Renal-cell carcinomas (RCC) are frequent in central and eastern Europe and the reasons remain unclear. Molecular mechanisms, except for VHL, have not been much investigated. We analysed 361 RCCs (334 clear-cell carcinomas) from a multi-centre case-control study for mutations in TP53 (exons 5-9 in the whole series and exons 4 and 10 in a pilot subset of 60 tumours) and a pilot 50 tumours for mutations in EGFR (exons 18-21) or KRAS (codon 12) in relation to VHL status. TP53 mutations were detected in 4% of clear-cell cases, independently of VHL mutations. In non-clear-cell carcinomas, they were detected in 11% of VHL-wild-type tumours and in 0% of tumours with VHL functional mutations. No mutations were found in EGFR or KRAS. We conclude that mutations in TP53, KRAS, or EGFR are not major contributors to the RCC development even in the absence of VHL inactivation. The prevalence of TP53 mutations in relation to VHL status may differ between clear-cell and other renal carcinomas.

There is compelling evidence from transgenic mouse studies and analysis of mutations in human carcinomas indicating that the TGF-β signal transduction pathway is tumor suppressive.We have shown that overexpression of TGF-β1 in mammary epithelial cells suppresses the development of carcinomas and that expression of a dominant negative type II TGF-β receptor (DNIIR) in mammary epithelial cells under control of the MMTV promoter/enhancer increases the incidence of mammary carcinomas.Studies of human tumors have demonstrated inactivating mutations in human tumors of genes encoding proteins involved in TGF-β signal transduction, including DPC4/Smad4, Smad2, and the type II TGF-β receptor (TβRII).There is also evidence that TGF-β can enhance the progression of tumors.This hypothesis is being tested in genetically modified mice.To attain complete loss of TβRII, we have generated mice with loxP sites flanking exon 2 of Tgfbr2 and crossed them with mice expressing Cre recombinase under control of the MMTV promoter/enhancer to obtain Tgfbr2 mgKO mice.These mice show lobuloalveolar hyperplasia.Mice are being followed for mammary tumor development.Tgfbr2 mgKO mice that also express polyoma virus middle T antigen under control of the MMTV promoter (MMTV-PyVmT) develop mammary tumors with a significantly shorter latency than MMTV-PyVmT mice and show a marked increase in pulmonary metastases.Our data do not support the hypothesis that TGF-β signaling in mammary carcinoma cells is important for invasion and metastasis, at least in this model system.The importance of stromal-epithelial interactions in mammary gland development and tumorigenesis is well established.These interactions probably involve autocrine and paracrine action of multiple growth factors, including members of the TGF-β family, which are expressed in both stroma and epithelium.Again, to accomplish complete knockout of the type II TGF-β receptor gene in mammary stromal cells, FSP1-Cre and Tgfbr2 flox/flox mice were crossed to attain Tgfbr2 fspKO mice.The loss of TGF-β responsiveness in fibroblasts resulted in intraepithelial neoplasia in prostate and invasive squamous cell carcinoma of the forestomach with high penetrance by 6 weeks of age.Both epithelial lesions were associated with an increased abundance of stromal cells.Activation of paracrine hepatocyte growth factor (HGF) signaling was identified as one possible mechanism for stimulation of epithelial proliferation.TGF-β signaling in fibroblasts thus modulates the growth and oncogenic potential of adjacent epithelia in selected tissues.More recently, we have examined the effects of Tgfbr2 fspKO fibroblasts on normal and transformed mammary epithelium.We analyzed the role of TGF-β signaling by stromal cells in mammary tumor progression.To avoid the possibility of endogenous wild-type fibroblasts masking potential effects of Tgfbr2 fspKO cells on tumor progression, we implanted PyVmT mammary carcinoma cells with Tgfbr2 fspKO or wildtype fibroblasts in the subrenal capsule of nude mice.Mammary tumor cells implanted with Tgfbr2 fspKO cells exhibited an increase in tumor growth and intravasation associated with an increase in tumor cell survival, proliferation and an increase in tumor angiogenesis compared with tumor cells implanted with control fibroblasts.We demonstrated increased expression of several growth factors by Tgfbr2 fspKO fibroblasts compared with control fibroblasts in primary culture.These included HGF, MSP and TGF-α.There was an increase in tumor cell activating phosphorylation of the cognate receptors, c-Met, RON, erbB1, and erbB2 in carcinomas accompanied by Tgfbr2 fspKO fibroblasts.The Tgfbr2 fspKO mouse model illustrates that a signaling pathway known to suppress cell-cycle progression when activated in epithelial cells can also have an indirect inhibitory effect on epithelial proliferation when activated in adjacent stromal fibroblasts in vivo.Loss of this inhibitory effect can result in increased epithelial proliferation and may even progress to invasive carcinoma in some tissues.

Advances in understanding cancer at the molecular level have identified numerous genomic and proteomic alterations associated with cancer development and progression. The efforts in evaluating these putative biomarkers in clinical studies of patients with cancer are increasing, reflecting the great potential of molecular phenotyping to improve screening, diagnosis, and treatment (1). The translation of new markers to the clinic is often hampered by the fact that they are typically evaluated in single institutional settings, making comparison with other markers difficult due to differences in study design, experimental methods, and data analysis. There are no well-recognized guides that the urologic researcher can use to develop marker studies.There is a clinical need for markers that will identify patients with a high risk for progression or predict tumor response to specific therapeutic regimens. Eighty percent of patients presenting with bladder cancer have superficial tumors that do not invade the muscular layer of the bladder wall and generally have a good prognosis (2). Nevertheless, 25% to 30% of these patients will eventually experience progression of their tumor into the muscle and 50% of them will die of their disease (3), translating into a mortality rate of 4 cases per 100,000 total population per year (4). Despite the description of high-risk and low-risk groups in superficial bladder cancer, clinical or pathologic prognostic factors cannot predict progression on an individual basis (5). Validated markers could enable patient-specific treatment decisions (6).The measurement of p53 is an example of the difficulty in marker development. Research on p53 in bladder cancer began with its discovery more than two decades ago (7, 8) and includes the original studies of its biological role in bladder cancer (9, 10), continuing to studies that examined the use of p53 to predict bladder cancer recurrence and progression (11–14) to the current use of p53 status to select patients for treatment within clinical trials (15). Several studies have shown that p53 alterations are present in a substantial proportion of bladder tumors. However, despite promising data suggesting that p53 expression by immunohistochemical analysis is a useful marker for bladder cancer (16, 17), the evaluation of this marker has been hampered by differences in study design, assay, and analysis (18, 19)The Bladder Cancer Marker Network, under the sponsorship of the National Cancer Institute, was formed to develop and assess biomarkers for diagnostic and prognostic use. This core of investigators initiated the first International Workshops on Diagnostic and Prognostic Markers in Bladder Cancer (20) resulting in the formation of the International Bladder Cancer Network (http://www.uni-essen.de/urologie/ibcn/index.html). This group defined four phases through which markers are developed.Phase 1 and 2 studies are often done at single institutions that can rapidly develop laboratory techniques and obtain preliminary information regarding the potential of new markers. However, it is rare that a single institution has the resources to achieve the required subject diversity or the large number of specimens to complete properly designed phase 3 or 4 studies. In addition, a multi-institutional approach offers advantages even in the initial phases of marker discovery, because samples obtained from multiple sources may help identify causes of variability. The essential interactions required by phase 3 and 4 evaluations and the need to gain access to greater resources are clearly facilitated by collaborative networks. Tumor banks and procedures are in place in many institutions, and there are several important existing networks and collaborations (21, 22) that have established procedures for data and tissue collection. However, only few groups have established general methodologic principles as guidelines and use discrete phases of marker development as an integral part of their collaboration.The formation of an International Bladder Cancer Bank (IBCB) was a logical but an ambitious proposal arising from the International Workshops on Diagnostic and Prognostic Markers in Bladder Cancer held in Barcelona and Trento. The IBCB is designed to (a) obtain access to tumor specimens from a large number of patients with bladder cancer, including rare entities; (b) standardize methods of tissue procurement and biomarker assay performance; (c) establish consistent methods of obtaining and coding patient information, end point definitions, and data management; and (d) serve as a resource for participating institutions to evaluate the biological and prognostic significance of potential “markers” in bladder cancer development and progression. It will coordinate and exploit the capabilities and resources offered by the current participating institutions while recruiting other new institutions to expand the resource and knowledge base, and it will complement efforts of cooperative groups by focusing on bladder cancer and collecting specimens from patients who are treated on standard regimens but are not part of large clinical trials.Tissue and data procurement are the fundamental components in this infrastructure, whereas execution of the biomarker assays—analysis and interpretation—is the core of any marker study. A detailed description of the IBCB was introduced recently (23). In brief, samples will be identified and stored at each participating institution using agreed-upon protocols and procedures. Two basic models are used to process tissue and perform assays in the multicenter setting. Identified samples are either transferred to a core facility for analysis or analyzed locally with predefined standardized protocols and techniques. For statistical analysis, results are transferred to a study core center. In addition, the use of tissue microarrays can be used to great advantage, as it allows tissue to be collected centrally with minimal loss of the tissue at the contributing institution (24). Common software will facilitate local data management of a standard, minimal set of patient and pathologic information and allow transfer to a central database. All contributing centers will have an overview of the available tissue within the bank and some of their clinical and pathologic characteristics. In summary, the advantages of this infrastructure include consistency among participating institutions regarding procurement and processing of specimens and collecting and managing the associated information. The IBCB will provide prospectively collected data sets with long-term follow-up that are linked to specimens. This will simplify the process of finding collaborating partners as well as provide accurate prediction of the number of available specimens to facilitate study design. As a result, studies can be completed much sooner than would be possible with a conventional prospective study in which follow-up begins at the time of specimen acquisition. For this multi-institutional approach to marker development and its supporting infrastructure, there is a recognized need to combine knowledge from different scientific fields, such as computer technology, statistics, epidemiology, pathology, ethics, oncology, and urology.The process to convert this concept into practice has already begun. The International Bladder Cancer Network established a collaboration with the National Cancer Institute Specialized Program of Research Excellence program and the Genitourinary Specialized Program of Research Excellence in Bladder Cancer at the University of Texas M.D. Anderson Cancer Center to create an international multi-institutional tissue resource and database by sharing bladder tumor specimens and establishing tissue microarrays to conduct highly powered multivariate biomarker studies to improve our understanding of the biology of bladder cancer. These efforts will enhance the development of more effective therapy. The IBCB/National Cancer Institute consortium will address pertinent translational research questions that cannot be adequately addressed by a single research institution. These investigations will foster the integration of new tools and strategies that will help standardize their use in pathologic and clinical applications.The recent publication of the multisector report entitled “National Biospecimen Network Blueprint” reflects a growing consensus on the need to establish a biorepository system that will provide investigators with the highest-quality biospecimens that are uniformly collected, stored, and annotated. The National Biospecimen Network Blueprint also includes a bioinformatics platform that would allow broad access to data and data analysis tools to the scientific community. This blueprint offers the opportunity to establish standardized operating procedures for specimen processing that would minimize preanalytic variability, facilitating the comparison of biomarker data derived from the analysis of specimens from different institutions. The development of an IBCB within the context of the National Biospecimen Network system may enhance the ability of public and private researchers to access many high-quality and well-annotated bladder cancer specimens without intellectual property restrictions.Modern genomics and proteomics are yielding an ever-increasing number of potential markers. The comprehensive infrastructure of the IBCB has the potential to be an effective resource that addresses the multiple methodologic difficulties that occur during all phases of marker development. Multi-institutional and interdisciplinary networks with a robust infrastructure, such as the proposed IBCB, will provide the resources enabling the evaluation of novel markers in an accurate and efficient fashion.

The remarkable generation of scores of increasingly sophisticated mouse models of mammary cancer over the past two decades has provided tremendous insights into molecular derangements that can lead to cancer.The relationships of these models to human breast cancer, however, remain problematic.Recent advances in genomic technologies offer significant opportunities to identify critical changes that occur during cancer evolution and to distinguish in a complex and comprehensive manner the key similarities and differences between mouse models and human cancer.Comparisons between mouse and human tumors are being performed using comparative genomic hybridization, gene expression profiling, and proteomic analyses.The appropriate use of genetically engineered mouse models of mammary cancer in preclinical studies remains an important challenge which may also be aided by genomic technologies.Genomic approaches to cancer are generating huge datasets that represent a complex system of underlying networks of genetic interactions.Mouse models offer a tremendous opportunity to identify such networks and how they relate to human cancer.The challenge of the future remains to decipher these networks in order to identify the genetic nodes of oncogenesis that may be important targets for chemoprevention and therapy.

BACKGROUND Clinical and histopathologic characteristics that may predict risks of recurrence in women with ductal carcinoma in situ (DCIS) have not been consistently identified. We identified factors associated with recurrence as DCIS versus invasive breast cancer and determined the 5-year absolute risks of recurrence as a function of these factors. METHODS We conducted a population-based cohort study among 1036 women in the San Francisco Bay Area who were aged 40 years or older when diagnosed with DCIS and treated by lumpectomy alone from January 1983 through December 1994. Standardized pathology reviews were conducted to determine disease recurrence, defined as DCIS or invasive breast cancer diagnosed in the ipsilateral breast containing the initial DCIS lesion or at a distant site more than 6 months after the initial diagnosis and treatment of DCIS. Conditional logistic regression models were used to determine factors associated with recurrence. All statistical significance tests were two-sided. RESULTS During a median follow-up of 77.9 months, 209 women (20.2%) experienced a recurrence. Overall, the 5-year risks of recurrence as invasive cancer and as DCIS were 8.2% (95% confidence interval [CI] = 6.6% to 9.8%) and 11.7% (95% CI = 9.9% to 13.3%), respectively. The 5-year risks of recurrence as invasive cancer and as DCIS were 4.8% (95% CI = 3.7% to 6.8%) and 4.8% (95% CI = 3.8% to 5.8%), respectively, for women with low-nuclear-grade DCIS; 11.8% (95% CI = 9.9% to 14.1%) and 17.1% (95% CI = 15.5% to 18.7%), respectively, for women with high-nuclear-grade DCIS; 11.6% (95% CI = 11.3% to 12.0%) and 8.6% (95% CI = 7.1% to 10.2%), respectively, for women whose initial DCIS lesion was detected by palpation; and 6.6% (95% CI = 6.2% to 7.1%) and 14.1% (95% CI = 11.4% to 17.8%), respectively, for women with DCIS detected by mammography alone. High- (versus low-) nuclear-grade DCIS lesions and detection of the initial DCIS lesion by palpation (versus mammography) were associated with recurrence as invasive cancer. High- (versus low-) nuclear-grade lesions; resection margins that were positive, uncertain, or less than 10 mm disease-free (versus > or = 10 mm disease-free); and age 40-49 years at diagnosis (versus > or =50 years) were associated with recurrence as DCIS. CONCLUSIONS Nuclear grade is strongly associated with recurrence but not with the type of recurrence. Women with high-nuclear-grade DCIS or DCIS detected by palpation who are treated by lumpectomy alone are at relatively high risk of having an invasive breast cancer recurrence, compared with women with low-nuclear-grade or mammographically detected DCIS, and may be appropriate candidates for additional treatment.

The genetics of renal cell tumors (RCT), which occur at a high frequency in patients with end-stage renal failure (ESRF), is not yet known. Using a fluorescence microsatellite assay and comparative genomic hybridization, 18 renal tumors obtained from nine patients with ESRF were analyzed for genetic alterations, which are known to be characteristic of common nonpapillary and papillary RCT in the general population. Deletion of chromosome 3p was detected in six nonpapillary tumors, whereas trisomies of 7 and 17 or 3, 8, and 16 were seen in four of 18 tumors. No alterations were found in four tumors, and another four tumors had unspecific changes. The fragile histidine triad (FHIT) gene is localized at the most common fragile site at chromosome 3p14.2. The FHIT and the p53 tumor suppressor gene are targets of different environmental agents. Because both toxic effect and genomic instability are implicated in the development of renal cysts in ESRF, the alteration of both genes in tumor cells was analyzed. No abnormal expression of the FHIT gene or mutation of the p53 gene were found. This study suggests that the genetics and also the morphology of some of the ESRF RCT differ from those known for RCT in the general population.

Studies using comparative genomic hybridization (CGH) indicate that portions of chromosome arm 8q from 8q12 to 8qter are present at an increased relative copy number in a broad range of solid tumors. In this study we define an approximately 1 Mb wide region that appears to be frequently abnormal in copy number or structure in breast cancer cell lines and primary tumors. This was accomplished by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs) mapped to 8q2-q22. Eleven breast cancer cell lines and ten primary tumors were analyzed. A minimal region of rearrangement was localized to the CEPH-YAC 928F9 in three breast cancer cell lines with unbalanced translocation breakpoints mapping in this region. Unbalanced translocations also were detected in two primary tumors mapping between CEPH-YAC clones 890C4 and 936B3, flanking 928F9. An increased copy number in the minimal region was detected in nine cell lines and in multiple primary tumors. This suggests the possibility that a single gene mapping to 928F9 is involved in breast cancer development or progression and may be deregulated by copy number increases in some tumors and by translocation in others. Four expressed sequence tags were mapped to YAC 928F9 and analyzed for rearrangements by Southern analysis and for abnormal expression by Northern analysis.