Novel fusion transcripts associate with progressive prostate cancer.

The mechanisms underlying the potential for aggressive behavior of prostate cancer (PCa) remain elusive. In this study, whole genome and/or transcriptome sequencing was performed on 19 specimens of PCa, matched adjacent benign prostate tissues, matched blood specimens, and organ donor prostates. A set of novel fusion transcripts was discovered in PCa. Eight of these fusion transcripts were validated through multiple approaches. The occurrence of these fusion transcripts was then analyzed in 289 prostate samples from three institutes, with clinical follow-up ranging from 1 to 15 years. The analyses indicated that most patients [69 (91%) of 76] positive for any of these fusion transcripts (TRMT11-GRIK2, SLC45A2-AMACR, MTOR-TP53BP1, LRRC59-FLJ60017, TMEM135-CCDC67, KDM4-AC011523.2, MAN2A1-FER, and CCNH-C5orf30) experienced PCa recurrence, metastases, and/or PCa-specific death after radical prostatectomy. These outcomes occurred in only 37% (58/157) of patients without carrying those fusion transcripts. Three fusion transcripts occurred exclusively in PCa samples from patients who experienced recurrence or PCaerelated death. The formation of these fusion transcripts may be the result of genome recombination. A combination of these fusion transcripts in PCa with Gleason's grading or with nomogram significantly improves the prediction rate of PCa recurrence. Our analyses suggest that formation of these fusion transcripts may underlie the aggressive behavior of PCa.

Whole-genome methylation sequencing reveals distinct impact of differential methylations on gene transcription in prostate cancer.

DNA methylation is one of the most important epigenetic mechanisms in regulating gene expression. Genome hypermethylation has been proposed as a critical mechanism in human malignancies. However, whole-genome quantification of DNA methylation of human malignancies has rarely been investigated, and the significance of the genome distribution of CpG methylation is unclear. We performed whole-genome methylation sequencing to investigate the methylation profiles of 13 prostate samples: 5 prostate cancers, 4 matched benign prostate tissues adjacent to tumor, and 4 age-matched organ-donor prostate tissues. Alterations of methylation patterns occurred in prostate cancer and in benign prostate tissues adjacent to tumor, in comparison with age-matched organ-donor prostates. More than 95% alterations of genome methylation occurred in sequences outside CpG islands. Only a small fraction of the methylated CpG islands had any effect on RNA expression. Both intragene and promoter CpG island methylations negatively affected gene expression. However, suppressions of RNA expression did not correlate with levels of CpG island methylation, suggesting that CpG island methylation alone might not be sufficient to shut down gene expression. Motif analysis revealed a consensus sequence containing Sp1 binding motif significantly enriched in the effective CpG islands.

High fidelity copy number analysis of formalin-fixed and paraffin-embedded tissues using Affymetrix Cytoscan HD chip.

Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging due to suboptimal DNA quality and failure to use appropriate baseline controls for such tissues. Here, we report a modified method in analyzing CNV in FFPE tissues using microarray with Affymetrix Cytoscan HD chips. Gel purification was applied to select DNA with good quality and data of fresh frozen and FFPE tissues from healthy individuals were included as baseline controls in our data analysis. Our analysis showed a 91% overlap between CNV detection by microarray with FFPE tissues and chromosomal abnormality detection by karyotyping with fresh tissues on 8 cases of lymphoma samples. The CNV overlap between matched frozen and FFPE tissues reached 93.8%. When the analyses were restricted to regions containing genes, 87.1% concordance between FFPE and fresh frozen tissues was found. The analysis was further validated by Fluorescence In Situ Hybridization on these samples using probes specific for BRAF and CITED2. The results suggested that the modified method using Affymetrix Cytoscan HD chip gave rise to a significant improvement over most of the previous methods in terms of accuracy in detecting CNV in FFPE tissues. This FFPE microarray methodology may hold promise for broad application of CNV analysis on clinical samples.

Development of a chemically defined medium and discovery of new mitogenic growth factors for mouse hepatocytes: mitogenic effects of FGF1/2 and PDGF.

Chemically defined serum-free media for rat hepatocytes have been useful in identifying EGFR ligands and HGF/MET signaling as direct mitogenic factors for rat hepatocytes. The absence of such media for mouse hepatocytes has prevented screening for discovery of such mitogens for mouse hepatocytes. We present results obtained by designing such a chemically defined medium for mouse hepatocytes and demonstrate that in addition to EGFR ligands and HGF, the growth factors FGF1 and FGF2 are also important mitogenic factors for mouse hepatocytes. Smaller mitogenic response was also noticed for PDGF AB. Mouse hepatocytes are more likely to enter into spontaneous proliferation in primary culture due to activation of cell cycle pathways resulting from collagenase perfusion. These results demonstrate unanticipated fundamental differences in growth biology of hepatocytes between the two rodent species.