Detection of recurrently mutated nucleotides identifies novel cancer hotspots in an analysis of >11,000 human tumor samples. Mutational hotspots indicate selective pressure across a population of tumor samples, but their prevalence within and across cancer types is incompletely characterized. An approach to detect significantly mutated residues, rather than methods that identify recurrently mutated genes, may uncover new biologically and therapeutically relevant driver mutations. Here, we developed a statistical algorithm to identify recurrently mutated residues in tumor samples. We applied the algorithm to 11,119 human tumors, spanning 41 cancer types, and identified 470 somatic substitution hotspots in 275 genes. We find that half of all human tumors possess one or more mutational hotspots with widespread lineage-, position- and mutant allele–specific differences, many of which are likely functional. In total, 243 hotspots were novel and appeared to affect a broad spectrum of molecular function, including hotspots at paralogous residues of Ras-related small GTPases RAC1 and RRAS2. Redefining hotspots at mutant amino acid resolution will help elucidate the allele-specific differences in their function and could have important therapeutic implications.

DNA methylation is an epigenetic modification involved in both normal developmental processes and disease states through the modulation of gene expression and the maintenance of genomic organization. Conventional methods of DNA methylation analysis, such as bisulfite sequencing, methylation sensitive restriction enzyme digestion and array-based detection techniques, have major limitations that impede high-throughput genome-wide analysis. We describe a novel technique, MBD-isolated Genome Sequencing (MiGS), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein and sequencing of the isolated DNA by a massively parallel sequencer. We utilized MiGS to study three isogenic cancer cell lines with varying degrees of DNA methylation. We successfully detected previously known methylated regions in these cells and identified hundreds of novel methylated regions. This technique is highly specific and sensitive and can be applied to any biological settings to identify differentially methylated regions at the genomic scale.

CpG island hypermethylation occurs in most cases of cancer, typically resulting in the transcriptional silencing of critical cancer genes. Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. We report here that procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication. At micromolar concentrations, procainamide was found to be a partial competitive inhibitor of DNMT1, reducing the affinity of the enzyme for its two substrates, hemimethylated DNA and S-adenosyl-l-methionine. By doing so, procainamide significantly decreased the processivity of DNMT1 on hemimethylated DNA. Procainamide was not a potent inhibitor of the de novo methyltransferases DNMT3a and DNMT3b2. As further evidence of the specificity of procainamide for DNMT1, procainamide failed to lower genomic 5-methyl-2′-deoxycytidine levels in HCT116 colorectal cancer cells when DNMT1 was genetically deleted but significantly reduced genomic 5-methyl-2′-deoxycytidine content in parental HCT116 cells and in HCT116 cells where DNMT3b was genetically deleted. Because many reports have strongly linked DNMT1 with epigenetic alterations in carcinogenesis, procainamide may be a useful drug in the prevention of cancer.

We used what is to our knowledge a new method to estimate volume loss after partial nephrectomy to assess the relative contributions of ischemic injury and volume loss on functional outcomes. We analyzed the records of 301 consecutive patients who underwent conventional partial nephrectomy between 2007 and 2010 with available data to meet inclusion criteria. Percent functional volume preservation was measured at a median of 1.4 years after surgery. Modification of diet in renal disease-2 estimated glomerular filtration rate was measured preoperatively and perioperatively, and a median of 1.2 years postoperatively. Statistical analysis was done to study associations. Hypothermia or warm ischemia 25 minutes or less was applied in 75% of cases. Median percent functional volume preservation was 91% (range 38%-107%). Percent glomerular filtration rate preservation at nadir and late time points was 77% and 90% of preoperative glomerular filtration rate, respectively. On multivariate analysis percent functional volume preservation and warm ischemia time were associated with nadir glomerular filtration rate while only percent functional volume preservation was associated with late glomerular filtration rate (each p <0.001). Late percent glomerular filtration rate preservation and percent functional volume preservation were directly associated (p <0.001). Recovery of function to 90% or greater of percent functional volume preservation predicted levels was observed in 86% of patients. In patients with de novo postoperative stage 3 or greater chronic kidney disease, percent functional volume preservation and Charlson score were associated with late percent glomerular filtration rate preservation. Warm ischemia time was not associated with late functional glomerular filtration rate decreases in patients considered high risk for ischemic injury. In this cohort volume loss and not ischemia time was the primary determinant of ultimate renal function after partial nephrectomy. Technical modifications aimed at minimizing volume loss during partial nephrectomy while still achieving negative margins may result in improved functional outcomes.

Purpose: Owing to its exquisite chemotherapy sensitivity, most patients with metastatic germ cell tumors (GCTs) are cured with cisplatin-based chemotherapy. However, up to 30% of patients with advanced GCT exhibit cisplatin resistance, which requires intensive salvage treatment, and have a 50% risk of cancer-related death. To identify a genetic basis for cisplatin resistance, we performed whole-exome and targeted sequencing of cisplatin-sensitive and cisplatin-resistant GCTs. Methods: Men with GCT who received a cisplatin-containing chemotherapy regimen and had available tumor tissue were eligible to participate in this study. Whole-exome sequencing or targeted exon-capture–based sequencing was performed on 180 tumors. Patients were categorized as cisplatin sensitive or cisplatin resistant by using a combination of postchemotherapy parameters, including serum tumor marker levels, radiology, and pathology at surgical resection of residual disease. Results: TP53 alterations were present exclusively in cisplatin-resistant tumors and were particularly prevalent among primary mediastinal nonseminomas (72%). TP53 pathway alterations including MDM2 amplifications were more common among patients with adverse clinical features, categorized as poor risk according to the International Germ Cell Cancer Collaborative Group (IGCCCG) model. Despite this association, TP53 and MDM2 alterations predicted adverse prognosis independent of the IGCCCG model. Actionable alterations, including novel RAC1 mutations, were detected in 55% of cisplatin-resistant GCTs. Conclusion: In GCT, TP53 and MDM2 alterations were associated with cisplatin resistance and inferior outcomes, independent of the IGCCCG model. The finding of frequent TP53 alterations among mediastinal primary nonseminomas may explain the more frequent chemoresistance observed with this tumor subtype. A substantial portion of cisplatin-resistant GCTs harbor actionable alterations, which might respond to targeted therapies. Genomic profiling of patients with advanced GCT could improve current risk stratification and identify novel therapeutic approaches for patients with cisplatin-resistant disease.