With 1.3 million newly diagnosed cases each year, colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related deaths.¹ Despite improved surgery, implementation of screening, and advances in treatment regimens, the 5-year mortality rate for patients with CRC remains high at approximately 40%, thereby representing a significant global health burden.^2,3

PURPOSE: Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS: We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS: Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence ( P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging ( P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION: ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.

Objective To develop an affordable and robust pipeline for selection of patient-specific somatic structural variants (SSVs) being informative about radicality of the primary resection, response to adjuvant therapy, incipient recurrence and response to treatment performed in relation to diagnosis of recurrence. Design We have established efficient procedures for identification of SSVs by next-generation sequencing and subsequent quantification of 3–6 SSVs in plasma. The consequence of intratumour heterogeneity on our approach was assessed. The level of circulating tumour DNA (ctDNA) was quantified in 151 serial plasma samples from six relapsing and five non-relapsing colorectal cancer (CRC) patients by droplet digital PCR, and correlated to clinical findings. Results Up to six personalised assays were designed for each patient. Our approach enabled efficient temporal assessment of disease status, response to surgical and oncological intervention, and early detection of incipient recurrence. Our approach provided 2–15 (mean 10) months' lead time on detection of metastatic recurrence compared to conventional follow-up. The sensitivity and specificity of the SSVs in terms of detecting postsurgery relapse were 100%. Conclusions We show that assessment of ctDNA is a non-invasive, exquisitely specific and highly sensitive approach for monitoring disease load, which has the potential to provide clinically relevant lead times compared with conventional methods. Furthermore, we provide a low-coverage protocol optimised for identifying SSVs with excellent correlation between SSVs identified in tumours and matched metastases. Application of ctDNA analysis has the potential to change clinical practice in the management of CRC.

TreeFam ( http://www.treefam.org ) was developed to provide curated phylogenetic trees for all animal gene families, as well as orthologue and paralogue assignments. Release 4.0 of TreeFam contains curated trees for 1314 families and automatically generated trees for another 14 351 families. We have expanded TreeFam to include 25 fully sequenced animal genomes, as well as four genomes from plant and fungal outgroup species. We have also introduced more accurate approaches for automatically grouping genes into families, for building phylogenetic trees, and for inferring orthologues and paralogues. The user interface for viewing phylogenetic trees and family information has been improved. Furthermore, a new perl API lets users easily extract data from the TreeFam mysql database.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70–80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.