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Molecular characterization of bladder tumors and studies of etiologic heterogeneity in the PLCO Trial

Principal Investigator
Name
Stella Koutros
Institution
National Cancer Institute
Position Title
Investigator
Email
About this CDAS Project
Study
PLCO (Learn more about this study)
Project ID
2016-0045
Initial CDAS Request Approval
Mar 14, 2017
Title
Molecular characterization of bladder tumors and studies of etiologic heterogeneity in the PLCO Trial
Summary
Recent high throughput advances in molecular techniques such as next-generation sequencing (NGS), have identified many new somatic and copy number alterations in well-defined series of urothelial cancer patients. Additional improvements have been made to identify heterogeneous bladder cancer subgroups defined by differential patterns of somatic alterations as well as protein expression. The incorporation of these tumor markers with important risk-factor data is still limited, however. Such studies provide valuable evidence to support exposure-disease relationships and strengthen causal inference when elevated disease risk is uniquely observed in one molecularly-defined tumor subgroups. DCEG has a long history of conducting molecular studies of bladder cancer etiology and more recently tumor heterogeneity which identified differences in genetic, epigenetic, and gene and protein expression in tumor tissue. Here, we propose to extend ongoing molecular pathology work in bladder cancer in the PLCO Trial. Results will be pooled with existing studies and will provide the opportunity to utilize high-quality questionnaire data collected prior to diagnosis. FFPE bladder tumor cores will be used for nucleic acid extraction. Tissue microarrays (TMAs) will be used to quantify protein expression using immunohistochemistry (IHC) methods for identification of specific markers. Questionnaire data will be used to explore etiologic heterogeneity by exposures to known/suspected bladder carcinogens and mechanistic pathways altered in bladder carcinogenesis. This interdisciplinary research team has a history of conducting molecular pathology studies of bladder cancer, most recently conducting pilot work utilizing the latest technologies for high throughput sensitive genomic analysis, as well as studies of genetic susceptibility and exposure-response associations for this tumor site. Thus, the research team is well poised to conduct the analyses described in this proposal.
Aims

The aims of this proposal are to utilize existing and novel methods to extend, replicate and pool results from ongoing investigations of bladder tumor molecular pathology to molecularly characterize bladder cancers among patients in the PLCO trial and to identify etiologic heterogeneous tumor subgroups associated with exposures to known/suspected risk factors for bladder cancer.
AIM 1: Characterization of Smatic Mutations and Protein expression
• Nucleic Acids (DNA/RNA) will be extracted from 175 patient tumors. DNA will be tested for contamination, quality and quantity to determine the amount of amplifiable DNA per subject. DNA will be used to characterize somatic mutations using a targeted panel of 50 genes previously developed by the investigators (Ampliseq, CGR) known to be mutated in bladder cancer. Feasibility of RNA extraction will be tested by assessing degradation and quality (determining the fragments size distribution using Agilent Bioanalyzer or similar system).
• Tissue microarray analyses (n=201 subjects, assuming all subjects are on one slide) A total of 15 slides (one for each marker plus a top and a bottom for H&E staining to identify areas of tumor) will be requested to assess protein expression in high-prior candidate genes and markers related to germline variants associated with bladder cancer risk.

AIM 2: Etiologic Heterogeneity

Molecular heterogeneity of bladder cancer defined by panels of tumor markers suggests that environmental exposures may have specific associations unique to molecularly defined tumors subgroups and provides potential to understand mechanisms through which exposures influence bladder carcinogenesis. In our previous work we have identified interesting etiologic heterogeneity for several important known and putative bladder carcinogens, most notably cigarette smoking. Thus, the PLCO study provides the opportunity to provide questionnaire data on: cigarette smoking, family history of bladder cancer, and non-steroidal anti-inflammatory use collected prior to cancer diagnosis that can be used to identify heterogeneity subgroups of tumors uniquely associated with risk factors of interest.

Collaborators

Stella Koutros (National Cancer Institute)
Mike Dean (NCI)
Debra Silverman (NCI)
Nat Rothman (NCI)
Ludmila Prokunina-Olsson (NCI)
Lee Moore (NCI)
Mike Nickerson (NCI)
Petra Lenz (NCI)