Expression of neuroendocrine cell markers L-dopa decarboxylase, chromogranin A, and dense core granules in human tumors of endocrine and nonendocrine origin

We evaluated the usefulness of L-dopa decarboxylase (DDC) as a tumor marker of neuroendocrine (NE) cell differentiation by measuring its expression in 432 human tumors of diverse types and origins. A subset of these tumors and cell lines derived from them also were studied for expression of two other general NE cell markers, chromogranin A (CgA) and dense core granules (DCG). High concentrations of DDC were present in 96 of 117 (82%) tumors recognized to be of NE or neural origin. As expected, endocrine tumors not recognized to be of NE cell origin, as well as leukemias, lymphomas, sarcomas, melanomas, and germ cell tumors, lacked DDC expression. Of interest, modest concentrations of DDC were present in 46 of 220 (21%) nonendocrine carcinomas, especially non-small cell lung and colorectal carcinomas. We studied concordant expression of the three NE cell markers in lung and colorectal tumors and cell lines. In both tumor types there was nearly 100% concordance between CgA and DCG expression. There was an excellent correlation between DDC and CgA expression in lung cancers, both small cell and non-small cell, but DDC positive colorectal carcinomas usually lacked CgA expression. We conclude: (a) DDC is an excellent cellular marker for tumors of the NE cell system; (b) about 20% of carcinomas not of NE cell origin, especially non-small cell lung and colorectal carcinomas, express DDC, suggesting a common endodermal origin of all of the respiratory and gastrointestinal mucosal cells; and (c) CgA and DCG are expressed concordantly, indicating that CgA expression may be used as a substitute for ultrastructural examination of tumors for DCG expression.     

Cross-linked envelope-related markers for squamous differentiation in human lung cancer cell lines

Lung carcinoma cell lines were analyzed in culture and in nude mouse xenograft for both morphological appearance and expression of specific proteins that participate in cross-linked envelope formation during normal squamous cell terminal differentiation. Cross-linked envelope formation, induced by artificial influx of millimolar Ca2+ into the cultured cells, was an exclusive trait of squamous, adenosquamous, and mucoepidermoid carcinomas. Small cell lung carcinoma and non-squamous non-small cell lung carcinoma lines, such as adenocarcinoma and large cell carcinoma, were uniformly negative for cross-linked envelope formation. Involucrin, which is incorporated into the cross-linked envelope by the enzyme transglutaminase, was expressed at highest levels in squamous tumors, but several of the non-squamous non-small cell lung carcinoma lines also expressed comparable amounts. On the other hand, transglutaminase activity was consistently higher in squamous as opposed to non-squamous lines, so that in cell culture, a clear contrast between the groups could be observed. A Mr 195,000 protein that is incorporated into cultured human epidermal cell cross-linked envelopes was also observed in some but not all of the squamous lines. Two forms of transglutaminase are expressed in cultured keratinocytes. One of them, tissue transglutaminase, was expressed in the majority of squamous cell lines even though it is not a normal product of squamous differentiation in vivo. Keratinocyte transglutaminase, which is distinct from the tissue form and is normally expressed during terminal differentiation in squamous epithelia. was measurably present in only one of the six squamous cell lines tested. In nude mouse xenografts, keratinocyte transglutaminase, localized immunohistochemically with a biotinylated mouse monoclonal antibody, was again present only in a minority of the squamous lines whereas involucrin was expressed in all. In contrast to involucrin, keratinocyte transglutaminase is not an obligatory component of squamous differentiation in the pulmonary carcinoma cell lines tested. Its expression may be of value in further refining their classification.     

Pro-gastrin-releasing peptide, neuron specific enolase and chromogranin A as serum markers of small cell lung cancer

BACKGROUND: Small cell lung cancer (SCLC) yields neuroendocrine properties. Pro-gastrin-releasing peptide (ProGRP), neuron specific enolase (NSE) and chromogranin A (CGA) are therefore putative serum markers in this disease. AIM: To assess any difference in the sensitivity-specificity relationship between these neuroendocrine markers regarding various control populations and disease extent. METHOD: A total of 146 patients were prospectively assessed clinically and biologically. Serum marker titrations were performed using commercial immunoradiometric assays (NSE, CGA) or ELISA (ProGRP). Areas under receiver operating characteristic curves (AUC-ROC) were calculated in order to assess the sensitivity-specificity relationship of each marker and to compare marker accuracy. Maximum Youden indices were used to determine marker thresholds able to produce the best overall diagnostic information. RESULTS: Assessing the sensitivity in the SCLC population and the specificity in benign lung disease, ProGRP demonstrated the best sensitivity relationship in as much as its AUC-ROC was significantly greater than the ones calculated using NSE and CGA (respective values, 0.95, 0.89, 0.70; two-tailed Z-test <0.05). The ProGRP threshold value, which offered the best sensitivity-specificity relationship was 53 pg/ml corresponding to a 0.80 sensitivity and a 0.96 specificity. In addition, when specificity was assessed in NSCLC and again the sensitivity in the whole SCLC population, ProGRP continued to demonstrate a greater AUC-ROC in comparison with other markers. Using the 53 pg/ml threshold the specificity of this marker was excellent with no false positives in NSCLC. On the other hand, none of the markers were able to discriminate limited from extensive SCLC as suggested by the fact that AUC-ROC, constructed when sensitivity was defined as a positive test in extensive disease and specificity as a true negative test in limited disease, did not reach the upper left octant (AUC 0.65, 0.71 and 0.63 for ProGRP, NSE and CGA, respectively). CONCLUSION: ProGRP yields the best sensitivity-specificity feature in SCLC, a result deserving further studies designed to evaluate the clinical applicability of this marker.     

Neuron-specific enolase and chromogranin A as markers of neuroendocrine tumours.

Circulating neuron-specific enolase (NSE) and chromogranin A (CgA) were measured in 128 patients with neuroendocrine tumours (NET) to compare their sensitivity and specificity, to investigate factors associated with elevated serum levels and to determine the usefulness of these markers in the follow-up of NET patients. NSE (Cispack NSE, Cis Bio International, Gif-sur-Yvette, France; normal <12.5 microg l(-1)), and chromogranin A (CgA-Riact, Cis Bio International, normal <100 microg l(-1)) were measured in 128 patients without renal insufficiency. There were 99 patients with gastroenteropancreatic (GEP) NET, 19 with medullary thyroid carcinoma and ten with phaeochromocytoma. Fifty-three patients with non-NET were studied as controls. Serum NSE and CgA levels were elevated in 48 (38%) and 76 (59%) of the 128 NET patients respectively. In all groups of NET patients, CgA proved to be more sensitive than NSE. NSE and CgA had a specificity of 73% and 68% respectively. Immunostaining for NSE was positive in three out of eight controls with elevated CgA levels, whereas immunostaining for CgA and synaptophysin was negative in all cases. Elevated CgA levels were significantly associated with two independent parameters, namely the presence of other secretions (P = 0.0001) and a heavy tumour burden (P = 0.001). Elevated NSE levels were exclusively associated with poor tumour differentiation (P = 0.01). Among six patients with NET followed for 11-37 months, CgA appeared to be a better marker of tumour evolution than NSE. We suggest that CgA ought to be the only general marker screened in NET patients.     

Chromogranin A, neuron-specific enolase and synaptophysin as neuroendocrine cell markers in the diagnosis of tumours of the gastro-entero-pancreatic system

Neuroendocrine (NE) tumours of the gastro-entero-pancreatic tract were analysed immunohistochemically for the expression of chromogranin A, neuron-specific enolase and synaptophysin. In all cases at least one marker was present and in 17 out of 19 investigated neoplasms, at least one of the three markers could be demonstrated in more than 75% of the NE tumour cells. Monoclonal antibody chromogranin A stained a much higher proportion of NE cells in tumours with hormonal activity than in hormonally inactive ones. Immunostaining of the primary tumour as compared to its respective metastases was almost identical. Thus, chromogranin A, neuron-specific enolase and synaptophysin identify NE tumours and their metastases regardless of their localization and their state of hormonal activity. As 'panendocrine' markers of NE tumours they are of special diagnostic value in NE tumours that do not produce hormones and peptides.     

Association between histological type and neuroendocrine differentiation on drug sensitivity of lung cancer cell lines.

Because most small-cell lung cancers (SCLC) are initially chemosensitive and express neuroendocrine (NE) cell markers and most non-SCLC tumors (NSCLC) are chemoresistant and do not express NE cell markers, we investigated the association between morphological type and NE cell differentiation with in vitro chemosensitivity. We tested a panel of 55 lung cancer cell lines established from previously untreated patients. These were tested against five cytotoxic drugs commonly used in the therapy of lung cancer, using the MTT assay. For comparative purposes, we also tested cell lines established from previously treated patients with SCLC and from colorectal tumors. The logarithms of the IC50 values of all of the cell lines were normally distributed, permitting the use of Student's t-test for assessment of differences. In general, the in vitro sensitivities of SCLC, NSCLC, and colorectal cell lines mirrored the clinical experience with these tumor types. Cell lines started from previously treated patients with SCLC were more resistant than those from previously untreated patients who responded to initial therapy. For all of the cell lines, the sensitivities to the five drugs tested were highly significantly correlated with each other. Thus, for comparative purposes, each group could be assigned an average standardized mean rank. About 15% of NSCLC tumors express multiple neuroendocrine (NE) cell markers and 4 of 5 lines from these NSCLC-NE tumors were relatively chemosensitive, similar to SCLC lines and significantly different from other NSCLC lines. Other NE cell lines tested included bronchial carcinoids and cell lines from small-cell carcinomas arising in extra-pulmonary locations (ExPuSC).(ABSTRACT TRUNCATED AT 250 WORDS)     

神经内分泌分化与p53对非小细胞肺癌预后的影响

目的探讨神经内分泌(neuroendocrine,NE)分化标志物嗜铬蛋白A(CgA)、突触素(Syn)、神经内分泌颗粒和p53对非小细胞肺癌(non-small cell lung carcinoma,NSCLC)预后的影响。方法62例NSCLC进行含铂类方案新辅助化疗1~2个周期,然后手术治疗,以免疫组化法检测术后标本中NE分化标志物CgA、Syn及p53,以电镜观察标本中神经内分泌颗粒,随访3年,结果采用SPSS11.0统计软件,进行生存率分析。结果p53及NE分化标志物联合p53对非小细胞肺癌患者的无病生存期在统计学上均有显著性差异(P<0.05),NE( )p53(-)患者生存率较高。说明p53和NE分化联合p53与非小细胞肺癌患者的预后有关。结论神经内分泌分化和p53两者联合,用于非小细胞肺癌患者无进展生存期评价具有一定的临床价值。     

Peripheral airway cell differentiation in human lung cancer cell lines

Clara cells and type II pneumocytes are the progenitor cells of the bronchioles and alveoli, respectively. These peripheral airway cells (PAC) contain characteristic cytoplasmic structures and express surfactant associated proteins. PAC cell markers are expressed by many pulmonary adenocarcinomas having papillary and/or lepidic growth patterns, which are characteristics of the bronchioloalveolar and papillary subtypes. We investigated the expression of PAC markers in a panel of 41 lung cancer cell lines. Ultrastructural studies demonstrated the presence of cytoplasmic structures characteristic of Clara cells or of type II pneumocytes in 9 of 34 (26%) non-small cell lung cancer cell lines, including 7 of 17 (41%) adenocarcinomas, one squamous cell carcinoma, and one large cell carcinoma. Of interest, the cytoplasmic structures were present in 5 of 6 (83%) cell lines initiated from papillolepidic adenocarcinomas. In addition, we examined the lines for expression of the surfactant associated proteins SP-A, SP-B, and SP-C. Eight of the nine cell lines containing cytoplasmic inclusions characteristic of PAC cells also expressed protein and/or RNA of SP-A, the major surfactant associated protein. Five of these lines expressed SP-B RNA (either constitutively or after dexamethasone induction), while a single line expressed SP-C only after dexamethasone induction. None of six small cell lung cancer cell lines examined expressed any of the PAC markers. Thus, PAC markers are expressed frequently (but not exclusively) in pulmonary adenocarcinoma cell lines, especially in those initiated from tumors having papillolepidic growth patterns. The establishment and identification of multiple cell lines expressing PAC features provide an important new resource for biological and preclinical therapeutic studies.     

小细胞肺癌神经内分泌起源标志物的研究进展

[摘要] 小细胞肺癌（small cell lung cancer,SCLC）具有生长分数高、倍增时间短、侵袭性极高的特点,往往在诊断该病的时候就已发生远处转移,若不对其病情进行及时有效的治疗,患者将在2~3 个月内死亡。如果SCLC能够在局限期得到诊断,那么将有20%～25%的患者在接受联合化疗与放疗后,可以获得一个较长生存期。因此,寻找特异性和敏感性高的血液肿瘤标志物,为SCLC早期诊断提供可靠、简便、安全、耐受性好等依据对治疗SCLC极其重要。本文就近年来SCLC神经内分泌起源标志物嗜铬粒蛋白A、神经特异性烯醇化酶、促胃泌素释放肽、脑型肌酸激酶同工酶BB、神经细胞黏附分子、突触素等的研究进展进行综述。     

肺癌神经内分泌分化与术后生存关系探讨

目的 探讨非小细胞肺癌神经内分泌(NSCLC—NE)分化与患者手术后生存关系。方法 收集1997年4月至1999年4月98例肺癌手术切除病理标本，采用免疫组化标记特异性烯醇化酶(NSE)及突触素(SY)，并按强弱区分为“ 、 、 ”。对同一手术病例标本采用电镜观察特异性NE颗粒。术后病例随访36月，最长60月。采用Cox多因素风险模型分析NSCLC-NE分化与患者术后生存的关系。结果 91例为非小细胞肺癌。非小细胞肺癌NF阳性表达率为63．7％(58/91)，其中NSE阳性表达54例(59．3％)，SY阳性表达22例(24．1％)，电镜观察NE持异性颗粒30例(33．0%)。结合免疫组化和电镜观察，NSCLC-NE分化44例(48．4％)。Cox模型多因素分析结果表明NSCLC-NE分化者术后生存时间明显缩短(P=0.048)。术后生存与肺癌细胞分化程度(P=0．006)、病理分期(P=0．001)、NE表达强弱(P=0．054)有密切关系。结论 NSCLC-NE分化与肿瘤细胞分化和患者术后生存有关。采用NE标志物标记肿瘤，并观察其强弱改变．对术后评估具有较重要的参考意义，可作为为临床判断患者预后指标之一。     

Radiosensitivity related to neuroendocrine and endodermal differentiation in lung carcinoma lines

A panel of human lung carcinoma lines was studied with respect to hormone production and intermediate filament expression to distinguish between endodermal and neuroendocrine differentiation. An index of the degree of neuroendocrine differentiation of each line was derived from the presence or absence of hormone production, cytokeratins, neurofilaments and an embryonic endodermal cell marker, which allowed identification of three groups showing high, intermediate or low neuroendocrine expression. This grouping correlated well with the in vitro radiosensitivity of the lines, those expressing pure neuroendocrine features being significantly more radiosensitive than those with an endodermal phenotype, with the intermediate group having intermediate sensitivity. Use of such an index might predict those patients likely to benefit from the use of radiotherapy in their management.     

The prognostic significance of circulating neuroendocrine markers chromogranin a, pro-gastrin-releasing peptide and neuron-specific enolase in patients with advanced non-small-cell lung cancer.

BACKGROUND: Chromogranin A (CGA), Pro-gastrin-releasing peptide (ProGRP) and neuron-specific enolase (NSE) are known as immunohistochemical tissue markers closely associated with neuroendocrine differentiation in non-small-cell lung carcinoma (NSCLC). The aim of the present study was to assess the value of serum levels of these markers in predicting response to chemotherapy and survival of patients with unresectable NSCLC. METHODS: The study included 67 patients with advanced NSCLC treated with chemotherapy. Before treatment, serum levels of CGA, ProGRP and NSE were measured with commercial kits. RESULTS: No association was found between serum NSE and age, gender, histology, performance status or extent of the disease. Distribution of serum CGA differed significantly according to gender and histology, with higher levels being found in men (p = 0.01) and in squamous cell carcinoma (p = 0.01). Serum ProGRP levels correlated with disease extent, being higher in patients with metastatic disease (M1) than in those with locoregional disease (M0; p = 0.02). The association of NSE, CGA and ProGRP levels with response to chemotherapy was not significant. While NSE had no impact on survival, the median survival was shorter for patients with elevated serum CGA and longer for patients with high ProGRP levels. Association with survival was significant when the Classification and Regression Tree (CART)-derived or median cutoff points were explored. On inclusion in multivariate Cox models, both CGA and ProGRP retained significance with high levels showing an opposite effect on survival [CART-derived cutoff points: CGA, relative risk (RR) -4.0; p < 0.001, and ProGRP, RR -0.4; p = 0.006, and median cutoff points: CGA, RR -1.8; p = 0.04, and ProGRP, RR -0.5; p = 0.03]. The combined use of CGA, ProGRP and NSE allowed for definition of two sets of patients with significantly different median survival times (25.2 vs. 8.8 months, p = 0.0001). CONCLUSIONS: In the circulation, CGA and Pro-GRP appear to bear important information related to the prognosis for NSCLC patients before chemotherapy. While a high CGA before treatment was found as an unfavorable prognostic determinant, a high ProGRP conferred a survival advantage. The combined use of serum CGA, ProGRP and NSE may supply additional information to prognosis.     

Establishment and characterization of a human large cell lung cancer cell line with neuroendocrine differentiation

Characterization of a human lung cancer cell line is reported. This cell line was established from a patient referred to Nemazi Hospital of Shiraz University of Medical Sciences with a diagnosis of poorly differentiated carcinoma. Sterile sample from peritoneal effusion was taken and immediately cultured in RPMI-1640 medium containing 20% FBS, at 37 degrees C with 5% CO2. This cell line has been in continuous culture for more than one year and has been named as Mehr-80. Several features of the cell line were investigated, including growth characteristics, electron microscopic features, cloning efficiency in soft agar, expression of various antigenic markers, chromosomal and DNA analysis. On the basis of morphological and immunohistochemical analysis of Mehr-80, it is possible to conclude that this cell line is characterized by features similar to those reported for large cell carcinoma with neuroendocrine differentiation (LCCND). This cell line will be a valuable in vitro tool for further studies on lung cancers.     

Hierarchical clustering of lung cancer cell lines using DNA methylation markers.

Recent analyses of global and gene-specific methylation patterns in cancer cells have suggested that cancers from different organs demonstrate distinct patterns of CpG island hypermethylation. Although certain CpG islands are frequently methylated in many different kinds of cancer, others are methylated only in specific tumor types. Because distinct patterns of CpG island hypermethylation can be seen in tumors from different organs, it seems likely that histological subtypes of cancer within a given organ may exhibit distinct methylation patterns as well. The goal of our study was to determine whether the patterns of CpG island hypermethylation could be used to distinguish between different histological subtypes of lung cancer. We analyzed the methylation status of 23 loci in 91 lung cancer cell lines using the quantitative real-time PCR method MethyLight. Genes PTGS2 (COX2), CALCA, MTHFR, ESR1, MGMT, MYOD1, and APC showed statistically significant differences in the level of CpG island methylation between small cell lung cancer (SCLC) and non-small cell lung cancer cell lines (NSCLC). Hierarchical clustering using a panel consisting of these seven loci yielded two major groups, one of which contained 78% of the SCLC lines. Within this group, a large cluster consisted almost exclusively of SCLC cell lines. Our results show that DNA methylation patterns differ between NSCLC and SCLC cell lines and suggest that these patterns could be developed into a powerful molecular marker to achieve accurate diagnosis of lung cancer.     

Immunohistochemical detection of cluster 1 small cell lung cancer antigen and chromogranin A in lung carcinomas.

Eighteen small cell lung cancers (SCLCs), 108 non-SCLCs (67 adenocarcinomas, 29 squamous cell carcinomas and 12 large cell carcinomas) were immunohistochemically examined for expressions of cluster 1 SCLC antigen/N-CAM and chromogranin A with monoclonal antibodies NCC-Lu-243 and anti-chromogranin A. The cell membranes of all the SCLCs and three of the 67 adenocarcinomas (4.5%) were stained for cluster 1 SCLC antigen/N-CAM. Eight of the 18 SCLCs (44.4%), and three of the 67 adenocarcinomas (4.5%) were stained for chromogranin A, but no squamous cell carcinoma or large cell carcinoma was stained for both antigens. Two of the three adenocarcinomas which expressed cluster 1 SCLC antigen/N-CAM had been suspected of being either SCLC or poorly-differentiated adenocarcinoma cytologically, and were resected after chemotherapy and radiotherapy. Histologically, they were poorly-differentiated adenocarcinoma with rosette-like tubules. The remaining one was moderately-differentiated papillary adenocarcinoma resembling bronchial surface epithelial cells without mucin (BSE type adenocarcinoma). The three adenocarcinomas which expressed chromogranin A were well- to moderately-differentiated BSE type adenocarcinomas, and stained tumor cells were distributed sparsely as neuroendocrine cells in the normal bronchial mucosa. One of them also expressed cluster 1 SCLC antigen/N-CAM. In the present study, we demonstrated the usefulness of NCC-Lu-243 in the immunohistochemical detection of adenocarcinomas with neuroendocrine features.     

Pulmonary large cell carcinoma expressing neuroendocrine markers: the morphological, biological, and neuroendocrine features of their cell lines and surgical cases.

A cell line expressing neuroendocrine (NE) markers, designated as KTS9, was established from a human large cell carcinoma of the lung using serum-free medium, ACL-3. KTS9 cells showed morphological characteristics of large cell undifferentiated carcinoma (LCUC) and expressed some general NE markers including neuron-specific enolase (NSE), protein gene product (PGP) 9.5, neural cell adhesion molecule (N-CAM), synaptophysin and neurofilaments (NF) of 200 kd. Some cells of this cell line were positive to chromogranin-A (CG-A), but did not express Leu7 or aromatic L-amino acid decarboxylase (AADC). Such a cell line derived from LCUC with NE properties has not previously been reported. The biological and NE properties of the KTS9 cell line were compared with those of 2 surgical cases of LCUC with NE markers and of the KTA7 cell line previously reported to derive from large cell carcinoma and to possess NE markers such as alpha-hCG, PGP9.5 N-CAM and AADC. Tumor cells of 2 large cell carcinomas expressed NSE, PGP9.5, N-CAM and NF. The KTS9 and KTA7 cell lines and 2 large cell carcinomas were thus considered to be LCUCs with NE differentiation. Both lines had the morphological characteristics of LCUC, relatively short doubling time and discordant expression of NE markers, indicating them to be closely related to the variant type of small cell carcinoma cell lines and thus possibly to represent high-grade malignancy. They may be useful for examining the biological behavior and NE features of large cell-type NE tumors of the lung.     

嗜铬素A和突触素在肾上腺肿瘤中的表达及其临床意义

目的探讨嗜铬素A（CgA）和突触素（Syn）在各型肾上腺肿瘤中的表达及其临床意义。方法对69例肾上腺肿瘤和4例正常肾上腺组织运用免疫组化方法进行组织染色,研究C外和Syn的表达,并采用x^2检验进行统计学分析。结果在正常肾上腺组织,CgA和Syn主要表达于髓质。CgA在全部25例嗜铬细胞瘤中均呈阳性表达,而在肾上腺皮质肿瘤中极少表达;Syn在肾上腺皮质腺瘤、皮质癌、嗜铬细胞瘤及肾上腺转移癌的阳性表达率分别为96．4％（27／28）、87．5％（7／8）、96．0％（24／25）和75．0％（6／8）。结论CgA在肾上腺皮质与髓质肿瘤间、嗜铬细胞瘤与恶性嗜铬细胞瘤间的表达率差异有统计学意义。CgA、Syn可作为标志物对各型肾上腺肿瘤进行鉴别诊断。     

Biomarkers for small cell lung cancer: neuroendocrine, epithelial and circulating tumour cells

Small cell lung cancer (SCLC) is characterised by an aggressive clinical course with invariable resistance to chemotherapy despite initially high response rates. There has been little improvement in outcome over the past few decades, with no breakthrough yet in targeted therapies. Recent preclinical data and studies of circulating tumour cells (CTCs) highlight distinct cellular heterogeneity within SCLC. Better understanding of how these phenotypes contribute to metastasis and tumour progression might pave the way for development of more successful targeted therapies. Here we review these studies, their implications for future research and for the incorporation of biomarkers reflecting neuroendocrine, epithelial and mesenchymal phenotypes in clinical studies.