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

Abstract 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 &amp;lt; 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 &amp;lt;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. </jats:sec

Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.

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

Correlation of <i>SHOX2</i> and <i>SEPT9</i> DNA methylation in PEs.

<p>Correlation of the methylation values of <i>SHOX2</i> and <i>SEPT9</i> in PEs from cancer patients. The p-value refers to a Pearson’s correlation.</p

<i>SHOX2</i> and <i>SEPT9</i> DNA methylation in PEs from cancer patients and patients with benign diseases.

<p>Methylation values of <i>SHOX2</i> and <i>SEPT9</i> in PEs from patients with cancer (cases) and patients with benign diseases (controls) determined by quantitative real-time PCR. The p-values refer to a Students t-test. A methylation cut-off was used to classify patient samples as <i>SHOX2</i> positive (above the cut-off).</p

Characteristics of the patient population.

<p>Clinical data of the 114 patients (58 cancer cases, 56 controls) included into the case control study.</p

Validation of cut-off (<i>SHOX2</i>).

<p>Statistical analysis of repeated measurements of pleural effusion samples and model DNA samples with different methylation levels close to the cut-off.</p

Diagnostic and Prognostic Value of <i>SHOX2</i> and <i>SEPT9</i> DNA Methylation and Cytology in Benign, Paramalignant and Malignant Pleural Effusions

<div><p>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 <i>SHOX2</i> and <i>SEPT9</i>. A quantitative real-time PCR assay was developed which enabled the accurate and sensitive detection of <i>SHOX2</i> and <i>SEPT9</i> 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 <i>SHOX2</i> and <i>SEPT9</i> methylation resulted in 100% specificity. 21% of the cases were cytologically positive and 26% were <i>SHOX2</i> or <i>SEPT9</i> 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 (<i>SHOX2</i>), p = 0.02 (<i>SEPT9</i>)). 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.</p></div

Survival analyses.

<p>Kaplan-Meier analysis of overall survival in 58 cancer patients stratified by the cytological diagnosis and the DNA methylation status of <i>SHOX2</i> and <i>SEPT9</i> in PEs. The p-values refer to the log rank test.</p

Cut-off validation.

<p>Cut-off validation for the <i>SHOX2</i> diagnostic assay. Model DNA with levels of unmethylated and artificially methylated DNA in addition to clinical samples were measured repeatedly in different runs. Methylation values of each sample and model DNA were measured in nine replicates.</p

Site (organ) specificity of malignant diseases in 58 cancer cases.

<p>one patient with lung, uterine cervix cancer and non-Hodgkin lymphoma, one patient suffering from lung and liver cancer, one patient with breast and bone cancer, and one patient with acute and chronic myeloid leukemia and breast cancer.</p