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Background This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment. Methods Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance. Results Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]). Conclusions Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.

Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test. Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n = 1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard. The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6-11 pg/mL) corresponding to <2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%-80%) and for stage I-III CRC, 64% (48%-77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%-82%). The Epi proColon test is a simple, real-time PCR-based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines.

Background: The presence of aberrantly methylated SEPT9 DNA in plasma is highly correlated with the occurrence of colorectal cancer. We report the development of a new SEPT9 biomarker assay and its validation in case–control studies. The development of such a minimally invasive blood-based test may help to reduce the current gap in screening coverage. Methods: A new SEPT9 DNA methylation assay was developed for plasma. The assay comprised plasma DNA extraction, bisulfite conversion of DNA, purification of bisulfite-converted DNA, quantification of converted DNA by real-time PCR, and measurement of SEPT9 methylation by real-time PCR. Performance of the SEPT9 assay was established in a study of 97 cases with verified colorectal cancer and 172 healthy controls as verified by colonoscopy. Performance based on predetermined algorithms was validated in an independent blinded study with 90 cases and 155 controls. Results: The SEPT9 assay workflow yielded 1.9 μg/L (CI 1.3–3.0) circulating plasma DNA following bisulfite conversion, a recovery of 45%–50% of genomic DNA, similar to yields in previous studies. The SEPT9 assay successfully identified 72% of cancers at a specificity of 93% in the training study and 68% of cancers at a specificity of 89% in the testing study. Conclusions: Circulating methylated SEPT9 DNA, as measured in the new mSEPT9 assay, is a valuable biomarker for minimally invasive detection of colorectal cancer. The new assay is amenable to automation and standardized use in the clinical laboratory.

Background: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. Methods: We first used restriction enzyme–based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. Results: Restriction enzyme–based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%–73%] of plasma samples from CRC patients and not detected in 69% (62%–76%) of the controls. The corresponding results for NGFR were 51% (42%–60%) and 84% (77%–89%); for SEPT9, the values were 69% (60%–77%) and 86% (80%–91%). Conclusions: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

Patients whose tumors showed methylation and decreased expression of the gene TFAP2E were more than five times as likely as patients with normal TFAP2E gene expression to have resistance to fluorouracil. The treatment options and prognosis for patients with advanced colorectal cancer have improved through the development of novel drugs. 1 , 2 However, studies of the molecular biology of cancer initiation and progression have so far provided scant knowledge of the molecular mechanisms contributing to chemotherapy resistance. 2 Epigenetic alterations underlying the pathogenesis of colorectal cancer have been reported by several groups. 3 These alterations include hypomethylation and hypermethylation of DNA as well as histone modifications, all of which have a profound effect on transcriptional gene regulation. The role of these molecular alterations in response prediction and treatment resistance are far less well known. . . .

Background DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. Results This combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. Conclusion The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

Background Tinurilimab is a humanized immunoglobulin G subclass 2 antibody that blocks carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), an immune checkpoint regulator that is overexpressed in several tumor types. Objectives This phase I study evaluated the safety, tolerability, pharmacokinetics, pharmacodynamics, and tumor response profile of tinurilimab in patients with advanced solid tumors with a described expression of CEACAM6. Patients and Methods In this first-in-human, dose-escalation and dose-expansion study, tinurilimab was administered as a 1-h intravenous infusion in 21-day cycles at a starting dose of 2.5 mg, with a planned escalation up to 1800 mg. Following observation of treatment toxicity (cytokine release syndrome in one patient and neutropenia in all patients treated at 30 mg), a premedication regimen was initiated that included dexamethasone 8 mg before and after dosing. Thirty patients received treatment across six dosing cohorts (2.5–100 mg with or without dexamethasone). Results The maximum tolerated dose was not determined, as the study was terminated due to an unfavorable benefit/risk assessment. All 30 patients (100%) treated with tinurilimab experienced at least one treatment-emergent adverse event of any grade, most commonly fatigue (36.7%), infusion-related reaction (30.0%), and neutropenia (26.7%). The most common grade ≥ 3 treatment-related adverse events were neutropenia (23.3%), followed by febrile neutropenia, cytokine release syndrome, increased hepatic enzymes, decreased lymphocyte count, hypophosphatemia, lactic acidosis, and acute kidney injury (3.3% each). No patients reported an objective response. Conclusions Following study termination, the clinical development program for tinurilimab was discontinued permanently. Clinical trial registration www.clinicaltrials.gov , NCT03596372.

Tinurilimab is a humanized immunoglobulin G subclass 2 antibody that blocks carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), an immune checkpoint regulator that is overexpressed in several tumor types. This phase I study evaluated the safety, tolerability, pharmacokinetics, pharmacodynamics, and tumor response profile of tinurilimab in patients with advanced solid tumors with a described expression of CEACAM6. In this first-in-human, dose-escalation and dose-expansion study, tinurilimab was administered as a 1-h intravenous infusion in 21-day cycles at a starting dose of 2.5 mg, with a planned escalation up to 1800 mg. Following observation of treatment toxicity (cytokine release syndrome in one patient and neutropenia in all patients treated at 30 mg), a premedication regimen was initiated that included dexamethasone 8 mg before and after dosing. Thirty patients received treatment across six dosing cohorts (2.5-100 mg with or without dexamethasone). The maximum tolerated dose was not determined, as the study was terminated due to an unfavorable benefit/risk assessment. All 30 patients (100%) treated with tinurilimab experienced at least one treatment-emergent adverse event of any grade, most commonly fatigue (36.7%), infusion-related reaction (30.0%), and neutropenia (26.7%). The most common grade ≥ 3 treatment-related adverse events were neutropenia (23.3%), followed by febrile neutropenia, cytokine release syndrome, increased hepatic enzymes, decreased lymphocyte count, hypophosphatemia, lactic acidosis, and acute kidney injury (3.3% each). No patients reported an objective response. Following study termination, the clinical development program for tinurilimab was discontinued permanently. www. gov , NCT03596372.