Blood based molecular diagnostic assay for noninvasive diagnosis of breast cancer Free

2689

Inflammation plays a significant role in both initiation and progression in cancer. We exploited this strategy and compared gene expression in blood, cancerous and normal tissuesfrom the same breast of breast cancer patients. We also hypothesized that inflammation is linked to abnormal cellular proliferation, which results in tissue injury in breast and activates the expression of breast specific gene such as Mammaglobin B. UsingReal Time PCR assay, the analysis for markers of inflammation (TNF-alpha, NFkB, Il-6, MMP-9, PPAR-alpha), cell cycle control (Skp2, Cyclin E) and breast specific Mammaglobin B was performed on20 paired tissues (cancerous and normal tissues) from the samebreast (obtained from CHTN) and blood and breast tissuesfrom patients in a blinded fashion (obtained from Fredtert MemorialLutheran Hospital/Medical College of Wisconsin. We also studiedmRNA expression for these markers in normal breast tissues obtainedfrom individuals undergoing breast reduction. RNA samples wereprepared from both the lymphocytes and tissue at the same time,reverse transcribed to cDNA and mRNA expression studied by realtime PCR assay. Results: The mRNA expression for MammaglobinB, PPAR-alpha, TNF-alpha, NFkB, Il-6, MMP-9, Skp2, and NFκB wassignificantly higher in cancerous tissues than normal tissuesfrom the same breast. The expression of mammaglobin B was observedonly in cancerous tissues not in normal tissues. The expressionof PPAR-alpha, Skp2, TNF-alpha and NFKB was also significantlyhigher in cancerous than paired normal breast tissues. However,the expression varied with the diagnosis. For example in a patientdiagnosed with infiltrating ductal carcinoma the fold differencefor TNF-alpha mRNA was 5 fold, with moderately differentiatingductal carcinoma 4 fold compared to invasive ductal carcinomawith 80% differentiation a 90 fold increase in mRNA was observed.Similar changes were observed with mRNA expression for othermarkers. More significantly, the mRNA expression for these markers was identicalin RNA isolated from and tissues from these patients and thequantitative differences for these markers were observed inpatients with different diagnosis. The mRNA expression for IL-6and TNF-alpha in tissue from a patient with benign Phyllodestumor was not different than normal controls. The mRNA expressionfor TNF-alpha in patients with Fibroadenoma was not detectable,however for IL-6 was detectable. In sharp contrast, the patientwith diagnosed with invasive ductal carcinoma exhibited significantlyhigher expression for IL-6 and TNF-alpha mRNA. These results demonstrate the ability of these molecular signaturesto differentiate malignant tissues from normal tissues and theusefulness of blood in detection of cancer. These resultsprovide potential clinical utility for the diagnosis of breast cancer patient with a noninvasive method using a multiplex PCR baseddiagnostic assay.