Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay

Background

Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC.

Methodology/Principal Findings

Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was ∼20%.

Conclusions/Significance

Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.     

The Detection of the Methylated Wif-1 Gene Is More Accurate than a Fecal Occult Blood Test for Colorectal Cancer Screening

Background

The clinical benefit of guaiac fecal occult blood tests (FOBT) is now well established for colorectal cancer screening. Growing evidence has demonstrated that epigenetic modifications and fecal microbiota changes, also known as dysbiosis, are associated with CRC pathogenesis and might be used as surrogate markers of CRC.

Patients and Methods

We performed a cross-sectional study that included all consecutive subjects that were referred (from 2003 to 2007) for screening colonoscopies. Prior to colonoscopy, effluents (fresh stools, sera-S and urine-U) were harvested and FOBTs performed. Methylation levels were measured in stools, S and U for 3 genes (Wif1, ALX-4, and Vimentin) selected from a panel of 63 genes; Kras mutations and seven dominant and subdominant bacterial populations in stools were quantified. Calibration was assessed with the Hosmer-Lemeshow chi-square, and discrimination was determined by calculating the C-statistic (Area Under Curve) and Net Reclassification Improvement index.

Results

There were 247 individuals (mean age 60.8±12.4 years, 52% of males) in the study group, and 90 (36%) of these individuals were patients with advanced polyps or invasive adenocarcinomas. A multivariate model adjusted for age and FOBT led to a C-statistic of 0.83 [0.77–0.88]. After supplementary sequential (one-by-one) adjustment, Wif-1 methylation (S or U) and fecal microbiota dysbiosis led to increases of the C-statistic to 0.90 [0.84–0.94] (p = 0.02) and 0.81 [0.74–0.86] (p = 0.49), respectively. When adjusted jointly for FOBT and Wif-1 methylation or fecal microbiota dysbiosis, the increase of the C-statistic was even more significant (0.91 and 0.85, p<0.001 and p = 0.10, respectively).

Conclusion

The detection of methylated Wif-1 in either S or U has a higher performance accuracy compared to guaiac FOBT for advanced colorectal neoplasia screening. Conversely, fecal microbiota dysbiosis detection was not more accurate. Blood and urine testing could be used in those individuals reluctant to undergo stool testing.     

Distinct High-Profile Methylated Genes in Colorectal Cancer

Background

Mutations and promoters'' methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes'' promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining.

Methodology/Principal Findings

The promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed.Seventy-seven and 34% of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was >65% for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was <50% for LGR6, RET, CD109, and RNF. The difference in methylation between the two populations was statistically significant for CHD5, ICAM5 and GPNMB. Thirty-one percent AA tumors showed MSI-H, compared to 28% in Iranians.

Conclusions/Significance

A significantly higher methylation rate was found for GPNMB, ICAM5, and CHD5 genes in AA patients compared to Iranians. These genes might play a role in the high incidence and aggressiveness of CRC in the AA population. The hypermethylation of the CAN genes can be considered as a marker of colon carcinogenesis.     

Detection of Methylated CDO1 in Plasma of Colorectal Cancer; A PCR Study

Background

Cysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1) in colorectal cancer (CRC). In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP) and extensive analysis of the PCR reaction.

Methods

DNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from “positive control” DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined.

Results

(1) The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2) DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3) By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20%) of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9%) or CA19-9 (23.1%) in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3%) as compared to CEA/CA19-9 (25%).

Conclusion

We have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients.     

Circulating Methylated XAF1 DNA Indicates Poor Prognosis for Gastric Cancer

Background

Methylated DNA in fluids may be a suitable biomarker for cancer patients. XAF1 has been shown to be frequently down-regulated in human gastric cancer (GC). Here, we investigated if XAF1 methylation in GC could be a useful biomarker.

Methods

Real-time RT-PCR was used to detect XAF1 mRNA expression; immunohistochemistry and western blot were used to examine XAF1 protein expression in GC tissues (n = 202) and their corresponding para-cancerous histological normal tissues (PCHNTs). Real-time methylation specific-PCR was used to investigate XAF1 promoter methylation in the same panel of GC tissues, their PCHNTs and sera.

Results

We confirmed frequent XAF1 down-regulation in both mRNA and protein levels in GC tissues as compared to normal controls and PCHNTs. XAF1 hypermethylation was evidenced in 83.2% (168/202) of GC tissues and 27.2% (55/202) of PCHNTs, while no methylation was detected in the 88 normal controls. The methylation level in GC tissues was significantly higher than that in PCHNTs (p<0.05). The hypermethylation of XAF1 significantly correlated with the down-regulation of XAF1 in GC tissues in both mRNA and protein levels (p<0.001 each). Moreover, we detected high frequency of XAF1 methylation (69.8%, 141 out of 202) in the sera DNAs from the same patients, while the sera DNAs from 88 non-tumor controls were negative for XAF1 methylation. The XAF1 methylation in both GC tissues and in the sera could be a good biomarker for diagnosis of GC (AUC = 0.85 for tissue and AUC = 0.91 for sera) and significantly correlated with poorer prognosis (p<0.001). In addition, after-surgery negative-to-positive transition of XAF1 methylation in sera strongly associated with tumor recurrence.

Conclusions

1) Dysfunction of XAF1 is frequent and is regulated through XAF1 promoter hypermethylation; 2) Detection of circulating methylated XAF1 DNAs in the serum may be a useful biomarker in diagnosis, evaluating patient’s outcome (prognosis and recurrence) for GC patients.     

Blood Muramidase Activity in Colorectal Cancer

The serum muramidase levels were measured in 128 patients with primary or metastatic colorectal cancer, 166 tumour-free patients after resection of a colorectal cancer, and 172 controls. Muramidase levels over 10 μg/ml were detected in 30%-39% of the tumour-bearing patients, in 8·2% of the tumour free, and in only 1·7% of the controls (normal level 6·68 ± 1·42 μg/ml). Long-term follow up indicated that raised levels may occur as a transient phenomenon in recurrent or metastatic disease. The likely relation of abnormal serum muramidase activity and stimulation of the reticuloendothelial system is discussed.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Methylated DNA labels for marking objects

We recently described a method for digitally labelling objects with DNA. Here we show that, using DNA methyltransferases to create polymorphic DNA templates, it is possible to significantly increase the number of labels that can be generated by this method. Nine double-stranded DNA templates of different length were methylated with either M.HaeIII or M.AluI methyltransferase, or both. Different mixtures of methylated and unmethylated versions of this template set were used to 'invisibly' label paper. The mixtures were eluted from the paper and the methylated status of the templates in each mixture successfully determined, and the labels read, by digestion with the complementary restriction endonuclease, followed by a polymerase chain reaction and agarose gel electrophoresis. One methylated DNA label was read after it had been left on paper for two months.     

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.     

A Sensitive Urinary Lipoarabinomannan Test for Tuberculosis

We have previously developed a diagnostic test for tuberculosis based on detection of mycobacterial lipoarabinomannan (LAM) in urine. The method depended on a laborious concentration step. We have now developed an easy to perform test based on a magnetic immunoassay platform, utilizing high avidity monoclonal antibodies for the detection of LAM in urine. With this method the analytical sensitivity of the assay was increased 50-100-fold compared to conventional ELISA. In a pilot study of HIV-negative patients with microbiologically verified TB (n=17) and healthy controls (n=22) the sensitivity of the test was 82% and the specificity 100%. This is in stark positive contrast to a range of studies using available commercial tests with polyclonal anti-LAM Abs where the sensitivity of the tests in HIV-negative TB patients was very low.     

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

Aim

Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.

Materials and Methods

To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.

Results

As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.

Conclusions

Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.     

Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N?=?32) and in colorectal carcinoma patients with localized (N?=?24) or metastatic (N?=?37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, in vitro analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.     

结/直肠癌的潜在治疗靶点

传统的结/直肠癌化疗药物,作用选择性较低,副反应明显,疗效欠佳,患者病死率居高不下.寻找新型治疗药物,十分必要.在结/直肠癌发病机制的探索过程中,研究者发现:一些分子的变化影响着疾病进程,对病情起指示作用,有可能成为治疗靶点.围绕这些分子设计药物,制定治疗方案,有望提高疗效,改善病人生存质量,降低不良反应的发生.本文就结/直肠癌的部分潜在治疗靶点:EGFR,VEGF,HADC,COX-2,PPARγ及galectin-3做一综述.     

Core Outcomes for Colorectal Cancer Surgery: A Consensus Study

BackgroundColorectal cancer (CRC) is a major cause of worldwide morbidity and mortality. Surgical treatment is common, and there is a great need to improve the delivery of such care. The gold standard for evaluating surgery is within well-designed randomized controlled trials (RCTs); however, the impact of RCTs is diminished by a lack of coordinated outcome measurement and reporting. A solution to these issues is to develop an agreed standard “core” set of outcomes to be measured in all trials to facilitate cross-study comparisons, meta-analysis, and minimize outcome reporting bias. This study defines a core outcome set for CRC surgery.ConclusionThis study used robust consensus methodology to develop a core outcome set for use in colorectal cancer surgical trials. It is now necessary to validate the use of this set in research practice.     

Circulating Free DNA as Biomarker and Source for Mutation Detection in Metastatic Colorectal Cancer

Background

Circulating cell-free DNA (cfDNA) in plasma has shown potential as biomarker in various cancers and could become an importance source for tumour mutation detection. The objectives of our study were to establish a normal range of cfDNA in a cohort of healthy individuals and to compare this with four cohorts of metastatic colorectal cancer (mCRC) patients. We also investigated the prognostic value of cfDNA and analysed the tumour-specific KRAS mutations in the plasma.

Methods

The study was a prospective biomarker evaluation in four consecutive Phase II trials, including 229 patients with chemotherapy refractory mCRC and 100 healthy individuals. Plasma was obtained from an EDTA blood-sample, and the total number of DNA alleles and KRAS mutated alleles were assessed using an in-house ARMS-qPCR as previously described.

Results

Median cfDNA levels were higher in mCRC compared to controls (p <0.0001). ROC analysis revealed an AUC of 0.9486 (p<0.00001). Data showed impaired OS with increasing levels of baseline cfDNA both when categorising patients by quartiles of cfDNA and into low or high cfDNA groups based on the upper normal range of the control group (Median OS 10.2 (8.3–11.7) and 5.2 (4.6–5.9) months, respectively, HR 1.78, p = 0.0006). Multivariate analysis confirmed an independent prognostic value of cfDNA (HR 1.5 (95% CI 1.3–1.7) for each increase in the cfDNA quartile). The overall concordance of KRAS mutations in plasma and tissue was high (85%).

Conclusions

These data confirm the prognostic value of cfDNA measurement in plasma and utility for mutation detection with the method presented.