The INK4A and the INK4B genes map to chromosome 9p21, an area frequently deleted in bladder neoplasms. In addition to the p16 protein, the INK4A encodes for a second product, termed p19(ARF). We analyzed tissues from 121 patients with initial Ta and T1 tumors. Deletions of the INK4A gene were observed in 17 of 121 (14.1%) cases. Point mutations were identified in 2 of 64 (3.1%) tumors. The INK4A-exon 1beta and the INK4B gene were codeleted with INK4A in all of the homozygously deleted cases analyzed. The p16 promoter underwent de novo methylation in 7 of 47 (14.9%) evaluable cases. The p16-positive phenotype was observed in 18 of 56 (32%) evaluable cases. p16 negative phenotype correlated with deletion and methylation status. A statistically significant association between INK4A homozygous deletions and tumor size was observed (P = 0.003). Patients bearing tumors with INK4A homozygous deletions had a lower recurrence-free survival (P = 0.040) than those with wild type INK4A. In conclusion, deletions and methylation of the INK4A gene occur frequently in superficial bladder tumors. However, only those deletions that affect both the p16 and the p19(ARF), deregulating both the pRb and p53 pathways, correlated with clinicopathological parameters of worse prognosis.

Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.The a,-fetoprotein (AFP) gene, a member of the albumin gene family, is expressed by fetal or malignant hepatocytes and repressed in normal mature hepatocytes. This forms the basis of a long-exploited model system to study cell differentiation and its impairment in neoplasia (1). Molecular genetics have brought the AFP model to molecular levels of cell differentiation, toward finely discerning how genes respond to or escape from developmental and growth signals (2,40,41). As yet, little is known about how differential regulation is exerted on the tandemly organized AFPalbumin locus and whether the two genes operate under shared elements of control. However, at least one wellcharacterized transcription factor, the glucocorticoid receptor, is known to selectively shut off the AFP gene in the developing liver (4, 21). This hormonal effect reaches into the mechanisms of neoplastic resistance to differentiation, because the AFP gene in malignant cells is generally refractory to glucocorticoids (2).Analyses of rat AFP gene and chromatin structures have identified domains in the 5' region of the locus potentially involved in its liver-specific, developmental stage-dependent, and glucocorticoid-regulated expression. A promoter domain spanning '230 nucleotides comprises a chromatin DNase I-hypersensitive (DH) site (32, 42, 44) that is selectively suppressed by dexamethasone (44), glucocorticoid receptor recognition sequences, and octamer motifs similar to those present in other genes under developmental and growth control (8). A distal domain (-2 to -4 kilobases [kb] relative to the AFP trans...

No abstract

The most common genetic alteration identi®ed in transitional cell carcinoma (TCC) of the bladder is loss of heterozygosity (LOH) on chromosome 9. However, localization of tumor suppressor genes on 9q has been hampered by the low frequency of subchromosomal deletions. We have analysed 139 primary, initial low stage TCC of the bladder using a panel of 28 microsatellite markers spanning chromosome 9 at an average distance of 5 Mb, following a primer-extension preampli®cation (PEP) technique. Sixty-seven (48%) tumors showed LOH at one or more loci and partial deletions were detected in 62 (45%) tumors; apparent monosomy 9 was detected in only ®ve (4%) tumors. Deletions were more frequent on 9q (44%) than on 9p (23%), the latter being mostly associated with 9q deletion, suggesting that alteration of genes on 9q may be an early event associated with super®cial papillary tumors. Combined data from the cases with partial 9q deletions displayed four candidate regions for tumor suppressor loci, based on the frequency of deletion observed and tumors with unique deletions at these sites. In two tumors, the unique partial deletion comprised D9S12 at 9q22.3, a region encompassing loci for the Gorlin syndrome and multiple self-healing squamous epithelioma gene. In two other tumors, the single LOH was identi®ed at the D9S172 locus at 9q31-32 where the dysautonia and Fukuyama-type congenital muscular dystrophy genes have been located. One tumor showed unique LOH at the GSN locus at 9q33, a region frequently deleted in other sporadic tumors while the fourth region of deletion was observed at 9q34 between ASS and ABL-1, in two tumors. This region is frequently deleted in tumors and encompasses the locus for the hereditary hemorrhagic telangiectasia gene. These ®ndings suggest four target regions on 9q within which suppressor genes for TCC may reside.

Cancer-testis (CT) genes encode proteins that are ideal targets for cancer immunotherapy because of their restricted expression in normal tissues and frequent expression in cancers. We previously observed that MAGE-A9 was one of the CT genes most frequently expressed in bladder tumors. To confirm that observation and evaluate the potential prognostic value of MAGE-A9 protein, we analyzed its expression by immunohistochemistry in 493 primary bladder tumors and 33 lymph node metastases, in comparison with MAGE-A4 protein, also frequently expressed in bladder tumors. Overall, MAGE-A4 and MAGE-A9 were observed, respectively, in 38% and 63% of nonmuscle-invasive tumors, 48% and 57% of muscle-invasive tumors, 65% and 84% of carcinomas in situ and in 73% and 85% of lymph node metastases. Expression was associated with higher grade (MAGE-A4, p 5 0.007; MAGE-A9, p 5 0.012). In multivariate Cox regression analyses, expression of MAGE-A9 in pTa tumors was associated with recurrence (HR 5 1.829; p 5 0.010). In univariate analyses, MAGE-A4 expression in these same tumors was associated with progression to muscle-invasive cancer (HR 5 7.417, p 5 0.013). MAGE-A9 expression was even more predictive of progression as all tumors that progressed expressed this antigen. In muscle-invasive bladder tumors, no association was found between expression of either MAGE and bladder cancer-specific death. In conclusion, MAGE-A9 is a target of choice for bladder cancer immunotherapy as it is expressed in 60% of bladder tumors, predominantly high-grade tumors, and at higher frequency in pTis and metastatic tumors. Moreover, in pTa tumors, an immunotherapy targeting MAGE-A9 could be protective against recurrence and progression to more advanced cancer. ' UICCKey words: bladder cancer; cancer-testis antigens; MAGE-A4; MAGE-A9; immunohistochemistry; prognosis Cancer-testis (CT) genes encode tumor-associated antigens that are attracting a lot of attention as potential targets for cancer vaccines.1,2 Their expression is mostly restricted to testicular germ cells in normal tissues, but they are aberrantly expressed in a large variety of cancers. Among these, bladder cancer, bladder cancer as melanoma, liver and nonsmall cell lung cancers, express CT genes in a large proportion of tumors. 3Characterization of the expression of various CT genes in bladder cancer has shown that members of the MAGE-A family are frequently expressed in these tumors. [4][5][6][7][8][9] Most of these studies used RT-PCR or, in a few cases, immunohistochemistry (IHC) to analyze MAGE-As expression. They suggested that MAGE-A4 had the highest incidence of expression in bladder tumors. However, we recently reported an RT-PCR analysis of 46 bladder tumors, showing that MAGE-A9 mRNA was expressed in 54% of the samples, whereas MAGE-A4 mRNA was found in 33% of them.10 A subgroup of these tumors was further studied by IHC using mAbs 14A11 and 57B recognizing, respectively, MAGE-A9 and MAGE-A4 proteins. MAGE-A9 protein expression was detected in 42% of the tumors while that of M...

Toll-like receptors (TLRs) have an important role in the activation of both innate and adaptive immunity in response to pathogens and danger signals. These receptors are expressed in immune cells and in some epithelia. They are expressed in the epithelium of the urinary bladder, where they actively participate in the fight against infection by uropathogens. TLR expression is decreased (although still evident) in bladder tumours, especially in non-muscle-invasive tumours. Intravesical immunotherapy with BCG to prevent recurrence of these tumours has been shown to involve the participation of three different TLRs (TLR2, TLR4, and TLR9). However, alternative therapies are needed as BCG fails in some patients and can sometimes cause severe adverse effects that are difficult to tolerate. In recent years, TLR2, TLR4, TLR7, and TLR9 agonists have been tested in vitro and in vivo for their ability to activate an antitumour immune response against bladder cancer. Promising results from these studies have led to the testing of TLR7 and TLR9 agonists in clinical trials.

Chromosome 9 alterations are the most frequently encountered cytologic anomalies in urothelial carcinoma (UC). We previously screened 139 low-stage UCs for loss of heterozygosity on chromosome 9, and identified five distinct regions likely to harbour tumour-suppressor genes. The present study focused on deletion mapping in the 9q22 region with 11 additional microsatellite markers. New deletions in the 9q22 region were found in five tumours. Deletion mapping allowed us to identify a 0.5 CM common minimal region of deletion between markers D9S280 and D9S1809, encompassing PATCHED (PTC), a gene identified as a tumour suppressor in basal cell carcinoma and in medulloblastoma. A marker located in the first intron of this gene showed the highest percentage of deletion (45%). cDNA sequencing in 15 tumours with deletion of PTC showed no mutation in the remaining allele. However, average expression of PTC mRNA measured by semiquantitative RT-PCR was significantly decreased in tumours with LOH in the 9q22 region, compared to normal urothelium (P ¼ 0.04), while it showed marked fluctuations in tumours without deletion. Our results suggest that the PTC gene is a putative suppressor at the 9q22 locus and that haploinsufficiency of this gene may be an early event in the development of papillary bladder tumours.

Toll-like receptors are expressed in normal urothelium and nonmuscle invasive bladder tumors. In cultured urothelial cells agonist inducible toll-like receptor 2 or constitutively expressed toll-like receptor 3 is functional. These data suggest the potential use of toll-like receptor agonists for antitumor immunotherapy of nonmuscle invasive tumors.