Some molecular-genetic markers defining the pathogenesis of superficial and invasive bladder cancer

We investigated deletions of 3p14, 9p21, 9q34, 17p13 (TP53) loci, activating FGFR3 mutations in exon 9, and aberrant methylation of RASSF1, RARβ, P16, P14, CDH1 genes with the aim of analyzing the pathways of the molecular pathogenesis of bladder cancer. FGFR3 activating mutations and 9p21 deletions were observed significantly more frequently in the group of noninvasive bladder cancer pTa than in minimally invasive cancers pT1 (p = 0.004 and 0.006, respectively). It was shown that groups of superficial and invasive bladder cancer differ significantly in the frequency of 17p13 (p = 0.006) and 9q34 (p = 0.04) deletions and in aberrant methylation of the gene P16 (p = 0.02). We revealed some molecular-genetic alterations in groups of superficial and invasive bladder cancers. Therefore, we propose that these two types of bladder cancer might have different pathways of development.     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Cell responses to FGFR3 signalling: growth, differentiation and apoptosis

FGFR3 is a receptor tyrosine kinase (RTK) of the FGF receptor family, known to have a negative regulatory effect on long bone growth. Fgfr3 knockout mice display longer bones and, accordingly, most germline-activating mutations in man are associated with dwarfism. Somatically, some of the same activating mutations are associated with the human cancers multiple myeloma, cervical carcinoma and carcinoma of the bladder. How signalling through FGFR3 can lead to either chondrocyte apoptosis or cancer cell proliferation is not fully understood. Although FGFR3 can be expressed as two main splice isoforms (IIIb or IIIc), there is no apparent link with specific cell responses, which may rather be associated with the cell type or its differentiation status. Depending on cell type, differential activation of STAT proteins has been observed. STAT1 phosphorylation seems to be involved in inhibition of chondrocyte proliferation while activation of the ERK pathway inhibits chondrocyte differentiation and B-cell proliferation (as in multiple myeloma). The role of FGFR3 in epithelial cancers (bladder and cervix) is not known. Some of the cell specificity may arise via modulation of signalling by crosstalk with other signalling pathways. Recently, inhibition of the ERK pathway in achondroplastic mice has provided hope for an approach to the treatment of dwarfism. Further understanding of the ability of FGFR3 to trigger different responses depending on cell type and cellular context may lead to treatments for both skeletal dysplasias and cancer.     

MGMT基因启动子甲基化与膀胱尿路上皮癌生物学行为关系的研究

目的 检测膀胱尿路上皮癌组织中O^6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因启动子甲基化状态,探讨MGMT甲基化与其蛋白表达水平以及肿瘤生物学行为之间的关系.方法 应用免疫组织化学SP法和甲基化特异性PCR（MSP）分别检测60例膀胱尿路上皮癌及15例正常膀胱黏膜组织中MGMT蛋白表达情况和MGMT基因启动子甲基化状态.结果 膀胱癌组织中MGMT蛋白阳性表达率为35.0 %（21/60）,低于正常膀胱组织（86.7 %,13/15,P〈0.01）.膀胱癌组织中MGMT甲基化阳性率为45.0 %（27/60）,明显高于正常膀胱组织（0.0 %,0/15,P〈0.01）;MGMT启动子甲基化与其蛋白表达呈负相关（r = -0.453,P〈0.01）;并且高级别膀胱癌中MGMT甲基化阳性率（70.6 %,12/17）要比低级别膀胱癌高（34.9 %,15/43）,（P〈0.05）,而MGMT甲基化与膀胱癌临床分期无明显关系.结论 MGMT启动子甲基化可能参与了膀胱尿路上皮癌的发生和肿瘤分化,MGMT启动子甲基化有望成为预判膀胱癌预后的重要标记.     

Disruption of the FA/BRCA pathway in bladder cancer

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.     

The cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes thanatophoric dysplasia types I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. The molecular pathways exploited by FGFR3 to stimulate abnormal proliferation during neoplasia are unclear. The nonreceptor protein-tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) has been shown previously to regulate apoptosis in multiple myeloma cells. Here we describe a novel interaction between FGFR3 and Pyk2, mediated by the juxtamembrane domain of FGFR3 and the kinase domain of Pyk2. Within the FGFR family, Pyk2 also interacted significantly with FGFR2. Overexpression of Pyk2 alone led to its spontaneous activation and tyrosine phosphorylation, resulting in activation of Stat5B, indicated by the reporter GFP-Stat5B. These effects were completely dependent upon Tyr(402), the autophosphorylation site of Pyk2, which allows recruitment of Src family members for further activating phosphorylations at other sites on Pyk2. In the presence of activated FGFR3, the activation of Pyk2 itself became independent of Tyr(402), indicating that FGFR3 activation circumvents the requirement for c-Src recruitment at Tyr(402) of Pyk2. We also examined the role of the tyrosine phosphatase Shp2 in antagonizing Pyk2 activation. Taken together, these results suggest that signaling pathways regulated by FGFR3 may converge with Pyk2-dependent pathways to provide maximal activation of Stat5B.     

Fibroblast growth factor family aberrations in cancers: clinical and molecular characteristics

Fibroblast growth factor ligands and receptors (FGF and FGFR) play critical roles in tumorigenesis, and several drugs have been developed to target them. We report the biologic correlates of FGF/FGFR abnormalities in diverse malignancies. The medical records of patients with cancers that underwent targeted next generation sequencing (182 or 236 cancer-related genes) were reviewed. The following FGF/FGFR genes were tested: FGF3, 4, 6, 7, 10, 12, 14, 19, 23 and FGFR1, 2, 3, and 4. Of 391 patients, 56 (14.3%) had aberrant FGF (N = 38, all amplifications) and/or FGFR (N = 22 including 5 mutations and one FGFR3-TACC3 fusion). FGF/FGFR aberrations were most frequent in breast cancers (26/81, 32.1%, p = 0.0003). In multivariate analysis, FGF/FGFR abnormalities were independently associated with CCND1/2, RICTOR, ZNF703, RPTOR, AKT2, and CDK8 alterations (all P < 0.02), as well as with an increased median number of alterations (P < 0.0001). FGF3, FGF4, FGF19 and CCND1 were co-amplified in 22 of 391 patients (5.6%, P < 0.0001), most likely because they co-localize on the same chromosomal region (11q13). There was no significant difference in time to metastasis or overall survival when comparing patients harboring FGF/FGFR alterations versus those not. Overall, FGF/FGFR was one of the most frequently aberrant pathways in our population comprising patients with diverse malignancies. These aberrations frequently co-exist with anomalies in a variety of other genes, suggesting that tailored combination therapy may be necessary in these patients.     

FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population

Bladder cancer accounts for nearly 5% of all newly diagnosed cancers in Jordan, with a much higher frequency in males. Recent studies have shown that activating mutations in FGFR3 are the most common findings in non-invasive low grade bladder tumors. In this study, we, retrospectively, investigated a cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes in FGFR3. Overexpression of FGFR3 was observed in 49%, 34%, 15%, and 2% of pTa, pT1, pT2, and pT3 cases, respectively. Further, FGFR3 expression was positive in 45%, 26%, and 30% of G1, G2 and G3 cases, respectively. Mutational analysis of exons 7, 10 and 15 of FGFR3 identified four previously reported mutations, namely R248C (n = 4; 10%), S249C (n = 23; 59%), Y375C (n = 7; 18%), G382R (n = 4; 10%), and one novel mutation, G382E (n = 1; 3%). Our results indicate that both mutations and overexpression of FGFR3 are correlated together, and are more prevalent in early stage (pTa and pT1) and low grade (G1 and G2) bladder tumors. Survival analysis showed no contribution of changes in FGFR3 on the patient's survival. Multivariate Cox proportional hazards model analysis of overall survival for the following variables: age, gender, stage and grade of tumor, and FGFR3 (expression and mutation) revealed that age, stage and grade of tumor are independent predictors of overall survival in patients with bladder cancer. Our work is the first to address the molecular status of FGFR3 in Jordanian patients with bladder cancer, and provides further support for FGFR3 as a key player in the initiation of bladder tumors.     

Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma

Similar to other malignancies, urothelial carcinoma (UC) is characterized by specific recurrent chromosomal aberrations and gene mutations. However, the interconnection between specific genomic alterations, and how patterns of chromosomal alterations adhere to different molecular subgroups of UC, is less clear. We applied tiling resolution array CGH to 146 cases of UC and identified a number of regions harboring recurrent focal genomic amplifications and deletions. Several potential oncogenes were included in the amplified regions, including known oncogenes like E2F3, CCND1, and CCNE1, as well as new candidate genes, such as SETDB1 (1q21), and BCL2L1 (20q11). We next combined genome profiling with global gene expression, gene mutation, and protein expression data and identified two major genomic circuits operating in urothelial carcinoma. The first circuit was characterized by FGFR3 alterations, overexpression of CCND1, and 9q and CDKN2A deletions. The second circuit was defined by E3F3 amplifications and RB1 deletions, as well as gains of 5p, deletions at PTEN and 2q36, 16q, 20q, and elevated CDKN2A levels. TP53/MDM2 alterations were common for advanced tumors within the two circuits. Our data also suggest a possible RAS/RAF circuit. The tumors with worst prognosis showed a gene expression profile that indicated a keratinized phenotype. Taken together, our integrative approach revealed at least two separate networks of genomic alterations linked to the molecular diversity seen in UC, and that these circuits may reflect distinct pathways of tumor development.     

Molecular Convergence of Neurodevelopmental Disorders

Neurodevelopmental disorders (NDDs) are caused by mutations in diverse genes involved in different cellular functions, although there can be crosstalk, or convergence, between molecular pathways affected by different NDDs. To assess molecular convergence, we generated human neural progenitor cell models of 9q34 deletion syndrome, caused by haploinsufficiency of EHMT1, and 18q21 deletion syndrome, caused by haploinsufficiency of TCF4. Using next-generation RNA sequencing, methylation sequencing, chromatin immunoprecipitation sequencing, and whole-genome miRNA analysis, we identified several levels of convergence. We found mRNA and miRNA expression patterns that were more characteristic of differentiating cells than of proliferating cells, and we identified CpG clusters that had similar methylation states in both models of reduced gene dosage. There was significant overlap of gene targets of TCF4 and EHMT1, whereby 8.3% of TCF4 gene targets and 4.2% of EHMT1 gene targets were identical. These data suggest that 18q21 and 9q34 deletion syndromes show significant molecular convergence but distinct expression and methylation profiles. Common intersection points might highlight the most salient features of disease and provide avenues for similar treatments for NDDs caused by different genetic mutations.     

Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects

Beckwith-Wiedemann syndrome (BWS) is a congenital cancer-predisposition syndrome associated with embryonal cancers, macroglossia, macrosomia, ear pits or ear creases, and midline abdominal-wall defects. The most common constitutional abnormalities in BWS are epigenetic, involving abnormal methylation of either H19 or LIT1, which encode untranslated RNAs on 11p15. We hypothesized that different epigenetic alterations would be associated with specific phenotypes in BWS. To test this hypothesis, we performed a case-cohort study, using the BWS Registry. The cohort consisted of 92 patients with BWS and molecular analysis of both H19 and LIT1, and these patients showed the same frequency of clinical phenotypes as those patients in the Registry from whom biological samples were not available. The frequency of altered DNA methylation of H19 in patients with cancer was significantly higher, 56% (9/16), than the frequency in patients without cancer, 17% (13/76; P=.002), and cancer was not associated with LIT1 alterations. Furthermore, the frequency of altered DNA methylation of LIT1 in patients with midline abdominal-wall defects and macrosomia was significantly higher, 65% (41/63) and 60% (46/77), respectively, than in patients without such defects, 34% (10/29) and 18% (2/11), respectively (P=.012 and P=.02, respectively). Additionally, paternal uniparental disomy (UPD) of 11p15 was associated with hemihypertrophy (P=.003), cancer (P=.03), and hypoglycemia (P=.05). These results define an epigenotype-phenotype relationship in BWS, in which aberrant methylation of H19 and LIT1 and UPD are strongly associated with cancer risk and specific birth defects.     

Preferential loss of 5q14-21 in intestinal-type gastric cancer with DNA aneuploidy

BACKGROUND: Little is known about the genetic changes associated with DNA ploidy in gastric cancer (GC). The aim of this study was to identify recurrent or specific chromosomal regions of DNA sequence copy number aberrations (DSCNAs) that might harbor genes associated with DNA aneuploidy in GC. METHODS: We analyzed DSCNAs with comparative genomic hybridization and DNA ploidy by laser scanning cytometry in 16 primary intestinal-type GCs. RESULTS: All GCs examined showed at least one DSCNA (loss or gain); eight were DNA diploid (DD) tumors and eight were DNA aneuploid (DA) tumors. The frequent (>30%) DSCNAs were loss of 5q14-21 and gains of 7p11-14, 8q, 20q, and Xq25-26. Recurrent amplifications (>10%) were detected at chromosomal regions 6p, 7p, and 13q. The overall number of DSCNAs was significantly greater in DA than in DD tumors (P = 0.006). Furthermore, the number of aberrations was clearly greater with 5q loss than without 5q loss (P = 0.002). Losses of 5q14-21, 9p21-pter, 16q, and 18q21-qter were preferentially detected in DA tumors. CONCLUSION: The present observations indicate that there is a close relationship between DSCNA and DNA ploidy in intestinal-type GC and that gene(s) at 5q14-21, 9p21-pter, 16q, and/or 18q21-qter may play important roles in acquisition of DNA aneuploidy.     

Somatic oncogenic mutations,benign skin lesions and cancer progression: Where to look next?

Somatic oncogenic activating mutations in FGFR3 and/or PIK3CA have recently been described in benign epithelial cutaneous lesions that never progress to malignancy (seborrheic keratoses and epidermal nevi). The same mutations have been observed in malignant neoplasms from other tissues (bladder carcinoma, cervix cancer, colorectal cancer, myeloma). However, many of the above-mentioned epithelial benign cutaneous tumors do not harbour mutations in FGFR3 or PIK3CA. In this review, we focus on new candidate genes for discovery and we outline the potential of the skin as a model to achieve a better understanding of cancer biology.     

Aberrant fibroblast growth factor receptor signaling in bladder and other cancers

Fibroblast growth factors (FGFs) are potent mitogens, morphogens, and inducers of angiogenesis, and FGF signaling governs the genesis of diverse tissues and organs from the earliest stages. With such fundamental embryonic and homeostatic roles, it follows that aberrant FGF signaling underlies a variety of diseases. Pathological modifications to FGF expression are known to cause salivary gland aplasia and autosomal dominant hypophosphatemic rickets, while mutations in FGF receptors (FGFRs) result in a range of skeletal dysplasias. Anomalous FGF signaling is also associated with cancer development and progression. Examples include the overexpression of FGF2 and FGF6 in prostate cancer, and FGF8 overexpression in breast and prostate cancers. Alterations in FGF signaling regulators also impact tumorigenesis, which is exemplified by the down-regulation of Sprouty 1, a negative regulator of FGF signaling, in prostate cancer. In addition, several FGFRs are mutated in human cancers (including FGFR2 in gastric cancer and FGFR3 in bladder cancer). We recently identified intriguing alterations in the FGF pathway in a novel model of bladder carcinoma that consists of a parental cell line (TSU-Pr1/T24) and two sublines with increasing metastatic potential (TSU-Pr1-B1 and TSU-Pr1-B2), which were derived successively through in vivo cycling. It was found that the increasingly metastatic sublines (TSU-Pr1-B1 and TSU-Pr1-B2) had undergone a mesenchymal to epithelial transition. FGFR2IIIc expression, which is normally expressed in mesenchymal cells, was increased in the epithelial-like TSU-Pr1-B1 and TSU-Pr1-B2 sublines and FGFR2 knock-down was associated with the reversion of cells from an epithelial to a mesenchymal phenotype. These observations suggest that modified FGF pathway signaling should be considered when studying other cancer types.     

Molecular genetic analysis of the intratumoral clonal heterogeneity of colorectal adenocarcinomas

Molecular genetic analysis of allelic deletions from the loci containing the tumor suppressor genes p16, p15, p19 (9p21), RB1 (13p14), PTEN (10q23), and TP53 (17p13); microsatellite instability; and activating mutations of K-RAS (codons 12 and 13) was performed in four different segments of sporadic colorectal cancer (CRC) in 11 patients. Intratumoral genetic heterogenity was detected in 9 out of 11 (81%) colorectal adenocarcinomas and was morphologically validated. Analysis of different segments of one tumor reported that not only intratumoral heterogeneity, but also the order of the appearance and distribution of molecular anomalies during tumorigenesis in sporadic CRC. K-RAS point mutations and anomalies of the p16-RB1-cyclin D pathway were assumed to occur prior to microsatellite instability and PTEN deletions during tumor progression.     

Genetic and Epigenetic Alterations in Primary–Progressive Paired Oligodendroglial Tumors

The aim of the present study was to identify genetic and epigenetic alterations involved in the progression of oligodendroglial tumors. We characterized 21 paired, World Health Organization (WHO) grade II and III oligodendroglial tumors from patients who received craniotomies for the partial or complete resection of primary and secondary oligodendroglial tumors. Tumor DNA was analyzed for alterations in selected genetic loci (1p36, 9p22, 10q23–24, 17p13, 19q13, 22q12), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2) and the CpG island methylation status of critical tumor-related genes (MGMT, P16, DAPK, PTEN, RASSF1A, Rb1). Alterations of these markers were common early in the tumorigenesis. In the primary tumors we identified 12 patients (57.1%) with 1p36 deletions, 17 (81.0%) with 19q13 deletions, 9 (42.9%) with 1p36/19q13 codeletions, 11 (52.3%) with 9p22 deletions, and 12 (57.1%) with IDH1 mutation. Epigenetic analysis detected promoter methylation of the MGMT, P16, DAPK, PTEN, RASSF1A, and Rb1 genes in 38.1%, 19.0%, 38.1%, 33.3%, 66.7%, and 14.3% of primary tumors, respectively. After progression, additional losses of 1p, 9p, 10q, 17p, 19q and 22q were observed in 3 (14.3%), 1 (4.8%), 3 (14.3%), 2 (9.5%), 1 (4.8%) and 3 (14.3%) cases, respectively. Additional methylations of the MGMT, P16, DAPK, PTEN, RASSF1A, and RB1 promoters was observed in 4 (19.0%), 2 (9.5%), 0 (0%), 6 (28.6%), 2(9.5%) and 3 (14.3%) cases, respectively. The status of IDH1 mutation remained unchanged in all tumors after progression. The primary tumors of three patients with subsequent progression to high-grade astrocytomas, all had 9p deletion, intact 1p, intact 10q and unmethylated MGMT. Whether this may represent a molecular signature of patients at-risk for the development of aggressive astrocytomas needs further investigation.