Chunde Li

Genomic Profiling Reveals Alternative Genetic Pathways of Prostate Tumorigenesis

Prostate cancer is clinically heterogeneous, ranging from indolent to lethal disease. Expression profiling previously defined three subtypes of prostate cancer, one (subtype-1) linked to clinically favorable behavior, and the others (subtypes-2 and -3) linked with a more aggressive form of the disease. To explore disease heterogeneity at the genomic level, we carried out array-based comparative genomic hybridization (array CGH) on 64 prostate tumor specimens, including 55 primary tumors and 9 pelvic lymph node metastases. Unsupervised cluster analysis of DNA copy number alterations (CNA) identified recurrent aberrations, including a 6q15-deletion group associated with subtype-1 gene expression patterns and decreased tumor recurrence. Supervised analysis further disclosed distinct patterns of CNA among gene-expression subtypes, where subtype-1 tumors exhibited characteristic deletions at 5q21 and 6q15, and subtype-2 cases harbored deletions at 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion). Lymph node metastases, predominantly subtype-3, displayed overall higher frequencies of CNA, and in particular gains at 8q24 (MYC) and 16p13, and loss at 10q23 (PTEN) and 16q23. Our findings reveal that prostate cancers develop via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating prostate cancer biology and explain in part the clinical heterogeneity of the disease.

Identification of two distinct deleted regions on chromosome 13 in prostate cancer.

Aberrations of 13q occur frequently in prostate cancer and this chromosome contains two known tumor suppressor genes, BRCA2 and Rb1. This study analysed 13q LOH, DNA ploidy, BRCA2 mutation and pRb expression in prostate cancers. In total, 13q deletions were found in 18 of 36 tumors but did not correlate with histological grade, stage or DNA ploidy. Two smallest regions of overlapping deletions were defined: one flanked by D13S218 and D13S153; the other flanked by D13S31 and D13S137. BRCA2 was less frequently deleted whereas Rb1 did have a high frequency of deletion. None of the two genes was located in any of these two regions. Furthermore, BRCA2 mutation was not found in the five tumors where deletions had involved the BRCA2 locus. Neither did the Rb1 deletion correlate with absent pRb expression. In addition, tetraploidy was found in 14 out of 25 tumors analysed and correlated with aberrant pRb expression. Our results indicate that 13q deletion is an early non-random event. Tumor suppressor genes other than BRCA2 or Rb1 may be the target of 13q deletions. Aberrant pRb expression may not reflect the two-hit Rb1 inactivation but may be involved in the tetraploidization of prostate cancer cells.

Combined LOH/CGH analysis proves the existence of interstitial 3p deletions in renal cell carcinoma.

We have recently developed an allele titration assay (ATA) to assess the sensitivity and influence of normal cell admixture in loss of heterozygosity (LOH) studies based on CA-repeat. The assay showed that these studies are biased by the size-dependent differential sensitivity of allele detection. Based on these data, we have set up new criteria for evaluation of LOH. By combining these new rules with comparative genome hybridization (CGH) we have shown the presence of interstitial deletions in renal cell carcinoma (RCC) biopsies and cell lines. At least three out of 11 analysed RCC cell lines and three out of 37 biopsies contain interstitial deletions on chromosome 3. Our study suggests the presence of several regions on human chromosome 3 that might contribute to tumor development by their loss: (i) 3p25-p26, around the VHL gene (D3S1317); (ii) 3p21.3-p22 (between D3S1260 and D3S1611); (iii) 3p21.2 (around D3S1235 and D3S1289); (iv) 3p13-p14 (around D3S1312 and D3S1285). For the first time, AP20 region (3p21.3-p22) was carefully tested for LOH in RCC. It was found that the AP20 region is the most frequently affected area. Our data also suggest that another tumor suppressor gene is located near the VHL gene in 3p25-p26.

Distinct deleted regions on chromosome segment 16q23–24 associated with metastases in prostate cancer

Cadherins (CDH) are cell adhesion molecules and their dysfunctions have been implicated in the development of cancer metastases. Several cadherin genes are tandemly located on 16q, which is frequently deleted in prostate cancer. We therefore used 22 markers on 16q to localize important deleted regions in metastases of this tumor. We found 16q deletions in 24/32 (75%) tumors. All lymph node and brain metastases showed extensive deletions, while 52% of primary tumors displayed limited deletions. Commonly deleted regions (CDRs) on 16q23–24, CDR2 (D16S515‐D16S516) and CDR4 (D16S520‐D13S3028), were strongly associated with metastases and increased Gleason score. Reduced CDH1 (E‐cadherin) expression was seen in 16/32 (50%) tumors, but the CDH1 gene is not within either of these two regions. Sequencing analysis for all 16 exons of the CDH1 gene did not reveal any mutations in 10 tumors, including three brain metastases with both 16q22.1 deletion and absent E‐cadherin expression. Our results implicate other, yet unidentified genes on 16q23–24 to be the frequent targets of mutations and deletions in prostate cancer metastases. Genes Chromosomes Cancer 24:175–182, 1999.

Links between genetic and environmental factors and prostate cancer risk

Genetic polymorphisms and expression of steroid receptors may explain why some individuals are more at risk of developing prostate cancer. Some risk factors often discussed are androgen stimulation, and vitamin A and D deficiency. Long CAG‐repeats in exon 1 of the androgen receptor (AR) gene on the X chromosome seem to have a protective role against androgen overstimulation. Likewise, long vitamin D receptor alleles in the poly‐A tract may prevent vitamin D stimulation.

Chromosome 16q24 Deletion and Decreased E-Cadherin Expression: Possible Association With Metastatic Potential in Prostate Cancer

Deletion of chromosome 16q is a frequent aberration in prostatic carcinoma, indicating the existence of candidate tumor suppressor genes involved in the pathogenesis of prostate cancer.

An expression signature at diagnosis to estimate prostate cancer patients’ overall survival

Background:This study aimed to identify biomarkers for estimating the overall and prostate cancer (PCa)-specific survival in PCa patients at diagnosis.Methods:To explore the importance of embryonic stem cell (ESC) gene signatures, we identified 641 ESC gene predictors (ESCGPs) using published microarray data sets. ESCGPs were selected in a stepwise manner, and were combined with reported genes. Selected genes were analyzed by multiplex quantitative polymerase chain reaction using prostate fine-needle aspiration samples taken at diagnosis from a Swedish cohort of 189 PCa patients diagnosed between 1986 and 2001. Of these patients, there was overall and PCa-specific survival data available for 97.9%, and 77.9% were primarily treated by hormone therapy only. Univariate and multivariate Cox proportional hazard ratios and Kaplan–Meier plots were used for the survival analysis, and a k-nearest neighbor (kNN) algorithm for estimating overall survival.Results:An expression signature of VGLL3, IGFBP3 and F3 was shown sufficient to categorize the patients into high-, intermediate- and low-risk subtypes. The median overall survival times of the subtypes were 3.23, 4.00 and 9.85 years, respectively. The difference corresponded to hazard ratios of 5.86 (95% confidence interval (CI): 2.91–11.78, P<0.001) for the high-risk subtype and 3.45 (95% CI: 1.79–6.66, P<0.001) for the intermediate-risk compared with the low-risk subtype. The kNN models that included the gene expression signature outperformed the one designed on clinical parameters alone.Conclusions:The expression signature can potentially be used to estimate overall survival time. When validated in future studies, it could be integrated in the routine clinical diagnostic and prognostic procedure of PCa for an optimal treatment decision based on the estimated survival benefit.

MUC1 as a Putative Prognostic Marker for Prostate Cancer

MUC1 is expressed on the apical surface of glandular epithelium. With functions including protection, adhesion and signaling, MUC1 has been implicated in prostate cancer. There are many splice variants, the best characterized of which are MUC1/1 and MUC1/2 which are determined by a SNP (rs4072037, 3506G>A). Blood DNA from the general population, BPH, sporadic and hereditary prostate cancer subjects were genotyped for the rs4072037 SNP. G allele frequencies were significantly reduced in hereditary prostate cancer (15%) compared to population, BPH or sporadic prostate cancer samples (27%, 39% and 26% respectively). In addition, the G allele was lost from 3 of 8 heterozygous sporadic prostate tumor samples compared to matched blood DNA. Bioinformatics analysis of MUC1 protein sequences provides insight into differences between the variants which may be functionally relevant. The literature indicates discrepancies between immunohistochemical studies, possibly due to the variety of MUC1 epitopes targeting diverse regions of the molecule. The contradictory findings in cell lines highlight the problem associated with inadequate experimental systems. This is the first report of genetic differences in MUC1 between blood and prostatic cancer tissue. This finding is important as proof of principle, given that many association studies focus on blood DNA rather than on the tumor DNA. As yet, potential functional differences between splice variants has been paid little attention. Antibodies which discriminate between the variants and standardization of methods would help to clarify whether there is a role for MUC1 as a prognostic marker.

Genotypic and phenotypic characterization of two newly established renal cell carcinoma cell lines.

Two new cell lines from human renal cell carcinoma are reported. Primary cell cultures from 75 consecutive cases of nephrectomy and metastatic surgery due to different stages of RCC during 4 years were studied. Two cell cultures could be propagated for more than 50 passages in vitro. HN4 was derived from a grade III clear cell carcinoma. HN51 originated from a metastatic brain lesion of a clear cell carcinoma grade III. Karyotype analysis of HN4 revealed triploidy with a clonal aberration, der(10)t(3;10)(q13;p12). HN51 also had a triploid pattern with different marker chromosomes but without any clonal aberration. Loss of heterozygosity studies revealed no loss of heterozygosity on 3p or other chromosomal markers in HN4 but LOH was found on one 3p marker and one 14q marker in addition to all 17 p and q markers in HN51. In vitro light microscopy showed distinctly different morphology in the two cell lines although they both had a typical epithelial growth pattern. Doubling times in vitro were low but slightly higher for HN51. Repeated tumorigenenic experiments in athymic mice only gave rise to subcutaneous tumors with HN51. On characterization by 2-dimensional gel electrophoresis, the two cell lines exhibited different polypeptide patterns with higher expression of proliferating cell nuclear antigen in HN51 and higher expression of glutathione-S-transferase in HN4 constituting the most prominent differences.

Improving the Prediction of Prostate Cancer Overall Survival by Supplementing Readily Available Clinical Data with Gene Expression Levels of IGFBP3 and F3 in Formalin-Fixed Paraffin Embedded Core Needle Biopsy Material

Background A previously reported expression signature of three genes (IGFBP3, F3 and VGLL3) was shown to have potential prognostic value in estimating overall and cancer-specific survivals at diagnosis of prostate cancer in a pilot cohort study using freshly frozen Fine Needle Aspiration (FNA) samples. Methods We carried out a new cohort study with 241 prostate cancer patients diagnosed from 2004–2007 with a follow-up exceeding 6 years in order to verify the prognostic value of gene expression signature in formalin fixed paraffin embedded (FFPE) prostate core needle biopsy tissue samples. The cohort consisted of four patient groups with different survival times and death causes. A four multiplex one-step RT-qPCR test kit, designed and optimized for measuring the expression signature in FFPE core needle biopsy samples, was used. In archive FFPE biopsy samples the expression differences of two genes (IGFBP3 and F3) were measured. The survival time predictions using the current clinical parameters only, such as age at diagnosis, Gleason score, PSA value and tumor stage, and clinical parameters supplemented with the expression levels of IGFBP3 and F3, were compared. Results When combined with currently used clinical parameters, the gene expression levels of IGFBP3 and F3 are improving the prediction of survival time as compared to using clinical parameters alone. Conclusion The assessment of IGFBP3 and F3 gene expression levels in FFPE prostate cancer tissue would provide an improved survival prediction for prostate cancer patients at the time of diagnosis.