Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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

Author(s): Kim K, Ylaya K, Perry C, Lee MY, Kim JW, Chung JY, Hewitt SM

Publication: Biopreserv Biobank, 2023, Vol. 21, Page 493-503

PubMed ID: 36264172 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper evaluated the stability of the HER-2 transcript by in situ hybridization (ISH) and estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 proteins by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) breast carcinoma sections stored for 1 d, 1 week, 1 month, and 3 months in humid (94-95% humidity) or dry conditions (slides were vacuum-sealed with a desiccant). A tissue microarray was created using cores (2 mm in diameter) from 97 cases of invasive carcinoma and 3 cases of invasive lobular carcinoma. Patient age ranged from 32-76 y and tumor size ranged from 0.7-5.7 cm. Details on collection, fixation, and tissue processing were not provided. Unstained TMA sections (5 µm thick) were slide-mounted, baked in an oven (temperature not specified) for 60 min and stored for 1 d (control), 1 week, 1 month, or 3 months in a dry (slides were vacuum sealed with an anhydrous calcium sulfate) or humid (94-96% humidity; slides were stored in a humidity chamber containing 100 mL of distilled water at room temperature) environment before analysis. Slides were stained by immunohistochemistry for ER, PR, HER-2, E-cadherin, and Ki-67; 3, 3’-diaminobenzidine (DAB) was used as the chromagen and slides were counterstained with hematoxylin and eosin. HER-2 transcripts were detected using the chromogen-based (DAB) RNAscope in situ Hybridization Kit. All stained slides were scanned with an Aperio system. ER and PR immunopositive staining was assessed using the Allred score (proportion of nuclear staining multiplied by staining intensity; 0=negative, 1=weak, 2=moderate, 3=strong). HER-2 and E-cadherin immunostaining were quantified by staining intensity and area. HER-2 positive/negative scoring was based on ASCO/CAP guidelines (0 or 1=negative, 2= equivocal, 3=positive). Ki-67 immunostaining was calculated as the proportion of tumor nuclei that demonstrated moderate or highly intense staining. HER-2 mRNA transcripts were quantified using Visiopharm software, and expression represented the mean number of brown dots per cell in user-defined areas.

   Summary of Findings:

   ER antigenicity was more stable in breast cancer TMA slides stored at room temperature under dry conditions (vacuum-sealed with desiccant) than those that stored under humid conditions (94-96% humidity). While the total score (TS) and the proportion score (PS) for ER remained stable when slides were stored for up to 3 months at room temperature under dry conditions, the ER intensity score (IS) declined significantly after 3 months of storage compared to fresh sections (1.99 versus 2.19, p<0.001).  Under humid slide storage conditions, significant declines in the PS of ER occurred after storage for 1 month and the IS, and TS beginning after 1 week. After 3 months of humid slide storage, the PS, IS, and TS for ER declined by 24.1%, 43,4%, and 30.9% relative to scores in fresh sections (p<0.001 for all). PR antigenicity was significantly reduced when slides were stored under dry conditions after ≥1 month (significant reductions in IS and TS, p< 0.001) and reductions in all three scores when slides were stored under humid conditions for ≥1 week (p<0.001).  The magnitude of declines in the extent and intensity of PR staining was more severe under wet slide storage conditions than dry; PR PS, IS, and TS values were 91.5%, 80.1%, and 87.7%, respectively, of those observed for fresh cut sections after 3 months of dry slide storage but were just 18%, 34.4%, and 23.4%, respectively, after 3 months of slide storage under humid conditions. Positive rates for ER and PR were not significantly altered by slide storage for up to 3 months under dry or humid conditions. HER-2 antigenicity was reduced by 12.6% relative to freshly cut sections when slides were stored under dry conditions for 1 month and by 28.9% when stored under humid conditions for 1 week (p<0.001).  The percentage of specimens that were classified as HER-2 positive, equivocal, and negative (15.6%, 42.7%, and 41.7% in freshly cut sections) changed significantly when slides were stored under dry conditions for 3 months (8.7%, 30.4%, 60.9%, respectively) or under humid conditions for 1 month (4.2%, 12.5%, and 83.3%) or 3 months (2.1%, 8.6%, and 89.3, respectively)(p<0.001 for all). HER-2 RNA expression exhibited a gradual decline relative to freshly cut sections when slides were stored under dry conditions (15.3%, 16.2%, and 26.3% after 1 week, 1 month, and 3 months of storage) but more robust declines under wet storage conditions (21.5%, 76.2%, and 83.8%, respectively). Reductions in the extent of E-cadherin positive staining occurred in slides stored under dry conditions after 3 months (a 7.6% decrease relative to freshly cut sections) and in slides stored under humid conditions after 1 week, 1 month, and 3 months (25.3%, 38.9%, and 53,5%, respectively); a reduction in the intensity of E-cadherin staining also occurred after 3 months of slide storage under humid conditions (23.4% decline).  Ki-67 staining also displayed a faster and more robust decline in antigenicity when slides were stored under humid compared to dry conditions (31.9% versus 9% decline after 3 months of storage relative to freshly cut sections).

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   RNA	Tissue microarray
   RNA	In situ hybridization
   Protein	Tissue microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1 day 1 week 1 month 3 months
   Immunohistochemistry Specific	Targeted peptide/protein	ER PR HER-2 E-cadherin Ki-67
   In situ hybridization Specific	Targeted nucleic acid	HER-2
   Storage	Storage conditions	Dry (vacuum packed with a desiccant) Wet (94-96% humidity)

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