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Abstract Background The majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint gene CTLA4 with regard to its predictive value. Methods We analyzed CTLA4 promoter methylation in tumors of HNSCC patients (N = 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort (N = 138) of patients that did not receive ICB with regard to CTLA4 promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine. Results Lower CTLA4 promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression. CTLA4 promoter methylation inversely correlated with infiltrates of CD3+, CD4+, CD8+, and CD45+ immune cells. CTLA4 methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreased CTLA4 methylation and an induction of CTLA4 mRNA and CTLA-4 protein expression in HNSCC cell lines. Conclusions Our results indicate that CTLA4 DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value of CTLA4 DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.

Abstract Background Inducible T cell costimulator ICOS is an emerging target in immuno-oncology. The aim of this study was to investigate the epigenetic regulation of ICOS in melanoma by DNA methylation. Methods We comprehensively investigate ICOS DNA methylation of specific CpG sites and expression pattern within the melanoma microenvironment with regard to immune correlates, differentiation, clinical outcomes, and immune checkpoint blockade (ICB) response. Results Our study revealed a sequence-contextual CpG methylation pattern consistent with an epigenetically regulated gene. We found a cell type-specific methylation pattern and locus-specific correlations and associations of CpG methylation with ICOS mRNA expression, immune infiltration, melanoma differentiation, prognosis, and response to ICB. High ICOS mRNA expression was identified as a surrogate for enriched immune cell infiltration and was associated with favorable overall survival (OS) in non-ICB-treated patients and predicted response and a prolonged progression-free survival (PFS) following ICB therapy initiation. ICOS hypomethylation, however, significantly correlated with poor OS in non-ICB patients but predicted higher response and prolonged PFS and OS in ICB-treated patients. Moreover, we observed cytoplasmic and sporadically nuclear tumor cell-intrinsic ICOS protein expression. Tumor cell-intrinsic ICOS protein and mRNA expression was inducible by pharmacological demethylation with decitabine. Conclusion Our study identified ICOS DNA methylation and mRNA expression as promising prognostic and predictive biomarkers for immunotherapy in melanoma and points towards a hitherto undescribed role of ICOS in tumor cells.

ABSTRACTPD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC.

Background: Metastatic prostate cancer (mPCA) poses challenges in treatment response assessment, particularly in cases where prostate-specific antigen (PSA) levels do not reliably indicate a response. Liquid biopsy, focusing on circulating cell-free DNA (ccfDNA) methylation analysis as a proxy for circulating tumor DNA, offers a non-invasive and cost-effective approach. This study explores the potential of two methylation markers, short stature homeobox 2 (SHOX2) and Septin 9 (SEPT9), as on-mPCA-treatment biomarkers. Methods: Plasma samples were collected from 11 mPCA patients undergoing various treatments. Quantitative assessment of hypermethylated SHOX2 (mSHOX2) and SEPT9 (mSEPT9) levels in ccfDNA was conducted through methylation-specific real-time PCR. Early and overall dynamics of PSA, mSHOX2, and mSEPT9 were analyzed. Statistical evaluation employed Wilcoxon tests. Results: mSHOX2 demonstrated a significant decline post-treatment in patients with a radiographic treatment response as well as in an early treatment setting. mSEPT9 and PSA exhibited non-significant declines. In individual cases, biomarker dynamics revealed unique patterns compared to PSA. Discussion: mSHOX2 and mSEPT9 exhibit dynamics on mPCA treatment. This proof-of-concept study lays the groundwork for further investigation into these markers as valuable additions to treatment response monitoring in mPCA. Further validation in larger cohorts is essential for establishing clinical utility.

CTLA-4 blocking therapeutic antibodies are currently under investigation in head and neck squamous cell carcinoma (HNSCC). A better understanding of the epigenetic regulation of the CD28 superfamily members CD28, CTLA-4, and ICOS and their B7 ligands, CD80 and CD86, could support the development of biomarkers for response prediction to anti-CTLA-4 immunotherapy. We investigated methylation of the encoding genes CD28, CTLA4, ICOS, CD80, and CD86 at single CpG resolution (51 CpG sites) in a cohort of HNSCC (N = 528) and normal adjacent tissue samples (N = 50) provided by The Cancer Genome Research Atlas, in isolated blood leukocytes from healthy individuals (N = 28), and HNSCC cell lines (N = 39). We analysed methylation levels with regard to mRNA expression, overall survival, mutational load, interferon-γ signature, and signatures of immune cell infiltrates. Depending on the location of the CpG sites (promoter, promoter flank, gene body, and intergenic sites), we found significant differences in methylation levels among isolated leukocytes, between leukocytes and HNSCC cell lines, and among HNSCCs. Methylation of all analysed genes correlated inversely or positively with mRNA expression, depending on the CpG site. CD28, CTLA4, and ICOS revealed almost identical correlation patterns. Furthermore, we found significant correlations with survival and features of response to immunotherapy, i.e. interferon-γ signature, signatures of tumour infiltrating immune cells, and mutational load. Our results suggest CD28, CTLA4, ICOS, CD80, and CD86 expression levels are epigenetically co-regulated by DNA methylation. This study provides rationale to test their DNA methylation as potential biomarker for prediction of response to CTLA-4 immune checkpoint inhibitors.

Background: The immune checkpoint, indoleamine 2,3-dioxygenase 1, is under investigation as target of novel immunotherapies for cancers, including head and neck squamous cell carcinomas (HNSCC). The aim of our study was to analyze DNA methylation of the encoding gene (IDO1) in HNSCC. Methods: Methylation of three CpG sites within the promoter, promoter flank, and gene body was investigated and correlated with mRNA expression, immune cell infiltration, mutational burden, human papillomavirus (HPV)-status, and overall survival in a cohort of N = 528 HNSCC patients obtained from The Cancer Genome Atlas. In addition, IDO1 immunohistochemistry and DNA methylation analysis was performed in an independent cohort of N = 138 HNSCC samples. Findings: Significant inverse correlations of IDO1 methylation and IDO1 mRNA expression were found in the promoter and promoter flank region (Spearman's ρ = −0.163 and ρ = −0.377, respectively) while a positive correlation was present in the gene body (ρ = 0.502; all P

Abstract Background Few diagnostic and prognostic biomarkers are available for head-and-neck squamous cell carcinoma (HNSCC). Long non-coding RNAs (lncRNAs) have shown promise as biomarkers in other cancer types and in some cases functionally contribute to tumor development and progression. Here, we searched for lncRNAs useful as biomarkers in HNSCC. Methods Public datasets were mined for lncRNA candidates. Two independent HNSCC tissue sets and a bladder cancer tissue set were analyzed by RT-qPCR. Effects of lncRNA overexpression or downregulation on cell proliferation, clonogenicity, migration and chemosensitivity were studied in HNSCC cell lines. Results Data mining revealed prominently CASC9, a lncRNA significantly overexpressed in HNSCC tumor tissues according to the TCGA RNAseq data. Overexpression was confirmed by RT-qPCR analyses of patient tissues from two independent cohorts. CASC9 expression discriminated tumors from normal tissues with even higher specificity than HOTAIR, a lncRNA previously suggested as an HNSCC biomarker. Specificity of HNSCC detection by CASC9 was further improved by combination with HOTAIR. Analysis of TCGA pan-cancer data revealed significant overexpression of CASC9 across different other entities including bladder, liver, lung and stomach cancers and especially in squamous cell carcinoma (SCC) of the lung. By RT-qPCR analysis we furthermore detected stronger CASC9 overexpression in pure SCC of the urinary bladder and mixed urothelial carcinoma with squamous differentiation than in pure urothelial carcinomas. Thus, CASC9 might represent a general diagnostic biomarker and particularly for SCCs. Unexpectedly, up- or downregulation of CASC9 expression in HNSCC cell lines with low or high CASC9 expression, respectively, did not result in significant changes of cell viability, clonogenicity, migration or chemosensitivity. Conclusions CASC9 is a promising biomarker for HNSCC detection. While regularly overexpressed, however, this lncRNA does not seem to act as a major driver of development or progression in this tumor.

Background: The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. Methods: We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). Findings: Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. Interpretation: Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Background: Immunotherapy, including checkpoint inhibition, has remarkably improved prognosis in advanced melanoma. Despite this success, acquired resistance is still a major challenge. The T cell costimulatory receptor TNFRSF9 (also known as 4-1BB and CD137) is a promising new target for immunotherapy and two agonistic antibodies are currently tested in clinical trials. However, little is known about epigenetic regulation of the encoding gene. In this study we investigate a possible correlation of TNFRSF9 DNA methylation with gene expression, clinicopathological parameters, molecular and immune correlates, and response to anti-PD-1 immunotherapy to assess the validity of TNFRSF9 methylation to serve as a biomarker. Methods: We performed a correlation analyses of methylation at twelve CpG sites within TNFRSF9 with regard to transcriptional activity, immune cell infiltration, mutation status, and survival in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas. Furthermore, we used quantitative methylation-specific PCR to confirm correlations in a cohort of N = 115 melanoma patients’ samples (UHB validation cohort). Finally, we tested the ability of TNFRSF9 methylation and expression to predict progression-free survival (PFS) and response to anti-PD-1 immunotherapy in a cohort comprised of N = 121 patients (mRNA transcription), (mRNA ICB cohort) and a case-control study including N = 48 patients (DNA methylation, UHB ICB cohort). Findings: We found a significant inverse correlation between TNFRSF9 DNA methylation and mRNA expression levels at six of twelve analyzed CpG sites (P ≤ 0.005), predominately located in the promoter flank region. Consistent with its role as costimulatory receptor in immune cells, TNFRSF9 mRNA expression and hypomethylation positively correlated with immune cell infiltrates and an interferon-γ signature. Furthermore, elevated TNFRSF9 mRNA expression and TNFRSF9 hypomethylation correlated with superior overall survival. In patients receiving anti-PD-1 immunotherapy (mRNA ICB cohort), we found that TNFRSF9 hypermethylation and reduced mRNA expression correlated with poor PFS and response. Interpretation: Our study suggests that TNFRSF9 mRNA expression is regulated via DNA methylation. The observed correlations between TNFRSF9 DNA methylation or mRNA expression with known features of response to immune checkpoint blockage suggest TNFRSF9 methylation could serve as a biomarker in the context of immunotherapies. Concordantly, we identified a correlation between TNFRSF9 DNA methylation and mRNA expression with disease progression in patients under immunotherapy. Our study provides rationale for further investigating TNFRSF9 DNA methylation as a predictive biomarker for response to immunotherapy. Funding: AF was partly funded by the Mildred Scheel Foundation. SF received funding from the University Hospital Bonn BONFOR program (O-105.0069). DN was funded in part by DFG Cluster of Excellence ImmunoSensation (EXC 1023). The funders had no role in study design, data collection and analysis, interpretation, decision to publish, or preparation of the manuscript; or any aspect pertinent to the study. Keywords: TNFRSF9, DNA methylation, Melanoma, Prognostic biomarker, Predictive biomarker, Immune cell infiltration, Immune checkpoint, Immunotherapy, Response prediction, 4-1BB

Immune checkpoints are important targets for immunotherapies. However, knowledge on the epigenetic modification of immune checkpoint genes is sparse. In the present study, we investigated promoter methylation of CTLA4, PD-L1, PD-L2, and PD-1 in diffuse lower-grade gliomas (LGG) harboring isocitrate dehydrogenase (IDH) mutations with regard to mRNA expression levels, clinicopathological parameters, previously established methylation subtypes, immune cell infiltrates, and survival in a cohort of 419 patients with IDH-mutated LGG provided by The Cancer Genome Atlas. PD-L1, PD-L2, and CTLA-4 mRNA expression levels showed a significant inverse correlation with promoter methylation (PD-L1: p = 0.005; PD-L2: p

Abstract Background SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. Methods Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9/SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. Results Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61–63% sensitivity/90–92% specificity (SEPT9/training), 53–57% sensitivity/87–90% specificity (SHOX2/training), and 64–65% sensitivity/90–91% specificity (mean SEPT9/SHOX2 /training). Results were confirmed in a testing cohort with 54–56% sensitivity/88–90% specificity (SEPT9/testing), 43–48% sensitivity/93–95% specificity (SHOX2/testing), and 49–58% sensitivity/88–94% specificity (mean SEPT9/SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals (SEPT9: AUCtraining = 0.79–0.80; AUCtesting = 0.74–0.75; SHOX2: AUCtraining = 0.78–0.81, AUCtesting = 0.77–0.79; mean SEPT9/SHOX2 : AUCtraining = 0.81–0.84, AUCtesting = 0.80). The accurate prediction of overall survival was possible with all three algorithms (training cohort: HR SEPT9 = 1.23-1.90, HR SHOX2 = 1.14-1.85, HRmeanSEPT9/SHOX2 =1.19-1.89 ; testing cohort: HR SEPT9 =1.22-1.67, HR SHOX2 = 1.15-1.71, HRmeanSEPT9/SHOX2 = 1.12-1.77). Conclusion The concordant clinical performance based on different quantification algorithms allows for the application of various diagnostic platforms for the analysis of ccfDNA methylation biomarkers.

Mitochondrial dysfunction is common in cancer and the mitochondrial electron transport chain is often affected in carcinogenesis. To date, little is known about the expression of the ATP synthase subunits in clear cell renal cell carcinoma (ccRCC). The NextBio database was used to determine an expression profile of the ATP synthase subunits based on published microarray studies. We observed down-regulation of 23 out of 29 subunits of the ATP synthase. Differential expression was validated exemplarily for 12 genes (ATP5A1, ATP5B, ATPAF1, ATP5C1, ATP5D, ATP5O, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5I, ATP5S; screening cohort ccRCC n = 18 and normal renal tissue n = 10) using real-time PCR. Additional eight genes (ATP5A1, ATP5B, ATPAF1, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5S) were internally validated within an enlarged cohort (ccRCC n = 74; normal renal tissue n = 36). Furthermore, down-regulation of ATP5A1, ATPAF1, ATP5G1/G2/G3 was confirmed on the protein level using Western Blot and immunohistochemistry. We observed that altered expression of ATPAF1 and ATP5G1/G2/G3 was correlated with overall survival in patients with ccRCC. In conclusion, down-regulation of many ATP Synthase subunits occurs in ccRCC and is the basis for the reduced activity of the mitochondrial electron chain. Alteration of the expression of ATP5A1, ATPAF1, and ATP5G1/G2/G3 is characteristic for ccRCC and may be prognostic for ccRCC patients' outcome.

We have recently shown that centrosomal protein 57 (CEP57) is overexpressed in a subset of human prostate cancers. CEP57 is involved in intracellular transport processes, and its overexpression causes mitotic defects as well as abnormal microtubule nucleation and bundling. In the present study, we further characterized the prognostic and functional role of CEP57 in prostate cancer. Unexpectedly, we found that high CEP57 expression is an independent prognostic factor for a more favorable biochemical recurrence-free survival in two large patient cohorts. To reconcile this finding with the ability of CEP57 to cause cell division errors and thus potentially promote malignant progression, we hypothesized that alterations of microtubule-associated transport processes, in particular nuclear translocation of the androgen receptor (AR), may play a role in our finding. However, CEP57 overexpression and microtubule bundling had, surprisingly, no effect on the nuclear translocation of the AR. Instead, we found a significant increase of cells with disarranged microtubules and a cellular morphology suggestive of a cytokinesis defect. Because mitotic dysfunction leads to a reduced daughter cell formation, it can explain the survival benefit of patients with increased CEP57 expression. In contrast, we show that a reduced expression of CEP57 is associated with malignant growth and metastasis. Taken together, our findings underscore that high CEP57 expression is associated with mitotic impairment and less aggressive tumor behavior. Because the CEP57-induced microtubule stabilization had no detectable effect on AR nuclear translocation, our results furthermore suggest that microtubule-targeting therapeutics used in advanced prostate cancer such as docetaxel may have modes of action that are at least in part independent of AR transport inhibition.

Background: The adenosine A2a receptor (A2aR) and the adenosine synthesizing enzyme CD73 have recently evolved as a novel immunotherapeutic target. However, little is known about epigenetic modification of the encoding genes ADORA2A and NT5E. Methods: In the present study, we evaluated methylation at 23 loci of ADORA2A and 17 loci of NT5E with regard to transcriptional activity, human papilloma virus (HPV) status, immune cell infiltration, and outcome in a cohort of 279 head and neck squamous carcinoma (HNSCC) patients obtained from The Cancer Genome Atlas (TCGA). Methylation and mRNA expression were generated by the Infinium HumanMethylation450 BeadChip and Illumina HiSeq 2000 RNA Sequencing Version 2 analysis (Illumina, Inc., San Diego, CA, USA). HPV status was assessed by RNA-Seq data analysis of the viral genes E6 and E7. Results: Thirteen out of 23 ADORA2A loci and 15/17 NT5E loci were significantly correlated with mRNA levels (p

BACKGROUND:Patients with squamous cell cancer of the head and neck region (HNSCC) are at risk for disease recurrence and metastases, even after initial successful therapy. A tissue-based biomarker could be beneficial to guide treatment as well as post-treatment surveillance. Gene methylation status has been recently identified as powerful prognostic biomarker in HNSCC. We therefore evaluated the methylation status of the homeobox gene PITX1 and the adjacent long intergenic non-coding RNA (lincRNA) C5orf66-AS1 in publicly available datasets. METHODS:Gene methylation and expression data from 528 patients with HNSCC included in The Cancer Genome Atlas (TCGA, there obtained by using the Infinium HumanMethylation450 BeadChip Kit) were evaluated and methylation and expression levels of PITX1 and lincRNA C5orf66-AS1 was correlated with overall survival and other parameters. Thus, ten beads targeting PITX1 exon 3 and three beads targeting lincRNA C5orf66-AS1 were identified as significant candidates. The mean methylation of these beads was used for further correlation and the median was employed for dichotomization. RESULTS:Both PITX1 exon 3 and lincRNA C5orf66-AS1 were significantly higher methylated in tumor tissue than in normal adjacent tissue (NAT) (PITX1 exon 3: tumor tissue 58.1%, NAT: 31.7%, p

Despite advances in combined modality therapy, outcomes in head and neck squamous cell cancer (HNSCC) remain dismal with five-year overall survival rates of less than 50%. Prognostic biomarkers are urgently needed to identify patients with a high risk of death after initial curative treatment. Methylation status of the paired-like homeodomain transcription factor 2 (PITX2) has recently emerged as a powerful prognostic biomarker in various cancers. In the present study, the clinical performance of PITX2 methylation was validated in a HNSCC cohort by means of an independent analytical platform (Infinium HumanMethylation450 BeadChip, Illumina, Inc.).A total of 528 HNSCC patients from The Cancer Genome Atlas (TCGA) were included in the study. Death was defined as primary endpoint. PITX2 methylation was correlated with overall survival and clinicopathological parameters.PITX2 methylation was significantly associated with sex, tumor site, p16 status, and grade. In univariate Cox proportional hazards analysis, PITX2 hypermethylation analyzed as continuous and dichotomized variable was significantly associated with prolonged overall survival of HNSCC patients (continuous: hazard ratio (HR) = 0.19 [95%CI: 0.04-0.88], p = 0.034; dichotomized: HR = 0.52 [95%CI: 0.33-0.84], p = 0.007). In multivariate Cox analysis including established clinicopathological parameters, PITX2 promoter methylation was confirmed as prognostic factor (HR = 0.28 [95%CI: 0.09-0.84], p = 0.023).Using an independent analytical platform, PITX2 methylation was validated as a prognostic biomarker in HNSCC patients, identifying patients that potentially benefit from intensified surveillance and/or administration of adjuvant/neodjuvant treatment, i.e. immunotherapy.

Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated.Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history.In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16-93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients

Biliary tract cancers (BTC) are rare but highly aggressive malignant epithelial tumors. In order to improve the outcome in this lethal disease, novel biomarkers for diagnosis, prognosis, and therapy response prediction are urgently needed. DNA promoter methylation of PITX2 variants (PITX2ab, PITX2c) and intragenic methylation of the PITX2 adjacent non-coding RNA (PANCR) were investigated by methylations-specific qPCR assays in formalin-fixed paraffin-embedded tissue from 80 patients after resection for BTC. Results were correlated with clinicopathologic data and outcome. PITX2 variants and PANCR showed significant hypermethylation in tumor vs. normal adjacent tissue (p < 0.001 and p = 0.015), respectively. In survival analysis, dichotomized DNA methylation of variant PITX2c and PANCR were significantly associated with overall survival (OS). Patients with high tumor methylation levels of PITX2c had a shorter OS compared to patients with low methylation (12 vs. 40 months OS; HR 2.48 [1.38-4.48], p = 0.002). In contrast, PANCR hypermethylation was associated with prolonged survival (25 vs. 19 months OS; HR 0.54 [0.30-0.94], p = 0.015) and qualified as an independent prognostic factor on multivariate analysis. The biomarkers investigated in this study may help to identify BTC subpopulations at risk for worse survival. Further studies are needed to evaluate if PITX2 might be a clinically useful biomarker for an optimized and individualized treatment.

DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.

PURPOSE:Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy regimens we need better methods for the assessment of a therapy response. MATERIAL AND METHODS:In a pilot study we prospectively enrolled 36 patients with advanced NSCLC and SCLC (34 stage IV, 2 stage IIIB) of whom 34 received standard platinum-based chemo/radiotherapy and two were treated with a tyrosine kinase inhibitor. We measured the levels of extracellular methylated SHOX2 DNA (mSHOX2) in plasma before and during therapy until re-staging. The mSHOX2 analysis was blinded with respect to the clinical data making it an observational study. RESULTS:According to the re-staging of 31 first-line patients, 19 patients were classified as non-responders while 12 patients were in the responder group. We observed a tight correlation between radiological data and the change of plasma mSHOX2 level as the equivalent for a therapy response. A ROC analysis showed a high discriminatory power for both patient groups already one week after therapy start (AUC 0.844). Additionally, a Kaplan-Meier and Cox Proportional Hazards analyses revealed a strong relationship between survival and plasma mSHOX2 value p ≤ 0.001 (hazard ratio 11.08) providing some evidence for mSHOX2 also being a predictive marker. CONCLUSION:The longitudinal measurement of extracellular plasma mSHOX2 DNA yields information about the response to cytotoxic treatment and allows an early assessment of treatment response for lung cancer patients. If confirmed in a larger study this would be a valuable tool for selecting and guiding a cytotoxic treatment.

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.

Pleural effusions (PE) are a common clinical problem. The discrimination between benign (BPE), malignant (MPE) and paramalignant (PPE) pleural effusions is highly important to ensure appropriate patient treatment. Today, cytology is the gold standard for diagnosing malignant pleural effusions. However, its sensitivity is limited due to the sometimes low abundance of tumor cells and the challenging assessment of cell morphology in cytological samples. This study aimed to develop and validate a diagnostic test, which allows for the highly specific detection of malignant cells in pleural effusions based on the DNA methylation biomarkers SHOX2 and SEPT9. A quantitative real-time PCR assay was developed which enabled the accurate and sensitive detection of SHOX2 and SEPT9 in PEs. Cytological and DNA methylation analyses were conducted in a case control study comprised of PEs from 114 patients (58 cases, 56 controls). Cytological analysis as well as SHOX2 and SEPT9 methylation resulted in 100% specificity. 21% of the cases were cytologically positive and 26% were SHOX2 or SEPT9 methylation positive. The combined analysis of cytology and DNA methylation resulted in an increase of 71% positively classified PEs from cancer patients as compared to cytological analysis alone. The absolute sensitivity of cytology and DNA methylation was not determinable due to the lack of an appropriate gold standard diagnostic for distinguishing between MPEs and PPEs. Therefore, it was unclear which PEs from cancer patients were malignant (containing tumor cells) and which PEs were paramalignant and resulted from benign conditions in cancer patients, respectively. Furthermore, DNA methylation analysis in PEs allowed the prognosis of the overall survival in cancer patients (Kaplan-Meier analysis, log rank test, p=0.02 (SHOX2), p=0.02 (SEPT9)). The developed test may be used as a diagnostic and prognostic adjunct to existing clinical and cytopathological investigations in patients with PEs of unclear etiology.

Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.