Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma

Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression

Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas

BACKGROUND: High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear. RESULTS: We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability. CONCLUSIONS: Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas

Migration rules: tumours are conglomerates of self-metastases

Tumours are heterogeneous populations composed of different cells types: stem cells with the capacity for self-renewal and more differentiated cells lacking such ability. The overall growth behaviour of a developing neoplasm is determined largely by the combined kinetic interactions of these cells. By tracking the fate of individual cancer cells using agent-based methods in silico, we apply basic rules for cell proliferation, migration and cell death to show how these kinetic parameters interact to control, and perhaps dictate defining spatial and temporal tumour growth dynamics in tumour development. When the migration rate is small, a single cancer stem cell can only generate a small, self-limited clone because of the finite life span of progeny and spatial constraints. By contrast, a high migration rate can break this equilibrium, seeding new clones at sites outside the expanse of older clones. In this manner, the tumour continually ‘self-metastasises'. Counterintuitively, when the proliferation capacity is low and the rate of cell death is high, tumour growth is accelerated because of the freeing up of space for self-metastatic expansion. Changes to proliferation and cell death that increase the rate at which cells migrate benefit tumour growth as a whole. The dominating influence of migration on tumour growth leads to unexpected dependencies of tumour growth on proliferation capacity and cell death. These dependencies stand to inform standard therapeutic approaches, which anticipate a positive response to cell killing and mitotic arrest

Monodispersal and deoxyribonucleic acid analysis of prostatic cell nuclei.

We collected prostatic glands from 50 unselected autopsies at the Pathology Institute and compared their histologic sections with cytologic preparations and with results of photometric measurements of isolated prostatic cells and isolated nuclei. The results obtained with single cell photometry and flow-through cytophotometry proved to be comparable with one another and with the results of the cytologic and histologic studies. With these methods of cytophotometry we could differentiate inflammatory conditions, microcarcinomas and frank carcinomas from normal and hyperplastic prostatic tissue. We had difficulties, however, preparing adequate suspensions of cell nuclei from chronic fibrosing prostatitis. Our results indicate that it should be possible for diagnostic purposes to combine the technique of fine needle biopsy of the prostate with that of flow-through cytophotometry and to use the combined techniques for studying diseases of other organs. </jats:p

Apoptosis resistance in pigmented villonodular synovitis

Objective: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. Methods: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multiparametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. Results: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p=0.037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expression of both caspase3 (p=0.017) and bcl-2 (p=0.0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found). Conclusion: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions

Comparative analysis of cell populations involved in the proliferative and inflammatory processes in diffuse and localised pigmented villonodular synovitis

The aim of the present study was a comparative quantitative evaluation of cell populations involved in the proliferative and inflammatory compartment in both localised and diffuse pigmented synovitis villonodularis (PVNS). 15 cases of each localised and diffuse PVNS were examined by flow cytometry, immunohistochemistry, double immunofluorescence and confocal microscopy with quantitative evaluation of CD3-, CD4-, CD8-, CD20-, CD57-, CD55-, CD68-, CD163- and h4Ph positive (+) cells. The proliferative compartment of localised and diffuse PVNS was mainly composed of double-positive CD68+/h4Ph+ (CD163+/CD55+) synoviocytes. The number of doublepositive synoviocytes for macrophage and fibroblast markers was significantly higher in diffuse compared to localised PVNS. The accompanying inflammatory infiltrate showed a predominance of cytotoxic cells (CD8+, CD57+), whereby the number of CD3+ and CD20+ cells was significantly higher in localised PVNS. The number of CD57+ NK cells was significantly higher in diffuse PVNS. The proliferating macrophage- like synovial cells and the cytotoxic lymphocytes could contribute to the aggressive behaviour of localised and diffuse PVNS. Moreover, with regard to the quantitative differences in cell composition between diffuse and localised PVNS and their different clinical behaviour, further studies should continue to analyse localised and diffuse PVNS separately