Genetic and Epigenetic Alterations in Urothelial Carcinoma




© Springer Science+Business Media New York 2015
Cristina Magi-Galluzzi and Christopher G. Przybycin (eds.)Genitourinary Pathology10.1007/978-1-4939-2044-0_20


20. Genetic and Epigenetic Alterations in Urothelial Carcinoma



Hikmat A. Al-Ahmadie  and Gopa Iyer 


(1)
Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

(2)
Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

 



 

Hikmat A. Al-Ahmadie (Corresponding author)



 

Gopa Iyer



Keywords
Somatic mutationsCopy number alterationsFusionsChromatin remodelingEpigenetic regulationMethylationHistone modification



Introduction


It is estimated that more than 70,000 new cancer cases are caused by urinary bladder cancer in 2013, resulting in nearly 18,000 cancer-related deaths [1]. Until recently, management approaches to these tumors have not incorporated molecular biomarkers in the diagnosis, risk stratification, and treatment, in contrast to what has become an integral component of the clinical management in other tumors such as lung, colon, and breast cancer. Recent major advances in cancer genetics and genomics are changing the landscape and rapidly affecting the clinical management of solid tumors, which undoubtedly includes cancers of the urinary bladder.


Genetic Alterations in Urothelial Carcinoma


Many genetic alterations have been described in bladder cancer including deletions and amplifications of chromosomal regions (wide or focal) as well as many mutations in significant cancer-related genes and pathways. In fact, in a recent comprehensive analysis by The Cancer Genome Atlas (TCGA) Project across several cancer types, bladder cancer (at least invasive into muscularis propria per TCGA inclusion criteria) was one of the cancers with the highest rate of somatic mutations [2]. On average, there were 302 exonic mutations, 204 segmental alterations in genomic copy number, and 22 genomic rearrangements per sample [3].

There is evidence to support viewing bladder cancer as developing through two distinct molecular pathways that correspond to two main groups of tumors with generally distinct treatment considerations: superficial bladder cancer, including noninvasive papillary and flat urothelial carcinoma (UC) and UC invasive into lamina propria and/or muscularis propria (detrusor muscle) [48].

Most studies of low-grade papillary UC show few molecular alterations in addition to deletions involving chromosome 9 and mutations of FGFR3 and HRAS [920]. These tumors are often near-diploid with loss of chromosome 9 being by far the most common cytogenetic finding [2123] . In a recent study utilizing whole-exome sequencing, it was reported that KDM6A (UTX), one of the genes involved in chromatin remodeling, was significantly more frequently mutated in low-grade and low-stage UC [24]. Another genetic aberration recently reported at a higher frequency in noninvasive papillary UC compared to invasive UC is an inactivating mutation in STAG2, a gene which regulates sister chromatid cohesion and segregation and has a role in controlling chromosome number and cell division [2527]. The majority of the mutations reported in STAG2 were truncating (~ 85 %) or missense (~ 15 %), and predicted to result in inactivation of the gene. It was suggested that STAG2 mutations represent an early event in the development of bladder cancer. It is worth noting that another recent publication reported stronger association between inactivating mutations in STAG2 and increased tumor aneuploidy and worse outcome compared to tumors without such aberrations [28]. This view, however, was not shared by other investigators [2527] .

Urothelial carcinoma in situ (CIS) is characterized by a high frequency of TP53 mutations but a relatively low frequency of chromosome 9 loss, unless it is associated with a papillary lesion, in which case loss of chromosome 9 is more frequent [29].

Many genetic alterations have been reported in invasive UC in addition to frequent chromosome 9 deletions, involving dysregulation of several oncogenes and tumor suppressor genes [46]. Multiple regions of somatic copy number alteration (CNA) have been reported including amplification of PPARG, E2F3, EGFR, CCND1, and MDM2, as well as loss of CDKN2A and RB1 [3032]. Sequencing of candidate pathways has identified recurrent mutations in TP53, FGFR3, PIK3CA, TSC1, RB1, and HRAS [30, 32]. It has been recently shown that tumors with aberrations in TP53, MDM2, RB1, and E2F3 are associated with more genomic instability compared to tumors without such aberrations [33].

The most comprehensive molecular analysis of invasive UC to date has been conducted by the TCGA which performed an integrated in-depth profiling of DNA copy number, somatic mutation, mRNA and miRNA (miR) expression, protein and phosphorylated protein expression, DNA methylation, and viral integration [3]. This study included samples from 19 tissue source sites and consisted of 131 chemotherapy naive, at least muscle-invasive (pT2), high-grade UC without significant amount of any divergent histology .

By assessing somatic CNAs (SCNAs), multiple aberration were identified including 22 significant arm-level copy number changes, 27 amplified, and 30 deleted recurrent focal SCNAs . The most common recurrent focal deletion contained CDKN2A (9p21.3, in 47 % of samples). Other focal deletions involved regions containing PDE4D, RB1, FHIT, CREBBP, IKZF2, FOXQ1, FAM190A, LRP1B, and WWOX. Focal amplifications involved genes previously reported to be altered in bladder cancer such as E2F3/SOX4, CCND1, CCNE1, EGFR, ERBB2, PPARG, and MDM2 [3032], but also some that have not been previously reported such as PVRL4, BCL2L1, and ZNF703.

Whole-exome sequencing of tumors along with matched germline samples identified 32 genes with statistically significant levels of recurrent somatic mutation. The most frequently mutated gene was TP53 (49 %), which was found to be altered in a mutually exclusive relationship with MDM2 amplification (9 %) or overexpression (29 %). Most RB1 mutations were inactivating and were mutually exclusive with CDKN2A deletions. PIK3CA mutations were also relatively common (20 %).

A number of genes involved in epigenetic regulation were significantly mutated such as MLL2, ARID1A, KDM6A, and EP300, with truncating mutations being the most common and indicating both a functional significance for these genes and a potential role in tumorigenesis. Other chromatin-regulating genes with less frequent mutations in UC include MLL3, MLL, CREBBP, CHD7, and SRCAP. Some of these mutations have previously been reported in bladder cancer [24, 28] .

When compared with other epithelial cancers in the TCGA Project, bladder cancer was found to be significantly more enriched for mutations in chromatin-regulatory genes. By using low-pass paired-end, whole-genome sequencing, and RNA sequencing, numerous structural aberrations including some that involve gene–gene fusions of different types were detected (e.g., interchromosomal, intrachromosomal, fusions resulting from inversions or deletions). One of the recurrent translocations of probable pathogenic significance was an intrachromosomal translocation on chromosome 4 involving FGFR3 and TACC3 in three tumors, which confirms a previously reported finding [28, 34, 35].

Integrated analysis of the mutation and copy number data revealed frequent dysregulation in major cancer pathways including cell cycle regulation (93 %), kinase and phosphatidylinositol-3-kinase (PI3K) signaling (72 %), and chromatin remodeling (histone-modifying genes, 89 % and the SWItch/Sucrose NonFermentable (SWI/SNF) nucleosome remodeling complex, 64 %). By applying network analysis, increased activity in other important signaling hubs was identified including MYC/MAX, FOXA2, SP1, and HSP90AA1.

Looking at the recently generated and reported data, it is notable that multiple druggable targets have been identified in UC [31, 32, 3439]. These targets include activating FGFR3 mutations, amplification and activating mutations in ERBB2 and ERBB3, and alterations in the PI3K/mTOR/AKT/TSC1 pathway. Possible targets also include alterations in epigenetic regulatory pathways due to the high frequency of such aberrations in UC and merit further investigation. In summary, the molecular profile of bladder cancer identified through TCGA and other efforts has opened a number of exciting therapeutic avenues in the treatment of this disease. Clinical trials based upon the genetics of bladder cancer are already underway in an attempt to exploit these aberrations .


Epigenetics of Bladder Cancer


Epigenetic changes are defined as heritable, reversible alterations in gene expression that are not due to DNA sequence alterations [4042] . This level of regulation of gene expression occurs in both normal and tumor cells and involves DNA methylation , typically at the cytosine 5 position within CpG repeat sequences, posttranslational modification of histones, and microRNA regulation [4346]. Early studies examined the methylation status of specific genes while the more recent advent of global methylation profiling technologies has helped to define the methylome in tumors of specific grades and stages of development [47, 48]. These investigations have identified both global hypomethylation and gene-specific promoter hyper- and hypomethylation as characteristic changes across numerous tumor types that impact tumor progression, invasion, and prognosis [49, 50].


Methylation


DNA hypermethylation of CpG repeats in the promoter region of specific genes with subsequent repression of expression has been reported in UC. In one study, methylation-specific polymerase chain reaction (PCR) of ten genes implicated in tumorigenesis was performed in 98 bladder tumors from both transurethral resection (TUR) and cystectomy specimens, revealing high methylation frequencies of > 20 % for CDH1, CDH13, RASSF1A, and APC [51]. CDH1 and FHIT methylation was associated with inferior survival, with CDH1 methylation status remaining an independent prognostic factor in a multivariate analysis; additionally, a methylation index was calculated for each tumor, representing the methylation fraction for all ten genes, and those tumors with a high methylation index displayed worse survival. Another study screened for methylation of seven genes commonly implicated in tumorigenesis in 98 bladder tumor specimens consisting of both primary and recurrent tumors removed by TUR [52]. Using methylation-specific PCR, RARβ, DAPK, CDH1, and p16Ink4a were found to be methylated from 26.5 to 87.8 %, and at least one of these four genes was methylated in all 98 samples. RARβ was methylated in three of seven normal urothelial samples obtained from patients without UC; none of the other genes was found to be methylated. Four CIS samples were analyzed in which DAPK, CDH1, and RARβ were found to be methylated at high frequency. No clinical or pathologic correlation was found based upon the methylation status of any of the genes screened. In this same study, the methylation status of DAPK1, CDH1, RARβ, and p16Ink4a was defined in 22 voided urine specimens. While the methylation frequency of these four genes was generally lower than in the corresponding tumor specimens, methylation of at least one of these four genes was detected in 90.9 % of samples. In contrast, cancer cells were detected by urine cytology in 45.5 % of these 22 samples, suggesting that detection of methylated genes in urine is more sensitive than traditional cytologic approaches as a screening tool for UC. Additional evidence for the utility of methylated gene detection within urine as a screening biomarker for the presence of urothelial tumors stems from another study of 51 bladder tumors and 47 matched urine samples [53]. The methylation status of four genes ( CDH1, p16, p14, and RASSF1A) was defined using methylation-specific PCR. The sensitivity of urine methylation marker detection was 83 % when using RASSF1A, p14, and CDH1 methylation status, while that for urine cytology was 28 %. Moreover, 90 % of superficial low-grade tumors that were not detected by urine cytology contained hypermethylation. Methylation-mediated inactivation of p16, which is part of the CDKN2A locus on chromosome 9p21, may contribute to loss of heterozygosity at this locus, since CDKN2A loss by both mutations and deletions is an early and common genetic alteration in UC. Loss of p16 function results in cell cycle dysregulation and uncontrolled proliferation. Furthermore, E-cadherin, the protein encoded by CDH1, is implicated in suppression of the Wnt/β-catenin pathway which is involved in promoting cell growth [54]; therefore, loss of E-cadherin expression by promoter methylation leads to constitutive activation of this pathway and excessive cell proliferation. Methylation patterns have been reported to vary based upon the location of urothelial tumors (upper tract vs. bladder) as well as stage and mortality [55]. Hypermethylation at CpG islands in the promoter regions of 11 genes was performed using methylation-specific PCR in 116 bladder tumors and 164 tumors of the upper tract. Promoter methylation was more common in upper tract tumors (94 vs. 76 %, p< 0.0001) as well as muscle-invasive tumors compared to pTa specimens. Notably, tumors harboring methylation at any of the 11 genes exhibited higher rates of progression, including pTa tumors with a high methylation index. Global hypomethylation has been correlated with noninvasive tumors while widespread hypermethylation seems to occur in invasive tumors [56]. Using the Illumina GoldenGate methylation platform, Wolff et al. interrogated 784 genes for methylation status in 49 noninvasive and 38 invasive tumors. Thirty-eight percent of loci were hypermethylated in invasive tumors versus 10 % in noninvasive tumors as compared to normal urothelial tissue from patients without UC. In contrast, hypomethylated loci were predominantly found in the noninvasive tumors (16 vs. 3 % invasive), and these regions of hypomethylation were frequently observed in non-CpG island regions of the genome. Notably, normal-appearing urothelium sampled at varying distances from invasive tumors also showed an abnormal pattern of hypermethylation in 12 % of loci with a significant overlap with the hypermethylated loci identified within invasive tumors, suggesting that epigenetic alterations may precede the development of histologic changes within the bladder. Such alterations may contribute to the well-described field effect in UC, an increased propensity for tumor development within the urinary tract of patients with disease.

Only gold members can continue reading. Log In or Register to continue

Stay updated, free articles. Join our Telegram channel

Oct 29, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Genetic and Epigenetic Alterations in Urothelial Carcinoma

Full access? Get Clinical Tree

Get Clinical Tree app for offline access