Peter J. Wild

WIF1, a component of the Wnt pathway, is down‐regulated in prostate, breast, lung, and bladder cancer

To detect novel Wnt‐pathway genes involved in tumourigenesis, this study analysed the RNA expression levels of 40 genes of the Wnt pathway by chip hybridization of microdissected matched pairs of 54 primary prostate carcinomas. Eleven genes showed greater than two‐fold differential expression in at least 10% of prostate cancers. Three of these genes encode extracellular components of the Wnt pathway (WNT2, WIF1, SFRP4); two are receptors (FZD4, FZD6); two belong to the intracellular signal cascade (DVL1, PPP2CB); one regulates transcription (TCF4); and three represent genes regulated by this pathway (CCND2, CD44, MYC). While SFRP4, FZD4, FZD6, DVL1, TCF4, and MYC are up‐regulated, WIF1, WNT2, PPP2CB, CCND2, and CD44 are down‐regulated in certain prostate cancer patients. Wnt inhibitory factor 1 (WIF1) and secreted frizzled related protein (SFRP4) showed the most significant aberrant expression at the RNA level. WIF1 was down‐regulated in 64% of primary prostate cancers, while SFRP4 was up‐regulated in 81% of the patients. Immunohistochemical analysis using a polyclonal antibody revealed strong cytoplasmic perinuclear WIF1 expression in normal epithelial cells of the prostate, breast, lung, and urinary bladder. Strong reduction of WIF1 protein expression was found in 23% of prostate carcinomas, but also in 60% of breast, 75% of non‐small cell lung (NSCLC), and 26% of bladder cancers analysed. No significant association between WIF1 down‐regulation and tumour stage or grade was observed for prostate, breast or non‐small cell lung carcinomas, indicating that loss of WIF1 expression may be an early event in tumourigenesis in these tissues. However, down‐regulation of WIF1 correlated with higher tumour stage in urinary bladder tumours (pTa versus pT1–pT4; p = 0.038). Copyright

Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer

Many molecular alterations are known to occur in urothelial carcinoma of the bladder, but their significance for tumor progression is poorly understood. Deletions of chromosome 8p are frequently found in several tumor types and are often associated with progressive disease. In all, 99 bladder tumors were screened for deletions at 8p using loss of heterozygosity (LOH) and multicolor fluorescence in situ hybridization FISH analyses. Allelic loss on chromosome 8p in at least one marker was found in 25/99 (25%) tumors. There was a significant correlation of 8p deletions with invasive tumor growth and a highly significant association with papillary growth pattern in patients with invasive disease. cDNA array analyses revealed that secreted Frizzled-related protein 1 (sFRP1), an antagonist of Frizzled receptors and Wnt pathway activation on chromosome 8p12–11.1, is frequently downregulated in bladder cancer. To investigate sFRP1 as a candidate for a putative progression-related gene on 8p, urothelial cell lines and primary urothelial carcinomas were screened for sFRP1 expression using quantitative real-time PCR, Northern blot, immunofluorescence and immunohistochemistry (IHC). Of the investigated bladder cancers, 38% showed loss of sFRP1 expression by quantitative RT-PCR. Evaluation of the protein expression by IHC using tissue microarrays containing 776 bladder cancers revealed loss or strong reduction of sFRP1 expression in 66% of cases. SFRP1 loss was associated with higher tumor stage and grade and shorter overall survival. In addition, loss of sFRP1 was an independent indicator of poor survival in patients with papillary but not with muscle invasive bladder cancer. There were neither mutations in the coding region of sFRP1 nor homozygous deletions at 8p12–11.21. However, promoter methylation was detected using methylation-specific PCR in 29% of cases. In conclusion, we could show a close correlation of chromosome 8p deletions and progression of papillary bladder tumors. The sFRP1 gene on chromosome 8p12–11.1 could be a candidate gene for the predicted, progression-related tumor suppressor gene in bladder cancer and could contribute to urothelial carcinogenesis.

Smoking and cancer-related gene expression in bronchial epithelium and non-small-cell lung cancers.

Tobacco smoking is the leading cause of lung cancer worldwide. Gene expression in surgically resected and microdissected samples of non‐small‐cell lung cancers (18 squamous cell carcinomas and nine adenocarcinomas), matched normal bronchial epithelium, and peripheral lung tissue from both smokers (n = 22) and non‐smokers (n = 5) was studied using the Affymetrix U133A array. A subset of 15 differentially regulated genes was validated by real‐time PCR or immunohistochemistry. Hierarchical cluster analysis clearly distinguished between benign and malignant tissue and between squamous cell carcinomas and adenocarcinomas. The bronchial epithelium and adenocarcinomas could be divided into the two subgroups of smokers and non‐smokers. By comparison of the gene expression profiles in the bronchial epithelium of non‐smokers, smokers, and matched cancer tissues, it was possible to identify a signature of 23 differentially expressed genes, which might reflect early cigarette smoke‐induced and cancer‐relevant molecular lesions in the central bronchial epithelium of smokers. Ten of these genes are involved in xenobiotic metabolism and redox stress (eg AKR1B10, AKR1C1, and MT1K). One gene is a tumour suppressor gene (HLF); two genes act as oncogenes (FGFR3 and LMO3); two genes are involved in matrix degradation (MMP12 and PTHLH); three genes are related to cell differentiation (SPRR1B, RTN1, and MUC7); and five genes have not been well characterized to date. By comparison of the tobacco‐exposed peripheral alveolar lung tissue of smokers with non‐smokers and with adenocarcinomas from smokers, it was possible to identify a signature of 27 other differentially expressed genes. These genes are involved in the metabolism of xenobiotics (eg GPX2 and FMO3) and may represent cigarette smoke‐induced, cancer‐related molecular targets that may be utilized to identify smokers with increased risk for lung cancer. Copyright

Induction of Hsp90 protein expression in malignant melanomas and melanoma metastases.

Abstract:  The heat‐shock protein Hsp90 has been shown to be essential for the functional integrity of the telomerase complex. The telomerase activity is enhanced in melanoma and stabilizes the chromosomal integrity in proliferating cells. Furthermore, overexpression of Hsp90 induces silencing of point mutations in transcription factors which, otherwise, would result in a loss‐of‐function phenotype. In melanocytic lesions there is a higher risk of mutations caused by the enhanced proliferation in melanocytic cells. By analyzing microdissected melanocytic tumors by semiquantitative PCR, we demonstrate an overexpression of Hsp90 mRNA in malignant melanomas (10/14) and in melanoma metastases (6/6) as well as in melanoma cell lines (9/9) when compared with melanocytic nevi (2/9). These results could be confirmed on protein level by immunohistochemistry. While melanocytic nevi show discrete Hsp90 expression only in a minor fraction (2/9), malignant melanomas and metastases show a positive Hsp90 immunohistochemistry in the majority of cases; (7/9) and (13/14), respectively. In addition, by analyzing melanoma metastases by flow cytometry we show that Hsp90 is expressed on the surface of tumor cells (7/8). From these data we conclude that Hsp90 is present in advanced malignant melanomas and may have a stabilizing effect on the cellular functions in proliferating cells of melanocytic lesions and could thereby be a prerequisite for the tumor progression. As Hsp90 is expressed on the cell surface, it might also be a potential immunorelevant target structure for immunotherapy of melanoma.

Elevated nuclear maspin expression is associated with microsatellite instability and high tumour grade in colorectal cancer.

Maspin, a member of the serpin family, has been reported to suppress metastasis and angiogenesis in breast and prostate cancers. Overexpression of maspin was associated with adverse prognostic features in several other tumours. In this study, expression of maspin was analysed in 41 colorectal carcinomas with high‐frequency microsatellite instability (MSI‐H) and 159 microsatellite stable colorectal cancers (MSS/MSI‐L) by immunohistochemistry (IHC) and partly by relative quantitative real‐time RT‐PCR and western blot analyses. Significant upregulation of maspin expression was found in MSI‐H tumours compared to both MSS/MSI‐L tumours and matched benign colonic mucosa. Increased maspin expression was also found in three MSI‐H colon cancer cell lines, but not in three MSS colon cancer cell lines by RT‐PCR and western blot analyses. Regulation of maspin expression depended on promoter methylation as tissue specimens and cell lines expressing maspin showed unmethylated maspin promoters, whereas promoter hypermethylation was found in specimens with loss of maspin expression. Intense nuclear maspin immunostaining was seen specifically in MSI‐H tumours (p = 0.013), de‐differentiated tumours (p = 0.006), and at the invasion front. These findings provide new insights into the role of maspin in colorectal cancer progression and may be useful for diagnosis and treatment strategies. Copyright

Expression levels of the putative zinc transporter LIV-1 are associated with a better outcome of breast cancer patients.

We investigated the expression pattern of the breast cancer associated gene LIV‐1 on mRNA and protein level in 111 human breast cancer patients by in situ hybridization as well as immunohistochemistry and focused on the unknown potential of LIV‐1 expression levels as a prognostic marker. To our knowledge, this is the first study on endogenous LIV‐1 protein expression. Results of our study indicate that LIV‐1 mRNA and protein expression levels are only weakly correlated, suggesting posttranscriptional regulatory mechanisms. Furthermore, LIV‐1 mRNA quantity in combination with a positive ER status seem to represent a better marker than the progesterone receptor status according to the prognostic significance for relapse free survival (RFS). A negative correlation of LIV‐1 protein levels with tumor size, grade and stage reflects an association of LIV‐1 protein expression with less aggressive tumors. High LIV‐1 protein expression seems to be associated with a longer relapse free and overall survival in breast cancer patients with invasive ductal carcinoma. This association, however, seems to be dependent from other prognostic markers. Our data suggest that LIV‐1 is a promising candidate for a novel marker for breast cancer patients with better outcome. Furthermore, our study presents a revised cDNA sequence of LIV‐1 and demonstrates the localization of endogenous LIV‐1 in the endoplasmic reticulum. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience. wiley.com/jpages/0020‐7136/suppmat/index.html).

Retinoblastoma-binding protein 2-homolog 1 : a retinoblastoma-binding protein downregulated in malignant melanomas

In malignant melanomas, the loss of cell cycle control is thought to be due to a lack of retinoblastoma protein (pRb)-activity. Members of the previously described family of retinoblastoma-binding proteins (RBPs) are supposed to act as pRb-modulating factors. Based on RNA-fingerprinting of normal human melanocytes, we previously described a new family member with high sequence homology to the retinoblastoma-binding protein-2 (RBP-2), termed RBP2-Homolog 1 (RBP2-H1). Based on its UVB responsiveness, it was hypothesized that this gene may also play a role in melanocytic tumors. In the present study, we can confirm by real-time RT-PCR (six common melanocytic nevi, five advanced nodular melanomas and seven melanoma metastases) and immunohistochemistry (tissue microarrays: 52 melanocytic nevi, 60 melanomas, 60 metastases; and conventional sections: five common nevi, four advanced nodular melanomas, five melanoma metastases) that RBP2-H1 expression is progressively downregulated in advanced and metastatic melanomas in vivo with a certain intratumoral heterogeneity. Whereas benign melanocytic nevi are RBP2-H1 positive in about 70% of the cases, a lack of RBP2-H1 expression was found in 90% of the primary malignant melanomas and 70% of the melanoma metastases, respectively. Interestingly, a similar deficiency can be found in glioblastomas, but not epithelial cancers. In accordance to the in vivo data, established melanoma cell lines exhibit low but heterogeneous levels of RBP2-H1 expression. By co-immunoprecipitation, we provide the first evidence that a subfraction of total RBP2-H1 can bind to pRb, which makes this protein a true pRb-interacting factor. We conclude that loss of RBP2-H1 is a common finding in the progression of malignant melanomas. Since a direct interaction of RBP2-H1 and pRb seems possible, the loss of RBP2-H1 may possibly contribute to uncontrolled growth in malignant melanomas.

Different types of microsatellite instability in ovarian carcinoma

Microsatellite instability at mono‐ and dinucleotide repeats is the hallmark of the hereditary non‐polyposis cancer syndrome (HNPCC) and is related to deficient DNA mismatch repair. In contrast, a distinct form of microsatellite instability at selective tetranucleotide repeats (EMAST or elevated microsatellite alterations at selected tetranucleotides) was described in several non‐HNPCC cancer types. EMAST is probably unrelated to mismatch repair defects. We investigated the frequency of microsatellite instability at mononucleotide, dinucleotide and tetranucleotide repeats in a series of 75 ovarian carcinomas (53 serous and 22 non‐serous). Microsatellite analysis was carried out using 5 mono‐ and dinucleotide markers from the National Cancer Institute Consensus Panel and 6 tetranucleotide markers, which have been reported as frequently unstable in the literature. High frequency of microsatellite instability (MSI‐H) at mono‐ and dinucleotide repeats was observed in 9% and a low frequency (MSI‐L) in 21% of serous carcinomas. MSI‐H was detected in 4% and MSI‐L in 18% of non‐serous carcinomas. Nine percent of serous carcinomas showed instability at multiple and 9% at single tetranucleotide loci. All non‐serous carcinomas were stable at tetranucleotide loci. In summary, EMAST (e.g., tumors with tetranucleotide instability without concomitant MSI‐H) was observed in 13% of ovarian serous carcinomas. All EMAST positive tumors were of advanced stage. We conclude that EMAST occurs as a distinct form of microsatellite instability in ovarian cancer. EMAST seems to be particularly frequent in advanced serous carcinomas. Its clinical significance needs to be investigated.

Morphological characterisation of Crohn's disease fistulae.

Background: Fistulae are a common complication in up to 35% of all patients with Crohn’s disease. Their therapy is difficult and frequently unsatisfactory. To date, no histological comparison of Crohn’s disease fistulae with non-inflammatory bowel disease fistulae has been performed. In addition, Crohn’s disease fistulae have not been well characterised morphologically. Methods: Eighty four fistulae from Crohn’s disease patients were compared with 13 fistulae from controls. Haematoxylin-eosin staining, electron microscopy, and immunohistochemistry for panCytokeratin (epithelial cells), CD20 (B cells), CD45R0 (T cells), and CD68 (macrophages) were performed according to standard techniques. In addition, histopathological findings were compared with clinical and laboratory data. Results: In 31.0% of controls and 27.4% of Crohn’s disease specimens, fistulae had a lining of flattened intestinal epithelium without goblet cells or, in the case of cutaneous/perianal disease, narrow squamous epithelium. Non-epithelialised fistulae were covered by a thin layer of (myo)fibroblasts, focally forming a new basement membrane, as demonstrated by electron microscopy. All fistulae were surrounded by granulation tissue. Crohn’s disease fistulae presented with central infiltration by CD45R0+ T cells, followed by a small band of CD68+ macrophages and dense accumulation of CD20+ B cells. In contrast, in controls, there was dense infiltration by CD68+ macrophages with only few CD20+ B cells and CD45R0+ T lymphocytes. Conclusions: Fistulae in Crohn’s disease differ markedly from non-Crohn’s disease fistulae with regard to their cellular composition. The presence of an epithelial lining in a subgroup of fistulae may be important for the therapeutic approach and healing process.

Nuclear Maspin expression is associated with response to adjuvant 5-fluorouracil based chemotherapy in patients with stage III colon cancer.

Maspin, a member of the Serpin protease inhibitor family, is overexpressed in poorly differentiated colorectal tumors and more frequently found in tumors with microsatellite instability. Immunohistochemical nuclear Maspin staining is predominantly seen in tumor cells at the invasion front of such cancers, suggesting that this molecule is associated with local tumor cell infiltration and aggressiveness. In a retrospective study, we studied nuclear Maspin expression as a potential prognostic tool in a total of 172 primary stage III colon cancers by immunohistochemistry. Of those 172 patients, 76 were treated by surgery only, and 96 patients received additional adjuvant 5‐fluorouracil (5‐FU) based chemotherapy. Nuclear Maspin expression was an independent adverse prognostic factor for overall survival in our patient cohort (hazard ratio 2.08; 95% CI, 1.13–3.81; p = 0.018). However, patients with primary tumors expressing Maspin in the nucleus showed a significant treatment benefit from 5‐FU chemotherapy (hazard ratio 0.384; 95% CI, 0.188–0.784; p = 0.009) compared to adjuvantly treated patients whose tumors did not express this molecule. Nuclear Maspin expression is highly predictive of 5‐FU chemotherapy response in patients with advanced stage colon cancer. Patients with negative immunhistochemical Maspin expression do not benefit from 5‐FU treatment and may be candidates for an alternative (non‐5‐FU based) adjuvant therapy regime.