Comparative analysis of neurofilaments and biomarkers of muscular damage in amyotrophic lateral sclerosis.

Diagnosis of the fatal neurodegenerative disease amyotrophic lateral sclerosis is challenging. Neurofilaments, indicative of neuronal damage, along with creatine kinase, creatinine, myoglobin, and troponin T, representing muscular damage, have been identified as promising fluid biomarkers. This study aims to comprehensively assess and compare their diagnostic and prognostic potential in a 'real-world' cohort of patients with amyotrophic lateral sclerosis. About 77 patients with amyotrophic lateral sclerosis and its clinical variants, and 26 age- and sex-matched controls with various neuromuscular and neurodegenerative diseases, were retrospectively included in this monocentric, cross-sectional study. Neurofilaments in cerebrospinal fluid and biomarkers of muscular damage in serum were measured and correlated with demographic features, motor function, survival time, clinical phenotypes, and the extent of upper and lower motor neuron involvement. Neurofilament, myoglobin, and troponin T concentrations were higher in patients with amyotrophic lateral sclerosis compared to disease controls. Higher neurofilament levels correlated with lower motor function and faster disease progression rate, while higher creatine kinase and creatinine concentrations were linked to preserved motor function. In contrast, troponin T elevation indicated poorer fine and gross motor functions. Increased neurofilament levels were associated with shorter survival, whereas biomarkers of muscular damage lacked survival correlation. Neurofilament concentrations were higher in classical amyotrophic lateral sclerosis than in progressive muscular atrophy, while myoglobin and troponin T levels were elevated in progressive muscular atrophy compared to primary lateral sclerosis. Neurofilaments were predominantly linked to upper motor neuron involvement. Our findings confirmed the robust diagnostic and prognostic value of neurofilaments in amyotrophic lateral sclerosis. Elevated neurofilament concentrations were associated with higher disease severity, faster disease progression, shorter survival, and predominant upper motor neuron degeneration. Biomarkers of muscular damage were inferior in distinguishing amyotrophic lateral sclerosis from other neuromuscular and neurodegenerative diseases. However, they may serve as complementary biomarkers and support in discriminating clinical variants of amyotrophic lateral sclerosis.

Detection of Subclinical Rejection in Pediatric Kidney Transplantation: Current and Future Practices.

INTRODUCTION: The successes in the field of pediatric kidney transplantation over the past 60 years have been extraordinary. Year over year, there have been significant improvements in short-term graft survival. However, improvements in longer-term outcomes have been much less apparent. One important contributor has been the phenomenon of low-level rejection in the absence of clinical manifestations-so-called subclinical rejection (SCR). METHODS: Traditionally, rejection has been diagnosed by changes in clinical parameters, including but not limited to serum creatinine and proteinuria. This review examines the shortcomings of this approach, the effects of SCR on kidney allograft outcome, the benefits and drawbacks of surveillance biopsies to identify SCR, and new urine and blood biomarkers that define the presence or absence of SCR. RESULTS: Serum creatinine is an unreliable index of SCR. Surveillance biopsies are the method most utilized to detect SCR. However, these have significant drawbacks. New biomarkers show promise. These biomarkers include blood gene expression profiles and donor derived-cell free DNA; urine gene expression profiles; urinary cytokines, chemokines, and metabolomics; and other promising blood and urine tests. CONCLUSION: Specific emphasis is placed on studies carried out in pediatric kidney transplant recipients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03719339.

Identification of novel snoRNA-based biomarkers for clear cell renal cell carcinoma from urine-derived extracellular vesicles.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC with high rates of metastasis. Targeted therapies such as tyrosine kinase and checkpoint inhibitors have improved treatment success, but therapy-related side effects and tumor recurrence remain a challenge. As a result, ccRCC still have a high mortality rate. Early detection before metastasis has great potential to improve outcomes, but no suitable biomarker specific for ccRCC is available so far. Therefore, molecular biomarkers derived from body fluids have been investigated over the past decade. Among them, RNAs from urine-derived extracellular vesicles (EVs) are very promising. METHODS: RNA was extracted from urine-derived EVs from a cohort of 78 subjects (54 ccRCC patients, 24 urolithiasis controls). RNA-seq was performed on the discovery cohort, a subset of the whole cohort (47 ccRCC, 16 urolithiasis). Reads were then mapped to the genome, and expression was quantified based on 100 nt long contiguous genomic regions. Cluster analysis and differential region expression analysis were performed with adjustment for age and gender. The candidate biomarkers were validated by qPCR in the entire cohort. Receiver operating characteristic, area under the curve and odds ratios were used to evaluate the diagnostic potential of the models. RESULTS: An initial cluster analysis of RNA-seq expression data showed separation by the subjects' gender, but not by tumor status. Therefore, the following analyses were done, adjusting for gender and age. The regions differentially expressed between ccRCC and urolithiasis patients mainly overlapped with small nucleolar RNAs (snoRNAs). The differential expression of four snoRNAs (SNORD99, SNORD22, SNORD26, SNORA50C) was validated by quantitative PCR. Confounder-adjusted regression models were then used to classify the validation cohort into ccRCC and tumor-free subjects. Corresponding accuracies ranged from 0.654 to 0.744. Models combining multiple genes and the risk factors obesity and hypertension showed improved diagnostic performance with an accuracy of up to 0.811 for SNORD99 and SNORA50C (p = 0.0091). CONCLUSIONS: Our study uncovered four previously unrecognized snoRNA biomarkers from urine-derived EVs, advancing the search for a robust, easy-to-use ccRCC screening method.

Personalized identification and characterization of genome-wide gene expression differences between patient-matched intracranial and extracranial melanoma metastasis pairs.

Melanoma is the most serious type of skin cancer that frequently spreads to other organs of the human body. Especially melanoma metastases to the brain (intracranial metastases) are hard to treat and a major cause of death of melanoma patients. Little is known about molecular alterations and altered mechanisms that distinguish intra- from extracranial melanoma metastases. So far, almost all existing studies compared intracranial metastases from one set of patients to extracranial metastases of an another set of melanoma patients. This neglects the important facts that each melanoma is highly individual and that intra- and extracranial melanoma metastases from the same patient are more similar to each other than to melanoma metastases from other patients in the same organ. To overcome this, we compared the gene expression profiles of 16 intracranial metastases to their corresponding 21 patient-matched extracranial metastases in a personalized way using a three-state Hidden Markov Model (HMM) to identify altered genes for each individual metastasis pair. This enabled three major findings by considering the predicted gene expression alterations across all patients: (i) most frequently altered pathways include cytokine-receptor interaction, calcium signaling, ECM-receptor interaction, cAMP signaling, Jak-STAT and PI3K/Akt signaling, (ii) immune-relevant signaling pathway genes were downregulated in intracranial metastases, and (iii) intracranial metastases were associated with a brain-like phenotype gene expression program. Further, the integration of all differentially expressed genes across the patient-matched melanoma metastasis pairs led to a set of 103 genes that were consistently down- or up-regulated in at least 11 of the 16 of the patients. This set of genes contained many genes involved in the regulation of immune responses, cell growth, cellular signaling and transport processes. An analysis of these genes in the TCGA melanoma cohort showed that the expression behavior of 11 genes was significantly associated with survival. Moreover, a comparison of the 103 genes to three closely related melanoma metastasis studies revealed a core set of eight genes that were consistently down- or upregulated in intra- compared to extracranial metastases in at least two of the three related studies (down: CILP, DPT, FGF7, LAMP3, MEOX2, TMEM119; up: GLDN, PMP2) including FGF7 that was also significantly associated with survival. Our findings contribute to a better characterization of genes and pathways that distinguish intra- from extracranial melanoma metastasis and provide important hints for future experimental studies to identify potential targets for new therapeutic approaches.

Network-based analysis of heterogeneous patient-matched brain and extracranial melanoma metastasis pairs reveals three homogeneous subgroups.

Melanoma, the deadliest form of skin cancer, can metastasize to different organs. Molecular differences between brain and extracranial melanoma metastases are poorly understood. Here, promoter methylation and gene expression of 11 heterogeneous patient-matched pairs of brain and extracranial metastases were analyzed using melanoma-specific gene regulatory networks learned from public transcriptome and methylome data followed by network-based impact propagation of patient-specific alterations. This innovative data analysis strategy allowed to predict potential impacts of patient-specific driver candidate genes on other genes and pathways. The patient-matched metastasis pairs clustered into three robust subgroups with specific downstream targets with known roles in cancer, including melanoma (SG1: RBM38, BCL11B, SG2: GATA3, FES, SG3: SLAMF6, PYCARD). Patient subgroups and ranking of target gene candidates were confirmed in a validation cohort. Summarizing, computational network-based impact analyses of heterogeneous metastasis pairs predicted individual regulatory differences in melanoma brain metastases, cumulating into three consistent subgroups with specific downstream target genes.

A multicenter prospective study to define the natural history of BK viral infections in kidney transplantation.

BACKGROUND: BK polyomavirus (BKV) can cause permanent loss of allograft function due to BKV-associated nephropathy (BKVN) in kidney transplant recipients. Besides immunosuppression reduction, there are no consistently effective interventions for BKV infection. Study purpose was to define natural history of BKV infection, identify risk factors for BKV reactivation and BKVN in kidney transplant recipients, and inform the design/conduct of future clinical trials of BKV-targeted therapeutics. METHODS: We conducted a multicenter prospective observational study of incident kidney transplant recipients at six U.S. transplant centers. Participants were monitored every 4 weeks for BKV reactivation and followed for up to 24 months post-transplant. We used regression models (logistic, survival, mixed models) to study relationships between BK viremia/BKVN, clinical characteristics, and allograft function. RESULTS: We enrolled 335 participants. Fifty-eight (17%) developed BK viremia, 6 (2%) developed biopsy-proven BKVN, and 29 (9%) developed suspected/presumed BKVN (defined as BKV viral load > 10,000 copies/mL without biopsy). Male donor sex was associated with lower odds for BK viremia, whereas recipient Black race was associated with two-fold increased odds for BK viremia. Recipient female sex was associated with more rapid clearance of BK viremia. Persistent BK viremia/BKVN was associated with poorer allograft function by 24 months post-transplant. CONCLUSIONS: We identified multiple donor and recipient demographic factors associated with risk for BKV infection and poorer allograft function by 24 months post-transplant. This may help design future clinical trials of therapies to prevent or mitigate the deleterious impact of BKV reactivation on kidney transplant outcomes.

Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome.

BACKGROUND: Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC). METHODS: The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and ß1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts. RESULTS: Fibroblast growth factor receptor (FGFR 1-4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with ß1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions. CONCLUSIONS: This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.

ß1 integrin mediates unresponsiveness to PI3Kα inhibition for radiochemosensitization of 3D HNSCC models.

Phosphoinositide 3-kinase (PI3K)-α represents a key intracellular signal transducer involved in the regulation of key cell functions such as cell survival and proliferation. Excessive activation of PI3Kα is considered one of the major determinants of cancer therapy resistance. Despite preclinical and clinical evaluation of PI3Kα inhibitors in various tumor entities, including head and neck squamous cell carcinoma (HNSCC), it remains elusive how conventional radiochemotherapy can be enhanced by concurrent PI3K inhibitors and how PI3K deactivation mechanistically exerts its effects. Here, we investigated the radiochemosensitizing potential and adaptation mechanisms of four PI3K inhibitors, Alpelisib, Copanlisib, AZD8186, and Idelalisib in eight HNSCC models grown under physiological, three-dimensional matrix conditions. We demonstrate that Alpelisib, Copanlisib and AZD8186 but not Idelalisib enhance radio- and radiochemosensitivity in the majority of HNSCC cell models (= responders) in a manner independent of PIK3CA mutation status. However, Alpelisib promotes MAPK signaling in non-responders compared to responders without profound impact on Akt, NFκB, TGFß, JAK/STAT signaling and DNA repair. Bioinformatic analyses identified unique gene mutations associated with extracellular matrix to be more frequent in non-responder cell models than in responders. Finally, we demonstrate that targeting of the cell adhesion molecule ß1 integrin on top of Alpelisib sensitizes non-responders to radiochemotherapy. Taken together, our study demonstrates the sensitizing potential of Alpelisib and other PI3K inhibitors in HNSCC models and uncovers a novel ß1 integrin-dependent mechanism that may prove useful in overcoming resistance to PI3K inhibitors.

Follow-up biopsies identify high rates of persistent rejection in pediatric kidney transplant recipients after treatment of T cell-mediated rejection.

BACKGROUND: Incomplete resolution of T cell-mediated rejection (TCMR) after treatment may not be detected with serum creatinine monitoring and is associated with donor-specific antibodies and chronic rejection. We evaluate the utility of follow-up biopsies (FUB) to identify and characterize rates of persistent TCMR after treatment in pediatric kidney transplant patients. METHODS: Patients from two pediatric transplant centers performing standard of care FUB at 1.5-2 months after treatment for TCMR were included. FUB were evaluated for extent of rejection resolution (complete vs. incomplete) and grade. Clinical data at time of FUB and later were reported, where available. RESULTS: Fifty-eight patients underwent FUB, at mean of 1.7 months (SD 0.7) post-index biopsy. Rejection grade on index biopsy was Banff borderline (≥i1t1 and

Prevalence of mycophenolate mofetil discontinuation and subsequent outcomes in pediatric kidney transplant recipients: A PNRC study.

BACKGROUND: Mycophenolate Mofetil (MMF) is an effective immunosuppressant used in kidney transplant recipients to prevent acute rejection. Complications such as diarrhea, leukopenia, and infections may necessitate the reduction or discontinuation of MMF. The objective of the study was to investigate the prevalence, timing, and reasons for MMF discontinuation and its association with outcomes in pediatric kidney transplant recipients. METHODS: Seven Pediatric Nephrology Research Consortium (PNRC) centers participated in a retrospective analysis of kidney transplant recipients <21 years of age. Characteristics and outcomes of patients in whom MMF was discontinued were compared to those who continued taking MMF throughout the first 2 years post-transplant. RESULTS: The study population included 288 participants (mean age 11.2 years) from 7 North American transplant centers. MMF was discontinued in 93/288 (32%) of participants. Common reasons for discontinuation included infections (35%), diarrhea (32%), leukopenia (15%), and others (18%). Increased cumulative alloimmunity (55% vs. 42%, p = .02), increased number of hospitalizations (82% vs. 67%, p = .01), and viral replications (79% vs. 47%, p < .0001) were observed in the MMF discontinuation group compared to the continuation group. Greater eGFR decline also occurred in the MMF discontinuation group over 2 years of follow-up (-7 vs. -1 mL/min/1.73 m2 , p = .05). CONCLUSIONS: Almost a third of pediatric kidney transplant recipients who begin MMF for maintenance immunosuppression have it discontinued within the first 2 years post-transplant, and this subset of patients is more likely to experience adverse outcomes. New strategies are needed to manage MMF therapy and improve post-transplant outcomes.

T-bet+ B Cells in Humans: Protective and Pathologic Functions.

The humoral immune system comprises B cells and plasma cells, which play important roles in organ transplantation, ranging from the production of both protective and injurious antibodies as well as cytokines that can promote operational tolerance. Recent data from conditions outside of transplantation have identified a novel human B-cell subset that expresses the transcription factor T-bet and exerts pleiotropic functions by disease state. Here, we review the generation, activation, and functions of the T-bet+ B-cell subset outside of allotransplantation, and consider the relevance of this subset as mediators of allograft injury.

A randomized controlled trial of preemptive rituximab to prevent recurrent focal segmental glomerulosclerosis post-kidney transplant (PRI-VENT FSGS): protocol and study design.

Background: Focal segmental glomerulosclerosis (FSGS) is a common cause of end-stage kidney disease requiring kidney transplantation and can recur in the allograft in 30-80% of recipients resulting in reduced graft survival. Plasmapheresis has shown efficacy in treating some cases of recurrent FSGS but isolated plasmapheresis has not demonstrated efficacy in preventing recurrent FSGS. Rituximab has had anecdotal success in preventing recurrence in a single center study but has not been studied in combination with plasmapheresis for preventing FSGS recurrence. Methods: We are conducting a randomized, controlled, multicenter clinical trial of adult and pediatric kidney transplant recipients with primary FSGS to assess whether plasmapheresis in combination with rituximab prevents recurrent disease post-transplantation. Discussion: Rituximab combined with plasmapheresis is a promising, novel therapy to prevent recurrent FSGS, a disease with limited therapeutic options and no consensus guidelines for prevention or treatment. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03763643, identifier NCT03763643.

Validation of a prediction system for risk of kidney allograft failure in pediatric kidney transplant recipients: An international observational study.

Predicting long-term kidney allograft failure is an unmet need for clinical care and clinical trial optimization in children. We aimed to validate a kidney allograft failure risk prediction system in a large international cohort of pediatric kidney transplant recipients. Patients from 20 centers in Europe and the United States, transplanted between 2004 and 2017, were included. Allograft assessment included estimated glomerular filtration rate, urine protein-to-creatinine ratio, circulating antihuman leukocyte antigen donor-specific antibody, and kidney allograft histology. Individual predictions of allograft failure were calculated using the integrative box (iBox) system. Prediction performances were assessed using discrimination and calibration. The allograft evaluations were performed in 706 kidney transplant recipients at a median time of 9.1 (interquartile range, 3.3-19.2) months posttransplant; mean estimated glomerular filtration rate was 68.7 ± 28.1 mL/min/1.73 m2, and median urine protein-to-creatinine ratio was 0.1 (0.0-0.4) g/g, and 134 (19.0%) patients had antihuman leukocyte antigen donor-specific antibodies. The iBox exhibited accurate calibration and discrimination for predicting the outcomes up to 10 years after evaluation, with a C-index of 0.81 (95% confidence interval, 0.75-0.87). This study confirms the generalizability of the iBox to predict long-term kidney allograft failure in children, with performances similar to those reported in adults. These results support the use of the iBox to improve patient monitoring and facilitate clinical trials in children.

Meta-analysis of expression and the targeting of cell adhesion associated genes in nine cancer types - A one research lab re-evaluation.

Cancer presents as a highly heterogeneous disease with partly overlapping and partly distinct (epi)genetic characteristics. These characteristics determine inherent and acquired resistance, which need to be overcome for improving patient survival. In line with the global efforts in identifying druggable resistance factors, extensive preclinical research of the Cordes lab and others designated the cancer adhesome as a critical and general therapy resistance mechanism with multiple druggable cancer targets. In our study, we addressed pancancer cell adhesion mechanisms by connecting the preclinical datasets generated in the Cordes lab with publicly available transcriptomic and patient survival data. We identified similarly changed differentially expressed genes (scDEGs) in nine cancers and their corresponding cell models relative to normal tissues. Those scDEGs interconnected with 212 molecular targets from Cordes lab datasets generated during two decades of research on adhesome and radiobiology. Intriguingly, integrative analysis of adhesion associated scDEGs, TCGA patient survival and protein-protein network reconstruction revealed a set of overexpressed genes adversely affecting overall cancer patient survival and specifically the survival in radiotherapy-treated cohorts. This pancancer gene set includes key integrins (e.g. ITGA6, ITGB1, ITGB4) and their interconnectors (e.g. SPP1, TGFBI), affirming their critical role in the cancer adhesion resistome. In summary, this meta-analysis demonstrates the importance of the adhesome in general, and integrins together with their interconnectors in particular, as potentially conserved determinants and therapeutic targets in cancer.

Secondary Sclerosing Cholangitis due to Severe COVID-19: An Emerging Disease Entity?

INTRODUCTION: Coronavirus disease 2019 (COVID-19) can lead to many extrapulmonary manifestations. In this case series, we report on 7 patients developing secondary sclerosing cholangitis (SSC) after severe COVID-19 with intensive care treatment. METHODS: Between March 2020 and November 2021, 544 patient cases with cholangitis treated at a German tertiary care centre were screened for SSC. Patients found to be suffering from SSC were assigned to COVID-19 group if SSC presented after a severe course of COVID-19 and to non-COVID-19 group if not. Peak liver parameters as well as intensive care treatment factors and data generated from liver elastography were compared between both groups. RESULTS: We identified 7 patients who developed SSC after a severe course of COVID-19. In the same period, 4 patients developed SSC due to other causes. Mean values of gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) were higher in the COVID-19 group than in the non-COVID-19 group (GGT: 2,689 U/L vs. 1,812 U/L and ALP: 1,445 U/L vs. 1,027 U/L), whereas intensive care treatment factors were comparable in both groups. Only the mean duration of mechanical ventilation was shorter in the COVID-19 group than in the non-COVID-19 group (22.1 days vs. 36.7 days). Liver elastography indicated a fast progression to liver cirrhosis with a mean liver stiffness of 17.3 kilopascals (kPa) in less than 12 weeks in the COVID-19 group. CONCLUSIONS: Our data suggest a more severe course of SSC when caused by SARS-CoV-2. Reasons for this are probably multifactorial, including a direct cytopathogenic effect of the virus.

Early Life Stress, Coping, and Cardiovascular Reactivity to Acute Social Stress.

OBJECTIVE: Early life stress (ELS) occurring during childhood and adolescence is an established risk factor for later cardiovascular disease and dysregulated reactivity to acute social stress. This study examined whether ELS associations with baseline cardiovascular functioning, cardiovascular stress reactivity and recovery, and emotional stress reactivity vary across levels of emotion-oriented, task-oriented, and avoidant coping styles. METHODS: The sample included 1027 adolescents and young adults (mean age = 19.29 years; 50% female; 64% Black, 34% non-Hispanic White) who reported on their ELS exposure and coping styles. Participants completed a standardized acute social stress test (the Trier Social Stress Test [TSST]), with heart rate (HR) and blood pressure (BP) measured before, during, and after the TSST. Self-reports of negative emotions during the TSST indexed emotional stress reactivity. RESULTS: Multiple regression models adjusting for demographic factors and body mass index showed that ELS was associated with lower HR stress reactivity, avoidant coping was related to lower systolic BP and diastolic BP during stress and lower systolic BP during recovery, and higher emotion-oriented coping and lower task-oriented coping predicted greater emotional stress reactivity. A consistent pattern emerged where emotion-oriented coping amplified the associations between ELS and maladaptive stress responses (blunted cardiovascular stress reactivity and recovery; enhanced emotional stress reactivity), whereas lower levels of emotion-oriented coping were associated with resilient profiles among those who experienced ELS (lower resting HR, lower emotional stress reactivity, average HR and BP stress reactivity and recovery). However, low levels of emotion-oriented coping also conferred a risk of higher BP during recovery for those with high levels of ELS. CONCLUSIONS: These results suggest that low to moderate levels of emotion-oriented coping promote optimal cardiovascular and emotional reactivity to acute stress among individuals exposed to ELS.

Identification of Epigenetically Regulated Genes Distinguishing Intracranial from Extracranial Melanoma Metastases.

Despite remarkable advances in treating patients with metastatic melanoma, the management of melanoma brain metastases remains challenging. Recent evidence suggests that epigenetic reprogramming is an important mechanism for the adaptation of melanoma cells to the brain environment. In this study, the methylomes and transcriptomes of a cohort of matched melanoma metastases were evaluated by integrated omics data analysis. The identified 38 candidate genes displayed distinct promoter methylation and corresponding gene expression changes in intracranial compared with extracranial metastases. The 11 most promising genes were validated on protein level in both tumor and surrounding normal tissue using immunohistochemistry. In accordance with the underlying promoter methylation and gene expression changes, a significantly different protein expression was confirmed for STK10, PDXK, WDR24, CSSP1, NMB, RASL11B, phosphorylated PRKCZ, PRKCZ, and phosphorylated GRB10 in the intracranial metastases. The observed changes imply a distinct intracranial phenotype with increased protein kinase B phosphorylation and a higher frequency of proliferating cells. Knockdown of PRKCZ or GRB10 altered the expression of phosphorylated protein kinase B and decreased the viability of a brain-specific melanoma cell line. In summary, epigenetically regulated cancer-relevant alterations were identified that provide insights into the molecular mechanisms that discriminate brain metastases from other organ metastases, which could be exploited by targeting the affected signaling pathways.

Patient-specific identification of genome-wide DNA-methylation differences between intracranial and extracranial melanoma metastases.

Melanomas frequently metastasize to distant organs and especially intracranial metastases still represent a major clinical challenge. Epigenetic reprogramming of intracranial metastases is thought to be involved in therapy failure, but so far only little is known about patient-specific DNA-methylation differences between intra- and extracranial melanoma metastases. Hierarchical clustering of the methylomes of 24 patient-matched intra- and extracranial melanoma metastases pairs revealed that intra- and extracranial metastases of individual patients were more similar to each other than to metastases in the same tissue from other patients. Therefore, a personalized analysis of each metastases pair was done by a Hidden Markov Model to classify methylation levels of individual CpGs as decreased, unchanged or increased in the intra- compared to the extracranial metastasis. The predicted DNA-methylation alterations were highly patient-specific differing in the number and methylation states of altered CpGs. Nevertheless, four important general observations were made: (i) intracranial metastases of most patients mainly showed a reduction of DNA-methylation, (ii) cytokine signaling was most frequently affected by differential methylation in individual metastases pairs, but also MAPK, PI3K/Akt and ECM signaling were often altered, (iii) frequently affected genes were mainly involved in signaling, growth, adhesion or apoptosis, and (iv) an enrichment of functional terms related to channel and transporter activities supports previous findings for a brain-like phenotype. In addition, the derived set of 17 signaling pathway genes that distinguished intra- from extracranial metastases in more than 50% of patients included well-known oncogenes (e.g. PRKCA, DUSP6, BMP4) and several other genes known from neuronal disorders (e.g. EIF4B, SGK1, CACNG8). Moreover, associations of gene body methylation alterations with corresponding gene expression changes revealed that especially the three signaling pathway genes JAK3, MECOM, and TNXB differ strongly in their expression between patient-matched intra- and extracranial metastases. Our analysis contributes to an in-depth characterization of DNA-methylation differences between patient-matched intra- and extracranial melanoma metastases and may provide a basis for future experimental studies to identify targets for new therapeutic approaches.

Adverse childhood experiences are associated with vascular changes in adolescents that are risk factors for future cardiovascular disease.

BACKGROUND: Adverse childhood experiences (ACEs), such as abuse, neglect, and household dysfunction, are associated with a higher risk of cardiovascular disease (CVD) and indicators of future CVD risk in adulthood, such as greater vascular stiffness. The impact of ACEs in adolescence is unclear, and understanding how ACEs relate to blood pressure (BP) and vascular function during early life is key for the development of prevention strategies to reduce CVD risk. We hypothesized that exposure to ACEs would be associated with changes in central hemodynamics such as increased vascular stiffness and higher BP during adolescence. METHODS: This pilot study enrolled 86 adolescents recruited from the Children's of Alabama. A validated ACE questionnaire was employed, and ACEs were modeled both as a continuous variable and a categorical variable (ACE ≥ 1 vs. ACE = 0). The primary outcomes used are considered to be indicators of future cardio-renal disease risk: aortic augmentation index normalized to 75 bpm (Alx75, a surrogate for vascular stiffness), carotid-femoral PWV (m/s), and ambulatory BP patterns. RESULTS: Adolescents with ACE ≥ 1 had significantly higher Alx75 (ACE: 5.2% ± 2.2 compared to no ACE: - 1.4% ± 3.0; p = 0.043). PWV only reflected this trend when adjustments were made for the body mass index. Adolescents with ACEs showed no differences in ambulatory BP patterns during the 24-h, wake, or sleep periods compared to adolescents with no ACEs. CONCLUSIONS: ACEs were associated with higher AIx75 in adolescence, which is a risk factor for future CVD. Adolescence could present an opportunity for early detections/interventions to mitigate adverse cardiovascular outcomes in adulthood. A higher resolution version of the Graphical abstract is available as Supplementary information.