Plasma bile acids in association with Crohn's disease.

BACKGROUND: In addition to facilitating lipid digestions, bile acids (BA) are signalling molecules acting on receptors on immune cells and along the gastrointestinal (GI) tract. The aim of this study was to assess if altered bile acid profiles in plasma are associated with Crohn's disease (CD). METHOD: This cross-sectional study included individuals (aged ≥18 years) referred for colonoscopy at a tertiary centre in Stockholm between 2016 and 2019. All participants received bowel preparation, completed a lifestyle questionnaire and provided blood samples for analysis. During colonoscopy, severity of disease was graded, and biopsies were taken from colonic mucosa. In the current substudy, 88 individuals with CD and 88 age-matched controls were selected for analysis of BA in plasma with ultra performance liquid chromatography (UPLC). Linear regression models were then used to compare mean bile acid concentrations and concentration ratios between CD and controls. RESULTS: Individuals with CD had lower plasma concentrations of the majority of secondary BA compared to controls, in total CD/CC ratio 0.60 (SE 0.12), p = 0.001. The most prominent observations were lower levels of deoxycolic acid derivates and lithocolic acid derivates among participants with CD. Moreover, plasma concentration for secondary BA among participants with active CD was significantly lower compared to those with CD in remission, CD active/CD remission ratio 0.65 (SE 0.11), p < 0.002. CONCLUSION: Crohn's disease may be associated with altered plasma bile acid composition. The significance of colonic bacterial diversity in this context needs to be investigated in further studies.

It is known that Crohn's disease is associated with dysbiosis in the gut microbiota and that primary bile acids are transformed to secondary bile acids by bacterial enzymes in the gut before reabsorbed and transported back to the liver.In this cross-sectional study, Crohn's disease was associated with lower concentrations of secondary bile acids in blood plasmaThe findings should encourage further studies the role of the gut microbiome and bile acid metabolism in development of Crohn's disease and bile acid profile as a biomarker for bowel inflammation.

Transcriptome-wide prediction of prostate cancer gene expression from histopathology images using co-expression-based convolutional neural networks.

MOTIVATION: Molecular phenotyping by gene expression profiling is central in contemporary cancer research and in molecular diagnostics but remains resource intense to implement. Changes in gene expression occurring in tumours cause morphological changes in tissue, which can be observed on the microscopic level. The relationship between morphological patterns and some of the molecular phenotypes can be exploited to predict molecular phenotypes from routine haematoxylin and eosin-stained whole slide images (WSIs) using convolutional neural networks (CNNs). In this study, we propose a new, computationally efficient approach to model relationships between morphology and gene expression. RESULTS: We conducted the first transcriptome-wide analysis in prostate cancer, using CNNs to predict bulk RNA-sequencing estimates from WSIs for 370 patients from the TCGA PRAD study. Out of 15 586 protein coding transcripts, 6618 had predicted expression significantly associated with RNA-seq estimates (FDR-adjusted P-value <1×10-4) in a cross-validation and 5419 (81.9%) of these associations were subsequently validated in a held-out test set. We furthermore predicted the prognostic cell-cycle progression score directly from WSIs. These findings suggest that contemporary computer vision models offer an inexpensive and scalable solution for prediction of gene expression phenotypes directly from WSIs, providing opportunity for cost-effective large-scale research studies and molecular diagnostics. AVAILABILITY AND IMPLEMENTATION: A self-contained example is available from http://github.com/phiwei/prostate_coexpression. Model predictions and metrics are available from doi.org/10.5281/zenodo.4739097. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Prognostic Value of Low-Pass Whole Genome Sequencing of Circulating Tumor DNA in Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Multiple treatments are available for metastatic castration-resistant prostate cancer (mCRPC), including androgen receptor signaling inhibitors (ARSI) enzalutamide and abiraterone, but therapy resistance remains a major clinical obstacle. We examined the clinical utility of low-pass whole-genome sequencing (LPWGS) of circulating tumor DNA (ctDNA) for prognostication in mCRPC. METHODS: A total of 200 plasma samples from 143 mCRPC patients collected at the start of first-line ARSI treatment (baseline) and at treatment termination (n = 57, matched) were analyzed by LPWGS (median: 0.50X) to access ctDNA% and copy number alteration (CNA) patterns. The best confirmed prostate specific antigen (PSA) response (≥50% decline [PSA50]), PSA progression-free survival (PFS), and overall survival (OS) were used as endpoints. For external validation, we used plasma LPWGS data from an independent cohort of 70 mCRPC patients receiving first-line ARSI. RESULTS: Baseline ctDNA% ranged from ≤3.0% to 73% (median: 6.6%) and CNA burden from 0% to 82% (median: 13.1%) in the discovery cohort. High ctDNA% and high CNA burden at baseline was associated with poor PSA50 response (P = 0.0123/0.0081), poor PFS (P < 0.0001), and poor OS (P < 0.0001). ctDNA% and CNA burden was higher at PSA progression than at baseline in 32.7% and 42.3% of the patients. High ctDNA% and high CNA burden at baseline was also associated with poor PFS and OS (P ≤ 0.0272) in the validation cohort. CONCLUSIONS: LPWGS of ctDNA provides clinically relevant information about the tumor genome in mCRPC patients. Using LPWGS data, we show that high ctDNA% and CNA burden at baseline is associated with short PFS and OS in 2 independent cohorts.

Ductal and acinar components of mixed prostatic adenocarcinoma frequently have a common clonal origin.

BACKGROUND: Ductal adenocarcinoma (DA) is an aggressive subtype of prostate cancer. It is most commonly seen in mixed tumors together with conventional acinar adenocarcinoma (AA). The genetic profile of DA and its clonal origin is not fully characterized. OBJECTIVE: To investigate whether DA represents a distinct genetic subtype and to investigate the somatic relationship between the ductal and acinar components of mixed cancers. DESIGN, SETTING, AND PARTICIPANTS: In 17 radical prostatectomy specimens ductal and acinar tumor components from the same tumor foci were dissected. DNA was extracted and genomic sequencing performed. After exclusion of two cases with low cell yield, 15 paired samples remained for analysis. RESULTS: In 12 of 15 cases a common somatic denominator was identified, while three cases had clonally separate components. In DA, TMPRSS2-ERG gene fusions were detected in 47% (7/15), clonal FOXA1 alterations in 33% (5/15) and SPOP alterations in 27% (4/15) of cases. In one case KIAA1549-BRAF fusion was identified. Genome doubling events, resulting in an increased ploidy, were identified in the DA in 53% (8/15) of cases, but not seen in any AA. PTEN and CTNNB1 alterations were enriched in DA (6/15) but not seen in any AA. No cancers showed microsatellite instability or high tumor mutation burden. CONCLUSIONS: Ductal and acinar prostate adenocarcinoma components of mixed tumors most often share the same origin and are clonally related. DA components in mixed tumor often exhibit genome doubling events resulting in aneuploidy, consistent with the aggressive nature of high grade prostate cancer.

Population pharmacokinetics of apramycin from first-in-human plasma and urine data to support prediction of efficacious dose.

BACKGROUND: Apramycin is under development for human use as EBL-1003, a crystalline free base of apramycin, in face of increasing incidence of multidrug-resistant bacteria. Both toxicity and cross-resistance, commonly seen for other aminoglycosides, appear relatively low owing to its distinct chemical structure. OBJECTIVES: To perform a population pharmacokinetic (PPK) analysis and predict an efficacious dose based on data from a first-in-human Phase I trial. METHODS: The drug was administered intravenously over 30 min in five ascending-dose groups ranging from 0.3 to 30 mg/kg. Plasma and urine samples were collected from 30 healthy volunteers. PPK model development was performed stepwise and the final model was used for PTA analysis. RESULTS: A mammillary four-compartment PPK model, with linear elimination and a renal fractional excretion of 90%, described the data. Apramycin clearance was proportional to the absolute estimated glomerular filtration rate (eGFR). All fixed effect parameters were allometrically scaled to total body weight (TBW). Clearance and steady-state volume of distribution were estimated to 5.5 L/h and 16 L, respectively, for a typical individual with absolute eGFR of 124 mL/min and TBW of 70 kg. PTA analyses demonstrated that the anticipated efficacious dose (30 mg/kg daily, 30 min intravenous infusion) reaches a probability of 96.4% for a free AUC/MIC target of 40, given an MIC of 8 mg/L, in a virtual Phase II patient population with an absolute eGFR extrapolated to 80 mL/min. CONCLUSIONS: The results support further Phase II clinical trials with apramycin at an anticipated efficacious dose of 30 mg/kg once daily.

Circulating tumor DNA (ctDNA) in adjuvant therapy of early stage colon cancer: current status and future perspectives.

BACKGROUND: This article reviews the current knowledge on circulating tumor DNA (ctDNA) in early stage colon cancer and ongoing trials on ctDNA-guided treatment in the adjuvant setting. METHODS: A literature search of Pubmed was performed to identify studies on ctDNA in early stage colon cancer and neoadjuvant or adjuvant treatment. For ongoing trials, we searched clinicaltrials.gov and the Australian New Zealand Clinical Trials Registry (ANZCTR). RESULTS: Several studies show that ctDNA is a strong predictor for recurrence and survival after surgery and adjuvant chemotherapy. The specificity of this marker is extremely high, and the sensitivity is increasing with the development of technology. Recurrences can be detected very early and the analysis can potentially be used to guide neoadjuvant and adjuvant treatment. Ongoing and planned studies are now looking into escalation and de-escalation of therapy according to ctDNA-status after surgery. CONCLUSION: Serial measurement of ctDNA shows great promise as a marker for both prognosis and response to treatment in early colon cancer. Future studies will show whether we can use this analysis for tailoring treatment for patients in the adjuvant and neoadjuvant setting. With improved technology, ctDNA has the potential of becoming a 'game-changer' in the treatment of early stage colon cancers.

Artificial intelligence for diagnosis and grading of prostate cancer in biopsies: a population-based, diagnostic study.

BACKGROUND: An increasing volume of prostate biopsies and a worldwide shortage of urological pathologists puts a strain on pathology departments. Additionally, the high intra-observer and inter-observer variability in grading can result in overtreatment and undertreatment of prostate cancer. To alleviate these problems, we aimed to develop an artificial intelligence (AI) system with clinically acceptable accuracy for prostate cancer detection, localisation, and Gleason grading. METHODS: We digitised 6682 slides from needle core biopsies from 976 randomly selected participants aged 50-69 in the Swedish prospective and population-based STHLM3 diagnostic study done between May 28, 2012, and Dec 30, 2014 (ISRCTN84445406), and another 271 from 93 men from outside the study. The resulting images were used to train deep neural networks for assessment of prostate biopsies. The networks were evaluated by predicting the presence, extent, and Gleason grade of malignant tissue for an independent test dataset comprising 1631 biopsies from 246 men from STHLM3 and an external validation dataset of 330 biopsies from 73 men. We also evaluated grading performance on 87 biopsies individually graded by 23 experienced urological pathologists from the International Society of Urological Pathology. We assessed discriminatory performance by receiver operating characteristics and tumour extent predictions by correlating predicted cancer length against measurements by the reporting pathologist. We quantified the concordance between grades assigned by the AI system and the expert urological pathologists using Cohen's kappa. FINDINGS: The AI achieved an area under the receiver operating characteristics curve of 0·997 (95% CI 0·994-0·999) for distinguishing between benign (n=910) and malignant (n=721) biopsy cores on the independent test dataset and 0·986 (0·972-0·996) on the external validation dataset (benign n=108, malignant n=222). The correlation between cancer length predicted by the AI and assigned by the reporting pathologist was 0·96 (95% CI 0·95-0·97) for the independent test dataset and 0·87 (0·84-0·90) for the external validation dataset. For assigning Gleason grades, the AI achieved a mean pairwise kappa of 0·62, which was within the range of the corresponding values for the expert pathologists (0·60-0·73). INTERPRETATION: An AI system can be trained to detect and grade cancer in prostate needle biopsy samples at a ranking comparable to that of international experts in prostate pathology. Clinical application could reduce pathology workload by reducing the assessment of benign biopsies and by automating the task of measuring cancer length in positive biopsy cores. An AI system with expert-level grading performance might contribute a second opinion, aid in standardising grading, and provide pathology expertise in parts of the world where it does not exist. FUNDING: Swedish Research Council, Swedish Cancer Society, Swedish eScience Research Center, EIT Health.

Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals-The ATHENA Project.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

Somatic alterations detected in diagnostic prostate biopsies provide an inadequate representation of multifocal prostate cancer.

BACKGROUND: The majority of clinical prostate cancers are multifocal with morphological and molecular heterogeneity. Adequate tissue representation is crucial for the clinical utility of multigene panel sequencing of core needle biopsies. The aim of this study was to evaluate the genomic heterogeneity in multifocal prostate cancer and to analyze how representative preoperative biopsies are of spatially separated tumor foci. METHODS: We analyzed at least 2 tumor foci and 1 to 3 preoperative biopsy cores from 11 patients. Diagnostic biopsies, as well as fresh frozen and formalin-fixed paraffin-embedded samples, from major tumor foci of radical prostatectomy specimens were macrodissected for the enrichment of tumor tissue. DNA was extracted and sequenced. We analyzed structural alterations, mutations, and copy number variations and compared the genomic profiles of tumor foci with those of preoperative biopsies. RESULTS: Alterations were rarely shared between foci, indicating a high degree of genomic heterogeneity. In 8 of 11 men at least 1 tumor focus was represented by the biopsies defined as harboring at least 1 common clonal somatic event. In only one case, somatic alterations from two spatially separate tumors were identified in the biopsies. Of the mutations and structural variants detected in fresh frozen or formalin-fixed paraffin-embedded prostatectomy material, only an average of 19% (range 0-44) and 55% (range 0-100), respectively, were found in preoperative biopsies where a common somatic origin was established. CONCLUSIONS: Multifocal prostate cancer is a somatically heterogeneous disease in which systematic needle biopsies do not provide sufficient molecular representation of the somatic alterations detected in spatially distinct tumor areas. Targeted biopsies, directed at separate tumor foci, could potentially improve tissue representation of these heterogeneous foci in preoperative biopsies.

Impact of first-line cancer treatment on the follicle quality in cryopreserved ovarian samples from girls and young women.

STUDY QUESTION: Does first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients? SUMMARY ANSWER: Exposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment. WHAT IS KNOWN ALREADY: First-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 43 patients (age 1-24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Patients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy. LIMITATIONS, REASONS FOR CAUTION: The two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients. WIDER IMPLICATION OF THE FINDINGS: This study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Swedish Childhood Cancer Foundation, the Finnish Cancer Society, the Finnish Pediatric Research Foundation, the Väre Foundation for Pediatric Cancer Research, The Swedish Research Council, the Stockholm County Council (ALF project) and Karolinska Institutet. The authors have no conflict of interest to declare.

Toxicokinetics of Perfluorinated Alkyl Acids Influences Their Toxic Potency in the Zebrafish Embryo ( Danio rerio).

Perfluorinated alkyl acids (PFAA) are highly persistent and bioaccumulative and have been associated with several adverse health effects. The chemical structure mainly differs in two ways: the length of the hydrophobic alkyl chain and the type of hydrophilic end group. Little is known how the chemical structure affects the toxicokinetics (TK) in different organisms. We studied the TK of four PFAA (PFOS, PFHxS, PFOA, and PFBA) with different chain lengths (4-8 carbons) and functional groups (sulfonic and carboxylic acid) in zebrafish ( Danio rerio) embryo. The time courses of the external (ambient water) and internal concentrations were determined at three exposure concentrations from 2 up to 120 h postfertilization (hpf). Three of the four PFAA showed a biphasic uptake pattern with slow uptake before hatching (around 48 hpf) and faster uptake thereafter. A two-compartment TK model adequately described the biphasic uptake pattern, suggesting that the chorion functions as an uptake barrier until 48 hpf. The bioconcentration factors (BCF) determined at 120 hpf varied widely between PFAA with averages of approximately 4000 (PFOS), 200 (PFHxS), 50 (PFOA), and 0.8 (PFBA) L kg dry weight-1, suggesting that both the alkyl chain length and the functional group influence the TK. The differences in toxic potency were reduced by 3 orders of magnitude when comparing internal effect concentrations instead of effective external concentrations.

A novel technique for biobanking of large sections of radical prostatectomy specimens.

AIMS: Harvesting of unfixed tissue from radical prostatectomy specimens for research purposes is challenging. Many prostate cancers cannot be identified at gross inspection, and this tumour is notoriously multifocal and heterogeneous. We aimed to develop a technique to allow detailed topographic analysis and the sampling of a sufficient amount of tumour without jeopardising clinical reporting. METHODS AND RESULTS: A custom-made double-bladed knife was utilised for cutting a 4-mm-thick horizontal section of the prostate. The slices were split into segments that were frozen in gel, cryosections were cut, and RNA integrity numbers (RINs) were analysed. Sections were cut from all blocks of 20 cases, and the cutting time was monitored. Slides were scanned, and the slices were digitally reconstructed. Cutting frozen sections of an entire slice took 79-253 min (mean 162 min). Tumour was detected in frozen sections of 85% (17/20) of cases and in 46% (72/155) of blocks. The morphological quality was determined to be excellent, and RIN values were high (mean 8.9). CONCLUSIONS: This novel protocol for biobanking of fresh tissue from prostatectomy specimens provides sufficient tumour material for research purposes, while also enabling reporting of histopathology. The harvesting of a full tissue slice facilitates studies of tumour multifocality and heterogeneity.

Resveratrol supports and alpha-naphthoflavone disrupts growth of human ovarian follicles in an in vitro tissue culture model.

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology.

Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.

BACKGROUND: Cancers arise from multiple acquired mutations, which presumably occur over many years. Early stages in cancer development might be present years before cancers become clinically apparent. METHODS: We analyzed data from whole-exome sequencing of DNA in peripheral-blood cells from 12,380 persons, unselected for cancer or hematologic phenotypes. We identified somatic mutations on the basis of unusual allelic fractions. We used data from Swedish national patient registers to follow health outcomes for 2 to 7 years after DNA sampling. RESULTS: Clonal hematopoiesis with somatic mutations was observed in 10% of persons older than 65 years of age but in only 1% of those younger than 50 years of age. Detectable clonal expansions most frequently involved somatic mutations in three genes (DNMT3A, ASXL1, and TET2) that have previously been implicated in hematologic cancers. Clonal hematopoiesis was a strong risk factor for subsequent hematologic cancer (hazard ratio, 12.9; 95% confidence interval, 5.8 to 28.7). Approximately 42% of hematologic cancers in this cohort arose in persons who had clonality at the time of DNA sampling, more than 6 months before a first diagnosis of cancer. Analysis of bone marrow-biopsy specimens obtained from two patients at the time of diagnosis of acute myeloid leukemia revealed that their cancers arose from the earlier clones. CONCLUSIONS: Clonal hematopoiesis with somatic mutations is readily detected by means of DNA sequencing, is increasingly common as people age, and is associated with increased risks of hematologic cancer and death. A subset of the genes that are mutated in patients with myeloid cancers is frequently mutated in apparently healthy persons; these mutations may represent characteristic early events in the development of hematologic cancers. (Funded by the National Human Genome Research Institute and others.).

Intra-tumor heterogeneity in breast cancer has limited impact on transcriptomic-based molecular profiling.

BACKGROUND: Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. METHODS: In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. RESULTS: Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. CONCLUSIONS: Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.

Elevated levels of circulating CDH5 and FABP1 in association with human drug-induced liver injury.

BACKGROUND & AIMS: The occurrence of drug-induced liver injury (DILI) is a major issue in all phases of drug development. To identify novel biomarker candidates associated with DILI, we utilised an affinity proteomics strategy, where antibody suspension bead arrays were applied to profile plasma and serum samples from human DILI cases and controls. METHODS: An initial screening was performed using 4594 randomly selected antibodies, representing 3450 human proteins. Resulting candidate proteins together with proposed DILI biomarker candidates generated a DILI array of 251 proteins for subsequent target analysis and verifications. In total, 1196 samples from 241 individuals across four independent cohorts were profiled: healthy volunteers receiving acetaminophen, patients with human immunodeficiency virus and/or tuberculosis receiving treatment, DILI cases originating from a wide spectrum of drugs, and healthy volunteers receiving heparins. RESULTS: We observed elevated levels of cadherin 5, type 2 (CDH5) and fatty acid-binding protein 1 (FABP1) in DILI cases. In the two longitudinal cohorts, CDH5 was elevated already at baseline. FABP1 was elevated after treatment initiation and seemed to respond more rapidly than alanine aminotransferase (ALT). The elevations were verified in the DILI cases treated with various drugs. In the heparin cohort, CDH5 was stable over time whereas FABP1 was elevated. CONCLUSIONS: These results suggest that CDH5 may have value as a susceptibility marker for DILI. FABP1 was identified as a biomarker candidate with superior characteristics regarding tissue distribution and kinetics compared to ALT but likely with limited predictive value for the development of severe DILI. Further studies are needed to determine the clinical utility of the proposed markers.

Determining breast cancer histological grade from RNA-sequencing data.

BACKGROUND: The histologic grade (HG) of breast cancer is an established prognostic factor. The grade is usually reported on a scale ranging from 1 to 3, where grade 3 tumours are the most aggressive. However, grade 2 is associated with an intermediate risk of recurrence, and carries limited information for clinical decision-making. Patients classified as grade 2 are at risk of both under- and over-treatment. METHODS: RNA-sequencing analysis was conducted in a cohort of 275 women diagnosed with invasive breast cancer. Multivariate prediction models were developed to classify tumours into high and low transcriptomic grade (TG) based on gene- and isoform-level expression data from RNA-sequencing. HG2 tumours were reclassified according to the prediction model and a recurrence-free survival analysis was performed by the multivariate Cox proportional hazards regression model to assess to what extent the TG model could be used to stratify patients. The prediction model was validated in N=487 breast cancer cases from the The Cancer Genome Atlas (TCGA) data set. Differentially expressed genes and isoforms associated with HGs were analysed using linear models. RESULTS: The classification of grade 1 and grade 3 tumours based on RNA-sequencing data achieved high accuracy (area under the receiver operating characteristic curve = 0.97). The association between recurrence-free survival rate and HGs was confirmed in the study population (hazard ratio of grade 3 versus 1 was 2.62 with 95 % confidence interval = 1.04-6.61). The TG model enabled us to reclassify grade 2 tumours as high TG and low TG gene or isoform grade. The risk of recurrence in the high TG group of grade 2 tumours was higher than in low TG group (hazard ratio = 2.43, 95 % confidence interval = 1.13-5.20). We found 8200 genes and 13,809 isoforms that were differentially expressed between HG1 and HG3 breast cancer tumours. CONCLUSIONS: Gene- and isoform-level expression data from RNA-sequencing could be utilised to differentiate HG1 and HG3 tumours with high accuracy. We identified a large number of novel genes and isoforms associated with HG. Grade 2 tumours could be reclassified as high and low TG, which has the potential to reduce over- and under-treatment if implemented clinically.

Prostate cancer screening in men aged 50-69 years (STHLM3): a prospective population-based diagnostic study.

BACKGROUND: The prostate-specific antigen (PSA) test is used to screen for prostate cancer but has a high false-positive rate that translates into unnecessary prostate biopsies and overdiagnosis of low-risk prostate cancers. We aimed to develop and validate a model to identify high-risk prostate cancer (with a Gleason score of at least 7) with better test characteristics than that provided by PSA screening alone. METHODS: The Stockholm 3 (STHLM3) study is a prospective, population-based, paired, screen-positive, diagnostic study of men without prostate cancer aged 50-69 years randomly invited by date of birth from the Swedish Population Register kept by the Swedish Tax Agency. Men with prostate cancer at enrolment were excluded from the study. The predefined STHLM3 model (a combination of plasma protein biomarkers [PSA, free PSA, intact PSA, hK2, MSMB, MIC1], genetic polymorphisms [232 SNPs], and clinical variables [age, family, history, previous prostate biopsy, prostate exam]), and PSA concentration were both tested in all participants enrolled. The primary aim was to increase the specificity compared with PSA without decreasing the sensitivity to diagnose high-risk prostate cancer. The primary outcomes were number of detected high-risk cancers (sensitivity) and the number of performed prostate biopsies (specificity). The STHLM3 training cohort was used to train the STHLM3 model, which was prospectively tested in the STHLM3 validation cohort. Logistic regression was used to test for associations between biomarkers and clinical variables and prostate cancer with a Gleason score of at least 7. This study is registered with ISCRTN.com, number ISRCTN84445406. FINDINGS: The STHLM3 model performed significantly better than PSA alone for detection of cancers with a Gleason score of at least 7 (p<0·0001), the area under the curve was 0·56 (95% CI 0·55-0·60) with PSA alone and 0·74 (95% CI 0·72-0·75) with the STHLM3 model. All variables used in the STHLM3 model were significantly associated with prostate cancers with a Gleason score of at least 7 (p<0·05) in a multiple logistic regression model. At the same level of sensitivity as the PSA test using a cutoff of ≥3 ng/mL to diagnose high risk prostate cancer, use of the STHLM3 model could reduce the number of biopsies by 32% (95% CI 24-39) and could avoid 44% (35-54) of benign biopsies. INTERPRETATION: The STHLM3 model could reduce unnecessary biopsies without compromising the ability to diagnose prostate cancer with a Gleason score of at least 7, and could be a step towards personalised risk-based prostate cancer diagnostic programmes. FUNDING: Stockholm County Council (Stockholms Läns Landsting).

Exome sequencing of contralateral breast cancer identifies metastatic disease.

Women with contralateral breast cancer (CBC) have significantly worse prognosis compared to women with unilateral cancer. A possible explanation of the poor prognosis of patients with CBC is that in a subset of patients, the second cancer is not a new primary tumor but a metastasis of the first cancer that has potentially obtained aggressive characteristics through selection of treatment. Exome and whole-genome sequencing of solid tumors has previously been used to investigate the clonal relationship between primary tumors and metastases in several diseases. In order to assess the relationship between the first and the second cancer, we performed exome sequencing to identify somatic mutations in both first and second cancers, and compared paired normal tissue of 25 patients with metachronous CBC. For three patients, we identified shared somatic mutations indicating a common clonal origin thereby demonstrating that the second tumor is a metastasis of the first cancer, rather than a new primary cancer. Accordingly, these patients all developed distant metastasis within 3 years of the second diagnosis, compared with 7 out of 22 patients with non-shared somatic profiles. Genomic profiling of both tumors help the clinicians distinguish between true CBCs and subsequent metastases.