Circulating cell-free DNA: A promising marker of regional lymphonode metastasis in breast cancer patients

Article type: Research Article

Authors: Agostini, M.^{a; b; c; *} | Enzo, M.V.^{a; c} | Bedin, C.^{a; c} | Belardinelli, V.^a | Goldin, E.^a | Del Bianco, P.^d | Maschietto, E.^{a; c} | D'Angelo, E.^{a; c} | Izzi, Leo^e | Saccani, A.^e | Zavagno, G.^a | Nitti, D.^a

Affiliations: [a] Department of Surgical, Oncological and Gastroenterological Sciences, 2nd Surgical Clinic, Padova, Italy | [b] The Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, TX, USA | [c] Istituto di Ricerca Pediatrica- Città della Speranza, Viale della Ricerca, Padova, Italy | [d] Clinical Trials and Biostatistics Unit, Veneto Oncological Institute – IRCSS, Padova, Italy | [e] Euroclone S.p.a, Pero (MI), Italy

Correspondence: [*] Corresponding author: Marco Agostini, PhD, Department of Oncological and Surgical Gastroenterological Sciences, Surgical Clinic II, University of Padova, Via Giustiniani 2, 35128 Padova, Italy. Tel.: +39 049 8214374, Fax: +39 049 651891, E-mail: [email protected].

Abstract: Purpose:We undertook the current study with untreated breast cancer to (1) role the variations in the plasma levels of cfDNA and the size distribution in early stage, (2) determine the frequency in plasma of methylation of three candidate genes, RASSF1A, MAL, and SFRP1, and (3) to determine whether detection of cfDNA variations and methylation changes in plasma might have specific clinical utility. Methods and materials:Thirty-nine patients woman patients (median age 64 years; range, 36–90 years) who underwent surgery for primary BR and 49 healthy females’ subjects (control group without any breast lesion) were evaluated. The cfDNA levels were analyzed using quantitative real-time polymerase chain reaction of β-globin. Based on the ALU repeats, the cfDNA was considered as either total (fragments of 115 bp, ALU115) or tumoral (fragments of 247 bp, ALU247). The association between the levels of the ALU247, ALU115 repeat, and ALU 247/115and the pathologic tumor characteristics was analyzed.Used methylight qPCR method, cfDNA from plasma samples of healthy donors and patients with breast cancer were evaluated for the diagnotic value of the methylation status of three genes (RASSF1A, MAL, SFRP1) frequently methylated in breast cancer. Results:The baseline levels of cfDNA were significantly higher in the patients with cancer, and the level of ALU247 was the most accurate circulating cfDNA marker in discriminating the cancer from non-cancer subjects.A high statistical significance was found by considering the T stage and patients with regional LN metastasis positive cancers showed significantly higher cfDNA level of ALU247. Moreover, patients with methylation of at least one of the gene under investigate showed a higher quantity of cfDNA ALU115 (p< 0.0001) and ALU247 level (p< 0.0001). Conclusions:We observed that necrosis could be a potential source of circulating tumour-specific cfDNA ALU247; and that cfDNA ALU247 and methylated cfDNA (RASSF1A, MAL and SFRP1) are both a phenotypic feature of tumour biology.

Keywords: Breast cancer, regional LN metastasis, cfDNA

DOI: 10.3233/CBM-2012-0263

Journal: Cancer Biomarkers, vol. 11, no. 2-3, pp. 89-98, 2012