RASSF1A Gene Research Articles

Assessment of Aberrant Promoter Hypermethylation of RASSF1a and P16 in Endometrial Carcinoma

Endometrial carcinoma is one of the most common gynecological malignancies known to affect around 142000 women worldwide. Aberrant promoter hypermethylation of tumor suppressor genes is a common epigenetic alteration reported in cancers. In the present study we report the aberrant promoter hypermethylation of RASSF1a and p16 in 78 endometrial cancer patients. Methods: Endometrial carcinoma samples were collected after pathological examination and DNA was extracted. The DNA was subjected to sodium bisulfite modification and then used to perform methylation specific PCR. The PCR was performed using a two step procedure of nested and methylation specific PCR. The PCR product was visualized using agarose gel electrophoresis. The results obtained were correlated with various clinicopathological parameters. Chi square test was used for statistical analysis and a p-value of <0.05 was considered statistically significant. Result: 60 of the 78 (76.92%) samples showed methylation for RASSF1a gene. 28 of the 78 (35.8%) samples showed methylation for p16 gene. A higher methylation frequency was observed for RASSF1a in the endometrioid and poorly differentiated histological subtypes and stage I and II of the disease. Conclusion: Aberrant promoter hypermethylation of RASSF1a and p16 is known to play an important role in endometrial cancer initiation and progression. The methylation patterns can be used as an important molecular marker for early diagnosis and prognosis of endometrial cancer. Keywords: Epigenetic alterations, DNA Hypermethylation, Endometrial cancer, RASSF1a, p16, Methylation-specific PCR.

Epigenetic Regulation of the Tumour Suppressor RASSF1A in Bone Cancer Cells: DNA Methylation Study

Objective: Osteosarcoma is a bone cancer that affects children and adolescents. The RASSF1A is a tumor suppressor capable of mediating the regulation of cell cycle arrest, migration, including apoptosis. It is the most continually silenced gene that contributes to human cancer. Furthermore, RASSF1A functions as a scaffold protein that can regulate microtubules network and bind apoptotic kinases MST1 and MST2 via the Sav-RASSF-Hippo domain. Epigenetic inactivation of genes by DNA methylation is a key factor regulating gene expression and genomic stability. Our aim was to study the RASSF1A gene promoter methylation in three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines.
 Materials and Methods: Three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines were used to study RASSF1A gene promoter methylation, using bisulphite conversion of DNA, followed by methylation-specific polymerase chain reaction (PCR)
 Results: The RASSFIA’s gene promoter methylation was established as a frequent event. Hypermethylation of RASSF1A promoter, was detected in five out of six studied cell lines.
 Conclusions: These results demonstrated that altering the Sav-RASSF1-Hippo may be accomplished through hypermethylation of RASSF1A and may play an essential role in Ewing’s sarcoma and Osteosarcoma. The methylation pattern of Sav-RASSF1-Hippo tumor suppressor pathway in human bone cancer along with RASSF1A expression with its effector proteins merits further investigation. This may reveal how the RASSFIA has a physiological signal transduction, including how the process of its deregulation can contribute to transformation of the cell, eventually leading to the incorporation of novel therapeutic options with improved prognosis for bone cancer.

RASSF1A Suppression as a Potential Regulator of Mechano-Pathobiology Associated with Mammographic Density in BRCA Mutation Carriers.

Simple SummaryHigh mammographic density (MD) is a significant risk factor for the development of breast cancer, as is inheritance of mutations in BRCA1 or BRCA2 tumour suppressor genes. High MD combined with BRCA1/2 gene mutations synergistically increases breast cancer risk, yet BRCA1/2 mutations alone or in combination do not increase MD or exacerbate the inherent tissue stiffness that high MD creates. The molecular basis for this additive effect therefore remains unclear. Our data indicate that the combinatory effect of high MD and BRCA mutations on breast cancer risk may be a product of repression of the tumour suppressor gene RASSF1A, in regions of increased tissue stiffness.High mammographic density (MD) increases breast cancer (BC) risk and creates a stiff tissue environment. BC risk is also increased in BRCA1/2 gene mutation carriers, which may be in part due to genetic disruption of the tumour suppressor gene Ras association domain family member 1 (RASSF1A), a gene that is also directly regulated by tissue stiffness. High MD combined with BRCA1/2 mutations further increase breast cancer risk, yet BRCA1/2 mutations alone or in combination do not increase MD. The molecular basis for this additive effect therefore remains unclear. We studied the interplay between MD, stiffness, and BRCA1/2 mutation status in human mammary tissue obtained after prophylactic mastectomy from women at risk of developing BC. Our results demonstrate that RASSF1A expression increased in MCF10DCIS.com cell cultures with matrix stiffness up until ranges corresponding with BiRADs 4 stiffnesses (~16 kPa), but decreased in higher stiffnesses approaching malignancy levels (>50 kPa). Similarly, higher RASSF1A protein was seen in these cells when co-cultivated with high MD tissue in murine biochambers. Conversely, local stiffness, as measured by collagen I versus III abundance, repressed RASSF1A protein expression in BRCA1, but not BRCA2 gene mutated tissues; regional density as measured radiographically repressed RASSF1A in both BRCA1/2 mutated tissues. The combinatory effect of high MD and BRCA mutations on breast cancer risk may be due to RASSF1A gene repression in regions of increased tissue stiffness.

Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress.

Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O2) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

Genistein From Fructus sophorae Protects Mice From Radiation-Induced Intestinal Injury.

The development of an effective pharmacological countermeasure is needed to reduce the morbidity and mortality in high-dose ionizing radiation-induced acute damage. Genistein has shown bioactivity in alleviating radiation damage and is currently synthesized by chemosynthetic methods. Due to concerns about chemical residues and high costs, the clinical application of genistein is still a major challenge. In this study, we aimed to establish an efficient method for the extraction of genistein from Fructus sophorae. The effects of extracted genistein (FSGen) on preventing intestinal injury from radiation were further investigated in this study. C57/BL mice were exposed to 7.5 Gy whole body irradiation with and without FSGen treatments. Histological analysis demonstrated significant structural and functional restitution of the intestine and bone marrow in FSGen-pretreated cohorts after irradiation. Through mRNA expression, protein expression, and small interfering RNA analyses, we demonstrated that FSGen protects IEC-6 cells against radiation damage by upregulating the Rassf1a and Ercc1 genes to effectively attenuate DNA irradiation damage. Together, our data established an effective method to extract genistein from the Fructus sophorae plant with high purity, and validated the beneficial roles of the FSGen in protecting the radiation damage. These results promise the future applications of Fructus sophorae extracted genistein in the protection of radiation related damages.

Methylation status of eight tumor suppressors in neuroendocrine pancreatic tumors.

e16197 Background: Aberrant DNA methylation is a characteristic feature of cancer, affecting gene expression and tumor phenotype. In this study, we quantified the methylation of promoters of eight tumor suppressor genes in pancreatic neuroendocrine tumors (Pan-NET). Methods: The method of pyrosequencing was used to quantity level (Met,%) of methylation of gene promoters - tumor suppressors AHRR, APC1A, DAPK, MGMT, MLH1, P16, RASSF1A, RUNX3 in tumor samples from 55 patients with pancreatic NET (G1-G3) and in the blood of 10 healthy donors. Met for each sample was calculated as the median methylation of CpG sites in triplicate. Results: Hypermethylation was observed for AHRR (75%), APC1A (25%), RASSF1A (30%). In contrast, DAPK, MGMT, MLH1, P16, RUNX3 had low methylation levels ( &lt; 20%). The median of methylation in the blood of healthy donors for AHRR was 91% (76-98); for all other loci it did not exceed 6%. A high incidence of methylation in excess of blood levels in healthy donors was identified for RASSF1A (0.96); AHRR (0.75); MGMT (0.65); RUNX3 (0.41), APC1A (0.25). For tumor suppressor P16, only one case of increased methylation was recorded (Met = 15%), despite the fact that this phenomenon is not uncommon for NETs of other localizations. In 66% of pancreatic NET cases, hypermethylation of more than two promoters of tumor suppressor genes was noted. An association tendency was found between the presence of MEN1 mutations and the RASSF1A methylation level (p = 0.08). Correlation analysis revealed a significant level of negative association between changes in methylation of MLH1 and AHRR (p &lt; 0.01); for the latter, the prognostic value of a high methylation status and a better prognosis for many malignant neoplasms were described. Conclusions: In the present study, significant methylation of the promoters of the APC1A, DAPK, MGMT, RASSF1A, and RUNX3 genes in well-differentiated pancreatic NETs was identified with a high frequency. At the same time, isolated cases of hypermethylation were noted for the well-known tumor suppressors MLH1 and P16.

Editor's Note: Epigenetic Modifications of RASSF1A Gene through Chromatin Remodeling in Prostate Cancer.

The editors are publishing this note to alert readers to a concern about this article (1): following an institutional review by the Veterans Affairs Medical Center, San Francisco and the University of California, San Francisco, the primary affiliations for several of the authors, it was determined that blank panels were used to represent negative results for AcH4 modification of the RASSF1A gene in LNCaP cells in Fig. 5A and AcH4 modification of the RASSF1A gene in cells untreated for 5-aza-dC and TSA in Fig. 6.

Aberrant Promoter Hypermethylation of p16 and RASSF1a Genes in Colorectal Cancer - Significance in Young Patients.

The clinical profile of colorectal cancers (CRC) in India is different from that described in western countries. Microsatellite instability and APC mutation explain the molecular biology of up to 50% of colorectal cancers. Global genome hypermethylation may be the cause in at least 20% of cases. Few studies from India have examined the epigenetic profile of colorectal cancers. This study was designed to study aberrant promoter hypermethylation of two select tumour suppressor genes (p16, RASSF1a) in patients with colorectal cancer and their association with clinicopathologic features. A total of 41 samples including controls were collected from colorectal cancer patients. DNA was isolated from tumour tissue, and methylation-specific PCR was performed for the 2 genes. p16 and RASSF1a promoter hypermethylation was found in 26% and 48% of CRC cases, respectively. RASSF1a promoter hypermethylation was more often seen in young CRC patients aged 40years or less, and this was found to be statistically significant (p value = 0.037). RASSF1a hypermethylation is peculiar to rectal cancers and left-sided colonic tumours in young patients. Large-scale population-based studies with extensive genetic and epigenetic characterization are required for a better understanding and further validation of our findings. For individuals diagnosed with sporadic CRC, these studies on specimen might help predict prognosis and response to therapy.

Impact of RASSF1A gene methylation on clinico-pathological features of tumor and non-tumor tissue of breast cancer

BackgroundBreast cancer is the most common malignancy in women caused by genetic and epigenetic changes. Promoter DNA methylation in tumor suppressor gene plays a major role in breast cancer. The study determined the association of promoter DNA methylation of RASSF1A gene with clinicopathological features in tumor and non-tumor tissue. Materials and methodsA cross sectional study was conducted in the Department of Pathology, Government Institute of Medical Sciences, Greater Noida and Molecular Pathology Laboratory, Department of Pathology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences. Two sections, one from tumor and the other from non-tumor tissue, were obtained and processed for DNA extraction and bisulphite conversion. Methylation specific PCR was done and results of RASSF1A promoter methylation were statistically correlated with clinicopathological features. ResultsOf the 27 breast cancer tissue, 22 showed invasive ductal carcinoma, one showed invasive lobular carcinoma, another showed ductal carcinoma in situ and three cases showed malignant phyllodes tumor of breast. DNA promoter methylation was found in all the cases. 93% of tumor tissue samples and 67% of the non-tumor tissue samples were found to be aberrantly methylated. Tumor size and histological grade were found to be significantly (p-val <0.05) associated with the RASSF1A gene promoter methylation. ConclusionA significant association of higher tumor size and tumor histological grade with promoter methylation of RASSF1A gene exists suggestive of its being an important determinant of prognostic staging. This critical event in tumorigenesis may be of clinical utility in assessing breast cancer progression. Micro abstractThe study focuses on the RASSF1A gene promoter methylation and its impact on the clinicopathological features in Indian breast cancer patients highlighting the differences from other genetically different population. We found that RASFF1A gene methylation has significant impact on tumor size and tumor grade. The work carries high significance because it addresses the DNA methylation of tumor suppressor gene in relevance of breast cancer. It may also be the first such report on Indian patients with breast cancer.

Cell-Free Foetal DNA as a Useful Marker for Preeclampsia Prediction.

Preeclampsia (PE) is a leading cause of maternal complications and is diagnosed by clinical manifestation. The aim of the study was to evaluate changes of cell-free DNA (cfDNA) and cell-free foetal DNA (cffDNA) concentration during uncomplicated pregnancy and PE in one group of women, and define the predictive value for PE. A total of 580 women were prospectively evaluated, 20 of them developed PE and were included in laboratory analysis, and 22 healthy pregnant with uncomplicated pregnancy were included as the laboratory control group. We determined cfDNA and cffDNA in maternal blood at 11-14, 24-26, and 30-32weeks. Level of cfDNA was evaluated by determining the RASSF1A gene using PCR analysis, cffDNA-by determining the hypermethylated part of RASSF1A gene. The concentration of cfDNA did not differ in the first and second trimesters but significantly increased at 30-32weeks in both groups. During uncomplicated pregnancy, median cffDNA level increased from 14.15 GE/ml to 24.87 GE/ml (p = 0.002) and 32.62 GE/ml (p = 0.005). In the PE group, an elevation in cffDNA level was significant only in the second half of pregnancy (from 54.85 to 96.72 (p > 0.05) and 158.30 GE/ml (p = 0.031) at 11-14, 24-26, and 30-32weeks, respectively). At all studied periods, cffDNA level in the PE group was significantly higher compared to uncomplicated pregnancy (р < 0.001). ROC analysis showed that a cut-off value of cffDNA concentration 22.54 GE/ml in maternal blood at 11-14weeks of pregnancy had the greatest predictive value for PE prediction, with 85.0% sensitivity and 81.8% specificity. CffDNA is a promising marker for PE prediction from the first trimester of pregnancy.

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients.

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer.

Cellular Cytotoxicity and Epigenetic Alteration in RP1 and RASSF1A Genes as Response for Anticancer Capabilities of Some Probiotic Bacteria in Breast Cancer

Cellular Cytotoxicity and Epigenetic Alteration in RP1 and RASSF1A Genes as Response for Anticancer Capabilities of Some Probiotic Bacteria in Breast Cancer

Methylation Profiling of Specific Genes in Ependymomas.

Ependymomas are neuroepithelial tumors of the central nervous system with heterogeneous biology and clinical course. The aim of the present study is to investigate the relationship between the methylation status and clinicopathological parameters in ependymomas. DNA methylation status of CDKN2A, RASSF1A, KLF4 and ZIC2 genes were quantitatively analyzed with pyrosequencing in 44 ependymoma tumor tissues. The relationship of methylation profiles with tumor subtype, histological grade and patient age was statistically analyzed. DNA methylation analyses for CDKN2A revealed no difference in methylation levels. Of the 31 included samples for optimal ZIC2 methylation analysis, 10 were hypermethylated (32.3%) and this change was significantly found in the adult spinal ependymomas (p=0.01). KLF4 hypermethylation was observed in 6 of the overall included 35 samples (17.1%); however, there was no statistically significant relation of the methylation status with tumor subtype, histological grade or age group. RASSF1A hypermethylation was observed in overall 40 included samples with varying methylation levels. Higher levels of hypermethylation were significantly related to the grade 3 histology (p=0.01) and spinal ependymomas (p=0.006). The pediatric cases with grade 3 ependymomas and ependymomas of adulthood showed significantly increased RASSF1A hypermethylation levels (p < 0.001 and p=0.001, respectively). DNA methylation changes are likely to have biological importance in ependymomas. Both ZIC2 and RASSF1A methylation status may be useful parameters in the subclassification of these tumors.

Methylation profiles of genes in breast cancer luminal HER2-negative primary tumor during regional lymphnode metastasis

Aberrant methylation is strongly associated with development of cancer, but limited data exist on correlation between methylation and regional lymph node metastasis (RLNM). The aim of this research was to study using of methylation levels of WIF1, RASSF1A, CDO1 and MEST aberrant methylated genes in a primary breast cancer for prediction of regional lymph node metastases. We used MS-HRM (Methylation Sensitive High Resolution Melting) to assess methylation levels. The results were confirmed by pyrosequencing. The study included 66 women with LumA and 46 women with HER2- (LumB-), 22 and 26 of them had metastasis in at least one lymph node respectively. It was found that methylation levels between LumA and LumB subtypes differed significantly in genes: WIF1 (p<0.001), CDO1 (p=0.002) and MEST (p=0.033). In the Lum A subtype statistically significant differences in level of methylation of WIF1 gene between patients with metastases in RLNM and patients without metastases were found (p=0.03). Analysis of tumors longer than 2 cm in the LumA subtype, revealed an increase of statistical significance of WIF1 gene - p=0.009 (AUC (95%CI) = 0.76 (0.59-0.93)). In LumB- subtype RASSF1A, CDO1 and MEST had statistically significant differences in methylation level between groups (p=0.03, p=0.048 and p=0.045 respectively). ROC analysis showed that combining of three genes by logistic regression, AUC (95%CI) was 0.74 (0.6-0.88). Analysis of tumors longer than 2 cm, did not increase statistical significance for these genes (p=0.046; p=0.089 and p=0.076, respectively). Thus, the study of methylation in primary tumors may be useful for prediction of lymph node metastasis, as well as for better understanding of biological process inside breast cancer.

Promoter Methylation Status and Expression Levels of RASSF1A Gene in Different Phases of Acute Lymphoblastic Leukemia (ALL)

Background: Although the precise pathogenesis of acute lymphoblastic leukemia (ALL) remains unclear, studying gene-regulating mechanisms during ALL pathogeneses may shed light on the underlying mechanisms driving malignant behavior. There is some evidence showing the promoter hypermethylation and silencing of RASSF1A tumor suppressor gene in ALL cells; however, there is a lack of evidence for whether the gene indeed alters during different phases of ALL or in response to therapy. Thus, the current study aimed to clarify this issue using groups of adult ALL patients who have been scarcely investigated regarding expression levels and promoter methylation status. Materials and Methods: In this case/control study, the expression levels and methylation status of the gene promoter was evaluated using quantitative real-time PCR and methylation-specific PCR (MSP), respectively in adults with ALL. The study included peripheral blood of patients with newly diagnosed ALL (n=10), complete remission (CR) (n=10), or relapse (n=10), and 10 control samples from healthy individuals. Results: MSP results revealed an unmethylated status for almost all patients and control samples, except a case with relapsing ALL, which showed a hemimethylated pattern. RASSF1A also showed no difference in terms of gene expression in the patients compared with the control group (p>0.05). Conclusion: The results revealed an up-regulation of RASSF1A tumor suppressor in adult ALL patients experiencing CR, suggesting this to be a marker of therapy response. However, further investigations using more sensitive methylation detecting tools with larger sample sizes may better clarify the involvement of the promoter methylation of RASSF1A in these patients.

Prenatal RHD genotyping in Croatia: preliminary results.

Croatian Institute of Transfusion Medicine (CITM) implemented non-invasive fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The diagnostic performance of in-house RT-PCR method for fetal RHD genotyping and preliminary results are analyzed. Evaluation included results of RHD genotyping for 205 RhD negative pregnant women, 12-36th week of gestation, whose samples were received in period between 2015 and 2020. QIAsymphony SP DSP Virus Midi Kit was used for cffDNA extraction on QIAsymphony SP platform (Qiagen, Germany). Fragments of RHD exons 7 and 10 and later exon 5 were RT-PCR amplified. As internal controls, amplification of SRY gene or RASSF1A fragment and β-actin genes digested with BsTUI were used. We identified 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes. We had one inconclusive result (0.50%) due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. When compared to newborns RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity and 95.08% specificity, while diagnostic accuracy was 98.48%. We were able to determine one case of fetal variant genotype RHD*04.04/*01N.01 inherited from the father. The negative and positive predictive test values were 100% and 97.86%, respectively. Automated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and fragments of SRY, RASSF1A genes represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly cleaning procedures are required.

Investigation of MLH1, MGMT, CDKN2A, and RASSF1A Gene Methylation in Thymomas From Patients With Myasthenia Gravis.

A feature of thymomas is their frequent association with myasthenia gravis (MG), an autoimmune disease characterized by the production of autoantibodies directed to different targets at the neuromuscular junction. Indeed, almost 30–40% of thymomas are found in patients with a type of MG termed thymoma-associated MG (TAMG). Recent studies suggest that TAMG-associated thymomas could represent a molecularly distinct subtype of thymic epithelial tumors (TETs), but few data are still available concerning the epigenetic modifications occurring in TAMG tissues. The promoter methylation levels of DNA repair (MLH1 and MGMT) and tumor suppressor genes (CDKN2A and RASSF1A) have been frequently investigated in TETs, but methylation data in TAMG tissues are scarce and controversial. To further address this issue, we investigated MLH1, MGMT, CDKN2A, and RASSF1A methylation levels in blood samples and surgically resected thymomas from 69 patients with TAMG and in the adjacent normal thymus available from 44 of them. Promoter methylation levels of MLH1, MGMT, CDKN2A, and RASSF1A genes were not increased in cancer with respect to healthy tissues and did not correlate with the histological or pathological features of the tumor or with the MG symptoms. The present study suggests that hypermethylation of these genes is not frequent in TAMG tissues.

DNA methylation patterns of RAR-β2 and RASSF1A gene promoters in FNAB samples from Greek population with benign or malignant breast lesions.

Promoter hypermethylation is common in Breast Cancer (BC) with studies mainly in histological specimens showing frequent methylation of tumor suppressor genes (TSGs) compared with normal tissues. The aim of this study was to estimate the frequency of promoter methylation of RAR-β2 and RASSF1A genes in breast FNAB material aiming to evaluate the methylation status of these two genes as biomarker for detecting BC in Greek population. FNAB material from 104 patients was collected for cytological evaluation and epigenetic analysis. DNA was extracted and subjected to bisulfite conversion. A methylation-specific PCR was carried out and the final products were separated with electrophoresis in 2% agarose gels. From 104 samples, RASSF1A hypermethylation was observed in 78 (75%) and RAR-β2 hypermethylation in 64 (61.6%). 84% and 78% of the cases diagnosed with breast malignancy (n = 50) were methylated for RASSF1A and RAR-β2, respectively. Methylated RASSF1A and RAR-β2 were also detected in 88.3% and 76.5% in samples diagnosed as suspicious for malignancy (n = 17) and in 57.2% of samples diagnosed with atypia (n = 14). The Odds Ratio for breast malignancy was 4.545 in patients with RASSF1A hypermethylation and 9.167 in patients with RAR-β2 hypermethylation underlying their promoter's methylation positive correlation with breast malignancy. To optimize the sensitivity and specificity of this epigenetic setting, more TSGs related to BC should be gradually imported in our evaluated methylation panel and be validated in a larger study sample with the aim that the obtained epigenetic profiles will provide clinicians with valuable tools for management of BC patients in Greece.

Abstract A13: DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA

Abstract Background: Accumulated evidence revealed that aberrant CpG island hypermethylation plays significant roles in carcinogenesis that can serve as a promising target for molecular detection in body fluids. Ovarian cancer remains the most lethal gynecologic malignancy and is characterized by few early symptoms, late stage presentation, and resulting poor survival, which thereby renders challenge to its prompt diagnosis. Despite a myriad of attempts at early diagnosis of ovarian cancer, this clinical aim still remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant circulating DNA methylation patterns provide highly specific cancer signals. Methods: To develop a DNA methylation-based screening assay for early diagnosis of ovarian cancer, we quantitatively assessed the promoter methylation of DAPK1, HOXA9, RASSF1A, and SOX1 genes in 105 ovarian cancer and 30 non-neoplastic ovarian specimens by means of a high-throughput quantitative, real-time PCR-based technique (MethyLight) and clonal bisulfite sequencing. The best-performing gene cassette was further evaluated for their methylation status in cell-free DNA from serum of matched 35 ovarian cancer patients and 20 normal controls. Area under the ROC (Receiver-operator characteristic) curve was used to evaluate the discriminatory performance of both individual and combined gene panel. Results: RASSF1A, HOXA9, DAPK1, and SOX1 displayed a methylation frequency of 72.72, 82.67, 68.0, and 80.0%, respectively, in tissue samples. We identified DNA methylation of HOXA9 and SOX1 as the best discriminator between cancer and non-neoplastic tissue. In the multiplex assay, the combined panel of HOXA9 and SOX1 achieved a sensitivity of 88.0% with a specificity of 86.7%, when either or both of the gene promoters showed methylation. These genes appear to have great potential to be evaluated for their methylation level in cell-free DNA as a noninvasive diagnostic marker for early diagnosis of ovarian cancer. In cell-free DNA, this novel panel achieved a sensitivity of 62.86.0% and a specificity of 95% with an area under the ROC curve of 0.81 for discriminating OC samples from normal control. The results of MethyLight were concordant with those of clonal bisulfite sequencing. Conclusion: Our findings revealed the potential of epigenetic biomarker for early detection of OC in cell-free DNA as a minimally invasive tool. Here, we have established that promoter hypermethylation can be detected in serum cell-free DNA from patients with early as well as advanced-stage ovarian cancer. Based on the multiplex MethyLight assay, study will focus on analyzing the clinical utility of HOXA9 and SOX1 as methylation biomarkers. These findings further underline the potential of multiplexing individual markers into a panel that helps to achieve higher sensitivity and specificity than individual marker and provides robustness to a noninvasive early screening test. Citation Format: Alka Singh, Jaydeep Aravindbhai Badarukhiya, Sameer Gupta, Manisha Sachan. DNA methylation of SOX1 and HOXA9 as a biomarker for early detection of ovarian cancer in cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A13.

Abstract A53: Hypermethylated RASSF1A as circulating tumor marker in pediatric and adolescent solid tumors

Abstract Background: The tumor suppressor gene RASSF1A is hypermethylated in many adult and pediatric cancers. With the absence of recurrent mutations or translocations in various pediatric and young adult (malignant) tumors, detection of hypermethylated RASSF1A (RASSF1A-M) is an attractive biomarker for diagnosis as well as follow-up. Previously, we showed that RASSF1A-M can be used to detect neuroblastoma cells in bone marrow. Here we provide proof of concept of the detection of RASSF1A-M in plasma and serum of patients with neuroblastoma, renal tumors, rhabdomyosarcoma and germ cell tumors. Method: Plasma was collected at diagnosis from patients with neuroblastoma (n=47), renal tumors (n=13), and rhabdomyosarcoma (n=19) and during treatment and follow-up for neuroblastoma (n=121). Serum was collected from patients with germ cell tumors at diagnosis (n=94). Initially, cell-free DNA (cfDNA) was isolated, bisulfite treated, and tested by qPCR for actin beta (ACTB), unmethylated and hypermethylated RASSF1A (35 diagnostic and 121 follow-up neuroblastoma samples, 19 rhabdomyosarcoma samples). Subsequently, a droplet digital PCR (ddPCR) method for RASSF1A-M and ACTB, including a methylation-specific restriction enzymatic digestion, was applied on the remaining samples to ensure accurate quantification and reducing bisulfite induced cfDNA loss. A threshold for detection of RASSF1A-M was established by testing plasma (73 by qPCR, 25 by ddPCR) and serum samples (21 by ddPCR) from healthy donors. Results: In plasma samples, the total cfDNA level (ng/mL; median) was significantly higher in patients with renal tumors and neuroblastoma (localized and metastatic disease), but not in rhabdomyosarcoma patients, when compared to healthy donors (66.4; 34.4; 80.9; 3.0 and 2.4, respectively). For diagnostic neuroblastoma samples, RASSF1A-M was detected in all 26 patients with metastatic disease and in 10/21 patients with localized disease (mean 71.4% and 8.4% RASSF1A-M, respectively). RASSF1A-M levels decreased during therapy and re-elevated at relapse. RASSF1A-M was detected in 10/13 renal tumor and 8/21 rhabdomyosarcoma diagnostic samples (18.6% and 19.7% RASSF1A-M, respectively). Total cfDNA levels in healthy donor samples were higher in serum when compared to plasma (39.9 versus 2.4 ng/mL). cfDNA levels were significantly higher in patients with germ cell tumors (230.20 ng/mL) compared to healthy donor serum. RASSF1A-M was detected across all subtypes, including 15/16 embryonal carcinomas, 16/18 seminomas, 2/2 yolk sac tumors, 10/11 teratomas, 1/1 choriocarcinoma, and 42/46 mixed tumors. Conclusion: Our findings demonstrate the value of RASSF1A-M as a molecular circulating tumor marker in various solid tumors. We developed a ddPCR-based sensitive and quantitative assay for RASSF1A-M detection. Based on the data presented, RASSF1A hypermethylation is an interesting biomarker for detection of minimal residual disease across various solid tumor types, including those lacking recurrent mutations/translocations. Citation Format: Lieke M. J. van Zogchel, João Lobo, Nathalie Lak, Esther M. van Wezel, Jalenka van Wijk, Janine Stutterheim, Lily Zappeij-Kannegieter, Leendert H.J. Looijenga, Ellen van der Schoot, Godelieve A. M. Tytgat. Hypermethylated RASSF1A as circulating tumor marker in pediatric and adolescent solid tumors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A53.