Gene-expression profiling reveals distinct expression patterns for Classic versus Variant Merkel cell phenotypes and new classifier genes to distinguish Merkel cell from small-cell lung carcinoma

Merkel cell carcinoma (MCC) is a rare aggressive skin tumor which shares histopathological and genetic features with small-cell lung carcinoma (SCLC), both are of neuroendocrine origin. Comparable to SCLC, MCC cell lines are classified into two different biochemical subgroups designated as 'Classic' and 'Variant'. With the aim to identify typical gene-expression signatures associated with these phenotypically different MCC cell lines subgroups and to search for differentially expressed genes between MCC and SCLC, we used cDNA arrays to profile 10 MCC cell lines and four SCLC cell lines. Using significance analysis of microarrays, we defined a set of 76 differentially expressed genes that allowed unequivocal identification of Classic and Variant MCC subgroups. We assume that the differential expression levels of some of these genes reflect, analogous to SCLC, the different biological and clinical properties of Classic and Variant MCC phenotypes. Therefore, they may serve as useful prognostic markers and potential targets for the development of new therapeutic interventions specific for each subgroup. Moreover, our analysis identified 17 powerful classifier genes capable of discriminating MCC from SCLC. Real-time quantitative RT-PCR analysis of these genes on 26 additional MCC and SCLC samples confirmed their diagnostic classification potential, opening opportunities for new investigations into these aggressive cancers.     

Characterization of cell lines established from merkel-cell (“small-cell”) carcinoma of the skin

Six cell lines have been established from different biopsies of the rare small-cell skin cancer of neuro-endocrine origin known as Merkel-cell carcinoma (MCC). These were established from metastatic lesions and have now been in culture for periods varying from 1 to 3 years. All lines grow as suspension cultures and exhibit typical MCC cytological features of small round cells with little cytoplasm, round nuclei and tight junctions. There was variation in the number and size of nucleoli and dense-core granules. We present details of their immunohistochemistry and growth characteristics in culture. The latter were similar to small-cell lung-cancer (SCLC) lines and can be subdivided, by their appearance, into type I, type II and type III in the same way as SCLC lines. This classification may be of significance for the prognosis and management of MCC.     

Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties

We have described the establishment and biochemical characterization of 50 small cell lung carcinoma (SCLC) cell lines. Further analysis of these data, combined with studies of morphology and growth characteristics, indicates that 35 (70%) of the lines retained typical morphology (SCLC, intermediate subtype), growth characteristics (growth as tightly packed floating cellular aggregates, long doubling times and low colony-forming efficiencies), and biochemical profile (presence of L-dopa decarboxylase, bombesin-like immunoreactivity, neuron-specific enolase, and high concentrations of brain isoenzyme of creatine kinase). They are referred to as classic SCLC lines. The remaining 15 (30%) lines had discordant expression of the biochemical markers; they retained high concentrations of brain isozyme of creatine kinase, but had significantly lower concentrations of neuron-specific enolase and lacked L-dopa decarboxylase and bombesin-like immunoreactivity. These cell lines are called variants. SCLC variant lines could further be divided into (a) biochemical variant lines having variant biochemical profile but retaining typical SCLC morphology and growth characteristics; and (b) morphological variant (SCLC-MV) lines having variant biochemical profile, altered morphology (features of large cell undifferentiated carcinoma) and altered growth characteristics (growth as loosely attached floating aggregates, relatively short doubling times and cloning efficiencies). Fifty-five clones derived from the three SCLC subclasses retained their parental phenotypes. In SCLC-MV lines there was a near constant relationship between variant morphology, altered growth characteristics and amplification of the c-myc oncogene; classic SCLC and biochemical variant SCLC lines were not amplified. Variant morphologies frequently are present in SCLC tumors at autopsy, and most SCLC-MV lines reflect changes that had occurred in the tumors from which they were derived. Because SCLC-MV tumors behave more virulently in the patient and are radioresistant in vitro, these findings are of considerable biological and clinical interest.     

Increased expression of differentiation markers can accompany laminin-induced attachment of small cell lung cancer cells.

We investigated the interaction between human lung cancer cells, laminin, and several differentiating agents. When grown on laminin coated substrate eight out of 11 small cell lung cancer (SCLC) cell lines exhibited attachment to laminin and three had extensive outgrowth of long neurite-like processes. Of seven non-small cell lung cancer cell lines, selected for their in vitro anchorage-independent growth, attachment was observed in only three cell lines, and process formation was far less extensive than in SCLC cell lines. Among several differentiating agents, only dcAMP, which alone induced attachment and some process formation, increased laminin-mediated attachment and process formation of two SCLC cell lines, NCI-N417 a variant cell line, and NCI-H345, a classic cell line. The expression of several neuroendocrine and neuronal markers was investigated in these two SCLC cell lines. The expression of the light subunit of neurofilaments increased in NCI-N417 within 3 to 4 days of seeding, while NCI-H345 exhibited approximately 5 fold increase in expression of the GRP gene and a 3 fold increase expression of the beta-actin gene. The expression of a number of other neuroendocrine and neuronal markers did not change following growth on laminin. The doubling times remained unchanged independent of the presence of and attachment to laminin while topoisomerase II gene expression levels in NCI-N417 cells decreased approximately 5 fold when cells were growing on laminin.     

Characterization of six Merkel cell polyomavirus-positive Merkel cell carcinoma cell lines: Integration pattern suggest that large T antigen truncating events occur before or during integration

Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor, is a polyomavirus-induced human cancer. To study the causal relationship of MCC carcinogenesis with the integrated Merkel cell polyomavirus (MCPyV) in detail, well-characterized MCC cell lines are needed. Consequently, in the current study, we established and characterized six MCPyV-positive MCC cell lines. Microarray-based comparative genomic hybridization revealed a stable genome carrying only a limited number of chromosomal gains and deletions. All cell lines expressed MCC markers Keratin-20 and neuron-specific enolase as well as truncated MCPyV-encoded large T antigen (LT). For five cell lines, we were able to identify the MCPyV-integration sites in introns of different genes. The LT-truncating stop codon mutations and integration sites were affirmed in the respective clinical patient samples. Inverse PCR suggested that three of the cell lines contained MCPyV genomes as concatemers. This notion was confirmed for the two cell lines with known integration sites. Importantly, our observation of distinct stop codon mutations in cell lines with concatemeric MCPyV integration indicates that these LT-truncating mutations occur before integration. In summary, we provide the detailed characterization of six MCPyV-positive MCC cell lines, which are likely to serve as valuable tools in future MCC research.     

Detection of Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines: Cell morphology and growth phenotype do not reflect presence of the virus

The recently discovered human polyomavirus (MCPyV) is frequently found in Merkel cell carcinoma (MCC) tissue and is believed to be causally linked to MCC pathogenesis. While cell lines established from MCC represent a valuable tool to study the contribution of MCPyV to MCC pathogenesis, hitherto only 1 MCPyV‐positive line has been described. We have analyzed 7 MCC cell lines for the presence, integration pattern and copy number of MCPyV. In 5 cell lines, MCPyV specific sequences were detected. In 3 of these lines, multiple copies of viral genomes per cell were detected, and sequencing of PCR amplificates identified distinct mutations predicted to lead to the expression of a truncated large T‐Antigen (LT‐Ag). In 1 cell line, clonal integration of concatamerized viral genomes was confirmed by Southern Blotting. MCC cell lines are conventionally categorized as “classic” or “variant” and further divided into 4 subtypes, based on expression of neuroendocrine markers and morphology. While it has been suggested that the presence of MCPyV might promote a classic phenotype, such a notion is not supported by our data. Instead, we find MCPyV‐positive as well as ‐negative lines of the classic variety, indicating that the distinguishing features are either inherently independent of viral infection or have become so in the course of tumorigenesis and/or cell line establishment. We therefore suggest a novel classification scheme based on MCPyV presence, integration patterns and T‐Ag mutations. The cell lines described here extend the repertoire of available MCPyV‐positive MCC‐lines and should aid in the elucidation of the role of MCPyV in the pathogenesis of MCC.     

Large cell neuroendocrine carcinoma of the lung: a comparison with large cell carcinoma with neuroendocrine morphology and small cell carcinoma

Large cell neuroendocrine carcinoma (LCNEC) of the lung is a malignant neuroendocrine tumor clinicopathologically similar to and falling in-between atypical carcinoid tumor and small cell lung carcinoma (SCLC). The diagnosis of LCNEC is based mainly on a characteristic neuroendocrine morphology and biological neuroendocrine differentiation. In order to know the discrepancy between morphological and biological neuroendocrine differentiation, LCNEC was immunohistochemically and molecular biologically compared with large cell carcinoma with neuroendocrine morphology (LCCNM), which lacked only biological neuroendocrine differentiation among the criteria of LCNEC. Immunohistochemically, disruption of the RB pathway, namely a lack of RB expression and simultaneous overexpression of p16 protein, was characteristic of LCNEC, but not LCCNM. In G2/M cell cycle regulation, 14-3-3 sigma expression was markedly reduced in LCNEC. Moreover, the antibody 34 beta E12 recognizing a set of large-sized keratin gave a different staining pattern between LCNEC and LCCNM. The immunohistochemical data suggested that LCNEC has a similar biological marker profile to SCLC and different from LCCNM. However, a loss of heterozygosity (LOH) analysis using microsatellite markers showed a high frequency of LOH at 3p in both LCNEC and LCCNM as well as in SCLC. Morphological neuroendocrine differentiation might not be identical to biological neuroendocrine differentiation in large cell carcinoma of the lung.     

Comparative proteomics of small cell lung carcinoma

Small cell lung carcinoma (SCLC) is an aggressive, highly metastatic cancer with a strong tendency for chemotherapy resistance. Identification of proteins uniquely expressed in SCLC cells, can facilitate the development of new diagnostic tools, improve immunotherapy, and deepen our understanding of the underlying mechanisms of the disease. Here we describe a comparative proteomics analysis of ten SCLC cell lines and three controls lines, while searching for proteins preferentially expressed in SCLC cells as potential disease markers. Total protein extracts were compared by two-dimensional gel electrophoresis and by two-dimensional liquid chromatography resulting in the identification of 1093 proteins, 202 of which were detected only in the SCLC cells. These include proteins of different cellular functions, including cellular proliferation and known tumor antigens. Since SCLC has a neuroendocrine origin, of major interest are the identified proteins involved in nerve and brain embryonic development. These proteins are potentially valuable as both tumor markers and as antigens for immunotherapy.     

A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines

Synaptophysin is a Mr 38,000 integral membrane glycoprotein expressed by a variety of normal and neoplastic neuroendocrine cells. We studied synaptophysin as an immunocytochemical marker for neuroendocrine differentiation in lung cancer and compared it to the immunocytochemical expression of chromogranin A, a marker for dense core (endocrine) granules, and the biochemical activity of L-dopa decarboxylase (DDC), the key amine-handling enzyme. Of the 250 cell lines available to us, we selected examples representative of the following cell types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell lung cancers (n = 18) whose neuroendocrine status had been previously determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of synaptophysin than chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (b) 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines expressed synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non-small cell lung cancer cell lines previously shown to express multiple neuroendocrine markers were positive for synaptophysin, chromogranin A, and DDC activity; (d) none of the other 11 non-small cell lung cancer cell lines expressed synaptophysin or chromogranin A; and (e) formalin fixation and paraffin embedding reduced synaptophysin immunoreactivity in 11 of 14 (79%) of the cell lines, as compared to freshly prepared specimens fixed in 95% ethanol. Western blot analysis using the synaptophysin antibody (SY38) demonstrated immunoreactive proteins ranging from Mr 43,000 to 45,000 in five representative cell lines. The concordance of expression of all three neuroendocrine markers was statistically significant when values for all cell lines were totalled. Synaptophysin was a more commonly expressed marker for variant SCLC cell lines, which rarely showed DDC activity. We conclude that synaptophysin may be a more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines which express only part of the neuroendocrine program.     

In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways

Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo- and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits alpha2, beta3 and beta4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylation-specific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kappaB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemo- and radio-resistance.     

Merkel cell carcinoma in a renal transplant patient

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer which seems to be common in transplant recipients. We describe the case of a renal transplant patient who developed a MCC on the right glutaeus eight years after transplantation.     

Demonstration of Chromogranin A in human neuroendocrine cell lines by immunohistology and immunoassay

We have used immunohistology and radioimmunoassay procedures to study Chromogranin A (CgA) in human neuroendocrine tumor cell lines, especially small cell lung cancers (SCLC). By immunohistology, CgA could be detected in 11 of 18 classical SCLC cell lines, in a medullary thyroid carcinoma (MTC) cell line, and in only one of 13 variant- or non-SCLC cell lines. By radioimmunoassay, CgA could be detected in the cells and culture media of all of the classical SCLC cell lines tested. Many of the classical SCLC cell lines also produced calcitonin (CT). These studies demonstrate that CgA production is a common feature of SCLC cell lines, especially those with neuroendocrine characteristics.     

Expression of neuroendocrine and epithelial markers in an adherent subline derived from a classic small cell lung cancer cell line.

Small cell lung cancer (SCLC) cell lines usually grow as floating aggregates, in contrast to the adherent monolayers formed by non small cell lung cancer (NSCLC). Induction of an adherent phenotype by a variety of methods has been the subject of a number of recent publications. In this study, cultivation of the classic SCLC cell line, NCI-H69, on a substratum provided by the pretreatment of tissue culture dishes with medium conditioned by the growth of a well differentiated squamous carcinoma cell line, HN5, induced an adherent phenotype with a variety of epithelioid morphologies, commencing within 24 hr of plating. From such cultures an adherent subline, H69A, has been established, which differs in its growth, morphological characteristics, and immunocytochemical marker expression from the parent NCI-H69 cells, and in its marker expression from other adherent SCLC cell lines. H69A retained expression of neural cell adhesion molecule (NCAM) and the neuroendocrine markers neuron specific enoclase, chromogranin A, and synaptophysin, but showed diminished expression of the epithelial cell surface markers AUA1, Ber-EP4, epithelial membrane antigen (EMA), and desmosomal protein. Compared to NCI-H69 cells, the amounts of cytokeratin 18 detected were elevated, while those of cytokeratin 19 were diminished in H69A cells. Focal expression of cytokeratin 4 was found in some H69A cells, indicative of a capacity for partial squamous differentiation. The expression of the cell surface glycoproteins detected by AUA1 and Ber-EP4 was reduced throughout cultivation of the H69A subline, while that of EMA and desmosomal protein was further diminished with continued passage. Changes in the expression of these markers and NCAM were evident in NCI-H69 cells grown on an HN5-derived substratum.     

Two novel cell surface antigens on small cell lung carcinoma defined by mouse monoclonal antibodies NE-25 and PE-35

Two mouse monoclonal antibodies, NE-25 and PE-35, defining novel cell surface antigens of small cell lung carcinoma (SCLC) were produced. The molecular weight of NE-25 and PE-35 antigens estimated by radioimmunoprecipitation was 25,000 and 35,000, respectively. NE-25 antigen was expressed on the majority of cell lines and tumor specimens of SCLC among lung carcinoma. These NE-25-positive cell lines showed typical growth morphology as SCLC classic lines and expressed high levels of neuroendocrine biomarkers, such as aromatic L-amino acid decarboxylase, while NE-25 antigen-negative lines lacked apparent neuroendocrine properties. This antigen was expressed also on a subset of neoplastic cells with (neuro)endocrine properties, including pulmonary carcinoid, and on various tumors of nervous tissues, such as neuroblastoma. Among the normal cells, Kulchitski cells of lung, thyroid gland, adrenal gland, Langerhans islet, and nervous tissues were positive. Thus, the expression of NE-25 antigen is closely associated with the neural and/or (neuro)endocrine differentiation state. On the contrary, PE-35 antigen was present on four major types of lung carcinomas as well as on squamous cell carcinoma and adenocarcinomas of various tissues, but it was absent from nervous tissue tumors. Thus, PE-35 antibody showed a "pan-epithelial" reactivity. Analysis by NE-25 and PE-35 antibodies provided evidence for the heterogeneities of SCLC by demonstrating four surface phenotypes, with the NE-25+/PE-35+ phenotype being most common. In addition, the results supported the current understanding that various histological types of lung carcinoma, including SCLC, are derived from a stem cell of the bronchial epithelium.     

Anti-HuC and -HuD autoantibodies are differential sero-diagnostic markers for small cell carcinoma from large cell neuroendocrine carcinoma of the lung

Aiming to identify novel sero-diagnostic markers for neuroendocrine carcinomas of the lung, the two-dimensional gel electrophoresis-immunoblot method was used to analyze tumor-associated autoantibodies in patients with small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). Several autoantigens were revealed and anti-HuC autoantibody was detected only in sera of SCLC patients. Since Hu family proteins including HuC are well-known causes of paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/SN), the expression of HuC as well as HuD mRNAs and their proteins was studied in 11 lung cancer cell lines. The expression of HuC and HuD mRNAs and proteins was only detected in SCLC- and LCNEC-derived cells. To validate the existence of anti-HuC and -HuD auto-antibodies, we studied a large number of sera including those from lung cancer patients employing dot blot analysis. Anti-HuC and -HuD autoantibodies were detected only in SCLC cases with or without PEM/SN, and not in the sera of LCNEC patients. The mechanism leading to different anti-HuC and -HuD autoantibody production between SCLC and LCNEC is unclear; however, the results from the present and previous studies suggest that anti-HuC and -HuD auto-antibodies are novel differential sero-diagnostic markers for SCLC from LCNEC.     

The neurotoxin 1-methyl-4-phenylpyridinium: a selective cytostatic agent in small-cell lung cancer cell lines with neuroendocrine properties.

BACKGROUND: Small-cell lung cancer (SCLC) is a common malignancy that is usually fatal, since it metastasizes and recurs even after aggressive chemotherapy. While the cellular origin of this cancer is not well established, the cells of certain tumors exhibit neuroendocrine markers, including L-dopa decarboxylase. PURPOSE: We designed in vitro and in vivo studies to investigate whether the neuroendocrine features in classic SCLC cell lines were sufficient to make them sensitive to 1-methyl-4-phenylpyridinium (MPP+), a known neurotoxin that destroys nigrostriatal dopaminergic neurons. METHODS: Both classic SCLC cell lines (NCI-H345, NCI-H510, NCI-H187, and NCI-H146) and variant SCLC cell lines (NCI-H417, NCI-H82, NCI-H446, and NCI-H524) were exposed to MPP+ (0-512 microM) for 3 days. Inhibition of DNA synthesis was determined by [3H]thymidine incorporation assays. In a related experiment, MPP+ was removed from the classic cell line culture, and the incorporation of [3H]thymidine was determined. In the in vivo study, male athymic nude mice received subcutaneous injections of 0.5 mL tumor cells with matrigel for 10 days to enhance tumor growth, followed by MPP+ at doses of 100-400 micrograms/d given intraperitoneally for 2 days. RESULTS: All four classic SCLC cell lines showed great sensitivity to MPP+, with detachment from laminin substrates and inhibition of DNA synthesis. MPP+ interfered with [3H]thymidine incorporation and, thus, with DNA synthesis in classic SCLC cell lines at low doses (median +/- SD, 12 +/- 4 microM), whereas much higher doses (median, > 512 microM) were required to inhibit [3H]thymidine incorporation in the variant lines. Treated cells excluded trypan blue dye, showing that inhibition of DNA synthesis was not due to cytotoxicity, and the cells incorporated [3H]thymidine when MPP+ was removed from the culture medium, demonstrating that the inhibition was reversible. MPP+ inhibited the growth of the classic NCI-H187 and variant NCI-H417 cell lines implanted in nude mice. CONCLUSIONS: These results suggest that MPP+ differentially interferes with DNA synthesis in SCLC cell lines in vitro; the selective inhibitory effect on classic cell lines suggests that the neuroendocrine properties expressed by classic SCLC cells may be responsible for the differential effect. IMPLICATIONS: MPP+ exerts a cytostatic effect on these cell lines, and the differential sensitivity observed in vitro is maintained in vivo, suggesting that MPP+ or other pyridinium compounds may be of therapeutic value in SCLC.     

Identification and characterisation in vitro of cells with a non-SCLC cell-like phenotype derived from a continuous SCLC cell line.

Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth rates of both the derivative lines were faster than the parental line with doubling times closer to non-SCLC cell lines in the derivative lines. Both H69V and H69VZ either express very low levels or do not express neuroendocrine cell markers including L-dopa-decarboxylase (DDC), creatine kinase-BB isoenzyme (CK-BB), bombesin-like immunoreactivity (BLI), neuron specific enolase (NSE), and neurosecretory type dense core granules (DGCs), compared to the parental cell line. All the lines stained positive for epithelial markers such as CAM5.2. LDH isoenzyme and chromosome analyses confirmed the human origin of all the cell lines. Therefore, it appears that cell line NCI-H69 contains stem cell subpopulation capable of generating cells of both small and non-small cell like phenotypes.