NECTIN4 Amplification Is Frequent in Solid Tumors and Predicts Enfortumab Vedotin Response in Metastatic Urothelial Cancer.

PURPOSE: The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC. MATERIALS AND METHODS: We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs. RESULTS: NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers. CONCLUSION: NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

Hypermethylated SHOX2 in circulating cell-free DNA post renal cell carcinoma surgery as TNM-independent biomarker for recurrence risk.

INTRODUCTION: Adjuvant immune checkpoint inhibitor trials in renal cell carcinoma (RCC) call for improved recurrence risk stratification. Due to limitations of circulating tumor DNA (ctDNA) use in RCC, the use of hypermethylated SHOX2 gene (mSHOX2) in circulating cell-free DNA is explored as a surrogate marker for identifying high-risk patients after RCC surgery. METHODS: Liquid biopsies were collected post-surgery from 45 RCC patients (mean duration 4.3 days). Real-time polymerase chain reaction was used to analyze SHOX2 methylation in circulating cell-free DNA. Patients were categorized as mSHOX2 positive or negative by cut-off. Metastasis-free survival (MFS), cancer-specific survival (CSS), and overall survival (OS) were assessed using Cox regression and Log-rank analyses (median follow-up time: 60 months). RESULTS: 17 patients were mSHOX2 positive, showing unfavorable OS/CSS (Log-rank P = 0.004 and 0.02) and nearly 6-fold higher recurrence risk (hazard ratio 5.89, 95% CI 1.46-23.8). Multivariable Cox analysis confirmed mSHOX2 as an independent recurrence risk factor, disregarding TNM-based stratification. CONCLUSIONS: mSHOX2 effectively identifies high-risk RCC patients post-surgery, indicating minimal residual disease. This easy to implement biomarker has potential for guiding of adjuvant therapy decisions.

Circulating Cell-Free SHOX2 DNA Methylation Is a Predictive, Prognostic, and Monitoring Biomarker in Adjuvant and Palliative Anti-PD-1-Treated Melanoma.

BACKGROUND: The majority of metastatic melanoma patients initially do not respond or acquire resistance to anti-programmed cell death 1 (PD-1) immunotherapy. Liquid biopsy biomarkers might provide useful early response information and allow for personalized treatment decisions. METHODS: We prospectively assessed circulating cell-free SHOX2 DNA methylation (SHOX2 ccfDNAm) levels and their dynamic changes in blood plasma of melanoma patients by quantitative methylation-specific polymerase chain reaction. Patients were treated with either palliative (n = 42) or adjuvant (n = 55) anti-PD-1 immunotherapy. Moreover, we included n = 126 control patients without evidence of malignant disease. We analyzed SHOX2 ccfDNAm status prior to and 4 weeks after palliative treatment initiation with regard to outcome [objective response, progression-free survival (PFS), and overall survival (OS)]. In the adjuvant setting, we associated longitudinal SHOX2 ccfDNAm status with disease recurrence. RESULTS: Sensitivity was 60% with 25/42 melanoma patients showing increased SHOX2 ccfDNAm levels, whereas specificity was 98% with 123/126 (P < 0.001) control patients having SHOX2 ccfDNAm levels below cut-off. Pretreatment SHOX2 ccfDNAm status did not correlate with outcome; however, SHOX2 ccfDNAm negativity 4 weeks after palliative treatment initiation was strongly associated with improved survival [PFS: hazard ratio (HR) = 0.25, P = 0.002; OS: HR = 0.12, P = 0.007]. Pretreatment positive patients who reached SHOX2 ccfDNAm clearance after 4 weeks of immunotherapy showed an exceptionally beneficial outcome. SHOX2 ccfDNAm testing allowed for an early detection of distant metastases in adjuvant-treated melanoma patients. CONCLUSIONS: Our study suggests SHOX2 ccfDNAm to be an early predictor of outcome in anti-PD-1 treated melanoma patients. SHOX2 ccfDNAm testing may aid individualized treatment decision-making.

Feasibility of Monitoring Response to Metastatic Prostate Cancer Treatment with a Methylation-Based Circulating Tumor DNA Approach.

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.

CD52 mRNA expression predicts prognosis and response to immune checkpoint blockade in melanoma.

The immune-modulating protein CD52 attenuates lymphocyte function and is associated with autoimmune disorders, for example, multiple sclerosis (MS). CD52 represents a therapeutic target in MS and chronic lymphocytic leukemia (CLL). Its expression has prognostic and predictive value in CLL and is prognostic in breast cancer. Its significance in melanoma is unclear. We analyzed CD52 mRNA expression data from tumor bulk tissues of N = 445 untreated melanoma patients from The Cancer Genome Atlas (TCGA) Research Network and of N = 121 melanoma patients undergoing anti-PD-1 immune checkpoint blockade (ICB) with regard to outcome (overall survival [OS], disease control [DC], and progression-free survival [PFS]), single-cell RNA-Seq data of N = 4645 cells from N = 19 melanoma tissues, and N = 15,457 cells from normal skin provided by N = 5 donors. Higher CD52 mRNA expression was associated with favorable OS (hazard ratio (HR) = 0.820, [95% CI 0.734-0.916], p < .001) in non-ICB-treated melanoma and with PFS (HR = 0.875, [95% CI 0.775-0.989], p = .033) and DC (p = .005) in ICB-treated melanoma. CD52 expression correlated significantly with distinct immune cell subsets and correlated negatively with immune checkpoint expression in T cells. Moreover, our results suggest CD52 expression by a certain type of tissue-resident macrophages. CD52 mRNA was expressed in a small subgroup (8%) of immune checkpoint coexpressing melanoma cells. CD52 expression is associated with features of ICB response in melanoma. Concomitant ICB and anti-CD52 treatment requires critical review.

PD-L1 (CD274) promoter hypomethylation predicts immunotherapy response in metastatic urothelial carcinoma.

PD-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.

Circulating Cell-Free SEPT9 DNA Methylation in Blood Is a Biomarker for Minimal Residual Disease Detection in Head and Neck Squamous Cell Carcinoma Patients.

BACKGROUND: Tumorous SEPT9 (septin 9, SEPTIN9) circulating cell-free DNA (ccfDNA) methylation in blood plasma is a powerful biomarker for diagnosis, molecular staging, prognosis, and recurrence monitoring in head and neck squamous cell carcinoma (HNSCC) patients. The present study aimed to evaluate the clinical performance of SEPT9 ccfDNA methylation to detect post-surgical minimal residual disease (MRD) in patients with localized or locally advanced HNSCC treated with curative intent. METHODS: We applied quasi-digital methylation-specific real-time PCR to quantify SEPT9 ccfDNA methylation levels 2 to 30 days post-surgically in plasma from n = 219 prospectively enrolled HNSCC patients. We tested the associations of SEPT9 ccfDNA methylation with clinicopathological parameters and used Kaplan-Meier and Cox proportional hazards analyses for univariate, pairwise bivariate, and multivariate analyses of disease-free survival. RESULTS: Of 219 patients, 26.5% (58/219) were post-surgically SEPT9 ccfDNA methylation positive. SEPT9 ccfDNA methylation positivity was significantly associated with tumor site, American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC; 8th edition) tumor stage, nodal category and extracapsular extension, lymphatic and vascular invasion, and surgical margin. Bivariate Cox proportional hazards analysis proved post-surgical SEPT9 ccfDNA methylation positivity to be an independent prognostic factor tested together with AJCC/UICC tumor stage (SEPT9: hazard ratio [HR] = 2.43, 95% CI, 1.37-4.30, P = 0.002; AJCC/UICC stage: HR = 1.48, 95% CI, 1.11-1.98, P = 0.008). CONCLUSIONS: Post-surgical SEPT9 ccfDNA methylation may aid to identify high-risk HNSCC patients who could benefit from an intensified adjuvant treatment and surveillance.

CTLA4 DNA methylation is associated with CTLA-4 expression and predicts response to immunotherapy in head and neck squamous cell carcinoma.

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.

ICOS DNA methylation regulates melanoma cell-intrinsic ICOS expression, is associated with melanoma differentiation, prognosis, and predicts response to immune checkpoint blockade.

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.

DNA methylation of GITR, OX40, 4-1BB, CD27 , and CD40 correlates with BAP1 aberrancy and prognosis in uveal melanoma.

Uveal melanoma represents an aggressive tumor that responds mostly poorly to established melanoma treatments. Comprehensive methylation profiling of the next-generation immunotherapeutic target genes, for example, members of the tumor necrosis factor receptor superfamily, might allow for the development of companion predictive biomarkers. We have analyzed CpG sites within the immune checkpoint genes GITR, OX40, 4-1BB, CD 27, and CD40 probed by the Illumina Infinium HumanMethylation450 BeadChip in N = 80 uveal melanomas included in The Cancer Genome Atlas with regard to BAP1 aberrancy, mRNA expression, and overall survival. In all analyzed immune checkpoint genes, BAP1 aberrancy was associated with decreased CpG methylation levels. We identified specific CpG sites that significantly correlated with BAP1 aberrancy, mRNA expression levels, and overall survival. Our results suggest epigenetic regulation of the analyzed immune checkpoint genes via DNA methylation in uveal melanoma and provide rationale for methylation testing in biomarker programs in clinical trials.

Nectin-4 is widely expressed in head and neck squamous cell carcinoma.

PURPOSE: Nectin-4 has been successfully established as a target molecule in locally advanced and metastatic bladder cancer. An antibody-drug conjugate (enfortumab-vedotin) directed against nectin-4 has shown marked tumor remission rates in this tumor type, which is known for high expression rates of nectin-4. As head and neck cancer and urothelial carcinomas share morphological and molecular similarities, we aimed to evaluate Nectin-4 expression in head and neck squamous cell carcinoma (HNSCC). MATERIAL AND METHODS: A previously described and clinically characterized cohort of HNSCC (n = 159) was analyzed by immunohistochemistry for Nectin-4 expression. The expression data was correlated to clinico-pathological parameters including patient outcome. RESULTS: Nectin-4 was found in 86.2% of HNSCC, with medium/high expression seen in 32.7% of cases. Non smokers and p16 positive HNSCC showed a higher expression of Nectin-4 (p < 0.005). There was no correlation of Nectin-4 with grading or tumor stage. Nectin-4 positive tumors showed a significant better survival (log rank p = 0.006). CONCLUSIONS: Similar to urothelial carcinoma, Nectin-4 is found in the majority of HNSCC, which clearly warrants further studies to clarify if HNSCC also respond to targeted therapy with enfortumab-vedotin. Moreover, expression of Nectin-4 is associated with HPV infection and may serve as a prognostic marker in HNSCC.

CTLA4 , PD-1 , PD-L1 , PD-L2 , TIM-3 , TIGIT , and LAG3 DNA Methylation Is Associated With BAP1 -Aberrancy, Transcriptional Activity, and Overall Survival in Uveal Melanoma.

Uveal melanoma (UM) is an aggressive disease with poor response to oncological treatment, including immunotherapy. Loss of the epigenetic modifier BRCA1-associated protein 1 (BAP1) function drives UM oncogenesis and is associated with an immune-suppressive tumor microenvironment, poor prognosis, and a distinct DNA methylation and gene expression profile. Our study aimed to analyze comprehensively the DNA methylation status of the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4, TIM-3 ( HAVCR2 ), TIGIT , and LAG3 and its association with mRNA expression, BAP1 -aberrancy, and patients' survival. We analyzed the DNA methylation landscape of immune checkpoint genes at single CpG resolution in N=80 UM samples provided by The Cancer Genome Atlas. We analyzed CpG methylation levels of the immune checkpoints with regard to their transcriptional signatures and patient outcomes.Methylation of specific CpG sites within the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4 , TIM-3 , TIGIT , and LAG3 correlated strongly with mRNA expression levels, indicating a strong regulation of gene expression through DNA methylation. Moreover, immune checkpoint gene methylation was strongly associated with BAP1 -mutation status and associated with overall survival in UM. Our data indicate an epigenetic regulation of immune checkpoints through DNA methylation in UM. Further, our data highlight the prognostic significance of DNA methylation of immune checkpoint genes in UM thereby providing a rationale for methylation testing as predictive biomarkers for immunotherapy response.

DNA methylation regulates TIGIT expression within the melanoma microenvironment, is prognostic for overall survival, and predicts progression-free survival in patients treated with anti-PD-1 immunotherapy.

BACKGROUND: TIGIT is an immune checkpoint under investigation as therapeutic target. Understanding the regulation of TIGIT on an epigenetic level might support the development of companion biomarkers. METHODS: We correlated TIGIT DNA methylation of single CpG sites with gene expression, signatures of immune infiltrates and interferon-γ, and survival in melanoma. We further analyzed methylation levels in immune cell subsets, melanocyte and melanoma cell lines. TIGIT expression patterns within components of the melanoma microenvironment were analyzed by single cell sequencing. We used quantitative methylation-specific PCR, flow cytometry, and immunohistochemistry for correlations between expression and methylation and to assess the effect of pharmacological demethylation of melanoma cells treated with 5-aza-2-deoxycytidine (decitabine). Finally, we investigated the association of patients' survival with TIGIT mRNA and methylation. RESULTS: Depending on the sequence context of the analyzed CpG site, we found a cell type-specific TIGIT gene locus methylation pattern and significant correlations of TIGIT methylation with mRNA expression, an interferon γ signature, and distinct immune cell infiltrates, including TIGIT+ lymphocytes. We detected a melanoma cell-intrinsic TIGIT protein expression. Pharmacological demethylation of the A375 melanoma cell line led to a constitutive TIGIT expression. Low promoter flank methylation and high mRNA expression was associated with patients' prognosis and predicted progression-free survival in patients treated with anti-PD-1 immunotherapy. A high TIGIT+ lymphocyte score was associated with better progression-free survival under anti-PD-1 immunotherapy. CONCLUSIONS: Our data demonstrate an epigenetic regulation of TIGIT expression via DNA methylation within the melanoma microenvironment. TIGIT DNA methylation and expression may serve as predictive biomarkers in the context of immunotherapies in melanoma.

DNA Methylation and mRNA Expression of OX40 (TNFRSF4) and GITR (TNFRSF18, AITR) in Head and Neck Squamous Cell Carcinoma Correlates With HPV Status, Mutational Load, an Interferon-γ Signature, Signatures of Immune Infiltrates, and Survival.

The tumor necrosis factor receptor superfamily members 4 (TNFRSF4, OX40) and 18 (TNFRSF18, GITR, AITR) are under investigation as targets for immunotherapy of various cancers, including head and neck squamous cell carcinomas. Understanding the regulation of OX40 and GITR, particularly on an epigenetic level, might help to develop companion predictive biomarkers. We conducted broad correlation analyses of DNA methylation of 46 CpG sites within the GITR/OX40 gene locus in head and neck squamous cell carcinomas and normal adjacent tissues provided by The Cancer Genome Atlas (TCGA) Research Network. We analyzed methylation levels with regard to transcriptional gene activity (mRNA expression), human papillomavirus (HPV) infection, differential methylation between tumors and normal adjacent tissues, signatures of immune cell infiltrates, an interferon-γ signature, mutational load, and overall survival. Moreover, we investigated methylation levels in HPV-positive and HPV-negative cell lines and in isolated monocytes, granulocytes, CD8+ and CD4+ T cells, and B cells from peripheral blood from healthy donors. Our results revealed a complex and sequence-contextual methylation pattern in accordance with features of epigenetic regulated genes. We detected significant methylation differences between normal adjacent and tumor tissues, between HPV-positive and HPV-negative tumors, between tumor and immune cells, and significant correlations between methylation and mRNA expression. We further found significant correlations of CpG methylation with overall survival, signatures of immune cell infiltrates, an interferon-γ signature, and mutational load. Our study provides a framework to prospectively test specific CpG sites as biomarkers, in particular in the context of immunotherapies.

Clonal Hematopoiesis-Associated Gene Mutations in a Clinical Cohort of 448 Patients With Ovarian Cancer.

BACKGROUND: Cancer patients are at risk of secondary therapy-related myeloid neoplasms (t-MNs). Acquired blood-specific mutations in clonal hematopoiesis (CH)-associated genes are t-MN risk factors, and their occurrence associated with cancer therapy and age. Patients with ovarian cancer (OC) showed a particularly high prevalence of CH-associated gene mutations, which may additionally be explained by the high proportion of a hereditary disease cause in this cancer entity. METHODS: We performed a retrospective analysis of 448 OC patients enrolled in the AGO-TR1 study; 249 were enrolled at primary diagnosis and 199 at platinum-sensitive recurrence. Analyses included the most frequently altered CH-associated genes (ASXL1, DNMT3A, GNAS, JAK2, PPM1D, SF3B1, SH2B3, SRSF2, TET2, TP53). Results were analyzed according to the BRCA1/2 germline (gBRCA1/2) mutation status. All statistical tests were 2-sided. RESULTS: Advanced age at blood draw and a high number of prior platinum-based chemotherapy lines were risk factors to acquire CH-associated gene mutations, with gene-specific effects observed. Binomial logistic regression suggested increased probabilities for gBRCA1/2 mutation carriers to acquire CH-associated PPM1D and TP53 gene mutations (PPM1D: odds ratio = 4.30, 95% confidence interval = 1.48 to 12.46, P = .007; TP53: odds ratio = 6.20, 95% confidence interval = 0.98 to 53.9, P = .06). This observation was due to a statistically significantly increased number of platinum-based chemotherapy lines in gBRCA1/2 mutation carriers vs noncarriers (PPM1D: mean [SD] = 2.04 [1.27] vs 1.04 [0.99], P < .001; TP53: mean [SD] = 2.83 [1.33] vs 1.07 [1.01], P < .001). No interaction between platinum-based chemotherapy and gBRCA1/2 mutation status with the occurrence of CH-associated gene mutations was observed. CONCLUSIONS: A positive gBRCA1/2 mutation status is not a risk factor to acquire CH-associated gene mutations. OC patients may benefit from monitoring CH-associated gene mutations, especially following carboplatin exposure. Future clinical studies are required to assess whether treatment regimen should be adapted according to individual t-MN risks.

Sensitivity and specificity of loss of heterozygosity analysis for the classification of rare germline variants in BRCA1/2: results of the observational AGO-TR1 study (NCT02222883).

Variant-specific loss of heterozygosity (LOH) analyses may be useful to classify BRCA1/2 germline variants of unknown significance (VUS). The sensitivity and specificity of this approach, however, remains unknown. We performed comparative next-generation sequencing analyses of the BRCA1/2 genes using blood-derived and tumour-derived DNA of 488 patients with ovarian cancer enrolled in the observational AGO-TR1 trial (NCT02222883). Overall, 94 pathogenic, 90 benign and 24 VUS were identified in the germline. A significantly increased variant fraction (VF) of a germline variant in the tumour indicates loss of the wild-type allele; a decreased VF indicates loss of the variant allele. We demonstrate that significantly increased VFs predict pathogenicity with high sensitivity (0.84, 95% CI 0.77 to 0.91), poor specificity (0.63, 95% CI 0.53 to 0.73) and poor positive predictive value (PPV; 0.71, 95% CI 0.62 to 0.79). Significantly decreased VFs predict benignity with low sensitivity (0.26, 95% CI 0.17 to 0.35), high specificity (1.0, 95% CI 0.96 to 1.00) and PPV (1.0, 95% CI 0.85 to 1.00). Variant classification based on significantly increased VFs results in an unacceptable proportion of false-positive results. A significantly decreased VF in the tumour may be exploited as a reliable predictor for benignity, with no false-negative result observed. When applying the latter approach, VUS identified in four patients can now be considered benign. Trial registration number NCT02222883.

Identification of cytokeratin24 as a tumor suppressor for the management of head and neck cancer.

To improve management of head and neck squamous cell carcinoma patients, we need to increase our understanding of carcinogenesis, to identify biomarkers, and drug targets. This study aimed to identify novel biomarkers by providing transcriptomics profiles of matched primary tumors, lymph node metastasis, and non-malignant tissue of 20 HNSCC patients as well as by bioinformatic analyses of a TCGA HNSCC cohort, comprising 554 patients. We provide cancer cell signaling networks differentially expressed in tumors versus metastases, such as mesenchymal-epithelial transition, and structural integrity networks. As a proof of principle study, we exploited the data sets and performed functional analyses of a novel cytokeratin, cytokeratin24 (cKRT24), which had not been described as biomarker for tumors before. Survival analysis revealed that low cKRT24 expression correlated with poor overall survival in HNSCC. Experimentally, downregulation of cKRT24 in primary tumors, metastases, and HNSCC cell lines was verified on mRNA and protein level. Cloning and ectopic overexpression of cKRT24 not only affected viability and growth of HNSSC cell lines, but also inhibited tumor growth in murine xenograft studies. We conclude that cKRT24 functions as a tumor suppressor in HNSCC, and may serve as an additional prognostic biomarker and novel target to support current HNSCC treatments.

Fibroblast growth factor (FGF), FGF receptor (FGFR), and cyclin D1 (CCND1) DNA methylation in head and neck squamous cell carcinomas is associated with transcriptional activity, gene amplification, human papillomavirus (HPV) status, and sensitivity to tyrosine kinase inhibitors.

BACKGROUND: Dysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies. METHODS: We performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(-) HNSCC cell lines. RESULTS: We found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3-selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1. CONCLUSIONS: Our results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials.

Targeting Cancer Chemotherapy Resistance by Precision Medicine-Driven Nanoparticle-Formulated Cisplatin.

Therapy resistance is the major cause of cancer death. As patients respond heterogeneously, precision/personalized medicine needs to be considered, including the application of nanoparticles (NPs). The success of therapeutic NPs requires to first identify clinically relevant resistance mechanisms and to define key players, followed by a rational design of biocompatible NPs capable to target resistance. Consequently, we employed a tiered experimental pipeline from in silico to analytical and in vitro to overcome cisplatin resistance. First, we generated cisplatin-resistant cancer cells and used next-generation sequencing together with CRISPR/Cas9 knockout technology to identify the ion channel LRRC8A as a critical component for cisplatin resistance. LRRC8A's cisplatin-specificity was verified by testing free as well as nanoformulated paclitaxel or doxorubicin. The clinical relevance of LRRC8A was demonstrated by its differential expression in a cohort of 500 head and neck cancer patients, correlating with patient survival under cisplatin therapy. To overcome LRRC8A-mediated cisplatin resistance, we constructed cisplatin-loaded, polysarcosine-based core cross-linked polymeric NPs (NPCis, Ø âˆ¼ 28 nm) with good colloidal stability, biocompatibility (low immunogenicity, low toxicity, prolonged in vivo circulation, no complement activation, no plasma protein aggregation), and low corona formation properties. 2D/3D-spheroid cell models were employed to demonstrate that, in contrast to standard of care cisplatin, NPCis significantly (p < 0.001) eradicated all cisplatin-resistant cells by circumventing the LRRC8A-transport pathway via the endocytic delivery route. We here identified LRRC8A as critical for cisplatin resistance and suggest LRRC8A-guided patient stratification for ongoing or prospective clinical studies assessing therapy resistance to nanoscale platinum drug nanoformulations versus current standard of care formulations.

Regulation of Endothelial Nitric Oxide Synthase in the Reticular Lamina of the Organ of Corti by a Nitric Oxide Donor.

In the vertebrate cochlea, the reticular lamina seals the organ of Corti against the endolymph filled scala media. After noise exposure, fast alterations in the endothelial nitric oxide synthase (eNOS) expression level were identified in this cochlear structure. Minor amounts of nitric oxide (NO) produced by eNOS or applied by NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP) might protect this vulnerable part of the organ of Corti, on the line of gap junctions of supporting cells and cochlear microcirculation. In n=5 anesthetized guinea pigs, SNAP was intravenously applied in two concentrations. Six untreated animals served as controls. The cochleae were removed and prepared for immunoelectron microscopy using specific gold-labeled anti-eNOS antibodies. The density of the gold particles was quantified for seven cellular regions in the reticular lamina at the ultrastructural level. Following SNAP application, a significant increase in eNOS expression (+176%) was detected compared with controls (p=0.012). The increase occurred mainly in actin-rich cuticular structures and the prominent microtubules bundles. Correlation analysis revealed three clear and five moderate cellular associations for controls, whereas only one clear and one moderate after SNAP application. Thus, application of the NO donor SNAP resulted in an increase in eNOS expression in distinct regions of the reticular lamina.