Methylated Septin Research Articles

The Fatty Liver, Cirrhosis, and Liver Cancer Study (TENDENCY): Protocol for a Multicenter Case-Control Study.

Hepatocellular cancer (HCC) is associated with high mortality, and early diagnosis leads to better survival. Patients with cirrhosis, especially due to nonalcoholic fatty liver disease and viral hepatitis, are at higher risk of developing HCC and form the main screening group. The current screening methods for HCC (6-monthly screening with serum alpha fetoprotein and ultrasound liver) have low sensitivity; hence, there is a need for better screening markers for HCC. Our study, TENDENCY, aims to validate the novel screening markers (methylated septin 9, urinary volatile organic compounds, and urinary peptides) for HCC diagnosis and study these noninvasive biomarkers in liver disease. This is a multicenter, nested case-control study, which involves comparing the plasma levels of methylated septin 9 between confirmed HCC cases and patients with cirrhosis (control group). It also includes the comparison of urine samples for the detection of HCC-specific volatile organic compounds and peptides. Based on the findings of a pilot study carried out at University Hospital Coventry & Warwickshire, we estimated our sample size to be 308 (n=88, 29% patients with HCC; n=220, 71% patients with cirrhosis). Urine and plasma samples will be collected from all participants and will be frozen at -80 °C until the end of recruitment. Gas chromatography-mass spectrometry will be used for urinary volatile organic compounds detection, and capillary electrophoresis-mass spectrometry will be used for urinary peptide identification. Real-time polymerase chain reaction will be used for the qualitative detection of plasma methylated septin 9. The study will be monitored by the Research and Development department at University Hospital Coventry & Warwickshire. The recruitment stage was completed in March 2023. The TENDENCY study is currently in the analysis stage, which is expected to finish by November 2023. There is lack of effective screening tests for hepatocellular cancer despite higher mortality rates. The application of more sensitive plasma and urinary biomarkers for hepatocellular cancer screening in clinical practice will allow us to detect the disease at earlier stages and hence, overall, improve HCC outcomes. DERR1-10.2196/44264.

Sensitive lateral flow assay for bisulfite-free DNA methylation detection based on the restriction endonuclease GlaI and rolling circle amplification

The detection of DNA methylation with high sensitivity and specificity is important for the early diagnosis of many human diseases, including cancers. Here, we integrated the high specificity of the methylation-dependent restriction endonuclease GlaI for methylation-dependent digestion and the high amplification efficiency of rolling circle amplification (RCA) for the detection of GlaI digestion products. GlaI can only digest a methylated template, leading to the generation of digestion products with specific ends. The specific digestion product can then be ligated to a ligation mediator with a dumbbell structure to generate a complete circular template for further RCA, and the final RCA amplicon can be detected using lateral flow detection (LFD) with the naked eye. The specificity of Gla-RCA not only depends on the specific methylation digestion of GlaI, but only the ligation process of RCA amplification. As a proof of principle, the sensitivity of GlaI-RCA assay was applied to methylated Septin 9 and showed a sensitivity of approximately 1% (50 copies of methylated template per reaction) and no cross-reactivity with 5000 copies of unmethylated DNA used as background. The application of GlaI-RCA was also evaluated with colorectal cancer tissue samples and showed great accordance with standard bisulfite sequencing. A bisulfite-free and LFD-based DNA methylation detection was successfully developed, promising high specificity and rapid visual detection and having a great potential to become a robust tool for DNA methylations analysis.

Septin 9 methylation analysis of lymph node micrometastases for predicting relapse of colorectal cancer

BackgroundMolecular markers for the detection of lymph node micrometastases of malignant tumors have been extensively investigated. However, epigenetic signatures have rarely been reported for identification of metastatic lymph nodes and disease relapse. Septin 9 is the most frequently reported hypermethylated gene in colorectal cancer (CRC). This study aimed to assess the clinical relevance of Septin 9 methylation in regional lymph nodes in recurrence/metastases of CRC. MethodsWe analyzed Septin 9 methylation of DNA from resected lymph nodes in 75 CRC patients with or without tumor recurrence using quantitative methylation-sensitive PCR (qMS-PCR). ResultsOf the 30 histologically negative lymph node CRC patients without recurrence (group 1), methylated Septin 9 was detected in 3 (10 %) cases. The positivity rate of methylated Septin 9 in group 2 containing 30 histologically node-negative CRC patients with recurrence was 30 % (9/30). For group 3, lymphatic invasion as well as tumor recurrence, 11 (73 %) out of 15 subjects had Septin 9 methylation-positive lymph nodes. Moreover, patients in group 3 had a higher level of methylated Septin 9 compared to subjects in group 1 and group 2 (p < 0.05). In addition, CRC patients with Septin 9 methylation in lymph nodes had significantly reduced survival (Log-rank P < 0.0001). ConclusionOur data support the predictive role of Septin 9 methylation analysis of lymph node micrometastases for tumor relapse after surgery.

Methylated Septin 9 as a Promising Biomarker in the Diagnosis and Recurrence Monitoring of Colorectal Cancer.

Purpose The clinical utility of plasma methylated septin 9 (mSEPT9) DNA in screening and recurrence monitoring for colorectal cancer (CRC) is highly promising. The present study was performed to determine the diagnostic value of mSEPT9 in CRC detection and recurrence monitoring in Chinese patients. Methods Overall, 616 patients newly diagnosed with CRC and 122 individuals with no evidence of disease were recruited from October 1, 2019, to May 31, 2021, at Fujian Medical University Union Hospital. Plasma and serum samples were collected for analyzing mSEPT9, carcinoembryonic antigen (CEA), and carbohydrate antigen-19-9 (CA19-9). Data on clinicopathological characteristics were collected and analyzed. Sensitivity and specificity were calculated to evaluate the diagnostic potential of each marker; the receiver operating characteristic (ROC) curve was applied for the assessment of diagnostic value, and comparisons among mSEPT9, CEA, CA19-9, and their combination were assessed via ROC curves. Results mSEPT9 achieved an overall sensitivity and specificity of 72.94% and 81.97%, respectively, with an area under the curve (AUC) value of 0.826, which were higher than those of CEA (sensitivity: 43.96%; specificity: 96.72%; AUC: 0.789) and CA19-9 (sensitivity: 14.99%; specificity: 96.61%; AUC: 0.590). The combination of mSEPT9, CEA, and CA19-9 further improved sensitivity, specificity, and AUC value (sensitivity: 78.43%; specificity: 86.07%; AUC: 0.878), respectively. Notably, the mSEPT9 positivity rate was significantly associated with TNM stage, T stage, N stage, tumor size, vascular invasion, and nerve invasion among patients with CRC. A 100% correlation was observed between the positive results of the mSEPT9 test and recurrence or metastasis in patients after therapeutic intervention. Conclusion Our findings suggest that mSEPT9 may represent a potential biomarker for the diagnosis and prognosis of CRC compared with CEA and CA19-9. Postoperative mSEPT9 status may represent the first noninvasive marker of CRC recurrence or metastasis.

Methylated septin 9 gene is an important prognostic marker in stage II and stage III colorectal cancer for evaluating local recurrence or distant metastasis after surgery

BackgroundAbnormal hypermethylation of the septin 9 gene was an inchoate incident in some cancers. Though latest several researches had paid attention to its value in prognosis, the consequences were not distinctly, especially in colorectal cancer (CRC) with stage II and stage III.PurposeThe aim of this research was to pick up the prognostic value of the methylated septin 9 gene (mSEPT9) in CRC patients, particularly in TNM stage II—III.MethodsBlood samples before surgery were obtained from 144 CRC patients, of which there were 94 with stage II and stage III. mSEPT9 was considered positive when the cycle number of the peak reaction (Ct) was lower than the threshold value (41.0) for two times during three times PCR test. mSEPT9 and other relative factors of prognosis were estimated by survival analysis. The level of septin 9 in tissues was tested by immunohistochemical (IHC).ResultsStage II and stage III patients with mSEPT9 positive (mSEPT9+) had a lower disease-free survival (DFS) rate than those with mSEPT9 negative (mSEPT9-) (2-year DFS rates, 52.1% vs 73.9%, P = 0.014). In multivariate regression analysis, mSEPT9 was also an independent predictor of prognosis (HR = 2.741, P = 0.009). The risk of local recurrence or distant metastasis in CRC patients after surgery was mSEPT9+ with stage III, mSEPT9- with stage III/mSEPT9+ with stage II, and mSEPT9- with stage II (P = 0.001), from highest to lowest. In addition, mSEPT9 was strongly associated with TNM staging, tumor immersion depth, distant metastasis, differentiation degree, vascular invasion and microsatellite. When we explored the associations between septin 9 protein level revealed by IHC and other elements, recurrence/progression (R = − 0.523, P = 0.001), mSEPT9 status (R = − 0.451, P = 0.004) and T stage (R = − 0.375, P = 0.017) showed significant correlations.ConclusionsPositive mSEPT9 is a poor prognostic marker for CRC patients in stage II and III. It is also a powerful complement to TNM staging in predicting postoperative DFS of CRC patients of stage II and III.

Application of droplet digital polymerase chain reaction of plasma methylated septin 9 on detection and early monitoring of colorectal cancer

Methylated septin 9 (SEPT9) has been approved for non-invasive screening of colorectal cancer (CRC), but data on monitoring of CRC is sparse. Droplet digital polymerase chain reaction (ddPCR), with higher detection precision and simpler quantification than conventional PCR, has not been applied in SEPT9 detection. We explored the role of SEPT9 ddPCR for CRC detection and to measure serial SEPT9 levels in blood samples of CRC patients before and 3-month after surgery. SEPT9 methylated ratio, methylated abundance, and CEA levels were all higher in CRC patients than normal controls (all P < 0.05). The area under the curve (AUC) for methylated ratio and abundance to detect CRC was 0.707 and 0.710, respectively. There was an increasing trend for SEPT9 methylated abundance from proximal to distal cancers (P = 0.017). At 3-month after surgery, both methylated abundance and ratio decreased (P = 0.005 and 0.053, respectively), especially methylated abundance in stage III and distal cancer (both P < 0.01). We have developed a ddPCR platform for the quantitative detection of plasma SEPT9 in CRC patients. SEPT9 methylated abundance had an early post-operative decline, which may be useful in monitoring of treatment response.

Identifying Novel Biomarkers Ready for Evaluation in Low-Prevalence Populations for the Early Detection of Lower Gastrointestinal Cancers: A Systematic Review and Meta-Analysis.

IntroductionLower gastrointestinal (GI) cancers are a major cause of cancer deaths worldwide. Prognosis improves with earlier diagnosis, and non-invasive biomarkers have the potential to aid with early detection. Substantial investment has been made into the development of biomarkers; however, studies are often carried out in specialist settings and few have been evaluated for low-prevalence populations.MethodsWe aimed to identify novel biomarkers for the detection of lower GI cancers that have the potential to be evaluated for use in primary care. MEDLINE, Embase, Emcare and Web of Science were systematically searched for studies published in English from January 2000 to October 2019. Reference lists of included studies were also assessed. Studies had to report on measures of diagnostic performance for biomarkers (single or in panels) used to detect colorectal or anal cancers. We included all designs and excluded studies with fewer than 50 cases/controls. Data were extracted from published studies on types of biomarkers, populations and outcomes. Narrative synthesis was used, and measures of specificity and sensitivity were meta-analysed where possible.ResultsWe identified 142 studies reporting on biomarkers for lower GI cancers, for 24,844 cases and 45,374 controls. A total of 378 unique biomarkers were identified. Heterogeneity of study design, population type and sample source precluded meta-analysis for all markers except methylated septin 9 (mSEPT9) and pyruvate kinase type tumour M2 (TuM2-PK). The estimated sensitivity and specificity of mSEPT9 was 80.6% (95% CI 76.6–84.0%) and 88.0% (95% CI 79.1–93.4%) respectively; TuM2-PK had an estimated sensitivity of 81.6% (95% CI 75.2–86.6%) and specificity of 80.1% (95% CI 76.7–83.0%).ConclusionTwo novel biomarkers (mSEPT9 and TuM2-PK) were identified from the literature with potential for use in lower-prevalence populations. Further research is needed to validate these biomarkers in primary care for screening and assessment of symptomatic patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12325-021-01645-6.

Role of methylated septin 9 as an adjunct diagnostic and prognostic biomarker in hepatocellular carcinoma

BackgroundMethylated septin 9 (mSEPT9) has a role in hepatocarcinogenesis. We evaluated mSEPT9 performance in patients with hepatocellular carcinoma (HCC) and those at risk of HCC MethodsUsing Epi-proColon® V2.0 assay adapted for 1 mL plasma, we investigated mSEPT9 sensitivity, specificity, associations with influential covariates and relation to death. ResultsOf 141 participants included, 136 had liver disease, 38 with HCC (mean-age 71 years) and 103 without HCC (mean-age 56.8 years), with further five without liver disease. 41 patients died (23 HCC) by the end of the study follow-up period. In HCC, mSEPT9 sensitivity and specificity were 89.47% (CI:75.20%–97.06%) and 81.55% (CI:72.70%–88.51%), whilst alpha fetoprotein (AFP) sensitivity and specificity were 50% (CI:33.38%–66.62%) and 97.09% (CI:91.72%–99.40%), respectively. Age-adjusted logistic regression showed mSEPT9 was associated with age, body mass index, HCC, liver cirrhosis, AFP, platelets, neutrophil-to-lymphocyte-ratio, albumin-bilirubin grade and fibrosis-4 index (p < 0.05). Odds for HCC patients to have positive mSEPT9 were 27.4 times more than those without HCC. Time-to-death was associated with mSEPT9 positivity (p < 0.05). Kaplan–Meier curves showed higher HCC survival with mSEPT9 compared to AFP. ConclusionsThe mSEPT9 offers potential diagnostic and prognostic biomarker for HCC. After adjusting for age, mSEPT9 remained associated with liver function, liver fibrosis and inflammatory surrogate markers.

Circulating methylated Septin 9 and ring finger protein 180 for noninvasive diagnosis of early gastric cancer.

BackgroundGastric cancer (GC) has a poor prognosis due to patients often being diagnosed at an advanced stage, when metastasis has already occurred. To improve the 5-year survival rate and reduce the number of cancer-related deaths in patients with GC, noninvasive methods for early detection need to be developed. This study aimed to evaluate the value of circulating methylated Septin 9 (SEPT9) and ring finger protein 180 (RNF180) for the early diagnosis of GC.MethodsSeventy-four patients with early GC, 99 patients with benign gastric diseases (BGD) (inflammation, polyps, intestinal metaplasia, ulcers, and erosion), and 57 cases with no evidence of disease (NED) were enrolled. Methylated SEPT9 and RNF180 in circulating cell-free DNA in blood samples from each group were detected, and the positivity rates were calculated. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), confidence interval (CI), and area under the curve (AUC) were determined for methylated SEPT9 and RNF180 in relation to early GC.ResultsAs a diagnostic target, methylated SEPT9 had a sensitivity of 28.3% (95% CI: 18.5–40.0%), specificity of 94.2% (95% CI: 89.3–97.3%), and AUC value of 0.616 (95% CI: 52.0–71.1%). Methylated RNF180 had a sensitivity of 32.4% (95% CI: 22.0–44.3%), specificity of 89.7% (95% CI: 83.9–94.0%), and AUC value of 0.636 (95% CI: 54.2–73.0%). A combination of the two yielded a sensitivity of 40.5% (95% CI: 29.3–52.6%), specificity of 85.3% (95% CI: 78.7–90.4%), and AUC value of 0.65 (95% CI: 55.7–74.4%).ConclusionsMethylated SEPT9 and RNF180 could be used as diagnostic biomarkers for early gastric cancer (EGC).

Plasma levels of methylated septin 9 are capable of detecting hepatocellular carcinoma and hepatic cirrhosis.

Hepatocellular carcinoma (HCC) was the third most common cause of cancer-associated mortality in China in 2015. Early detection of HCC and hepatic cirrhosis (HC) can serve a crucial role in the prevention and therapeutic intervention of these diseases. Current early detection methods rely on less sensitive imaging modalities compared with the pathological examination. In the present study, a total of 64 patients with HCC, 44 patients with HC and 298 individuals with no evidence of disease (NED) were recruited, and the ability of methylated septin 9 (mSEPT9) in diagnosing HCC and HC was investigated. The overall detection sensitivity of mSEPT9 for HCC and HC was 76.7 and 34.1%, respectively, with a 95.9% specificity (HCC vs. NED). The sensitivity of mSEPT9 for HCC was significantly higher than that of α-fetoprotein (AFP; χ2 test; 56.7%; P<0.05). The areas under the curve from the receiver operating characteristic curves of mSEPT9 for detection of HCC vs. NED, HC vs. NED and HCC vs. HC were 0.85, 0.77 and 0.66, respectively, while those of AFP for the same groups were 0.80, 0.55 and 0.77, respectively. Although both markers exhibited stage-dependent sensitivity in HCC, mSEPT9 was demonstrated to be more sensitive than AFP. The net reclassification index of mSPET9 for HCC detection was 0.212 compared with AFP, suggesting an improved diagnostic performance of mSEPT9 compared with AFP. In addition, Kaplan-Meier survival analysis revealed that mSEPT9 is able to predict the long-term survival of patients with HCC. Further analysis suggested that patients >50 years of age exhibited higher sensitivity compared with those <50 years old in mSEPT9, but not in AFP. No significant difference in sensitivity was observed between compensated and decompensated patients with HC, and in patients with HC with a history of hepatitis B or C virus infection. No difference was observed between male and female subjects in the HC and HCC groups for mSEPT9 and AFP. In conclusion, mSEPT9 may detect HCC with an overall improved sensitivity compared with AFP and may help in predicting the long-term survival of patients with HCC. The present clinical study was retrospectively registered to the Chinese Clinical Trial Registry on April 4, 2020 (http://www.chictr.org.cn/enIndex.aspx; registration no. ChiCTR2000031547).

Biomarkers for detecting colorectal cancer non-invasively: DNA, RNA or proteins?

Colorectal cancer (CRC) is a global problem affecting millions of people worldwide. This disease is unique because of its slow progress that makes it preventable and often curable. CRC symptoms usually emerge only at advanced stages of the disease, consequently its early detection can be achieved only through active population screening, which markedly reduces mortality due to this cancer. CRC screening tests that employ non-invasively detectable biomarkers are currently being actively developed and, in most cases, samples of either stool or blood are used. However, alternative biological substances that can be collected non-invasively (colorectal mucus, urine, saliva, exhaled air) have now emerged as new sources of diagnostic biomarkers. The main categories of currently explored CRC biomarkers are: (1) Proteins (comprising widely used haemoglobin); (2) DNA (including mutations and methylation markers); (3) RNA (in particular microRNAs); (4) Low molecular weight metabolites (comprising volatile organic compounds) detectable by metabolomic techniques; and (5) Shifts in gut microbiome composition. Numerous tests for early CRC detection employing such non-invasive biomarkers have been proposed and clinically studied. While some of these studies generated promising early results, very few of the proposed tests have been transformed into clinically validated diagnostic/screening techniques. Such DNA-based tests as Food and Drug Administration-approved multitarget stool test (marketed as Cologuard®) or blood test for methylated septin 9 (marketed as Epi proColon® 2.0 CE) show good diagnostic performance but remain too expensive and technically complex to become effective CRC screening tools. It can be concluded that, despite its deficiencies, the protein (haemoglobin) detection-based faecal immunochemical test (FIT) today presents the most cost-effective option for non-invasive CRC screening. The combination of non-invasive FIT and confirmatory invasive colonoscopy is the current strategy of choice for CRC screening. However, continuing intense research in the area promises the emergence of new superior non-invasive CRC screening tests that will allow the development of improved disease prevention strategies.

Integrating multiple “omics” analyses, on a triage concept, for effective case selection followed by diagnostic colonoscopy.

244 Background: Implementation of population screening for colorectal cancer (CRC) before colonoscopy can reduce the challenge of the overall capacity of bowel examination and improve survival. Blood based CRC assessment biomarkers, on a triage concept, can lead to improved selection to colonoscopy and cost-effective CRC care. Methods: Innovative multi-omics approaches, with global and targeted LCMS data production (metabolomics, lipidomics, and 2D proteomics) and integrative data analytics, were applied to discover serological biomarkers to assess nonadvanced adenoma and identify stage I/II colorectal bowel lesions. A cohort of 2396 normal, 660 adenoma, 953 stage I, and 101 stage II blood samples, was constructed to discover screening biomarkers to support case finding of patients at high risk for nonadvanced adenoma and stage I/II cancer for subsequent diagnostic colonoscopy. Results: A three-analyte mProbe panel was constructed which outperformed the commercial assays of plasma methylated septin 9 and fecal Cologuard tests. Sensitivity: (1) nonadvanced adenoma–Cologuard 17.2%, mProbe 76.0%; (2) stage I-III-Cologuard 93.3%, stage I-II Septin 9 (ARUP laboratories) 77%, stage I-II mProbe: 92.3%. Specificity–Cologuard 89.8%, Septin 9 (ARUP laboratories) 88%, mProbe 90.7%. Conclusions: mProbe triage concept of a blood-based protein biomarker panel promises the precision to allow future CRC screening, and reduce the low-risk utilization of unnecessary, unpleasant and risk-associated bowel examinations.

Methylated Septin 9 and Carcinoembryonic Antigen for Serological Diagnosis and Monitoring of Patients with Colorectal Cancer After Surgery

With the increasing incidence and mortality of colorectal cancer (CRC), early and accurate diagnosis is of paramount priority to combat this cancer. Epigenetic alterations such as DNA methylation are innovative biomarkers for CRC, due to their stability, frequency, and accessibility in bodily fluids. In this study, blood samples were prospectively collected from patients before and after operation for CRC for determination of methylated septin 9 (mSEPT9) and compared to carcinoembryonic antigen (CEA). The sensitivity of using mSEPT9 methylation status for diagnosing CRC was significantly higher than using elevated CEA levels (73.2% vs 48.2%; p value < 0.001). The sensitivities of both tests increased with higher tumor staging (P = 0.004 and 0.04 respectively). Combined mSEPT9 and CEA had higher accuracy than single CEA or mSEPT9 (P = 0.009 and 0.532 separately). An increase in the methylation level of mSEPT9 detected in the post-operative samples was associated with a higher mortality rate (15.2% vs 1.8%; P = 0.024) and the presence of metastasis (27.3% vs 7.0%; P = 0.013). mSEPT9 was more sensitive than CEA for diagnosing CRC, and combined mSEPT9 and CEA was more accurate. After curative resection, detection of increased mSEPT9 methylation level may indicate adverse outcomes.

Clinical value of preoperative methylated septin 9 in Chinese colorectal cancer patients

BACKGROUNDThe methylated septin 9 (mSEPT9) assay was the first blood-based test approved by the United States Food and Drug Administration as a colorectal screening test. However, the diagnostic and prognostic role of preoperative mSEPT9 for colorectal cancer (CRC) in Chinese patients is still unknown.AIMTo improve the understanding of diagnostic and prognostic factors, serum mSEPT9 was detected in Chinese CRC patients.METHODSA retrospective analysis of 354 cases, of which 300 had CRC and 54 were normal, was performed in China. Patients’ characteristics, treatments, and laboratory data, including age, the date of surgery, Union for International Cancer Control (UICC) stages, distant metastasis (M), and so on, were collected. Methylation levels of SEPT9 were quantified by quantitative, methylation-specific polymerase chain reaction before surgery. In addition, the effects of mSEPT9 on the occurrence and prognosis of 330 CRC cases from The Cancer Genome Atlas (TCGA) database were evaluated using bioinformatics analyses. Potential prognostic factors for overall survival (OS) and progression-free survival (PFS) were evaluated by Kaplan-Meier univariate analysis.RESULTSIn Chinese CRC patients, positive mSEPT9 was strongly associated with advanced UICC stages, deeper invasion by the primary tumor, and more distant metastasis. Methylation levels of SEPT9 were stage-dependent and showed a stepwise increase in UICC stages (I–IV), primary tumor categories (T1–T4), regional node categories (N0–N2), and distant metastasis categories (M0–M1). The patients with positive mSEPT9 showed a tendency toward lower PFS. After analyzing TCGA clinical data, the high mSEPT9 group was found to be obviously correlated only with more distant metastasis. The patients with high mSEPT9 levels showed a tendency toward lower OS. Besides, nine meaningful mSEPT9 sites were found to provide guidance for the follow-up studies.CONCLUSIONMSEPT9 analysis may add valuable information to current tumor staging. Serum mSEPT9 in Chinese CRC patients appears to offer promising novel prognostic markers and might be considered for monitoring CRC recurrence.

Methylated septin 9 gene for noninvasive diagnosis and therapy monitoring of breast cancer

Background: Blood-based biomarkers specific for breast cancer is urgently demanded to facilitate the screening and reduce breast cancer-related deaths. This study evaluated the potential of methylated septin 9 gene ( mSEPT9 ) as a biomarker for breast cancer. Methods: A total of 86 breast biopsy samples were collected from 59 patients with breast cancer and from 27 patients with benign breast tumor. Two hundred and seven plasma samples were collected from 80 patients with breast cancer, 27 patients with benign breast tumor patients, and 100 healthy volunteers. mSEPT9 was detected using the Epi proColon 2.0 CE test. Additionally, 40 tissue cases of the tumor samples were analysed by real time PCR for SEPT9_v2 mRNA expression and by immunohistochemical staining for SEPT9 protein expression. Serum CEA, CK19 and CA15-3 concentrations were analyzed using an Electrochemiluminescence Immunoassay Kit. Results: The sensitivity of plasma mSEPT9 for detecting breast cancer was 26.3% with a specificity of 97.6%. The plasma mSEPT9 -positive rate was higher in patients with later stage lymph node metastasis (P=0.018), intravascular tumor thrombus (P=0.035), ki67-positive (P=0.033) and p53-positive (P=0.044). Despite its limitation in sensitivity, conversion of plasma mSEPT9 to negative status in mSEPT9 -positive breast cancer patients following cancer treatment was correlated with favorable responses, while persistent positivity was associated with poor responses. mSEPT9 performed comparably with CK19 (P>0.05) and better than CEA (P=0.002) and CA15-3 (P mSEPT9 was detected in 83.1% (49/59) of the breast cancer tissues, whereas 8.0% (2/25) of the benign breast tumor tissues were mSEPT9 positive (P mSEPT9 in breast cancer tissue was associated with tumor size of >2 cm (P=0.026) and with invasion of adipose tissue (P=0.015). Conclusions: Our results indicate that mSEPT9 test has the potential to become a valuable tool in the diagnosis and treatment monitoring of breast cancer for Chinese patients.

Methylated septin 9 gene for noninvasive diagnosis and therapy monitoring of breast cancer

Background: Blood-based biomarkers specific for breast cancer is urgently demanded to facilitate the screening and reduce breast cancer-related deaths. This study evaluated the potential of methylated septin 9 gene (mSEPT9) as a biomarker for breast cancer.

Diagnostic Performance of DNA Hypermethylation Markers in Peripheral Blood for the Detection of Colorectal Cancer: A Meta-Analysis and Systematic Review

DNA hypermethylation in blood is becoming an attractive candidate marker for colorectal cancer (CRC) detection. To assess the diagnostic accuracy of blood hypermethylation markers for CRC in different clinical settings, we conducted a meta-analysis of published reports. Of 485 publications obtained in the initial literature search, 39 studies were included in the meta-analysis. Hypermethylation markers in peripheral blood showed a high degree of accuracy for the detection of CRC. The summary sensitivity was 0.62 [95% confidence interval (CI), 0.56–0.67] and specificity was 0.91 (95% CI, 0.89–0.93). Subgroup analysis showed significantly greater sensitivity for the methylated Septin 9 gene (SEPT9) subgroup (0.75; 95% CI, 0.67–0.81) than for the non-methylated SEPT9 subgroup (0.58; 95% CI, 0.52–0.64). Sensitivity and specificity were not affected significantly by target gene number, CRC staging, study region, or methylation analysis method. These findings show that hypermethylation markers in blood are highly sensitive and specific for CRC detection, with methylated SEPT9 being particularly robust. The diagnostic performance of hypermethylation markers, which have varied across different studies, can be improved by marker optimization. Future research should examine variation in diagnostic accuracy according to non-neoplastic factors.

Value of methylated Septin 9 detection in screening colorectal cancer in elderly people

Objective To investigate the value of detecting methylated SEPT9 gene (mSEPT9) in serum and stool in screening colorectal cancer in elderly people. Methods Subjects were divided into three groups, the colorectal cancer group (n=82), the colorectal polyps group (n=80) and the healthy control group (n=100). Real-time quantitative polymerase chain reaction (PCR) was used to detect mSEPT9 in peripheral blood and feces. The relationship between mSEPT9 and clinical and pathological characteristics of colorectal cancer was analyzed. The agreement between serum and stool results was analyzed. Results The positive rate of serum mSEPT9 was 73.2% in the colorectal cancer group, 6.3% in the colorectal polyps group and 4.0% in the healthy control group, with statistical significance between the colorectal cancer group and the other two groups (P<0.01). The sensitivity and specificity for plasma mSEPT9 detection in screening colorectal cancer were 73.2% and 95.6%, respectively. The positive rate was higher in patients with tumor maximum diameter over 3.0 cm, invasion of the entire serosa layer, lower differentiation or advanced clinical staging (P<0.05). The detection results of plasma mSEPT9 were in complete agreement with those of stool mSEPT9 from the same patients. Conclusions The expression of mSEPT9 is high in elderly patients with colorectal cancer and has a high sensitivity and specificity for PCR detection. Complete agreement can be seen in results from plasma and feces. Detecting mSEPT9 can be a valuable approach to screening colorectal cancer in the elderly. Key words: SEPT9; Colorectal cancer; Methylation

Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA.

BackgroundDetermination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported.MethodsPlasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry.ResultsThe percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors.ConclusionsMethylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.

Abstract 1850: The detection of methylated Septin 9 in tissue and plasma of colorectal neoplasia and its relationship to the amount of free circulating DNA

Abstract Background The determination of methylated Septin 9 (SEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, it has not been investigated how the methylated DNA detected in plasma relates to the occurrence of methylated DNA in colon tissue. The goal of this study was to quantitatively compare levels of methylated SEPT9 level in matched plasma and tissue samples of healthy, adenoma and CRC cases; and to determine the of amount of circulating free DNA (cfDNA) and the expression of Septin-9 protein in tissue. Methods Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 adenomas and 34 CRC. Following bisulfite conversion of DNA a commercial real time PCR assay was used to determine the total amount of DNA in each sample and the portion of DNA methylated at a specific locus of the Septin 9 gene. The presence of Septin-9 protein was determined using immunohistochemistry in healthy (n=10), adenoma (n=14) and CRC (n=13) samples. Results Similar concentrations of cfDNA were detected in NED (49.72 ng/ml), adenoma (45.39 ng/ml) and CRC (70.32 ng/ml) plasma cases. In tissue samples, percent methylated reference (PMR) values of SEPT9 above a selected PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC. PMR differences found between NED vs. adenoma and NED vs. CRC comparisons were highly significant (p&amp;lt;0.001). The protein expression of Septin-9 in tissues determined by IHC inversely correlated to SEPT9 methylation levels with abundant expression in normals and diminished expression levels in adenomas and tumors. In matching plasma samples SEPT9 PMR values, using a cut-off level higher than 0.01%, were detected in 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC cases. Highly significant PMR difference were observed in comparisons between NED vs. CRC (p&amp;lt;0.01) and adenoma vs. CRC (p&amp;lt;0.01). Conclusions Methylated SEPT9 was detected in all tissue samples, at very low levels in the NED group, but at significantly elevated levels in both adenoma and CRC. In plasma samples, elevated mSEPT9 values were only detected in the CRC group, but not in the adenoma group. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Parameters like the degree of vascularization of the lesions, the amount of cfDNA in plasma and probably additional factors seem to be equally important. Citation Format: Alexandra Kalmar, Kinga Toth, Reinhold Wasserkort, Ferenc Sipos, Barnabas Wichmann, Gabor Valcz, Zsolt Tulassay, Bela Molnar. The detection of methylated Septin 9 in tissue and plasma of colorectal neoplasia and its relationship to the amount of free circulating DNA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2014-1850