Decline in Antigenicity of Tumor Markers by Storage Time Using Pathology Sections Cut From Tissue Microarrays.
Author(s): Blows FM, Ali HR, Dawson SJ, Le Quesne J, Provenzano E, Caldas C, Pharoah PD
Publication: Appl Immunohistochem Mol Morphol, 2016, Vol. 24, Page 221-6
PubMed ID: 26067143 PubMed Review Paper? No
Purpose of Paper
This paper explored how immunohistochemical staining of breast cancer-relevant antigens is affected by the duration of tissue microarray (TMA) block and slide storage and whether retention of antigenicity is improved by coating TMA slides in paraffin before storage.
Conclusion of Paper
When immunohistochemical scores were plotted against the storage duration of individual TMA cores, antigenicity displayed a mean change over time for 52 of the 54 markers evaluated, 40 of which displayed a significant decline in immunostaining with storage duration (P<0.05). Although most changes in immunostaining were small, the nuclear androgen receptor marker displayed the largest change, with an average decline in immunostaining score of 0.88 units/year. Declines in immunostaining scores for cytoplasmic intensity over time were greater than declines in the proportion of cytoplasmic staining, intensity of nuclear staining, and the proportion of nuclear staining, which were all of similar magnitudes.
When freshly sectioned TMA slides were compared to those dipped in paraffin or left undipped before 12 months of room temperature storage, similar weighted kappa values indicated a lack of effect associated with dipping TMA slides in paraffin for both progesterone receptor (PGR, PR) (proportion: 0.89 versus 0.91, intensity: 0.80 versus 0.80, respectively) and Marker Of Proliferation Ki-67 (MKI67) (proportion: 0.64 versus 0.72, intensity: 0.44 versus 0.54, respectively), but did indicate marker-specific differences in antigen stability over slide storage.
Studies
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Study Purpose
Study Purpose
This study explored how immunohistochemical staining of breast cancer-relevant antigens is affected by the duration of tissue microarray (TMA) block and slide storage and whether retention of antigenicity is improved by coating TMA slides in paraffin prior to storage. In total, a single 0.6 mm core from tumors from each of 4,125 breast cancer patients under the age of 70 y was used to construct TMAs. Blocks that cores were sampled from had been stored for various durations (2-1,897 d; storage conditions were not specified) prior to TMA construction, which included three cycles of heating TMA blocks to 42°C for 30 min and cooling them to room temperature. Sections (3 µm-thick) were cut, mounted on glass slides, and deparaffinized in clearene, rehydrated in a graded alcohol series, and immunohistochemically stained with an autostainer for the following antigens: aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), ALDH 1 family member A3 (ALDH1A3), AURKA, Geminin DNA Replication Inhibitor (GMNN), MKI67, Minichromosome Maintenance Complex Component 2 (MCM2), polo-like kinase 1 (PLK1), estrogen receptor (ER), PGR, epidermal growth factor receptor (EGFR), cytokeratin 5/6 (CK5/6), ASMA, cytokeratin 14 (CK14), E-cadherin(CDH1), GATA binding protein 3 (GATA3), androgen receptor (AR), beta-catenin (CTNNB1), Fibroblast growth factor receptor 2 (FGFR2), forkhead box P3 (FOXP3), KIT, mitogen-activated protein kinase kinase kinase 1 (MAP3K1), MYB, N-acetyltransferase 1 (NAT1), Programmed Cell Death 4 (PDCD4), phosphatase and tensin homolog (PTEN), Solute Carrier Family 7 Member 5 (SLC7A5), tumor protein p53 (TP53). Immunopositive staining was scored based on the cellular compartment (nucleus, cytoplasm, membrane), the intensity of stain, and the extent of staining.
To explore effects associated with slide storage conditions, six 3 µm-thick sections were cut from each of the two TMAs constructed and mounted on glass slides; two sections each were (i) untreated and stored in the dark for 12 months at room temperature, (ii) coated in paraffin and stored in the dark for 12 months at room temperature, and (iii) analyzed immediately after sectioning; slides were analyzed by immunohistochemistry for PGR and MKI67 and scored using a 0-4 scale for intensity (0=no staining, 1= weak, 2=moderate, 3=strong) and a 0-6 scale for the percentage of immunopositive nuclei (0=0%, 1=<1%, 2=1 to <10%, 3= 10 to <34%, 4=34 to <67%, 5=67 to 100%).
Summary of Findings:
When immunohistochemical scores were plotted against the storage duration of individual TMA cores, antigenicity displayed a mean change over time for 52 of the 54 markers evaluated, 40 of which displayed a significant decline in immunostaining with storage duration (P<0.05). Although most changes in immunostaining were small, the nuclear androgen receptor marker displayed the largest change, with an average decline in immunostaining score of 0.88 units/year. Declines in immunostaining scores for cytoplasmic intensity over time were greater than for the declines observed for the proportion of cytoplasmic staining, intensity of nuclear staining, and the proportion of nuclear staining, which were all of similar magnitudes.
When freshly sectioned TMA slides were compared to those dipped in paraffin or left undipped before 12 months of room temperature storage, similar weighted kappa values indicated a lack of effect associated with dipping TMA slides in paraffin for both PGR (proportion: 0.89 versus 0.91, intensity: 0.80 versus 0.80, respectively) and MKI67 markers (proportion: 0.64 versus 0.72, intensity: 0.44 versus 0.54, respectively), but did indicate marker-specific differences in antigen stability over slide storage.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Tissue microarray Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 2- 1897 days
Immunohistochemistry Specific Targeted peptide/protein PGR
MKI67
Storage Storage conditions Paraffin-coated slides
Untreated slides
Freshly sectioned slides