Quantitative methylation-specific PCR for the detection of aberrant DNA methylation in liquid-based Pap tests

Abstract Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents, and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. To investigate if the miR-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. An upstream CpG island of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, cell xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2’-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma.

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Study of the DNA - Methylation Status in Primary Breast Cancer Using Bioinformatics and Methylation Specific PCR

Ain Shams Medical Journal ◽

10.12816/0013824 ◽

2013 ◽

Vol 64 (1-3) ◽

pp. 91-98

Author(s):

Samar K. Kasem ◽

Marwa Mahmoud ◽

Maha M. Sallam ◽

Hanan H. Shehata

Keyword(s):

Breast Cancer ◽

Dna Methylation ◽

Primary Breast Cancer ◽

Methylation Status ◽

Methylation Specific Pcr ◽

Specific Pcr

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Quantitation of DNA Methylation by Quantitative Multiplex Methylation-Specific PCR (QM-MSP) Assay

Methods in Molecular Biology - DNA Methylation Protocols ◽

10.1007/978-1-4939-7481-8_24 ◽

2017 ◽

pp. 473-496 ◽

Cited By ~ 2

Author(s):

Mary Jo Fackler ◽

Saraswati Sukumar

Keyword(s):

Dna Methylation ◽

Methylation Specific Pcr ◽

Specific Pcr

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

Cancers ◽

10.3390/cancers10100357 ◽

2018 ◽

Vol 10 (10) ◽

pp. 357 ◽

Cited By ~ 15

Author(s):

Sandra Nunes ◽

Catarina Moreira-Barbosa ◽

Sofia Salta ◽

Susana Palma de Sousa ◽

Inês Pousa ◽

...

Keyword(s):

Dna Methylation ◽

Early Stage ◽

Simultaneous Detection ◽

Clinicopathological Parameters ◽

Plasma Sodium ◽

Cell Free Dna ◽

Early Stage Disease ◽

Specific Pcr ◽

Free Dna ◽

Aberrant Dna Methylation

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.

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Hypermethylation of SH2-Containing Phosphatase-1 (SHP-1) and Suppressor of Cytokine Signaling-1 and -3 (SOCS-1 and -3) in Philadelphia Negative Chronic Myeloproliferative Disorders (Ph-CMPD).

Blood ◽

10.1182/blood.v108.11.118.118 ◽

2006 ◽

Vol 108 (11) ◽

pp. 118-118

Author(s):

Davide Rossi ◽

Clara Deambrogi ◽

Tiziana Lischetti ◽

Michaela Cerri ◽

Marianna Spunton ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Methylation Status ◽

Jak2v617f Mutation ◽

Cytokine Signaling ◽

Aberrant Methylation ◽

Normal Bone Marrow ◽

Normal Bone ◽

Specific Pcr ◽

Aberrant Dna Methylation

Abstract A fraction of Ph- CMPD is characterized by JAK2F617F mutation leading to constitutive JAK-STAT activation. The negative regulators of cytokine signaling SHP-1, SOCS-1 and SOCS-3 have a crucial function in the negative regulation of JAK2 activation/phosphorylation in response to EPO, G-CSF and a subset of cytokines. SHP-1, SOCS-1 and SOCS-3 may be silenced by aberrant DNA methylation in human malignancies. Here we tested chronic phase Ph- CMPD and acute myeloid leukaemia (AML) from Ph- CMPD for aberrant DNA methylation of SHP-1, SOCS-1 and SOCS-3. The study was based on: i) 85 Ph- CMPD, including 35 essential thrombocythemia (ET), 20 polycythemia vera (PV), 15 idiopathic myelofibrosis (IMF), 6 chronic myelomonocytic leukemia (CMML) and 9 Ph- chronic myeloid leukemia (Ph- CML); and on ii) 19 AML from Ph- CMPD, including 4 AML from PV, 10 AML from ET and 5 AML from IMF. Cases were analysed for SHP-1, SOCS-1 and SOCS-3 aberrant methylation by methylation-specific PCR and for JAK2V617F mutation by allele specific PCR. For comparison, 10 samples of normal bone marrow hematopoietic cells were also investigated. Among Ph- CMPD, methylation of SHP-1 occurred in 4/20 (20%) PV and 4/35 (11%) ET, while it was absent in IMF (0/15), CMML (0/6) and Ph- CML (0/9). Methylation of SOCS-1 occurred in 5/20 (25%) PV, 5/35 (14%) ET, 2/15 (13%) IMF and in 1/9 (11%) Ph- CML while it was absent in CMML (0/6). Methylation of SOCS-3 occurred in 11/20 (55%) PV, 13/35 (37%) ET, 4/15 (26%) IMF, 3/6 (50%) CMML and 3/9 (33%) Ph- CML. JAK2V617F mutation was detected in 47/85 (55%) Ph-CMPD, including 17/20 (85%) PV, 18/35 (51%) ET, 12/15 (80%) IMF, 0/6 CMML and 0/9 Ph- CML. SHP-1, SOCS-1 and SOCS-3 methylation was analysed according to JAK2 mutation status in PV, ET and IMF. In this group of patients, SHP-1, SOCS-1 and SOCS-3 methylation occurred in both JAK2 mutated cases (6/47, 13% for SHP-1; 10/47, 21% for SOCS-1 and 18/47, 38% for SOCS-3) and germline cases (2/38, 5% for SHP-1; 2/38, 5% for SOCS-1 and 10/38, 26% for SOCS-3). By combining the results of SHP-1, SOCS-1 and SOCS-3 methylation status, 21/47 (45%) JAK2 mutated cases carried SHP-1 and/or SOCS-1 and/or SOCS-3 methylation as opposed to 12/38 (31%) germline cases. This pattern of SHP-1 and SOCS-1 methylation was conserved also when the analysis was restricted to PV, ET and IMF each as a single group and after stratification for JAK2V617F mutation. Among AML from Ph- CMPD, methylation of SHP-1 occurred in 1/10 (10%) AML from ET, while it was absent in AML from PV and AML from IMF. Methylation of SOCS-1 occurred in 1/4 (25%) AML from PV and 1/10 (10%) AML from ET. One ET patient acquired SHP-1 methylation at transformation to AML. All normal bone marrow samples (n=10) scored negative for SHP-1, SOCS-1 and SOCS-3 methylation. The implications of these results are threefold. First, inactivation by aberrant methylation of SHP-1, SOCS-1 and SOCS-3 is involved in the pathogenesis of Ph- CMPD and is selectively associated with neoplastic hemopiesis. Second, among PV, ET and IMF, methylation of SHP-1, SOCS-1 and SOCS-3 may occur in both JAK2V617F positive and negative cases. Third, the low prevalence of SHP-1 and SOCS-1 methylation in AML from Ph- CMPD suggests that SHP-1 and SOCS-1 silencing is not involved in leukemic transformation.

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