ABSTRACT: Dysfunction in type I interferon (IFN) signaling occurs in patients with stage II or more advanced cancer. After screening the effects of a panel of 12 melanoma cell lines on PBMCs of healthy volunteers of IFNalpha signal pathway, two groups of melanoma cell lines could be identified one with stronger suppression (low pSTAT-1 group) than the other (high pSTAT-1 group). Comparative genomic hybridization (CGH) identified consistent amplification of 12q22-24 as a genomic marker for the immune suppressive melanoma cell lines. This region corresponded to higher transcription of the NOS1 gene located in that region in the low pSTAT1 group and NOS1 expression was identified as causal factor in melanoma induced immune suppression. Twelve melanoma cell lines were analyzed. Genomic DNA of the cell line sample was labeled with Cy5 and reference normal PBMC DNA was labeled with Cy3.
Project description:In this study, we sought to indentify stable individual traits in developing metastatic melanoma, assess their stability in time, and analyze typical dynamics and common trends of genomic changes in individual cases of late stage metastatic cancer. To this end, we compared consecutive recurrent metastases developed by a special group of eight individuals with advanced metastatic melanoma, all developing several recurrent metastases over several months to years of natural disease evolution. Twenty-six recurrent melanoma metastases were surgically isolated from 8 patients experiencing relapse after one or more successful treatment intervention(s) with no signs of residual disease. Patients experiencing recurrent metastases were labeled with capital letters; “A”, “B”, “C”, etc., their subsequent metastases as “A/1”, “A/2”, “B/1”, “B/2”, etc., while synchronous metastases in a given patient were labeled as “A/1a”, “A/1b” etc. Another 22 melanoma cell lines isolated and maintained as above were expanded from melanoma patients with rapid disease curse, for whom only one metastasis was available. As no extended follow up was possible in these cases, the cell lines are considered representative of random time points in the natural course of metastatic melanoma. These cell lines were labeled with Arabic numbers, as “1”, “2”, ”3”, etc.
Project description:Dysfunction in type I interferon (IFN) signaling occurs in patients with stage II or more advanced cancer. After screening the effects of a panel of 12 melanoma cell lines on PBMCs of healthy volunteers of IFNalpha signal pathway, two groups of melanoma cell lines could be identified one with stronger suppression (low pSTAT-1 group) than the other (high pSTAT-1 group). Comparative global gene expression between two groups identified 6771 differential expression genes. This gene list indicated down regulation of IFNalpha signal in immune suppressive melanoma cells. To evaluate this gene list for predictive power on IFNalpha signal modulatory function, we analyzed gene expression 41 independent melanoma cell lines and heat map clusters these cell lines into two groups, one with strong immune suppressive function and other with less effect. Fifty-three melanoma cell lines were analyzed with twelve technical repeats.
Project description:Search for copy number imbalances and allelotype in 971 early stage breast cancer using molecular inversion probe arrays Affymetrix MIP arrays were performed according to the manufacturer's directions on DNA extracted from paraffin embedded formalin fixed Stage I and II Breast Cancers Copy number analysis of Affymetrix Oncoscan Version 1 MIP arrays was performed for 971 Stage I and II breast cancers.
Project description:Background: Ovarian carcinomas consist of at least five distinct diseases: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies. Methods: We have focused on the identification of clear cell carcinoma cell line models. A panel of 32 “ovarian cancer” cell lines has been classified into histological types using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis. Results: Many described ovarian clear cell lines have characteristic mutations (including ARID1A and PIK3CA) and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements. Conclusions: As with primary ovarian tumors, mutation status of cancer genes like ARID1A and TP53 and a general immuno-profile serve well for establishing histological type of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic “ovarian carcinoma” cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of SKOV3 and A2780 as models of high-grade serous carcinoma. The DNA copy number of 10 ovarian cancer cell lines was examined and changes in copy number of genes whose expression is assumed to be critical to the phenotype of ovarian clear cell carcinoma was evaluated. Copy number data was estimated from signal intensity on Affmetrix SNP 6.0 arrays. Copy number ratio values were generated in Partek Genomics Suite (v 6.6) using a Partek corporation distributed baseline file of normal (2N) genomic DNA and default parameters. Post import values were corrected for localized GC content using the inbuilt Partek feature based on methods described in Diskin et al. (Nucleic Acids Research. 2008. 36:19). The characteristic "literature reported histotype" is the reported histological subtype for each cell line from the originating laboratory or cell bank (repository). The characteristic "Predicted histology" is based on parameters described in Anglesio et al. (PLOS ONE 2013. in press), including immunohistochemical phenotype, presence of typical mutations, consistency in growth characteristics, and DNA copy number. All cell lines were grown under recomended conditions, collected near confluence (80%) and not subjected to any experimental treatments or modifications.
Project description:DNA copy number profiling for all primary tumor samples and cell lines was performed by ROMA, a form of comparative genomic hybridization. The aim was to identify commonly amplifiied and deleted regions. 538 primary tumor samples and 253 human cell lines were analyzed.
Project description:DNA copy number profiling for all primary tumor samples and HCC cell lines was performed by ROMA, a form of comparative genomic hybridization. The aim was to identify commonly amplifiied and deleted regions across human HCC. 88 primary HCC samples and 12 human HCC cell lines were analyzed.
Project description:Human pluripotent stem cells commonly undergo adaptive changes during prolonged passaging in vitro. We have developed a new laminin-521 based method for self-renewal of human pluripotent stem cells. SNP6.0 array was used to assess genetic stability of several human ES cell lines cultured in the new system with two lines (H1 and HS401) cultured on feeders (standard conditions) Two main objectives in this study: To compare the number of the CNVs in human ES cells at early and late passages; for example, samples HS980 p6 and HS980 p31 represent HS980 cells at passages 6 and 31, respectively. The overall conclusion is that the observed number of CNVs is similar in the cells at early and late passages. This analysis can be remade without the reference samples. To compare the number of CNVs in control human ES cells (H1 and HS401) grown under standard protocol with that in cells (HS980, HS983a, HS999, HS916, and HS1001) grown under a new, developed by us, protocol. Because the cells were from different individuals, we could not compare them directly. Instead, we compared them to a set of samples from 75 healthy individuals, and then with each other. For the Reference These samples had been hybridized in the same lab as our samples, but for a different project; and the data were kindly provided by Prof. Kere. However, the same overall conclusion would result from an other sufficiently large dataset of healthy human individuals. Please note that 75 healthy individual samples had been hybridized in the same lab as our samples and the data were kindly provided by Prof. Kere, which is linked as a Series supplementary file ('75_samples_ref.REF'). It is a Reference Model File that Affymetrix Genotyping Console software has created based on data from 75 healthy individuals (i.e. processed file based on the CEL files of the 75 healthy individuals). The file derives from the "CN/LOH Reference Model File Creation and Analysis (Batch Sample Mode)" process according to the Genotyping Console manual. It contains SNP-wise information on the observed distribution of hybridization intensities of a set of samples that are assumed to be diploid in copy number (for most part of their genomes at least). This file is then used to compare the intensities observed in the CEL files of interest to infer deviations from the reference as CNVs. Thus, in combination with the CEL files (from each ES cell line), this file enables the exact replication of the CNV analyses done for the various human ES cell lines study. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted cultured human ES cells. Several lines were analysed at different time points of the experiment. The analysis was done using default parameters and comparing each sample against a reference set of 75 healthy individuals provided by Juha Kere.
Project description:Analysis of DNA from fixed tissues specimens of 58 primary uveal melanomas, with known clinical outcome, to determine gene copy number variations that were associated with survival. Abstract: Uveal melanomas can be stratified into subgroups with high or low risk of metastatic death, according to the presence of gross chromosomal abnormalities. Where a monosomy 3 uveal melanoma is detected, patient survival at three years is reduced to 50%. However, approximately 5% of patients with a disomy 3 tumour ultimately develop metastasis, and a further 5% of monosomy 3 uveal melanoma patients’ exhibit disease-free survival for more than five years. Despite extensive knowledge of the chromosomal abnormalities occurring in uveal melanoma, the genes driving metastasis are not well defined. Gene copy number variations occurring in a well-characterised cohort of 58 formalin-fixed, paraffin-embedded uveal melanoma samples were identified using the Affymetrix SNP 6.0 whole genome microarray. Four genetic sub-groups of primary uveal melanoma were represented in the patient cohort: 1) disomy 3 with long-term survival; 2) metastasizing disomy 3; 3) metastasizing monosomy 3; and 4) monosomy 3 with long-term survival. Cox regression and Kaplan-Meier survival analysis identified three genes that were associated with differences in patient survival. Patients with an amplification of CNKSR3 (6q) or RIPK1 (6p) demonstrated longer survival than those with gene deletions or no copy number change (log rank, p=0.022 and p<0.001, respectively). Conversely, those patients with an amplification of PENK (8q) showed reduced survival (log rank p<0.001). CNKSR3, RIPK1 and PENK are novel candidate metastasis regulatory genes in uveal melanoma. This is the first report of amplification of a specific gene on 6p that is associated with improved uveal melanoma patient survival and suggests that the development of uveal melanomas with a propensity to metastasise may be limited by genes on 6p. 58 samples in total. Ten disomy 3 with long-term survival. Fifteen disomy 3 with metastasising. Seventeen monosomy 3 with long-term survival. Sixteen monosomy 3 metastasising.
Project description:Melanoma cell lines were genotyped to evaluate copy number differences between nodular melanoma (NM) and superficial spreading melanoma (SSM). Cell lines were also evaluated for copy number alterations in the SKP2/p27 axis. Affymetrix SNP arrays were performed according to manufacturer's instructions using DNA extracted from 18 melanoma cell lines and 4 melanocyte controls. Affymetrix SNP6.0 Array data for melanoma cell lines Copy number analysis of Affymetrix SNP 6.0 arrays was performed on 18 melanoma cell lines including 2 primary superficial spreading melanoma, 2 primary nodular melanoma, 2 metastatic nodular melanoma, and 12 metastatic cell lines. Four melanocyte control lines were also evaluated including 2 immortalized melanocyte cell lines (Hermes 1 and 2B) and 2 normal melanocyte lines cultured from neonatal foreskin (HEM-N and HEM-LP) that were used to construct the baseline for copy number analysis.
Project description:Circulating tumor cells (CTCs) are critical in the development of distant organ tumor metastasis, and are associated with advanced cancer stage and poor patient outcome. Here, we present the first genome-wide nucleotide-level characterization of CTCs. Our single-nucleotide polymorphism (SNP) analysis in patients with melanoma involved: 1) global comparative genomic analysis of CTCs and matched regional metastases, 2) identification of key genomic aberrations in CTCs, 3) verification of these target genes in aggressive distant tumor metastases, and 4) evidence of selective expression and functional consequence of CTC-associated genes in melanomas. We report 131 aberrant loci in CTCs that are potentially pro-metastatic, and show that such expression of a 5-marker gene panel (CSMD2, CNTNAP5, FLJ14051, ADAM6, TRPM2) in melanomas confers prognostic utility. Successful treatment of melanoma requires understanding of the metastatic process and identification of patients with tumors most likely to develop aggressive metastatic disease. Melanomas are heterogeneous, and CTCs have long been recognized as vehicles for cancer spread, representing particularly aggressive tumor clones that can evolve into successful clinical metastases. Elucidation of genomic aberrations in CTCs will aid in the development of prognostic biomarkers and therapeutic strategies to target CTCs to prevent or control distant cancer spread. This study provides the first detailed genomic confirmation of the close relation between CTCs and tumor metastases, and illustrates how CTCs can be utilized as a novel approach and rational source for identification of pro-metastatic genes in cancer research. Three individual patient cohorts were utilized in the study. CNV and LOH loci were evaluated initially in metastatic melanoma patients (n=13) in a discovery cohort. SNP loci that harbored CNV/LOH in CTCs were then separately verified for: (a) presence in distant organ metastasis (AJCC Stage IV melanoma) (n=27), and (b) relevance to prognosis in regional melanoma metastasis (AJCC Stage III melanoma) (n=35). The first discovery patient cohort group was utilized for capture of CTCs, and consisted of peripheral blood mononuclear cell (PBMC) and tumor specimens from metastatic melanoma patients (n=13). CTC-related loci were verified in a second cohort of patients with Stage IV distant organ metastases (n=27), including 15 brain, 4 lung, and 8 gastrointestinal (bowel, liver) metastases. The third patient cohort consisted of early passage (<12) established melanoma cell lines derived from 35 regional melanoma metastases (Stage III) for evaluation of the prognostic utility of the CTC-associated aberrant loci.