Abstract
One-step nucleic acid amplification (OSNA) is an established method for intraoperative diagnosis of breast cancer metastasis in sentinel lymph nodes, based on quantification of CK19 mRNA, specific to breast epithelial cells. Inhibitors interfere with the PCR amplification process of PCR. Thus, OSNA, based on gene amplification without RNA purification, might be impacted by numerous factors persisting in a sample, and thereby potentially acting as PCR inhibitors. However, neither the characteristics of breast cancers showing inhibitory effects during OSNA, nor any of the possible inhibitors, have as yet been identified. Inhibitory effects detected during OSNA in 72 metastatic lymph nodes and the patients’ clinicopathological features were examined. Left-over OSNA samples were analyzed with mass spectrometry to identify proteins possibly acting as inhibitors. Most tumors showed inhibitory effects, though to varying degrees. Large tumor, young age and high tumor-infiltrating lymphocyte counts were related to stronger inhibitory effects. Proteome analysis revealed elevations in RPB9 protein and EIF2 signaling upregulation in samples showing strong inhibitory effects. Tumors showing strong inhibitory effects had clinically relevant characteristics, including large size and extensive tumor-infiltrating lymphocyte involvement. Identifying inhibitors in OSNA might provide new insights into breast cancer biology as well as advancing the current technology.
Highlights
Assay (Sysmex Corporation, Japan) is an established method for intraoperative diagnosis of breast cancer metastasis in axillary sentinel lymph nodes (SLNs)[1]
A node will be judged as harboring breast cancer metastasis when amplification of cytokeratin 19 (CK19) mRNA is detected, because CK19 expression is specific to breast epithelial cells and theoretically does not exist in normal lymph nodes
We confirmed that the inhibitory effect is observed in most tumors, though to varying degrees
Summary
Assay (Sysmex Corporation, Japan) is an established method for intraoperative diagnosis of breast cancer metastasis in axillary sentinel lymph nodes (SLNs)[1]. This assay is performed based on quantification of cytokeratin 19 (CK19) mRNA and has been shown to be more diagnostically accurate than classical pathological diagnosis using frozen sections[2,3]. It is widely known that the amplification process of PCR can be inhibited or prevented by several substances known as “inhibitors”[12] and that inhibition is one of the most common causes of PCR failure[13] The mechanism underlying this phenomenon is poorly understood, though multiple steps during the PCR process have been suggested to be involved (e.g. interference in DNA extraction, nucleic acid degradation, inhibition of polymerase activity)[12]. It should be noted that even in highly purified samples, the effects of inhibitors might persist without significantly altering the PCR reaction
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