Systematic Assessment of Tumor Purity and Its Clinical Implications.

PURPOSE: The tumor microenvironment is complex, comprising heterogeneous cellular populations. As molecular profiles are frequently generated using bulk tissue sections, they represent an admixture of multiple cell types (including immune, stromal, and cancer cells) interacting with each other. Therefore, these molecular profiles are confounded by signals emanating from many cell types. Accurate assessment of residual cancer cell fraction is crucial for parameterization and interpretation of genomic analyses, as well as for accurately interpreting the clinical properties of the tumor. MATERIALS AND METHODS: To benchmark cancer cell fraction estimation methods, 10 estimators were applied to a clinical cohort of 333 patients with prostate cancer. These methods include gold-standard multiobserver pathology estimates, as well as estimates inferred from genome, epigenome, and transcriptome data. In addition, two methods based on genomic and transcriptomic profiles were used to quantify tumor purity in 4,497 tumors across 12 cancer types. Bulk mRNA and microRNA profiles were subject to in silico deconvolution to estimate cancer cell-specific mRNA and microRNA profiles. RESULTS: We present a systematic comparison of 10 tumor purity estimation methods on a cohort of 333 prostate tumors. We quantify variation among purity estimation methods and demonstrate how this influences interpretation of clinico-genomic analyses. Our data show poor concordance between pathologic and molecular purity estimates, necessitating caution when interpreting molecular results. Limited concordance between DNA- and mRNA-derived purity estimates remained a general pan-cancer phenomenon when tested in an additional 4,497 tumors spanning 12 cancer types. CONCLUSION: The choice of tumor purity estimation method may have a profound impact on the interpretation of genomic assays. Taken together, these data highlight the need for improved assessment of tumor purity and quantitation of its influences on the molecular hallmarks of cancers.

Repeated assessment by high-throughput assay demonstrates that sperm DNA methylation levels are highly reproducible.

OBJECTIVE: To assess reliability of a high-throughput assay of sperm DNA methylation. DESIGN: Observational study comparing DNA methylation of sperm isolated from 3 divided and 12 longitudinally collected semen samples. SETTING: Academic medical center. PATIENT(S): One man undergoing screening semen analysis during evaluation of an infertile couple and 2 healthy fertile male volunteers. INTERVENTION(S): Spermatozoa were separated from seminal plasma and somatic cells using gradient separation. DNA was extracted from spermatozoa, and DNA methylation was assessed at 1,505 DNA sequence-specific sites. MAIN OUTCOME MEASURE(S): Repeatability of sperm DNA methylation measures, estimated by correlation coefficients. RESULT(S): DNA methylation levels were highly correlated within matched sets of divided samples (all r ≥ 0.97) and longitudinal samples (average r = 0.97). CONCLUSION(S): The described methodology reliably assessed methylation of sperm DNA at large numbers of sites. Methylation profiles were consistent over time. High-throughput assessment of sperm DNA methylation is a promising tool for studying the role of epigenetic state in male fertility.