Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of circulating tumor cells (CTCs) and plasma-circulating tumor DNA (ctDNA). USP44 is a critical gene which plays an important role in cell proliferation; however, its accurate role in other cellular networks is under research. USP44 promoter methylation has been so far reported in colorectal neoplasia and metastatic breast cancer. In this study, we examined for the first time USP44 promoter methylation in plasma cell-free DNA (cfDNA) of patients with prostate cancer (early stage n = 32, metastatic n = 39) and 10 healthy donors (HD). USP44 promoter methylation was detected in plasma cell-free DNA by a newly developed highly specific and sensitive real-time MSP method. Our findings indicate that USP44 promoter is methylated in plasma cell-free DNA of metastatic prostate cancer patients and that detection of USP44 promoter methylation is significantly associated with overall survival (OS) (p = 0.008). We report for the first time that detection of USP44 promoter methylation in plasma cell free DNA provides significant prognostic information in metastatic prostate cancer.

Osimertinib has been an effective second-line treatment in EGFR mutant NSCLC patients; however, resistance inevitably occurs. DNA methylation has been previously implicated in NSCLC progression and often in therapy resistance, however its distinct role in osimertinib resistance is not elucidated as yet. In the present study, we directly compared DNA methylation of nine selected genes (RASSF1A, RASSF10, APC, WIF-1, BRMS1, SLFN11, RARβ, SHISA3, and FOXA1) in plasma-cfDNA and paired CTCs of NSCLC patients who were longitudinally monitored during osimertinib treatment. Peripheral blood (PB) from 42 NSCLC patients was obtained at two time points: (a) baseline: before treatment with osimertinib and (b) at progression of disease (PD). DNA methylation of the selected genes was detected in plasma-cfDNA (n = 80) and in paired CTCs (n = 74). Direct comparison of DNA methylation of six genes between plasma-cfDNA and paired CTC samples (n = 70) revealed a low concordance, indicating that CTCs and cfDNA give complementary information. DNA methylation analysis of plasma-cfDNA and CTCs indicated that when at least one of these genes was methylated there was a statistically significant increase at PD compared to baseline (p = 0.031). For the first time, DNA methylation analysis in plasma-cfDNA and paired CTCs of NSCLC patients during osimertinib therapy indicated that DNA methylation of these genes could be a possible resistance mechanism.

Background: Epigenetic alterations in ctDNA are highly promising as a source of novel potential liquid biopsy biomarkers and comprise a very promising liquid biopsy approach in ovarian cancer, for early diagnosis, prognosis and response to treatment. Methods: In the present study, we examined the methylation status of six gene promoters (BRCA1, CST6, MGMT, RASSF10, SLFN11 and USP44) in high-grade serous ovarian cancer (HGSOC). We evaluated the prognostic significance of DNA methylation of these six gene promoters in primary tumors (FFPEs) and plasma cfDNA samples from patients with early, advanced and metastatic HGSOC. Results: We report for the first time that the DNA methylation of SLFN11 in plasma cfDNA was significantly correlated with worse PFS (p = 0.045) in advanced stage HGSOC. Conclusions: Our results strongly indicate that SLFN11 epigenetic inactivation could be a predictor of resistance to platinum drugs in ovarian cancer. Our results should be further validated in studies based on a larger cohort of patients, in order to further explore whether the DNA methylation of SLFN11 promoter could serve as a potential prognostic DNA methylation biomarker and a predictor of resistance to platinum-based chemotherapy in ovarian cancer.

Background: Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis represents a liquid biopsy approach for real-time monitoring of tumor evolution. DNA methylation is considered to be an early event in the process of cancer development and progression. The aim of the present study was to evaluate whether detection of DNA methylation of selected tumour suppressor genes in size-based CTC fraction and matched ctDNA could provide prognostic information in early stage NSCLC. Methods: The methylation status of five selected gene promoters (APC, RASSFIA1, FOXA1, SLFN11, SHOX2) was examined by highly specific and sensitive real time methylation specific PCR assays in: a) a training group of 35 primary tumours and their corresponding adjacent non-cancerous tissues of early stage NSCLC patients, b) a validation group of 22 primary FFPEs tumor tissues and 42 peripheral blood samples of early stage NSCLC patients that gDNA was isolated from FFPEs, sized-based CTCs and plasma, and c) a control group of healthy blood donors (n=12). Results: All five gene promoters tested were highly methylated in the training group of primary tumours; methylation of SHOX2 promoter in the primary tissues was associated with unfavorable outcome. RASSFIA and APC were found methylated in plasma-cfDNA samples at 14.3% and 11.9% respectively, whereas in the corresponding size-based CTC-enriched fractions SLFN11 and APC promoters were methylated in 7.1%. The incidence of relapse was higher in patients with i) promoter methylation of APC and SLFN11 in plasma-cfDNA (P=0.037 and P=0.042 respectively) and ii) at least one gene promoter was methylated in the sized-based CTC fraction or plasma-cfDNA. Conclusions: Although in liquid biopsy components methylation of these gene promoters was significantly lower than in the primary tumors, the combination of DNA methylation analysis in sized-based CTC fraction and plasma-cfDNA revealed clinical relevance. Additional studies are required to validate our findings in a large cohort of early stage NSCLC patients. Citation Format: Athina N. Markou, Dora Londra, Victoria Tserpeli, John Kollias, Emilia Tsaroucha, Ioannis Pateras, K Potaris, Ioannis Vamvakaris, Athanasios Kotsakis, Vasilis Georgoulias, Evi Lianidou. Combined DNA methylation analysis in size based CTC fraction and matched plasma-cfDNA provides prognostic information in early stage NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3384.

Purpose: We assessed whether preoperativemutational analyses of circulating tumor cells (CTCs) and plasma-cfDNA could be used as minimally invasive biomarkers and as complimentary tools for early prediction of relapse in early-stage non-small -cell lung cancer (NSCLC). Experimental Design: Using ddPCR assays, hotspot mutations of BRAF, KRAS, EGFR and PIK3CA were identified in plasma-cfDNA samples and size-based enriched CTCs isolated from the same blood samples of 49 early-stage NSCLC patients before surgery and in a control group of healthy blood donors (n = 22). Direct concordance of the mutational spectrum was further evaluated in 27 patient-matched plasma-cfDNA and CTC-derived DNA in comparison to tissue-derived DNA. Results: The prevalence of detectable mutations of the four tested genes was higher in CTC-derived DNA than in the corresponding plasma-cfDNA (38.8% and 24.5%, respectively).The most commonly mutated gene was PIK3CA, in both CTCs and plasma-cfDNA at baseline and at the time of relapse. Direct comparison of the mutation status of selected drug-responsive genes in CTC-derived DNA, corresponding plasma-cfDNA and paired primary FFPE tissues clearly showed the impact of heterogeneity both within a sample type, as well as between different sample components. The incidence of relapse was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA compared with patients in whom no mutation was detected (p = 0.023). Univariate analysis showed a significantly higher risk of progression (HR: 2.716; 95% CI, 1.030–7.165; p = 0.043) in patients with detectable mutations in plasma-cfDNA compared with patients with undetectable mutations, whereas the hazard ratio was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA (HR: 3.375; 95% CI, 1.098–10.375; p = 0.034). Conclusions: Simultaneous mutational analyses of plasma-cfDNA and CTC-derived DNA provided complementary molecular information from the same blood sample and greater diversity in genomic information for cancer treatment and prognosis. The detection of specific mutations in ctDNA and CTCs in patients with early-stage NSCLC before surgery was independently associated with disease recurrence, which represents an important stratification factor for future trials.