Tiling resolution array CGH and high density expression profiling of urothelial carcinomas delineate genomic amplicons and candidate target genes specific for advanced tumors.

ABSTRACT: BACKGROUND: Urothelial carcinoma (UC) is characterized by nonrandom chromosomal aberrations, varying from one or a few changes in early-stage and low-grade tumors, to highly rearranged karyotypes in muscle-invasive lesions. Recent array-CGH analyses have shed further light on the genomic changes underlying the neoplastic development of UC, and have facilitated the molecular delineation amplified and deleted regions to the level of specific candidate genes. In the present investigation we combine detailed genomic information with expression information to identify putative target genes for genomic amplifications. METHODS: We analyzed 38 urothelial carcinomas by whole-genome tiling resolution array-CGH and high density expression profiling to identify putative target genes in common genomic amplifications. When necessary expression profiling was complemented with Q-PCR of individual genes. RESULTS: Three genomic segments were frequently and exclusively amplified in high grade tumors; 1q23, 6p22 and 8q22, respectively. Detailed mapping of the 1q23 segment showed a heterogeneous amplification pattern and no obvious commonly amplified region. The 6p22 amplicon was defined by a 1.8 Mb core region present in all amplifications, flanked both distally and proximally by segments amplified to a lesser extent. By combining genomic profiles with expression profiles we could show that amplification of E2F3, CDKAL1, SOX4, and MBOAT1 as well as NUP153, AOF1, FAM8A1 and DEK in 6p22 was associated with increased gene expression. Amplification of the 8q22 segment was primarily associated with YWHAZ (14-3-3-zeta) and POLR2K over expression. The possible importance of the YWHA genes in the development of urothelial carcinomas was supported by another recurrent amplicon paralogous to 8q22, in 2p25, where increased copy numbers lead to enhanced expression of YWHAQ (14-3-3-theta). Homozygous deletions were identified at 10 different genomic locations, most frequently affecting CDKN2A/CDKN2B in 9p21 (32%). Notably, the latter occurred mutually exclusive with 6p22 amplifications. CONCLUSION: The presented data indicates 6p22 as a composite amplicon with more than one possible target gene. The data also suggests that amplification of 6p22 and homozygous deletions of 9p21 may have complementary roles. Furthermore, the analysis of paralogous regions that showed genomic amplification indicated altered expression of YWHA (14-3-3) genes as important events in the development of UC

A Molecular Taxonomy for Urothelial Carcinoma.

PURPOSE: Even though urothelial cancer is the fourth most common tumor type among males, progress in treatment has been scarce. A problem in day-to-day clinical practice is that precise assessment of individual tumors is still fairly uncertain; consequently efforts have been undertaken to complement tumor evaluation with molecular biomarkers. An extension of this approach would be to base tumor classification primarily on molecular features. Here, we present a molecular taxonomy for urothelial carcinoma based on integrated genomics. EXPERIMENTAL DESIGN: We use gene expression profiles from 308 tumor cases to define five major urothelial carcinoma subtypes: urobasal A, genomically unstable, urobasal B, squamous cell carcinoma like, and an infiltrated class of tumors. Tumor subtypes were validated in three independent publically available data sets. The expression of 11 key genes was validated at the protein level by immunohistochemistry. RESULTS: The subtypes show distinct clinical outcomes and differ with respect to expression of cell-cycle genes, receptor tyrosine kinases particularly FGFR3, ERBB2, and EGFR, cytokeratins, and cell adhesion genes, as well as with respect to FGFR3, PIK3CA, and TP53 mutation frequency. The molecular subtypes cut across pathologic classification, and class-defining gene signatures show coordinated expression irrespective of pathologic stage and grade, suggesting the molecular phenotypes as intrinsic properties of the tumors. Available data indicate that susceptibility to specific drugs is more likely to be associated with the molecular stratification than with pathologic classification. CONCLUSIONS: We anticipate that the molecular taxonomy will be useful in future clinical investigations. Clin Cancer Res; 1-10. ©2012 AACR

Molecular changes during progression from nonmuscle invasive to advanced urothelial carcinoma

Molecular changes occurring during invasion and clinical progression of cancer are difficult to study longitudinally in patient-derived material. A unique feature of urothelial bladder cancer (UBC) is that patients frequently develop multiple nonmuscle invasive tumors, some of which may eventually progress to invade the muscle of the bladder wall. Here, we use a cohort of 73 patients that experienced a total of 357 UBC diagnoses to study the stability or change in detected molecular alterations during cancer progression. The tumors were subtyped by gene expression profiling and analyzed for hotspot mutations in FGFR3, PIK3CA and TERT, the most frequent early driver mutations in this tumor type. TP53 alterations, frequent in advanced UBC, were inferred from p53 staining pattern, and potential genomic alterations were inferred by gene expression pattern

Recurrent and multiple bladder tumors show conserved expression profiles

<p>Abstract</p> <p>Background</p> <p>Urothelial carcinomas originate from the epithelial cells of the inner lining of the bladder and may appear as single or as multiple synchronous tumors. Patients with urothelial carcinomas frequently show recurrences after treatment making follow-up necessary. The leading hypothesis explaining the origin of meta- and synchronous tumors assumes a monoclonal origin. However, the genetic relationship among consecutive tumors has been shown to be complex in as much as the genetic evolution does not adhere to the chronological appearance of the metachronous tumors. Consequently, genetically less evolved tumors may appear chronologically later than genetically related but more evolved tumors.</p> <p>Methods</p> <p>Forty-nine meta- or synchronous urothelial tumors from 22 patients were analyzed using expression profiling, conventional CGH, LOH, and mutation analyses.</p> <p>Results</p> <p>We show by CGH that partial chromosomal losses in the initial tumors may not be present in the recurring tumors, by LOH that different haplotypes may be lost and that detected regions of LOH may be smaller in recurring tumors, and that mutations present in the initial tumor may not be present in the recurring ones. In contrast we show that despite apparent genomic differences, the recurrent and multiple bladder tumors from the same patients display remarkably similar expression profiles.</p> <p>Conclusion</p> <p>Our findings show that even though the vast majority of the analyzed meta- and synchronous tumors from the same patients are not likely to have originated directly from the preceding tumor they still show remarkably similar expressions profiles. The presented data suggests that an expression profile is established early in tumor development and that this profile is stable and maintained in recurring tumors.</p

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

A Systematic Study of Gene Mutations in Urothelial Carcinoma; Inactivating Mutations in TSC2 and PIK3R1

Abstract BACKGROUND: Urothelial carcinoma (UC) is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations. METHODOLOGY/PRINCIPAL FINDINGS: We performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC

HER2-testning för bröstcancer. Kvalitetssäkrad analys av tillväxtfaktor erbjuds nu på svenska laboratorier

Between 20 and 30 per cent of all breast cancers show an overexpression of the growth factor HER2 and this implies a worse prognosis. Metastasising breast cancer that overexpress HER2 can effectively be treated with trastuzumab (Herceptin). In order to determine whether this treatment is indicated, HER2 expression must be quantified with immunohistochemical or molecular biological methods. Several factors may influence the laboratory analysis of HER2, such as choice of antibodies and probes, the reliability of the test performance itself and the performance of the pathologist(s) interpreting the test. National guidelines for HER2 testing have been published in several countries. In this article a series of quality assurance projects leading to the formulation of Swedish guidelines for HER2 testing is described. These projects have demonstrated that several Swedish pathology laboratories can reliably perform and interpret analysis of HER2 status today

Urothelial carcinoma in the prostatic urethra and prostate: current controversies

We reviewed the literature on urothelial carcinoma in the prostatic urethra and prostate. We concluded that the incidence of urothelial carcinoma in the prostatic urethra and prostate is probably underestimated. This fact warrants thorough follow-up of patients with high-risk bladder cancers and also whole-mount examination of the prostate after cystectomy to recognize the true incidence and extent of such tumor involvement. Resectoscope loop biopsy is the method of choice to detect urothelial carcinoma in the prostatic: urethra/prostate and such biopsies should include the area around the verumontanum to ensure optimal sensitivity. Carcinoma in situ in the prostatic urethra should be treated with intravesical Bacillus Calmette-Guerin and a transurethral resection of the prostate prior to that treatment might increase the contact of Bacillus Calmette-Guerin with the prostatic urethra, improve staging and in itself treat the prostatic involvement. Conservative treatment of carcinoma in situ in the prostatic ducts is an option, although radical surgery is probably best for treating extensive intraductal involvement, since data on the former strategy are inconclusive. Patients with stromal invasion should undergo radical surgery. It is necessary to take the route of prostatic involvement into account when estimating prognosis in each individual patient, since contiguous growth into the prostate is associated with worse prognosis. Prospective studies using a whole-mount technique to investigate the prostate are needed to clarity both the role of different routes of prostate invasion and the prognostic significance of different degrees of prostate invasion. At cystectomy, when urothelial carcinoma is present in the prostatic urethra and/or prostate, it is necessary to balance the risk of urethral recurrence and decreased sexual function against opinion and expectations expressed by the patient during preoperative counseling regarding urinary diversion and primary urethrectomy