Identification, validation and characterization of novel DNA methylation biomarkers for liquid biopsy based early breast cancer detection and therapy monitoring in non-small cell lung cancer

  • Identifizierung, Validierung und Charakterisierung neuartiger DNA-Methylierungs-Biomarker für die auf Flüssigbiopsie basierende Früherkennung von Brustkrebs und Therapieüberwachung in nicht-kleinzelligem Lungenkrebs

Mijnes, Jolein; Dahl, Edgar (Thesis advisor); Panstruga, Ralph (Thesis advisor); Pradel, Gabriele (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Background: Breast cancer remains the most frequently diagnosed cancer and leading cause of cancer deaths amongst women. Mammography is currently employed for early breast cancer detection, nevertheless it shows important limitations. Lung cancer is the most frequently diagnosed cancer in men and the third most common cancer in women. NSCLC frequently and rapidly recurs after therapy, requiring accurate disease monitoring. Blood-based approaches provide innovative, minimally invasive tools that can be used to characterize epigenetic alterations of tumour suppressor genes. Therefore, detection of cell-free DNA (cfDNA) hypermethylation could serve as a liquid biopsy, complementing mammography and monitoring of NSCLC patients. Methods: Potential biomarkers for early breast cancer detection, SPAG6, PER1 and NKX2-6, were identified from The Cancer Genome Atlas (TCGA), using HumanMethylation450-BeadChip data. ITIH5 was included on basis of previous results. In the here presented study validation of these markers was pursued in both serum (n = 467) and plasma (n = 50). SPAG6 was not previously described in breast cancer. Immunohistochemistry on a formalin-fixed paraffin embedded (FFPE) tissue cohort (n = 92) and a tissue microarray (TMA, n = 776) was performed. SPAG6-GFP overexpressing MDA-MB-231 and T-47D breast cancer cell lines were used in in vitro cell culture assays to test for effects on proliferation, apoptosis, migration, colony formation and invasion. In addition, immunolabeling on fixated cells was performed. For the identification of hypermethylation biomarkers for the monitoring of NSCLC patients, reduced representation bisulfite sequencing (RRBS) was, as a proof-of-principle, established on the analyte cfDNA. To this end serial collected blood samples of NSCLC patients (n = 13) were analysed. COPD patients were used as controls. Results: On basis of SPAG6 and ITIH5, ductal carcinoma in situ (DCIS) could be detected with a 63% sensitivity and early invasive breast cancer at 51% sensitivity in a serum test cohort (80% specificity). Sensitivity for DCIS could be increased to 70% by adding PER1 and NKX2-6 to the panel. Breast cancer detection in a serum validation cohort proved challenging, mainly due to pre-analytical conditions. Taking these pre-analytical conditions into account, sensitivity for breast cancer detection was increased to 81% (83% specificity) in a plasma cohort using all four genes. After successfully establishing an RRBS protocol for cfDNA it was possible to identify differentially methylated CpGs between NSCLC- and COPD patients: genes MYO1D and XRRA1 revealed a higher methylation frequency in NSCLC as did six RNAs (BC017002 (mRNA), BC070106 (mRNA), MG828791 (lncRNA), MG828792 (lncRNA), JB175072 (piRNA) and RNA5-8SP2 (rRNA). The identified regions were not previously described in literature. Clinical data indicated an association of SPAG6 with more aggressive tumours. The same trend was found in cell culture experiments: migration was increased by SPAG6 overexpression and EMT was induced. In addition, apoptosis was influenced by SPAG6. Immunolabeling revealed an overlap between SPAG6-GFP and microtubules. Conclusion: Despite the challenges associated with liquid biopsy applications we were able to identify SPAG6, PER1 and NKX2-6 as blood-borne markers for early breast cancer detection. Quantification of promoter methylation in cfDNA isolated from plasma might in the future be a sensitive and specific tool to complement current breast cancer detection strategies. Even though promising, further technical development and clinical validation is required. Implementation of the RRBS protocol, as well as the bioinformatic analysis pipeline and evaluation of the data were successfully established and completed. In general, SPAG6 seemingly associates with more advanced and aggressive breast tumours, were it appears to have an oncogenic role by stimulating migration and EMT. SPAG6 might have different functions in different stages of tumour development.


  • [528001-2]
  • Department of Biology [160000]
  • Unit of Plant Molecular Cell Biology [161920]