Real-world outcomes upon second-line treatment in patients with chronic lymphocytic leukaemia.

For chronic lymphocytic leukaemia (CLL), targeted drugs have become the standard of care, in particular for second-line treatment. In this study, overall survival (OS), treatment-free survival (TFS) and adverse events (AE) were registered retrospectively in a Danish population-based cohort upon second-line treatment for CLL. Data were collected from medical records and the Danish National CLL register. For 286 patients receiving second-line treatment, three-year TFS was higher upon targeted treatment (ibrutinib/venetoclax/idelalisib) [63%, 95% confidence interval (CI) 50%-76%] compared with fludarabine, cyclophosphamide and rituximab or bendamustine and rituximab (FCR/BR) (37%, CI: 26%-48%) and chlorambucil+/-CD20-antibody (CD20Clb/Clb) (22%, CI: 10%-33%). Upon targeted treatment, three-year OS estimates were higher for targeted treatment (79%, CI: 68%-91%) compared with FCR/BR (70%, CI: 60%-81%) or CD20Clb/Clb (60%, CI: 47%-74%). The most common AEs were infections and haematological AEs; 92% of patients treated with targeted drugs had AEs, 53% of which were severe. Upon FCR/BR and CD20Clb/Clb, AEs were present for 75% and 53% respectively, of which 63% and 31% were severe. These real-world data demonstrate higher TFS and a tendency towards higher OS following targeted second-line treatment for CLL compared to chemoimmunotherapy, also for patients who may be frailer and more comorbid.

Randomised controlled trial of in- versus out-patient management of benign hemithyroidectomy.

INTRODUCTION: Outpatient (OPT) thyroid surgery is increasing, with patient selection being pivotal for safety. While numerous studies exist, most are retrospective and encompass both benign and malignant cases. METHODS: We conducted a randomised clinical trial on patients undergoing hemithyroidectomy for benign thyroid disease. Participants were assigned to OPT or inpatient groups. We collected data on complications, failure to discharge on surgery day, post-operative pain, nausea, sleep quality and patient satisfaction. RESULTS: Among 97 patients, 27.5% (14/51) in the OPT group could not be discharged on the day of surgery due to minor complications, primarily nausea (36%) and neck swelling (29%). No reoperations were needed. Though OPT patients exhibited a higher rate of minor complications (29%), they reported less post-operative nausea, better sleep and a faster return to normal activity. CONCLUSIONS: Discharge on the day of surgery is not always possible with OPT thyroid surgery. However, our findings suggest that OPT hemithyroidectomy for benign cases can be both safe and feasible for a selected group of patients. FUNDING: None TRIAL REGISTRATION. CLINICALTRIALS: gov Identifier: NCT02891252.

Methylation microarray-based detection of clinical copy-number aberrations in CLL benchmarked to standard FISH analysis.

Copy-number aberrations (CNAs) are assessed using FISH analysis in diagnostics of chronic lymphocytic leukemia (CLL), but CNAs can also be extrapolated from Illumina BeadChips developed for genome-wide methylation microarray screening. Increasing numbers of microarray data-sets are available from diagnostic samples, making it useful to assess the potential in CNA diagnostics. We benchmarked the limitations of CNA testing from two Illumina BeadChips (EPIC and 450k) and using two common packages for analysis (conumee and ChAMP) to FISH-based assessment of 11q, 13q, and 17p deletions in 202 CLL samples. Overall, the two packages predicted CNAs with similar accuracy regardless of the microarray type, but lower than FISH-based assessment. We showed that the bioinformatics analysis needs to be adjusted to the specific CNA, as no general settings were identified. Altogether, we were able to predict CNAs using methylation microarray data, however, with limited accuracy, making FISH-based assessment of deletions the superior diagnostic choice.

Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM).

Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays' specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves.

Influence of Unequal Amplification of Methylated and Non-Methylated Template on Performance of Pyrosequencing.

Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.

IGHV-associated methylation signatures more accurately predict clinical outcomes of chronic lymphocytic leukemia patients than IGHV mutation load.

Currently, no molecular biomarker indices are used in standard care to make treatment decisions at diagnosis of chronic lymphocytic leukemia (CLL). We used Infinium MethylationEPIC array data from diagnostic blood samples of 114 CLL patients and developed a procedure to stratify patients based on methylation signatures associated with mutation load of the IGHV gene. This procedure allowed us to predict the time to treatment with a hazard ratio (HR) of 8.34 (95% confidence interval [CI]: 4.54-15.30), as opposed to a HR of 4.35 (95% CI: 2.60-7.28) using IGHV mutation status. Detailed evaluation of 17 cases for which the two classification procedures gave discrepant results showed that these cases were incorrectly classified using IGHV status. Moreover, methylation-based classification stratified patients with different overall survival (HR=1.82; 95% CI: 1.07-3.09), which was not possible using IGHV status. Furthermore, we assessed the performance of the developed classification procedure using published HumanMethylation450 array data for 159 patients for whom information on time to treatment, overall survival and relapse was available. Despite 450K array methylation data not containing all the biomarkers used in our classification procedure, methylation signatures again stratified patients with significantly better accuracy than did IGHV mutation load regarding all available clinical outcomes. Thus, stratification using IGHV-associated methylation signatures may provide better prognostic power than IGHV mutation status.

Nonclinical safety evaluation, pharmacokinetics, and target engagement of Lu AF82422, a monoclonal IgG1 antibody against alpha-synuclein in development for treatment of synucleinopathies.

Alpha-synuclein is a 15 kDa protein associated with neurodegenerative diseases such as Parkinson disease and multiple-system atrophy where pathological forms of alpha-synuclein aggregate and become neurotoxic. Here we describe the nonclinical program to support a first-in-human (FIH) single ascending dose (SAD) study for Lu AF82422, a human recombinant, anti-alpha-synuclein monoclonal antibody (mAb) in development for treatment of synucleinopathies. Alpha-synuclein is primarily expressed in brain, peripheral nerves and in blood cells. A tissue cross-reactivity assessment showed that Lu AF82422 binding was generally restricted to nervous tissues. Flow cytometry analysis did not show extracellular surface binding of Lu AF82422 to human platelets, erythrocytes, granulocytes, or lymphocytes, but to a low fraction of monocytes, without any functional consequences on activation or phagocytic capacity. A single dose pharmacokinetic (PK) study in cynomolgus monkeys with dose levels of 1-30 mg/kg confirmed PK properties in the expected range for a mAb with a soluble target, and target engagement was shown as a decrease in free alpha-synuclein in plasma. Four-week repeat-dose toxicity studies were conducted in rats and cynomolgus monkeys at doses up to 600 mg/kg administered intravenously every 10 days. Results showed no treatment-related adverse findings and the no-observed-adverse-effect-level was the highest dose tested. Target engagement was shown in plasma and cerebrospinal fluid. Taken together, the nonclinical data indicated no safety signal of concern and provided adequate safety margins between observed safe doses in animals and the planned dose levels in the FIH SAD study.

Bortezomib induces methylation changes in neuroblastoma cells that appear to play a significant role in resistance development to this compound.

The anticancer activity of bortezomib (BTZ) has been increasingly studied in a number of indications and promising results for the use of this treatment have been shown in neuroblastoma. As BTZ treatment is usually administered in cycles, the development of resistance and side effects in patients undergoing therapy with BTZ remains a major challenge for the clinical usage of this compound. Common resistance development also means that certain cells are able to survive BTZ treatment and bypass molecular mechanisms that render BTZ anticancer activity. We studied the methylome of neuroblastoma cells that survived BTZ treatment. Our results indicate that BTZ induces pronounced genome wide methylation changes in cells which recovered from the treatment. Functional analyses of identified methylation changes demonstrated they were involved in key cancer pathology pathways. These changes may allow the cells to bypass the primary anticancer activity of BTZ and develop a treatment resistant and proliferative phenotype. To study whether cells surviving BTZ treatment acquire a proliferative phenotype, we repeatedly treated cells which recovered from the first round of BTZ treatment. The repetitive treatment led to induction of the extraordinary proliferative potential of the cells, that increased with subsequent treatments. As we did not observe similar effects in cells that survived treatment with lenalidomide, and non-treated cells cultured under the same experimental conditions, this phenomenon seems to be BTZ specific. Overall, our results indicate that methylation changes may play major role in the development of BTZ resistance.

miR-151a enhances Slug dependent angiogenesis.

MicroRNAs (miRs) are small non-coding RNAs, that modulate cognate gene expression either by inducing mRNA degradation or by blocking translation, and play crucial and complex roles in tissue homeostasis and during disease initiation and progression. The sprouting of new blood vessels by angiogenesis is critical in vascular development and homeostasis and aberrant angiogenesis is associated with pathological conditions such as ischemia and cancer. We have previously established that miR-151a functions as an onco-miR in non-small cell lung cancer (NSCLC) cells by inducing partial EMT and enhancing tumor growth. Here, we identify anti-miR-151a as a molecule that promotes endothelial cell contacts and barrier properties, suggesting that miR-151a regulates cell-cell junctions. We find that induced miR-151a expression enhances endothelial cell motility and angiogenesis and these functions depend on miR-151a-induced Slug levels. Moreover, we show that miR-151a overexpression enhances tumor-associated angiogenesis in 3D vascularized tumor spheroid assays. Finally, we verify that miR-151a is expressed in the vasculature of normal lung and NSCLC tissue. Our results suggest that miR-151a plays multi-faceted roles in the lung, by regulating multiple functions (cell growth, motility, partial EMT and angiogenesis) in distinct cell types.

Disentangling public preferences for health gains at end-of-life: Further evidence of no support of an end-of-life premium.

In many countries, it has been publicly debated whether health gains for patients at end-of-life (EoL) should be valued higher than health gains for other patients. This has led to a range of stated preference studies examining the justification for an EoL premium on the basis of public preferences - so far with mixed findings. In the present study, we seek to extend this literature. We apply a simple stated preference approach with illustrative binary choices to elicit both individual and social preferences for several types of health gains. More specifically, we investigate whether health gains at EoL, resulting from either an improvement in quality of life (QoL) or life expectancy (LE) are valued differently from similarly sized health gains from preventive treatment and treatment of a temporary disease. Furthermore, we examine whether social preferences are affected by the age of beneficiaries. A web-based survey was conducted in 2015 using a random sample of 1047 members of the general public in Denmark. Overall, we do not find evidence to support an EoL premium compared to other health gains, neither when preferences are elicited from a social nor an individual perspective. Furthermore, our results demonstrate that the type of the health gain received matters to preferences for treatment at EoL with more weight given to gains in QoL than gains in LE. Finally, we find heterogeneity in preferences according to respondent characteristics, perspectives and age of beneficiaries.

Publisher Correction: Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Chronic lymphocytic leukemia patients with heterogeneously or fully methylated LPL promotor display longer time to treatment.

AIM: We investigated whether DNA methylation regulates expression of LPL and PI3K complex genes in chronic lymphocytic leukemia (CLL) and evaluated the prognostic significance of LPL promoter methylation in CLL patients. Patients & methods: Methylation of LPL promoter was assessed in 112 patients using methylation-sensitive high-resolution melting (MS-HRM). RESULTS: Patients with a fully or heterogeneously methylated LPL promoter had significantly longer median time to treatment (p < 0.001) and 75% lower (hazard ratio: 0.25; 95% CI: 0.15-0.42; p < 0.001) risk of requirement for treatment as opposed to patients with nonmethylated promoter. Multivariate modeling confirmed independent prognostic value of these findings. CONCLUSION: Chronic lymphocytic leukemia patients with a fully or heterogeneously methylated LPL gene promoter display indolent disease course and acquisition of heterogeneous methylation of LPL promoter is insufficient to induce gene expression.

Methylation-Sensitive High Resolution Melting (MS-HRM).

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

miRNA profiling identifies deregulated miRNAs associated with osteosarcoma development and time to metastasis in two large cohorts.

Osteosarcoma (OS) is an aggressive bone tumor primarily affecting children and adolescents. The etiology of OS is not fully understood. Thus, there is a great need to obtain a better understanding of OS development and progression. Alterations in miRNA expression contribute to the required molecular alterations for neoplastic initiation and progression. This study is the first to investigate miRNA expression in OS in a large discovery and validation cohort comprising a total of 101 OS samples. We established the signature of altered miRNA expression in OS by profiling the expression level of 752 miRNAs in 23 OS samples using sensitive LNA-enhanced qPCR assays. The identified miRNA expression changes were correlated with gene expression in the same samples. Furthermore, miRNA expression changes were validated in a second independent cohort consisting of 78 OS samples. Analysis of 752 miRNAs in the discovery cohort led to the identification of 33 deregulated miRNAs in OS. Twenty-nine miRNAs were validated with statistical significance in the second cohort comprising 78 OS samples. miRNA/mRNA targets were determined, and 361 genes with an inverse expression of the target miRNA were identified. Both the miRNAs and the identified target genes were associated with multiple pathways related to cancer as well as bone cell biology, thereby correlating the deregulated miRNAs with OS tumorigenesis. An analysis of the prognostic value of the 29 miRNAs identified miR-221/miR-222 to be significantly associated with time to metastasis in both cohorts. This study contributes to a more profound understanding of OS tumorigenesis, by substantiating the importance of miRNA deregulation. We have identified and validated 29 deregulated miRNAs in the - to our knowledge - largest discovery and validation cohorts used so far for miRNA analyses in OS. Two of the miRNAs showed a promising potential as prognostic biomarkers for the aggressiveness of OS.

The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle.

BACKGROUND: Transplanting myogenic cells and scaffolds for tissue engineering in skeletal muscle have shown inconsistent results. One of the limiting factors is neovascularization at the recipient site. Low intensity shockwave therapy (Li-SWT) has been linked to increased tissue regeneration and vascularization, both integral to survival and integration of transplanted cells. This study was conducted to demonstrate the response of myoblasts and skeletal muscle to Li-SWT. METHOD: Primary isolated human myoblasts and explants were treated with low intensity shockwaves and subsequently cell viability, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after cardiotoxin injection.. The animals were followed up and documented up to 21 days after cardiotoxin injury. RESULTS: Li-SWT had no significant effect on cell death, proliferation, differentiation and migration, the explants however showed decreased adhesion. In the animal experiments, qPCR studies revealed a significantly increased expression of apoptotic, angiogenic and myogenic genes; expression of Bax, Bcl2, Casp3, eNOS, Pax7, Myf5 and Met was increased in the early phase of regeneration in the Li-SWT treated hind limbs. Furthermore, a late accumulative angiogenic effect was demonstrated in the Li-SWT treated limbs by a significantly increased expression of Angpt1, eNOS, iNOS, Vegfa, and Pecam1. CONCLUSION: Treatment was associated with an early upregulation in expression of selected apoptotic, pro-inflammatory, angiogenic and satellite cell activating genes after muscle injury. It also showed a late incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro.

Associations between the structural and functional aspects of social relations and poor mental health: a cross-sectional register study.

BACKGROUND: Social relations influence mental health through different pathways. To capture the complexity of social relations, it is beneficial to consider both the structural (e.g., reachability of social network and social integration) and functional (e.g., instrumental and emotional support) aspects of the concept. Both aspects are rarely investigated simultaneously. This study aimed to examine the association between the structural and functional aspects of social relations and poor mental health. METHODS: The study was designed as a cross-sectional register study. We used data on mental health and social relations from 15,839 individuals aged 16-92 years with a mean age of 49.0 years (SD 17.9) who responded to The North Denmark Region Health Survey 2013 among residents in Northern Jutland, Denmark. The 12-Item Short-Form Health Survey measured mental health; a cut-off point of 44.5 was used to dichotomize participants into poor and good mental health. The categorization of social relations was inspired by Berkman et al.'s conceptual model of social relations and health. The analyses were performed with survey logistic regression. RESULTS: We found that 21.6% (n = 3422) of participants reported poor mental health, and 59% (n = 2020) of these were women. Being in contact with family and friends less than once a month statistically significantly increased the risk for poor mental health (Family OR = 1.78, 95% CI = 1.51-2.10 and Friends OR = 2.65, 95% CI = 2.30-3.06). The individuals who were not in contact with their network as often as they liked had a significantly higher risk for poor mental health (OR = 2.40, 95% CI = 2.20-2.62). Lack of instrumental support was associated with a higher risk for poor mental health (OR = 2.81, 95% CI = 2.26-3.48). We found an interaction between age and emotional support; the youngest population had the highest risk for poor mental health when they did not have access to emotional support (Young OR = 5.26, 95% CI = 3.91-7.09; Adult OR = 3.69, 95% CI = 3.17-4.30; and Elderly OR = 2.73, 95% CI = 2.23-3.34). CONCLUSIONS: Both structural and functional aspects of social relations were associated with poor mental health in our study. Rarely being in contact with friends and a lack of network reachability were associated with poor mental health. Likewise, low levels of emotional and instrumental support were associated with poor mental health.

Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations.

This is the first study to compare genome-wide DNA methylation profiles of sorted blood cells from myelofibrosis (MF) patients and healthy controls. We found that differentially methylated CpG sites located to genes involved in 'cancer' and 'embryonic development' in MF CD34+ cells, in 'inflammatory disease' in MF mononuclear cells, and in 'immunological diseases' in MF granulocytes. Only few differentially methylated CpG sites were common among the three cell populations. Mutations in the epigenetic regulators ASXL1 (47%) and TET2 (20%) were not associated with a specific DNA methylation pattern using an unsupervised approach. However, in a supervised analysis of ASXL1 mutated versus wild-type cases, differentially methylated CpG sites were enriched in regions marked by histone H3K4me1, histone H3K27me3, and the bivalent histone mark H3K27me3 + H3K4me3 in human CD34+ cells. Hypermethylation of selected CpG sites was confirmed in a separate validation cohort of 30 MF patients by pyrosequencing. Altogether, we show that individual MF cell populations have distinct differentially methylated genes relative to their normal counterparts, which likely contribute to the phenotypic characteristics of MF. Furthermore, differentially methylated CpG sites in ASXL1 mutated MF cases are found in regulatory regions that could be associated with aberrant gene expression of ASXL1 target genes.

The Common Follicle-Stimulating Hormone Receptor (FSHR) Promoter Polymorphism FSHR -29G > A Affects Androgen Production in Normal Human Small Antral Follicles.

Follicle-stimulating hormone receptors (FSHRs) are almost exclusively expressed on granulosa cells, and FSH action is probably most clearly reflected in intrafollicular hormone milieu of antral follicles. Little is known about the possible effects of the common single nucleotide polymorphism (SNP) FSHR -29G > A (rs1394205) on hormonal conditions in humsan small antral follicles (hSAFs) obtained from women in the natural menstrual cycle. This study investigated the follicle fluid (FF) concentrations of anti-Müllerian hormone, estradiol, progesterone, androstenedione, and testosterone in hSAF in relation to the different genotypes of FSHR -29G > A. FF from 362 follicles was collected in 95 women undergoing fertility preservation, who did not suffer from a disease that directly affected ovarian function. The testosterone levels of the minor A/A genotype were significantly increased compared to the A/G and the G/G genotype. Furthermore, significantly reduced androstenedione levels were observed for the G/G genotype, as compared to the A/G genotype, while the other hormones did not show statistical significant differences. In conclusion, the androgen levels of hSAF were significantly elevated in the minor SNP genotype in the FSHR promoter polymorphism FSHR -29G > A.

Small RNA sequencing reveals metastasis-related microRNAs in lung adenocarcinoma.

The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed that decreased expression of miR-30a-3p and increased expression of miR-210-3p were significantly associated with the presence of distant metastases. miR-210-3p tumor cell specificity was evaluated by in situ hybridization and its biomarker potential was confirmed by ROC curve analysis (AUC = 0.839). Lastly, agreement between miRNA-seq and RT-qPCR for FFPE-derived RNA was evaluated and a high level of concordance was determined. In conclusion, this study has identified and validated metastasis-related miRNAs in LAC.

The association between miR-34 dysregulation and distant metastases formation in lung adenocarcinoma.

Lung cancer has the highest mortality rate amongst human cancers and the majority of deaths can be attributed to metastatic spread. The miR-34 family includes three tumor suppressive miRs: miR-34a, miR-34b and miR-34c. miR-34 downregulation is a frequent observation in human malignancies and is often attributed to hypermethylation of the miR-34a and miR-34b/c promoters. Here, the potential association between aberrant miR-34 expression and promoter methylation and distant metastases formation in lung adenocarcinoma (LAC) is investigated. The expression levels of miR-34a, miR-34b and miR-34c, as well as the methylation status of the miR-34a and miR-34b/c promoters were determined in a LAC patient cohort comprising 26 non-metastasizing and 26 metastasizing primary LACs, as well as 24 paired distant metastases and 25 tumor-adjacent normal lung samples using RT-qPCR and Methylation-Sensitive High Resolution Melting (MS-HRM) analysis. No difference in expression was observed for miR-34a when comparing metastasizing and non-metastasizing LACs (p=0.793). For both miR-34b and miR-34c, a significantly lower expression level was determined in metastasizing LACs compared to non-metastasizing LACs (p=0.0005 and p=0.002) with similarly decreased expression levels observed in the paired distant metastases. Hypermethylation was detected in 35/51 LACs compared to 0/25 tumor-adjacent normal lungs for the miR-34a promoter (p<0.0001). Similarly, 18/51 LACs compared to 1/25 tumor-adjacent normal lungs showed hypermethylation of the miR-34b/c promoter (p=0.003). No difference in methylation was observed between metastasizing and non-metastasizing LACs for neither the miR-34a (p=0.832) nor the miR-34b/c (p=0.900) promoter. In conclusion, miR-34a and miR-34b/c promoter hypermethylation is a frequent event in LAC occurring in 68.7% and 35.3% of tested cases (n=51), respectively. Low miR-34b and miR-34c expression was associated with distant metastases formation in LAC. These changes can be targeted as novel biomarkers in LAC.