Molecular alterations of neuroendocrine tumours of the lung

Neuroendocrine tumours of the lung comprise low [typical carcinoid (TC)], intermediate [atypical carcinoid (AC)] and high‐grade [small‐cell lung cancer (SCLC) and large‐cell neuroendocrine carcinoma (LCNEC)] malignancies, while a pre‐invasive lesion [diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH)] may generate a subset of peripheral carcinoid tumours. These neoplasms are differentiated conventionally based on mitotic rate, presence of necrosis and cytological details, according to the 2015 World Health Organisation (WHO) classification. Clinical data and molecular alterations distinguish carcinoids and high‐grade carcinomas into two separate categories. Previous studies have demonstrated a significantly higher rate of chromosomal aberrations in carcinomas (e.g. 3p and 17p deletions), but restriction of multiple endocrine neoplasia type 1 (MEN1) mutations to carcinoids. High‐grade carcinomas are also characterised by TP53 and RB1 gene inactivation. In this review, a critical analysis of the diagnostic and prognostic role of Ki67 labelling index and a concise discussion of the most relevant findings regarding molecular characterisation of lung neuroendocrine neoplasms are reported. In addition, we illustrate how the development of promising therapeutic strategies based on the identification of molecular targets (mTOR inhibitors in carcinoids and targeting of the Notch ligand DLL3 in SCLC) may require the assessment of predictive biomarkers, even in the group of neuroendocrine tumours of the lung.     

Immunohistochemical markers of small cell carcinoma and related neuroendocrine tumours of the lung

A selected group of 263 pulmonary neuroendocrine tumours comprised 156 small cell carcinomas, five combined cell carcinomas, nine atypical carcinoid/small cell carcinomas, 32 atypical carcinoids, ten large cell/small cell carcinomas, and 51 carcinoid tumours. These were compared with a group of 109 non-small cell carcinomas, using four markers of neuroendocrine differentiation to determine differences in reactivity between the two groups and among the variants of neuroendocrine tumour. The antibodies used were neuron-specific enolase (NSE), protein gene product (PGP) 9.5, human bombesin, and the C-terminal flanking peptide of human bombesin (CTP). Most small cell carcinomas, carcinoid tumours, and atypical carcinoid variants showed immunoreactivity for both NSE and PGP 9.5 but a significant number of non-small cell carcinomas, mainly squamous cell carcinomas, were also positive (11 and 35 per cent, respectively). Bombesin was specific for neuroendocrine tumours, being demonstrable in 35 per cent carcinoids and 24 per cent small cell carcinomas, but staining was focal and often confined to scattered cells. Diffuse strongly positive immunoreactivity for CTP was seen in the majority of malignant neuroendocrine tumours, but only 12 per cent of carcinoid tumours were positive and non-small cell carcinomas were negative. CTP is therefore of potential value as a specific marker of malignant neuroendocrine tumours, particularly if the amount of biopsy material is limited and the tumour is an unusual variant, such as atypical carcinoid or large cell-small cell carcinoma.     

LKB1 protein expression in neuroendocrine tumors of the lung

During a recent investigation of LKB1 gene abnormality in lung lesions, strong expression of LKB1 protein in normal neuroendocrine (NE) cells of the bronchial epithelium was found. Because LKB1 functions as a tumor suppressor gene, the question of whether alteration of LKB1 expression is related to the development of pulmonary NE tumors of various grades was investigated. LKB1 immunohistochemistry was examined in a total of 68 primary pulmonary NE tumors consisting of 30 specimens of small cell lung carcinoma (SCLC), 23 large cell neuroendocrine carcinomas (LCNEC), two atypical carcinoids, and 13 typical carcinoids. Loss or low expression (<20% immunoreactive cells) of LKB1 protein expression was more frequently observed in high-grade NE tumors (SCLC and LCNEC; 45/53, 84.9%) than in typical and atypical carcinoids (3/15; 20%). The difference in LKB1 immunoreactivity between the high-grade NE tumors and the carcinoid group was statistically significant ( P  < 0.0001). In conclusion, marked reduction of LKB1 expression in high-grade NE tumors of the lung suggests a possible role of LKB1 inactivation in its tumorigenesis. Although a few previous studies indicated rare genetic alterations of LKB1 in SCLC, further studies including analysis of other NE tumors and focusing on epigenetic abnormalities of LKB1 gene are warranted.     

Malignancy-associated X chromosome allelic losses in foregut endocrine neoplasms: further evidence from lung tumors.

Association of X chromosome allelic losses with tumor malignancy has been identified in foregut but not in midgut endocrine neoplasms. The aim of this study was to investigate the association of deletions on X chromosome with malignancy in lung neuroendocrine tumors, another family of foregut neoplasms comprising four categories with increased malignancy: typical and atypical carcinoids, large cell neuroendocrine and small cell lung carcinomas. To evaluate loss of heterozygosity, DNA extracted from nine typical carcinoids, 17 atypical carcinoids, six large cell neuroendocrine carcinomas and five small cell lung carcinomas was PCR-amplified for 18 microsatellite markers spanning the whole X chromosome. All tissue samples were formalin-fixed and paraffin-embedded. X chromosome losses were absent in typical carcinoids, whereas they were found in nine out of 17 atypical carcinoids and in five out of six large cell neuroendocrine carcinomas (involving 28 and 70% of informative loci, respectively). On the contrary, deletions on X chromosome were an extremely rare event in small cell lung carcinomas. In atypical carcinoids, the presence of losses was associated with larger tumor size, higher pT status and advanced stage. No death occurred in atypical carcinoid patients without deletions on X chromosome, whereas all atypical carcinoid patients who had died from disease showed allelic losses. In conclusion, X chromosome allelic losses, absent in benign 'typical' carcinoids, progressively increased in frequency from intermediate-grade 'atypical' carcinoids to high-grade large cell neuroendocrine carcinomas. These results extend the association of deletions on X chromosome with malignancy, already demonstrated in other foregut endocrine neoplasms, to lung neuroendocrine tumors. The absence of X chromosome allelic losses in small cell lung carcinomas underlines a striking difference from large cell neuroendocrine carcinomas, possibly linked to different pathogenetic mechanisms of these two highly aggressive neuroendocrine lung tumors.     

Abnormal p53 expression in lung neuroendocrine tumors. Diagnostic and prognostic implications.

The diagnostic and prognostic implications of p53 immunostaining have been investigated in 59 pulmonary neuroendocrine tumors, including typical carcinoids (n = 15), so-called "atypical carcinoids" (n = 22), and small cell lung carcinomas (SCLCs; n = 22). Immunocytochemistry was performed on formalin-fixed, paraffin-embedded samples using the monoclonal antibody PAb1801, which has been shown to be suitable for staining fixed and embedded tissue sections. p53 immunoreactivity was restricted to atypical carcinoids (45% of the cases being immunoreactive) and to SCLCs (which were positively stained in 59% of the cases), whereas it was consistently lacking in typical carcinoid tumors. When the group of the so-called "atypical carcinoids" was further reclassified, p53 immunostaining was strictly confined to those cases belonging to the histologically more aggressive subsets (well differentiated neuroendocrine carcinoma subsets II and III). Within the same tumor type, however, p53 immunoreactivity did not correlate with the clinical outcome of the disease and was not predictive of the length of survival. The data indicate that abnormal p53 expression (which is strictly dependent on structural abnormalities of the p53 gene) is detectable in the majority of neuroendocrine carcinomas of the lung and might represent a useful adjunct in the differential diagnosis of pulmonary neuroendocrine neoplasms, particularly in routinely fixed and embedded small bronchoscopic biopsies.     

Quantitative expansion of structural genomic alterations in the spectrum of neuroendocrine lung carcinomas

The pathogenesis and interrelationships of neuroendocrine lung carcinomas are not well understood. Tissue macro-arrays prepared from surgical resection specimens from 35 patients with typical carcinoid (TC), six with atypical carcinoid (AC), 13 with large cell neuroendocrine carcinoma (LCNEC), and 15 with small cell lung carcinoma (SCLC) were investigated by fluorescence in situ hybridization (FISH) and immunohistochemistry. Hybridizations with locus-specific DNA probes demonstrated a high incidence of deletion for the tumour suppressor genes p53 and retinoblastoma (Rb), and for the oncogene cyclin D1, comparable in all carcinoma types. Similarly, an increase of DNA copy number for the Her-2/neu and c-myc oncogenes was noted in all neoplasms. A more detailed quantitative analysis of the results, however, demonstrated increasing numbers of cells harbouring these genomic alterations, from low-grade TC to highly malignant SCLC, with the exception of cyclin D1 deletion. Mutations of the p53 and Rb genes, as assayed by immunohistochemical studies, were observed at high incidence in high-grade carcinomas, compared with a low incidence in the low-grade carcinomas. Conversely, in all carcinoma types, neither membrane-bound Her-2/neu nor nuclear cyclin D1 was detected. It is concluded that structural genomic alterations are frequent in neuroendocrine lung carcinomas and that their occurrence may be underestimated by immunohistochemical studies alone. The quantitative expansion of the Rb, p53, c-myc, and Her-2/neu alterations towards high-grade carcinomas suggests common pathogenetic mechanisms in the spectrum of these neoplasms.     

Prealbumin in the diagnosis of bronchopulmonary carcinoid tumours.

The reliability of prealbumin as a diagnostic marker was studied in 60 cases of bronchopulmonary carcinoid tumours. There were differences in the incidence of positivity between typical and atypical carcinoids (well differentiated neuroendocrine carcinomas). Seventy five per cent of the carcinoid tumours were positive for prealbumin; (86.7% typical and 63.3% atypical carcinoids). In 15 cases, which were Grimelius negative, 10 were prealbumin positive. Only 8.3% carcinoids were negative with both prealbumin and Grimelius stains. Ten squamous, 10 adeno- and 10 small cell carcinomas showed only occasional scattered prealbumin positive cells. It is concluded that prealbumin is a useful marker for bronchopulmonary carcinoid tumours. It is cheap, readily available, and should be considered part of routine diagnostic procedures for the diagnosis of carcinoid tumours.     

High-grade neuroendocrine carcinomas of the lung express K homology domain containing protein overexpressed in cancer but carcinoid tumors do not

K homology domain containing protein overexpressed in cancer (KOC) is a member of the insulin-like growth factor (IGF) messenger RNA-binding protein family and is expressed during embryogenesis and in certain malignancies. KOC, known as L523S and IGF messenger RNA-binding protein 3, was shown to be frequently expressed in high-grade neuroendocrine carcinomas of the lung in our immunohistochemical studies using a monoclonal antibody against human KOC. Specifically, all 10 small cell lung carcinomas (SCLCs) exhibited strong cytoplasmic staining, 9 with diffuse positivity and 1 with focal positivity. Among 14 large cell neuroendocrine carcinomas (LCNECs), 9 exhibited strong and diffuse cytoplasmic staining, and 5 cases showed focal immunoreactivity. In contrast, no KOC was detected in 21 typical and atypical carcinoids, except for one atypical carcinoid with oncocytic cells showing weak cytoplasmic staining. Although SCLCs exhibited a strong and diffuse staining pattern more frequently (90%) than LCNECs (64%), the difference did not reach statistical significance (P = .3408). Interestingly, our immunohistochemical studies demonstrated that IGF-II, reportedly regulated by KOC, was comparably expressed in SCLC, LCNEC, and typical and atypical carcinoids, irrespective of KOC expression status of the tumors. These results support the formulation that KOC may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas. In addition, detection of KOC expression may be diagnostically useful in distinguishing high-grade neuroendocrine carcinomas from carcinoid tumors. Our findings of equivalent IGF-II expression in KOC-positive SCLC and LCNEC and KOC-negative carcinoid tumors suggest different regulatory mechanisms involved in the control of IGF-II expression in these tumors.     

Differential expression of neural-cadherin in pulmonary epithelial tumours

Aims:  Neural (N)-cadherin belongs to a group of transmembrane molecules with a crucial role in tissue morphogenesis and maintenance of an epithelioid phenotype and increased N-cadherin expression is implicated in tumour progression and dedifferentiation. The aim was to determine whether evaluation of N-cadherin in pulmonary tumours might assist in identifying lesions with more aggressive potential.
Methods and results:  One hundred and fifty-five pulmonary lesions were analysed for N-cadherin expression using immunohistochemistry, including neuroendocrine hyperplasia ( n  = 3), typical carcinoid ( n  = 59), atypical carcinoid ( n  = 12), small cell lung carcinoma ( n  = 11), large cell neuroendocrine carcinoma ( n  = 12), adenocarcinoma ( n  = 35) and squamous cell carcinoma ( n  = 23). Lymph node status was correlated with immunohistochemical expression. N-cadherin expression was demonstrated in all cases of neuroendocrine hyperplasia, 96% of typical carcinoids, 83% of atypical carcinoids, 63% of the small cell lung carcinomas and 32% of large cell neuroendocrine carcinomas. Over 90% of the adenocarcinomas and 100% of the squamous cell carcinomas were negative. Increased N-cadherin expression in typical carcinoids was associated with negative lymph node status ( P  < 0.001).
Discussion:  N-cadherin is differentially expressed in pulmonary tumours and is predominantly observed in neuroendocrine lung lesions with high expression in typical and atypical pulmonary carcinoids. The level of expression of N-cadherin between types of lung tumours does not appear to indicate malignant potential or aggressive behaviour.     

Divergent differentiation in neuroendocrine lung tumors

The classification of neuroendocrine (NE) lung tumors has been revised in the 1999 World Health Organization (WHO) classification of lung tumors, allowing sharp morphological definition of typical versus atypical carcinoids, and atypical carcinoids versus large cell neuroendocrine carcinoma (LCNEC), a newly described class of high-grade NE lung tumors which differs from small-cell lung cancer by a large-cell phenotype. Divergent differentiation accounts for the high frequency of glandular differentiation with mucin production, and ultrastructural features in carcinoids and LCNEC, and low frequency of squamous differentiation in both LCNEC and SCLC. Specific NE markers (chromogranin, synaptophysin, neural cell adhesion molecule) and epithelial markers consistently negative in neuroendocrine components (cytokeratins 1, 5, 10, 14; epidermal growth factor (EGF)-receptor, human leukocyte antigen beta 2 (HLA-beta2) microglobuline) help to recognize divergent differentiation in NE tumors. At morphological level, divergent differentiation in NE tumors is recognized in WHO classification as variants: combined SCLC and combined LCNEC. The derivation of all lung tumors from a common endodermal stem cell and adoption of amine precursor uptake and decarboxylation properties by this endodermal stem cell explains divergent differentiation in NE lung tumors and the occurrence of NE subsets in NSCLC.     

High-grade neuroendocrine carcinomas of the lung highly express enhancer of zeste homolog 2, but carcinoids do not

Enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, is a histone methyltransferase and plays an important role in cell proliferation and cell cycle regulation. It has been shown to be overexpressed in a number of malignant neoplasms. This study aimed to determine the expression pattern of enhancer of zeste homolog 2 in neuroendocrine tumors of the lung and the potential of enhancer of zeste homolog 2 to serve as a biomarker to segregate carcinoids from high-grade neuroendocrine carcinomas. Fifty-four cases, including 25 typical carcinoids, 7 atypical carcinoids, 9 large-cell neuroendocrine carcinomas, and 13 small-cell lung carcinomas, were immunohistochemically studied using a monoclonal antibody against enhancer of zeste homolog 2. All 13 small-cell lung carcinomas demonstrated moderate to strong nuclear staining with 12 exhibiting more than 90% of tumor cells staining. All 9 large-cell neuroendocrine carcinomas were moderately to strongly positive for enhancer of zeste homolog 2, with 6 cases having staining in more than 80% of tumor cells. In contrast, all 25 typical carcinoids and 6 atypical carcinoids showed only rare scattered enhancer of zeste homolog 2-positive tumor cells, with 1 case of atypical carcinoid exhibiting moderate staining in 40% of tumor cells. A subsequent validation study of the 14 specimens of lung or mediastinal lymph node biopsy and fine-needle aspiration, including 6 small-cell lung carcinomas, 2 large-cell neuroendocrine carcinomas, 5 typical carcinoids, and 1 atypical carcinoid, was performed. Enhancer of zeste homolog 2 was diffusely and strongly positive in all small-cell lung carcinomas and large-cell neuroendocrine carcinomas, even with severe crush artifact, whereas it was only positive in rare tumor cells in carcinoids. These findings support the formulation that enhancer of zeste homolog 2 may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas and as a diagnostically useful marker in distinguishing high-grade neuroendocrine carcinomas from carcinoids.     

A case of Lambert-Eaton myasthenic syndrome associated with atypical bronchopulmonary carcinoid tumor

The Lambert-Eaton myasthenic syndrome (LEMS) is typically recognized as a paraneoplastic syndrome associated with a small cell lung carcinoma (SCLC), whereas LEMS with other neuroendocrine lung tumors, including carcinoids or large cell lung carcinoma, are highly unusual. Here, we report a rare case of LEMS with atypical bronchopulmonary carcinoid tumor: a 65-yr-old man presented with progressive leg weakness and a diagnosis of LEMS was made by serial repetitive nerve stimulation test. Chest CT revealed a lung nodule with enlargement of paratracheal lymph nodes, and surgically resected lesion showed pathological features of atypical carcinoid tumor. We concluded that LEMS could be associated with rare pulmonary neuroendocrine tumor other than SCLC, which necessitates pathologic confirmation followed by aggressive treatment for optimal management in these rare cases.     

Analysis of p53, K-ras-2, and C-raf-1 in pulmonary neuroendocrine tumors. Correlation with histological subtype and clinical outcome.

Neuroendocrine tumors of lung, including typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC) constitute a spectrum of malignancies in which the pathologist at times has difficulty in discerning tumor subtype and aggressiveness in a reproducible fashion. Therefore, 59 primary neuroendocrine lung tumors including 10 TCs, 26 ACs, 15 LCNECs, and 8 SCLCs were selected from cases collected from 1976 to 1988 and immunostained for p53 protein. All of these tumors were also genotyped for specific point mutational damage affecting p53 (exons 5, 7, and 8; with ACs additionally sequenced for p53 exon 6); 13 tumors for K-ras-2 (exon 1); and 31 tumors for c-raf-1 (exon 15) growth-regulatory genes. Genotyping was performed on topographically selected, minute tumor samples removed from unstained formalin-fixed, paraffin-embedded tissue sections (topographic genotyping) using polymerase chain reaction and direct sequencing. The distribution of p53 immunohistochemical staining had four patterns: negative in TCs, one-half of ACs, 3 of 15 LCNECs, and 1 of 8 SCLCs; less than 10% but more than five tumor cells per 10 high power fields (focal) in a subset (7 of 26) of aggressive ACs; 10 to 49% of tumor cells (patchy) in a subset (6 of 26) of ACs with a higher grade of aggressiveness; and 50 to 100% of tumor cells (diffuse), exclusively seen in LCNECs (12 of 15) and SCLCs (7 of 8). Three patterns of immunohistochemical staining intensity of p53 protein were seen: negative, weak or mild, and moderate to marked. SCLCs and LCNECs accounted for cases of moderate to marked staining and were the only ones to have mutations in p53 exons 5, 7, or 8. No mutations were found in AC and TC, showing absent to weak staining and no staining, respectively. The difference in distribution and staining intensities between LCNEC and SCLC compared with AC and TC was statistically significant (P < 0.001). Patients having AC with patchy p53 immunostaining usually had survival limited to 3 years, whereas those having AC with focal p53 immunostaining subsequently developed metastatic or recurrence of AC disease (P < 0.05). The absence of point mutations in cases with patchy or focal immunostaining suggests increased expression of wild-type p53 tumor suppressor protein likely in response to growth deregulation in a more aggressive subtype of AC. A novel hypothesis is presented in regard to these findings. K-ras-2 and c-raf-1 gene sequence analysis showed no evidence of point mutational change in any of the tumors studied. The TC and AC categories are therefore genetically distinct from the higher grade neuroendocrine SCLC and LCNEC. Immunohistochemistry for p53 on AC lung tumors may be helpful to delineate cases at higher risk for aggressive behavior. Additionally, although LCNEC is categorized as a non-small-cell carcinoma, it is more akin genetically and immunohistochemically to SCLC.     

Differential retinoblastoma protein expression in neuroendocrine tumors of the lung. Potential diagnostic implications.

Neuroendocrine lung tumors have been considered by some to be a continuum ranging from relatively benign typical carcinoids to highly malignant small-cell carcinomas. Histopathological diagnosis may sometimes be difficult because of their overlapping features. Correct classification, however, carries important prognostic and therapeutic significance. To determine the clinicopathological implications of retinoblastoma (RB) protein expression in these neoplasms, we examined the RB status in a series of neuroendocrine tumors by immunohistochemical analysis of paraffin-embedded tissue sections. A total of 105 tumors were studied. All 44 typical and 15 atypical carcinoids, one of which was initially misdiagnosed as a small-cell carcinoma, manifested a heterogeneous RB-positive staining pattern. Atypical carcinoids in general showed an increase in the number of tumor cells with strong nuclear staining compared to typical carcinoids. In contrast, all 40 small-cell and 6 large-cell neuroendocrine carcinomas failed to show RB staining in any tumor nuclei, indicating loss of RB function. Our results suggest that RB status as measured by immunohistochemical staining can be used as a marker to distinguish typical and atypical carcinoids from small-cell and large-cell neuroendocrine carcinomas.     

Carcinoids of the gastrointestinal tract: importance of determining differentiation and proliferation markers

The group of 35 carcinoid tumours obtained from 34 patients was reviewed according to recent histopathological criteria. Consequently, evaluation of the Grimelius staining and immunohistochemical detection of chromogranin A (CgA), Leu-7 (CD-57), synaptophysin, neuron-specific enolase (NSE), (beta-III tubulin, Ki-67 and proliferating cell nuclear antigen (PCNA) was performed. The majority of tumours (29, i.e. 83%) were classified as typical carcinoids composed predominantly of mixed solid and trabecular or solid and tubular growth patterns. Six tumours (17%) revealed more prominent cytological abnormalities corresponding with the diagnosis of atypical carcinoid. The majority of tumours (31, i.e. 93.9%) showed granular cytoplasmic positivity in Grimelius staining and diffuse cytoplasmic positivity of NSE (34, i.e. 97.1%). All of the 32 stained tumour samples showed positive immunoreactivity for synaptophysin. A high percentage of tumours (32, i.e. 91.4%) revealed also a positive reaction with antibody TU-20 detecting (beta-III tubulin, a marker of an early stage of neuronal differentiation. Thirty-four tumours (97.1%) showed granular cytoplasmic positivity for both markers of neuroendocrine granules (CgA and Leu-7). One tumour (2.9%) was positive only for Leu-7. Tumour cells revealed predominantly low proliferative activity evaluated by PCNA and Ki-67 immunodetection. Higher degree of proliferation was observed especially in atypical carcinoids.     

hASH1基因与肺神经内分泌肿瘤的关系

肺神经内分泌肿瘤(neuroendocrine tumor，NET)是由具有多向分化潜能的肿瘤细胞组成，这些肿瘤细胞具有分泌多种活性激素的功能。肺NET的发生、发展与hASH1(human achaete-scute homologue 1), MEN1(multiple endocrine neoplasia type 1), pRB和E2F1等基因相关，其中hASH1基因成为近年来研究的热点，该基因能促进肺NET的内分泌分化，降低肿瘤的分化程度，并且有临床资料显示hASH1基因的表达还与小细胞肺癌的低生存率有关，可以作为临床预后的一个标准。