Vissers, K.J.

Activation of c-MET induces a Stem-Like phenotype in human prostate cancer (Article)

2011-11-14T00:00:00Z

Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model) or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF) receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway. Gene-expression profiling of DU145 prostate cancer cells stimulated with HGF revealed induction of a molecular signature associated with stem cells, characterized by up-regulation of CD49b, CD49f, CD44 and SOX9, and down-regulation of CD24 ('stem-like signature'). We confirmed the acquisition of a stem-like phenotype by quantitative PCR, FACS analysis and Western blotting. Further, HGF led to activation of the stem cell related Notch pathway by up-regulation of its ligands Jagged-1 and Delta-like 4. Small molecules SU11274 and PHA665752 targeting c-MET activity were both able to block the molecular and biologic effects of HGF. Knock-down of c-MET by shRNA infection resulted in significant reduction and delay of orthotopic tumour-formation in male NMRI mice. Immunohistochemical analysis in prostatectomies revealed significant enrichment of c-MET positive cells at the invasive front, and demonstrated co-expression of c-MET with stem-like markers CD49b and CD49f. In conclusion, activation of c-MET in prostate cancer cells induced a stem-like phenotype, indicating a dynamic relation between differentiated and stem-like cells in this malignancy. Its mediation of efficient tumour-formation in vivo and predominant receptor expression at the invasive front implicate that c-MET regulates tumour infiltration in surrounding tissues putatively by acquisition of a stem-like phenotype.

Cysteine-rich secretory protein 3 and β-microseminoprotein on prostate cancer needle biopsies do not have predictive value for subsequent prostatectomy outcome (Article)

2011-10-01T00:00:00Z

What's known on the subject? and What does the study add? Cysteine-rich secretory protein 3 (CRISP-3) and β-microseminoprotein (β-MSP) both have independent prognostic value for biochemical recurrence of prostate cancer after radical prostatectomy. The study investigates whether CRISP-3 and β-MSP have prognostic value on diagnostic prostate needle-biopsies, which are more relevant for therapeutic decision-making. On needle-biopsies CRISP-3 and β-MSP do not have significant prognostic value. OBJECTIVES • To investigate whether cysteine-rich secretory protein 3 (CRISP-3) and/or β-microseminoprotein (β-MSP) expression in diagnostic prostate needle biopsies have predictive value for prostate cancer (PC) on radical prostatecomy (RP). • To evaluate their potential clinical implementation in a preoperative setting. PATIENTS AND METHODS • In total, 174 participants from the European Randomized Study of Screening for Prostate Cancer, Rotterdam section, treated by RP for PC were included in the present study. • CRISP-3 and β-MSP immunohistochemistry was performed on corresponding diagnostic needle biopsies. • Outcome was correlated with clinicopathological parameters (prostate-specific-antigen, PSA; number of positive biopsies; Gleason score, GS; pT-stage; surgical margins at RP) and significant PC at RP (pT3/4, or GS > 6, or tumour volume ≥0.5 mL) in the total cohort (n= 174) and in a subgroup with low-risk features at biopsy (PSA ≤ 10 ng/ml, cT a;circ 2, PSA density <0.20 ng/mL/g, GS < 7 and ≤2 positive biopsy cores; n= 87). RESULTS âcent β-MSP and CRISP-3 expression in PC tissue was heterogeneous, with variable staining intensities occurring in the same tissue specimen. âcent High expression of β-MSP significantly correlated with GS < 7 at RP; it was not a predictor for significant PC at RP neither in the total group (n= 174; odds ratio, OR, 0.319; 95% confidence interval, CI, 0.060-1.695; P= 0.180), nor in the low-risk group (n= 87; OR, 0.227; 95% CI, 0.040-1.274; P= 0.092). âcent CRISP-3 expression was not related to clinicopathological parameters, and did not predict significant PC at RP in the total group (n= 174; OR, 1.056; 95% CI, 0.438-2.545; P= 0.904) or the low-risk group (n= 87; OR, 1.856; 95% CI, 0.626-5.506; P= 0.265). CONCLUSIONS âcent High β-MSP expression correlated with low GS in subsequent RP specimens, supporting the view that β-MSP exerts a tumour-suppressive effect. âcent No significant prognostic value of β-MSP or CRISP-3 in prostate needle biopsies for significant PC at RP was found. âcent β-MSP or CRISP-3 do not have additional value in the therapeutic stratification of patients with PC.

The value of EZH2, p27kip1, BMI-1 and MIB-1 on biopsy specimens with low-risk prostate cancer in selecting men with significant prostate cancer at prostatectomy (Article)

2010-07-01T00:00:00Z

OBJECTIVE To assess the additional prognostic value of the molecular markers EZH2, MIB-1, p27kip1 and BMI-1 on needle biopsies from men with low-risk prostate cancer, as this disease in needle biopsies shows a heterogeneous clinical outcome, and while it is known that the expression of these tissue markers is predictive of the clinical outcome after radical prostatectomy (RP) their value in prostate biopsies is largely unknown. PATIENTS AND METHODS The study included men participating in a screening study, diagnosed with low-risk prostate cancer and subsequently treated with RP. Immunohistochemical staining for EZH2, MIB-1, p27kip1 and BMI-1 on the needle biopsies were (semi)quantitatively scored and expression levels were related to significant disease at RP. Clinical low-risk prostate cancer was defined as a prostate-specific antigen (PSA) level of ≤10 ng/mL, clinical T-stage ≤2, biopsy Gleason score ≤6, a PSA density of <0.20 ng/mL/g and two or fewer positive cores. Significant PC at RP was defined as presence of any of extracapsular extension, Gleason pattern 4/5, or tumour volume ≥0.5 mL. RESULTS In all, 86 biopsy specimens were included; there was high EZH2 expression (>1.0%) in 42% and a low p27kip expression (<90%) in 63%. Significant disease was present in 44 (51%) RP specimens. A high EZH2 (odds ratio 3.19, P = 0.043) and a low p27kip1 (4.69, P = 0.036) were independent predictors for significant prostate cancer at RP. CONCLUSIONS The determination of EZH2 and p27kip1 on diagnostic needle biopsies supports the selection of men with indolent prostate cancer at RP. Especially p27kip1 could improve the pretreatment risk assessment of patients with low-risk prostate cancer.

False-negative prostate needle biopsies: Frequency, histopathologic features, and follow-up (Article)

2010-01-01T00:00:00Z

Little is known about the frequency, histopathologic characteristics, and clinical consequences of false-negative prostate biopsies, that is, biopsies classified as benign but containing adenocarcinoma or atypical suspicious glands [atypical small acinar proliferations (ASAP)]. Objective of this study was to evaluate false-negative prostate biopsy in a prostate cancer screening setting. Prostate biopsy sets of 196 participants of a screening trial, which had been reported as "benign" at initial diagnosis, followed by a diagnosis of adenocarcinoma in a subsequent screening round were reviewed by 2 urologic pathologists. Adenocarcinoma was identified in 19 biopsy cores corresponding to 16 (8.2%) patients and ASAP in 24 cores, corresponding to 19 patients (9.7%). All missed prostate cancers were Gleason score 6 (3+3). After correction for patient selection, the overall false-negative biopsy rate was estimated to be 2.4%; 1.1% for prostate cancer; and 1.3% for ASAP. Clinicopathologic features at the time of initial biopsy and of subsequent prostate cancer diagnosis did not differ between patients with a false-negative or true benign biopsy. Relatively low number of atypical glands (<10 glands), intense intermingling with preexistent glands or lack of architectural disorganization were the most prominent risk factors for a false-negative diagnosis. Another potential pitfall was the presence of prostate cancer variants, as 1 adenocarcinoma was of foamy gland type and 3 of pseudohyperplastic type. Routine examination of at least 1 level of prostate biopsy sets at high magnification and awareness of histologic prostate cancer variants might reduce the risk of missing or misinterpreting a relevant lesion at prostate biopsy evaluation.

Array comparative genomic hybridization, expression array, and protein analysis of critical regions on chromosome arms 1q, 7q, and 8p in adenocarcinomas of the gastroesophageal junction (Article)

2009-02-01T00:00:00Z

Survival rates of adenocarcinomas of the gastroesophageal junction (GEJ) are low, because these tumors are generally in an advanced stage by the time they are detected. Chromosomal regions 1q32, 7q21, and 8p22 display critical alterations in GEJ cancers; however, the genes underlying alterations in these genomic areas are largely unknown. To delineate overexpressed genes, we performed array comparative genomic hybridization (aCGH) and mRNA expression analysis of 15 GEJ adenocarcinoma samples using a fine-tiling cDNA array covering chromosome segments 1q31.3∼q41 (193.9-215.8 Mb: 21.9 Mb), 7q11.23∼q22.1 (72.3-103.0 Mb: 30.7 Mb), and 8p23.1∼p21.3 (11.1-20.7 Mb: 9.6 Mb). Based on a mRNA overexpression criterion, 11 genes were selected: ELF3 and SLC45A3 on 1q; CLDN12, CDK6, SMURF1, ARPC1B, ZKSCAN1, MCM7, and COPS6 on 7q; and FDFT1 and CTSB on 8p. The protein expression levels were subsequently determined by immunohistochemical analysis of the cancer samples. There was a significant correlation between genomic amplification, mRNA, and protein expression or overexpression for CDK6, a cell cycle regulator on 7q21.2 (92.1 Mb; P < 0.01); other genes showed less stringent associations. In conclusion, using a straightforward approach we constructed a targeted gene profile for GEJ adenocarcinomas.

Immunohistochemical evaluation of a panel of tumor cell markers during malignant progression in Barrett esophagus (Article)

2008-11-01T00:00:00Z

Histopathologic grading of dysplasia in Barrett esophagus (BE) shows substantial interobserver and intraobserver variation. We used immunohistochemical analysis with a set of tumor cell markers, ie, epidermal growth factor receptor (EGFR), ERBB2 (HER2/neu), MYC, CDKN2A (p16), SMAD4, MET, CCND1 (cyclin D1), CTNNB1 (β-catenin), and TP53 (p53), in histologic sections of endoscopic biopsies of 86 patients with BE in various stages of neoplastic progression. The markers, except SMAD4, were scored as 0 (<1% of cells stained), 1 (1%-25%), 2 (26%-50%), or 3 (>50%). All markers, except EGFR, showed a significant trend for immunohistochemical protein overexpression during malignant progression in BE (P < .01). When the successive stages along the metaplasia-low-grade dysplasia (LGD)-high-grade dysplasia (HGD)-adenocarcinoma axis were compared, protein overexpression of β-catenin separated LGD from metaplasia, whereas protein overexpression of cyclin D1 and p53 discriminated HGD from LGD (all P < .001). β-Catenin can be helpful for a diagnosis of LGD in BE, although it stains positively in a subset only, whereas p53 remains an appropriate marker to define HGD. In case of doubt, cyclin D1 can be added to separate LGD from HGD in BE.

Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levels (Article)

2008-08-01T00:00:00Z

Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22-q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin-dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto-oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barrett's dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high-resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas.

High-resolution array comparative genomic hybridization of chromosome 8q: Evaluation of putative progression markers for gastroesophageal junction adenocarcinomas (Article)

2007-11-01T00:00:00Z

Amplification of 8q is frequently found in gastroesophageal junction (GEJ) cancer. It is usually detected in high-grade, high-stage GEJ adenocarcinomas. Moreover, it has been implicated in tumor progression in other cancer types. In this study, a detailed genomic analysis of 8q was performed on a series of GEJ adenocarcinomas, including 22 primary adenocarcinomas, 13 cell lines and two xenografts, by array comparative genomic hybridization (aCGH) with a whole chromosome 8q contig array. Of the 37 specimens, 21 originated from the esophagus and 16 were derived from the gastric cardia. Commonly overrepresented regions were identified at distal 8q, i.e. 124-125 Mb (8q24.13), at 127-128 Mb (8q24.21), and at 141-142 Mb (8q24.3). From these regions six genes were selected with putative relevance to cancer: ANXA13, MTSS1, FAM84B (alias NSE2), MYC, C8orf17 (alias MOST-1) and PTK2 (alias FAK). In addition, the gene EXT1 was selected since it was found in a specific amplification in cell line SK-GT-5. Quantitative RT-PCR analysis of these seven genes was subsequently performed on a panel of 24 gastroesophageal samples, including 13 cell lines, two xenografts and nine normal stomach controls. Significant overexpression was found for MYC and EXT1 in GEJ adenocarcinoma cell lines and xenografts compared to normal controls. Expression of the genes MTSS1, FAM84B and C8orf17 was found to be significantly decreased in this set of cell lines and xenografts. We conclude that, firstly, there are other genes than MYC involved in the 8q amplification in GEJ cancer. Secondly, the differential expression of these genes contributes to unravel the biology of GEJ adenocarcinomas. Copyright

Wnt pathway-related gene expression during malignant progression in ulcerative colitis (Article)

2007-08-01T00:00:00Z

Long-standing ulcerative colitis (UC) has been associated with a high risk of developing colonic adenocarcinoma. Importantly, both low- and high-grade dysplasia are strongly related to the presence or development of malignancy. The canonical Wnt/β-catenin signaling pathway is of crucial importance in cancer development and progression, but its role in UC-related carcinogenesis remains to be determined. We evaluated the immunolabeling patterns of β-catenin, as well as the products of Wnt-associated cancer genes E-cadherin, cyclin D1 and c-myc, along the dysplasia-carcinoma pathway in UC. For this purpose, immunohistochemistry (IHC) was performed on 18 adenocarcinomas and 17 dysplasias, derived from 21 patients. We found that intracellular β-catenin accumulation, the hallmark of Wnt signaling activation, is observed in dysplasia, together with enhanced labeling of nuclear protein cyclin D1 and reduction of membranous labeling of E-cadherin. c-myc displayed moderate immunolabeling in the (pre)malignant lesions. Thus, the Wnt pathway is activated in early stages of malignant progression in UC. Furthermore, upregulation of the oncogene cyclin D1 and downregulation of tumor suppressor E-cadherin also occurs in the (pre)neoplastic state. This may contribute to the high potential for malignant degeneration of dysplasia in UC-related colitis.

Genomic alterations in malignant transformation of Barrett's esophagus (Article)

2001-01-01T00:00:00Z

The incidence of adenocarcinoma in Barrett's esophagus has been increasing rapidly over the past decades. Neoplastic progression is characterized by three well-defined premalignant stages: metaplasia, low-grade dysplasia, and high-grade dysplasia. A genome-wide overview, based on comparative genomic hybridization, was performed, evaluating 30 Barrett's adenocarcinomas and 25 adjacent precursors, i.e., 6 metaplasias, 9 low-grade dysplasias, and 10 high-grade dysplasias. The frequency of losses and gains significantly increased in the subsequent stages of malignant transformation. Losses of 5q21-q23, 9p21, 17p12-13.1, 18q21, and Y were revealed in low-grade dysplasias. This was followed by loss of 7q33-q35 and gains of 7p12-p15, 7q21-q22, and 17q21 in high-grade dysplasias along with high-level amplification (HLA) of 7q21 and 17q21. In the invasive cancers, additional losses of 3p14-p21, 4p, 4q, 8p21, 13q14-q31, 14q24.3-q31, 16q21-q22, and 22q as well as gains of 3q25-q27, 8q23-24.1, 12p11.2-12, 15q22-q24, and 20q11.2-q13.1 were distinguished along with HLAs of 8p12-p22 and 20q11.2-q13.1. Approximately one-third of the alterations in the dysplasias were also found in the adjacent adenocarcinomas, illustrating that multiple clonal lineages can be present in Barrett's esophagus. Novel findings include loss on 7q, gain on 12p, and the observation of several HLAs in high-grade dysplasias. Furthermore, loss of 7q33-q35 was found to represent a significant distinction between low-grade and high-grade dysplasia (P = 0.01), whereas loss of 16q21-q22 and gain of 20q11.2-q13.1 were disclosed to significantly discriminate between high-grade dysplasia and adenocarcinoma (P = 0.02 and P = 0.03, respectively). This inventory of genetic aberrations increases our understanding of malignant transformation in Barrett's esophagus and might provide useful biomarkers for disease progression.

Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization. EDTA is highly preferable to a routinely used acid decalcifier (Article)

1999-01-01T00:00:00Z

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e. , one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques.