Project description:MicroRNAs are important negative regulators of protein coding gene expression, and have been studied intensively over the last few years. To this purpose, different measurement platforms to determine their RNA abundance levels in biological samples have been developed. In this study, we have systematically compared 12 commercially available microRNA expression platforms by measuring an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples, and synthetic spikes from homologous microRNA family members. We developed novel quality metrics in order to objectively assess platform performance of very different technologies such as small RNA sequencing, RT-qPCR and (microarray) hybridization. We assessed reproducibility, sensitivity, quantitative performance, and specificity. The results indicate that each method has its strengths and weaknesses, which helps guiding informed selection of a quantitative microRNA gene expression platform in function of particular study goals.
Project description:Cancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression. We combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) to study somatic genetic alterations in cervical cancer-associated stromal cell fractions (n = 58) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to study LOH. By flow cytometry we found no proof for DNA aneuploidy in the vimentin-positive stromal cell fractions of any samples (CV G0G1 population 3.7% +/- 1.2; S-phase 1.4% +/- 1.8). The genotype concordance between the stromal cells and the paired normal endometrium samples was > 99.9%. No evidence for CNAs or LOH was found in the stromal cell fractions. In contrast, high frequencies of DNA content abnormalities (43/57), a significant higher S-phase (14.6% +/- 8.5)(p = 0.0001) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions (CV G0G1 population 4.1% +/- 1.0). Smooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. LOH hot-spots on chromosomes 3p, 4p and 6p were confirmed by microsatellite analysis but the stromal cell fractions showed retention of heterozygosity only. From our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available. Paired experiment, Endometrial (non-tumor) cells vs stromal cells from cervical tumors. Biological replicates: 58 patients. From 5 tumors also the tumor fraction was profiled.
Project description:Recurrent non-medullary thyroid carcinoma (NMTC) is a rare disease. We initially characterized 27 recurrent NMTC: 13 papillary thyroid cancers (PTC), 10 oncocytic follicular carcinomas (FTC-OV), and 4 non-oncocytic follicular carcinomas (FTC). A validation cohort composed of benign and malignant (both recurrent and non-recurrent) thyroid tumours was subsequently analysed (n = 20). Methods Data from genome-wide SNP arrays and flow cytometry were combined to determine the chromosomal dosage (allelic state) in these tumours, including mutation analysis of components of PIK3CA/AKT and MAPK pathways. Results All FTC-OVs showed a very distinct pattern of genomic alterations. Ten out of 10 FTC-OV cases showed near-haploidisation with or without subsequent genome endoreduplication. Near-haploidisation was seen in 5/10 as extensive chromosome-wide monosomy (allelic state [A]) with near-haploid DNA indices and retention of especially chromosome 7 (seen as a heterozygous allelic state [AB]). In the remaining 5/10 chromosomal allelic states AA with near diploid DNA indices were seen with allelic state AABB of chromosome 7, suggesting endoreduplication after preceding haploidisation. The latter was supported by the presence of both near-haploid and endoreduplicated tumour fractions in some of the cases. Results were confirmed using FISH analysis. Relatively to FTC-OV limited numbers of genomic alterations were identified in other types of recurrent NMTC studied, except for chromosome 22q which showed alterations in 6 of 13 PTCs. Only two HRAS, but no mutations of EGFR or BRAF were found in FTC-OV. The validation cohort showed two additional tumours with the distinct pattern of genomic alterations (both with oncocytic features and recurrent). Conclusions We demonstrate that recurrent FTC-OV is frequently characterised by genome-wide DNA haploidisation, heterozygous retention of chromosome 7, and endoreduplication of a near-haploid genome. Whether normal gene dosage on especially chromosome 7 (containing EGFR, BRAF, cMET) is crucial for FTC-OV tumour survival is an important topic for future research. 28 thyroid tumors from 27 patients were profiled by SNP array. Comparisons between different types were made.
Project description:BackgroundEpithelial ovarian cancer remains one of the leading causes of cancer deaths among women worldwide, and advanced epithelial ovarian cancer frequently metastasizes to the omentum. The characteristics of metastatic cancer may differ from those of primary ovarian cancer and reflect the unique omental microenvironment. This study investigated metabolomic differences in epithelial ovarian cancers.MethodsPatients with advanced epithelial ovarian cancer were eligible for this study. Five patients underwent surgery and resection of paired primary ovarian and omental metastatic cancer at Nagoya University. Metabolome analysis was performed in these paired cancer and metastatic cancer tissues through a facility service (C-SCOPE) at Human Metabolome Technologies, Inc. The concentrations of 116 compounds were measured by CE-TOFMS and CE-QqQMS, and 30 metabolic parameters were calculated. For statistical analyses, Welch's t-test was used for comparisons between two independent groups.ResultsMetabolite profiles were all different, which reflects diversity among these cancer tissues. Of the measured compounds, urea was the only metabolite that was significantly decreased in omental metastatic cancers compared with the primary cancers (p = 0.031). Moreover, in omental metastatic cancers, the pentose phosphate pathway was more dominant than glycolysis. Furthermore, in some cases, lactic acids in omental metastatic cancers were markedly decreased compared with primary cancers. With regard to histological subtype, the total levels of amino acids, especially the percentage of glutamine, were significantly enriched in serous carcinomas compared with nonserous carcinomas (p = 0.004 and p = 0.001). Moreover, the reduced forms of glutathione and polyamines were also more abundant in serous carcinomas than in nonserous carcinomas (p = 0.025 and 0.048).ConclusionsThe metabolite profiles differed depending on tumor location and histological subtype. Metabolome analysis may be a useful tool for identifying cancer diagnostic and prognostic markers.
Project description:Non-invasive prognostic markers are needed to improve survival of colorectal cancer (CRC) patients. Towards this goal, we here apply integrative systems glycobiology approaches to tumour tissues and PBMCs from CRC patients and matching controls as well as a CRC patient-derived cell line. The untargeted -omics-driven approaches revealed that non-canonical paucimannosidic proteins from monocytic and cancer cell origins are prominent signatures in CRC tumour tissues, and that their expression associates with CRC progression. Guided by these novel relationships, we then show in vitro that N-acetyl-β-D-hexosaminidase (Hex) drives paucimannosidic protein biosynthesis in CRC cells, and is intimately involved in processes underpinning CRC metastasis (adhesion, migration, invasion). Importantly, Hex activity was elevated in PBMCs and plasma from patients with advanced CRC relative to those with early-stage disease. Notably, we show that plasma Hex activity accurately informs on CRC patient survival. Our study opens new avenues for effective prognostication and therapeutic intervention in CRC.
Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).
Project description:BackgroundAlthough microbiota in prostatic tissues of patients with prostate cancer have been studied, results of different studies have been inconsistent. Different ethnicity of study subjects, different study designs, and potential contaminations during sample collection and experiments might have influenced microbiome results of prostatic tissues. In this study, we analyzed microbiota and their potential functions in benign and malignant tissues of prostate cancer considering possible contaminants and host variables.Materials and methodsA total of 118 tissue samples (59 benign tissues and 59 malignant tissues) obtained by robot-assisted laparoscopic radical prostatectomy were analyzed and 64 negative controls (from sampling to sequencing processes) were included to reduce potential contaminants.ResultsAlteration of the microbiome in prostate tissues was detected only in patients with diabetes. Furthermore, the influence of diabetes on microbiome was significant in malignant tissues. The microbiome in malignant tissues of patients with diabetes was influenced by pathologic stages. The relative abundance of Cutibacterium was reduced in the high pathologic group compared to that in the intermediate group. This reduction was related to microbial pathways increased in the high pathologic group.ConclusionResults of this study indicate that diabetes can influence the progression of prostate cancer with microbiome alteration in prostate tissues. Although further studies are necessary to confirm findings of this study, this study can help us understand tissue microbiome in prostate cancer and improve clinical therapy strategies.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:Glycobiology plays a central role in prostate cancer (PCa), as evidenced by the aberrant glycosylation in PCa-derived glycoproteins. Understanding the protein glycosylation changes underling PCa onset and progression may open opportunities for the discovery of novel biomarkers for PCa detection and stratification as well as targets for PCa metastasis. Advances in mass spectrometry-based glycomics and glycoproteomics have opened up exciting avenues for deep characterisation of the immensely, yet poorly understood complex glycoproteome. In this study, we have used integrated glycomics and glycoproteomics to explore the protein, cell and tumour-grade specific N- and O- glycosylation signatures in surgically-removed fresh PCa tissues grouped into five PCa disease grades and tissues from benign prostatic hyperplasia patients (BPH). Porous graphitised carbon–liquid chromatography (PGC–LC)-MS/MS analysis was used to profile the N-and O-glycome, revealing PCa grade specific N and O glycosylation changes, including oligomannosidic and paucimannosidic, bisecting-GlcNAc and highly branched tri- and tetra-antennary fucosylated and sialylated N-glycans as well as sialylated core 1 and 2 type O-glycans. High resolution LC-MS/MS was then employed to capture the micro and macroheterogeneity of 1,085 N-glycosites (>7,400 unique N-glycopeptides) from 540 N-glycoproteins and 360 O-glycosites (>500 unique O-glycopeptides) from 178 O-glycoproteins and reveal protein and cell specific N- and O- glycosylation changes associated with PCa progression. We also showed PCa grade-specific increase in the expression of oligosaccharyltransferase (OST) subunits that correlate with changes in the site occupancy of N-glycoproteins. In conclusion, this study shows that integrated glycomics and glycoproteomics can provide unprecedented insight into key molecular features and site-specific glycan heterogeneity contributing to the highly diverse tumour-microenvironment and allow us to deconstruct the regulation of the protein, cell and tumour-grade-specific glycosylation associated with PCa onset and development.
Project description:Benign prostate hyperplasia (BPH) is a frequent condition in aging men, which affects life quality, causing principally lower urinary tract symptoms. Epidemiologic studies suggest that BPH may raise the risk of developing prostate cancer (PCa), most likely promoting a chronic inflammatory environment. Studies aiming at elucidating the link and risk factors that connect BPH and PCa are urgently needed to develop prevention strategies. The BPH microenvironment, similar to the PCa one, increases immune infiltration of the prostate, but, in contrast to PCa, immunosuppression may not be established yet. In this study, we found that prostate-infiltrating lymphocytes (PILs) expanded from hyperplastic prostate tissue recognized tumor-associated antigens (TAA) and autologous tissue, regardless of the presence of tumor cells. PILs expanded from BPH samples of patients with PCa, however, seem to respond more strongly to autologous tissue. Phenotypic characterization of the infiltrating PILs revealed a trend towards better expanding CD4+ T cells in infiltrates derived from PCa, but no significant differences were found. These findings suggest that T cell tolerance is compromised in BPH-affected prostates, likely due to qualitative or quantitative alterations of the antigenic landscape. Our data support the hypothesis that BPH increases the risk of PCa and may pave the way for new personalized preventive vaccine strategies for these patients.