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Background: Combination chemotherapy provides significant survival advantage in patients with advanced gastro-oesophageal adenocarcinoma compared with best supportive care. Peri-operative chemotherapy is standard of care for patients with operable disease. We hypothesised that biomarkers of genomic instability and inflammation may have clinical utility in these patients.

Methods: We initiated open-label, non-randomised biomarker studies in patients with advanced disease due to receive Epirubicin, Cisplatin and Capecitabine (ECX)/Epirubicin, Cisplatin and Fluorouracil (ECF) or Epirubicin, Oxaliplatin and Capecitabine (EOX)/Epirubicin, Oxaliplatin and Fluorouracil (EOF) regimens (advanced study, n = 375), and in patients planned to receive perioperative chemotherapy with the same regimen (peri-operative study, n = 306). Relative telomere length (RTL) in peripheral blood mononuclear cells (PBMCs) and plasma levels of 10 inflammatory cytokines were analysed to determine association with progression-free and overall survival, and response. Blood samples were collected prior to treatment and on each treatment cycle. Both studies comprised biomarker discovery and validation cohorts. Here we report analysis of the discovery cohorts.

Results: In advanced disease, high pre-treatment levels of IL8 and IL10 associated with poor Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis, and IL6 with poor OS. In multivariate analysis, IL6 and IL8 remained associated with OS, and IL8 with PFS. In the perioperative study, cytokine levels were significantly lower and no relationships were observed. There was no association between RTL and any endpoint in either study.

Conclusions: Pre-treatment RTL was not prognostic, although IL6/IL8 were negative prognostic factors in advanced disease. Levels of these were lower in patients with localised disease, suggesting an association with disease progression. Further analysis of systemic inflammatory status in gastro-oesophageal adenocarcinoma may be promising for development of future predictive biomarker signatures.

Air pollution is increasingly discussed as a risk factor for dementia, but the biological mechanisms are not yet fully understood. Biological markers like telomere length are relevant to study with air pollution, as they are associated with aging and dementia. The study aimed to investigate the relationship between source-specific air pollution exposure and telomere length in a low-level air pollution area, and whether this potential relationship depended on future dementia status. The data originated from the Betula study in Northern Sweden, where 509 participants recruited between 1988 and 1995 were included to investigate the association between annual mean air pollution concentrations at the participants’ residences and relative leukocyte telomere length using a linear regression model. No association was observed between air pollution and telomere length, with regression slope estimates close to zero and p-values > 0.10 (e.g. PM2.5_total: β = 0.01 (-0.011, 0.025) and BC_total: β = 0.03 (95% CI: -0.046, 0.114). There were indications of a positive association between longer telomere length and higher exposure to air pollution among individuals later diagnosed with dementia (N = 74), but these findings were not conclusive (p-values > 0.10) (PM2.5_total: β = 0.03, p-value = 0.12; BC_total: β = 0.11, p-value = 0.17). Although not statistically significant, our findings contribute to the evidence from low-exposure settings, and it is important to report these types of findings for a balanced understanding of potential health effects.

Current intensive treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has substantial side effects, highlighting a need for novel biomarkers to improve risk stratification. Canonical biomarkers, such as genetics and immunophenotype, are largely not used in pediatric T-ALL stratification. This study aimed to validate the prognostic relevance of DNA methylation CpG island methylator phenotype (CIMP) risk stratification in 2 pediatric T-ALL patient cohorts: the Nordic Society of Paediatric Haematology (NOPHO) ALL2008 T-ALL study cohort (n = 192) and the Dutch Childhood Oncology Group (DCOG) ALL-10/ALL-11 validation cohorts (n = 156). Both cohorts revealed that combining CIMP classification at diagnosis with measurable residual disease (MRD) at treatment day 29 (D29) or 33 (D33) significantly improved outcome prediction. The poor prognosis subgroup, characterized by CIMP low/D29 or D33 MRD ≥ 0.1%, had a cumulative incidence of relapse (pCIR5yr) of 29% and 23% and overall survival (pOS5yr) of 59.7% and 65.4%, in NOPHO and DCOG, respectively. Conversely, a good prognosis subgroup was also identified representing CIMP high/D29 or D33 MRD < 0.1% with pCIR5yr of 0% and 3.4% and pOS5yr of 98.2% and 94.8%, in NOPHO and DCOG, respectively. For NOPHO, MRD was also evaluated on D15, and the relapse prediction accuracy of CIMP/D29 MRD (0.79) and CIMP/D15 MRD (0.75) classification was comparable, indicating potential for earlier stratification. The evaluation of the biology behind the CIMP subgroups revealed associations with transcriptome profiles, genomic aberrations, and mitotic history, suggesting distinct routes for leukemia development. In conclusion, integrating MRD assessment with the novel CIMP biomarker has the potential to improve risk stratification in pediatric T-ALL and guide future therapeutic decisions.

The telomerase enzyme is essential for telomere maintenance. Pathogenic variants in telomere-associated genes have been associated with critical telomere shortening, resulting in telomere biology disorders (TBD) such as bone marrow failure, idiopathic pulmonary fibrosis, and dyskeratosis congenita. The TBDs are clinically heterogeneous and families with TBD often experience an earlier onset and increased symptom severity for each generation. Consensus guidelines have identified certain genetic variants as pathogenic or likely pathogenic, but many are classified as variants of uncertain significance (VUS) in the absence of additional supporting evidence. The pathogenicity of a VUS in genes encoding the telomerase complex could be evaluated by in vitro telomerase activity (TA) measurement. We have developed a functional TA assay in patient-derived T-cells based on the Telomeric Repeat Amplification Protocol (TRAP) combined with qPCR. TA was significantly lower in six TBD patients with a TERT or TERC variant compared to controls (0.11 versus 0.54, p < 0.001). Four patients had a TA of more than three standard deviations below the mean of controls, strongly supporting pathogenicity of the variants. In summary, functional analysis of TA in patient-derived cells could support pathogenic evaluation in clinical diagnostics and reduce the number of reported VUS for TBD patients.

Relapsed and refractory disease in children with T-cell acute lymphoblastic leukemia (R/R T-ALL) remains a major clinical challenge. Outcomes for children who relapse or exhibit resistance to initial treatments are dismal, with survival rates frequently below 25% despite aggressive therapy. To minimize toxicities and improve outcomes, individualized precision medicine approaches targeting the underlying biology of R/R T-ALL are especially important, considering that T-ALL is characterized by genetic, epigenetic and posttranscriptional heterogeneity, and organ and niche specificities (e.g. the central nervous system), all of which underlie disease progression and therapy resistance. Here, we summarize the current understanding of the complexity of pediatric T-ALL biology and how such knowledge may be clinically leveraged, emphasizing the need for innovative therapeutic routes to improve outcomes for children with R/R T-ALL. Emerging approaches that hold promise or show palpable results include proteasome inhibitors, BCL-2 antagonists, and JAK (for JAK- and IL-7R-driven cases), ABL and SRC family tyrosine kinase (for LCK-activated cases), MEK or PI3K-mTOR inhibitors. MYC-targeting agents, DNA demethylating agents, histone deacetylase inhibitors, splicing modulators, or drugs exploring T-ALL metabolic vulnerabilities, are other examples for potential pharmacological intervention. Immunotherapies, particularly CAR T-cell products targeting CD7 and other markers, but also biologics (e.g. targeting CD38), are under development and increasing interest. These agents should be rationally integrated into precision medicine combination therapies informed by genetic, epigenetic, and posttranscriptional insights that will be essential to refine risk stratification and minimize the risk of resistance. Novel strategies leveraging artificial intelligence and machine learning could accelerate discovery and optimize treatment frameworks.

Background: Large B-cell lymphoma (LBCL) is the most common lymphoma and is known to be a biologically heterogeneous disease regarding genetic, phenotypic, and clinical features. Although the prognosis is good, one-third has a primary refractory or relapsing disease which underscores the importance of developing predictive biological markers capable of identifying high- and low-risk patients. DNA methylation (DNAm) and telomere maintenance alterations are hallmarks of cancer and aging. Both these alterations may contribute to the heterogeneity of the disease, and potentially influence the prognosis of LBCL.

Results: We studied the DNAm profiles (Infinium MethylationEPIC BeadChip) and relative telomere lengths (RTL) with qPCR of 93 LBCL cases: Diffuse large B-cell lymphoma not otherwise specified (DLBCL, n = 66), High-grade B-cell lymphoma (n = 7), Primary CNS lymphoma (n = 8), and transformation of indolent B-cell lymphoma (n = 12). There was a substantial methylation heterogeneity in DLBCL and other LBCL entities compared to normal cells and other B-cell neoplasms. LBCL cases had a particularly aberrant semimethylated pattern (0.15 ≤ β ≤ 0.8) with large intertumor variation and overall low hypermethylation (β > 0.8). DNAm patterns could not be used to distinguish between germinal center B-cell-like (GC) and non-GC DLBCL cases. In cases treated with R-CHOP-like regimens, a high percentage of global hypomethylation (β < 0.15) was in multivariable analysis associated with worse disease-specific survival (DSS) (HR 6.920, 95% CI 1.499–31.943) and progression-free survival (PFS) (HR 4.923, 95% CI 1.286–18.849) in DLBCL and with worse DSS (HR 5.147, 95% CI 1.239–21.388) in LBCL. These cases with a high percentage of global hypomethylation also had a higher degree of CpG island methylation, including islands in promoter-associated regions, than the cases with less hypomethylation. Additionally, telomere length was heterogenous in LBCL, with a subset of the DLBCL-GC cases accounting for the longest RTL. Short RTL was independently associated with worse DSS (HR 6.011, 95% CI 1.319–27.397) and PFS (HR 4.689, 95% CI 1.102–19.963) in LBCL treated with R-CHOP-like regimens.

Conclusion: We hypothesize that subclones with high global hypomethylation and hypermethylated CpG islands could have advantages in tumor progression, e.g. by inactivating tumor suppressor genes or promoting treatment resistance. Our findings suggest that cases with high global hypomethylation and thus poor prognosis could be candidates for alternative treatment regimens including hypomethylating drugs.

Late-onset Fuchs endothelial corneal dystrophy (FECD) is a disease affecting the corneal endothelium (CE), associated with a cytosine-thymine-guanine repeat expansion at the CTG18.1 locus in the transcription factor 4 (TCF4) gene. It is unknown whether CTG18.1 expansions affect global methylation including TCF4 gene in CE or whether global CE methylation changes at advanced age. Using genome-wide DNA methylation array, we investigated methylation in CE from FECD patients with CTG18.1 expansions and studied the methylation in healthy CE at different ages. The most revealing DNA methylation findings were analyzed by gene expression and protein analysis. 3488 CpGs had significantly altered methylation pattern in FECD though no substantial changes were found in TCF4. The most hypermethylated site was in a predicted promoter of aquaporin 1 (AQP1) gene, and the most hypomethylated site was in a predicted promoter of coagulation factor V (F5 for gene, FV for protein). In FECD, AQP1 mRNA expression was variable, while F5 gene expression showed a ~ 23-fold increase. FV protein was present in both healthy and affected CE. Further gene expression analysis of coagulation factors interacting with FV revealed a ~ 34-fold increase of thrombomodulin (THBD). THBD protein was detected only in CE from FECD patients. Additionally, we observed an age-dependent hypomethylation in elderly healthy CE.Thus, tissue-specific genome-wide and gene-specific methylation changes associated with altered gene expression were discovered in FECD. TCF4 pathological methylation in FECD because of CTG18.1 expansion was ruled out.

Telomere Biology Disorders (TBDs) are characterized by mutations in telomere-related genes leading to short telomeres and premature aging but with no strict correlation between telomere length and disease severity. Epigenetic alterations are also markers of aging and we aimed to evaluate whether DNA methylation (DNAm) could be part of the pathogenesis of TBDs. In blood from 35 TBD cases, genome-wide DNAm were analyzed and the cases were grouped based on relative telomere length (RTL): short (S), with RTL close to normal controls, and extremely short (ES). TBD cases had increased epigenetic age and DNAm alterations were most prominent in the ES-RTL group. Thus, the differentially methylated (DM) CpG sites could be markers of short telomeres but could also be one of the mechanisms contributing to disease phenotype since DNAm alterations were observed in symptomatic, but not asymptomatic, cases with S-RTL. Furthermore, two or more DM-CpGs were identified in four genes previously linked to TBD or telomere length (PRDM8, SMC4, VARS, and WNT6) and in three genes that were novel in telomere biology (MAS1L, NAV2, and TM4FS1). The DM-CpGs in these genes could be markers of aging in hematological cells, but they could also be of relevance for the progression of TBD.

T-cell lymphoblastic lymphoma (T-LBL) is a rare and aggressive lymphatic cancer, often diagnosed at a young age. Patients are treated with intensive chemotherapy, potentially followed by a hematopoietic stem cell transplantation. Although prognosis of T-LBL has improved with intensified treatment protocols, they are associated with side effects and 10–20% of patients still die from relapsed or refractory disease. Given this, the search toward less toxic anti-lymphoma therapies is ongoing. Here, we targeted the recently described DNA hypermethylated profile in T-LBL with the DNA hypomethylating agent decitabine. We evaluated the anti-lymphoma properties and downstream effects of decitabine, using patient derived xenograft (PDX) models. Decitabine treatment resulted in prolonged lymphoma-free survival in all T-LBL PDX models, which was associated with downregulation of the oncogenic MYC pathway. However, some PDX models showed more benefit of decitabine treatment compared to others. In more sensitive models, differentially methylated CpG regions resulted in more differentially expressed genes in open chromatin regions. This resulted in stronger downregulation of cell cycle genes and upregulation of immune response activating transcripts. Finally, we suggest a gene signature for high decitabine sensitivity in T-LBL. Altogether, we here delivered pre-clinical proof of the potential use of decitabine as a new therapeutic agent in T-LBL.

Background: DNA methylation (DNAm), an epigenetic mark reflecting both inherited and environmental influences, hasshown promise for Alzheimer’s disease (AD) prediction.Objective: Testing long-term predictive ability (>15 years) of existing DNAm-based epigenetic age acceleration (EAA)measures and identifying novel early blood-based DNAm AD-prediction biomarkers.

Methods: EAA measures calculated from Illumina EPIC data from blood were tested with linear mixed-effects models(LMMs) in a longitudinal case-control sample (50 late-onset AD cases; 51 matched controls) with prospective data up to 16years before clinical onset, and post-onset follow-up. NovelDNAmbiomarkers were generated with epigenome-wide LMMs,and Sparse Partial Least Squares Discriminant Analysis applied at pre- (10–16 years), and post-AD-onset time-points.

Results: EAA did not differentiate cases from controls during the follow-up time (p > 0.05). Three new DNA biomarkersshowed in-sample predictive ability on average 8 years pre-onset, after adjustment for age, sex, and white blood cell proportions(p-values: 0.022-<0.00001). Our longitudinally-derived panel replicated nominally (p = 0.012) in an external cohort (n = 146cases, 324 controls). However, its effect size and discriminatory accuracy were limited compared to APOE 4-carriership(OR = 1.38 per 1 SD DNAmscore increase versus OR= 13.58 for 4-allele carriage; AUCs = 77.2% versus 87.0%). Literaturereview showed low overlap (n = 4) across 3275 AD-associated CpGs from 8 published studies, and no overlap with ouridentified CpGs.

Conclusion: The limited predictive value of EAA for AD extends prior findings by considering a longer follow-up time, andwith appropriate control for age, sex, APOE, and blood-cell proportions. Results also highlight challenges with replicatingdiscriminatory or predictive CpGs across studies.