Occupational exposure to polycyclic aromatic hydrocarbons influenced neither the frequency nor the spectrum of FGFR3 mutations in bladder urothelial carcinoma

Occupational exposure to polycyclic aromatic hydrocarbons (PAH) is associated with an increased risk of urothelial carcinoma (UC). FGFR3 is found mutated in about 70% of Ta tumors, which represent the major group at diagnosis. The influence of PAH on FGFR3 mutations and whether it is related to the emergence or shaping of these mutations is not yet known. We investigated the influence of occupational PAH on the frequency and spectrum of FGFR3 mutations. We included on 170 primary urothelial tumors from five hospitals from France. Patients (median age, 64 yr) were interviewed to gather data on occupational exposure to PAH, revealing 104 non‐ and possibly PAH exposed patients, 66 probably and definitely exposed patients. Tumors were classified as follows: 75 pTa, 52 pT1, and 43 ≥pT2. Tumor grades were as follows: 6 low malignant potential neoplasms (LMPN) and 41 low‐grade and 123 high‐grade carcinomas. The SnaPshot method was used to screen for the following FGFR3 mutations: R248C, S249C, G372C, Y375C, A393E, K652E, K652Q, K652M, and K652T. Occupational PAH exposure was not associated with a particular stage or grade of tumors. Thirty‐nine percent of the tumors harbored FGFR3 mutations. After adjustment for smoking, occupational exposure to PAH did not influence the frequency [OR, 1.10; 95% CI, 0.78–1.52], or spectrum of FGFR3 mutations. Occupational exposure to PAH influenced neither the frequency nor the spectrum of FGFR3 mutations and there was no direct relationship between these mutations and this occupational hazard. © 2009 Wiley‐Liss, Inc.     

RUNX3 methylation in normal surrounding urothelium of patients with non-muscle-invasive bladder cancer: Potential role in the prediction of tumor progression

Purpose

Previously, we reported a causal relationship between RUNX3 methylation and bladder tumor development. Thus, in order to clarify its role in tumorigenesis, this study aims to identify the function of RUNX3 methylation in normal adjacent urothelium of patients with non-muscle invasive bladder cancer (NMIBC).

Methods

Tumor tissue and donor-matched normal adjacent tissue from 55 patients who underwent transurethral resection (TUR) were selected for the study, and RUNX3 promoter methylation was assessed using methylation-specific polymerase chain reaction (MS-PCR).

Results

RUNX3 promoter methylation occurred more frequently in tumor samples than in histologically normal urothelium in patients with NMIBC (P = 0.02). The methylation rates for the RUNX3 promoter in normal adjacent urothelium and tumor tissue were 47% and 69%, respectively. Interestingly, RUNX3 methylation in normal adjacent urothelium was associated with tumor number (P = 0.022) and progression (P = 0.035). Kaplan–Meier estimates revealed that RUNX3 methylation in normal urothelium showed a significant association with time to progression (P = 0.017) in NMIBC patients. Stratifying the patients into ‘both methylation’, ‘one methylation’ and ‘no methylation’ groups for tumors and normal urothelium revealed that no progression occurred in the ‘no methylation’ group during follow-up. Multivariate Cox regression analysis demonstrated that RUNX3 methylation in normal urothelium [hazards ratio (HR): 5.692, P = 0.042] was an independent predictor of progression.

Conclusions

RUNX3 methylation was associated with transition from normal urothelium to bladder tumor. More importantly, RUNX3 methylation in normal adjacent urothelium may predict progression in NMIBC patients who have undergone TUR.     

Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events

The bladder cancer genome harbors numerous oncogenic mutations and aberrantly methylated gene promoters. The aim of our study was to generate a profile of these alterations and investigate their use as biomarkers in urine sediments for noninvasive detection of bladder cancer. We systematically screened FGFR3, PIK3CA, TP53, HRAS, NRAS and KRAS for mutations and quantitatively assessed the methylation status of APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5 and WIF1 in a prospective series of tumor biopsies (N = 105) and urine samples (N = 113) from 118 bladder tumor patients. We also analyzed urine samples from 33 patients with noncancerous urinary lesions. A total of 95 oncogenic mutations and 189 hypermethylation events were detected in the 105 tumor biopsies. The total panel of markers provided a sensitivity of 93%, whereas mutation and methylation markers alone provided sensitivities of 72% and 70%, respectively. In urine samples, the sensitivity was 70% for all markers, 50% for mutation markers and 52% for methylation markers. FGFR3 mutations occurred more frequently in tumors with no methylation events than in tumors with one or more methylation events (78% vs. 33%; p < 0.0001). FGFR3 mutation in combination with three methylation markers (APC, RASSF1A and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. These results suggest an inverse correlation between FGFR3 mutations and hypermethylation events, which may be used to improve noninvasive, DNA‐based detection of bladder cancer.     

Chromosome 9 deletions are more frequent than FGFR3 mutations in flat urothelial hyperplasias of the bladder

Flat urothelial hyperplasias (FUHs) in patients with papillary bladder tumours frequently show deletions of chromosome 9, suggesting that FUH could be the first neoplastic step in the development of papillary bladder cancer. FGFR3 mutations are frequent in non-invasive papillary tumours with low risk of progression. Our aim was to investigate the frequency of FGFR3 mutations and deletions of chromosomes 9p/q and 8p/q in FUH. Thirty FUH and 9 simultaneous or consecutive tumours were detected by 5-ALA-based photodynamic cystoscopy. DNA was isolated from frozen sections and whole genome amplification was done by I-PEP-PCR, followed by LOH analysis on chromosomes 8p/q and 9p/q. FGFR3 mutations were detected by SNaPshot analysis. LOH analysis on FUH revealed deletions at 9p/q (11/30, 37%) and 8p/q (3/30, 10%). FGFR3 mutations were found in 7/30 FUH (23%). Only 2 FUH showed an FGFR3 mutation without deletions of chromosome 9. In contrast, 6 FUH revealed chromosome 9 deletions but wild type FGFR3 (p = 0.03). These results suggest that chromosome 9 deletions are the earliest genetic alterations in bladder cancer. The detection of FGFR3 mutations in FUH further supports the role of this lesion as precursor of papillary bladder cancer.     

Phase 2 trial of dovitinib in patients with progressive FGFR3-mutated or FGFR3 wild-type advanced urothelial carcinoma

BackgroundSecond-line treatment options for patients with advanced urothelial carcinoma (UC) are limited. Fibroblast growth factor receptor 3 (FGFR3) is dysregulated in UC by activating mutations or protein overexpression in non-mutant tumours. In this study, the efficacy, pharmacodynamics and safety of dovitinib—a broad-targeted inhibitor of tyrosine kinases, including FGFR3—were evaluated in patients with previously treated advanced UC with and without FGFR3 mutations.MethodsForty-four adults with advanced UC who had progressed after one to three platinum-based and/or combination chemotherapy regimens were classified as having mutant (FGFR3^MUT; n = 12), wild-type (FGFR3^WT; n = 31), or unknown (n = 1) FGFR3 status. Patients received 500 mg dovitinib once daily on a 5-days-on/2-days-off schedule. The primary end-point of this two-stage study was the investigator-assessed overall response rate (ORR).ResultsMost of the patients were men (75%) and over half of the patients were aged ⩾65 years (61%). All patients had received ⩾1 prior antineoplastic therapy for UC. The study was terminated at the end of stage 1, when it was determined by investigator review that the ORR of both the FGFR3^MUT (0%; 95% confidence interval [CI], 0.0–26.5) and FGFR3^WT (3.2%; 95% CI, 0.1–16.7) groups did not meet the criteria to continue to stage 2. The most common grade 3/4 adverse events, suspected to be study-drug related, included thrombocytopenia (9%), fatigue (9%), and asthenia (9%).ConclusionAlthough generally well tolerated, dovitinib has very limited single-agent activity in patients with previously treated advanced UC, regardless of FGFR3 mutation status. clinicaltrials.gov NCT00790426.     

膀胱尿路上皮癌组织中EGFL7蛋白的表达及微血管密度的意义

目的:研究膀胱尿路上皮癌组织中EGFL7蛋白的表达及微血管密度（microvessel density,MVD）,探讨EGFL7及MVD与膀胱尿路上皮癌生物学特征的内在联系。方法:应用免疫组化SP法,对63例膀胱尿路上皮癌组织及10例正常人膀胱组织中的EGFL7蛋白进行检测,同时应用八因子抗体对膀胱尿路上皮癌组织中微血管进行染色,结合Meta-Morph显微荧光图像分析系统测定并分析63例膀胱尿路上皮癌中微血管密度（MVD）及其与EGFL7和膀胱尿路上皮癌的病理分期、临床分级之间的关系。结果:EGFL7在正常膀胱组织均呈阴性表达,40例膀胱尿路上皮癌EGFL7蛋白表达阳性,阳性率为63%（P<0.05）。EGFL7蛋白的表达在膀胱尿路上皮癌不同肿瘤组织学分级、临床分期组间比较差异具有统计学意义(P<0.05)。MVD值在膀胱尿路上皮癌组织中不同病理分级、临床分期组间有显著性差异（P<0.05）。结论:EGFL7和MVD在膀胱尿路上皮癌组织中表达与肿瘤病理分级及临床分期密切相关,在膀胱尿路上皮癌浸润转移过程中起重要作用。     

Upregulation of TRAG3 gene in urothelial carcinoma of the bladder

Conventional chemotherapy is commonly used for advanced stages of bladder cancer with modest success and high morbidity. Identifying markers of resistance will allow clinicians to tailor treatment to a specific patient population. T24‐tumorigenic cell line was grown orthotopically in nude mice and monitored using bioluminescence imaging and microcomputed tomography until they developed metastases. Stable sublines were then developed from primary bladder (T24‐P), lung (T24‐L) and bone (T24‐B) tissues. Chromosomal analysis and DNA microarray were used to characterize these sublines. Real‐time quantitative polymerase chain reaction and immunohistochemistry were used for validation. Epigenetic modifiers were used to study gene regulation. The cell viability was quantified with MTT assay. Chromosomal analysis revealed multiple alterations in metastatic cell lines compared to T24‐P. DNA microarray analysis showed that taxol resistance‐associated gene (TRAG) 3 was the most upregulated gene. From real‐time quantitative polymerase chain reaction and immunohistochemistry, TRAG3 was significantly higher in T24‐L and T24‐B than T24‐P. TRAG3 gene expression is likely controlled by DNA methylation but not histone acetylation. Interestingly, T24‐B and T24‐L cells were more resistant than T24‐P to treatment with antimicrotubule agents such as docetaxel, paclitaxel and vinblastine. TRAG3 mRNA expression was higher in 20% of patients with ≤pT2 (n = 10) and 60% of patients with ≥pT3 (n = 20) compared to normal adjacent tissue (p = 0.05). In addition, the median TRAG3 expression was 6.7‐fold higher in ≥pT3 tumors compared to ≤pT2 tumors. Knowing the status of TRAG3 expression could help clinicians tailor treatment to a particular patient population that could benefit from treatment, while allocating patients with resistant tumors to new experimental therapies.     

ASP5878, a selective FGFR inhibitor,to treat FGFR3‐dependent urothelial cancer with or without chemoresistance

FGF/FGFR gene aberrations such as amplification, mutation and fusion are associated with many types of human cancers including urothelial cancer. FGFR kinase inhibitors are expected to be a targeted therapy for urothelial cancer harboring FGFR3 gene alternations. ASP5878, a selective inhibitor of FGFR1, 2, 3 and 4 under clinical investigation, selectively inhibited cell proliferation of urothelial cancer cell lines harboring FGFR3 point mutation or fusion (UM‐UC‐14, RT‐112, RT4 and SW 780) among 23 urothelial cancer cell lines. Furthermore, ASP5878 inhibited cell proliferation of adriamycin‐resistant UM‐UC‐14 cell line harboring MDR1 overexpression and gemcitabine‐resistant RT‐112 cell line. The protein expression of c‐MYC, an oncoprotein, in gemcitabine‐resistant RT‐112 cell line was higher than that in RT‐112 parental cell line and ASP5878 decreased the c‐MYC expression in both RT‐112 parental and gemcitabine‐resistant RT‐112 cell lines. Once‐daily oral administration of ASP5878 exerted potent antitumor activities in UM‐UC‐14, RT‐112 and gemcitabine‐resistant RT‐112 xenograft models without affecting body weight. These findings suggest that ASP5878 has the potential to be an oral targeted therapy against urothelial cancer harboring FGFR3 fusion or FGFR3 point mutation after the acquisition of gemcitabine‐ or adriamycin‐resistance.     

Antitumor effects and molecular mechanisms of ponatinib on endometrial cancer cells harboring activating FGFR2 mutations

Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC₅₀ of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs.     

Constitutive activating mutation of the FGFR3b in oral squamous cell carcinomas

A G to T mutation at nucleotide position 2128 in the human FGFR3b coding region resulting in a Cys for Gly substitution (G697C) in the tyrosine kinase domain was observed in 62% (44/71) of oral squamous cell carcinomas (OSCC) examined. Immunostained FGFR3b was found in the cytoplasm of prickle cells in normal epithelia, and FGFR3b was localized in the cytoplasm and nucleus in non-FGFR3b mutant OSCC. Overexpressed FGFR3b protein on plasma membranes was noted in OSCC bearing the FGFR3b mutation. Enhanced tyrosine kinase activity of G697CFGFR3b was confirmed. Our results indicate that G697C is an activating mutation causing constitutive ligand-independent FGFR3b signaling. This mutation may be involved in the progression of OSCC and thus the FGFR3b coding sequence may have diagnostic or prognostic value for OSCC.     

p53 mutations as a marker of malignancy in bladder washing samples from patients with bladder cancer

The diagnosis and follow-up of patients with bladder cancer rely on invasive procedures (cystoscopy with biopsy). Polymerase chain reaction (PCR)-based technologies may allow the sensitive detection of cancer-related genetic mutations in exfoliated tumour material, potentially allowing the development of less invasive techniques. This pilot study investigated the feasibility of detecting mutations in exons 5-8 of the p53 gene using single-stranded conformational polymorphism (SSCP) analysis in bladder-washing specimens from patients with bladder cancer. Bladder-washing samples (31) were collected from patients (27) with bladder cancer. An abnormal additional SSCP band was detected in five samples from five different patients suggesting the presence of a p53 mutation. In all five cases the same abnormal SSCP pattern was demonstrated in samples of the corresponding bladder tumour. In one case bladder washings were available from the same patient on two separate occasions with one washing demonstrating a mutation and the other not. In two further cases a mutation was demonstrated in the bladder tumour but not in the corresponding washing. It is concluded that it is possible to detect and characterize p53 mutations in bladder-washing samples from patients with bladder cancer. Improved sensitivity in detecting mutations in a sample containing a mixture of normal and malignant cells may lead to the clinical applicability of molecular methods of disease monitoring.     

FGFR3和PIK3CA基因突变影响膀胱癌的预后

背景与目的：成纤维细胞生长因子受体3（fibroblast growth factor receptor 3,FGFR3）和磷脂酰肌醇3-激酶催化亚基ɑ（phosphoinositide 3 kinase catalytic alpha polypeptide,PIK3CA）基因突变与多种肿瘤的发生、发展密切相关。然而,其临床病理意义尚未明确。探讨FGFR3和PIK3CA基因突变对膀胱癌预后的影响。方法：采用Sanger基因测序技术分析于南京中医药大学张家港附属医院行手术治疗的63例膀胱癌患者中FGFR3（外显子7、10、15）和PIK3CA（外显子9、20）基因突变,同时采用免疫组织化学方法检测FGFR3和PIK3CA蛋白水平。分析FGFR3和PIK3CA基因突变与各临床参数的关系,应用Kaplan-Meier方法进行生存分析,通过多因素COX模型回归分析方法对影响膀胱癌预后的因素进行分析。结果：FGFR3基因突变率为17.46%（11/63）,蛋白表达率为33.33%（21/63）,PIK3CA基因突变率为7.93%（5/63）,蛋白表达率为55.56%（35/63）。FGFR3基因突变与年龄、肿瘤是否复发显著相关（P<0.05）,PIK3CA基因突变与各临床参数均无显著相关性（P>0.05）;11例FGFR3 ^mut 中2例PIK3CA ^mut ,52例FGFR3 ^wt 中3例PIK3CA ^mut ,两者比较,差异有统计学意义（χ ² =4.61,P=0.03）。FGFR3基因突变型和野生型的无进展生存期（progression-free survival,PFS）分别为29.13和46.25个月,差异有统计学意义（P=0.021）,而两组的总生存期（overall survival,OS）差异无统计学意义（P>0.05）;PIK3CA基因突变型和野生型比较OS和PFS,差异均无统计学意义（P>0.05）。多参数COX回归分析影响膀胱癌的因素中,FGFR3和PIK3CA基因突变和蛋白表达均不是影响OS的主要因素,病理学分级是影响OS的主要因素,吸烟史和FGFR3基因突变则是影响PFS的主要因素。结论：FGFR3基因突变是膀胱癌预后的不利因素,PIK3CA更易在FGFR3突变的膀胱癌中突变,可能促进FGFR3突变型膀胱癌的恶性行为。     

Association of FGFR3 and FGFR4 gene polymorphisms with breast cancer in Chinese women of Heilongjiang province

Background

The fibroblast growth factor (FGF) receptor pathway is activated in many tumors. FGFR2 has been identified as a breast cancer susceptibility gene. Common variation in other FGF receptors might also affect breast cancer risk. We carried out a case-control study to investigate associations of variants in FGFR3 and FGFR4 with breast cancer in women from Heilongjiang Province.

Methods

SNP rs2234909 and rs3135848 in FGFR3 and rs1966265 and rs351855 in FGFR4 were successfully genotyped in 747 breast cancer patients and 716 healthy controls using the SNaPshot method. The associations between SNPs and breast cancer were examined by logistic regression. The associations between SNPs and disease characteristics were examined by chi-square tests or one-way ANOVA as needed.

Results

The minor alleles of rs1966265 and rs351855 in FGFR4 were strongly associated with breast cancer in the population, with odds ratios of 1.335 (95%CI = 1.154-1.545) and 1.364 (95%CI = 1.177-1.580), respectively. However, no significant associations were detected between other SNPs and breast cancer. Analyses of the disease characteristics showed that SNP rs351855 was associated with lymph-node-positive breast cancer with a dose-dependent effect of the minor allele (P = 0.008).

Conclusions

SNPs rs1966265 and rs351855 in FGFR4 were associated with breast cancer in a northern Chinese population.     

FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder

FGFR3 and TP53 mutations are frequent in superficial papillary and invasive disease, respectively. We used denaturing high-performance liquid chromatography and sequencing to screen for FGFR3 and TP53 mutations in 81 newly diagnosed urothelial cell carcinomas. Tumors were classified as follows: 31 pTa, 1 carcinoma in situ, 30 pT1, and 19 pT2-T4. Tumor grades were as follows: 10 G1, 29 G2, and 42 G3. FGFR3 mutations were associated with low-stage (P < 0.0001), low-grade (P < 0.008) tumors, whereas TP53 mutations were associated with high-stage (P < 0.003), high-grade (P < 0.02) tumors. Mutations in these two genes were almost mutually exclusive. Our results suggest that FGFR3 and TP53 mutations define separate pathways at initial diagnosis of urothelial cell carcinoma.     

Association of the PIG3 promoter polymorphism with invasive bladder cancer in a Japanese population

PIG3 (p53-induced gene 3) is one of the targets of TP53 and is involved in apoptosis. The promoter of PIG3 contains a variable number of tandem repeats (VNTRs) of pentanucleotides (TGYCC)n (Y = C or T) and the number of VNTRs was reported to be correlated with the activation by TP53. In this study, the clinical significance of the PIG3 promoter VNTRs was analyzed in the bladder cancer patients using the genome DNAs from 338 controls and 273 bladder cancer patients. There was no significant difference in the allele frequency of the PIG3 promoter VNTRs between them. However, the presence of 14 or less repeats allele was associated with higher cancer grade (P = 0.038) and higher stage in relative risk (adjusted odds ratio = 2.31, 95% confidence interval = 1.05-5.90). These data suggested that the PIG3 promoter VNTRs was associated with generation of invasive bladder cancer.     

The clinicopathological characteristics of muscle-invasive bladder recurrence in upper tract urothelial carcinoma

This study aimed to clarify the clinical characteristics and oncological outcomes of patients with upper tract urothelial carcinoma (UTUC) who developed muscle-invasive bladder cancer (MIBC) after radical nephroureterectomy (RNU). We identified 966 pTa-4N0-2M0 patients with UTUC who underwent RNU and clarified the risk factors for MIBC progression after initial intravesical recurrence (IVR). We also identified 318 patients with primary pT2-4N0-2M0 MIBC to compare the oncological outcomes with those of patients with UTUC who developed or progressed to MIBC. Furthermore, immunohistochemical examination of p53 and FGFR3 expression in tumor specimens was performed to compare UTUC of MIBC origin with primary MIBC. In total, 392 (40.6%) patients developed IVR after RNU and 46 (4.8%) developed MIBC at initial IVR or thereafter. As a result, pT1 stage on the initial IVR specimen, concomitant carcinoma in situ on the initial IVR specimen, and no intravesical adjuvant therapy after IVR were independent factors for MIBC progression. After propensity score matching adjustment, primary UTUC was a favorable indicator for cancer-specific death compared with primary MIBC. Subgroup molecular analysis revealed high FGFR3 expression in non-MIBC and MIBC specimens from primary UTUC, whereas low FGFR3 but high p53 expression was observed in specimens from primary MIBC tissue. In conclusion, our study demonstrated that patients with UTUC who develop MIBC recurrence after RNU exhibited the clinical characteristics of subsequent IVR more than those of primary UTUC. Of note, MIBC subsequent to UTUC may have favorable outcomes, probably due to the different molecular biological background compared with primary MIBC.