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.

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.

Cutaneous Lupus Erythematosus: An Update on Pathogenesis and Future Therapeutic Directions.

Lupus erythematosus comprises a spectrum of autoimmune diseases that may affect various organs (systemic lupus erythematosus [SLE]) or the skin only (cutaneous lupus erythematosus [CLE]). Typical combinations of clinical, histological and serological findings define clinical subtypes of CLE, yet there is high interindividual variation. Skin lesions arise in the course of triggers such as ultraviolet (UV) light exposure, smoking or drugs; keratinocytes, cytotoxic T cells and plasmacytoid dendritic cells (pDCs) establish a self-perpetuating interplay between the innate and adaptive immune system that is pivotal for the pathogenesis of CLE. Therefore, treatment relies on avoidance of triggers and UV protection, topical therapies (glucocorticosteroids, calcineurin inhibitors) and rather unspecific immunosuppressive or immunomodulatory drugs. Yet, the advent of licensed targeted therapies for SLE might also open new perspectives in the management of CLE. The heterogeneity of CLE might be attributable to individual variables and we speculate that the prevailing inflammatory signature defined by either T cells, B cells, pDCs, a strong lesional type I interferon (IFN) response, or combinations of the above might be suitable to predict therapeutic response to targeted treatment. Therefore, pretherapeutic histological assessment of the inflammatory infiltrate could stratify patients with refractory CLE for T-cell-directed therapies (e.g. dapirolizumab pegol), B-cell-directed therapies (e.g. belimumab), pDC-directed therapies (e.g. litifilimab) or IFN-directed therapies (e.g. anifrolumab). Moreover, Janus kinase (JAK) and spleen tyrosine kinase (SYK) inhibitors might broaden the therapeutic armamentarium in the near future. A close interdisciplinary exchange with rheumatologists and nephrologists is mandatory for optimal treatment of lupus patients to define the best therapeutic strategy.

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.

Characterization of B cells in lupus erythematosus skin biopsies in the context of different immune cell infiltration patterns.

Cutaneous lesions in lupus erythematosus (LE) subtypes are heterogenous. In line with the heterogeneity of the clinical presentation, the underlying lesional inflammation in LE skin samples is defined by different immune cell infiltrates. Pathophysiologically, lesional inflammation is driven by autoreactive cytotoxic T cells, targeting keratinocytes; plasmacytoid dendritic cells (pDCs), producing large amounts of interferon (IFN); and B cells, whose function in cutaneous LE is still unclear. This study aims to (a) classify inflammatory patterns with regard to the dominating cell type or cytokine expression and (b) investigating the specific role of B cells in LE skin lesions. Therefore, the immunohistological expression of inflammatory surrogates (CD20, CD123, MXA) in skin samples of n = 119 LE (subtypes: subacute cutaneous LE, chronic discoid LE, chilblain LE, LE tumidus, other LE) and n = 17 patients with inflammatory skin diseases (atopic dermatitis, psoriasis) were assessed. Samples were classified with regard to inflammatory groups. In addition multiplex-immunohistochemical analyses of n = 17 LE skin samples focusing on lesional B cells were conducted. In this study, we show that cutaneous lesions present with eight different inflammatory groups dominated by B cells, pDCs, a strong IFN expression, or overlapping patterns. Altogether, LE subtypes show heterogenous infiltration regardless of LE subtype, certain subtypes display a preference for infiltration groups. Furthermore, lesional B cells either form diffuse infiltrates or pseudofollicular structures, wherein they show antigen-presenting and T cell-activating properties. Altogether, in the light of emerging targeted therapeutic options, we suggest histological assessment in regard to B-cell or pDC preponderance to allow tailored treatment decisions.

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.

Current Concepts on Pathogenic Mechanisms and Histopathology in Cutaneous Lupus Erythematosus.

Cutaneous lupus erythematosus (CLE) is an interferon (IFN)-driven autoimmune disease that may be limited to the skin or can be associated with systemic lupus erythematosus (SLE). CLE occurs in several morphologic subtypes ranging from isolated, disc-shaped plaques to disseminated skin lesions. The typical histopathologic pattern of skin lesions is named interface dermatitis and characterized by a lymphocytic infiltrate and necroptotic keratinocytes at the dermo-epidermal junction. Other histopathologic patterns primarily involve the dermis or subcutis, depending on the subtype. One critical mechanism in CLE is the chronic reactivation of innate and adaptive immune pathways. An important step in this process is the recognition of endogenous nucleic acids released from dying cells by various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and other cytosolic receptors. Crucial cells in CLE pathogenesis comprise plasmacytoid dendritic cells (pDCs) as major producers of type I IFN, T cells exerting cytotoxic effects, and B cells, previously believed to contribute via secretion of autoantibodies. However, B cells are increasingly considered to have additional functions, supported by studies finding them to occur in highest numbers in chronic discoid lupus erythematosus (CDLE), a subtype in which autoantibodies are often absent. More precise knowledge of how CLE subtypes differ pathophysiologically may allow a tailored pharmacotherapy in the future, taking into account the specific molecular signature in relation to the morphologic subtype.

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.

Annular plaques mimicking Rowell's syndrome in the course of coronavirus disease 2019 mRNA vaccines: An overlooked phenomenon?

Effective vaccines for prevention of severe course and lethal outcome of coronavirus disease 2019 have been developed and approved in regulatory rolling and fast-track procedures; they are now widely distributed worldwide. Data about cutaneous side-effects of the new mRNA-type vaccines is scant, however. We herein report two similar cases of cutaneous adverse drug reactions (ADR) mimicking Rowell's syndrome that occurred after the first dose of BNT162b2 and mRNA-1273, respectively. Both patients achieved prompt clinical improvement with a short pulse of oral prednisolone and non-steroidal inflammatory drugs. We suspect this phenomenon to occur in a timeframe of 7-14 days after vaccination due to an interferon-γ-driven shift towards type I immunity in susceptible individuals. As rheumatic patients were excluded from phase III clinical trials and as most countries prioritized the elderly population to receive the vaccinations first, cutaneous ADR might become more frequent once the younger part of the population is vaccinated over the course of 2021. Atypical cutaneous ADR might be misinterpreted or overlooked by non-dermatologists. Further studies are required to determine the best suitable vaccine types for individual groups of patients.

Grzybowski's Generalized Eruptive Keratoacanthomas in a Patient with Terminal Kidney Disease-An Unmet Medical Need Equally Ameliorated by Topical Imiquimod Cream and Lapacho Tea Wraps: A Case Report.

INTRODUCTION: Development of singular keratoacanthoma (KA) is generally considered a benign condition as it has a tendency to regress spontaneously in spite of histological similarity to squamous cell carcinoma. Most KAs undergo excision to rule out differential diagnoses. Several alternative treatment modalities (keratinolytic, ablative, immunomodulating, antiproliferative, or targeted therapy) have been described in the past with varying success, underlining the therapeutic challenges associated with large or multiple lesions. Isomorphic response (Koebner phenomenon) may limit the efficacy of ablative options, and comorbidity may limit the use of systemic treatments. Less aggressive topical immunomodulatory treatment options represent an alternative with varying therapeutic success. CASE REPORT: Here, we describe the clinical course of a 51-year-old male patient with terminal kidney disease who suffered from the rare benign pruritic condition of Grzybowski's generalized eruptive keratoacanthomas (GEKA) and experienced a significant reduction of lesions and symptoms upon topical therapy with imiquimod 5% cream and lapacho tea dressings alike. CONCLUSIONS: Very little is known about the potential antiinflammatory or antiproliferative effects on the epidermis of the popular phytotherapeutic agent lapacho tea. More studies are warranted considering both the etiology and treatment of GEKA and topical use of phytotherapeutics in dermatology in general. Management of large or multiple KAs remains challenging.

Treatment Response Monitoring in Patients with Advanced Malignancies Using Cell-Free SHOX2 and SEPT9 DNA Methylation in Blood: An Observational Prospective Study.

Patients with incurable cancer usually receive palliative treatment with significant toxicity and limited efficacy. Methylation analysis of circulating cell-free DNA (ccfDNA) in blood from cancer patients represents a promising approach for minimally invasive, real-time monitoring of treatment response. Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) methylation was analyzed in N = 8865 malignant and N = 746 normal adjacent tissues across 33 different malignancies from The Cancer Genome Atlas. Furthermore, we performed quantitative SHOX2 and SEPT9 ccfDNA methylation analysis in plasma obtained before and consecutively during treatment from prospectively enrolled N = 115 patients with various advanced cancers. SHOX2 and/or SEPT9 hypermethylation in malignant tissues is present in various carcinomas, sarcoma, melanoma, brain tumors, mesothelioma, and hematopoietic malignancies. Among the prospectively enrolled cancer patients, 61% (70/115) of patients had a baseline-positive blood cumulative ccfDNA methylation score (CMS) and were eligible for response monitoring. Dynamic changes of CMS during treatment were strongly associated with treatment response. A CMS increase indicated response up to 80 days before conventional monitoring. SHOX2 and SEPT9 ccfDNA methylation represents a pan-cancer biomarker and has the potential to be a powerful tool for monitoring treatment response in patients with solid tumors and lymphomas. The early identification of nonresponders might allow for a timely change of treatment regimen.

The landscape of CD28, CD80, CD86, CTLA4, and ICOS DNA methylation in head and neck squamous cell carcinomas.

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.

Molecular and immune correlates of TIM-3 (HAVCR2) and galectin 9 (LGALS9) mRNA expression and DNA methylation in melanoma.

BACKGROUND: The T cell immunoglobulin and mucin-domain containing-3 receptor TIM-3 (also known as hepatitis A virus cellular receptor 2, encoded by HAVCR2) and its ligand galectin 9 (LGALS9) are promising targets for immune checkpoint inhibition immunotherapies. However, little is known about epigenetic regulation of the encoding genes. This study aimed to investigate the association of TIM-3 and LGALS9 DNA methylation with gene expression, patients' survival, as well as molecular and immune correlates in malignant melanoma. RESULTS: Methylation of all six TIM-3 CpGs correlated significantly with TIM-3 mRNA levels (P ≤ 0.05). A strong inverse correlation (Spearman's ρ = - 0.49) was found in promoter regions, while a strong positive correlation (ρ = 0.63) was present in the gene body of TIM-3. High TIM-3 mRNA expression (hazard ratio (HR) = 0.88, 95% confidence interval (CI) [0.81-0.97], P = 0.007) was significantly associated with better overall survival. Seven of the eight LGALS9 CpG sites correlated significantly with LGALS9 mRNA levels (P ≤ 0.003). Methylation at five CpG sites showed a strong inverse correlation (Spearman's ρ = - 0.67) and at two sites a weak positive correlation (Spearman's ρ = 0.15). High LGALS9 mRNA expression was significantly associated with increased overall survival (HR = 0.83, 95%CI [0.75-0.93], P = 0.001). In addition, we found significant correlations between TIM-3 and LGALS9 methylation and mRNA expression with immune cell infiltrates and significant differences among distinct immune cell subsets. CONCLUSIONS: Our study points toward an epigenetic regulation of TIM-3 and LGALS9 via DNA methylation and might provide an avenue for the development of a predictive biomarker for response to immune checkpoint blockade.

Cell-Free SHOX2 DNA Methylation in Blood as a Molecular Staging Parameter for Risk Stratification in Renal Cell Carcinoma Patients: A Prospective Observational Cohort Study.

BACKGROUND: Novel targeted treatments and immunotherapies have substantially changed therapeutic options for advanced and metastatic renal cell carcinomas (RCCs). However, accurate diagnostic tests for the identification of high-risk patients are urgently needed. Here, we analyzed SHOX2 mRNA expression in RCC tissues and SHOX2 gene body methylation quantitatively in circulating cell-free DNA (ccfDNA) and RCC tissues with regard to risk stratification. METHODS: The clinical performance of SHOX2 methylation was tested retrospectively and prospectively in a training and testing cohort of RCC tissue samples (n = 760 in total). SHOX2 mRNA expression analysis was included in the training cohort. In matched blood plasma samples from the testing cohort (n = 100), we prospectively examined the capability of pretherapeutic quantitative SHOX2 ccfDNA methylation to assess disease stage and identify patients at high risk of death. RESULTS: SHOX2 gene body methylation was positively correlated with mRNA expression in RCC tissues (training cohort: Spearman ρ = 0.23, P < 0.001). SHOX2 methylation in tissue and plasma strongly correlated with an advanced disease stage (training cohort: ρ = 0.28, P < 0.001; testing cohort/tissue: ρ = 0.40, P < 0.001; testing cohort/plasma: ρ = 0.34, P = 0.001) and risk of death after initial partial or radical nephrectomy [training cohort: hazard ratio (HR) = 1.40 (95% CI, 1.24-1.57), P < 0.001; testing cohort/tissue: HR = 1.16 (95% CI, 1.07-1.27), P = 0.001; testing cohort/plasma: HR = 1.50 (95% CI, 1.29-1.74), P < 0.001]. CONCLUSIONS: Pretherapeutic SHOX2 ccfDNA methylation testing allows for the identification of RCC patients at high risk of death after nephrectomy. These patients might benefit from an adjuvant treatment or early initiation of a palliative treatment.

Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients.

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.

Free-Circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis, and Monitoring of Head and Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort Study.

BACKGROUND: Circulating cell-free DNA methylation testing in blood has recently received regulatory approval for screening of colorectal cancer. Its application in other clinical settings, including staging, prognosis, prediction, and recurrence monitoring is highly promising, and of particular interest in head and neck squamous cell carcinomas (HNSCCs) that represent a heterogeneous group of cancers with unsatisfactory treatment guidelines. METHODS: Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) DNA methylation in plasma from 649 prospectively enrolled patients (training study: 284 HNSCC/122 control patients; testing study: 141 HNSCC/102 control patients) was quantified before treatment and longitudinally during surveillance. RESULTS: In the training study, 59% of HNSCC patients were methylation-positive at 96% specificity. Methylation levels correlated with tumor and nodal category (P < 0.001). Initially increased methylation levels were associated with a higher risk of death [SEPT9: hazard ratio (HR) = 5.27, P = 0.001; SHOX2: HR = 2.32, P = 0.024]. Disease recurrence/metastases were detected in 47% of patients up to 377 days earlier compared to current clinical practice. The onset of second cancers was detected up to 343 days earlier. In the testing study, sensitivity (52%), specificity (95%), prediction of overall survival (SEPT9: HR = 2.78, P = 0.022; SHOX2: HR = 2.50, P = 0.026), and correlation with tumor and nodal category (P <0.001) were successfully validated. CONCLUSIONS: Methylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients' outcomes.

PDCD1 (PD-1) promoter methylation predicts outcome in head and neck squamous cell carcinoma patients.

BACKGROUND: Biomarkers that facilitate the prediction of disease recurrence in head and neck squamous cell carcinoma (HNSCC) may enable physicians to personalize treatment. In the current study, DNA promoter methylation of programmed cell death 1 (PDCD1, PD-1) was evaluated as a prognostic biomarker in HNSCC patients. RESULTS: High PDCD1 methylation (mPDCD1) was associated with a significantly shorter overall survival after surgical resection in both the discovery (HR = 2.24 [95%CI: 1.08-4.64], p = 0.029) and the validation cohort (HR = 1.54 [95%CI: 1.08-2.21], p = 0.017). In multivariate Cox proportional hazards analysis, PDCD1 methylation remained a significant prognostic factor for HNSCC (HR = 2.14 [95%CI: 1.19-3.84], p = 0.011). Further, mPDCD1 was strongly associated with the human papilloma virus (HPV) status. MATERIALS AND METHODS: mPDCD1 was assessed retrospectively in a discovery cohort of 120 HNSCC patients treated at the University Hospital of Bonn and a validation cohort of 527 HNSCC cases analyzed by The Cancer Genome Atlas Research Network. CONCLUSIONS: PDCD1 methylation might aid the identification of HNSCC patients potentially benefitting from a radical or alternative treatment, particularly in the context of immunotherapies targeting PD-1/PD-L1.

PITX2 and PANCR DNA methylation predicts overall survival in patients with head and neck squamous cell carcinoma.

BACKGROUND: Squamous cell carcinoma of the head and neck region (HNSCC) is a common malignant disease accompanied by a high risk of local or distant recurrence after curative-intent treatment. Biomarkers that allow for the prediction of disease outcome can guide clinicians with respect to treatment and surveillance strategies. Here, the methylation status of PITX2 and an adjacent lncRNA (PANCR) were evaluated for their ability to predict overall survival in HNSCC patients. RESULTS: PITX2 hypermethylation was associated with a better overall survival (hazard ratio, HR = 0.51, 95%CI: 0.35-0.74, p<0.001), while PANCR hypermethylation was significantly associated with an increased risk of death (HR = 1.64, 95%CI: 1.12-2.39, p=0.010). METHODS: Quantitative, methylation-specific real-time PCR assays for PITX2 and PANCR were employed to measure bisulfite-converted DNA from formalin-fixed, paraffin-embedded (FFPE) tissues in a cohort of 399 patients with localized or locally advanced HNSCC who received curative-intent treatment (surgery with optional adjuvant radiochemotherapy or definite radiochemotherapy). CONCLUSIONS: PITX2 and PANCR methylation status were shown to be independent predictors for overall survival in HNSCC patients. Tissue-based methylation testing could therefore potentially be employed to identify patients with a high risk for death who might benefit from a more radical or alternative treatment.