Quality Assessment of Proteins and RNA Following Storage in Archival Formalin-Fixed Paraffin-Embedded Human Breast Cancer Tissue Microarray Sections

Abstract

Although the immunogenicity of formalin-fixed paraffin-embedded tissue sections can decrease during storage and transport, the exact mechanism of antigenic loss and how to prevent it are not clear. Herein, we investigated changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 in human breast tissue microarray (TMA) tissue sections stored for up to 3 months in dry and wet conditions. The positive rates of ER and PR expression were minimally changed after 3 months of storage, but the Allred scores of ER and PR stored in humid conditions decreased remarkably in comparison to fresh-cut tissue. The HER-2 antigenicity and RNA integrity of breast TMA sections stored in dry conditions diminished gradually with storage time, whereas the immunoreactivity and RNA quality of HER-2 in humid conditions decreased sharply as storage length increased. The area and intensity of E-cadherin staining in tissue sections stored in dry conditions did not change significantly and were minimally changed after 3 months, respectively. In contrast, the area and intensity of E-cadherin staining in tissue sections stored in humid conditions decreased significantly as storage length increased. Finally, the Ki-67 labeling index of tissue sections stored for 3 months in dry (9% decrease) and wet (31.9% decrease) conditions was decreased in comparison to fresh sections. In conclusion, these results indicate that water is a crucial factor for protein and RNA degradation in stored tissue sections, and detailed guidelines are required in the clinic.

Keywords: antigenicity; formalin fixed; immunohistochemistry; in situ hybridization; storage condition; tissue microarray.

FIG. 1.

Immunohistochemical assessment of ER and PR according to storage time in both dry and wet conditions. Representative images of ER (A) and PR (B) immunohistochemical staining in human breast tissue. High magnification images are shown in the inset. Scale bar, 100 μm. Quantitative analysis of ER (C) and PR (D) immunohistochemical staining. Data are presented as mean ± SD. ER, estrogen receptor; PR, progesterone receptor; SD, standard deviation.

FIG. 2.

Evaluation of HER-2 immunochemical staining according to storage time in both dry and wet conditions. (A) Representative images of HER-2 immunohistochemical staining in human breast tissue. High magnification images are shown in the inset. Scale bar, 100 μm. (B) Quantitative analysis of HER-2 immunohistochemical staining. The data are presented as mean ± SD. (C) Change of HER-2-positive rate according to storage time in both dry and wet conditions. HER-2, human epidermal growth factor receptor 2.

FIG. 3.

RNA quality according to storage time in both dry and wet conditions. RNA quality was assessed by RNAScope in situ hybridization. (A) Representative RNAScope in situ hybridization images of ERBB2 in human breast tissue. High magnification images are shown in the inset. Scale bar, 100 μm. (B) Quantitative analysis of ERBB2 expression. Data are presented as mean ± SD. ERBB2, Erb-B2 receptor tyrosine kinase 2.

FIG. 4.

E-cadherin immunohistochemical staining of human breast cancer tissue for slides stored over time and in dry and wet storage conditions. (A) Representative immunohistochemical images of E-cadherin before and after storage in both dry and wet conditions. High magnification images are shown in the inset. Scale bar, 100 μm. (B) Quantitative analysis of the immunohistochemical stained area and intensity of E-cadherin according to storage conditions. Data are presented as mean ± SD.

FIG. 5.

Ki-67 labeling index of human breast cancer tissue for slides stored over time and in dry and wet storage conditions. (A) Representative immunohistochemical images of Ki-67 before and after storage in dry or wet conditions. High magnification images are shown in the inset. Scale bar, 100 μm. (B) Quantitative analysis of the Ki-67 labeling index according to storage conditions. Ki-67 labeling index was defined as the percentage of Ki-67 antigen-positive cells. Data are presented as mean ± SD.