Hélène LaRue

Four tumor suppressor loci on chromosome 9q in bladder cancer : evidence for two novel candidate regions at 9q22.3 and 9q31

The most common genetic alteration identified 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 preamplification (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 five (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 superficial 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 identified 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 findings suggest four target regions on 9q within which suppressor genes for TCC may reside.

Bladder Tumor Infiltrating Mature Dendritic Cells and Macrophages as Predictors of Response to Bacillus Calmette-Guérin Immunotherapy

BACKGROUND The clinical significance of tumor-infiltrating dendritic cells (TIDCs) and tumor-associated macrophages (TAMs) as markers of immune response has been reported for many cancers. OBJECTIVE To measure tumor infiltration by CD83(+) dendritic cells (DCs) and CD68(+) macrophages in non-muscle-invasive urothelial cancer (NMIUC) prior to bacillus Calmette-Guérin (BCG) immunotherapy and to evaluate their significance in the response to immunotherapy. DESIGN, SETTING, AND PARTICIPANTS Patients with NMIUC at high risk of recurrence and progression were recruited for a study on markers of the response to BCG. INTERVENTION Patients were treated by transurethral resection followed by maintenance BCG. MEASUREMENTS Immunohistochemical staining with anti-CD83 and anti-CD68 monoclonal antibodies on 53 and 46 NMIUC tumors, respectively, prior to BCG treatment. A scoring index was calculated based on the average density of positive cells within the papillary axis, the stroma, lymphoid aggregates, and infiltration into tumors. RESULTS AND LIMITATIONS CD83(+) TIDCs were observed mostly within lymphoid aggregates. Multivariate Cox regression analysis showed that maintenance BCG (more than one maintenance cycle) was highly effective in patients with a low level of CD83(+) TIDCs at time of resection (hazard ratio [HR]: 0.035; p=0.002) but showed reduced efficacy in patients with a high level of CD83(+) TIDCs (HR: 0.87; p=0.810). A high level of infiltration by CD83(+) TIDCs slightly decreased the risk of recurrence in patients treated with one or no maintenance BCG cycle (HR: 0.4; p=0.117). In the same population, a strong infiltration of CD68(+) TAMs was associated with an increased risk of recurrence (HR: 3.8; p=0.013). CONCLUSIONS These results suggest that patients with a high level of infiltration by CD83(+) TIDCs or CD68(+) TAMs do not respond as well to BCG immunotherapy. If confirmed in larger cohorts, the pretreatment level of infiltration by these cells may be useful to influence the choice of treatment strategy.

Functional analysis of developmentally regulated chromatin-hypersensitive domains carrying the alpha 1-fetoprotein gene promoter and the albumin/alpha 1-fetoprotein intergenic enhancer.

During liver development, the tandem alpha 1-fetoprotein (AFP)/albumin locus is triggered at the AFP end and then asymmetrically enhanced; this is followed by autonomous repression of the AFP-encoding gene. To understand this regulation better, we characterized the two early developmental stage-specific DNase I-hypersensitive (DH) sites so far identified in rat liver AFP/albumin chromatin: an intergenic DH-enhancer site and the AFP DH-promoter site. Mutation-transfection analyses circumscribed the DH-enhancer domain to a 200-bp DNA segment stringently conserved among species. Targeted mutations, DNA-protein-binding assays, and coexpression experiments pinpointed C/EBP as the major activatory component of the intergenic enhancer. Structure-function relationships at the AFP DH-promoter site defined a discrete glucocorticoid-regulated domain activated cooperatively by HNF1 and a highly specific AFP transcription factor, FTF, which binds to a steroid receptor recognition motif. The HNF1/FTF/DNA complex is deactivated by glucocorticoid receptors or by the ubiquitous factor NF1, which eliminates HNF1 by competition at an overlapping, high-affinity binding site. We propose that the HNF1-NF1 site might serve as a developmental switch to direct autonomous AFP gene repression in late liver development. We also conclude that the intergenic enhancer is driven by C/EBP alpha primarily to fulfill albumin gene activation functions at early developmental stages. Factor FTF seems to be the key regulator of AFP gene-specific functions in carcinoembryonic states.

High frequency of MAGE-A4 and MAGE-A9 expression in high-risk bladder cancer

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 = 0.007; MAGE‐A9, p = 0.012). In multivariate Cox regression analyses, expression of MAGE‐A9 in pTa tumors was associated with recurrence (HR = 1.829; p = 0.010). In univariate analyses, MAGE‐A4 expression in these same tumors was associated with progression to muscle‐invasive cancer (HR = 7.417, p = 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.

Alteration of the PATCHED locus in superficial bladder cancer

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=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.

Chromosome 9 deletions and recurrence of superficial bladder cancer: identification of four regions of prognostic interest.

In a previous study, loss of heterozygosity (LOH) of 28 chromosome 9 microsatellite markers was assessed on 139 Ta/T1 bladder tumors. LOH at one or more loci was detected in 67 tumors, 62 presenting subchromosomal deletions. One hundred and thirty-three of these patients have now been followed for up to 8 years. The purpose of the present study was to evaluate the potential biological significance of chromosome 9 deletions in superficial bladder tumors at initial diagnosis. High grade was associated with LOH (P=0.004). Large tumors carried more frequently 9p deletions (P=0.022). Female patients had more chromosome 9q LOH than male patients did (P=0.010). Chromosome 9 LOH at all loci was associated with an elevated risk of recurrence but four regions were associated with a particularly high risk of recurrence. Multivariate analysis taking into account grade, stage, size and number of tumors showed that tumors deleted in the regions 9ptr-p22, 9q22.3, 9q33, and 9q34 recurred significantly more rapidly than those without deletions (Recurrence rate ratio=2.32, 2.53, 2.52 and 2.43 respectively). Log-rank statistics comparing Kaplan-Meier survival curves for the same chromosomal regions confirmed the correlation (P=0.0002, 0.010, 0.002 and 0.009 respectively). Only four patients progressed to muscle-invasive disease. They all had extensive deletions on 9q but none had deletions at 9ptr-p22. This study suggests a link between chromosome 9 anomalies and recurrence of superficial bladder cancer.

Prognostic value of the proliferative index determined by Ki-67 immunostaining in superficial bladder tumors.

The biological behavior of urothelial carcinomas remains unpredictable. The objective of this study was to determine the prognostic value of Ki-67 index in superficial papillary bladder tumors and to correlate it with the S-phase fraction (SPF) measured by flow cytometry. Three hundred nineteen patients with newly diagnosed superficial (pTa, pT1) bladder tumors were included between September 1990 and April 1992. Patients with bladder carcinoma in situ alone were excluded. We observed 255 pTa tumors and 64 pT1 tumors, whereas 111 lesions were classified as grade G1 and 208 as grade G2-G3. Ki-67 immunostaining was performed on paraffin-embedded material using a 3-step immunoperoxidase procedure with the murine monoclonal antibody MiB1. The relation between Ki-67 expression and prognostic variables (stage, grade, tumor size, multifocality, age, and sex) was investigated by the chi-square test. Cox regression was used to describe the association between Ki-67 and tumor recurrence in 308 patients with follow-up while adjusting for potentially confounding prognostic variables. The frequency of high Ki-67 expression (> or =10%) increased with stage (P = .005) and grade (P = .001), but not with tumor size or multifocality. Two hundred one patients experienced tumor recurrence in a median follow-up of 68 months. Stage, grade, tumor size, and multifocality were all independent predictors of recurrence. Ki-67 index greater than 10% was found to be an independent predictor of tumor recurrence among patients with tumors larger than 3 cm in diameter (HR = 2.05, CI = 1.18-3.55), but not those with smaller size tumors. With regards to the DNA index, a significant but weak correlation was observed between Ki-67 expression and the SPF (Spearmans correlation coefficient = 0.23, P = .004). In addition, aneuploid tumors had significantly higher expression of Ki-67 (22.5%) than diploid tumors (10.1%) (P = .0006). Moreover, patients with DNA aneuploid bladder tumors were more likely to have more than 10% Ki-67-positive cells than those with diploid tumors. In patients with newly diagnosed pTa or pT1 bladder tumors, a Ki-67 index above 10% is an independent predictor of shorter time to recurrence only in those with tumors larger than 3 cm.

MAGE-A9 mRNA and protein expression in bladder cancer.

In a previous analysis, we showed that MAGE‐As were the most frequently expressed cancer‐testis antigens in human bladder tumours. Here, we further characterized by RT‐PCR the expression of this family of genes by analyzing specifically MAGE‐A3, ‐A4, ‐A8 and ‐A9 mRNAs in 46 bladder tumours and 10 normal urothelia. We found that they were expressed in 30, 33, 56 and 54% of tumours, respectively. Although MAGE‐A8 was the most frequent, its expression was low and was also found in most normal urothelia. The other MAGE‐A mRNAs were all tumour‐specific but MAGE‐A9 mRNA was expressed at a higher level and was two times more frequent in superficial than in invasive tumours. To study the expression of the protein, we produced 2 MAGE‐A9‐specific monoclonal antibodies (mAbs) presenting no cross‐reactivity with other MAGE‐A proteins. MAb 14A11, was used to analyse the expression of the antigen in testis and tumour samples by immunohistochemistry. In testis, MAGE‐A9 expression was restricted to primary spermatocytes. Most bladder tumours that expressed the MAGE‐A9 transcript were positive with mAb 14A11. Staining was heterogeneous but half of the tumours showed over 75% positive cells. Finally, we showed that treatment of bladder cancer cells with the methylation inhibitor, 5‐aza‐2′‐deoxycytidine, alone or in combination with the histone deacetylase inhibitors MS‐275 and 4‐phenylbutyrate could strongly induce the expression of MAGE‐A9. These results show that MAGE‐A9 is frequently expressed in superficial bladder cancer and could be a relevant target for immunotherapy or chemoimmunotherapy because its expression can be induced by chemotherapeutic drugs.

Maintenance bacillus Calmette-Guérin in high-risk nonmuscle-invasive bladder cancer: how much is enough?

Intravesical bacillus Calmette‐Guérin (BCG) immunotherapy is effective in preventing recurrence and progression in nonmuscle‐invasive bladder cancer but the dosing schedule and duration of treatment remain empirical. The outcome of BCG therapy was prospectively evaluated on patients according to the number of maintenance cycles received.

mdr1 mRNA expression by RT-PCR in patients with primary breast cancer submitted to neoadjuvant therapy

Abstractmdr1 expression by reverse transcription and polymerase chain reaction(RT-PCR) has been compared to P-glycoprotein (Pgp) expression byimmunohistochemistry (IHC) and correlated with clinical response toneoadjuvant therapy. RNA has been recovered from glass slide smears offine-needle aspiration from 57 untreated primary breast cancers prior toneoadjuvant chemotherapy (33 cases), hormone therapy (23 cases), or both (1case). Furthermore, mdr1 mRNA has been analyzed in 6 cases after 2 months oftreatment. The neoadjuvant therapy consisted of 4 cycles of adriamycin andcyclophosphamide or tamoxifen. Of 57 tumor specimens, an interpretableresult was obtained in 52 cases, indicating the feasibility of the analysisby RT-PCR with very small tumor specimens. The presence of mdr1 mRNA hasbeen documented in 44/52 (84%) tumor samples with a spectrum ofexpression levels. The expression of mdr1 mRNA was compared withP-glycoprotein (Pgp) expression by IHC using JSB-1, 4E3, and C494 monoclonalantibodies in 48 of the 52 interpretable tumor samples. 12/48 (25%)expressed Pgp by IHC. All tumors expressing Pgp by IHC were also positive byRT-PCR. The results confirm the higher prevalence of mdr1 mRNA compared tothe protein expression. However, mdr1 mRNA expression was found to correlatesignificantly with resistance to neoadjuvant hormone therapy only while Pgpexpression detected by JSB-1 immunostaining only correlated withchemoresistance. The lack of convincing correlation with chemoresistancesuggests that mRNA and Pgp may not be directly or solely responsible forclinical response to drugs. Further studies should focus on theposttranslational modulation of P-glycoprotein and other mechanisms of drugresistance.