Signatures of copy number alterations in human cancer.

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage-fusion-bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3-5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

Neoantigen-directed immune escape in lung cancer evolution.

The interplay between an evolving cancer and a dynamic immune microenvironment remains unclear. Here we analyse 258 regions from 88 early-stage, untreated non-small-cell lung cancers using RNA sequencing and histopathology-assessed tumour-infiltrating lymphocyte estimates. Immune infiltration varied both between and within tumours, with different mechanisms of neoantigen presentation dysfunction enriched in distinct immune microenvironments. Sparsely infiltrated tumours exhibited a waning of neoantigen editing during tumour evolution, indicative of historical immune editing, or copy-number loss of previously clonal neoantigens. Immune-infiltrated tumour regions exhibited ongoing immunoediting, with either loss of heterozygosity in human leukocyte antigens or depletion of expressed neoantigens. We identified promoter hypermethylation of genes that contain neoantigenic mutations as an epigenetic mechanism of immunoediting. Our results suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated non-small-cell lung cancers that produces multiple routes to immune evasion, which are clinically relevant and forecast poor disease-free survival.

Effect of Robot-Assisted Radical Cystectomy With Intracorporeal Urinary Diversion vs Open Radical Cystectomy on 90-Day Morbidity and Mortality Among Patients With Bladder Cancer: A Randomized Clinical Trial.

Importance: Robot-assisted radical cystectomy is being performed with increasing frequency, but it is unclear whether total intracorporeal surgery improves recovery compared with open radical cystectomy for bladder cancer. Objectives: To compare recovery and morbidity after robot-assisted radical cystectomy with intracorporeal reconstruction vs open radical cystectomy. Design, Setting, and Participants: Randomized clinical trial of patients with nonmetastatic bladder cancer recruited at 9 sites in the UK, from March 2017-March 2020. Follow-up was conducted at 90 days, 6 months, and 12 months, with final follow-up on September 23, 2021. Interventions: Participants were randomized to receive robot-assisted radical cystectomy with intracorporeal reconstruction (n = 169) or open radical cystectomy (n = 169). Main Outcomes and Measures: The primary outcome was the number of days alive and out of the hospital within 90 days of surgery. There were 20 secondary outcomes, including complications, quality of life, disability, stamina, activity levels, and survival. Analyses were adjusted for the type of diversion and center. Results: Among 338 randomized participants, 317 underwent radical cystectomy (mean age, 69 years; 67 women [21%]; 107 [34%] received neoadjuvant chemotherapy; 282 [89%] underwent ileal conduit reconstruction); the primary outcome was analyzed in 305 (96%). The median number of days alive and out of the hospital within 90 days of surgery was 82 (IQR, 76-84) for patients undergoing robotic surgery vs 80 (IQR, 72-83) for open surgery (adjusted difference, 2.2 days [95% CI, 0.50-3.85]; P = .01). Thromboembolic complications (1.9% vs 8.3%; difference, -6.5% [95% CI, -11.4% to -1.4%]) and wound complications (5.6% vs 16.0%; difference, -11.7% [95% CI, -18.6% to -4.6%]) were less common with robotic surgery than open surgery. Participants undergoing open surgery reported worse quality of life vs robotic surgery at 5 weeks (difference in mean European Quality of Life 5-Dimension, 5-Level instrument scores, -0.07 [95% CI, -0.11 to -0.03]; P = .003) and greater disability at 5 weeks (difference in World Health Organization Disability Assessment Schedule 2.0 scores, 0.48 [95% CI, 0.15-0.73]; P = .003) and at 12 weeks (difference in WHODAS 2.0 scores, 0.38 [95% CI, 0.09-0.68]; P = .01); the differences were not significant after 12 weeks. There were no statistically significant differences in cancer recurrence (29/161 [18%] vs 25/156 [16%] after robotic and open surgery, respectively) and overall mortality (23/161 [14.3%] vs 23/156 [14.7%]), respectively) at median follow-up of 18.4 months (IQR, 12.8-21.1). Conclusions and Relevance: Among patients with nonmetastatic bladder cancer undergoing radical cystectomy, treatment with robot-assisted radical cystectomy with intracorporeal urinary diversion vs open radical cystectomy resulted in a statistically significant increase in days alive and out of the hospital over 90 days. However, the clinical importance of these findings remains uncertain. Trial Registration: ISRCTN Identifier: ISRCTN13680280; ClinicalTrials.gov Identifier: NCT03049410.

Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner.

Epigenetic changes are frequently observed in cancer. However, their role in establishing or sustaining the malignant state has been difficult to determine due to the lack of experimental tools that enable resetting of epigenetic abnormalities. To address this, we applied induced pluripotent stem cell (iPSC) reprogramming techniques to invoke widespread epigenetic resetting of glioblastoma (GBM)-derived neural stem (GNS) cells. GBM iPSCs (GiPSCs) were subsequently redifferentiated to the neural lineage to assess the impact of cancer-specific epigenetic abnormalities on tumorigenicity. GiPSCs and their differentiating derivatives display widespread resetting of common GBM-associated changes, such as DNA hypermethylation of promoter regions of the cell motility regulator TES (testis-derived transcript), the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), and many polycomb-repressive complex 2 (PRC2) target genes (e.g., SFRP2). Surprisingly, despite such global epigenetic reconfiguration, GiPSC-derived neural progenitors remained highly malignant upon xenotransplantation. Only when GiPSCs were directed to nonneural cell types did we observe sustained expression of reactivated tumor suppressors and reduced infiltrative behavior. These data suggest that imposing an epigenome associated with an alternative developmental lineage can suppress malignant behavior. However, in the context of the neural lineage, widespread resetting of GBM-associated epigenetic abnormalities is not sufficient to override the cancer genome.

Exploring patients' experience and perception of being diagnosed with bladder cancer: a mixed-methods approach.

OBJECTIVE: To determine patient experience and perception following a diagnosis of non-muscle-invasive bladder cancer (NMIBC). PATIENT AND METHODS: Patients were part of a prospective multicentre observational study recruiting patients with NMIBC for a urine biomarker study (DETECT II; ClinicalTrials.gov: NCT02781428). A mixed-methods approach comprising: (i) the Brief Illness Perception Questionnaire (Brief-IPQ) and (ii) semi-structured interviews to explore patients' experience of having haematuria, and initial and subsequent experience with a NMIBC diagnosis. Both assessments were completed at 6 months after NMIBC diagnosis. RESULTS: A total of 213 patients completed the Brief-IPQ. Patients felt that they had minimal symptoms (median [interquartile range, IQR] score 2 [0-5]) and were not particularly affected emotionally (median [IQR] score 3 [1-6]) with a minimal effect to their daily life (median [IQR] score 2 [0-5]). However, they remained concerned about their cancer diagnosis (median [IQR] score 5 [3-8]) and felt that they had no personal control over the cancer (median [IQR] score 2 [2-5]) and believed that their illness would affect them for some time (median [IQR] score 6 [3-10]). A significant association with a lower personal control of the disease (P < 0.05) and a poorer understanding of the management of NMIBC (P < 0.05) was seen in patients aged >70 years. Many patients were uncertain about the cause of bladder cancer. Qualitative analysis found that at initial presentation of haematuria, most patients were not aware of the risk of bladder cancer. Patients were most anxious and psychologically affected between the interval of cystoscopy diagnosis and transurethral resection of bladder tumour (TURBT). Following TURBT, most patients were positive about their cancer prognosis. CONCLUSION: Patients with NMIBC have a poor perception of disease control and believe that their disease will continue over a prolonged period of time. This is particularly more pertinent in the elderly. Patients are most psychologically affected during the interval between cancer diagnosis following cystoscopy and TURBT. Health awareness about bladder cancer remained poor with a significant number of patients unaware of the causes of bladder cancer. Psychological support and prompt TURBT following bladder cancer diagnosis would help improve the mental health of patients with NMIBC.

CALIBER: a phase II randomized feasibility trial of chemoablation with mitomycin-C vs surgical management in low-risk non-muscle-invasive bladder cancer.

OBJECTIVES: To evaluate the activity of intravesical mitomycin-C (MMC) to ablate recurrent low-risk non-muscle-invasive bladder cancer (NMIBC) and assess whether it may enable patients to avoid surgical intervention for treatment of recurrence. PATIENTS AND METHODS: CALIBER is a phase II feasibility study. Participants were randomized (2:1) to treatment with four once-weekly MMC 40-mg intravesical instillations (chemoablation arm) or to surgical management. The surgical group was included to assess the feasibility of randomization. The primary endpoint was complete response to intravesical MMC in the chemoablation arm at 3 months, reported with exact 95% confidence intervals (CIs). Secondary endpoints included time to subsequent recurrence, summarized by Kaplan-Meier methods. RESULTS: Between February 2015 and August 2017, 82 patients with visual diagnosis of recurrent low-risk NMIBC were enrolled from 24 UK hospitals (chemoablation, n = 54; surgical management, n =28). The median follow-up was 24 months. Complete response at 3 months was 37.0% (20/54; 95% CI 24.3-51.3) with chemoablation and 80.8% (21/26; 95% CI 60.6-93.4) with surgical management. Amongst patients with complete response at 3 months, a similar proportion was recurrence-free by 12 months in both groups (84%). Amongst those with residual disease at 3 months, the 12-month recurrence-free proportion was lower in the surgical management group (40.0%) than in the chemoablation group (84%). Recruitment stopped early as chemoablation did not meet the prespecified threshold of 45% complete responses at 3 months. CONCLUSION: Intravesical chemoablation in low-risk NMIBC is feasible and safe, but did not demonstrate sufficient response in the present trial. After chemoablation there may be a reduction in recurrence rate, even in non-responders, that is greater than with surgery alone. Further research is required to investigate the role and optimal schedule of neoadjuvant intravesical chemotherapy prior to surgery for NMIBC.

PEOPLE: PatiEnt prOstate samPLes for rEsearch, a tissue collection pathway utilizing magnetic resonance imaging data to target tumor and benign tissue in fresh radical prostatectomy specimens.

BACKGROUND: Over 1 million men are diagnosed with prostate cancer each year worldwide, with a wide range of research programs requiring access to patient tissue samples for development of improved diagnoses and treatments. A random sampling of prostate tissue is sufficient for certain research studies; however, there is growing research need to target areas of the aggressive tumor as fresh tissue. Here we set out to develop a new pathway "PEOPLE: PatiEnt prOstate samPLes for rEsearch" to collect high-quality fresh tissue for research use, using magnetic resonance imaging (MRI) to target areas of tumor and benign tissue. METHODS: Prostate tissue was sampled following robotic radical prostatectomy, using MRI data to target areas of benign and tumor tissue. Initially, 25 cases were sampled using MRI information from clinical notes. A further 59 cases were sampled using an optimized method that included specific MRI measurements of tumor location along with additional exclusion criteria. All cases were reviewed in batches with detailed clinical and histopathological data recorded. For one subset of samples, DNA was extracted and underwent quality control. Ex vivo culture was carried out using the gelatin sponge method for an additional subset. RESULTS: Tumor was successfully fully or partially targeted in 64% of the initial cohort and 70% of the optimized cohort. DNA of high quality and concentration was isolated from 39 tumor samples, and ex vivo culture was successfully carried out in three cases with tissue morphology, proliferation, and apoptosis remaining comparable before and after 72 hours culture. CONCLUSION: Here we report initial data from the PEOPLE pathway; using a method for targeting areas of tumor within prostate samples using MRI. This method operates alongside the standard clinical pathway and minimizes additional input from surgical, radiological, and pathological teams, while preserving surgical margins and diagnostic tissue.

Mixed-methods approach to exploring patients' perspectives on the acceptability of a urinary biomarker test in replacing cystoscopy for bladder cancer surveillance.

OBJECTIVES: To determine the minimal accepted sensitivity (MAS) of a urine biomarker that patients are willing to accept to replace cystoscopy and to assess qualitatively their views and reasons. PATIENTS AND METHODS: Patients were part of a prospective multicentre observational study recruiting people with bladder cancer for a urine biomarker study (DETECT II; ClinicalTrials.gov: NCT02781428). A mixed-methods approach comprising (1) a questionnaire to assess patients' experience with cystoscopy and patients' preference for cystoscopy vs urinary biomarker, and (2) semi-structured interviews to understand patient views, choice and reasons for their preference. RESULTS: A urine biomarker with an MAS of 90% would be accepted by 75.8% of patients. This was despite a high self-reported prevalence of haematuria (51.0%), dysuria/lower urinary tract symptoms (69.1%) and urinary tract infection requiring antibiotics (25.8%). There was no association between MAS with patient demographics, adverse events experienced, cancer characteristics or distance of patients' home to hospital. The qualitative analysis suggested that patients acknowledge that cystoscopy is invasive, embarrassing and associated with adverse events but are willing to tolerate the procedure because of its high sensitivity. Patients have confidence in cystoscopy and appreciate the visual diagnosis of cancer. Both low- and high-risk patients would consider a biomarker with a reported sensitivity similar to that of cystoscopy. CONCLUSION: Patients value the high sensitivity of cystoscopy despite the reported discomfort and adverse events experienced after it. The sensitivity of a urinary biomarker must be close to cystoscopy to gain patients' acceptance.

Does urinary cytology have a role in haematuria investigations?

OBJECTIVES: To determine the diagnostic accuracy of urinary cytology to diagnose bladder cancer and upper tract urothelial cancer (UTUC) as well as the outcome of patients with a positive urine cytology and normal haematuria investigations in patients in a multicentre prospective observational study of patients investigated for haematuria. PATIENT AND METHODS: The DETECT I study (clinicaltrials.gov NCT02676180) recruited patients presenting with haematuria following referral to secondary case at 40 hospitals. All patients had a cystoscopy and upper tract imaging (renal bladder ultrasound [RBUS] and/ or CT urogram [CTU]). Patients, where urine cytology were performed, were sub-analysed. The reference standard for the diagnosis of bladder cancer and UTUC was histological confirmation of cancer. A positive urine cytology was defined as a urine cytology suspicious for neoplastic cells or atypical cells. RESULTS: Of the 3 556 patients recruited, urine cytology was performed in 567 (15.9%) patients from nine hospitals. Median time between positive urine cytology and endoscopic tumour resection was 27 (IQR: 21.3-33.8) days. Bladder cancer was diagnosed in 39 (6.9%) patients and UTUC in 8 (1.4%) patients. The accuracy of urinary cytology for the diagnosis of bladder cancer and UTUC was: sensitivity 43.5%, specificity 95.7%, positive predictive value (PPV) 47.6% and negative predictive value (NPV) 94.9%. A total of 21 bladder cancers and 5 UTUC were missed. Bladder cancers missed according to grade and stage were as follows: 4 (19%) were ≥ pT2, 2 (9.5%) were G3 pT1, 10 (47.6%) were G3/2 pTa and 5 (23.8%) were G1 pTa. High-risk cancer was confirmed in 8 (38%) patients. There was a marginal improvement in sensitivity (57.7%) for high-risk cancers. When urine cytology was combined with imaging, the diagnostic performance improved with CTU (sensitivity 90.2%, specificity 94.9%) superior to RBUS (sensitivity 66.7%, specificity 96.7%). False positive cytology results were confirmed in 22 patients, of which 12 (54.5%) had further invasive tests and 5 (22.7%) had a repeat cytology. No cancer was identified in these patients during follow-up. CONCLUSIONS: Urine cytology will miss a significant number of muscle-invasive bladder cancer and high-risk disease. Our results suggest that urine cytology should not be routinely performed as part of haematuria investigations. The role of urine cytology in select cases should be considered in the context of the impact of a false positive result leading to further potentially invasive tests conducted under general anaesthesia.

ChAMP: updated methylation analysis pipeline for Illumina BeadChips.

SUMMARY: The Illumina Infinium HumanMethylationEPIC BeadChip is the new platform for high-throughput DNA methylation analysis, effectively doubling the coverage compared to the older 450 K array. Here we present a significantly updated and improved version of the Bioconductor package ChAMP, which can be used to analyze EPIC and 450k data. Many enhanced functionalities have been added, including correction for cell-type heterogeneity, network analysis and a series of interactive graphical user interfaces. AVAILABILITY AND IMPLEMENTATION: ChAMP is a BioC package available from https://bioconductor.org/packages/release/bioc/html/ChAMP.html. CONTACT: a.teschendorff@ucl.ac.uk or s.beck@ucl.ac.uk or a.feber@ucl.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Can Renal and Bladder Ultrasound Replace Computerized Tomography Urogram in Patients Investigated for Microscopic Hematuria?

PURPOSE: Computerized tomography urogram is recommended when investigating patients with hematuria. We determined the incidence of urinary tract cancer and compared the diagnostic accuracy of computerized tomography urogram to that of renal and bladder ultrasound for identifying urinary tract cancer. MATERIALS AND METHODS: The DETECT (Detecting Bladder Cancer Using the UroMark Test) I study is a prospective observational study recruiting patients 18 years old or older following presentation with macroscopic or microscopic hematuria at a total of 40 hospitals. All patients underwent cystoscopy and upper tract imaging comprising computerized tomography urogram and/or renal and bladder ultrasound. RESULTS: A total of 3,556 patients with a median age of 68 years were recruited in this study, of whom 2,166 underwent renal and bladder ultrasound, and 1,692 underwent computerized tomography urogram in addition to cystoscopy. The incidence of bladder, renal and upper tract urothelial cancer was 11.0%, 1.4% and 0.8%, respectively, in macroscopic hematuria cases. Patients with microscopic hematuria had a 2.7%, 0.4% and 0% incidence of bladder, renal and upper tract urothelial cancer, respectively. The sensitivity and negative predictive value of renal and bladder ultrasound to detect renal cancer were 85.7% and 99.9% but they were 14.3% and 99.7%, respectively, to detect upper tract urothelial cancer. Renal and bladder ultrasound was poor at identifying renal calculi. Renal and bladder ultrasound sensitivity was lower than that of computerized tomography urogram to detect bladder cancer (each less than 85%). Cystoscopy had 98.3% specificity and 83.9% positive predictive value. CONCLUSIONS: Computerized tomography urogram can be safely replaced by renal and bladder ultrasound in patients who have microscopic hematuria. The incidence of upper tract urothelial cancer is 0.8% in patients with macroscopic hematuria and computerized tomography urogram is recommended. Patients with suspected renal calculi require noncontrast renal tract computerized tomography. Imaging cannot replace cystoscopy to diagnose bladder cancer.

Variable outcome and methylation status according to CEBPA mutant type in double-mutated acute myeloid leukemia patients and the possible implications for treatment.

Although CEBPA double-mutated (CEBPADM) acute myeloid leukemia is considered to be a favorable-risk disease, relapse remains a major cause of treatment failure. Most CEBPADM patients have a classic biallelic mutant combination with an N-terminal mutation leading to production of p30 protein plus a C-terminal loss-of-function in-frame indel mutation (CEBPAClassic-DM), but approximately one-third of cases have one or more non-classic mutations, with diverse combinations reported, and there is little information on the consequences of such mutants. We evaluated outcome in a cohort of 104 CEBPADM patients, 79 CEBPAClassic-DM and 25 with non-classic mutants, and found that the latter may have poorer survival (5-year overall survival 64% vs. 46%; P=0.05), particularly post relapse (41% vs. 0%; P=0.02). However, for this analysis, all non-classic cases were grouped together, irrespective of mutant combination. As CEBPADM cases have been reported to be hypermethylated, we used methylation profiling to assess whether this could segregate the different mutants. We developed a CEBPAClassic-DM methylation signature from a preliminary cohort of 10 CEBPADM (including 8 CEBPAClassic-DM) and 30 CEBPA wild-type (CEBPAWT) samples, and independently validated the signature in 17 CEBPAClassic-DM cases. Assessment of the signature in 16 CEBPADM cases with different non-classic mutant combinations showed that only 31% had a methylation profile equivalent to CEBPAClassic-DM whereas for 69% the profile was either intermediate between CEBPAClassic-DM and CEBPAWT or equivalent to CEBPAWT These results suggest that CEBPADM cases with non-classic mutants may be functionally different from those with CEBPAClassic-DM mutants, and should not automatically be included in the same prognostic group. (AML12 is registered under ISRCTN17833622 and AML15 under ISRCTN17161961).

Quantification of tumour evolution and heterogeneity via Bayesian epiallele detection.

BACKGROUND: Epigenetic heterogeneity within a tumour can play an important role in tumour evolution and the emergence of resistance to treatment. It is increasingly recognised that the study of DNA methylation (DNAm) patterns along the genome - so-called 'epialleles' - offers greater insight into epigenetic dynamics than conventional analyses which examine DNAm marks individually. RESULTS: We have developed a Bayesian model to infer which epialleles are present in multiple regions of the same tumour. We apply our method to reduced representation bisulfite sequencing (RRBS) data from multiple regions of one lung cancer tumour and a matched normal sample. The model borrows information from all tumour regions to leverage greater statistical power. The total number of epialleles, the epiallele DNAm patterns, and a noise hyperparameter are all automatically inferred from the data. Uncertainty as to which epiallele an observed sequencing read originated from is explicitly incorporated by marginalising over the appropriate posterior densities. The degree to which tumour samples are contaminated with normal tissue can be estimated and corrected for. By tracing the distribution of epialleles throughout the tumour we can infer the phylogenetic history of the tumour, identify epialleles that differ between normal and cancer tissue, and define a measure of global epigenetic disorder. CONCLUSIONS: Detection and comparison of epialleles within multiple tumour regions enables phylogenetic analyses, identification of differentially expressed epialleles, and provides a measure of epigenetic heterogeneity. R code is available at github.com/james-e-barrett.

DETECT I & DETECT II: a study protocol for a prospective multicentre observational study to validate the UroMark assay for the detection of bladder cancer from urinary cells.

BACKGROUND: Haematuria is a common finding in general practice which requires visual inspection of the bladder by cystoscopy as well as upper tract imaging. In addition, patients with non-muscle invasive bladder cancer (NMIBC) often require surveillance cystoscopy as often as three monthly depending on disease risk. However, cystoscopy is an invasive procedure which is uncomfortable, requires hospital attendance and is associated with a risk of urinary tract infection. We have developed the UroMark assay, which can detect 150 methylation specific alteration specific to bladder cancer using DNA from urinary sediment cells. METHODS: DETECT I and DETECT II are two multi-centre prospective observational studies designed to conduct a robust validation of the UroMark assay. DETECT I will recruit patients having diagnostic investigations for haematuria to determine the negative predictive value of the UroMark to rule out the presence of bladder cancer. DETECT II will recruit patients with new or recurrent bladder cancer to determine the sensitivity of the UroMark in detecting low, intermediate and high grade bladder cancer. NMIBC patients in DETECT II will be followed up with three monthly urine sample collection for 24 months while having surveillance cystoscopy. DETECT II will include a qualitative analysis of semi-structured interviews to explore patients' experience of being diagnosed with bladder cancer and having cystoscopy and a urinary test for bladder cancer surveillance. Results of the UroMark will be compared to cystoscopy findings and histopathological results in patients with bladder cancer. DISCUSSION: A sensitive and specific urinary biomarker will revolutionise the haematuria diagnostic pathway and surveillance strategies for NMIBC patients. None of the six approved US Food and Drug Administration urinary test are recommended as a standalone test. The UroMark assay is based on next generation sequencing technology which interrogates 150 loci and represents a step change compared to other biomarker panels. This enhances the sensitivity of the test and by using a random forest classifier approach, where the UroMark results are derived from a cut off generated from known outcomes of previous samples, addresses many shortcomings of previous assays. TRIAL REGISTRATION: Both trails are registered on clinicaltrials.gov. DETECT I: NCT02676180 (18th December 2015). DETECT II: NCT02781428 (11th May 2016).

Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

The aim of this study was to test the utility of AIMP3, an upstream regulator of DNA damage response following genotoxic stress, as a clinical biomarker in muscle-invasive bladder cancer (MIBC). AIMP3 was identified from a meta-analysis of a global gene-expression dataset. AIMP3 protein expression was determined by immunohistochemistry on a customised bladder cancer tissue-microarray (TMA). The mechanism of gene silencing was probed using methylation-specific PCR. The association between AIMP3 expression, Tp53 transactivity and genomic stability was analysed. In vitro AIMP3 translocation to the nucleus in response to ionising radiation was demonstrated using immunofluorescence. Radiosensitisation effects of siRNA-mediated AIMP3-knockdown were measured using colony forming assays. TMAs derived from patients enrolled in BCON, a Phase III multicentre radiotherapy trial in bladder cancer (ISRCTN45938399) were used to evaluate the association between AIMP3 expression and survival. The prognostic value of AIMP3 expression was determined in a TMA derived from patients treated by radical cystectomy. Loss of AIMP3 expression was frequent in MIBC and associated with impaired Tp53 transactivity and genomic instability. AIMP3-knockdown was associated with an increase in radioresistance. Loss of AIMP3 expression was associated with survival in MIBC patients following radiotherapy (HR = 0.53; 95% CI: 0.36 to 0.78, p = 0.002) but was not prognostic in the cystectomy set. In conclusion, AIMP3 expression is lost in a subset of bladder cancers and is significantly predictive of survival following radiotherapy in MIBC patients.

A functional methylome map of ulcerative colitis.

The etiology of inflammatory bowel diseases is only partially explained by the current genetic risk map. It is hypothesized that environmental factors modulate the epigenetic landscape and thus contribute to disease susceptibility, manifestation, and progression. To test this, we analyzed DNA methylation (DNAm), a fundamental mechanism of epigenetic long-term modulation of gene expression. We report a three-layer epigenome-wide association study (EWAS) using intestinal biopsies from 10 monozygotic twin pairs (n = 20 individuals) discordant for manifestation of ulcerative colitis (UC). Genome-wide expression scans were generated using Affymetrix UG 133 Plus 2.0 arrays (layer 1). Genome-wide DNAm scans were carried out using Illumina 27k Infinium Bead Arrays to identify methylation variable positions (MVPs, layer 2), and MeDIP-chip on Nimblegen custom 385k Tiling Arrays to identify differentially methylated regions (DMRs, layer 3). Identified MVPs and DMRs were validated in two independent patient populations by quantitative real-time PCR and bisulfite-pyrosequencing (n = 185). The EWAS identified 61 disease-associated loci harboring differential DNAm in cis of a differentially expressed transcript. All constitute novel candidate risk loci for UC not previously identified by GWAS. Among them are several that have been functionally implicated in inflammatory processes, e.g., complement factor CFI, the serine protease inhibitor SPINK4, and the adhesion molecule THY1 (also known as CD90). Our study design excludes nondisease inflammation as a cause of the identified changes in DNAm. This study represents the first replicated EWAS of UC integrated with transcriptional signatures in the affected tissue and demonstrates the power of EWAS to uncover unexplained disease risk and molecular events of disease manifestation.

ChAMP: 450k Chip Analysis Methylation Pipeline.

UNLABELLED: The Illumina Infinium HumanMethylation450 BeadChip is a new platform for high-throughput DNA methylation analysis. Several methods for normalization and processing of these data have been published recently. Here we present an integrated analysis pipeline offering a choice of the most popular normalization methods while also introducing new methods for calling differentially methylated regions and detecting copy number aberrations. AVAILABILITY AND IMPLEMENTATION: ChAMP is implemented as a Bioconductor package in R. The package and the vignette can be downloaded at bioconductor.org

Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors.

Aberrant DNA methylation (DNAm) was first linked to cancer over 25 yr ago. Since then, many studies have associated hypermethylation of tumor suppressor genes and hypomethylation of oncogenes to the tumorigenic process. However, most of these studies have been limited to the analysis of promoters and CpG islands (CGIs). Recently, new technologies for whole-genome DNAm (methylome) analysis have been developed, enabling unbiased analysis of cancer methylomes. By using MeDIP-seq, we report a sequencing-based comparative methylome analysis of malignant peripheral nerve sheath tumors (MPNSTs), benign neurofibromas, and normal Schwann cells. Analysis of these methylomes revealed a complex landscape of DNAm alterations. In contrast to what has been reported for other tumor types, no significant global hypomethylation was observed in MPNSTs using methylome analysis by MeDIP-seq. However, a highly significant (P < 10(-100)) directional difference in DNAm was found in satellite repeats, suggesting these repeats to be the main target for hypomethylation in MPNSTs. Comparative analysis of the MPNST and Schwann cell methylomes identified 101,466 cancer-associated differentially methylated regions (cDMRs). Analysis showed these cDMRs to be significantly enriched for two satellite repeat types (SATR1 and ARLα) and suggests an association between aberrant DNAm of these sequences and transition from healthy cells to malignant disease. Significant enrichment of hypermethylated cDMRs in CGI shores (P < 10(-60)), non-CGI-associated promoters (P < 10(-4)) and hypomethylated cDMRs in SINE repeats (P < 10(-100)) was also identified. Integration of DNAm and gene expression data showed that the expression pattern of genes associated with CGI shore cDMRs was able to discriminate between disease phenotypes. This study establishes MeDIP-seq as an effective method to analyze cancer methylomes.

Donor whole blood DNA methylation is not a strong predictor of acute graft versus host disease in unrelated donor allogeneic haematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (HCT) is used to treat many blood-based disorders and malignancies, however it can also result in serious adverse events, such as the development of acute graft-versus-host disease (aGVHD). This study aimed to develop a donor-specific epigenetic classifier to reduce incidence of aGVHD by improving donor selection. Genome-wide DNA methylation was assessed in a discovery cohort of 288 HCT donors selected based on recipient aGVHD outcome; this cohort consisted of 144 cases with aGVHD grades III-IV and 144 controls with no aGVHD. We applied a machine learning algorithm to identify CpG sites predictive of aGVHD. Receiver operating characteristic (ROC) curve analysis of these sites resulted in a classifier with an encouraging area under the ROC curve (AUC) of 0.91. To test this classifier, we used an independent validation cohort (n = 288) selected using the same criteria as the discovery cohort. Attempts to validate the classifier failed with the AUC falling to 0.51. These results indicate that donor DNA methylation may not be a suitable predictor of aGVHD in an HCT setting involving unrelated donors, despite the initial promising results in the discovery cohort. Our work highlights the importance of independent validation of machine learning classifiers, particularly when developing classifiers intended for clinical use.

Minimal Residual Disease Monitoring in Radically Treated Non-Small Cell Lung Cancer: Challenges and Future Directions.

Circulating tumor DNA (ctDNA) analysis can identify patients with residual disease before it is clinically or radiologically evident. Minimal residual disease (MRD) is an advancing area in the management of radically treated solid tumors. Which MRD assay is optimum and when it should be used is still not defined. Whilst promising, the clinical utility of this technology to guide patient care is still investigational in non-small cell lung cancer (NSCLC) and has not entered routine care. Once technically and clinically optimized, MRD may be utilized to personalize adjuvant therapy, detect disease relapse earlier and improve cure rates. In this review, we discuss the current status of MRD monitoring in NSCLC by summarizing frequently used MRD assays and their associated evidence in NSCLC. We discuss the potential applications of these technologies and the challenge of demonstrating MRD clinical utility in trials.