Methylation of NRIP3 Is a Synthetic Lethal Marker for Combined PI3K and ATR/ATM Inhibitors in Colorectal Cancer.

INTRODUCTION: The aim of this study was to investigate the epigenetic regulation and underlying mechanism of NRIP3 in colorectal cancer (CRC). METHODS: Eight cell lines (SW480, SW620, DKO, LOVO, HT29, HCT116, DLD1, and RKO), 187 resected margin samples from colorectal cancer tissue, 146 cases with colorectal adenomatous polyps, and 308 colorectal cancer samples were used. Methylation-specific PCR, Western blotting, RNA interference assay, and a xenograft mouse model were used. RESULTS: NRIP3 exhibited methylation in 2.7% (5/187) of resected margin samples from colorectal cancer tissue, 32.2% (47/146) of colorectal adenomatous polyps, and 50.6% (156/308) of CRC samples, and the expression of NRIP3 was regulated by promoter region methylation. The methylation of NRIP3 was found to be significantly associated with late onset (at age 50 years or older), poor tumor differentiation, lymph node metastasis, and poor 5-year overall survival in CRC (all P < 0.05). In addition, NRIP3 methylation was an independent poor prognostic marker ( P < 0.05). NRIP3 inhibited cell proliferation, colony formation, invasion, and migration, while induced G1/S arrest. NRIP3 suppressed CRC growth by inhibiting PI3K-AKT signaling both in vitro and in vivo . Methylation of NRIP3 sensitized CRC cells to combined PI3K and ATR/ATM inhibitors. DISCUSSION: NRIP3 was frequently methylated in both colorectal adenomatous polyps and CRC. The methylation of NRIP3 may potentially serve as an early detection, late-onset, and poor prognostic marker in CRC. NRIP3 is a potential tumor suppressor. NRIP3 methylation is a potential synthetic lethal marker for combined PI3K and ATR/ATM inhibitors.

RASSF1A promotes ATM signaling and RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

Aim: The mechanism of RASSF1A in DNA damage repair remains to be further clarified for applying to synthetic lethal strategy. Materials & methods: Eight esophageal cancer cell lines, 181 cases of esophageal dysplasia and 1066 cases of primary esophageal squamous cell carcinoma (ESCC) were employed. Methylation-specific PCR, the CRISPR/Cas9 technique, immunoprecipitation assay and a xenograft mouse model were used. Results: RASSF1A was methylated in 2.21% of esophageal dysplasia and 11.73% of ESCC. RASSF1A was also involved in DNA damage repair through activating Hippo signaling. Loss of RASSF1A expression sensitized esophageal cancer cell lines to ataxia telangiectasia mutated and rad3-related (ATR) inhibitor (VE-822) both in vitro and in vivo. Conclusion: RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.

Improving lung cancer diagnosis with cancer, fungal, and imaging biomarkers.

OBJECTIVE: Indeterminate pulmonary nodules (IPNs) represent a significant diagnostic burden in health care. We aimed to compare a combination clinical prediction model (Mayo Clinic model), fungal (histoplasmosis serology), imaging (computed tomography [CT] radiomics), and cancer (high-sensitivity cytokeratin fraction 21; hsCYFRA 21-1) biomarker approach to a validated prediction model in diagnosing lung cancer. METHODS: A prospective specimen collection, retrospective blinded evaluation study was performed in 3 independent cohorts with 6- to 30-mm IPNs (n = 281). Serum histoplasmosis immunoglobulin G and immunoglobulin M antibodies and hsCYFRA 21-1 levels were measured and a validated CT radiomic score was calculated. Multivariable logistic regression models were estimated with Mayo Clinic model variables, histoplasmosis antibody levels, CT radiomic score, and hsCYFRA 21-1. Diagnostic performance of the combination model was compared with that of the Mayo Clinic model. Bias-corrected clinical net reclassification index (cNRI) was used to estimate the clinical utility of a combination biomarker approach. RESULTS: A total of 281 patients were included (111 from a histoplasmosis-endemic region). The combination biomarker model including the Mayo Clinic model score, histoplasmosis antibody levels, radiomics, and hsCYFRA 21-1 level showed improved diagnostic accuracy for IPNs compared with the Mayo Clinic model alone with an area under the receiver operating characteristics curve of 0.80 (95% CI, 0.76-0.84) versus 0.72 (95% CI, 0.66-0.78). Use of this combination model correctly reclassified intermediate risk IPNs into low- or high-risk category (cNRI benign = 0.11 and cNRI malignant = 0.16). CONCLUSIONS: The addition of cancer, fungal, and imaging biomarkers improves the diagnostic accuracy for IPNs. Integrating a combination biomarker approach into the diagnostic algorithm of IPNs might decrease unnecessary invasive testing of benign nodules and reduce time to diagnosis for cancer.

Response to Neoadjuvant Targeted Therapy in Operable Head and Neck Cancer Confers Survival Benefit.

PURPOSE: Neoadjuvant targeted therapy provides a brief, preoperative window of opportunity that can be exploited to individualize cancer care based on treatment response. We investigated whether response to neoadjuvant therapy during the preoperative window confers survival benefit in patients with operable head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: A pooled analysis of treatment-naïve patients with operable HNSCC enrolled in one of three clinical trials from 2009 to 2020 (NCT00779389, NCT01218048, NCT02473731). Neoadjuvant regimens consisted of EGFR inhibitors (n = 83) or anti-ErbB3 antibody therapy (n = 9) within 28 days of surgery. Clinical to pathologic stage migration was compared with disease-free survival (DFS) and overall survival (OS) while adjusting for confounding factors using multivariable Cox regression. Circulating tumor markers validated in other solid tumor models were analyzed. RESULTS: 92 of 118 patients were analyzed; all patients underwent surgery following neoadjuvant therapy. Clinical to pathologic downstaging was more frequent in patients undergoing neoadjuvant targeted therapy compared with control cohort (P = 0.048). Patients with pathologic downstage migration had the highest OS [89.5%; 95% confidence interval (CI), 75.7-100] compared with those with no stage change (58%; 95% CI, 46.2-69.8) or upstage (40%; 95% CI, 9.6-70.4; P = 0.003). Downstage migration remained a positive prognostic factor for OS (HR, 0.22; 95% CI, 0.05-0.90) while adjusting for measured confounders. Downstage migration correlated with decreased circulating tumor markers, SOX17 and TAC1 (P = 0.0078). CONCLUSIONS: Brief neoadjuvant therapy achieved pathologic downstaging in a subset of patients and was associated with significantly better DFS and OS as well as decreased circulating methylated SOX17 and TAC1.

Epigenetic silencing of JAM3 promotes esophageal cancer development by activating Wnt signaling.

BACKGROUND: The role of JAM3 in different tumors is controversial. The epigenetic regulation and the mechanism of JAM3 remain to be elucidated in human esophageal cancer (EC). METHODS: Eleven EC cell lines, 49 cases of esophageal intraepithelial neoplasia (EIN) and 760 cases of primary EC samples were employed. Methylation-specific polymerase chain reaction, immunohistochemistry, MTT, western blot and xenograft mouse models were applied in this study. RESULTS: The inverse association between RNA expression and promoter region methylation of JAM3 was found by analyzing 185 cases of EC samples extracted from the TCGA database (p < 0.05). JAM3 was highly expressed in KYSE450, KYSE520, TE1 and YES2 cells, low level expressed in KYSE70 cells and unexpressed in KYSE30, KYSE150, KYSE410, KYSE510, TE13 and BIC1 cells. JAM3 was unmethylated in KYSE450, KYSE520, TE1 and YES2 cells, partial methylated in KYSE70 cells and completely methylated in KYSE30, KYSE150, KYSE410, KYSE510, TE13 and BIC1 cells. The expression of JAM3 is correlated with methylation status. The levels of JAM3 were unchanged in KYSE450, KYSE520, TE1 and YES2 cells, increased in KYSE70 cells and restored expression in KYSE30, KYSE150, KYSE410, KYSE510, TE13 and BIC1 cells after 5-aza-2'-deoxycytidine treatment, suggesting that the expression of JAM3 is regulated by promoter region methylation. JAM3 was methylated in 26.5% (13/49) of EIN and 51.1% (388/760) of primary EC, and methylation of JAM3 was associated significantly with tumor differentiation and family history (all p < 0.05). Methylation of JAM3 is an independent prognostic factor of poor 5-year overall survival (p < 0.05). JAM3 suppresses cell proliferation, colony formation, migration and invasion and induces G1/S arrest and apoptosis in EC. Further study demonstrated that JAM3 suppressed EC cells and xenograft tumor growth by inhibiting Wnt/ß-catenin signaling. CONCLUSION: JAM3 is frequently methylated in human EC, and the expression of JAM3 is regulated by promoter region methylation. JAM3 methylation is an early detection and prognostic marker of EC. JAM3 suppresses EC growth both in vitro and in vivo by inhibiting Wnt signaling.

Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells.

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Lessons From a Systematic Literature Search on Diagnostic DNA Methylation Biomarkers for Colorectal Cancer: How to Increase Research Value and Decrease Research Waste?

OBJECTIVES: To improve colorectal cancer (CRC) survival and lower incidence rates, colonoscopy and/or fecal immunochemical test screening are widely implemented. Although candidate DNA methylation biomarkers have been published to improve or complement the fecal immunochemical test, clinical translation is limited. We describe technical and methodological problems encountered after a systematic literature search and provide recommendations to increase (clinical) value and decrease research waste in biomarker research. In addition, we present current evidence for diagnostic CRC DNA methylation biomarkers. METHODS: A systematic literature search identified 331 diagnostic DNA methylation marker studies published before November 2020 in PubMed, EMBASE, Cochrane Library, and Google Scholar. For 136 bodily fluid studies, extended data extraction was performed. STARD criteria and level of evidence were registered to assess reporting quality and strength for clinical translation. RESULTS: Our systematic literature search revealed multiple issues that hamper the development of DNA methylation biomarkers for CRC diagnosis, including methodological and technical heterogeneity and lack of validation or clinical translation. For example, clinical translation and independent validation were limited, with 100 of 434 markers (23%) studied in bodily fluids, 3 of 434 markers (0.7%) translated into clinical tests, and independent validation for 92 of 411 tissue markers (22%) and 59 of 100 bodily fluids markers (59%). DISCUSSION: This systematic literature search revealed that major requirements to develop clinically relevant diagnostic CRC DNA methylation markers are often lacking. To avoid the resulting research waste, clinical needs, intended biomarker use, and independent validation should be better considered before study design. In addition, improved reporting quality would facilitate meta-analysis, thereby increasing the level of evidence and enabling clinical translation.

Technical considerations in PCR-based assay design for diagnostic DNA methylation cancer biomarkers.

BACKGROUND: DNA methylation biomarkers for early detection, risk stratification and treatment response in cancer have been of great interest over the past decades. Nevertheless, clinical implementation of these biomarkers is limited, as only < 1% of the identified biomarkers is translated into a clinical or commercial setting. Technical factors such as a suboptimal genomic location of the assay and inefficient primer or probe design have been emphasized as important pitfalls in biomarker research. Here, we use eleven diagnostic DNA methylation biomarkers for colorectal cancer (ALX4, APC, CDKN2A, MGMT, MLH1, NDRG4, SDC2, SFRP1, SFRP2, TFPI1 and VIM), previously described in a systematic literature search, to evaluate these pitfalls. RESULTS: To assess the genomic assay location, the optimal genomic locations according to TCGA data were extracted and compared to the genomic locations used in the published assays for all eleven biomarkers. In addition, all primers and probes were technically evaluated according to several criteria, based on literature and expert opinion. Both assay location and assay design quality varied widely among studies. CONCLUSIONS: Large variation in both assay location and design hinders the development of future DNA methylation biomarkers as well as inter-study comparability.

Pembrolizumab plus azacitidine in patients with chemotherapy refractory metastatic colorectal cancer: a single-arm phase 2 trial and correlative biomarker analysis.

BACKGROUND: DNA mismatch repair proficient (pMMR) metastatic colorectal cancer (mCRC) is not responsive to pembrolizumab monotherapy. DNA methyltransferase inhibitors can promote antitumor immune responses. This clinical trial investigated whether concurrent treatment with azacitidine enhances the antitumor activity of pembrolizumab in mCRC. METHODS: We conducted a phase 2 single-arm trial evaluating activity and tolerability of pembrolizumab plus azacitidine in patients with chemotherapy-refractory mCRC (NCT02260440). Patients received pembrolizumab 200 mg IV on day 1 and azacitidine 100 mg SQ on days 1-5, every 3 weeks. A low fixed dose of azacitidine was chosen in order to reduce the possibility of a direct cytotoxic effect of the drug, since the main focus of this study was to investigate its potential immunomodulatory effect. The primary endpoint of this study was overall response rate (ORR) using RECIST v1.1., and secondary endpoints were progression-free survival (PFS) and overall survival (OS). Tumor tissue was collected pre- and on-treatment for correlative studies. RESULTS: Thirty chemotherapy-refractory patients received a median of three cycles of therapy. One patient achieved partial response (PR), and one patient had stable disease (SD) as best confirmed response. The ORR was 3%, median PFS was 1.9 months, and median OS was 6.3 months. The combination regimen was well-tolerated, and 96% of treatment-related adverse events (TRAEs) were grade 1/2. This trial was terminated prior to the accrual target of 40 patients due to lack of clinical efficacy. DNA methylation on-treatment as compared to pre-treatment decreased genome wide in 10 of 15 patients with paired biopsies and was significantly lower in gene promoter regions after treatment. These promoter demethylated genes represented a higher proportion of upregulated genes, including several immune gene sets, endogenous retroviral elements, and cancer-testis antigens. CD8+ TIL density trended higher on-treatment compared to pre-treatment. Higher CD8+ TIL density at baseline was associated with greater likelihood of benefit from treatment. On-treatment tumor demethylation correlated with the increases in tumor CD8+ TIL density. CONCLUSIONS: The combination of pembrolizumab and azacitidine is safe and tolerable with modest clinical activity in the treatment for chemotherapy-refractory mCRC. Correlative studies suggest that tumor DNA demethylation and immunomodulation occurs. An association between tumor DNA demethylation and tumor-immune modulation suggests immune modulation and may result from treatment with azacitidine. Trial registration ClinicalTrials.gov, NCT02260440. Registered 9 October 2014, https://clinicaltrials.gov/ct2/show/NCT02260440 .

High-throughput sample processing for methylation analysis in an automated, enclosed environment.

Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated. Thus, sample processing methods still require numerous manual steps that may reduce sample throughput and precision, while increasing the risk of contamination and human error. In this work, we present an integrated, automated solution for performing the entire sample preparation process, including DNA extraction, purification, bisulfite conversion and PCR plate preparation within in an enclosed environment. The method employs silica-coated magnetic particles that eliminate the need for a centrifuge or vacuum manifold, thereby reducing the complexity and cost of the required automation platform. Toward this end, we also compare commercial DNA extraction and bisulfite conversion kits to identify a protocol suitable for automation to significantly improve genomic and bisulfite-treated DNA yields over manufacturer protocols. Overall, this research demonstrated development of an automated protocol that offers the ability to generate high-quality, bisulfite-treated DNA samples in a high-throughput and clean environment with minimal user intervention and comparable yields to manual processing.

DNA-methylation for the detection and distinction of 19 human malignancies.

The contribution of DNA-methylation based gene silencing to carcinogenesis is well established. Increasingly, DNA-methylation is examined using genome-wide techniques, with recent public efforts yielding immense data sets of diverse malignancies representing the vast majority of human cancer related disease burden. Whereas mutation events may group preferentially or in high frequency with a given histology, mutations are poor classifiers of tumour type. Here we examine the hypothesis that cancer-specific DNA-methylation reflects the tissue of origin or carcinogenic risk factor, and these methylation abnormalities may be used to faithfully classify tumours according to histology. We present an analysis of 7427 tumours representing 19 human malignancies and 708 normal samples demonstrating that specific tumour changes in methylation can correctly determine site of origin and tumour histology with 86% overall accuracy. Examination of misclassified tumours reveals underlying shared biology as the source of misclassifications, including common cell of origin or risk factors.

Methylation of NRN1 is a novel synthetic lethal marker of PI3K-Akt-mTOR and ATR inhibitors in esophageal cancer.

Wnt, PI3K-Akt-mTOR, and NF-κB pathways were reported to be involved in DNA damage repair (DDR). DDR-deficient cancers become critically dependent on backup DNA repair pathways. Neuritin 1 (NRN1) is reported to be involved in PI3K-Akt-mTOR, and its role in DDR remains unclear. Methylation-specific PCR, siRNA, flow cytometry, esophageal cancer cell lines, and xenograft mouse models were used to examine the role of NRN1 in esophageal cancer. The expression of NRN1 is frequently repressed by promoter region methylation in human esophageal cancer cells. NRN1 was methylated in 50.4% (510/1012) of primary esophageal cancer samples. NRN1 methylation is associated significantly with age (P < .001), tumor size (P < .01), TNM stage (P < .001), differentiation (P < .001) and alcohol consumption (P < .05). We found that NRN1 methylation is an independent prognostic factor for poor 5-y overall survival (P < .001). NRN1 inhibits colony formation, cell proliferation, migration, and invasion, and induces apoptosis and G1/S arrest in esophageal cancer cells. NRN1 suppresses KYSE150 and KYSE30 cells xenografts growth in nude mice. PI3K signaling is reported to activate ATR signaling by targeting CHK1, the downstream component of ATR. By analyzing the synthetic efficiency of NVP-BEZ235 (PI3K inhibitor) and VE-822 (an ATR inhibitor), we found that the combination of NVP-BEZ235 and VE-822 increased cytotoxicity in NRN1 methylated esophageal cancer cells, as well as KYSE150 cell xenografts. In conclusion, NRN1 suppresses esophageal cancer growth both in vitro and in vivo by inhibiting PI3K-Akt-mTOR signaling. Methylation of NRN1 is a novel synthetic lethal marker for PI3K-Akt-mTOR and ATR inhibitors in human esophageal cancer.

A phase 2 trial of gemcitabine and docetaxel in patients with metastatic colorectal adenocarcinoma with methylated checkpoint with forkhead and ring finger domain promoter and/or microsatellite instability phenotype.

We previously reported CHFR methylation in a subset of colorectal cancer (CRC; ∼30%) with high concordance with microsatellite instability (MSI). We also showed that CHFR methylation predicted for sensitivity to docetaxel, whereas the MSI-high phenotypes were sensitive to gemcitabine. We hypothesized that this subset of patients with CRC would be selectively sensitive to gemcitabine and docetaxel. We enrolled a Phase 2 trial of gemcitabine and docetaxel in patients with MSI-high and/or CHFR methylated CRC. The primary objective was Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 response rate. Enrolled patients were treated with gemcitabine 800 mg/m2 on days 1 and 8 and docetaxel 70 mg/m2 on day 8 of each 21-day cycle. A total of 6 patients with CHFR-methylated, MSI-high CRC were enrolled from September 2012 to August 2016. The study was closed in September of 2017 due to poor accrual prior to reaching the first interim assessment of response rate, which would have occurred at 10 patients. No RECIST criteria tumor responses were observed, with 3 patients (50%) having stable disease as best response, 1 lasting more than 9 months. Median progression-free survival (PFS) was 1.79 months (95% confidence interval [CI] = 1.28, not available [NA]) and median overall survival (OS) was 15.67 months (95% CI = 4.24, NA). Common grade 3 toxicities were lymphopenia (67%), leukopenia (33%), and anemia (33%). Although negative, this study establishes a proof-of-concept for the implementation of epigenetic biomarkers (CHFR methylation/MSI) as inclusion criteria in a prospective clinical trial to optimize combinatorial strategies in the era of personalized medicine. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? CHFR silencing via DNA methylation has been suggested to be predictive of taxane sensitivity in diverse tumors. The frequent association of CHFR methylation with microsatellite instability (MSI) suggested a possible combination therapy with gemcitabine, because the MSI phenotype may result in sensitivity to nucleoside analogues. WHAT QUESTION DID THIS STUDY ADDRESS? We hypothesized that metastatic colorectal cancer (mCRC), which have CHFR methylation and MSI phenotype were sensitive to gemcitabine and docetaxel, and have designed this Phase 2 trial in biomarker-selected mCRC to test this prediction. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? The study enrolled a molecularly defined subgroup of patients with colorectal cancer (CRC) and showed that the combination is safe in this population. Nevertheless, due to poor enrollment and early termination, no conclusions on the primary and secondary end points could be made. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? This study supports the feasibility of implementing DNA methylation markers in a prospective clinical trial and further efforts toward their application as predictive biomarkers for therapeutic agents in defined subsets of patients are warranted.

Identification of DNA methylation markers for early detection of CRC indicates a role for nervous system-related genes in CRC.

PURPOSE: Colonoscopy and the fecal immunochemical test (FIT) are currently the most widely used screening modalities for colorectal cancer (CRC), however, both with their own limitations. Here we aim to identify and validate stool-based DNA methylation markers for the early detection of CRC and investigate the biological pathways prone to DNA methylation. METHODS: DNA methylation marker discovery was performed using The Cancer Genome Atlas (TCGA) colon adenocarcinoma data set consisting of normal and primary colon adenocarcinoma tissue. The performance of the five best candidate markers and a previously identified marker, NDRG4, was evaluated on tissues and whole stool samples of healthy subjects and CRC patients using quantitative MSP assays. The results were compared and combined with FIT data. Finally, pathway and gene ontology enrichment analyses were performed using ToppFun, GOrilla and clusterProfiler. RESULTS: GDNF, HAND2, SLC35F3, SNAP91 and SORCS1 were ranked as the best performing markers. Gene combinations of all five markers, NDRG4 and FIT were evaluated to establish the biomarker panel with the highest diagnostic potential, resulting in the identification of GDNF/SNAP91/NDRG4/FIT as the best performing marker panel. Pathway and gene ontology enrichment analyses revealed that genes associated with the nervous system were enriched in the set of best performing CRC-specific biomarkers. CONCLUSION: In silico discovery analysis using TCGA-derived data yielded a novel DNA-methylation-based assay for the early detection of CRC, potentially improving current screening modalities. Additionally, nervous system-related pathways were enriched in the identified genes, indicating an epigenetic regulation of neuronal genes in CRC.

Noninvasive Diagnostics for Early Detection of Lung Cancer: Challenges and Potential with a Focus on Changes in DNA Methylation.

Lung cancer remains the leading cause of cancer deaths in the United States and the world. Early detection of this disease can reduce mortality, as demonstrated for low-dose computed tomography (LDCT) screening. However, there remains a need for improvements in lung cancer detection to complement LDCT screening and to increase adoption of screening. Molecular changes in the tumor, and the patient's response to the presence of the tumor, have been examined as potential biomarkers for diagnosing lung cancer. There are significant challenges to developing an effective biomarker with sufficient sensitivity and specificity for the early detection of lung cancer, particularly the detection of circulating tumor DNA, which is present in very small quantities. We will review approaches to develop biomarkers for the early detection of lung cancer, with special consideration to detection of rare tumor events, focus on the use of DNA methylation-based detection in plasma and sputum, and discuss the promise and challenges of lung cancer early detection. Plasma-based detection of lung cancer DNA methylation may provide a simple cost-effective method for the early detection of lung cancer.See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible."

Detection of Promoter DNA Methylation in Urine and Plasma Aids the Detection of Non-Small Cell Lung Cancer.

PURPOSE: Low-dose CT screening can reduce lung cancer-related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation-based detection of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: This nested case-control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation-specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42). RESULTS: DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively. CONCLUSIONS: DNA methylation-based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

Ultrasensitive DNA hypermethylation detection using plasma for early detection of NSCLC: a study in Chinese patients with very small nodules.

PURPOSE: We had previously developed highly sensitive DNA methylation detection to diagnose lung cancer in patients with pulmonary nodules. To validate this approach and determine clinical utility in Chinese patients with indeterminate pulmonary nodules, we assessed the diagnostic accuracy for early stage lung cancer in plasma samples. EXPERIMENTAL DESIGN: Patients with CT-detected small lung nodules (diameter ≤ 3.0 cm) were included. Cases (n = 163) had staged IA or IB non-small cell lung cancer (NSCLC), while controls (n = 83) had non-cancerous lesions. Promoter methylation of eight lung cancer-specific genes (CDO1, TAC1, SOX17, HOXA7, HOXA9, GATA4, GATA5, and PAX5) was detected using nanoparticle-based DNA extraction (MOB) followed by qMSP. RESULTS: Methylation detection for CDO1, TAC1, SOX17, and HOXA7 in plasma was significantly higher in cases compared with the benign group (p < 0.001). The sensitivity and specificity for lung cancer diagnosis using individual gene was 41-69% and 49-82%. A three-gene combination of the best individual genes has sensitivity and specificity of 90% and 71%, with area under the receiver operating curve (AUC) of 0.88, (95% CI 0.84-0.93). Furthermore, three-gene combinations detected even the smallest lung nodules, with the combination of CDO1, SOX17, and HOXA7 having the overall best performance, while the combination of CDO1, TAC1, and SOX17 was best in tumor sizes less than 1.0 cm. CONCLUSIONS: Using modified MOB-qMSP, high sensitivity and specificity, for the detection of circulating tumor DNA was obtained for early stage NSCLC. This strategy has great potential to identify patients at high risk and improve the diagnosis of lung cancer at an earlier stage.

PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

Signal Transducers and Activators of Transcription-3 (STAT3), a potent oncogenic transcription factor, is constitutively activated in lung cancer, but mutations in pathway genes are infrequent. Protein Tyrosine Phosphatase Receptor-T (PTPRT) is an endogenous inhibitor of STAT3 and PTPRT loss-of-function represents one potential mechanism of STAT3 hyperactivation as observed in other malignancies. We determined the role of PTPRT promoter methylation and sensitivity to STAT3 pathway inhibitors in non-small cell lung cancer (NSCLC). TCGA and Pittsburgh lung cancer cohort methylation data revealed hypermethylation of PTPRT associated with diminished mRNA expression in a subset of NSCLC patients. We report frequent hypermethylation of the PTPRT promoter which correlates with transcriptional silencing of PTPRT and increased STAT3 phosphorylation (Y705) as determined by methylation-specific PCR (MSP) and real time quantitative reverse transcription (RT)-PCR in NSCLC cell lines. Silencing of PTPRT using siRNA in H520 lung cancer cell line resulted in increased pSTAT3Tyr705 and upregulation of STAT3 target genes such as Cyclin D1 and Bcl-XL expression. We show this association of PRPRT methylation with upregulation of the STAT3 target genes Cyclin D1 and Bcl-XL in patient derived lung tumour samples. We further demonstrate that PTPRT promoter methylation associated with different levels of pSTAT3Ty705 in lung cancer cell lines had selective sensitivity to STAT3 pathway small molecule inhibitors (SID 864,669 and SID 4,248,543). Our data strongly suggest that silencing of PTPRT by promoter hypermethylation is an important mechanism of STAT3 hyperactivation and targeting STAT3 may be an effective approach for the development of new lung cancer therapeutics.

The association between BMI and BSA-temozolomide-induced myelosuppression toxicities: a correlative analysis of NRG oncology RTOG 0525.

BACKGROUND: Fearing increased myelotoxicity, many practitioners adjust the body surface area (BSA)-calculated doses in obese patients. Regarding temozolomide (TMZ), a prior study suggested men with a BSA >2 m2 may experience increased toxicity; however, surprisingly, the inverse observation was noted in women, ie, BSA <2 m2 was associated with higher toxicity. To further clarify this issue, data derived from a large clinical trial were analyzed. METHODS: The incidence of grade 3 and 4 myelotoxicity in a newly diagnosed glioblastoma phase 3 trial (RTOG 0525) was statistically correlated with BMI and separately with BSA. All patients received radiation and TMZ followed by adjuvant standard dose TMZ vs dose-dense TMZ; dosing regimen-associated myelotoxicity and BMI/BSA were analyzed separately. Obesity was defined as a BMI ≥30. RESULTS: There was no statistically significant correlation between gender and BSA and the occurrence of myotoxicities. For the standard arm, surprisingly the incidence of grade 3/4 myotoxicities in patients with a BMI <30 was significantly higher than in patients with a BMI ≥30 (12% vs 1%, odds ratio [OR] 12.5, P < .001). There was no significant difference between obese and nonobese patients (BMI "cut-point" of 30) in the dose-dense arm (OR = 0.9, 95% confidence interval: 0.4-1.6). The grade hematological 3/4 toxicity rate was significantly higher in women vs men (14% vs 8%) P = .009 in spite of the lack of association between gender and BSA or BMI. CONCLUSION: TMZ dosing based on actual BSA is recommended with the caveat that woman are likely at higher toxicity risk.