Epidemiologic associations of HPV-positive oropharyngeal cancer and (pre)cancerous cervical lesions.

Human papillomavirus (HPV)-induced oropharyngeal squamous cell carcinoma (OPSCC) remains increasing worldwide. We aimed to investigate if the HPV-prevalence of OPSCC in the Netherlands is rising as well, also in female patients. In addition, we evaluated the association between HPV-positive OPSCC and suspicious Pap results of the cervix in these female patients. Patients with OPSCC treated in the period 2000-2015 at the VU University Medical Center Amsterdam, were included (n = 926). The presence of an oncogenic HPV infection was determined by p16-immunostaining, followed by a high-risk HPV general primer 5+/6+ DNA PCR on the p16-immunopositive cases. A review of pathology reports in all female patients (n = 305) was undertaken to identify cytological signs of HPV-related (pre)cancer of the cervix. In total 281 of 926 (30.3%) OPSCCs were HPV-positive. Moreover, a significant increase in the prevalence of HPV-positive OPSCCs was observed from 14.0% in 2000 to 48.1% in 2015 (p < 0.001). Among the female patients with an HPV-positive OPSSC (n = 70), the results of cervical smears were available in 56 of 70 patients (80.0%). Of the female patients with HPV-positive OPSCC, 9 of 56 (16.1%) patients had a vaginal cuff Papanicolaou (Pap) test ≥3b in their medical history compared to 7 of 168 (4.2%) in the HPV-negative group (p = 0.003). In conclusion, a continuous increase in the HPV-attributable fraction of OPSCC was demonstrated in the period 2000-2015 in the Amsterdam region. HPV-positive OPSCC has a significant association with a history of suspicious Pap results of the cervix in female patients.

Evaluation of the eighth TNM classification on p16-positive oropharyngeal squamous cell carcinomas in the Netherlands and the importance of additional HPV DNA testing.

Background: Oropharyngeal squamous cell carcinomas (OPSCCs) are traditionally caused by smoking and excessive alcohol consumption. However, in the last decades high-risk human papillomavirus (HPV) infections play an increasingly important role in tumorigenesis. HPV-driven OPSCCs are known to have a more favorable prognosis, which has led to important and marked changes in the recently released TNM-8. In this 8th edition, OPSCCs are divided based on p16 immunostaining, with p16 overexpression as surrogate marker for the presence of HPV. The aims of this study are to evaluate TNM-8 on a Dutch consecutive cohort of patients with p16-positive OPSCC and to determine the relevance of additional HPV DNA testing. Patients and methods: All OPSCC patients without distant metastases at diagnosis and treated with curative intent at VU University Medical Center (2000-2015) and Erasmus Medical Center (2000-2006) were included (N = 1204). HPV status was determined by p16 immunostaining followed by HPV DNA PCR on the p16-immunopositive cases. We compared TNM-7 and TNM-8 using the Harrell's C index. Results: In total, 388 of 1204 (32.2%) patients were p16-immunopositive. In these patients, TNM-8 had a markedly better predictive prognostic power than TNM-7 (Harrell's C index 0.63 versus 0.53). Of the 388 p16-positive OPSCCs, 48 tumors (12.4%) were HPV DNA-negative. This subgroup had distinct demographic, clinical and morphologic characteristics and showed a significantly worse five-year overall survival compared with the HPV DNA-positive tumors (P < 0.001). Conclusions: TNM-8 has a better predictive prognostic power than TNM-7 in patients with p16-positive OPSCC. However, within p16-positive OPSCCs, there is an HPV DNA-negative subgroup with distinct features and a worse overall survival, indicating the importance to perform additional HPV DNA testing when predicting prognosis and particularly for selecting patients for de-intensified treatment regimens.

Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.

BACKGROUND: Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation--constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. METHODS: Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. RESULTS: In vitro, many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. CONCLUSIONS: These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

The role of inflammation and infection in prostate cancer: Importance in prevention, diagnosis and treatment.

It is currently estimated that infections and inflammatory responses are linked to 15-20% of all deaths from cancer worldwide. Many studies point to an important role of inflammation in prostate growth, although the contribution of inflammation to benign prostatic hyperplasia and prostate cancer is not completely understood. There is an unmet need for epidemiologic and molecular pathologic approaches to address the issue of inflammation and prostate cancer. Here we review the published evidence with respect to the involvement of inflammation and infection in prostate cancer. We also present an overarching hypothesis that chronic inflammation associated with aging and infection may play an important role in the etiology and progression of prostate cancer. As such, chronic inflammation may represent an important therapeutic target in prostate cancer.

Impact of summer droughts on water quality of the Rhine River - a preview of climate change?

It is generally recognized that climate change will affect the discharge regime of the Rhine River. Especially the anticipated increase in extreme river discharges (floods and droughts) poses serious problems to water management, both with regard to water quantity and water quality. Water quality effects of climate change are not sufficiently recognized, however. The purpose of this study is to investigate the impact of droughts on the water quality of the River Rhine. Time series of river flow and water quality were analyzed for station Lobith, located at the Dutch-German border. Over the past three decades, three major droughts were identified, occurring in the years 1976, 1991, and 2003. The water quality during these dry years was compared with the water quality in reference years, characterized by average hydrological conditions and similar chemical pollution. Four groups of water quality parameters were investigated: 1, general variables (water temperature, dissolved oxygen, chlorophyll-a); 2, major ions (chloride, sodium, sulfate, fluoride, bromide); 3, nutrients; and 4, heavy metals. It was found that water quality is negatively influenced by (summer) droughts, with respect to water temperature, eutrophication, major ions and heavy metals. Effects on nutrient concentrations were small for ammonium and could not be demonstrated for nitrate, nitrite and phosphate. The decline in water quality during summer droughts is both related to the high water temperatures and to low river discharges (limited dilution of the chemical load from point sources). Moreover, the impact of the 1976 drought on water quality was far more important than that of the 2003 drought, indicating that the impact of droughts on water quality will be greater when the water quality is already poor.

The prognostic value of E-cadherin and the cadherin-associated molecules alpha-, beta-, gamma-catenin and p120ctn in prostate cancer specific survival: a long-term follow-up study.

OBJECTIVES: To determine the value of loss of expression of E-cadherin and cadherin associated molecules as prognostic markers for prostate cancer patients in a long-term follow-up study. METHODS: Sixty-five prostate cancer specimens, obtained from patients with different stages of prostate cancer who underwent a radical prostatectomy or TUR-P between 1987 and 1991, were used for immunohistochemical analysis of the expression pattern of E-cadherin, alpha-, beta-, gamma-catenin and p120(ctn). Clinical records of these patients were studied for follow-up data and the prognostic value of expression of these adhesion molecules was determined by Kaplan-Meier survival analyses and multivariable proportional hazard regression analysis. RESULTS: Normal staining patterns were found in 36 cases (55.4%) for E-cadherin, 37 cases (56.9%) for alpha-catenin, 40 cases (61.5%) for beta-catenin, 25 cases (38.5%) for gamma-catenin, and 40 cases (61.5%) for p120(ctn). Overall, a strong correlation was found between the expression of E-cadherin and other cadherin-associated molecules. The 5-year survival rates for each staining were as follows: E-cadherin (normal 79.2%, aberrant 26.8%), alpha-catenin (normal 79.2%, aberrant 26.8%), beta-catenin (normal 73.1%, aberrant 27.3%), gamma-catenin (normal 86.4%, aberrant 37.1%), and p120(ctn) (normal 72.8%, aberrant 30.0%). There was a significant difference in survival between normal and aberrant expression in each staining (log rank P < 0.0001). The proportional hazard regression model including tumor stage and Gleason score revealed alpha-catenin expression as the best prognostic marker for patients with prostate cancer. CONCLUSIONS: Our data revealed a strong correlation between E-cadherin expression and other cadherin-associated molecules. Among these markers, alpha-catenin seems the best prognostic marker for prostate cancer specific survival. Larger studies are needed to confirm this result.

TSU-Pr1 and JCA-1 cells are derivatives of T24 bladder carcinoma cells and are not of prostatic origin.

We have shown previously that the putative prostate carcinoma cell lines TSU-Pr1 and JCA-1 share a common origin. The observation that these cell lines have p53 and Ha-ras mutations identical to those in bladder carcinoma cell line T24 prompted us to investigate their possible interrelations. We used cytogenetics and DNA profiling to compare the genetic backgrounds of the three cell lines. At least 12 structural chromosomal abnormalities are shared between T24, TSU-Pr1, and JCA-1 cells. DNA profiles were identical for all three cell lines. These results clearly indicate that the cell lines TSU-Pr1 and JCA-1 are not of prostatic origin but are derivatives of the bladder carcinoma cell line T24. TSU-Pr1 and, to a lesser extent, JCA-1 are frequently used as models in prostate cancer research, and numerous publications have appeared based on these lines. Several other T24 cross-contaminants have been identified in the past, and some of these, such as ECV304, continue to be used under the wrong identity. Our findings highlight the insidious problem that can occur when information regarding cross-contamination does not reach individual researchers and/or the importance of the problem is not fully acknowledged.

Widely used prostate carcinoma cell lines share common origins.

BACKGROUND: Cross-contamination is a persistent problem in the establishment and maintenance of mammalian cell lines. The observation that the cell lines PC-3, ALVA-31, and PPC-1 all have a homozygous deletion of the alpha-catenin gene prompted us to investigate the uniqueness of these and several other widely used prostate carcinoma cell lines. METHODS: The genetic backgrounds of the putative human prostate cell lines (ALVA-31, ALVA-41, BPH-1, DU 145, JCA-1, LAPC-4, LNCaP, NCI-H660, ND-1, PC-3, PC-3MM2, PC-346C, PPC-1, and TSU-Pr1) were analyzed by cytogenetics, mutation analysis, and DNA profiling. RESULTS: Similarities between several groups of cell lines were found. ALVA-31, ALVA-41, PC-3, PC-3MM2, and PPC-1 all have a deletion of a C in codon 138 of the p53 gene and show almost identical DNA profiles. The ND-1 cell line has two p53 mutations that are identical to the mutations found in DU 145. These two cell lines also share a high number of structural chromosomal abnormalities and nearly identical DNA profiles. The cell lines TSU-Pr1 and JCA-1 share an identical p53 mutation in exon 5 and identical DNA profiles. CONCLUSIONS: Several widely used prostate carcinoma cell lines apparently have identities in common. The knowledge that some of these cell lines are derivatives of one another prompts re-evaluation of previously obtained results.

The effect of fibroblast growth factor 8, isoform b, on the biology of prostate carcinoma cells and their interaction with stromal cells.

Fibroblast growth factor 8, isoform b (FGF8b), has been implicated in the oncogenesis of the prostate and mammary epithelia. We examined whether overexpression of FGF8b in a weakly tumorigenic prostate carcinoma cell line, LNCaP, could alter the growth and tumorigenic properties of these cells. LNCaP cells were infected with a lentivirus vector carrying FGF8b cDNA and the green fluorescent protein (GFP) cDNA in the same construct, and the infected cell population was sorted on the basis of GFP protein expression. It was demonstrated that, in comparison with the cells transduced with GFP-vector alone, LNCaP cells with FGF8b-GFP expression manifested an increased growth rate, higher soft agar clonogenic efficiency, enhanced in vitro invasion, and increased in vivo tumorigenesis. Most strikingly, whereas parental or vector-control LNCaP cells failed to grow at all in an in vivo tumorigenesis/diaphragm invasion assay in nude mice, the cells overexpressing FGF8b proliferated as deposits of tumor cells on the diaphragm, frequently with indications of tumor cell invasion into the diaphragm. Coculturing of primary prostatic or non-prostatic stromal cells with the infected LNCaP cells led us to observe that: (a) stromal cells, irrespective of tissue origin, strongly suppressed LNCaP cell growth; (b) FGF8b producing LNCaP cells could partially evade the stromal inhibition, perhaps from the autocrine stimulatory effect of FGF8b; and (c) production of FGF8b in the coculture had a stimulatory effect on the proliferation of the stromal cells, prostatic or non-prostatic. This stimulation was not attributable to the direct action of FGF8b on stromal cells. Instead, it appears that epithelial-stromal cell-cell contact and some unknown soluble factors secreted by LNCaP cells upon stimulation of FGF8b are required for the maximal effect. Together, these results suggest that the growth rate and biological behavior of prostatic cancer cells can be altered to a more aggressive phenotype by up-regulation of FGF8b expression. These changes in phenotype also influence the interaction of the affected cells with stromal cells. The data obtained may have direct relevance to the progression of prostate cancer, recognizing that FGF8b is naturally overexpressed in advanced disease.

Isolation and characterization of the promoter of the human prostate cancer-specific DD3 gene.

Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific genes described to date (Bussemakers, M. J. G., van Bokhoven, A., Verhaegh, G. W., Smit, F. P., Karthaus, H. F. M., Schalken, J. A., Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 5975-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer. To identify the promoter elements that are responsible for the prostate cancer-specific expression of DD3, we have isolated and characterized the DD3 promoter. Sequence analysis of the DD3 5'-flanking region was performed and several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter construct, we could detect promoter activity in LNCaP cells, which was not affected by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprint analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-protected areas and recruits another, yet unidentified, protein to the DD3 promoter in LNCaP cells.

Cadherin switching in human prostate cancer progression.

The progression of carcinomas is associated with the loss of epithelial morphology and a concomitant acquisition of a more mesenchymal phenotype, which in turn is thought to contribute to the invasive and/or metastatic behavior of the malignant process. Changes in the expression of cadherins, "cadherin switching," plays a critical role during embryogenesis, particularly in morphogenetic processes. Loss of E-cadherin is reported to be associated with a poor prognosis; however, thus far, evidence (R. Umbas, et al., Cancer Res. 54: 3929-3933, 1994) for up-regulation of other cadherins has only been reported in vitro, ie., we have found evidence (M. J. G. Bussemakers et al., Int. J. Cancer, 85: 446-450, 2000) for cadherin switching in prostate cancer cell lines (up-regulation of N-cadherin and cadherin-11, two mesenchymal cadherins, in cell lines that lack a functional E-cadherin-catenin adhesion complex). Here, we report on the immunohistochemical analysis of the expression of N-cadherin and cadherin-11 in human prostate cancer specimens. N-cadherin was not expressed in normal prostate tissue; however, in prostatic cancer, N-cadherin was found to be expressed in the poorly differentiated areas, which showed mainly aberrant or negative E-cadherin staining. Cadherin-11 is expressed in the stroma of all prostatic tumors, in the area where stromal and epithelial cells are found. In addition, cadherin-11 is also expressed in a dotted pattern or at the membrane of the epithelial cells of high-grade cancers. In a number of metastatic lesions, N-cadherin and cadherin-11 are expressed homogeneously. These data raise the possibility that cadherin switching plays an important role in prostate cancer metastasis.

Partial sequencing and tissue distribution of the canine isoforms of steroid 5alpha-reductase type I and type II.

BACKGROUND: The dog is regarded to be a valid model to test the effects of 5alpha-reductase inhibitors on prostatic growth. However, limited information is available on the characteristics or even existence of 5alpha-reductase isozymes in this species. METHODS: Here, we set out to clone the cDNA of the dog isoforms of 5alpha-reductase type I and type II by a degenerate cloning strategy and to assess the tissue distribution of both transcripts and the enzymatic activity of the isozymes. RESULTS: We identified two clones with homology to the human 5alpha-reductase isoforms type I and type II to be expressed in dog prostate. At the amino-acid level, these partial clones were found to exhibit a homology with their human counterparts of 83% and 88%, respectively. The expression levels of 5alpha-reductase mRNA were screened by RT-PCR in a number of dog tissues. No correlation was found between tissue mRNA expression and enzymatic 5alpha-reductase activities. CONCLUSIONS: The present study describes the partial cloning of the dog 5alpha-reductase isozymes and their tissue distribution. These results provide additional data for the use of the dog as an animal model to investigate the role of 5alpha-reductase isozymes in steroid metabolism.

Real-time quantification of human telomerase reverse transcriptase mRNA in tumors and healthy tissues.

BACKGROUND: Expression of the hTERT gene, which codes for the catalytic subunit of telomerase, is associated with malignancy. We recently developed a real-time reverse transcription-PCR assay, based on TaqMan technology, for accurate and reproducible determination of hTERT mRNA expression (Lab Investig 1999;79:911-2). This method may be of interest for molecular tumor diagnostics in tissues and corresponding body fluids, washings, or brushes. METHODS: In this study, we measured hTERT expression in a subset of healthy tissues and tumors to select those tumor types with the best potential for quantification of hTERT in corresponding body fluids. To demonstrate the use of the method in body fluids, we quantified hTERT expression in voided urine of patients with bladder cancer and controls. RESULTS: Real-time measurement of hTERT expression could discriminate between all healthy and malignant tissue samples from pancreas, lung, esophagus, and bladder, but not for colon tissues. Moreover, in five of nine (55%) urine samples, hTERT could be quantified. CONCLUSIONS: The present study demonstrates that accurate quantitative measurement of hTERT expression has high potential for discrimination between healthy and tumor cells in tissues and urine and supports future measurements in pancreatic fluid, bronchoalveolar lavage fluid, esophageal brushings, and urine or bladder washings.

Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250.

The molecular cloning of the cDNA and gene encoding the renal cell carcinoma (RCC)-associated protein G250 is described. This protein is one of the best markers for clear cell RCC: all clear-cell RCC express this protein, whereas no expression can be detected in normal kidney and most other normal tissue. Antibody studies have indicated that this molecule might serve as a therapeutic target. In view of the induction/up-regulation of G250 antigen in RCC, its restricted tissue expression and its possible role in therapy, we set out to molecularly define the G250 antigen, which we identified as a transmembrane protein identical to the tumor-associated antigen MN/CAIX. We determined, by FISH analysis, that the G250/MN/CAIX gene is located on chromosome 9p12-13. In view of the relative immunogenicity of RCC, we investigated whether the G250 antigen can be recognized by TIL derived from RCC patients. The initial characterization of 18 different TIL cultures suggests that anti-G250 reactivity is rare.

Coordinate recruitment of E-cadherin and ALCAM to cell-cell contacts by alpha-catenin.

Here we report on the role of alpha-catenin in the cellular localization of activated leukocyte cell adhesion molecule, ALCAM, and cadherin-mediated cell adhesion in human prostate cancer cells. Cell lines that have a functional E-cadherin-mediated cell adhesion (DU-145 and LNCaP) show ALCAM staining at cell-cell contacts. In contrast, in cell lines that lack alpha-catenin expression (ALVA-31, PC-3, and PPC-1), E-cadherin-mediated adhesion is disturbed and ALCAM staining is cytoplasmic. A role of alpha-catenin in the recruitment of E-cadherin and ALCAM to cell-cell contacts was established by transfection of an alpha-N-catenin construct into cell lines ALVA-31 and PC-3. This resulted not only in the correct assembly of E-cadherin/alpha-catenin complexes at the cell membrane but also in localization of ALCAM to cell-cell contacts, indicating that indeed alpha-catenin affects ALCAM localization.

Complex cadherin expression in human prostate cancer cells.

Changes in cell-cell interactions are critical in the process of cancer progression. Likewise, it has been shown that loss of expression of the cell adhesion molecule E-cadherin is associated with grade, stage, and prognosis in many carcinomas, including prostate cancer. Impaired E-cadherin-mediated interactions result in an invasive phenotype; however, the mere loss of cell-cell contact and communication is not the sole explanation for the observed correlation between loss of E-cadherin-mediated adhesion and poor clinical outcome. Using a degenerate cloning strategy for sequences that are highly conserved between the various cadherins, we found several other cadherins (N- and P-cadherin and cadherin-4, -6, and -11) to be expressed in human prostate cancer cells. Our data suggest that besides loss of E-cadherin function, also (upregulation of) expression of other cadherins is involved in the acquisition of an invasive and/or metastatic phenotype. Especially, changes in the expression of N-cadherin and cadherin-11 may play an important role in prostate cancer progression.

DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the Western male population. Despite the tremendous efforts that have been made to improve the early detection of this disease and to design new treatment modalities, there is still an urgent need for new markers and therapeutic targets for the management of prostate cancer patients. Using differential display analysis to compare the mRNA expression patterns of normal versus tumor tissue of the human prostate, we identified a cDNA, DD3, which is highly overexpressed in 53 of 56 prostatic tumors in comparison to nonneoplastic prostatic tissue of the same patients. Reverse transcription-PCR analysis using DD3-specific primers indicated that the expression of DD3 is very prostate specific because no product could be amplified in 18 different normal human tissues studied. Also, in a sampling of other tumor types and a large number of cell lines, no expression of DD3 could be detected. Molecular characterization of the DD3 transcription unit revealed that alternative splicing and alternative polyadenylation occur. The fact that no extensive open reading frame could be found suggests that DD3 may function as a noncoding RNA. The DD3 gene was mapped to chromosome 9q21-22, and no homology of DD3 to any gene present in the computer databases was found. Our data indicate that DD3 is one of the most prostate cancer-specific genes yet described, and this makes DD3 a promising marker for the early diagnosis of prostate cancer and provides a powerful tool for the development of new treatment strategies for prostate cancer patients.

Molecular analysis of multifocal prostate cancer lesions.

To analyse the origin of multifocal prostate cancer lesions, radical prostatectomy specimens from 17 patients were examined. As a marker of genetic lineage, the allelotype based on 33 microsatellite loci was compared between the different tumours present in a given case. Some results provide evidence suggestive of a clonal origin of multiple tumours in a subset of the prostates. In five cases, for example, comparison of multifocal tumour lesions within a given case revealed at least two concordant changes in allelic imbalance (AI) sequence dosages at different loci. In addition, considerable heterogeneity of allelotype was found within and among tumour foci of a given case. In five of the six tumours analysed for intratumour heterogeneity, for example, more than five discordant AI changes were found in one tumour region but not in the other. Conclusions regarding the clonality of such heterogeneous lesions are difficult to draw. A high frequency of AI changes in four lesions exhibiting prostatic intraepithelial neoplasia (mean 6.5 changes per lesion, range 3-6) was found, compared with eight primary tumours present in the same cases (mean 5.8 changes per lesion, range 3-6). The interpretation of AI associated with clinically detected prostate cancer remains a highly complex issue. The fact that no clear evidence was obtained for either a clonal or a non-clonal origin of multiple lesions in a given prostate indicates that several different mechanisms are likely to operate in establishing the allelotype and that additional evidence from unique mutations or selective gene inactivation may be necessary to obtain definitive results.

Expression of cadherin-6 as a novel diagnostic tool to predict prognosis of patients with E-cadherin-absent renal cell carcinoma.

In many carcinomas, E-cadherin is considered to be a prognostic marker for patient survivals, and its decreased expression is associated with metastatic disease. Among renal cell carcinomas (RCCs), however, only 20% of tumors express E-cadherin, whereas a much higher percentage express other cadherins, e.g., N-cadherin and cadherin-6 (T. Shimazui et al, Cancer Res., 56: 3234-3237, 1996). Among these cadherins expressed in RCCs, cadherin-6 has been identified as a major cadherin in the renal proximal tubules and in the tumors themselves. Hence, we have investigated the relationship between prognosis and cadherin-6 expression in tumor cells in 43 patients with RCC. Expression of cadherin-6, E-cadherin, and alpha-catenin was detected immunohistochemically and evaluated microscopically as normal, heterogeneous, or absent. Normal, heterogeneous, and absent expression of cadherin-6 were observed in 19, 16, and 8 of 43 cases, respectively. Coexpression of E-cadherin and cadherin-6 was detected in only 10 cases. Among 30 tumors in which E-cadherin expression was absent, 24 expressed cadherin-6. In addition, the expression pattern of alpha-catenin correlated more highly with that of cadherin-6 than it did with E-cadherin (P = 0.0003 versus 0.025). In survival analyses, aberrant expression of cadherin-6 correlated with poor survivals both among all patients (P = 0.0009) and in those with E-cadherin-absent RCC (P = 0.0008). These results suggest that cadherin-6 is a major cadherin playing an essential role in cell-cell adhesion in E-cadherin-absent RCC.

Role of E boxes in the repression of E-cadherin expression.

Decreased expression of the intercellular adhesion molecule E-cadherin correlates with tumor aggressiveness and invasion capacity of cell lines. The decrease of E-cadherin expression results primarily from reduced mRNA expression. We show here that the activity of a human E-cadherin promoter construct is cell specific and correlates with E-cadherin mRNA expression. This spectrum of activity is conserved by a region as short as 81 bp obtained after 5' deletion. Mutation analysis revealed that two E box elements of this minimal promoter are involved in the silencing of E-cadherin promoter activity occurring in cancer cells. E boxes are mainly known as target sequences for bHLH transcription factors that are involved in the control of tissue differentiation and are antagonised by HLH proteins. However, mutation data and cotransfection experiments using HLH protein expression vectors indicate that bHLH transcription factors are not significantly involved in the E box mediated silencing of this E-cadherin promoter fragment.