Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Loss of antigenicity with tissue age in breast cancer.

Author(s): Combs SE, Han G, Mani N, Beruti S, Nerenberg M, Rimm DL

Publication: Lab Invest, 2016, Vol. 96, Page 264-9

PubMed ID: 26568292 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study evaluated and quantified the loss of antigenicity for estrogen receptor (ER) human epidermal growth receptor 2 (HER2), Ki67, and cytokeratin (CK) in invasive breast carcinoma tumor specimens that were stored as FFPE blocks for 7-53 years before creation and analysis of tissue microarrays (TMA) by automated quantitative analysis (AQUA) of fluorescence immunohistochemistry (fIHC).  A total of 1,330 invasive breast carcinoma specimens collected via surgical resection from 1,295 patients between 1961 and 2006 were used for the study.  All specimens were formalin-fixed and paraffin-embedded “according to standard operating procedures” and were stored as FFPE blocks; no additional details on fixation, processing, or storage were provided.  Four TMAs were created from cores (0.6 mm) collected from each patient’s tumor block, and 23 duplication cores were collected.  TMAs were sectioned (thickness not specified), mounted on slides, and stained by fluorescence immunohistochemistry (fIHC) the same day as sectioning for ER, HER2, and Ki67.  Immunopositive/targeted staining was visualized with Cy5-conjugated tyramide, tumor cells were visualized by CK staining, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).  An automated quantitative analysis (AQUA) score (the target compartment pixel intensities divided by either the pixel intensities of the tumor or nuclear compartment) was determined for each TMA core using a HistoRX PM2000 microscope. Given the aim of the study was to estimate loss of antigenicity, specimens that stained negative were excluded from the dataset.

   Summary of Findings:

   Tissue and ifIHC signals were sufficient to calculate AQUA scores for ER, HER2, Ki67, and CK for 630, 630, 707, and 647 patients, respectively. Based on Pearson’s correlations, there was a significant but very weak correlation between FFPE block storage duration and ER (r²=0.06, P<0.0001), HER2 (r²=0.11, P=0.0034), Ki67 (r²=0.22, P<0.0001), and CK (r²=0.03, P<0.0001) AQUA scores. Based on the rate of decrease in AQUA score that occurred with storage, the authors predicted a 10% loss in signal after 9.9 y of FFPE block storage for ER, 8.5 y for HER2, 4.5 y for Ki67, and 22.5 y for CK. Based on random effects models, the decline in staining with progressive FFPE block storage was linear for ER (P<0.001), HER2 (P=0.004), and CK (P<0.0001).  Conversely, the best-fit line for Ki67 staining over time was represented by a quadratic equation (p(x²)=0.0387), which matches the observed greater loss of antigenicity over the first three decades before reaching a plateau.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Tissue microarray
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Immunohistochemistry Specific	Targeted peptide/protein	ER HER2 Ki67 CK
   Storage	Storage duration	7-53 years

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