Fig 1.
TMA map and HER2 IHC scoring by DAB staining.
(a) 3,3-diaminobenzidine (DAB) immunohistochemistry (IHC) staining of HER2 in the tissue microarray cohort. (b) IHC score by experienced pathologists. The excluded core has been partially blacked out. (c) Comparison of IHC scores between experienced pathologists. PV%: Percentage of positive values.
Fig 2.
Breast tumor tissue microarray by PID staining.
Representative image of TMA-stained HER2 IHC assay by phosphor-integrated dots (PID), including four spots of the negative spot (IHC 0, A2), low expresser (IHC 1+, H5), medium expresser (IHC 2+, G5), and high expresser (IHC 3+, C8). The staining images are 3× digital image magnifications on a Nanozoomer S60.
Fig 3.
HER2-expressing breast cancer specimen stained with hematoxylin-eosin (HE) to the upper, HER2 IHC by DAB to the middle, and HER2 IHC by PID to the lower.
Tissues were classified as follows: (a) 3+, (b) 2+, (c) 1+, (d) 0. The staining images are 20× digital image magnifications on a Nanozoomer S60.
Fig 4.
Calculation method of the integrated fluorescence intensity of HER2 expression in invasive ductal carcinoma.
(a) Calculation method of PID fluorescence intensity in the invasive area. The PID value of the invasive cancer area was calculated by superimposing the invasive cancer area extracted from the hematoxylin image and PID image. (b) To measure PID fluorescence intensity, the fluorescence image of the invasive cancer region was divided into 12-μm square sections, and each section was quantified by integrating the fluorescence intensity.
Fig 5.
HER2 concordance between PID and DAB IHC.
(a) PID value map (×103) on the invasive cancer area. (b) Comparison of PID and DAB IHC (R2 = 0.93). The pink line shows the cut point (lowest values of 1+ (4.33 × 103), 2+ (13.12 × 103), or 3+ (32.17 × 103)). The asterisks represent statistical significance based on two-tailed paired Student’s t-test between denoted samples (*P < 0.001). (c) Raw categorical data displaying the graph in (b). PV%: Percentage of positive values.
Fig 6.
Separation ability of the low HER2 expression region.
(a) HER2 concordance between DAB and PID re-evaluated based on low HER2 expression. (b) A PID-stained image of tissues judged as having low HER2 expression by DAB staining. The PID value is an integral of the fluorescence intensity of PID in the invasive cancer area. Clear positive signals were detected in all cores, classified as HER2 1+, which is difficult to determine by DAB staining. The staining images are 3× digital image magnifications on a NanoZoomer S60.
Fig 7.
Schematic representation in the case of applying tentative PID IHC score to DAB IHC diagnosis.
This shows the relationship between current HER2 DAB IHC diagnosis and PID IHC score in breast cancer. PID IHC helps classify HER2 positive, low HER2 and HER2 negative. ISH: in situ hybridization.