Effects of Slide Storage on Detection of Molecular Markers by IHC and FISH in Endometrial Cancer Tissues From a Clinical Trial: An NRG Oncology/GOG Pilot Study.
Author(s): Grushko TA, Filiaci VL, Montag AG, Apushkin M, Gomez MJ, Monovich L, Ramirez NC, Schwab C, Kesterson JP, Seward SM, Method MW, Olopade OI, Fleming GF, Birrer MJ
Publication: Appl Immunohistochem Mol Morphol, 2022, Vol. 30, Page 27-35
PubMed ID: 34224438 PubMed Review Paper? No
Purpose of Paper
This paper compared human epidermal growth factor receptor 2 (HER2), topoisomerase 2A (TOP2A) and Ki67 staining via immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) endometrial tumors stored for >10 years at room temperature either as an FFPE block or as slide-mounted, unstained sections (of the same block). Freshly cut sections of FFPE blocks were stored for up to 3 weeks before IHC staining. HER2 fluorescent in situ hybridization (FISH) staining was compared among FFPE slide-mounted sections that were stained prior to or after 22 months of storage at room temperature.
Conclusion of Paper
Archival FFPE slides stored for longer than 10 y had, on average, 12% fewer TOP2A immunopositive cells (P=0.03) and approximately 10% fewer Ki67 immunopositive cells (P=0.01) than freshly cut sections from the same FFPE block. Concordance between freshly cut sections and archival slides for the extent and intensity of IHC staining was moderate for TOP2A (κ=0.57) and was substantial for Ki67 using a threshold of 10% (κ=0.61) but was considered moderate (κ=0.48) when a 13% threshold was applied. Although HER2 staining was concordant for 93% of cases, only two cases were HER2+ positive among freshly cut sections. In one of these cases, HER-2 IHC staining was scored as moderate (2+) in both the freshly cut and archival sections, while in the other case, IHC staining was strong (3+) in the freshly cut section and faint (1+) in the archival section. HER2 FISH staining was comparable in original fresh-cut sections and FFPE slide-mounted sections stored for 22 months. The authors conclude that TOP2A and Ki67 staining may be slightly underestimated in archival FFPE slides relative to freshly cut sections of archival blocks, but that HER2 amplification by FISH is unaffected by storage of FFPE slide-mounted sections for up to 22 months at room temperature.
Studies
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Study Purpose
This study compared HER2, TOP2 and Ki67 IHC staining in FFPE endometrial tumors stored for >10 years at room temperature either as an FFPE block or as slide-mounted, unstained sections (of the same block). HER2 fluorescent in situ hybridization (FISH) staining was compared between FFPE slide-mounted sections stained before or after 22 months of storage at room temperature. Case-matched FFPE blocks and slide-mounted, unstained sections of 15 primary endometrial tumors (3 cases were grade 1 endometrial, 2 cases were grade 2 endometrial, 6 cases were grade 3 endometrial, 1 case was clear cell, 2 cases were mixed carcinoma and 1 case was undifferentiated carcinoma) that had been stored for >10 years at room temperature were obtained. Fresh sections were cut from the block and stored <3 weeks (temperature and conditions not specified) before IHC staining. Matched fresh and archival sections of 15 specimens were deparaffinized in xylene, and immunohistochemically stained with antibodies against Ki67, TOP2A and HER2 after antigen retrieval. Staining was evaluated by two blinded pathologists. TOP2A staining was scored based on nuclear intensity and percentage of positive cells. Ki67 staining was scored based on the percentage of positive cells using different cut-off values (10%, 13% and 1%). HER2 status was analyzed by FISH in aged slide-mounted sections (stored for 22 months at room temperature) of 21 FFPE endometrial tumors with HER2 amplification and was compared to the previously determined HER2 status.
Summary of Findings:
The percentage of cells that were TOP2A-immunopositive was, on average, 12% lower in archival FFPE slide-mounted sections than in freshly cut sections from the same archived block (P=0.03). Levels of TOP2A nuclear staining were concordant between matched fresh and archival sections in 53% of cases, but this increased to 87% when slides were categorized as none/weak versus moderate/strong. Overall concordance in TOP2A expression was considered moderate between fresh and archived slides (κ=0.57). Ki67 staining was observed in 1-60% of cells in freshly cut sections from the archival block compared to 0-50% of cells in the case-matched archival sections; 7 of 15 archival sections had a complete loss of staining compared to 0 of 15 freshly cut sections from an archival block. Freshly cut sections from FFPE blocks with an archival slide showing complete loss of staining had a lower percentage of immunopositive cells than freshly cut sections from other blocks (1-5% versus ≥30%). The percentage of Ki67-positive cells was consistently lower (by approximately 10%) when specimens were stored as slide-mounted cut sections than specimens stored as FFPE blocks (P<0.01). Using a threshold of 10% for negative (low/no proliferation) versus positive (high proliferation), there was substantial agreement in Ki67 staining between archived and freshly cut sections (κ=0.61), with concordant interpretation in 80% of cases. However, when the more stringent and recently imposed cut off of 13% was applied, agreement in Ki67 staining between archived and freshly cut sections was moderate (κ=0.48). When Ki67 staining was categorized as none, low and high, concordance was even lower. Importantly, loss of TOP2A and Ki67 staining, relative to fresh sections from the case-matched archival block, occurred in archived sections of the same specimens. HER-2 staining was concordant between case-matched freshly cut and archival sections of the same block in 14 of 15 cases (93%). The single discordant case was stained strongly in the freshly cut section and faintly in the case-matched archival section. However, 13 of 15 pairs had HER2+ scores of 0/1+ in both the freshly cut and archived sections.
The number of HER-2 FISH signals per cell in the slide analyzed prior to storage was comparable to the case-matched FFPE slide stored for 22 months before staining (2.69-62.66 versus 2.53-48.07). The baseline and maximal HER-2/ chromosome 17 centromere enumeration (CEP17) ratios were also similar in slides stained before storage (2.1 and 26.78, respectively) and those stained after 22 months of storage (2.1 and 21.52, respectively). Finally, there was no difference in the HER-2 amplification ratio in tumor cells between slides stained before or after 22 months of storage.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA FISH Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage conditions As unstained slide
As FFPE block