Multiplex Methylation-specific PCR Research Articles

Methylation Status of SOCS1 and SOCS3 Genes in Patients with Acute Lymphoid Leukemia

Background: Acute Lymphoid Leukemia (ALL) is the leading childhood cancer with a high mortality and morbidity. Studies have suggested an association of epigenetic transformations with prognosis, recurrence and immunophenotypes of ALL. SOCS1 and SOCS3 are tumor suppressors inhibiting JAK/STAT signaling pathway and the resultant aberrant cell proliferation.
 Method: We aimed to assess the association between methylation status and ALL, using bone marrow and peripheral blood samples. 18 patients with ALL and 13 children with no malignancies were included. Using Bisulfite conversion, quantitative multiplex methylation-specific PCR and 2^(-∆∆Ct) formula, the methylated DNA in the promoters of SOCS1 and SOCS3 were measured.
 Results: ALL patients had higher mean methylation in SOCS1 promoter and lower mean methylation in SOCS3 promoter, compared to the control group. However, neither of these mean differences were statistically significant.
 Conclusion: This finding can set the foundation for further large-sample studies with the use of healthy children as a control group to strengthen the hypothetical association of the methylation status of SOCS1 and SOCS3 with ALL.

Analytical validation and diagnostic performance of the ASCL1/ZNF582 methylation test for detection of high-grade anal intraepithelial neoplasia and anal cancer

DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN).For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10−8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity.In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.

B-382 Accurate and Early Detection of Colorectal Cancer Using a Multilocus DNA Methylation Markers-based Testing in Peripheral Blood Mononuclear Cells

Abstract Background An effective blood-based method for the diagnosis of colorectal cancer (CRC) has not yet been developed. Molecular alterations of immune cells occur early in tumorigenesis, providing the theoretical underpinning for early cancer diagnosis. Therefore, we aimed to develop an effective multi-molecular simultaneous detection method based on peripheral blood mononuclear cells (PBMCs) to improve the diagnosis of early-stage CRC. Methods Candidate DNA methylation markers were first identified from PBMCs using the Infinium MethylationEPIC array in the discovery phase (Shandong cohort I, n = 100) and further validated via pyrosequencing and targeted bisulfite sequencing in validation phase (Shandong cohort II, n = 202). Then, a single-tube multiplex methylation-specific quantitative PCR assay (multi-msqPCR) for simultaneous detection of five markers was established in a pilot study. After that, a CRC prediction model (CPM) based on DNA methylation and multi-msqPCR method was construct and its diagnostic performance were evaluated using the area under the receiver operating characteristic curve (AUROC) in our multicenter cohort (n = 595). Results Five discriminative DNA methylation markers identified by Illumina 850K microarray were successfully validated to diagnose early-stage CRC. Multi-msqPCR showed a better discriminative performance and 10-time lower detection limit than single-molecule detection in early-stage CRC. The cross-validated AUROC for CPM for early-stage CRC was 0.91 (sensitivity = 81.18%; specificity = 89.39%), significantly higher than CEA (AUROC = 0.62). CPM assay also yielded a high degree of discrimination for advanced adenoma (AA) cases (AUROC = 0.85; sensitivity = 63.04%; specificity = 89.39%). Besides, detecting CPM in multiple cancer types implied a CRC-specific diagnostic value. Our follow-up data also demonstrated that CPM could detect early-stage CRC up to 2 years before current traditional diagnostic methods. Conclusions CPM, in combination with epigenetic biomarkers and the multi-msqPCR method, was promising, cost-effective, and easily implementable for routine clinical diagnosis of early-stage CRC.

B-381 A Multiplex Blood-based Assay Targeting DNA methylation in Peripheral Blood Mononuclear Cells Enables Early Detection of Breast Cancer

Abstract Background The immune system can monitor the development of tumors, and the DNA methylation is involved in the body immune response to tumors. Therefore, the DNA from peripheral blood mononuclear cell (PBMC) that carries cancer-specific epigenetic aberrations may serve as a non-invasive marker for early detection of breast cancer (BC). Methods Genome-wide DNA methylation profiling on PBMC DNA from BC patients and healthy controls (Cohort I, n = 80) was performed in the discovery phase by Infinium 850K BeadChips. The candidate CpG sites were selected and validated in a multistep validation phase (Cohort II, n = 200) via pyrosequencing and targeted bisulfite sequencing. After that, the multiplex quantitative methylation-specific PCR assay (mBC-MSP) was established to detect BC, and the performance of this diagnostic method was analyzed in a multicenter cohort (Cohort III, n = 501). Results A total of 289 differentially methylated CpG positions (DMPs) between BC patients and healthy controls were identified. Among them, eight significant DMPs markers were selected and further validated using pyrosequencing and targeted bisulfite sequencing, and four hyper-methylated DMPs (cg18637238, cg16652347, cg13828440, cg11754974) passed the final validation. Then, we developed a mBC-MSP assay based on the four methylated markers. This assay exhibited excellent performance in distinguishing BC patients, with an AUC of 0.925, sensitivity of 83.1%, and specificity of 90.4%. More importantly, compared with CA153, CA125 and CEA, the mBC-MSP assay had higher sensitivity for early-stage BC (stage 0, 88.2% vs 0%, 0%, 5.9%) and minimal tumor with diameter ≤1.5 cm (91.7% vs 0%, 2.1%, 0%). Conclusions The mBC-MSP assay based on the four identified DNA methylation markers from PBMC can serve as a noninvasive, accurate, rapid, and high-throughput method for early diagnosis of BC, and it is more sensitive than traditional tumor markers for detecting early-stage BC and minimal tumor.

Abnormal Promoter Methylation of Nucleotide-Binding Oligomerization Domain Containing 2 (NOD2) Gene in the Pathogenesis of Crohn’s Disease

Changes in the expression of nucleotide-binding oligomerization domain containing 2 (NOD2) play an important role in the pathogenesis of several autoimmune diseases, such as inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC). It seems that epigenetic modifications, including DNA methylation, have an important role in the suppression of gene activity. In this study, the relationship between DNA methylation patterns of the promoter region of the NOD2 gene and the pathogenesis of CD was assessed. Colonic mucosal samples were obtained from 15 Iranian patients with CD and 15 matched healthy controls with no history of autoimmune diseases. After bisulfite conversion of genomic DNA, the DNA methylation status of three CpG sites in the promoter region of the NOD2 gene was determined by the real-time quantitative multiplex methylation-specific PCR assay. Using this approach, we identified a decreased level of methylation of the NOD2 promoter in the colonic mucosa of patients with CD (0.128±0.093 vs. 0.025±0.016, unmethylated DNA in CD vs. healthy controls, respectively, P&lt;0.000). According to our findings, promoter hypomethylation of the NOD2 gene in the colonic mucosa might contribute to the development and severity of CD.

A multiplex blood-based assay targeting DNA methylation in PBMCs enables early detection of breast cancer

The immune system can monitor tumor development, and DNA methylation is involved in the body’s immune response to tumors. In this work, we investigate whether DNA methylation alterations in peripheral blood mononuclear cells (PBMCs) could be used as markers for early detection of breast cancer (BC) from the perspective of tumor immune alterations. We identify four BC-specific methylation markers by combining Infinium 850 K BeadChips, pyrosequencing and targeted bisulfite sequencing. Based on the four methylation markers in PBMCs of BC, we develop an efficient and convenient multiplex methylation-specific quantitative PCR assay for the detection of BC and validate its diagnostic performance in a multicenter cohort. This assay was able to distinguish early-stage BC patients from normal controls, with an AUC of 0.940, sensitivity of 93.2%, and specificity of 90.4%. More importantly, this assay outperformed existing clinical diagnostic methods, especially in the detection of early-stage and minimal tumors.

Methylation testing for the detection of recurrent cervical intraepithelial neoplasia.

Women treated for CIN2/3 remain at increased risk of recurrent CIN and cervical cancer, and therefore posttreatment surveillance is recommended. This post hoc analysis evaluates the potential of methylation markers ASCL1/LHX8 and FAM19A4/miR124-2 for posttreatment detection of recurrent CIN2/3. Cervical scrapes taken at 6 and 12 months posttreatment of 364 women treated for CIN2/3 were tested for methylation of ASCL1/LHX8 and FAM19A4/miR124-2 using quantitative multiplex methylation-specific PCR. Performance of the methylation tests were calculated and compared with the performance of HPV and/or cytology. Methylation levels of recurrent CIN were compared between women with a persistent HPV infection, and women with an incident HPV infection or without HPV infection. Recurrent CIN2/3 was detected in 42 women (11.5%), including 28 women with CIN2 and 14 with CIN3. ASCL1/LHX8 tested positive in 13/14 (92.9%) of recurrent CIN3 and 13/27 (48.1%) of recurrent CIN2. FAM19A4/miR124-2 tested positive in 14/14 (100%) of recurrent CIN3 and 10/27 (37.0%) of recurrent CIN2. Combined HPV and/or methylation testing showed similar positivity rates as HPV and/or cytology. The CIN2/3 risk at 12 months posttreatment was 30.8% after a positive ASCL1/LHX8 result at 6 months posttreatment. Methylation levels of CIN2/3 in women with a persistent HPV infection were significantly higher compared with women with an incident or no HPV infection. In conclusion, posttreatment monitoring by methylation analysis of ASCL1/LHX8 and FAM19A4/miR124-2 showed a good performance for the detection of recurrent CIN. DNA methylation testing can help to identify women with recurrent CIN that require re-treatment.

Quantitative Evaluation of NOD2 Promoter Methylation Profiling in Colon Biopsy Samples from Ulcerative Colitis Patients and Non-Colitis Controls

Background: Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the colon with evidence addressing the role of epigenetic changes. The intention of this study was to detect the association of DNA methylation levels of NOD2 gene with UC and to evaluate whether any of these changes might be a useful biomarker for detecting patients with UC.&#x0D; Methods: The methylation status of the promoter CpG islands (CGIs) of NOD2 gene was examined in the colonic mucosae of 15 cancer-free patients with UC and 15 age- and sex-matched healthy controls by the real-time quantitative multiplex-methylation specific PCR (QM-MSP) assay. Methylation-specific melting curve analysis (MS-MCA) were used to analyze the data.&#x0D; Results: The median unmethylated DNA index was significantly higher in cases compared to controls, and hypormethylation of NOD2 gene was significantly correlated with UC (Unmethylated DNA in UC vs. HC; 0.102±0.055 vs. 0.025±0.016, P = 0.000).&#x0D; Conclusion: The NOD2 gene that was differentially methylated in UC patients, providing new insights into the pathogenesis of UC, with a view to making steps toward the development of accurate biomarkers for diagnostic tools in UC.

Evaluation of DNA methylation biomarkers ASCL1 and LHX8 on HPV-positive self-collected samples from primary HPV-based screening

BackgroundHost-cell DNA methylation analysis can be used to triage women with high-risk human papillomavirus (HPV)-positive self-collected cervicovaginal samples, but current data are restricted to under-/never-screened women and referral populations. This study evaluated triage performance in women who were offered primary HPV self-sampling for cervical cancer screening.MethodsSelf-collected samples from 593 HPV-positive women who participated in a primary HPV self-sampling trial (IMPROVE study; NTR5078), were tested for the DNA methylation markers ASCL1 and LHX8 using quantitative multiplex methylation-specific PCR (qMSP). The diagnostic performance for CIN3 and cervical cancer (CIN3 + ) was evaluated and compared with that of paired HPV-positive clinician-collected cervical samples.ResultsSignificantly higher methylation levels were found in HPV-positive self-collected samples of women with CIN3 + than control women with no evidence of disease (P values <0.0001). The marker panel ASCL1/LHX8 yielded a sensitivity for CIN3 + detection of 73.3% (63/86; 95% CI 63.9–82.6%), with a corresponding specificity of 61.1% (310/507; 95% CI 56.9–65.4%). The relative sensitivity for detecting CIN3+ was 0.95 (95% CI 0.82–1.10) for self-collection versus clinician-collection, and the relative specificity was 0.82 (95% CI 0.75–0.90).ConclusionsThe ASCL1/LHX8 methylation marker panel constitutes a feasible direct triage method for the detection of CIN3 + in HPV-positive women participating in routine screening by self-sampling.

Clinical validation of methylation biomarkers for optimal detection of high-grade vulvar intraepithelial neoplasia.

The precursor lesions of vulvar squamous cell carcinoma (VSCC) include human papillomavirus (HPV)-associated and HPV-independent squamous neoplasia with a varying cancer risk. Our study aimed to validate the accuracy of previously identified DNA methylation markers for detection of such high-grade vulvar intraepithelial neoplasia (VIN). A large clinical series of 751 vulvar lesions, originally diagnosed as high-grade VIN, were reassessed and categorized into HPV-associated or HPV-independent vulvar disease categories. Together with 113 healthy vulvar controls, all samples were tested for 12 methylation markers with quantitative multiplex methylation-specific PCR (qMSP). Performance of individual markers and selection of an optimal marker panel for detection of high-grade VIN was determined by logistic regression analysis. SST was the best-performing individual marker (AUC 0.90), detecting 80% of high-grade VIN cases, with excellent detection of HPV-independent VIN (95%), known to have the highest cancer risk. Merely 2% of controls tested methylation positive for SST. Selection of a marker panel, including ZNF582, SST and miR124-2, resulted in a comparably high accuracy for detection of high-grade VIN (AUC 0.89). In conclusion, we clinically validated the accuracy of 12 DNA methylation markers for detection of high-grade VIN. SST, as a sole marker or in a panel, provides an optimal diagnostic tool to distinguish high-grade VIN in need of treatment, particularly HPV-independent VIN, from low-grade or reactive vulvar lesions. These findings warrant further prognostic validation of methylation biomarkers for cancer risk stratification of patients with VIN.

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.

DNA Methylation of JAK2 Gene in Intestinal Biopsy and Peripheral Blood Samples of Patients with Ulcerative Colitis

Background: Comprised of two main subtypes (Ulcerative Colitis (UC) and Crohn), inflammatory bowel disease is caused by an interaction between genetic and environmental factors. As of the important role of innate immunity and JAK/STAT signaling pathway, the current study was designed to investigate the methylation status JAK2 gene in blood and tissue samples of patients with UC.&#x0D; Methods: Genomic DNA was extracted from blood and intestinal biopsy samples of 28 UC patients and 28 controls. After bisulfite DNA conversion, real-time quantitative multiplex methylation specific PCR (QM-MSP) method was applied in order to assess JAK2 promotor methylation status.&#x0D; Results: The JAK2 promotor in the intestinal biopsy samples was significantly hypermethylated in UC as the mean of unmethylated DNA was 1.255±1.865 in the patients group, while it was 1.292±4.726 in control group.&#x0D; Conclusion: Hypermathylation of JAK2 gene may play a part in pathophysiology of UC which could result in gene silencing.

Association between Promotor hypomethylation of TFF1 and Crohn's Disease.

Background and aim:Epigenetic modifications exhibit promising evidence in etiology and prognosis of important diseases such as inflammatory bowel diseases (IBD). In addition to complex factors involved in IBD, a trend toward better prognosis have been reported in older ages of disease onset. Gastrointestinal mucous layer is one of the important components which is disturbed in the disease course. Integrity of this layer is maintained with an anti-inflammatory factor called trefoil factors (TFF). We investigated the methylation status of TFF1 gene in IBD patients alongside with correlation of its alteration level with age of disease onset.Methods:We analyzed the promoter methylation status of TFF1 gene, using the real-time quantitative multiplex methylation specific PCR (QM-MSP). DNA was extracted from colorectal biopsies of 15 Crohn disease cases and 15 healthy controls. Correlation analysis was performed between unmethylated DNA level and age through Pearson correlation coefficient (PPC) test and simple linear regression models.Results:Our data didn’t provide significant positive correlation of age and TFF1 hypomethylation in Crohn patients (r = .518, p = .058).Conclusions:In conclusion, our case-control study didn’t show significant alteration in TFF1 methylation status in CD patients. (www.actabiomedica.it)

Association of Aberrant Promoter Methylation Changes in the Suppressor of Cytokine Signaling 3 (SOCS3) Gene with Susceptibility to Crohn's Disease.

Background:Growing evidence supports that changes in the methylation state of Inflammatory Bowel Disease (IBD)-associated genes could significantly alter levels of gene expression, potentially contributing to disease onset and progression. We supposed that alterations in DNA methylation status at promoter region within the suppressor of cytokine signaling 3 (SOCS3) gene in intestinal tissues may be involved in the susceptibility to Crohn's Disease (CD).Methods:DNA methylation status in the promoter region of the human SOCS3 gene of intestinal tissues from 15 patients with CD and 15 age- and sex-matched healthy controls were profiled using the real-time Quantitative Multiplex Methylation Specific PCR (QM-MSP) assay.Results:Based on methylation assay data profiling, we found that patients with CD showed a higher degree of methylation of the SOCS3 gene promoter region than did the healthy controls (unmethylated DNA in CD vs. healthy controls; 0.00048±0.0011 vs. 0.07±0.142, p<0.000).Conclusion:The data presented here demonstrate that aberrant methylation of the CpG islands within promoter regions of SOCS3 gene in colonic mucosa of CD was associated with mucosal inflammatory status, providing insights into the involvement of methylation could contribute to the initiation of the inflammatory process and development of CD.

High performance methylated DNA markers for detection of colon adenocarcinoma

BackgroundColon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA.MethodsHierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann–Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy.ResultsIn the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue.ConclusionsWe identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.

Glutathione S transferase T1 gene polymorphism and its promoter methylation are associated with breast cancer susceptibility in Egyptian women.

Breast cancer (BC) is one of the leading causes of cancer mortality in women. Glutathione S-transferase (GSTT1) is involved in activation of detoxification reactions and catalysis of chemicals conjugation with glutathione. GSTT1 genotype is a limiting factor for some environmental diseases. Epigenetic changes have an essential role in BC through inappropriate interaction between genomic and environmental risk factors. This study was directed to explore the association of BC risk with GSTT1 genetic variations and its methylation status in Egyptian women. This study included 100 healthy women as the control group and 100 patients were clinically and histologically diagnosed with breast cancer. All blood samples were used for genomic DNA extraction. GSTT1 genotyping was accomplished by multiplex PCR and methylation-specific PCR was used to analyze the GSTT1 promoter methylation status. Breast cancer patients showed significant incidence of null GSTT1 in relation to controls (p = 0.004). GSTT1 gene promoter methylation status showed significant difference between hypermethylated and unmethylated patients when compared with healthy subjects (p = 0.005). GSTT1 promoter methylation status was not significantly associated with null genotype. There was no significant association between GSTT1-null genotypes and BC stage in cases with or without family history, but for promotor methylation, there was significant association with stage III and IV breast cancer disease. GSTT1 null genetic variant and promoter hypermethylation in the GSTT region of the gene may be considered as critical risk factors for BC in Egyptian women.

A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients \u2013 Design of the Urodiag\xae PCR kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

BackgroundWe have recently developed a highly accurate urine-based test, named Urodiag®, associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating a cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine.MethodsComparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays.ResultsComplete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at − 20 °C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA.ConclusionsWe developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

Abstract P4-02-06: Automated molecular diagnosis of suspicious axillary lymph nodes in breast cancer patients

Abstract Background: Axillary lymph node metastasis is associated with a high risk of breast cancer recurrence and is the single most powerful predictor of patient prognosis. Various imaging tests alone or in combination with preoperative fine needle aspiration (FNA) cytology are used to evaluate suspicious lymph nodes (LNs). However, these methods have low sensitivity (70%) and their utility varies depending on the local surgical practice and experience of the cytologists. In previous work, we have demonstrated higher than 90% sensitivity/specificity of a panel of ten hypermethylated biomarkers in FNA to distinguish between cancer and benign breast lesions. Analysis of methylated genes in FNA of suspicious LNs may improve sensitivity of cancer detection and assist in staging disease more accurately. Methods: We used Quantitative Multiplex Methylation-Specific PCR (QM-MSP) to determine the utility of the 10-gene panel for detecting tumor cells in LN FNAs. Two studies were performed to test the accuracy of a newly developed automated, cartridge-based GeneXpert® Breast Cancer Detection Assay (RUO*). First, we conducted a prospective case-control study in China in breast cancer patients with palpable LNs undergoing sentinel lymph node biopsy. FNA specimens were collected intraoperatively from a single palpable LN and assayed. Histopathology of the same LN (88 metastatic and 104 benign), was used as the gold standard for comparison. Next, using the automated assay, a pilot validation study was conducted on archival ultrasound-guided FNAs (69 metastatic and 53 benign by cytopathology) collected preoperatively from enlarged axillary LNs in patients with and without breast cancer in the outpatient centers in China and U.S.A. We calculated sensitivity, specificity and area under the receiver operating characteristic curve (AUC) for the marker panel tested by the GeneXpert® assay compared to histopathology for the case control study, and cytopathology for the pilot validation study. We also compared its performance to the highly sensitive laboratory assay, QM-MSP. Results: In the case-control study in China, compared to histopathology, cytopathology of the palpable enlarged LNs achieved cancer detection sensitivity of 85% and specificity of 95% (ROC; AUC=0.898, 95% CI: 0.843-0.953). Using the GeneXpert® assay on the same samples, compared to histopathology, the methylated biomarker panel showed a sensitivity of 92.0% and a specificity of 98.1% based on receiver operating characteristic statistics (ROC; AUC = 0.960, 95% CI: 0.928-0.993). QM-MSP yielded a similar sensitivity of 91.8% and a specificity of 97.7% (ROC; AUC = 0.966, 95% CI: 0.934-0.998). In the pilot validation set, compared to cytopathology, the automated assay achieved a sensitivity of 94.2% and a specificity of 92.5% (ROC; AUC=0.976 (95%CI: 0.950-1.001). Conclusions: We have developed and piloted a 5-hour, automated GeneXpert® Breast Cancer Detection Assay (RUO*) on FNA to detect cancer in suspicious LNs. This assay performs with an accuracy equivalent to that of the highly sensitive but labor-intensive assay, QM-MSP. This test has the potential to determine axillary involvement with higher sensitivity than cytopathology within a very short time in the outpatient setting. A positive test preoperatively could provide guidance for decision-making regarding eligibility of patients for neoadjuvant treatment, post-mastectomy radiotherapy and breast reconstruction. Further evaluation of this automated assay in larger, blinded, prospective studies is warranted. Citation Format: Juanjuan Li, Bradley Downs, Leslie Cope, Xiuyun Zhang, Chuan-gui Song, Kejing Zhang, Yong Han, Mary Jo Fackler, Edwin Lai, Suzana Tulac, Neesha Venkatesan, Timothy Guzman, Chuang Chen, Jingping Yuan, Saraswati Sukumar. Automated molecular diagnosis of suspicious axillary lymph nodes in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-02-06.

Abstract 3324: Non-invasive detection of aberrant DNA methylation in colorectal cancer by multiplex methylation specific PCR

Abstract Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide, with an increasing prevalence. The 5-year relative survival rate for advanced stages is very low, with only 14% for stage IV. However, that of the early stage of CRC is about 90%. Therefore, it is important to screen CRC in the early stage. Epigenetic alterations linked to the carcinogenesis of CRC have been shown to occur earlier and more frequently than genetic alterations in CRC. Here, our study aims to develop a CRC screening methodology by applying the multiplex methylation specific PCR (MMSP) assay to detect CRC-specific methylation biomarkers, which has been pre-selected from public databases and subsequently validated in CRC samples of the Chinese population. Experimentally, cell free DNA (cfDNA) is extracted from ~4ml peripheral blood plasma. Following bisulfite conversion of the cfDNA, MMSP were applied to amplify and detect CRC-specific methylated CpG sites within the cfDNA. From the results of 28 CRCs and 52 control specimens, a sensitivity of 85.7% and the a specificity of 92.3% was achieved. Current standard and most widely used method of detecting CRC is colonoscopy screening. However, colonoscopy requires bowel preparation, and the sedation for patients is a complex and time-consuming procedure with high cost. Additionally, this invasive method also increases the possibility of infection and complications and patient compliance still largely remains a problem. Our non-invasive MMSP assay therefore provides an alternative non-invasive screening option that is cost-effective, meeting the urgent demand of early CRC detection. In addition, because colonoscopy screening currently has low adherence in Chinese population, our method holds great potential for increasing CRC screening rate in China. Citation Format: Lili Ye, Chunting Zheng, Yuan Jie, Bin Li, Yuan Li, Kunling Hu, Liuhong Zeng, Yuying Wang, Mao Mao, Peirong Ding, Taiping Shi, Mingzhi Ye. Non-invasive detection of aberrant DNA methylation in colorectal cancer by multiplex methylation specific PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3324.

Cell-Free DNA Methylation of Selected Genes Allows for Early Detection of the Major Cancers in Women.

Background: Breast (BrC), colorectal (CRC) and lung (LC) cancers are the three most common and deadly cancers in women. Cancer screening entails an increase in early stage disease detection but is hampered by high false-positive rates and overdiagnosis/overtreatment. Aberrant DNA methylation occurs early in cancer and may be detected in circulating cell-free DNA (ccfDNA), constituting a valuable biomarker and enabling non-invasive testing for cancer detection. We aimed to develop a ccfDNA methylation-based test for simultaneous detection of BrC, CRC and LC. Methods: CcfDNA from BrC, CRC and LC patients and asymptomatic controls were extracted from plasma, sodium-bisulfite modified and whole-genome amplified. APC, FOXA1, MGMT, RARβ2, RASSF1A, SCGB3A1, SEPT9, SHOX2 and SOX17 promoter methylation levels were determined by multiplex quantitative methylation-specific PCR. Associations between methylation and standard clinicopathological parameters were assessed. Biomarkers’ diagnostic performance was also evaluated. Results: A “PanCancer” panel (APC, FOXA1, RASSF1A) detected the three major cancers with 72% sensitivity and 74% specificity, whereas a “CancerType” panel (SCGB3A1, SEPT9 and SOX17) indicated the most likely cancer topography, with over 80% specificity, although with limited sensitivity. Conclusions: CcfDNA’s methylation assessment allows for simultaneous screening of BrC, CRC and LC, complementing current modalities, perfecting cancer suspects’ triage, increasing compliance and cost-effectiveness.