Highly sensitive detection of sentinel lymph node metastasis of breast cancer by digital PCR for RASSF1A methylation.

One­step nucleic acid amplification (OSNA) targeting cytokeratin 19 (CK19) mRNA expression and pathological examination are widely used for the intraoperative diagnosis of sentinel node (SN) metastasis. The aim of the present study was to develop a novel assay for detecting SN metastasis by targeting Ras association domain­containing protein 1 (RASSF1A) methylation in tumor cells, and to compare its performance with OSNA. Using digital PCR with methylation­specific restriction enzymes (RE­dMSP), our assay was able to detect ≥3 copies of methylated DNA per well, and was ≥10 times more sensitive than real­time PCR with bisulfite modification. OSNA lysates were examined using RE­dMSP and digital PCR for PIK3CA mutation, in the event that primary tumors were PIK3CA mutation­positive. RE­dMSP revealed a high concordance of 95.0% (153/161) with OSNA, and 100% (59/59) with PIK3CA mutation for detecting SN metastasis. In 11 breast cancer cell lines, the variation in methylated RASSF1A copy number was significantly lower than that of CK19 mRNA (2.8 vs. 10.5­fold; P<0.01). RE­dMSP has the potential to more accurately detect SN metastasis, and to more precisely estimate total tumor loads in SN, compared with OSNA.

Each liver X receptor (LXR) type has a different purpose in different situations.

LXRs, which are nuclear receptors, have 2 isoforms-LXRα and LXRß. Generally, LXRα is expressed in the liver, kidney, and a limited number of other organs, whereas LXRß is thought to be expressed ubiquitously. Nevertheless, no clear consensus has been reached on the role of each in kidney lipid metabolism. Many researchers have reported that lipids accumulate in renal tubular epithelial cells during nephrosis. The nephrosis model we used showed the presence of urinary protein 4 days after the induction of illness. Additionally, the model maintained high levels of urinary protein from day 7-14. Lipid accumulation was clearly verified at day 4 and extreme accumulation was observed at day 7. We observed increased expression of LXRα from an early stage of nephrosis. To explore the role of increased LXRα in diseased kidney in vitro, NRK52E, normal kidney tubular epithelial cells, were forced to overexpress LXRα. These cells showed significantly lower lipid accumulation than mock cells did. In contrast, LXRß knockdown lead to increased lipid accumulation in mock cells, and constancy in overexpressing cells. In normal kidneys, LXRß is expressed stably to control mainly the intracellular lipids. However, with increasing intracellular lipid accumulation, expression of LXRα and its downstream gene, ABCA1, was upregulated, followed by lipid excretion in an LXRα-dependent manner. This phenomenon strongly suggests the importance of LXRα in lipid metabolism in the diseased kidney.