Tumor heterogeneity of fibroblast growth factor receptor 3 (FGFR3) mutations in invasive bladder cancer: implications for perioperative anti-FGFR3 treatment.

BACKGROUND: Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response. PATIENTS AND METHODS: We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201). RESULTS: We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type. CONCLUSIONS: FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting.

Locomotor performance in highly-trained young soccer players: does body size always matter?

To examine the effects of body size on locomotor performance, 807 15-year-old French and 64 Qatari soccer players participated in the present study. They performed a 40-m sprint and an incremental running test to assess maximal sprinting (MSS) and aerobic speeds, respectively. French players were advanced in maturity, taller, heavier, faster and fitter than their Qatari counterparts (e.g., Cohen's d=+1.3 and + 0.5 for body mass and MSS). However, when adjusted for body mass (BM), Qatari players had possibly greater MSS than French players (d=+0.2). A relative age effect was observed within both countries, with the players born in the first quarter of the year being taller, heavier and faster that those born during the fourth quarter (e.g., d=+0.2 for MSS in French players). When directly adjusted for BM, these MSS differences remained (d=+0.2). Finally, in both countries, players selected in National teams were taller, heavier, faster and fitter than their non-selected counterparts (e.g., d=+0.6 for MSS in French players), even after adjustments for body size (d=+0.5). Differences in locomotor performances between players with different phenotypes are likely mediated by differences in body size. However, when considering more homogeneous player groups, body dimensions are unlikely to substantially explain the superior locomotor performances of older and/or international players.

Milk fat globule--epidermal growth factor--factor VIII (MFGE8)/lactadherin promotes bladder tumor development.

Milk fat globule-epidermal growth factor-factor VIII (MFGE8), also called lactadherin or SED1, is a secreted integrin-binding protein that promotes elimination of apoptotic cells by phagocytes leading to tolerogenic immune responses, and vascular endothelial growth factor (VEGF)-induced angiogenesis: two important processes for cancer development. Here, by transcriptomic analysis of 228 biopsies of bladder carcinomas, we observed overexpression of MFGE8 during tumor development, correlated with expression of genes involved in cell adhesion or migration and in immune responses, but not in VEGF-mediated angiogenesis. To test whether MFGE8 expression was instrumental in bladder tumor development, or a simple consequence of this development, we used genetic ablation in a mouse model of carcinogen-induced bladder carcinoma. We showed that Mfge8 was also upregulated in mouse carcinoma, and that in its absence, Mfge8-deficient animals developed less advanced tumors. Angiogenesis was similar in carcinogen-treated Mfge8-expressing or -deficient bladders, thus ruling out a major role of the proangiogenic function of Mfge8 for its protumoral role. By contrast, the tumor-promoting role of Mfge8 was not observed anymore in mice devoid of adaptive immune system, and human tumors overexpressing MFGE8 where invaded with macrophages and regulatory T cells, thus suggesting that MFGE8/lactadherin favors development of bladder tumors at least partly by an immune system-dependent mechanism. Our observations suggest future use of MFGE8-inhibiting molecules as therapies of bladder carcinomas, and of a limited number of other human cancers, in which our analysis of public databases also revealed overexpression of MFGE8.

Increased expression of class III beta-tubulin in castration-resistant human prostate cancer.

BACKGROUND: Class III beta-tubulin (betaIII-tubulin) is expressed in tissues of neuronal lineage and also in several human malignancies, including non-small-cell lung carcinoma, breast and ovarian cancer. Overexpression of betaIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies. At present, betaIII-tubulin expression remains largely uncharacterised in prostate cancer. METHODS: In this report, we evaluated the expression of betaIII-tubulin in 138 different human prostate tumour specimens by immunohistochemistry from patients with hormone-treated or hormone-untreated prostate cancer. betaIII-tubulin expression was also examined in various prostatic cancer cell lines including in androgen-sensitive human prostate cancer cells, LNCaP, grown in androgen-depleted medium in 2D cultures or as tumour xenografts when the host mouse was castrated. RESULTS: Whereas moderate-to-strong betaIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of betaIII-tubulin. These findings were supported by in vitro and in vivo settings. CONCLUSION: Our data indicate that betaIII-tubulin expression is augmented in prostate cancer by androgen ablation and that the expression of this beta-tubulin isoform is associated with the progression of prostate cancer to the castration-resistant state, a stage largely responsible for mortality from prostate cancer.

Comparative expression of Hedgehog ligands at different stages of prostate carcinoma progression.

Recent studies have revealed the potential involvement of Hedgehog (Hh) signalling in proliferation and invasive behaviour of prostate carcinoma (PCa). The aim of this study was to specify the role of Sonic Hh (Shh), Desert Hh (Dhh) and Indian Hh (Ihh) in the natural history of PCa. Hh ligands expression was compared in primary hormone-naive PCa (HNPC), hormone-treated PCa (HTPC) and hormone-refractory PCa (HRPC), using immunohistochemistry. Shh and Dhh were expressed by both epithelial and stromal cells of prostate tissues. Ihh was only expressed by stromal cells. For the three ligands, mRNA and immunostaining were not correlated. In HNPC, Shh epithelial expression was significantly associated with high Gleason scores (p = 0.03), metastatic lymph nodes (p = 0.004) and Dhh epithelial staining was associated with high pT stages (p = 0.003), seminal vesicle invasion (p = 0.03) and bladder neck invasion (p = 0.0008). Negative Shh staining in stromal cells was associated with high Gleason scores (p = 0.015), high pT stages (p = 0.01) and bladder neck invasion (p = 0.04). Concomitant absence of Shh and Dhh expression in stromal cells was an independent prognostic parameter for biological recurrence on multivariate analysis (p = 0.01). Epithelial expression of Shh and Dhh was increased in HTPC compared to HNPC (p = 0.02 and p = 0.04). Interestingly, in vitro, transcript analysis also showed increased expression of these 2 Hh ligands when androgen-sensitive LNCaP cells were maintained in androgen-free medium mimicking hormonal therapy. Epithelial expression of Dhh was increased (p < 0.0001) in HRPC compared to HNPC, while stromal expression of Shh and Dhh was decreased (p < 0.0001). In conclusion, the Hh signalling pathway is associated with pejorative pathological parameters in HNPC and is up-regulated in epithelial cells of HTPC and HRPC. Moreover, the lack of Hh molecules in stromal cells seems to be associated with invasive and hormone-refractory behaviours and suggests specific changes in stromal-epithelial crosstalks during PCa progression.

T helper 1/2 lymphocyte urinary cytokine profiles in responding and nonresponding patients after 1 and 2 courses of bacillus Calmette-Guerin for superficial bladder cancer.

PURPOSE: Interleukin (IL)-2 and interferon-gamma are released during T helper 1 lymphocyte responses and IL-10 is released during T helper 2 lymphocyte responses. We have previously reported that a T helper 1 lymphocyte urinary cytokine profile is associated with a favorable prognosis after bacillus Calmette-Guerin (BCG) treatment. We evaluated the T helper 1/2 lymphocyte cytokine profiles during courses 1 and 2 of 6 weekly BCG instillations. MATERIALS AND METHODS: Urinary interferon-gamma, IL-2 and IL-10 were measured by enzyme-linked immunosorbent assay after each of 6 weekly instillations of 150 mg. BCG, Pasteur strain, in 19 patients with superficial stages Ta and T1 bladder cancer, and carcinoma in situ. The 11 patients who did not respond to course 1 were re-treated according to the same schedule and reevaluated. RESULTS: During course 1 interferon-gamma was higher than during course 2 (p <0.001), which was associated with nonrecurrence (p <0.001). In contrast, IL-2 cytokine was higher after course 2 (p <0.01), which was associated with a BCG response (p = 0.01). Interferon-gamma and IL-10 correlated during courses 1 and 2 (p = 0.04 and 0.0004, respectively). We distinguished groups 1-immediate T helper 1 lymphocyte profile consisting of responders to course 1 with high interferon-gamma, IL-2 and IL-10, 2-delayed T helper 1 lymphocyte profile consisting of responders to course 2 with early high IL-2 and 3-consisting of nonresponders to the 2 courses with low interferon-gamma, IL-2 and IL-10. CONCLUSIONS: A T helper 1 lymphocyte urinary cytokine profile was associated with a clinical response to BCG. A repeat BCG course induces a favorable immune response in a subset of patients, suggesting that maintenance therapy may be beneficial.

Expression of transforming growth factor beta1 and its receptors in normal human urothelium and human transitional cell carcinomas.

Previous studies indicated that transforming growth factor beta1 (TGFbeta1) is expressed by normal urothelial cells and exerts regulatory autocrine functions in urothelial maintenance and wound healing. However, little is known about the expression patterns of TGFbeta1 and its receptors in bladder tumors. Therefore, we studied the protein and mRNA localization of TGFbeta1 and TGFbeta receptor types I and II (TGFbetaRI and TGFbetaRII) in normal human urothelium and transitional cell carcinomas (TCCs) of different grades and stages. Expression of TGFbeta1 and its receptors was examined by immunocytochemistry and mRNA in situ hybridization in normal urothelium and TCCs using a semiquantitative method. By immunocytochemistry, the expression of TGFbeta1 and TGFbetaRII was higher in superficial and basal cell layers of normal urothelium than in the intermediate layer. A similar localization was seen in superficial TCCs. TGFbetaRI was mainly present in basal and intermediate cell layers of normal urothelium and superficial TCCs. In contrast, in muscle invasive TCCs, all tumor cells stained intensely for all three proteins. No correlation was found between immunostaining and TCC grade. In situ hybridization pointed out that all cell layers in normal urothelium exhibit similar TGFbeta1 mRNA levels. Elevated TGFbeta1 mRNA levels were noted in TCCs irrespective of grade or stage. In conclusion, these data indicate that in normal urothelium TGFbeta1, TGFbetaRI, and TGFbetaRII expression depend on maturation and differentiation. This pattern is particularly lost in muscle invasive TCCs, in which the expression of the three proteins is enhanced. These data suggest autocrine TGFbeta1 mechanisms in human TCC cells that may be more pronounced in muscle invasive TCC cells.

Tumor suppressor INK4: determination of the solution structure of p18INK4C and demonstration of the functional significance of loops in p18INK4C and p16INK4A.

Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclin-dependent kinase 4) family have been reported recently, the detailed structures and the functional roles of the loops have drawn considerable interest. This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects. First, the solution structure of p18INK4C was determined by NMR, and the loop structures were analyzed in detail. The loops adapt nascent antiparallel beta-sheet structures, but the positions are slightly different from those in the crystal structure. A detailed comparison between the solution structures of p16 and p18 has also been presented. The determination of the p18 solution structure made such detailed comparisons possible for the first time. Second, the [1H,15N]HSQC NMR experiment was used to probe the interactions between p18INK4C and other proteins. The results suggest that p18INK4C interacts very weakly with dna K and glutathione S-transferase via the loops. The third aspect employed site-specific mutagenesis and functional assays. Three mutants of p18 and 11 mutants of p16 were constructed to test functional importance of loops and helices. The results suggest that loop 2 is likely to be part of the recognition surface of p18INK4C or p16INK4A for CDK4, and they provide quantitative functional contributions of specific residues. Overall, our results enhance understanding of the structural and functional roles of the loops in INK4 tumor suppressors in particular and in ankyrin-repeat proteins in general.

Tumor suppressor p16INK4A: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4.

The solution structure of the tumor suppressor p16INK4A has been determined by NMR, and important recognition regions of both cdk4 and p16INK4A have been identified. The tertiary structure of p16INK4A contains four helix-turn-helix motifs linked by three loops. Twelve tumorigenic mutants of p16INK4A have been constructed and analyzed for their structure and activity, and new mutants have been designed rationally. A fragment of 58 residues at the N terminus of cdk4 important for p16INK4A binding has been identified. The importance of this region was further verified by mutational analysis of cdk4. These results and docking experiments have been used to assess possible modes of binding between p16INK4A and cdk4.

Clonality of tumor-infiltrating lymphocytes in human urinary bladder carcinoma.

The immune system has been implicated in the control of bladder tumor growth. To evaluate the clonality of bladder tumor-infiltrating T lymphocytes (TILs) in vivo, we studied the T-cell antigen receptor (TCR) repertoire in tumor biopsy specimens from 10 patients with transitional-cell carcinoma (TCC) of the bladder. Nine patients had a primary tumor, and one had a multifocal disease, consisting of two bladder tumors and three bilateral upper urinary tract sites of involvement. The following specimens from the nine patients with a primary tumor also were analyzed: a recurrent tumor from four patients, a metastatic lymph node from one patient, and peripheral blood from five patients. We used a high-resolution polymerase chain reaction (PCR) method to determine CDR3 (complementarity-determining region 3) size lengths of TCR beta-chain transcripts. Oligoclonal T-cell expansion was identified in all specimens, with a larger number of expanded clones in the tumors than in peripheral blood. Expanded clones were identified in several beta-chain variable region (BV) subfamilies and varied from one patient to the next and also in different specimens from the same patient. However, a number of clones with the same VJ combination and the same CDR3 size were identified in a given patient (in specimens collected either simultaneously or at different times), suggesting homogeneity in the immunogenic environment. Clonal T-cell expansion in patients with bladder cancer may reflect prolonged exposure of T lymphocytes to tumor antigens. Our findings provide a basis for functional studies to elucidate T lymphocyte-bladder tumor cell interactions.

Evaluation of local immune response after intravesical bacille Calmette-Guérin treatment for superficial bladder cancer.

OBJECTIVE: To help define the optimal protocol of Bacille Calmette-Guérin (BCG) treatment for transitional cell carcinoma (TCC) of the bladder by examining cytokine production and antigen presentation to effector cells after instillations of BCG in patients with bladder TCC. PATIENTS AND METHODS: Sixty-four urine samples from 11 patients were tested for the production of interferon gamma (IFN-gamma) using a modified commercial enzyme-linked immunosorbent assay (ELISA) kit. Urine was collected before and at intervals up to 24 h after the intravesical instillation of BCG. Immunohistological studies were also carried out using a two-step alkaline phosphatase technique to explore the expression of tumour-associated antigens (TAAs) (E7, 19A211, T138), major histocompatibility complex (MHC) molecules and lymphocyte subset infiltrates (CD3, CD4, CD8) in bladder biopsies before and 3 weeks after the completion of treatment in seven patients. RESULTS: IFN-gamma was only detected 4, 6 and 8 h after instillation, with a maximum concentration at 6 h (2.9-34.7 IU/mL in 10 patients). During a 6-week course of BCG, IFN-gamma was barely detectable after the first two instillations, but gradually increased from the third instillation onwards. TAA and MHC II antigens, which were absent or faintly expressed on normal urothelial cells before treatment, were expressed strongly in five patients after treatment. The local recruitment of immunocompetent cells was detected in all patients. CONCLUSION: These results suggest that the local immune response after the intravesical instillation of BCG can be quantified using simple ELISA tests and could be useful in defining objective criteria for rationalizing treatment (dose and duration), and in determining the relation between the immune response and anti-tumour activity. There is evidence that antigen presentation is enhanced after BCG instillations, suggesting that a T cell-MHC restricted pathway might be involved in the anti-tumour response. This study supports the search for tumour-rejection antigens in bladder cancer.