonstrated focal immunostaining. The lack of CK34βE12 staining in SCLCs reported by Zhang et al
provided further evidence of association between neuroendocrine differentiation and negative IHC staining for CK34βE12 [36
Therefore, CK34βE12 should be included in the routine diagnostic panel of antibodies in the differential diagnosis of lung cancers, particularly when the differential diagnosis involves both neuroendocrine and non-neuroendocrine neoplasms. This antibody may also be useful in resolving questions of basaloid carcinoma versus SCLC, SCLC versus poorly differentiated squamous cell carcinoma, LCNEC versus other poorly differentiated NSCLCs, and carcinoid versus NSCLC excluding LCNEC.