The eTHINK Study: Cognitive and Behavioral Outcomes in Children with Hemophilia.

OBJECTIVE: To assess cognitive, behavioral, and adaptive functions in children and young adults with hemophilia treated according to contemporary standards of care. STUDY DESIGN: Evolving Treatment of Hemophilia's Impact on Neurodevelopment, Intelligence, and Other Cognitive Functions (eTHINK) is a US-based, prospective, cross-sectional, observational study (September 2018 through October 2019). Males (aged 1-21 years) with hemophilia A or B of any severity, with or without inhibitors, were eligible. Participants underwent neurologic examinations and age-appropriate neuropsychological assessments, including standardized tests/ratings scales of early development, cognition, emotional/behavioral adjustment, and adaptive skills. RESULTS: Five hundred and fifty-one males with hemophilia A (n = 433) or B (n = 101) were enrolled. Performance on cognitive tests was largely comparable with that of age-matched US population norms, although participants in certain age groups (4-5 and 10-21 years) performed worse on measures of attention and processing speed. Furthermore, adolescents and young adults and those with comorbid attention-deficit/hyperactivity disorder (ADHD; n = 64) reported more adaptive and executive function problems in daily life. Incidence of ADHD in adolescents (21%) was higher than expected in the general population. CONCLUSIONS: In general, males with hemophilia demonstrated age-appropriate intellectual, behavioral, and adaptive development. However, specific patient/age groups showed poorer attention performance and concerns for executive and adaptive development. This study established a normative data set for monitoring neurodevelopment in individuals with hemophilia and highlight the importance of screening and intervention for challenges with cognitive and adaptive skills in this population. CLINICAL TRIAL REGISTRATION: Evolving Treatment of Hemophilia's Impact on Neurodevelopment, Intelligence, and Other Cognitive Functions (eTHINK); NCT03660774; https://clinicaltrials.gov/ct2/show/NCT03660774.

Renoprotective effects of genetically proxied fibroblast growth factor 21: Mendelian randomization, proteome-wide and metabolome-wide association study.

BACKGROUND: Fibroblast growth factor 21 (FGF21) has demonstrated efficacy for reducing liver fat and reversing non-alcoholic steatohepatitis in phase 2 clinical trials. It is also postulated to have anti-fibrotic effects and therefore may be amenable to repurposing for the prevention and treatment of chronic kidney disease (CKD). METHODS: We leverage a missense genetic variant, rs739320 in the FGF21 gene, that associates with magnetic resonance imaging-derived liver fat as a clinically validated and biologically plausible instrumental variable for studying the effects of FGF21 analogs. Performing Mendelian randomization, we ascertain associations between instrumented FGF21 and kidney phenotypes, cardiometabolic disease risk factors, as well as the circulating proteome (Somalogic, 4907 aptamers) and metabolome (Nightingale platform, 249 metabolites). RESULTS: We report consistent renoprotective associations of genetically proxied FGF21 effect, including higher glomerular filtration rates (p = 1.9 × 10-4), higher urinary sodium excretion (p = 5.1 × 10-11), and lower urine albumin-creatinine ratio (p = 3.6 × 10-5). These favorable effects translated to lower CKD risk (odds ratio per rs739320 C-allele, 0.96; 95%CI, 0.94-0.98; p = 3.2 × 10-4). Genetically proxied FGF21 effect was also associated with lower fasting insulin, waist-to-hip ratio, blood pressure (systolic and diastolic BP, p < 1.0 × 10-07) and blood lipid (low-density lipoprotein cholesterol, triglycerides and apolipoprotein B, p < 6.5 × 10-24) profiles. The latter associations are replicated in our metabolome-wide association study. Proteomic perturbations associated with genetically predicted FGF21 effect were consistent with fibrosis reduction. CONCLUSION: This study highlights the pleiotropic effects of genetically proxied FGF21 and supports a re-purposing opportunity for the treatment and prevention of kidney disease specifically. Further work is required to triangulate these findings, towards possible clinical development of FGF21 towards the treatment and prevention of kidney disease.

A higher non-severe hypoglycaemia rate is associated with an increased risk of subsequent severe hypoglycaemia and major adverse cardiovascular events in individuals with type 2 diabetes in the LEADER study.

AIMS/HYPOTHESIS: Hypoglycaemia is a common side effect of insulin and some other antihyperglycaemic agents used to treat diabetes. Severe hypoglycaemia has been associated with adverse cardiovascular events in trials of intensive glycaemic control in type 2 diabetes. The relationship between non-severe hypoglycaemic episodes (NSHEs) and severe hypoglycaemia in type 2 diabetes has been documented. However, an association between more frequent NSHEs and cardiovascular events has not been verified. This post hoc analysis of the LEADER (Liraglutide Effect and Action in Diabetes: Evaluation of Cardiovascular Outcome Results) trial aimed to confirm whether there is an association between NSHEs and severe hypoglycaemic episodes in individuals with type 2 diabetes. In addition, the possible association between NSHEs and major adverse cardiac events (MACE), cardiovascular death and all-cause mortality was investigated. METHODS: LEADER was a double-blind, multicentre, placebo-controlled trial that found that liraglutide significantly reduced the risk of MACE compared with the placebo. In this post hoc analysis, we explored, in all LEADER participants, whether the annual rate of NSHEs (defined as self-measured plasma glucose <3.1 mmol/l [56 mg/dl]) was associated with time to first severe hypoglycaemic episode (defined as an episode requiring the assistance of another person), time to first MACE, time to cardiovascular death and time to all-cause mortality. Participants with <2 NSHEs per year were used as reference for HR estimates. Cox regression with a time-varying covariate was used. RESULTS: We demonstrate that there is an association between NSHEs (2-11 NSHEs per year and ≥12 NSHEs per year) and severe hypoglycaemic episodes (unadjusted HRs 1.98 [95% CI 1.43, 2.75] and 5.01 [95% CI 2.84, 8.84], respectively), which was consistent when baseline characteristics were accounted for. Additionally, while no association was found between participants with 2-11 NSHEs per year and adverse cardiovascular outcomes, higher rates of NSHEs (≥12 episodes per year) were associated with higher risk of MACE (HR 1.50 [95% CI 1.01, 2.23]), cardiovascular death (HR 2.08 [95% CI 1.17, 3.70]) and overall death (HR 1.80 [95% CI 1.11, 2.92]). CONCLUSIONS/INTERPRETATION: The analysis of data from the LEADER trial demonstrated that higher rates of NSHEs were associated with both a higher risk of severe hypoglycaemia and adverse cardiovascular outcomes in individuals with type 2 diabetes. Therefore, irrespective of the cause of this association, it is important that individuals with high rates of hypoglycaemia are identified so that the potentially increased risk of cardiovascular events can be managed and steps can be taken to reduce NSHEs. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01179048).

Surrogate endpoint evaluation using data from one large global randomized controlled trial.

BACKGROUND: Robust identification of surrogate endpoints can help accelerate the development of pharmacotherapies for diseases traditionally evaluated using true endpoints associated with prolonged follow-up. The meta-analysis-based surrogate endpoint evaluation (SEE) integrates data from multiple, usually smaller, trials to statistically confirm a surrogate endpoint as a robust proxy for the true endpoint. To test the applicability of SEE when only a single, larger trial is available, we analysed the cardiovascular (CV) survival endpoint from the large multinational trial LEADER (9340 subjects) that confirmed the CV safety of a diabetes drug (liraglutide). We evaluated if using country as a trial unit adequately facilitated the meta-analysis and calculation of R2 by country group. METHODS: Data were grouped by country, ensuring at least 30 CV deaths (497 in total) in each of the nine resulting by-country groups. In a two-step SEE on the grouped dataset, we first fitted the group-specific Cox proportional hazard models; next, on the trial-level, we regressed the estimated hazard ratio (HR; liraglutide vs placebo) of the true endpoints (CV death: 497 events, or all-cause death: 828 events) on the HR of the surrogate endpoint (major CV adverse event [MACE]: 1302 events) and derived the group-specific R2 and its 95% confidence interval (CI). RESULTS: Group-level surrogacy of MACE was supported for CV death but not for all-cause death, with [Formula: see text] values of 0.85 [0.63;1.00]95% CI and 0.23 [0.00;0.67]95% CI, respectively. Sensitivity analyses using different grouping approaches (e.g. grouping by region) corroborated the robustness of the conclusions as well as the appropriateness of the data-grouping approaches. CONCLUSIONS: We derived a specific grouping approach to successfully apply SEE on data from a single trial. This may allow for the statistically robust identification and validation of surrogate endpoints based on the abundance of large monolithic outcome trials conducted as part of drug development programmes in, for example, diabetes.

A polymorphism in the promoter of FRAS1 is a candidate SNP associated with metastatic prostate cancer.

BACKGROUND: Inflammation and one of its mediators, NF-kappa B (NFκB), have been implicated in prostate cancer carcinogenesis. We assessed whether germline polymorphisms associated with NFκB are associated with the risk of developing lethal disease (metastases or death from prostate cancer). METHODS: Using a Bayesian approach leveraging NFκB biology with integration of publicly available datasets we used a previously defined genome-wide functional association network specific to NFκB and lethal prostate cancer. A dense-module-searching method identified modules enriched with significant genes from a genome-wide association study (GWAS) study in a discovery data set, Physicians' Health Study and Health Professionals Follow-up Study (PHS/HPFS). The top 48 candidate single nucleotide polymorphisms (SNPs) from the dense-module-searching method were then assessed in an independent prostate cancer cohort and the one SNP reproducibly associated with lethality was tested in a third cohort. Logistic regression models evaluated the association between each SNP and lethal prostate cancer. The candidate SNP was assessed for association with lethal prostate cancer in 6 of 28 studies in the prostate cancer association group to investigate cancer associated alterations in the genome (PRACTICAL) Consortium where there was some medical record review for death ascertainment which also had SNP data from the ONCOARRAY platform. All men self-identified as Caucasian. RESULTS: The rs1910301 SNP which was reproducibly associated with lethal disease was nominally associated with lethal disease (odds ratio [OR] = 1.40; p = .02) in the discovery cohort and the minor allele was also associated with lethal disease in two independent cohorts (OR = 1.35; p = .04 and OR = 1.35; p = .07). Fixed effects meta-analysis of all three cohorts found an association: OR = 1.37 (95% confidence interval [CI]: 1.15-1.62, p = .0003). This SNP is in the promoter region of FRAS1, a gene involved in epidermal-basement membrane adhesion and is present at a higher frequency in men with African ancestry. No association was found in the subset of studies from the PRACTICAL consortium studies which had a total of 106 deaths out total of 3263 patients and a median follow-up of 4.4 years. CONCLUSIONS: Through its connection with the NFκB pathway, a candidate SNP with a higher frequency in men of African ancestry without cancer was found to be associated with lethal prostate cancer across three well-annotated independent cohorts of Caucasian men.

Long-term safety and efficacy of N8-GP in previously treated adults and adolescents with hemophilia A: Final results from pathfinder2.

BACKGROUND: N8-GP (turoctocog alfa pegol; Esperoct® , Novo Nordisk A/S, Bagsvaerd, Denmark) is a glycoPEGylated human recombinant factor VIII with a half-life of ~1.6-fold of standard FVIII products. pathfinder2 (NCT01480180) was a multi-national, open-label trial of N8-GP in previously treated adolescent and adult patients with severe hemophilia A. OBJECTIVE: We report end-of-trial efficacy and safety of N8-GP from pathfinder2. METHODS: pathfinder2 main phase and extension phase part 1 results have been previously reported. During extension phase part 2, patients could switch from N8-GP prophylaxis 50 IU/kg every fourth day (Q4D) or 75 IU/kg once weekly (Q7D), depending on bleeding status. Extension phase part 2 collected long-term safety and efficacy data for all regimens until trial end (first patient in main phase, 30 January 2012; trial end, 10 December 2018). RESULTS: Overall, 186 patients were exposed to N8-GP for up to 6.6 years (median 5.4 years). The estimated annualized bleeding rate (ABR) was 2.14 (median 0.84) for the Q4D prophylaxis arm and 1.31 (median 1.67) for the Q7D prophylaxis arm. Nearly 30% of patients experienced zero bleeds throughout the entire duration of the trial, the hemostatic response was 83.2% across all treatment arms, and patient-reported outcomes were maintained or slightly improved. No safety concerns were detected. CONCLUSION: Data from the completed pathfinder2 trial, one of the largest and longest-running clinical trials to investigate treatment of severe hemophilia A, demonstrate the efficacy and safety of N8-GP in previously treated adolescent and adult patients.

A digital mRNA expression signature to classify challenging Spitzoid melanocytic neoplasms.

Spitzoid neoplasms are a challenging group of cutaneous melanocytic proliferations. They are characterized by epithelioid and/or spindle-shaped melanocytes and classified as benign Spitz nevi (SN), atypical Spitz tumors (AST), or malignant Spitz tumors (MST). The intermediate AST category represents a diagnostically challenging group since on purely histopathological grounds, their benign or malignant character remains unpredictable. This results in uncertainties in patient treatment and prognosis. The molecular properties of Spitzoid lesions, especially their transcriptomic landscape, remain poorly understood, and genomic alterations in melanoma-associated oncogenes are typically absent. The aim of this study was to characterize their transcriptome with digital mRNA expression profiling. Formalin-fixed paraffin-embedded samples (including 27 SN, 10 AST, and 14 MST) were analyzed using the NanoString nCounter PanCancer Pathways Panel. The number of significantly differentially expressed genes in SN vs. MST, SN vs. AST, and AST vs. MST was 68, 167, and 18, respectively. Gene set enrichment analysis revealed upregulation of pathways related to epithelial-mesenchymal transition and immunomodulatory-, angiogenesis-, hormonal-, and myogenesis-associated processes in AST and MST. A molecular signature of SN vs. MST was discovered based on the top-ranked most informative genes: NRAS, NF1, BMP2, EIF2B4, IFNA17, and FZD9. The AST samples showed intermediate levels of the identified signature. This implies that the gene signature can potentially be used to distinguish high-grade from low-grade AST with a larger study cohort in the future. This combined histopathological and transcriptomic methodology is promising for prospective diagnostics of Spitzoid neoplasms and patient management in dermatological oncology.

Validation of distinct type 2 diabetes clusters and their association with diabetes complications in the DEVOTE, LEADER and SUSTAIN-6 cardiovascular outcomes trials.

AIMS: To validate the clusters of Swedish individuals with recent-onset diabetes at differential risk of complications, which were identified in a previous study, in three global populations with long-standing type 2 diabetes (T2D) who were at high cardiovascular risk, and to test for differences in the risk of major diabetes complications and survival endpoints. MATERIALS AND METHODS: We assigned participants from recent global outcomes trials (DEVOTE [n = 7637], LEADER [n = 9340] and SUSTAIN-6 [n = 3297]) to the previously defined clusters according to age at diabetes diagnosis, baseline glycated haemoglobin (HbA1c) and body mass index (BMI). Outcomes were assessed using Kaplan-Meier analysis and log-rank tests. RESULTS: The T2D clusters were consistently replicated across the three trial cohorts. The risk of major adverse cardiovascular events and cardiovascular death differed significantly, in all trials, across clusters over a median follow-up duration of 2.0, 3.8 and 2.1 years, respectively, and was highest for the cluster of participants with high HbA1c and low BMI (P < 0.05 in DEVOTE and LEADER). In LEADER and SUSTAIN-6, the risk of nephropathy differed across clusters (P < 0.0001 and P = 0.003, respectively). The risk of severe hypoglycaemia differed in DEVOTE (P = 0.006). CONCLUSIONS: Previously identified clusters can be replicated in three geographically diverse cohorts of long-standing T2D and are associated with cluster-specific risk profiles for additional clinical and survival outcomes, providing further validation of the clustering methodology. The external validity and stability of clusters across cohorts provides a premise for future work to optimize the clustering approach to yield T2D subgroups with maximum predictive validity who may benefit from subtype-specific treatment paradigms.

A nutrient-wide association study for risk of prostate cancer in the European Prospective Investigation into Cancer and Nutrition and the Netherlands Cohort Study.

PURPOSE: The evidence from the literature regarding the association of dietary factors and risk of prostate cancer is inconclusive. METHODS: A nutrient-wide association study was conducted to systematically and comprehensively evaluate the associations between 92 foods or nutrients and risk of prostate cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC). Cox proportional hazard regression models adjusted for total energy intake, smoking status, body mass index, physical activity, diabetes and education were used to estimate hazard ratios and 95% confidence intervals for standardized dietary intakes. As in genome-wide association studies, correction for multiple comparisons was applied using the false discovery rate (FDR < 5%) method and suggested results were replicated in an independent cohort, the Netherlands Cohort Study (NLCS). RESULTS: A total of 5916 and 3842 incident cases of prostate cancer were diagnosed during a mean follow-up of 14 and 20 years in EPIC and NLCS, respectively. None of the dietary factors was associated with the risk of total prostate cancer in EPIC (minimum FDR-corrected P, 0.37). Null associations were also observed by disease stage, grade and fatality, except for positive associations observed for intake of dry cakes/biscuits with low-grade and butter with aggressive prostate cancer, respectively, out of which the intake of dry cakes/biscuits was replicated in the NLCS. CONCLUSIONS: Our findings provide little support for an association for the majority of the 92 examined dietary factors and risk of prostate cancer. The association of dry cakes/biscuits with low-grade prostate cancer warrants further replication given the scarcity in the literature.

Total nut, tree nut, peanut, and peanut butter intake and the risk of prostate cancer in the Netherlands Cohort Study.

BACKGROUND: The consumption of nuts has been associated with a reduction of cancer risk, but only a few studies have examined the effects of nuts on prostate cancer risk. The current study prospectively investigated the association between the consumption of total nuts, tree nuts, peanuts, and peanut butter and the risk of total, advanced, and non-advanced prostate cancer. METHODS: The association between nuts and prostate cancer was evaluated in the Netherlands Cohort Study, which was conducted among 58,279 men aged 55-69 year at baseline. A case-cohort approach was used for data processing and analyses. After 20.3 years of follow-up, 3868 incident prostate cancer cases and 1979 subcohort members were available for multivariable Cox regression analyses. RESULTS: For total, advanced, and non-advanced prostate cancer, no significant associations were found for total nuts (total prostate cancer: hazard ratio (HR) (95%CI) for 10+ g/day vs. non-consumers = 1.09 (0.92-1.29), Ptrend = 0.409). No significant associations were observed for tree nuts and peanuts for total, advanced, and non-advanced prostate cancer risk. Peanut butter consumption was associated with a significantly increased risk of non-advanced prostate cancer (HR (95%CI) for 5+ g/day vs. non-consumers = 1.33 (1.08-1.63), Ptrend = 0.008), but not with total or advanced prostate cancer. CONCLUSIONS: No significant associations were found between total nut, tree nut, and peanut consumption and total, advanced, and non-advanced prostate cancer. Peanut butter might be associated with an increased non-advanced prostate cancer risk.

DNA methylation profiles in African American prostate cancer patients in relation to disease progression.

This study examined whether differential DNA methylation is associated with clinical features of more aggressive disease at diagnosis and prostate cancer recurrence in African American men, who are more likely to die from prostate cancer than other populations. Tumor tissues from 76 African Americans diagnosed with prostate cancer who had radical prostatectomy as their primary treatment were profiled for epigenome-wide DNA methylation levels. Long-term follow-up identified 19 patients with prostate cancer recurrence. Twenty-three CpGs were differentially methylated (FDR q≤0.25, mean methylation difference≥0.10) in patients with vs. without recurrence, including CpGs in GCK, CDKL2, PRDM13, and ZFR2. Methylation differences were also observed between men with metastatic-lethal prostate cancer vs. no recurrence (five CpGs), regional vs. local pathological stage (two CpGs), and higher vs. lower tumor aggressiveness (one CpG). These results indicate that differentially methylated CpG sites identified in tumor tissues of African American men may contribute to prostate cancer aggressiveness.

Molecular profiling of Spitz nevi identified by digital RNA counting.

The molecular properties of benign melanocytic lesions are poorly understood. Only a few studies have been carried out on specific nevi subtypes, including common nevocellular nevi (NCN) or Spitz nevi (SN). Genomic alterations in melanoma-associated oncogenes are typically absent in SN. In the present study, mRNA expressions of 25 SN and 15 NCN were analyzed. Molecular profiling was performed using the RNA NanoString nCounter Gene Expression Platform (number of genes=770). Marker discovery was performed with a training set consisting of seven SN and seven NCN samples from the same patients, and validation was performed using a second set consisting of 18 SN and eight NCN samples. Using the training set, 197 differentially expressed genes were identified in SN versus NCN. Of these, 74 genes were validated in the validation set (false discovery rate q≤0.13). In addition, using random forest and least absolute shrinkage and selection operator feature selection, a molecular signature of SN versus NCN was identified including 15 top-ranked genes. The present study identified a distinct molecular expression profile in SN compared with NCN, even when lesions were obtained from the same patients. Gene set analysis showed upregulation of gene pathways with increased expression of transcripts related to immunomodulatory, inflammatory, and extracellular matrix interactions as well as angiogenesis-associated processes in SN. These findings strongly indicate that SN represent a distinct group of melanocytic neoplasms and evolve differentially and not sequentially from NCN.

A five-CpG DNA methylation score to predict metastatic-lethal outcomes in men treated with radical prostatectomy for localized prostate cancer.

BACKGROUND: Prognostic biomarkers for localized prostate cancer (PCa) could improve personalized medicine. Our group previously identified a panel of differentially methylated CpGs in primary tumor tissue that predict disease aggressiveness, and here we further validate these biomarkers. METHODS: Pyrosequencing was used to assess CpG methylation of eight biomarkers previously identified using the HumanMethylation450 array; CpGs with strongly correlated (r >0.70) results were considered technically validated. Logistic regression incorporating the validated CpGs and Gleason sum was used to define and lock a final model to stratify men with metastatic-lethal versus non-recurrent PCa in a training dataset. Coefficients from the final model were then used to construct a DNA methylation score, which was evaluated by logistic regression and Receiver Operating Characteristic (ROC) curve analyses in an independent testing dataset. RESULTS: Five CpGs were technically validated and all were retained (P < 0.05) in the final model. The 5-CpG and Gleason sum coefficients were used to calculate a methylation score, which was higher in men with metastatic-lethal progression (P = 6.8 × 10-6 ) in the testing dataset. For each unit increase in the score there was a four-fold increase in risk of metastatic-lethal events (odds ratio, OR = 4.0, 95%CI = 1.8-14.3). At 95% specificity, sensitivity was 74% for the score compared to 53% for Gleason sum alone. The score demonstrated better prediction performance (AUC = 0.91; pAUC = 0.037) compared to Gleason sum alone (AUC = 0.87; pAUC = 0.025). CONCLUSIONS: The DNA methylation score improved upon Gleason sum for predicting metastatic-lethal progression and holds promise for risk stratification of men with aggressive tumors. This prognostic score warrants further evaluation as a tool for improving patient outcomes.

Genetic Mechanisms of Immune Evasion in Colorectal Cancer.

To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability-high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/ß-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/ß-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion.Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730-49. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Genomic Landscape of Spitzoid Neoplasms Impacting Patient Management.

Spitzoid neoplasms are a distinct group of melanocytic proliferations characterized by epithelioid and/ or spindle shaped melanocytes. Intermediate forms that share features of both benign Spitz nevi (SN) and Spitz melanoma, i.e., malignant Spitz tumor (MST) represent a diagnostically and clinically challenging group of melanocytic lesions. A multitude of descriptive diagnostic terms exist for these ambiguous lesions with atypical Spitz tumor (AST) or Spitz tumor of uncertain malignant potential (STUMP) just naming two of them. This diagnostic gray zone creates confusion and high insecurity in clinicians and in patients. Biological behavior and clinical course of this intermediate group still remains largely unknown, often leading to difficulties with uncertainties in clinical management and prognosis. Consequently, a better stratification of Spitzoid neoplasms in benign and malignant forms is required thereby keeping the diagnostic group of AST/STUMP as small as possible. Ancillary diagnostic techniques such as immunohistochemistry, comparative genomic hybridization, fluorescence in situ hybridization, next generation sequencing, micro RNA and mRNA analysis as well as mass spectrometry imaging offer new opportunities for the distinct diagnosis, thereby allowing the best clinical management of Spitzoid neoplasms. This review gives an overview on these additional diagnostic techniques and the recent developments in the field of molecular genetic alterations in Spitzoid neoplasms. We also discuss how the recent findings might facilitate the diagnosis and stratification of atypical Spitzoid neoplasms and how these findings will impact the diagnostic work up as well as patient management. We suggest a stepwise implementation of ancillary diagnostic techniques thereby integrating immunohistochemistry and molecular pathology findings in the diagnosis of challenging ambiguous Spitzoid neoplasms. Finally, we will give an outlook on pending future research objectives in the field of Spitzoid melanocytic lesions.

PTEN loss is associated with prostate cancer recurrence and alterations in tumor DNA methylation profiles.

BACKGROUND: Prostate cancer (PCa) with loss of the tumor suppressor gene PTEN has an unfavorable prognosis. DNA methylation profiles associated with PTEN loss may provide further insights into the mechanisms underlying these more aggressive, clinically relevant tumors. METHODS: The cohort included patients with clinically localized PCa. Samples taken from the primary tumor were used to determine PTEN genomic deletions using FISH, and to analyze epigenome-wide DNA methylation profiles. Patients were followed for PCa recurrence on average for 8 years after diagnosis. RESULTS: The study included 471 patients with data on PTEN loss, and the frequency of hemi- and homozygous PTEN loss was 10.0% and 4.5%, respectively. Loss of PTEN was associated with a significantly higher risk of recurrence (any vs. no PTEN loss; HR = 1.74; 95% CI: 1.03-2.93). Hazard ratios for hemi- and homozygous loss were 1.39 (95% CI: 0.73-2.64) and 2.84 (95% CI: 1.30-6.19), respectively. Epigenome-wide methylation profiling identified 4,208 differentially methylated CpGs (FDR Q-value < 0.01) in tumors with any versus no PTEN loss. There were no genome-wide significant differentially methylated CpGs in homo- versus hemizygous deleted tumors. Tumor methylation data were used to build a methylation signature of PTEN loss in our cohort, which was confirmed in TCGA, and included CpGs in ATP11A, GDNF, JAK1, JAM3, and VAPA. CONCLUSION: Loss of PTEN was positively associated with PCa recurrence. Prostate tumors with PTEN loss harbor a distinct methylation signature, and these aberrantly methylated CpG sites may mediate tumor progression when PTEN is deleted.

Gene expression signature of Gleason score is associated with prostate cancer outcomes in a radical prostatectomy cohort.

Prostate cancer (PCa) is a leading cause of cancer-related mortality worldwide. Gleason score (GS) is one of the best predictors of PCa aggressiveness, but additional tumor biomarkers may improve its prognostic accuracy. We developed a gene expression signature of GS to enhance the prediction of PCa outcomes. Elastic net was used to construct a gene expression signature by contrasting GS 8-10 vs. ≤6 tumors in The Cancer Genome Atlas (TCGA) dataset. The constructed signature was then evaluated for its ability to predict recurrence and metastatic-lethal (ML) progression in a Fred Hutchinson (FH) patient cohort (N=408; NRecurrence=109; NMLprogression=27). The expression signature included transcripts representing 49 genes. In the FH cohort, a 25% increase in the signature was associated with a hazard ratio (HR) of 1.51 (P=2.7×10-5) for recurrence. The signature's area under the curve (AUC) for predicting recurrence and ML progression was 0.68 and 0.76, respectively. Compared to a model with age at diagnosis, pathological stage and GS, the gene expression signature improved the AUC for recurrence (3%) and ML progression (6%). Higher levels of the signature were associated with increased expression of genes in cell cycle-related pathways and decreased expression of genes in androgen response, estrogen response, oxidative phosphorylation, and apoptosis. This gene expression signature based on GS may improve the prediction of recurrence as well as ML progression in PCa patients after radical prostatectomy.

Gene expression panel predicts metastatic-lethal prostate cancer outcomes in men diagnosed with clinically localized prostate cancer.

Prognostic biomarkers are needed to distinguish patients with clinically localized prostate cancer (PCa) who are at high risk of metastatic progression. The tumor transcriptome may reveal its aggressiveness potential and have utility for predicting adverse patient outcomes. Genomewide gene expression levels were measured in primary tumor samples of 383 patients in a population-based discovery cohort, and from an independent clinical validation dataset of 78 patients. Patients were followed for ≥ 5 years after radical prostatectomy to ascertain outcomes. Area under the receiver-operating characteristic curve (AUC), partial AUC (pAUC, 95% specificity), and P-value criteria were used to detect and validate the differentially expressed transcripts. Twenty-three differentially expressed transcripts in patients with metastatic-lethal compared with nonrecurrent PCa were validated (P < 0.05; false discovery rate < 0.20) in the independent dataset. The addition of each validated transcript to a model with Gleason score showed that 17 transcripts significantly improved the AUC (range: 0.83-0.88; all P-values < 0.05). These differentially expressed mRNAs represent genes with diverse cellular functions related to tumor aggressiveness. This study validated 23 gene transcripts for predicting metastatic-lethal PCa in patients surgically treated for clinically localized disease. Several of these mRNA biomarkers have clinical potential for identifying the subset of PCa patients with more aggressive tumors who would benefit from closer monitoring and adjuvant therapy.

Epigenome-Wide Tumor DNA Methylation Profiling Identifies Novel Prognostic Biomarkers of Metastatic-Lethal Progression in Men Diagnosed with Clinically Localized Prostate Cancer.

PURPOSE: Aside from Gleason sum, few factors accurately identify the subset of prostate cancer patients at high risk for metastatic progression. We hypothesized that epigenetic alterations could distinguish prostate tumors with life-threatening potential. EXPERIMENTAL DESIGN: Epigenome-wide DNA methylation profiling was performed in surgically resected primary tumor tissues from a population-based (n = 430) and a replication (n = 80) cohort of prostate cancer patients followed prospectively for at least 5 years. Metastasis was confirmed by positive bone scan, MRI, CT, or biopsy, and death certificates confirmed cause of death. AUC, partial AUC (pAUC, 95% specificity), and P value criteria were used to select differentially methylated CpG sites that robustly stratify patients with metastatic-lethal from nonrecurrent tumors, and which were complementary to Gleason sum. RESULTS: Forty-two CpG biomarkers stratified patients with metastatic-lethal versus nonrecurrent prostate cancer in the discovery cohort, and eight of these CpGs replicated in the validation cohort based on a significant (P < 0.05) AUC (range, 0.66-0.75) or pAUC (range, 0.007-0.009). The biomarkers that improved discrimination of patients with metastatic-lethal prostate cancer include CpGs in five genes (ALKBH5, ATP11A, FHAD1, KLHL8, and PI15) and three intergenic regions. In the validation dataset, the AUC for Gleason sum alone (0.82) significantly increased with the addition of four individual CpGs (range, 0.86-0.89; all P <0.05). CONCLUSIONS: Eight differentially methylated CpGs that distinguish patients with metastatic-lethal from nonrecurrent tumors were validated. These novel epigenetic biomarkers warrant further investigation as they may improve prognostic classification of patients with clinically localized prostate cancer and provide new insights on tumor aggressiveness. Clin Cancer Res; 23(1); 311-9. ©2016 AACR.

Investigating the possible causal role of coffee consumption with prostate cancer risk and progression using Mendelian randomization analysis.

Coffee consumption has been shown in some studies to be associated with lower risk of prostate cancer. However, it is unclear if this association is causal or due to confounding or reverse causality. We conducted a Mendelian randomisation analysis to investigate the causal effects of coffee consumption on prostate cancer risk and progression. We used two genetic variants robustly associated with caffeine intake (rs4410790 and rs2472297) as proxies for coffee consumption in a sample of 46,687 men of European ancestry from 25 studies in the PRACTICAL consortium. Associations between genetic variants and prostate cancer case status, stage and grade were assessed by logistic regression and with all-cause and prostate cancer-specific mortality using Cox proportional hazards regression. There was no clear evidence that a genetic risk score combining rs4410790 and rs2472297 was associated with prostate cancer risk (OR per additional coffee increasing allele: 1.01, 95% CI: 0.98,1.03) or having high-grade compared to low-grade disease (OR: 1.01, 95% CI: 0.97,1.04). There was some evidence that the genetic risk score was associated with higher odds of having nonlocalised compared to localised stage disease (OR: 1.03, 95% CI: 1.01, 1.06). Amongst men with prostate cancer, there was no clear association between the genetic risk score and all-cause mortality (HR: 1.00, 95% CI: 0.97,1.04) or prostate cancer-specific mortality (HR: 1.03, 95% CI: 0.98,1.08). These results, which should have less bias from confounding than observational estimates, are not consistent with a substantial effect of coffee consumption on reducing prostate cancer incidence or progression.