Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Exploring the tumor genomic landscape of aggressive prostate cancer by whole-genome sequencing of tissue or liquid biopsies.

Treatment resistance remains a major issue in aggressive prostate cancer (PC), and novel genomic biomarkers may guide better treatment selection. Circulating tumor DNA (ctDNA) can provide minimally invasive information about tumor genomes, but the genomic landscape of aggressive PC based on whole-genome sequencing (WGS) of ctDNA remains incompletely characterized. Thus, we here performed WGS of tumor tissue (n = 31) or plasma ctDNA (n = 10) from a total of 41 aggressive PC patients, including 11 hormone-naïve, 15 hormone-sensitive, and 15 castration-resistant patients. Across all variant types, we found progressively more altered tumor genomic profiles in later stages of aggressive PC. The potential driver genes most frequently affected by single-nucleotide variants or insertions/deletions included the known PC-related genes TP53, CDK12, and PTEN and the novel genes COL13A1, KCNH3, and SENP3. Etiologically, aggressive PC was associated with age-related and DNA repair-related mutational signatures. Copy number variants most frequently affected 14q11.2 and 8p21.2, where no well-recognized PC-related genes are located, and also frequently affected regions near the known PC-related genes MYC, AR, TP53, PTEN, and BRCA1. Structural variants most frequently involved not only the known PC-related genes TMPRSS2 and ERG but also the less extensively studied gene in this context, PTPRD. Finally, clinically actionable variants were detected throughout all stages of aggressive PC and in both plasma and tissue samples, emphasizing the potential clinical applicability of WGS of minimally invasive plasma samples. Overall, our study highlights the feasibility of using liquid biopsies for comprehensive genomic characterization as an alternative to tissue biopsies in advanced/aggressive PC.

Two independent and primitive envelopes of the bilobate nucleus of comet 67P.

The factors shaping cometary nuclei are still largely unknown, but could be the result of concurrent effects of evolutionary and primordial processes. The peculiar bilobed shape of comet 67P/Churyumov-Gerasimenko may be the result of the fusion of two objects that were once separate or the result of a localized excavation by outgassing at the interface between the two lobes. Here we report that the comet's major lobe is enveloped by a nearly continuous set of strata, up to 650 metres thick, which are independent of an analogous stratified envelope on the minor lobe. Gravity vectors computed for the two lobes separately are closer to perpendicular to the strata than those calculated for the entire nucleus and adjacent to the neck separating the two lobes. Therefore comet 67P/Churyumov-Gerasimenko is an accreted body of two distinct objects with 'onion-like' stratification, which formed before they merged. We conclude that gentle, low-velocity collisions occurred between two fully formed kilometre-sized cometesimals in the early stages of the Solar System. The notable structural similarities between the two lobes of comet 67P/Churyumov-Gerasimenko indicate that the early-forming cometesimals experienced similar primordial stratified accretion, even though they formed independently.

Large heterogeneities in comet 67P as revealed by active pits from sinkhole collapse.

Pits have been observed on many cometary nuclei mapped by spacecraft. It has been argued that cometary pits are a signature of endogenic activity, rather than impact craters such as those on planetary and asteroid surfaces. Impact experiments and models cannot reproduce the shapes of most of the observed cometary pits, and the predicted collision rates imply that few of the pits are related to impacts. Alternative mechanisms like explosive activity have been suggested, but the driving process remains unknown. Here we report that pits on comet 67P/Churyumov-Gerasimenko are active, and probably created by a sinkhole process, possibly accompanied by outbursts. We argue that after formation, pits expand slowly in diameter, owing to sublimation-driven retreat of the walls. Therefore, pits characterize how eroded the surface is: a fresh cometary surface will have a ragged structure with many pits, while an evolved surface will look smoother. The size and spatial distribution of pits imply that large heterogeneities exist in the physical, structural or compositional properties of the first few hundred metres below the current nucleus surface.

Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery.

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.

A collision in 2009 as the origin of the debris trail of asteroid P/2010 A2.

The peculiar object P/2010 A2 was discovered in January 2010 and given a cometary designation because of the presence of a trail of material, although there was no central condensation or coma. The appearance of this object, in an asteroidal orbit (small eccentricity and inclination) in the inner main asteroid belt attracted attention as a potential new member of the recently recognized class of main-belt comets. If confirmed, this new object would expand the range in heliocentric distance over which main-belt comets are found. Here we report observations of P/2010 A2 by the Rosetta spacecraft. We conclude that the trail arose from a single event, rather than a period of cometary activity, in agreement with independent results. The trail is made up of relatively large particles of millimetre to centimetre size that remain close to the parent asteroid. The shape of the trail can be explained by an initial impact ejecting large clumps of debris that disintegrated and dispersed almost immediately. We determine that this was an asteroid collision that occurred around 10 February 2009.

Field Cancerization Is Associated with Tumor Development, T-cell Exhaustion, and Clinical Outcomes in Bladder Cancer.

BACKGROUND: Field cancerization is characterized by areas of normal tissue affected by mutated clones. Bladder field cancerization may explain the development and recurrence of bladder cancer and may be associated with treatment outcomes. OBJECTIVE: To investigate the predictive and prognostic roles of field cancerization in patients with high-risk non-muscle-invasive bladder cancer (NMIBC) treated with bacillus Calmette-Guérin (BCG). DESIGN, SETTING, AND PARTICIPANTS: We conducted comprehensive genomic and proteomic analyses for 751 bladder biopsies and 234 urine samples from 136 patients with NMIBC. The samples were collected at multiple time points during the disease course. Field cancerization in normal-appearing bladder biopsies was measured using deep-targeted sequencing and error correction models. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Endpoints included the rates of recurrence and progression. Cox regression and Wilcoxon rank-sum and Fisher's exact tests were used. RESULTS AND LIMITATIONS: A high level of field cancerization was associated with high tumor mutational burden (p = 0.007), high tumor neoantigen load (p = 0.029), and high tumor-associated CD8 T-cell exhaustion (p = 0.017). In addition, high field cancerization was associated with worse short-term outcomes (p = 0.029). Nonsynonymous mutations in bladder cancer-associated genes such as KDM6A, ARID1A, and TP53 were identified as early disease drivers already found in normal-appearing bladder biopsies. Urinary tumor DNA (utDNA) levels reflected the bladder tumor burden and originated from tumors and field cancerization. High levels of utDNA after BCG were associated with worse clinical outcomes (p = 0.027) and with disease progression (p = 0.003). High field cancerization resulted in high urinary levels of proteins associated with angiogenesis and proliferation. Limitations include variation in the number of biopsies and time points analyzed. CONCLUSIONS: Field cancerization levels are associated with tumor development, immune responses, and clinical outcomes. utDNA measurements can be used to monitor disease status and treatment response. PATIENT SUMMARY: Molecular changes in the tissue lining the bladder result in tumor recurrence. Urinary measurements may be used to monitor bladder cancer status and treatment responses.

Whole-genome Mutational Analysis for Tumor-informed Detection of Circulating Tumor DNA in Patients with Urothelial Carcinoma.

BACKGROUND AND OBJECTIVE: Circulating tumor DNA (ctDNA) can be used for sensitive detection of minimal residual disease (MRD). However, the probability of detecting ctDNA in settings of low tumor burden is limited by the number of mutations analyzed and the plasma volume available. We used a whole-genome sequencing (WGS) approach for ctDNA detection in patients with urothelial carcinoma. METHODS: We used a tumor-informed WGS approach for ctDNA-based detection of MRD and evaluation of treatment responses. We analyzed 916 longitudinally collected plasma samples from 112 patients with localized muscle-invasive bladder cancer who received neoadjuvant chemotherapy (NAC) before radical cystectomy. Recurrence-free survival (primary endpoint), overall survival, and ctDNA dynamics during NAC were assessed. KEY FINDINGS AND LIMITATIONS: We found that WGS-based ctDNA detection is prognostic for patient outcomes with a median lead time of 131 d over radiographic imaging. WGS-based ctDNA assessment after radical cystectomy identified recurrence with sensitivity of 91% and specificity of 92%. In addition, genomic characterization of post-treatment plasma samples with a high ctDNA level revealed acquisition of platinum therapy-associated mutational signatures and copy number variations not present in the primary tumors. The sequencing depth is a limitation for studying tumor evolution. CONCLUSIONS AND CLINICAL IMPLICATIONS: Our results support the use of WGS for ultrasensitive ctDNA detection and highlight the possibility of plasma-based tracking of tumor evolution. WGS-based ctDNA detection represents a promising option for clinical use owing to the low volume of plasma needed and the ease of performing WGS, eliminating the need for personalized assay design. PATIENT SUMMARY: Detection of tumor DNA in blood samples from patients with cancer of the urinary tract is associated with poorer outcomes. Disease recurrence after surgery can be identified by the presence of tumor DNA in blood before it can be detected on radiography scans.

Non-CpG island promoter hypomethylation and miR-149 regulate the expression of SRPX2 in colorectal cancer.

Gene silencing by DNA hypermethylation of CpG islands is a well-characterized phenomenon in cancer. The effect of hypomethylation in particular of non-CpG island genes is much less well described. By genome-wide screening, we identified 105 genes in microsatellite stable (MSS) colorectal adenocarcinomas with an inverse correlation (Spearman's ρ ≤ -0.40) between methylation and expression. Of these, 35 (33%) were hypomethylated non-CpG island genes and two of them, APOLD1 (Spearman's ρ = -0.82) and SRPX2 (Spearman's ρ = -0.80) were selected for further analyses. Hypomethylation of both genes were localized events not shared by adjacent genes. A set of 662 FFPE DNA samples not only confirmed that APOLD1 and SRPX2 are hypomethylated in CRC but also revealed hypomethylation to be significantly (p < 0.01) associated with tumors being localized in the left side, CpG island methylator phenotype negative, MSS, BRAF wt, undifferentiated and of adenocarcinoma histosubtype. Demethylation experiments supported SRPX2 being epigenetically regulated via DNA methylation, whereas other mechanisms in addition to DNA methylation seem to be involved in the regulation of APOLD1. We further identified miR-149 as a potential novel post-transcriptional regulator of SRPX2. In carcinoma tissue, miR-149 was downregulated and inversely correlated to SRPX2 (ρ = -0.77). Furthermore, ectopic expression of miR-149 significantly reduced SRPX2 transcript levels. Our study highlights that in colorectal tumors, hypomethylation of non-CpG island-associated promoters deregulate gene expression nearly as frequent as do CpG-island hypermethylation. The hypomethylation of SRPX2 is focal and not part of a large block. Furthermore, it often translates to an increased expression level, which may be modulated by miR-149.

MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.

Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies.

This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection: sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed that a combination of IgePal lysis buffer, tissue dissection by sectioning, and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well-described clinical and histopathological information and known clinical outcomes with snRNA-seq.

Single-nucleus and Spatially Resolved Intratumor Subtype Heterogeneity in Bladder Cancer.

Background: Current bulk transcriptomic classification systems for bladder cancer do not consider the level of intratumor subtype heterogeneity. Objective: To investigate the extent and possible clinical impact of intratumor subtype heterogeneity across early and more advanced stages of bladder cancer. Design setting and participants: We performed single-nucleus RNA sequencing (RNA-seq) of 48 bladder tumors and additional spatial transcriptomics for four of these tumors. Total bulk RNA-seq and spatial proteomics data were available from the same tumors for comparison, along with detailed clinical follow-up of the patients. Outcome measurements and statistical analysis: The primary outcome was progression-free survival for non-muscle-invasive bladder cancer. Cox regression analysis, log-rank tests, Wilcoxon rank-sum tests, Spearman correlation, and Pearson correlation were used for statistical analysis. Results and limitations: We found that the tumors exhibited varying levels of intratumor subtype heterogeneity and that the level of subtype heterogeneity can be estimated from both single-nucleus and bulk RNA-seq data, with high concordance between the two. We found that a higher class 2a weight estimated from bulk RNA-seq data is associated with worse outcome for patients with molecular high-risk class 2a tumors. The sparsity of the data generated using the DroNc-seq sequencing protocol is a limitation. Conclusions: Our results indicate that discrete subtype assignments from bulk RNA-seq data may lack biological granularity and that continuous class scores may improve clinical risk stratification of patients with bladder cancer. Patient summary: We found that several molecular subtypes can exist within a single bladder tumor and that continuous subtype scores can be used to identify a subgroup of patients with poor outcomes. Use of these subtype scores may improve risk stratification for patients with bladder cancer, which can help in making decisions on treatment.

Cell-Free Urine and Plasma DNA Mutational Analysis Predicts Neoadjuvant Chemotherapy Response and Outcome in Patients with Muscle-Invasive Bladder Cancer.

PURPOSE: To investigate the use of plasma and urine DNA mutation analysis for predicting neoadjuvant chemotherapy (NAC) response and oncological outcome in patients with muscle-invasive bladder cancer. EXPERIMENTAL DESIGN: Whole-exome sequencing of tumor and germline DNA was performed for 92 patients treated with NAC followed by radical cystectomy (RC). A custom NGS-panel capturing approximately 50 mutations per patient was designed and used to track mutated tumor DNA in plasma and urine. A total of 447 plasma samples, 281 urine supernatants, and 123 urine pellets collected before, during, and after treatment were analyzed. Patients were enrolled from 2013 to 2019, with a median follow-up time of 41.3 months after RC. RESULTS: We identified tumor DNA before NAC in 89% of urine supernatants, 85% of urine pellets, and 43% of plasma samples. Tumor DNA levels were higher in urine supernatants and urine pellets compared with plasma samples (P < 0.001). In plasma, detection of circulating tumor DNA (ctDNA) before NAC was associated with a lower NAC response rate (P < 0.001). Detection of tumor DNA after NAC was associated with lower response rates in plasma, urine supernatant, and urine pellet (P < 0.001, P = 0.03, P = 0.002). Tumor DNA dynamics during NAC was predictive of NAC response and outcome in urine supernatant and plasma (P = 0.006 and P = 0.002). A combined measure from plasma and urine supernatant tumor DNA dynamics stratified patients by outcome (P = 0.003). CONCLUSIONS: Analysis of tumor DNA in plasma and urine samples both separately and combined has a potential to predict treatment response and outcome.

Circulating Tumor DNA Analysis in Advanced Urothelial Carcinoma: Insights from Biological Analysis and Extended Clinical Follow-up.

PURPOSE: To investigate whether circulating tumor DNA (ctDNA) assessment in patients with muscle-invasive bladder cancer predicts treatment response and provides early detection of metastatic disease. EXPERIMENTAL DESIGN: We present full follow-up results (median follow-up: 68 months) from a previously described cohort of 68 neoadjuvant chemotherapy (NAC)-treated patients who underwent longitudinal ctDNA testing (712 plasma samples). In addition, we performed ctDNA evaluation of 153 plasma samples collected before and after radical cystectomy (RC) in a separate cohort of 102 NAC-naïve patients (median follow-up: 72 months). Total RNA sequencing of tumors was performed to investigate biological characteristics of ctDNA shedding tumors. RESULTS: Assessment of ctDNA after RC identified metastatic relapse with a sensitivity of 94% and specificity of 98% using the expanded follow-up data for the NAC-treated patients. ctDNA dynamics during NAC was independently associated with patient outcomes when adjusted for pathologic downstaging (HR = 4.7; P = 0.029). For the NAC-naïve patients, ctDNA was a prognostic predictor before (HR = 3.4; P = 0.0005) and after RC (HR = 17.8; P = 0.0002). No statistically significant difference in recurrence-free survival for patients without detectable ctDNA at diagnosis was observed between the cohorts. Baseline ctDNA positivity was associated with the Basal/Squamous (Ba/Sq) subtype and enrichment of epithelial-to-mesenchymal transition and cell cycle-associated gene sets. CONCLUSIONS: ctDNA is prognostic in NAC-treated and NAC-naïve patients with more than 5 years follow-up and outperforms pathologic downstaging in predicting treatment efficacy. Patients without detectable ctDNA at diagnosis may benefit significantly less from NAC, but additional studies are needed.

Genome-wide Association Study of Bladder Cancer Reveals New Biological and Translational Insights.

BACKGROUND: Genomic regions identified by genome-wide association studies (GWAS) for bladder cancer risk provide new insights into etiology. OBJECTIVE: To identify new susceptibility variants for bladder cancer in a meta-analysis of new and existing genome-wide genotype data. DESIGN, SETTING, AND PARTICIPANTS: Data from 32 studies that includes 13,790 bladder cancer cases and 343,502 controls of European ancestry were used for meta-analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSES: Log-additive associations of genetic variants were assessed using logistic regression models. A fixed-effects model was used for meta-analysis of the results. Stratified analyses were conducted to evaluate effect modification by sex and smoking status. A polygenic risk score (PRS) was generated on the basis of known and novel susceptibility variants and tested for interaction with smoking. RESULTS AND LIMITATIONS: Multiple novel bladder cancer susceptibility loci (6p.22.3, 7q36.3, 8q21.13, 9p21.3, 10q22.1, 19q13.33) as well as improved signals in three known regions (4p16.3, 5p15.33, 11p15.5) were identified, bringing the number of independent markers at genome-wide significance (p < 5 × 10-8) to 24. The 4p16.3 (FGFR3/TACC3) locus was associated with a stronger risk for women than for men (p-interaction = 0.002). Bladder cancer risk was increased by interactions between smoking status and genetic variants at 8p22 (NAT2; multiplicative p value for interaction [pM-I] = 0.004), 8q21.13 (PAG1; pM-I = 0.01), and 9p21.3 (LOC107987026/MTAP/CDKN2A; pM-I = 0.02). The PRS based on the 24 independent GWAS markers (odds ratio per standard deviation increase 1.49, 95% confidence interval 1.44-1.53), which also showed comparable results in two prospective cohorts (UK Biobank, PLCO trial), revealed an approximately fourfold difference in the lifetime risk of bladder cancer according to the PRS (e.g., 1st vs 10th decile) for both smokers and nonsmokers. CONCLUSIONS: We report novel loci associated with risk of bladder cancer that provide clues to its biological underpinnings. Using 24 independent markers, we constructed a PRS to stratify lifetime risk. The PRS combined with smoking history, and other established risk factors, has the potential to inform future screening efforts for bladder cancer. PATIENT SUMMARY: We identified new genetic markers that provide biological insights into the genetic causes of bladder cancer. These genetic risk factors combined with lifestyle risk factors, such as smoking, may inform future preventive and screening strategies for bladder cancer.

Rseg--an R package to optimize segmentation of SNP array data.

UNLABELLED: The use of high-density SNP arrays for investigating copy number alterations in clinical tumor samples, with intra tumor heterogeneity and varying degrees of normal cell contamination, imposes several problems for commonly used segmentation algorithms. This calls for flexibility when setting thresholds for calling gains and losses. In addition, sample normalization can induce artifacts in the copy-number ratios for the non-changed genomic elements in the tumor samples. RESULTS: We present an open source R package, Rseg, which allows the user to define sample-specific thresholds to call gains and losses. It also allows the user to correct for normalization artifacts. AVAILABILITY: The R package, Rseg, is available at: http://www.cs.au.dk/~plamy/Rseg/ and runs on Linux and MS-Windows.

A review of software for microarray genotyping.

The focus of this review is software for the genotyping of microarray single nucleotide polymorphisms, in particular software for Affymetrix and Illumina arrays. Different statistical principles and ideas have been applied to the construction of genotyping algorithms - for example, likelihood versus Bayesian modelling, and whether to genotype one or all arrays at a time. The release of new arrays is generally followed by new, or updated, algorithms.

Immune Contexture and Differentiation Features Predict Outcome in Bladder Cancer.

BACKGROUND: An improved risk assessment of patients with bladder cancer (BC) is important to optimize clinical management. OBJECTIVE: To identify whether immune cell subpopulations and cancer cell-intrinsic features are associated with outcome and response to first-line chemotherapy in BC. DESIGN, SETTING, AND PARTICIPANTS: Primary tumor tissue from 785 patients with BC (stage Ta-T4b) were stained using multiplex immunofluorescence (CD3, CD8, FOXP3, CD20, CD68, CD163, and MHC-I) and immunohistochemistry (pancytokeratin, CK5/6, GATA3, programmed death 1 [PD-1], and programmed death ligand 1 [PD-L1]). A digital image analysis quantified staining results within the carcinoma cell and stromal part of the tumor. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary endpoints were progression-free survival, recurrence-free survival, and response to first-line chemotherapy. Optimal cutoff values for investigated markers were estimated using maximally selected rank statistics and receiver operating characteristic for each primary endpoint. Time-to-event analyses were performed using Cox regression analyses. RESULTS AND LIMITATIONS: Several immune subpopulations were independently associated with clinical outcomes. Especially, high PD-1 and PD-L1 expression was independently associated with an increased risk of recurrence and progression in non-muscle-invasive tumors, but with a lower risk of recurrence in muscle-invasive tumors. Furthermore, we observed a lower likelihood of response to first-line chemotherapy in patients with basal differentiation features. Finally, a model combining clinical risk factors with our most evident prognosticator improved prediction accuracy compared with clinical risk factors alone for progression in non-muscle-invasive BC and recurrence in muscle-invasive BC. The use of tissue microarrays and a long inclusion period are limitations to this study. CONCLUSIONS: Immune cell subpopulations and cancer cell-intrinsic features are associated with different clinical outcomes in BC. PATIENT SUMMARY: Immune cells play an important role in cancer development and treatment outcomes. Infiltration with specific immune cells and the presence of markers associated with immune evasion in the tumor predict clinical outcomes in bladder cancer.

Elevated T-cell Exhaustion and Urinary Tumor DNA Levels Are Associated with Bacillus Calmette-Guérin Failure in Patients with Non-muscle-invasive Bladder Cancer.

BACKGROUND: The functional status of immune cells in the tumor microenvironment and tumor characteristics may explain bacillus Calmette-Guérin (BCG) failure in high-risk non-muscle-invasive bladder cancer (NMIBC). OBJECTIVE: To characterize molecular correlates of post-BCG high-grade (HG) recurrence using multiomics analysis. DESIGN, SETTING, AND PARTICIPANTS: Patients with BCG-treated NMIBC (n = 156) were included in the study. Metachronous tumors were analyzed using RNA sequencing (n = 170) and whole-exome sequencing (n = 195). Urine samples were analyzed for immuno-oncology-related proteins (n = 190) and tumor-derived DNA (tdDNA; n = 187). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was post-BCG HG recurrence. Cox regression and Wilcoxon rank-sum, t, and Fisher's exact tests were used for analyses. RESULTS AND LIMITATIONS: BCG induced activation of the immune system regardless of clinical response; however, immunoinhibitory proteins were observed in the urine of patients with post-BCG HG recurrence (CD70, PD1, CD5). Post-BCG HG recurrence was associated with post-BCG T-cell exhaustion (p = 0.002). Pre-BCG tumors from patients with post-BCG T-cell exhaustion had high expression of genes related to cell division and immune function. A high predicted post-BCG exhaustion score for pre-BCG tumors was associated with worse post-BCG HG recurrence-free survival (HGRFS; p = 0.002). This was validated in independent cohorts. Pre-BCG class 2a and 2b tumors (UROMOL2021 scheme) were associated with worse post-BCG HGRFS (p = 0.015). Post-BCG exhaustion was observed in patients with high pre-BCG neoantigen load (p = 0.017) and MUC4 mutations (p = 0.002). Finally, the absence of post-BCG tdDNA clearance identified patients at high risk of recurrence (p = 0.018). The retrospective design and partial overlap for analyses are study limitations. CONCLUSIONS: Post-BCG HG recurrence may be caused by T-cell exhaustion. Tumor subtype and pre-BCG tumor characteristics may identify patients at high risk of post-BCG HG recurrence. Urinary measurements have potential for real-time assessment of treatment response. PATIENT SUMMARY: A dysfunctional immune response to bacillus Calmette-Guérin (BCG) therapy may explain high-grade recurrences of bladder cancer.

Genome-wide Meta-analysis Identifies Novel Genes Associated with Recurrence and Progression in Non-muscle-invasive Bladder Cancer.

BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) is characterized by frequent recurrences and a risk of progression in stage and grade. Increased knowledge of underlying biological mechanisms is needed. OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with recurrence-free (RFS) and progression-free (PFS) survival in NMIBC. DESIGN, SETTING, AND PARTICIPANTS: We analyzed outcome data from 3400 newly diagnosed NMIBC patients from the Netherlands, the UK, Canada, and Spain. We generated genome-wide germline SNP data using Illumina OmniExpress and Infinium Global Screening Array in combination with genotype imputation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cohort-specific genome-wide association studies (GWASs) for RFS and PFS were performed using a Cox proportional hazard model. Results were combined in a fixed-effect inverse-variance weighted meta-analysis. Candidate genes for the identified SNP associations were prioritized using functional annotation, gene-based analysis, expression quantitative trait locus analysis, and transcription factor binding site databases. Tumor expression levels of prioritized genes were tested for association with RFS and PFS in an independent NMIBC cohort. RESULTS AND LIMITATIONS: This meta-analysis revealed a genome-wide significant locus for RFS on chromosome 14 (lead SNP rs12885353, hazard ratio [HR] C vs T allele 1.55, 95% confidence interval [CI] 1.33-1.82, p = 4.0 × 10-8), containing genes G2E3 and SCFD1. Higher expression of SCFD1 was associated with increased RFS (HR 0.70, 95% CI 0.59-0.84, pFDR = 0.003). Twelve other loci were suggestively associated with RFS (p < 10-5), pointing toward 18 additional candidate genes. For PFS, ten loci showed suggestive evidence of association, indicating 36 candidate genes. Expression levels of ten of these genes were statistically significantly associated with PFS, of which four (IFT140, UBE2I, FAHD1, and NME3) showed directional consistency with our meta-analysis results and published literature. CONCLUSIONS: In this first prognostic GWAS in NMIBC, we identified several novel candidate loci and five genes that showed convincing associations with recurrence or progression. PATIENT SUMMARY: In this study, we searched for inherited DNA changes that affect the outcome of non-muscle-invasive bladder cancer (NMIBC). We identified several genes that are associated with disease recurrence and progression. The roles and mechanisms of these genes in NMIBC prognosis should be investigated in future studies.