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The proper handling of unstained paraffin slides for immunohistochemistry has been a matter of debate, with several studies demonstrating loss of antigenicity with prolonged storage at room temperature, 4°C and -20°C. The purpose of this study was to determine whether long-term storage of unstained slides at -80°C would impact the staining intensity and expression distribution of markers used to molecularly subtype breast cancer specimens [estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), cytokeratin 5 (CK5), epidermal growth factor receptor (EGFR), and Ki67]. The staining pattern of previously unstained breast tumor slides (n=39 to 64) stored at -80°C for a minimum of 9.93 years (avg., 12.8 y) was compared with the staining pattern of fresh cut slides from the same tumors. The Allred scoring method was used to score ER (0 to 2, negative; 3 to 8, positive), CK5 (≥4, positive), and EGFR (≥4, positive). ASCO/CAP guidelines were used to assess HER2 (0/1+, 2+, or 3+). Ki67 scores were determined based on the proportion of cells stained of any intensity, with 20% staining used as a cut-off. Agreement was assessed using concordance rates and chance-corrected agreement statistics. The chance-corrected agreements were as follows: 0.94 (38/39) for ER, 0.92 (53/55) for CK5, 0.87 (61/64) for EGFR, 0.86 (37/39) for HER2, and 0.67 (46/54) for Ki67. Long-term storage of cut unstained slides at -80°C does not significantly impact the scoring interpretation of ER, CK5, EGFR, and HER2.

Fresh Cut Versus Stored Cut Paraffin-embedded Tissue: Effect on Immunohistochemical Staining for Common Breast Cancer Markers