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Abstract
The mechanism of transition of ductal carcinoma in-situ (DCIS) to invasive cancer is poorly understood.
Summary
We hypothesised that during the evolution of DCIS, myoepithelial cells undergo a switch in gene expression profile that compromises their suppressor function and may lead to promotion of tumour progression.
Our study included initially 5 cases of DCIS and 4 cases of normal breast tissue. We used optimized immuno-LCM technique to dissect pure population of MECs from fresh frozen tissue of these cases followed by cDNA microarray using Affymetrix platform. This was followed by validation step by real time PCR and immunohistochemistry.
The optimization step revealed that the most sensitive and most specific markers for labelling of MECs are P63 and calponin, and the best technique for RNA extraction from small amount of tissue is the Qiagen RNeasy kits protocol.
Ingenuity Pathways software analysis showed clustering of most of the altered genes in cancer, cell death and cell-cell signalling networks, with the Wnt/β-catenin pathway as the top affected canonical pathway. Validation using QRT-PCR revealed 71.4% correlation rate with the array results with up-regulation of FN-1, and NPNT and down-regulation of PTHLH, FGFR2, ADAMTS5, TGFBR3, CAV2, ITGβ8, IGFBP5 and DSG2 in DCIS associated MECs. The most dramatic change was up-regulation of Fibronectin-1 (FN-1) in DCIS associated MECs. IHC analysis for FN, PTHLH and FGFR2 on normal and DCIS tissues with and without associated invasion confirmed a strong correlation between FN protein expression by MECs and DCIS (p<0.0001) and statistically highly significant difference between normal MECs and DCIS associated MECs regarding PTHLH protein expression (P<0.0001), and
FGFR2 was expressed in normal MECs and absent from DCIS associated MECs. FN in pure DCIS cases and DCIS cases that have progressed to IDC and found a statistically significant correlation between high/wide expression (>52%) and presence of invasion (p=0.006).