Search

Clinical data and follow-up data

Supplementary Fig. 1. Overview of sample availability and in-silico analysis of the limit of detection for the applied methodology.Supplementary Fig. 2. Comparison between mutations observed in primary and residual tumors at radical cystectomy (RC).Supplementary Fig. 3. Mean sample VAF levels for plasma, urine pellet and urine supernatant split by tdDNA call status in other paired sample types from the same clinical visit.Supplementary Fig. 4. Association between mean sample VAF.Supplementary Fig. 5. Association between mean VAFSupplementary Fig. 6. Urine dipstick measurements compared to the amount of DNA extracted from urine supernatants collected at the same time (+/- 1 day).Supplementary Fig. 7. Urine dipstick measurements compared to the urinary tumor DNA (utDNA) call status in urine supernatants collected at the same time (+/- 1 day).Supplementary Fig. 8. Urine dipstick measurements compared to the urinary tumor DNA (utDNA) level in urine supernatants collected at the same time (+/- 1 day).Supplementary Fig. 9. Mean sample VAF levels for all patients with detectable tumor DNA split by sample type. Tumor stage evaluation was based on the TURB specimen and tumor DNA level based on samples collected before TURB.Supplementary Fig. 10. Kaplan-Meier survival analysis of recurrence-free survival (RFS) stratified by tumor DNA status for all sample types.Supplementary Fig. 11. Tumor DNA levels compared to NAC response.Supplementary Fig. 12. Kaplan-Meier survival analysis of recurrence-free survival (RFS) stratified by tumor DNA level for all sample types.Supplementary Fig. 13. a-b) Association between tumor DNA dynamics and neoadjuvant chemotherapy (NAC) response for a) urine pellet and b) urine supernatant. Tumor DNA clearance was defined as tumor DNA going from detectable to non-detectable and tumor DNA remains was defined as tumor DNA remaining detectable. c-d) Kaplan-Meier survival analysis of tumor DNA dynamics groups and recurrence-free survival (RFS) for c) urine pellet and d) urine supernatant.

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.

Supplementary data

The cGAS-STING pathway serves a critical role in anticancer therapy. Particularly, response to immunotherapy is likely driven by both active cGAS-STING signaling that attracts immune cells, and by the presence of cancer neoantigens that presents as targets for cytotoxic T cells. Chromosomal instability (CIN) is a hallmark of cancer, but also leads to an accumulation of cytosolic DNA that in turn results in increased cGAS-STING signaling. To avoid triggering the cGAS-STING pathway, it is commonly disrupted by cancer cells, either through mutations in the pathway or through transcriptional silencing. Given its effect on the immune system, determining the cGAS-STING activation status prior to treatment initiation is likely of clinical relevance. Here, we used combined expression data from 2,307 tumors from five cancer types from The Cancer Genome Atlas to define a novel cGAS-STING activity score based on eight genes with a known role in the pathway. Using unsupervised clustering, four distinct categories of cGAS-STING activation were identified. In multivariate models, the cGAS-STING active tumors show improved prognosis. Importantly, in an independent bladder cancer immunotherapy-treated cohort, patients with low cGAS-STING expression showed limited response to treatment, while patients with high expression showed improved response and prognosis, particularly among patients with high CIN and more neoantigens. In a multivariate model, a significant interaction was observed between CIN, neoantigens, and cGAS-STING activation. Together, this suggests a potential role of cGAS-STING activity as a predictive biomarker for the application of immunotherapy.Significance:The cGAS-STING pathway is induced by CIN, triggers inflammation and is often deficient in cancer. We provide a tool to evaluate cGAS-STING activity and demonstrate clinical significance in immunotherapy response.

Detection of ctDNA in pre-operative plasma samples according to stage.

Supplementary Table 1 to 6

Purpose: We investigated whether detection of ctDNA after resection of colorectal cancer identifies the patients with the highest risk of relapse and, furthermore, whether longitudinal ctDNA analysis allows early detection of relapse and informs about response to intervention.Experimental Design: In this longitudinal cohort study, we used massively parallel sequencing to identify somatic mutations and used these as ctDNA markers to detect minimal residual disease and to monitor changes in tumor burden during a 3-year follow-up period.Results: A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA postoperatively in all relapsing patients (n = 14), but not in any of the nonrelapsing patients. ctDNA detected relapse with an average lead time of 9.4 months compared with CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months after surgery. All six later relapsed compared with four of the remaining patients [HR, 37.7; 95% confidence interval (CI), 4.2–335.5; P < 0.001]. The ability of a 3-month ctDNA analysis to predict relapse was confirmed in 23 liver metastasis patients (HR 4.9; 95% CI, 1.5–15.7; P = 0.007). Changes in ctDNA levels induced by relapse intervention (n = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman ρ = 0.4).Conclusions: Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance enables early detection of relapse and informs about response to intervention. These observations have implications for the postoperative management of colorectal cancer patients. Clin Cancer Res; 23(18); 5437–45. ©2017 AACR.

Supplementary Figure legends

Bladder field cancerization may be associated with disease outcome in patients with bladder cancer. To investigate this, we analyzed biopsies from bladder urothelium and urine samples by genomics and proteomics analyses. Samples were procured from multiple timepoints from 134 patients with early stage bladder cancer and detailed long term follow-up. We measured the field cancerization in normal-appearing bladder biopsies and found that high levels were associated with high tumor mutational burden, high neoantigen load, and high tumor-associated CD8 T-cell exhaustion. Non-synonymous mutations in known bladder cancer driver genes such asKDM6AandTP53were identified as early disease drivers in normal urothelium. High field cancerization was associated with worse outcome but not with response to BCG. The level of urinary tumor DNA (utDNA) reflected the bladder tumor burden and originated from both tumors and field cancerization. High utDNA levels after BCG were associated with worse clinical outcomes for the patients. Our results indicate that the level of field cancerization may affect clinical outcome, tumor development and immune responses. utDNA measurements have significant prognostic value and reflect the disease status of the bladder.

OBJECTIVES: Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders. Prenatal or preconception CF screening is offered in some countries. A maternal blood sample in early pregnancy can provide circulating trophoblasts and offers a DNA source for genetic analysis of both the mother and the fetus. This study aimed to develop a cell-based noninvasive prenatal test (NIPT) to screen for the 50 most common CF variants.METHODS: Blood samples were collected from 30 pregnancies undergoing invasive diagnostics and circulating trophoblasts were harvested in 27. Cystic fibrosis testing was conducted using two different methods: by fragment length analysis and by our newly developed NGS-based CF analysis.RESULTS: In all 27 cases, cell-based NIPT provided a result using both methods in agreement with the invasive test result.CONCLUSION: This study shows that cell-based NIPT for CF screening provides a reliable result without the need for partner- and proband samples.

SummaryCurrent transcriptomic classification systems for bladder cancer do not consider the level of intra-tumor subtype heterogeneity. Here we present an investigation of the extent and possible clinical impact of intra-tumor heterogeneity across early and more advanced disease stages of bladder cancer. We performed single nucleus RNA-sequencing of 48 bladder tumors and four of these tumors were additionally analyzed using spatial transcriptomics. Total bulk RNA-sequencing and spatial proteomics data were available from the same tumors for comparison, along with detailed clinical follow-up of the patients. We demonstrate that tumors display varying levels of intra-tumor subtype heterogeneity and show that a higher class 2a weight estimated from bulk RNA-sequencing data is associated with worse outcome in patients with molecular high-risk class 2a tumors. Our results indicate that discrete subtype assignments from bulk RNA-sequencing data may lack biological granularity and continuous class scores could improve clinical risk stratification of patients.HighlightsSingle nucleus RNA-sequencing of tumors from 48 bladder cancer patients.Tumors display varying levels of intra-tumor subtype heterogeneity at single nucleus and bulk tumor level.The level of subtype heterogeneity could be estimated from both single nucleus and bulk RNA-sequencing data with a high concordance between the two.High class 2a weight estimated from bulk RNA-sequencing data is associated with worse outcome in patients with molecular high-risk class 2a tumors.

PurposeInvestigate and compare the use of plasma- and urine DNA mutation analysis for predicting neoadjuvant chemotherapy (NAC) response and long-term oncological outcome in patients with muscle invasive bladder cancer.Experimental DesignWhole 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 approx. 50 mutations per patient was designed and utilized to track tumor-derived DNA (tdDNA) in liquid biopsies. 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 2013-2019 with a median follow-up time of 41.3 months after RC.ResultsWe identified tdDNA before initiation of NAC in 89% of urine supernatants, 85% of urine pellets and 43% of plasma samples. tdDNA levels were higher in urine supernatants and urine pellets compared to plasma samples (pp=0.006,p=0.002). A combined measure from plasma and urine supernatant tdDNA dynamics stratified patients by outcome (p=0.003).ConclusionsAnalysis of tdDNA in plasma and urine samples both separately and combined has potential to predict treatment response and outcome.

The cGAS-STING pathway serves a critical role in anticancer therapy. Particularly, response to immunotherapy is likely driven by both active cGAS-STING signaling that attracts immune cells, and by the presence of cancer neoantigens that presents as targets for cytotoxic T cells. Chromosomal instability (CIN) is a hallmark of cancer, but also leads to an accumulation of cytosolic DNA that in turn results in increased cGAS-STING signaling. To avoid triggering the cGAS-STING pathway, it is commonly disrupted by cancer cells, either through mutations in the pathway or through transcriptional silencing. Given its effect on the immune system, determining the cGAS-STING activation status prior to treatment initiation is likely of clinical relevance. Here, we used combined expression data from 2,307 tumors from five cancer types from The Cancer Genome Atlas to define a novel cGAS-STING activity score based on eight genes with a known role in the pathway. Using unsupervised clustering, four distinct categories of cGAS-STING activation were identified. In multivariate models, the cGAS-STING active tumors show improved prognosis. Importantly, in an independent bladder cancer immunotherapy-treated cohort, patients with low cGAS-STING expression showed limited response to treatment, while patients with high expression showed improved response and prognosis, particularly among patients with high CIN and more neoantigens. In a multivariate model, a significant interaction was observed between CIN, neoantigens, and cGAS-STING activation. Together, this suggests a potential role of cGAS-STING activity as a predictive biomarker for the application of immunotherapy. Significance: The cGAS-STING pathway is induced by CIN, triggers inflammation and is often deficient in cancer. We provide a tool to evaluate cGAS-STING activity and demonstrate clinical significance in immunotherapy response.

Overtreatment with cisplatin-based chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC), and currently none of the reported biomarkers for predicting response have been implemented in the clinic. Here we perform a comprehensive multi-omics analysis (genomics, transcriptomics, epigenomics and proteomics) of 300 MIBC patients treated with chemotherapy (neoadjuvant or first-line) to identify molecular changes associated with treatment response. DNA-based associations with response converge on genomic instability driven by a high number of chromosomal alterations, indels, signature 5 mutations and/or BRCA2 mutations. Expression data identifies the basal/squamous gene expression subtype to be associated with poor response. Immune cell infiltration and high PD-1 protein expression are associated with treatment response. Through integration of genomic and transcriptomic data, we demonstrate patient stratification to groups of low and high likelihood of cisplatin-based response. This could pave the way for future patient selection following validation in prospective clinical trials., There are currently only a few biomarkers to predict the response of muscle invasive bladder cancer to therapy. Here, the authors analyse 300 tumors using exome and RNA sequencing and find that tumors with a high degree of genomic instability and a non-basal/squamous gene expression subtype are most likely to respond to treatment.

The recurrence rate of colorectal liver metastases (CRLM) patients treated with curative intent is above 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging as well as carcinoembryonic antigen (CEA) measurements. Nonetheless, relapse detection often happens too late to resume curative treatment. This longitudinal cohort study enrolled 115 patients with plasma samples (N = 439) prospectively collected before surgery, postoperatively at day 30 and every third month for up to 3 years. Droplet digital PCR (ddPCR) was used to monitor serial plasma samples for somatic mutations. Assessment of ctDNA status either immediately after surgery, or serially during surveillance, stratified the patients into groups of high and low recurrence risk (hazard ratio [HR], 7.6; 95% CI, 3.0-19.7; P .0001; and HR, 4.3; 95% CI, 2.3-8.1; P .0001, respectively). The positive predictive value (PPV) of ctDNA was 100% in all postoperative analyses. In multivariable analyses, postoperative ctDNA status was the only consistently significant risk marker associated with relapse (P .0001). Indeterminate CT findings were observed for 30.8% (21/68) of patients. All patients (9/21) that were ctDNA positive at the time of the indeterminate CT scan later relapsed, contrasting 42.6% (5/12) of those ctDNA negative (P = .0046). Recurrence diagnoses in patients with indeterminate CT findings were delayed (median 2.8 months, P .0001). ctDNA status is strongly associated with detection of minimal residual disease and early detection of relapse. Furthermore, ctDNA status can potentially contribute to clinical decision-making in case of indeterminate CT findings, reducing time-to-intervention.

Introduction: Single cell technologies now make it possible to study tumor ecosystems at single cell resolution and may improve our biological understanding of disease aggressiveness and tumor heterogeneity. Whereas single-cell RNA-sequencing of clinical tumor biopsies requires immediate processing after tissue acquisition, single-nucleus RNA-sequencing (snRNA-seq) allows profiling of single nuclei isolated from frozen tumor tissue from patients with long-term follow-up and known clinical outcomes. Methods: We performed snRNA-seq of frozen tumors from 48 bladder cancer (BC) patients (10 Ta, 13 T1, 25 T2-4) using an optimized DroNc-seq protocol. Nuclei were isolated from frozen biopsies using IgePal lysis buffer and droplets were created using the Dolomite Bio platform followed by library generation and sequencing on an Illumina NovaSeq 6000. All epithelial nuclei were classified according to the UROMOL classes of non-muscle-invasive BC or the consensus classes of muscle-invasive BC. Bulk total RNA-sequencing (RNA-seq) was available for 44 of the tumors for comparison. Three tumors were additionally analyzed using 10x Chromium for validation and four tumors were analyzed using 10x Visium Spatial Transcriptomics. Results: After pre-processing the raw sequencing data, we obtained data from 117,653 nuclei in total and 59,201 nuclei remained after quality control filtering (1,233 nuclei per tumor and 529 expressed genes per nuclei on average). We focused our analysis on the epithelial compartment, as it constituted the bulk of the tumors (99% of all nuclei). UMAP visualization and clustering of all tumors were mainly driven by patient origin indicating a high level of inter-tumor heterogeneity. To explore intra-tumor heterogeneity and the association to disease aggressiveness, we characterized the tumors individually using hallmark BC gene signatures. Finally, we explored the composition of transcriptomic classes for each tumor and found that 52% of tumors displayed profound intra-tumor class heterogeneity with less than 70% of all nuclei belonging to a single class. The dominating transcriptomic class of single nuclei was only consistent in 44% of the tumors when compared to the overall transcriptomic class from bulk RNA-seq. This may be explained by several levels of heterogeneity and method differences, including the technical challenges of applying a bulk classifier to single nuclei data. We are currently investigating whether specific epithelial subpopulations are associated to outcome and whether signatures derived from snRNA-seq data can be recovered in bulk RNA-seq data and used as prognostic predictors. Conclusion: Our results highlight the biological complexity of bladder tumors and underline the importance of considering the extent of intra-tumor heterogeneity in the clinical management of BC patients. Citation Format: Sia V. Lindskrog, Sofie S. Schmøkel, Iver Nordentoft, Philippe Lamy, Michael Knudsen, Jørgen B. Jensen, Lars Dyrskjøt. Single-nucleus RNA-sequencing of human bladder tumors delineates intra-tumor cellular and subtype heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3483.

Background Standard treatment in patients with localized muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy (NAC) followed by radical cystectomy (CX). Importantly, there is currently no clinically validated way to estimate response to NAC before CX, where a pathological assessment of tumor down-staging is used as a proxy for response. Identification and tracking of circulating tumor DNA (ctDNA) has been shown to reflect prognosis and treatment response in several cancers, including bladder cancer. Here, we aim to investigate the capability of a combined plasma, urine supernatant and urine pellet mutation analysis by deep targeted sequencing for detecting residual tumor and treatment response before CX. Methods In total, 92 patients with localized MIBC treated with NAC followed by CX were included in the study. DNA extracted from primary tumors and leukocytes were subjected to whole exome sequencing for identification of approx. 50 mutations per patient that were used to design a custom NGS panel. Urine supernatants (n=281), urine pellets (n=114) and plasma samples (n=167), collected before CX, were subjected to deep targeted sequencing employing unique molecular identifiers (UMI). Mutations were assessed using the Shearwater algorithm and samples were further assessed using Fisher’s Method and bootstrapping of random mutations. In-silico analyses identified the limit of detection to be approx. 0.1%. For 56 patients, plasma mutation data (n=288 samples) were obtained from a previously published study by our group. Results Tumor derived DNA levels in paired urine supernatants and pellets were correlated (rho=0.8), however with higher levels in pellets when adjusting for the extracted amount of DNA (p Conclusions Tumor derived DNA was more frequently observed in urine samples and at higher levels compared to plasma samples. Interestingly, we show that tumor derived DNA dynamics in urine supernatants might better reflect the local tumor environment and plasma-based tumor derived DNA better reflects outcome following surgery, i.e. disease dissemination. Citation Format: Emil Christensen, Iver Nordentoft, Sara K. Elbæk, Karin Birkenkamp-Demtröder, Ann Taber, Michael Knudsen, Philippe Lamy, Mads Agerbæk, Jørgen B. Jensen, Lars Dyrskjøt. Treatment response and outcome prediction guided by deep targeted sequencing of urine and plasma samples in patients with muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1275.

Background: Prostate cancer (PC) is the fifth most lethal male malignancy worldwide, as advanced metastatic castration resistant PC (mCRPC) remains incurable. Genomic biomarkers that can predict treatment response are urgently needed to facilitate personalized mCRPC treatment. Such biomarkers can be identified by whole genome sequencing (WGS) of tumor biopsies. Blood plasma can serve as a non-invasive liquid tumor biopsy as it contains cell free DNA (cfDNA), a subset of which in cancer patients is tumor-derived (circulating tumor DNA; ctDNA). The biomarker discovery potential of WGS of cfDNA in mCRPC remains largely undescribed, as most WGS studies to date have focused on tissue biopsies. Thus, we here aimed to characterize the genomic tumor landscape of mCRPC using WGS of cfDNA. Methods: We previously performed low-pass WGS (mean coverage: 0.5X) of cfDNA sampled prior to initiation of first-line mCRPC treatment from 143 mCRPC patients (mean ctDNA fraction: 0.15). From these, 10 patients with high (>0.35) ctDNA fractions who received enzalutamide as first-line mCRPC treatment were selected for deeper WGS here. Matched germline DNA from buffy coat (peripheral blood mononuclear cells) was also sequenced. Single-nucleotide variants (SNVs) were called with Mutect2 and CaVEMan, indels with Mutect2 and Pindel, copy-number variants (CNVs) with ASCAT, and structural variants (SVs) with BRASS. For the final analysis, we considered only SNVs and indels called by both tools. Results: We sequenced germline samples to a mean coverage of 25X (range: 19-28X) and cfDNA samples to a mean coverage of 32X (range: 23-43X). We identified a median of 5,241 SNVs/indels (range: 3,422-48,314) per patient and the mean tumor mutation burden was 1.7 mutations/Mb. One sample had >9 times more SNVs/indels than the median, suggesting microsatellite instability. Among the most recurrently mutated genes were LRP1B (7/10 patients), ARSB (5/10 patients), and TP53 (4/10 patients). COSMIC mutational signature analysis revealed that the clock-like signatures 1, 5, and 40 were most frequent. In contrast, the hypermutated sample was driven primarily by the defective DNA mismatch repair signatures 15, 26, and 44. CNVs affected a mean of 40.9% of the genome. Common PC CNVs were observed, including gains at chromosome 8 (MYC) in 8/10 patients and losses at chromosome 10 (PTEN) in 5/10 patients. Recurrent focal amplifications (defined as >8 copies in regions Conclusion: This study highlights that WGS of cfDNA contributes to the identification of genomic aberrations that may serve as potential biomarkers to guide personalized treatment of mCRPC in the future. Citation Format: Simone Weiss, Philippe Lamy, Maibritt Nørgaard, Michael Knudsen, Jørgen B. Jensen, Jakob S. Pedersen, Michael Borre, Karina D. Sørensen. Whole genome sequencing of liquid tumor biopsies (ctDNA) from men with metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3409.

Previous studies have identified a significant potential in using economic model predictive control for space heating. This type of control requires a thermodynamic model of the controlled building that maps certain controllable inputs (heat power) and measured disturbances (ambient temperature and solar irradiation) to the controlled output variable (room temperature). Occupancy related disturbances, such as people heat gains and venting through windows, are often completely ignored or assumed to be fully known (measured) in these studies. However, this assumption is usually not fulfilled in practice and the current simulation study investigated the consequences thereof. The results indicate that the predictive performance (root mean square errors) of a black-box state-space model is not significantly affected by ignoring people heat gains. On the other hand, the predictive performance was significantly improved by including window opening status as a model input. The performance of black-box models for MPC of space heating could therefore benefit from having inputs from sensors that tracks window opening.

Of the patients undergoing radical cystectomy, 20-80% experience relapse. Minimally invasive methods for early detection of metastatic relapse after cystectomy and for monitoring ongoing therapy are urgently needed to improve individualised follow-up and treatment. Therefore, we evaluated the use of circulating tumour DNA (ctDNA) in plasma and urine to detect metastatic relapse after cystectomy and measure treatment efficacy. We exome sequenced tumour and germline DNA from patients with muscle-invasive bladder cancer and monitored ctDNA in 370 liquid biopsies throughout the disease courses by 84 personalised digital droplet polymerase chain reaction assays targeting 61 genes. Patients were prospectively recruited between 2013 and 2017. Patients with metastatic relapse had significantly higher ctDNA levels compared with disease-free patients (p

Previous studies have identified significant demand response (DR) potentials in using economic model predictive control (E-MPC) of space heating to exploit the inherent thermal mass in residential buildings for short-term energy storage. However, the economically viable realisation of E-MPC in residential buildings requires an effort to minimise the need for additional equipment and labour-intensive modelling processes. This paper reports on an experiment where a novel E-MPC setup was used for thermostatically control of a hydronic radiator in a highly-insulated residential building located on the NTNU Campus in Trondheim, Norway. The E-MPC utilized data from a heating meter, two temperature sensors and an existing weather forecast web service to train a linear black-box model. The results showed that the precision of model trained on excitation data that was generated using setpoints of either 21 or 24 °C was sufficient to obtain good control of the indoor air temperature while shifting consumption from high to low price periods. The findings of the experiment indicate that a minimal E-MPC setup is able to realize the significant DR potential that lies in utilizing the inherent thermal mass in residential buildings.

Purpose: We investigated whether detection of ctDNA after resection of colorectal cancer identifies the patients with the highest risk of relapse and, furthermore, whether longitudinal ctDNA analysis allows early detection of relapse and informs about response to intervention. Experimental Design: In this longitudinal cohort study, we used massively parallel sequencing to identify somatic mutations and used these as ctDNA markers to detect minimal residual disease and to monitor changes in tumor burden during a 3-year follow-up period. Results: A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA postoperatively in all relapsing patients (n = 14), but not in any of the nonrelapsing patients. ctDNA detected relapse with an average lead time of 9.4 months compared with CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months after surgery. All six later relapsed compared with four of the remaining patients [HR, 37.7; 95% confidence interval (CI), 4.2–335.5; P < 0.001]. The ability of a 3-month ctDNA analysis to predict relapse was confirmed in 23 liver metastasis patients (HR 4.9; 95% CI, 1.5–15.7; P = 0.007). Changes in ctDNA levels induced by relapse intervention (n = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman ρ = 0.4). Conclusions: Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance enables early detection of relapse and informs about response to intervention. These observations have implications for the postoperative management of colorectal cancer patients. Clin Cancer Res; 23(18); 5437–45. ©2017 AACR.

Model predictive control is a promising control scheme to utilize space heating in buildings for price-based demand response. It is, however, crucial to have an adequate thermal model of the building in order to make this work. It is often very time-consuming to construct these models and common system identification approaches suggest experimental input signals that are difficult to obtain in practice, which often leads to thermal discomfort. This paper proposes an alternative approach where an initial model is identified from historical data and then later re-identified based on control input generated from a model predictive control using the initial model.

Gonadoblastoma and malignant transformations thereof can occur in females with Turner syndrome (TS) and Y chromosomal material. However, in females with TS and no Y chromosomal material, this is rarely seen. We report a female with an apparent 45,X karyotype (in blood and tumor) who was diagnosed with a metastatic embryonal carcinoma. Exome sequencing of blood and the tumor was done, and no Y chromosomal material was detected, while predicted deleterious mutations in KIT (likely driver), AKT1, and ZNF358 were identified in the tumor. The patient was treated with chemotherapy (first-line: cisplatin, etoposide, and bleomycin; second-line: paclitaxel and gemcitabine), and after that surgical debulking was performed. She is currently well and without signs of relapse. We conclude that embryonal carcinoma can apparently occur in 45,X TS without signs of Y chromosomal material.

Background Microsatellite instability (MSI), resulting from a defective mismatch repair system, occurs in approximately 15% of sporadic colorectal cancers (CRC). Since MSI is associated with a poor response to 5-fluorouracile based chemotherapy and is a positive predictive marker of immunotherapy, it is routine practice to evaluate the MSI status of resected tumors in CRC patients. MSIsensor is a novel computational tool for determining MSI status using Next Generation Sequencing. However, it is not widely used in the clinic and has not been independently validated in exome data from CRC. To facilitate clinical implementation of computational determination of MSI status, we compared MSIsensor to current gold standard methods for MSI testing. Methods MSI status was determined for 130 CRC patients (UICC stage I-IV) using immunohistochemistry, PCR based microsatellite stability testing and by applying MSIsensor to exome sequenced tumors and paired germline DNA. Furthermore, we investigated correlation between MSI status, mutational load and mutational signatures. Results Eighteen out of 130 (13.8%) patients were microsatellite instable. We found a 100% agreement between MSIsensor and gold standard methods for MSI testing. All MSI tumors were hypermutated. In addition, two microsatellite stable (MSS) tumors were hypermutated, which was explained by a dominant POLE signature and pathogenic POLE mutations (p.Pro286Arg and p.Ser459Phe). Conclusion MSIsensor is a robust tool, which can be used to determine MSI status of tumor samples from exome sequenced CRC patients.

Chemotherapy resistance remains a challenge in the clinical management of metastatic colorectal cancer (mCRC). Here, early changes in cell-free circulating tumour DNA (ctDNA) levels were explored as a marker of therapeutic efficacy. Twenty-four mCRC patients were enrolled and treated with FOLFIRI based first-line therapy. Blood samples collected pre-treatment, at day 7, 14, 21, 60 and at progression were analysed for cell-free DNA (cfDNA) and ctDNA levels using digital droplet PCR. A subset of samples were additionally analysed by targeted sequencing. Patients with high pre-treatment ctDNA or cfDNA levels (≥75th centile) had significantly shorter progression free survival (PFS) than patients with lower levels. Despite an overall decline in ctDNA levels from pre-treatment to first CT-scan, serial analysis identified seven patients with temporary increases in ctDNA consistent with growth of resistant cells. These patients had shorter PFS and shorter overall survival. Targeted sequencing analyses of cfDNA revealed dramatic changes in the clonal composition in response to treatment. Our study suggests that increasing ctDNA levels during the first cycles of first-line FOLFIRI treatment is a predictor of incipient progressive disease and poorer survival. Thus, we demonstrate the importance of monitoring ctDNA levels as early as one week after treatment onset to enable early detection of treatment failure.

Importance: Novel sensitive methods for detection and monitoring of residual disease can improve postoperative risk stratification with implications for patient selection for adjuvant chemotherapy (ACT), ACT duration, intensity of radiologic surveillance, and, ultimately, outcome for patients with colorectal cancer (CRC). Objective: To investigate the association of circulating tumor DNA (ctDNA) with recurrence using longitudinal data from ultradeep sequencing of plasma cell-free DNA in patients with CRC before and after surgery, during and after ACT, and during surveillance. Design, Setting, and Participants: In this prospective, multicenter cohort study, ctDNA was quantified in the preoperative and postoperative settings of stages I to III CRC by personalized multiplex, polymerase chain reaction-based, next-generation sequencing. The study enrolled 130 patients at the surgical departments of Aarhus University Hospital, Randers Hospital, and Herning Hospital in Denmark from May 1, 2014, to January 31, 2017. Plasma samples (n = 829) were collected before surgery, postoperatively at day 30, and every third month for up to 3 years. Main Outcomes and Measures: Outcomes were ctDNA measurement, clinical recurrence, and recurrence-free survival. Results: A total of 130 patients with stages I to III CRC (mean [SD] age, 67.9 [10.1] years; 74 [56.9%] male) were enrolled in the study; 5 patients discontinued participation, leaving 125 patients for analysis. Preoperatively, ctDNA was detectable in 108 of 122 patients (88.5%). After definitive treatment, longitudinal ctDNA analysis identified 14 of 16 relapses (87.5%). At postoperative day 30, ctDNA-positive patients were 7 times more likely to relapse than ctDNA-negative patients (hazard ratio [HR], 7.2; 95% CI, 2.7-19.0; P

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.

Sensitivity analysis (SA) can be applied to building energy models (BEM) to identify which input parameters that drive the majority of the model output variation. The screening-based Morris method is often applied for this purpose; however, consideration regarding the effect of the user-defined number of levels (p) and trajectories (r) on the obtained results are rare. This paper investigates how the choice of p and r affects the outcome of a SA using the Morris method on a high fidelity BEM. The results indicates that the Morris method was not able to replicate the ranking from the variance-based Sobol’ method no matter the choice of r and p. It was, however, able to identify groups of input parameters (parameter clusters) most sensitive to the model output variability, but it required significantly more r than usually applied in studies featuring the Morris method. The reason is that marginal differences in absolute values of elementary effects (the sensitivity indices of the Morris method) for some input parameters may lead to a change in ranking position several times as the number of r increases. Users of the Morris method must therefore not be predetermined on the size of the parameter cluster; instead, they must make a visual assessment of the convergence of the parameter ranking to qualitatively determine the appropriate size of parameter cluster. The final recommendation for future studies deploying the Morris method for SA applied to a high fidelity BEM is to choose p ≥ 4 as it seems to lead the analysis towards a more truthful ranking, and then run simulations in steps of r = 100 when making the visual assessment to determine convergence and the size of parameter cluster. The identified need for more r questions the general notion that the Morris method is a computationally efficient screening method in terms of absolute time use. However, the Morris method is still much more computational efficient than a Sobol’-based analysis if the purpose of the SA is to identify a cluster of input parameters most sensitive to the model output variability.

A cancer of unknown primary (CUP) is a metastatic cancer for which standard diagnostic tests fail to locate the primary cancer. As standard treatments are based on the cancer type, such cases are hard to treat and have very poor prognosis. Using molecular data from the metastatic cancer to predict the primary site can make treatment choice easier and enable targeted therapy. In this article, we first examine the ability to predict cancer type using different types of omics data. Methylation data lead to slightly better prediction than gene expression and both these are superior to classification using somatic mutations. After using 3 data types independently, we notice some differences between the classes that tend to be misclassified, suggesting that integrating the data might improve accuracy. In light of the different levels of information provided by different omics types and to be able to handle missing data, we perform multi-omics classification by hierarchically combining the classifiers. The proposed hierarchical method first classifies based on the most informative type of omics data and then uses the other types of omics data to classify samples that did not get a high confidence classification in the first step. The resulting hierarchical classifier has higher accuracy than any of the single omics classifiers and thus proves that the combination of different data types is beneficial. Our results show that using multi-omics data can improve the classification of cancer types. We confirm this by testing our method on metastatic cancers from the MET500 dataset.

Occupancy controlled heating, ventilation and air condition in offices are a mean to avoid energy waste due to over-conditioning of rooms. Occupancy detection methods based on sensor data that is already available in a building for other reasons than occupancy detection are therefore of great interest. However, such methods need to be validated according to a ground truth. This paper presents an image-based method for automatic establishment of a reliable and anonymous ground truth at entrance areas where the room height only allow the camera to be installed at 2.3–3.0 m above the floor. The method deploys various image processing techniques to infrared depthframe images recorded with a ceiling-mounted Kinect camera to anonymously detect and track persons entering or leaving a room and, consequently, counting the number of occupants in the room. The method demonstrated an accuracy of more than 98% in a stress test, 100% in a range of relevant function test and 99% in a three week long room occupancy test.

This paper reports on a simulation-based study that investigated the demand response potential of a model predictive controller (MPC) for space heating defined to minimize a weighted sum of electricity costs and CO2 emissions. The performance of the MPC was compared to a traditional controller and the results showed that an MPC with no weight on CO2 emissions reduced the total electricity costs, shifted consumption from high to low load periods and reduced consumption in the hour with the yearly maximum grid load; but it could also cause an increase in CO2 emissions. Contrary, the MPC with no weight on electricity costs reduced CO2 emissions; but it only reduced total costs marginally, it could cause a shift of consumption from low to high load periods and it increased consumption in the hour with the yearly maximum grid load. Finally, if the MPC used a weighted sum of electricity costs and CO2 emissions a range of intermediate results were obtained. The weighting factor can thus be used either to balance the performance of the MPC with respect to all performance indicators or to maximise it with respect to one indicator of particular interest.

Dynamic solar shading devices are an effective mean to avoid overheating in buildings due to excessive solar heat gains. They can also be used to affect the heat balance of buildings; they can be on e.g. during nighttime to reduce space heating or retracted to increase night cooling. The shading control system can utilize this feature to obtain energy cost savings and reduce peak consumption. However, it is difficult to define optimal rule-based control strategies that minimize overheating and energy costs simultaneously. In this paper, we therefore propose an economic model predictive control (E-MPC) scheme for space heating where solar shading is included as an additional control variable. The proposed scheme employs black-box models, as they are ‘cheap’ to create from data; this is important for a widespread deployment of E-MPC in practice. The study was based on co-simulation experiments where EnergyPlus models represented the actual building and it was hereby demonstrated that the proposed E-MPC outperformed two rule-based controllers with respect to reducing overheating, energy costs and peak consumption. Shading also turned out to be valuable for north facing rooms where it reduced costs and peak consumptions compared to an MPC without shading. The energy-related benefits of the proposed E-MPC scheme can be regarded as ‘an added value’ that can be used for economic justification of an investment in dynamic solar shading devices.

Several studies have indicated that Model Predictive Control (MPC) of space heating systems can utilize the thermal mass of residential buildings as short-term thermal storage for various demand response purposes. Realization of this potential relies heavily on the accuracy of the model used to represent the thermodynamics of the building. Such models, whether they are grey box or black box, are calibrated using relevant data obtained from initial measurements, and the performance of the calibrated model is validated using data from a subsequent period. However, many studies use validation periods with weather conditions similar to those of the calibration period. Only a few studies investigate whether the calibrated model performs satisfactory when subjected to significantly different conditions. This paper presents data from a simulation-based study on the effect of seasonal weather changes on the performance of a black-box model. The study was conducted using 11 years of Danish weather data (2008-2018). The results indicate that the performance of the black-box model deteriorate as the weather data conditions become increasingly different from those used in the initial model calibration. Further, the results show that calibration in heating season leads to satisfactory model performance through the heating season, but lower performance in transitional seasons (especially spring). Results also show that calibration in February led to highest model performance through heating season, while calibration in March led to satisfactory model performance in the whole heating and fall season.

Model-based control schemes such as model predictive control (MPC) can assist smart-energy systems in achieving higher efficiency and utilization of renewable energy sources. A practical barrier for deploying such control schemes for space heating of residential buildings is the costs related to obtaining the weather data measurements needed for identifying a model that describes the dynamic behaviour of the building. Therefore, this paper reports on a simulation-based study investigating whether there is a significant impact on the performance of MPC schemes when substituting these weather measurements with data from meteorological weather services. Since access to weather forecasts is necessary during the operation of the MPC scheme, this implementation approach draws on data already available to remove the need for weather measurements. The results indicated that this approach only led to a minor performance impact in that heating savings were reduced by 4% while comfort violations increased by less than 0.1 Kh per day on average. The results thereby suggest that the use of data from meteorological forecast services for model identification may constitute a cost-efficient alternative to on-site or near-by weather measurements.

Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g. antibiotics. There is thus an interest in predicting the compound synthesized by an NRPS from its primary structure (amino acid sequence) alone, as this would enable an in silico search of whole genomes for NRPS enzymes capable of synthesizing potentially useful compounds. Results: NRPS synthesis happens in a conveyor belt-like fashion where each individual NRPS module is responsible for incorporating a specific substrate (typically an amino acid) into the final product. Here, we present a new method for predicting substrate specificities of individual NRPS modules based on occurrences of motifs in their primary structures. We compare our classifier with existing methods and discuss possible biological explanations of how the motifs might relate to substrate specificity. Availability and implementation: SEQL-NRPS is available as a web service implemented in Python with Flask at http://services.birc.au.dk/seql-nrps and source code available at https://bitbucket.org/dansondergaard/seql-nrps/. Contact: micknudsen@gmail.com or cstorm@birc.au.dk Supplementary information: Supplementary data are available at Bioinformatics online.

We present Bioconda (https://bioconda.github.io), a distribution of bioinformatics software for the lightweight, multiplatform and language-agnostic package manager Conda. Currently, Bioconda offers a collection of over 3000 software packages, which is continuously maintained, updated, and extended by a growing global community of more than 200 contributors. Bioconda improves analysis reproducibility by allowing users to define isolated environments with defined software versions, all of which are easily installed and managed without administrative privileges.

Patient-derived in vitro cultures of colorectal cancer (CRC) may help guide treatment strategies prior to patient treatment. However, most previous studies have been performed on a single biopsy per tumor. The purpose of this study was to analyze multiple spatially distinct biopsies from CRCs and see how well intratumor heterogeneity (ITH) was recapitulated in matching patient-derived spheroids. Three to five biopsies were collected from six CRC tumors. Each biopsy was split in two; one half was used for spheroid culturing, while the other half was used for DNA and RNA purification. For two patients, lymph node metastases were analyzed. Somatic mutations were called from whole exome sequencing data. Each tumor contained mutations shared across all biopsies and spheroids, including major CRC drivers such as APC, KRAS, and TP53. At the same time, all tumors exhibited ITH on both mutation and copy number level. The concordance between biopsies and spheroids ranged between 40 and 70% for coding mutations. For three patients, the biopsy and spheroid from matching areas clustered together, meaning that the spheroid resembled the area of origin more than the other areas. However, all biopsies and spheroids contained private mutations. Therefore, multiple cultures from spatially distinct sites of the tumor increase the insight into the genetic profile of the entire tumor. Molecular subtypes were called from RNA sequencing data. When based on transcripts from both cancer and noncancerous cells, the subtypes were largely independent of sampling site. In contrast, subtyping based on cancer cell transcripts alone was dependent on sample site and genetic ITH. In conclusion, all examined CRC tumors showed genetic ITH. Spheroid cultures partly reflected this ITH, and having multiple cultures from distinct tumor sites improved the representation of the genetic tumor subclones. This should be taken into account when establishing patient-derived models for drug screening. Patient-derived in vitro cultures of colorectal cancer (CRC) may help guide treatment strategies prior to patient treatment. However, most previous studies have been performed on a single biopsy per tumor. The purpose of this study was to analyze multiple spatially distinct biopsies from CRCs and see how well intratumor heterogeneity (ITH) was recapitulated in matching patient-derived spheroids. Three to five biopsies were collected from six CRC tumors. Each biopsy was split in two; one half was used for spheroid culturing, while the other half was used for DNA and RNA purification. For two patients, lymph node metastases were analyzed. Somatic mutations were called from whole exome sequencing data. Each tumor contained mutations shared across all biopsies and spheroids, including major CRC drivers such as APC, KRAS, and TP53. At the same time, all tumors exhibited ITH on both mutation and copy number level. The concordance between biopsies and spheroids ranged between 40 and 70% for coding mutations. For three patients, the biopsy and spheroid from matching areas clustered together, meaning that the spheroid resembled the area of origin more than the other areas. However, all biopsies and spheroids contained private mutations. Therefore, multiple cultures from spatially distinct sites of the tumor increase the insight into the genetic profile of the entire tumor. Molecular subtypes were called from RNA sequencing data. When based on transcripts from both cancer and noncancerous cells, the subtypes were largely independent of sampling site. In contrast, subtyping based on cancer cell transcripts alone was dependent on sample site and genetic ITH. In conclusion, all examined CRC tumors showed genetic ITH. Spheroid cultures partly reflected this ITH, and having multiple cultures from distinct tumor sites improved the representation of the genetic tumor subclones. This should be taken into account when establishing patient-derived models for drug screening.

Gonadoblastoma and malignant transformations thereof can occur in females with Turner syndrome (TS) and Y chromosomal material. However, in females with TS and no Y chromosomal material, this is rarely seen. We report a female with an apparent 45,X karyotype (in blood and tumor) who was diagnosed with a metastatic embryonal carcinoma. Exome sequencing of blood and the tumor was done, and no Y chromosomal material was detected, while predicted deleterious mutations in KIT (likely driver), AKT1, and ZNF358 were identified in the tumor. The patient was treated with chemotherapy (first-line: cisplatin, etoposide, and bleomycin; second-line: paclitaxel and gemcitabine), and after that surgical debulking was performed. She is currently well and without signs of relapse. We conclude that embryonal carcinoma can apparently occur in 45,X TS without signs of Y chromosomal material.