Lack of prognostic significance of neuroendocrine differentiation and stem cell antigen co-expression in resected early-stage non-small cell lung cancer.

BACKGROUND: Neuroendocrine (NE) carcinomas of the lung exhibit expression of various stem cell antigens, and except for carcinoid tumours, carry a poor prognosis. Despite the fact that 10%-30% of all non-small cell lung carcinomas (NSCLC) which are not classified as NE carcinomas also show expression of NE markers, data on their prognostic significance are conflicting and analyses of the expression and relevance of stem cell antigens in this subgroup are lacking. MATERIALS AND METHODS: Tissue specimens of 100 resected early-stage NSCLC were analyzed by immunohistochemistry for the expression and prognostic significance of NE markers. Moreover, the subgroup of NSCLC with NE differentiation (ND) were assessed for the expression and prognostic significance of the stem cell antigens CD117, CD133 and breast cancer resistance protein-1 (ABCG2). RESULTS: ND correlated significantly with adenocarcinoma histology (p=0.035), but not with prognosis. In the subgroup of ND-NSCLC (n=80), the stem cell antigens CD117, CD133 and ABCG2 were expressed in 51%, 14% and 33% of the cases, but likewise, showed no association with prognosis or clinicopathological characteristics. CONCLUSION: This study indicates that neither ND, nor co-expression of the stem cell antigens CD117, CD133 or ABCG2, have a prognostic significance in resected early-stage NSCLC.

Mesenchymal stem cells in non-small cell lung cancer--different from others? Insights from comparative molecular and functional analyses.

BACKGROUND: Cancer-associated fibroblasts (CAF) play a vital role in lung cancer initiation and progression. Although mesenchymal stem cells (MSC) are considered progenitor cells of fibroblasts and show cancer modulating abilities themselves, analyses on their presence and properties in lung cancer are lacking so far. METHODS: We performed a comparative molecular and functional analysis of MSC derived from non-small cell lung cancer (NSCLC) and corresponding normal lung tissue (NLT) of a total of 15 patients. MSC were identified and selected according to their mesenchymal multilineage differentiation capability and surface marker profile. RESULTS: Compared to NLT-MSC, NSCLC-MSC showed accelerated growth kinetics and reduced sensitivity to cisplatin. Karyotyping, comparative genomic hybridization and multiplex fluorescence in situ hybridization revealed no chromosomal aberrations. However, gene expression profiling of NSCLC- and NLT-MSC indicated variable expression of 62 genes involved in proliferation, DNA repair, apoptosis, extracellular matrix synthesis, tissue remodeling and angiogenesis. Differential expression of the selected candidate genes butyrylcholinesterase, clusterin and quiescin Q6 sulfhydryl oxidase 1 was validated by quantitative real-time PCR and, on protein level, by immunohistochemical analyses of original tumor tissue. Upon exposure to tumor cell-conditioned medium or transforming growth factor-ß, both, NSCLC-MSC and NLT-MSC acquired expression of α-smooth muscle actin (α-SMA), a major characteristics of CAF. CONCLUSIONS: This study indicates that NSCLC tissue contains MSC with specific molecular and functional properties. These cells might represent a progenitor reservoir for CAF and thus crucially contribute to lung cancer progression.

Stage-specific embryonic antigen-4 is expressed in basaloid lung cancer and associated with poor prognosis.

Basaloid carcinoma represents a rare variant of nonsmall cell lung cancer (NSCLC), which has shown a poor prognosis in a number of studies. Although it is considered to derive from a pluri- or multipotent pulmonary stem cells, little is known about the expression and clinical significance of stem cell antigens in this variant. Stage-specific embryonic antigen-4 (SSEA-4) was analysed by immunohistochemistry in 38 patients with resected early-stage basaloid NSCLC who had a median follow-up of 72.9 months. The expression of SSEA-4 was related to clinico-pathological characteristics, to the expression of the adult stem cell antigens CD117, CD133 and breast cancer resistance protein 1 (BCRP1), and to prognosis. SSEA-4 was positive in 37% of the specimens and showed no association with clinico-pathological characteristics or the expression of adult stem cell antigens. Cox proportional hazards regression analysis revealed a 6.0-fold increased risk of relapse (p = 0.001) and a 4.2-fold increased risk of disease-related mortality (p = 0.017) in SSEA-4-positive patients, while SSEA-4-negative patients showed a prognosis comparable with that of other early-stage NSCLC. SSEA-4 is expressed in a fraction of basaloid NSCLC and is associated with poor prognosis.

Are we missing the target? Cancer stem cells and drug resistance in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in Western countries. Despite novel molecular therapies, the majority of patients with advanced or metastatic disease show rapid progression and a median survival time of not more than 18 months. In the last decade, there has been increasing evidence that cancer stem cells (CSC) play a pivotal role in drug resistance, tumour regeneration and metastasis of various cancer entities including lung cancer. In this review, we discuss the evidence for stem cells in NSCLC, their predictive and prognostic significance, their specific mechanisms of resistance and potential targets and strategies for eradication of these cells. Consideration of the specific properties of CSC in lung cancer therapy might substantially contribute to increased response and prolonged survival rates in this disease.

The gefitinib long-term responder (LTR)--a cancer stem-like cell story? Insights from molecular analyses of German long-term responders treated in the IRESSA expanded access program (EAP).

BACKGROUND: In selected patients with advanced non-small cell lung cancer (NSCLC) the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor (TKI) gefitinib (IRESSA) shows response rates of ≥ 70% and a significant prolongation of progression free survival (PFS). However, cogent biomarkers predicting long-term response to EGFR-TKIs are yet lacking. Cancer stem-like cells (CSC) are thought to play a pivotal role in tumor regeneration and appear to be influenced by the EGFR-pathway. This makes them a promising candidate for predicting long-term response to EGFR-TKIs. MATERIALS AND METHODS: We analyzed pre-therapeutic tissue specimens of a rare and specific subset of previously treated German patients with advanced NSCLC who experienced ≥ 3 year response to gefitinib within the International IRESSA EAP. 11/20 identified long-term responders (LTRs) had appropriate tissue specimens available. Those were analyzed for EGFR and k-ras (Kirsten rat sarcoma) mutations, EGFR and c-met (met proto-oncogene) amplifications and protein expression of EGFR, E-cadherin/vimentin and the CSC antigens CD133 and BCRP1 (breast cancer resistance protein 1). The results were compared to primary resistant patients (RPs) and intermediate responders (IRs) showing a median response of 8.6 months. RESULTS: Each group consisted of 6 women and 5 men, with 1 squamous cell carcinoma (SCC) and 10 adenocarcinoma (AC). Along the LTRs, all but the SCC had EGFR mutations, whereas the RPs had no EGFR, but k-ras mutations in 5/11 cases. 8/11 IRs had EGFR and 3/11 k-ras mutations, of which 2 occurred concomitantly. One patient of each group had an EGFR and/or c-met amplification. EGFR and E-cadherin/vimentin expression was not different between the groups, whereas CD133 was expressed only in 4/10 LTRs and BCRP1 predominantly in responders. The LTRs showed a substantially longer mean PFS to previous therapies, a substantially lower number of metastatic sites and almost exclusively pulmonary or pleural metastasis. CONCLUSION: LTRs display established properties of EGFR-TKI responders. Antigens characterizing CSC might identify a fraction of LTRs and matter of interest for further evaluation.

Establishment and comparative characterization of novel squamous cell non-small cell lung cancer cell lines and their corresponding tumor tissue.

BACKGROUND: Cell lines play an important role for studying tumor biology and novel therapeutic agents. Particularly in pulmonary squamous cell carcinoma (SCC) the availability of cell lines is limited and knowledge about their representativeness for corresponding tumor tissue is scanty. MATERIALS AND METHODS: We established three novel SCC cell lines from fresh tumor tissue of 28 donors, including 8 SCC. Two cell lines were derived from different localizations of the same donor, i.e. primary tumor and lymph node metastasis. This represents a so far unique combination in lung cancer. The genotypes, gene expression profiles and mutational status of epidermal growth factor receptor (EGF-R) and Kirsten rat sarcoma (k-ras) of the cell lines and their corresponding tumor tissue were analyzed and compared. Moreover, the molecular characteristics were related to functional properties of the cell lines. Those comprised proliferation, motility and chemosensitivity. The cell lines were authenticated by single tandem repeat DNA typing. Tumorigenicity was analyzed in a murine xenograft model. RESULTS: Comparative genomic hybridization and multiplex fluorescence in situ hybridization revealed essential genetic similarities between the cell lines and their corresponding tumor tissue, but indicated also some genetic evolution and clonal selection. EGF-R or k-ras mutations were not detected. Gene expression profiling showed various differences between tumor tissue and cell lines affecting gene clusters associated with immune response, adhesion, proliferation, differentiation and angiogenesis. However, there were also common gene expression patterns reflecting the relationship between cell lines and their corresponding tumor tissue. Moreover, the molecular characteristics of the tumor tissue and the descendent cell line were associated with functional properties of the latter. All cell lines showed a unique, heterozygous human DNA profile and one cell line displayed rapid tumor formation in mice. CONCLUSIONS: Here, we demonstrate that cell lines represent a useful in vitro system for studying basic mechanisms in lung cancer, but cover only distinct molecular characteristics of the original tumor. Moreover, we present three novel, comprehensively characterized SCC cell lines.

The cancer stem cell antigens CD133, BCRP1/ABCG2 and CD117/c-KIT are not associated with prognosis in resected early-stage non-small cell lung cancer.

BACKGROUND: In various tumor entities, expression of cancer stem cell (CSC) antigens has been proven to be prognostically unfavorable. However, for lung cancer, the data are scant and conflicting. PATIENTS AND METHODS: The CSC antigens CD117/c-KIT, CD133 and breast cancer resistance protein-1 (BCRP1/ABCG2) were immunohistochemically analyzed in tissues from a total of 133 completely resected stage I/II non-small cell lung cancer (NSCLC) patients with a median follow-up time of 53.8 months. Their expression was related to clinicopathological characteristics, angiogenic features and prognosis. RESULTS: Cox proportional hazards regression analysis revealed no association between CSC antigens, disease-free survival or overall survival (OS). However, in the subgroup of patients with relapse and tumors >3 cm, there was a trend towards worse OS upon expression of CD117 (hazard ratio=2.6, 95%, confidence interval=0.8-8.3, p=0.080). Except for CD133, which was overrepresented in T1 tumors (p=0.001), the CSC antigens were not linked to clinico-pathological characteristics or angiogenic features. CONCLUSION: In resected early-stage NSCLC, CSC antigens show no association with prognosis. However, in patients with relapse and tumors >3 cm, expression of CD117 might predict worse OS.

Correlation of EGFR mutations with chromosomal alterations and expression of EGFR, ErbB3 and VEGF in tumor samples of lung adenocarcinoma patients.

INTRODUCTION: Mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are frequently detected in lung adenocarcinomas with bronchioloalveolar (BAC) differentiation and have been associated with increased response to small molecule EGFR inhibitors in some clinical studies. However, further molecular characterization of tumor cells carrying EGFR mutations (EGFR-mut) is warranted. METHODS: By DNA sequencing, 120 patients with lung adenocarcinomas (70 tumors with BAC components) were screened for EGFR mutations within exons 18-21. Performing comparative genomic hybridization (CGH) and immunohistochemistry, chromosomal imbalances and protein expression levels of EGFR, ErbB3 and VEGF (vascular endothelial growth factor) were analyzed, respectively. RESULTS: EGFR mutations were detected in 20/120 tumors. Tumors with BAC components carried more frequently EGFR mutations compared to adenocarcinomas without BAC histology (17/70=24% vs 3/50=6.0%; p=0.012). In a subsequent matched-pair analysis, CGH-analysis demonstrated similar mean numbers of chromosomal imbalances for EGFR mutated and wild-type tumors (8.6 vs 7.8 gains; 2.4 vs 2.7 losses), respectively. Furthermore, tumors with mutated EGFR demonstrated gains in chromosomes 7p, 16p and 20q and losses in chromosome 8p. Interestingly, EGFR mutated tumors showed higher VEGF expression (p=0.03) while differences in EGFR expression were not statistically significant. CONCLUSION: EGFR gene mutations are frequently seen in lung adenocarcinomas with BAC differentiation and can be linked to chromosomal imbalances and increased VEGF expression.

Primitive and committed human hematopoietic progenitor cells interact with primary murine neural cells and are induced to undergo self-renewing cell divisions.

OBJECTIVE: Studies in animal models have indicated that hematopoietic progenitor cells (HPC) migrate and home to the central nervous system and might acquire neural features under specific circumstances. The interaction between HPC and the neural environment and the functional effect on hematopoiesis have not yet been defined. METHODS: CD34(+)133(+) cells from mobilized peripheral blood were cocultured with primary murine neurons or astrocytes. Chemotaxis and adhesive interactions were studied by applying beta(1)- and beta(2)-integrin function-blocking anibodies. The impact of neural feeder layers on integrin expression of HPC and the presence of appropriate adhesion ligands on neural cells were determined by immunostaining and flow cytometry. The hematopoietic long-term fate was monitored by time-lapse microscopy of individual cell-division history followed by long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) assays. Neural differentiation was assessed by immunostaining against specific neuronal and glial antigens. RESULTS: The 23.0% +/- 4.9% of HPC showed stromal cell-derived factor-1-induced migration toward neural cells, and 20.2% +/- 1.6% displayed firm beta(1)-integrin-mediated adhesion to astrocytes. The latter expressed appropriate adhesion ligands, stabilized beta(1)-integrin expression, and increased beta(2)-integrin expression of HPC. Neural differentiation of HPC could not be identified but astrocytes were able to induce limited self-renewing cell divisions of HPC and thus maintain 25.8% +/- 3.4% of the initial LTC-IC and 80.7% +/- 1.9% of the initial CFC. CONCLUSION: Human HPC are able to interact with neural cells and interaction maintains, albeit to a limited extent, the self-renewal capability of HPC.

Human mesenchymal stromal cells regulate initial self-renewing divisions of hematopoietic progenitor cells by a beta1-integrin-dependent mechanism.

In previous reports, we have demonstrated that only direct cell-cell contact with stromal cells, such as the murine stromal cell line AFT024, was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment, we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose, we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours, human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g., fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC, thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism.