Elucidation of inhibitory effects on metastatic sentinel lymph nodes of breast cancer during One-Step Nucleic Acid Amplification

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.

Figure 1

Inhibitory effects in the 72 patients. Orange bar indicates plus I cases, while dotted horizontal line indicates the median CK19-D/CK19 ratio, 0.41. Group A includes eleven cases showing no inhibitory effect, with a CK19-D/CK19 value of less than 0.1. Group B is comprised of 12 cases showing a strong inhibitory effect, as indicated by a CK19-D/CK19 value of 1.0 or more.

Figure 2

Volcano plots of proteins differed between the two groups. All 1228 proteins tested are plotted. The red circle indicates RPB9 protein, the only protein in Group B present at a statistically significantly higher amount. The orange circle indicates perforin-1 and the alpha-crystallin B chain, the expressions of which were reduced.

Figure 3

Canonical pathways varied in Group 2. Orange indicates an “activated” canonical pathway, while blue means “inactivated”. White: activation not predictable in any particular direction, grey: not covered for predictive function, Ratio: ratio of the molecules examined to those that are already known.