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

Abstract

We performed a pilot study in anticipation of using long-aged precut formalin-fixed paraffin-embedded tissue sections stored in real-world conditions for translational biomarker studies of topoisomerase 2A (TOP2A), Ki67, and human epidermal growth factor receptor 2 (HER2) in endometrial cancer. Formalin-fixed paraffin-embedded tissue blocks or unstained slides or both from GOG-0177 were collected centrally (1999-2000) and stored at room temperature. During 2004 to 2011 specimens were stored at 4°C. Matched pairs of stored slides and freshly cut slides from stored blocks were analyzed for TOP2A (KiS1), Ki67 (MIB1), and HER2 (HercepTest) proteins. To assess DNA stability (HER2 PathVision), fluorescence in situ hybridization (FISH) was repeated on stored slides from 21 cases previously shown to be HER2 amplified. Immunohistochemistry (IHC) staining intensity and extent, mean FISH copies/cell, and copy number ratios were compared using the κ statistic for concordance or signed rank test for differences in old cut versus new cut slides. IHC results reflected some protein degradation in stored slides. The proportion of cells with TOP2A staining was lower on average by 12% in older sections (P=0.03). The proportion of Ki67-positive cells was lower in stored slides by an average of 10% (P<0.01). Too few cases in the IHC cohort were FISH positive for any conclusions. HER2 amplification by FISH was unaffected by slide storage. We conclude that use of aged stored slides for proliferation markers TOP2A and Ki67 is feasible but may modestly underestimate true values in endometrial cancer. Pilot studies for particular storage conditions/durations/antigens to be used in translational studies are warranted.

Trial registration: ClinicalTrials.gov NCT01164735.

Conflict of interest statement

V.L.F.: grants from NIH during the conduct of this study and additional funding from GOG Foundation Inc. outside the submitted work for other gynecologic clinical trials. S.M.S.: speakers Burea for GSK (Zejula), AstraZeneca (Olaparib), and Merk (Keytruda+lenvatanib). T.A.G.: current employee of Abbott. M.W.M.: current employee of Lilly and own Lilly stock. G.F.F.: Ad board for GSK, institutional PI for industry trials of Roche, Syros, GSK, Iovance, Sanofi, Sermonix, Incyte, Compugen, Abbvie, Eisai, Celldex, Astra Zeneca, Corcept, Merck, Plexxicon. The remaining authors declare no conflict of interest.

Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.

Figures

Fig. 1.

Flow chart depicting the tumor samples collection, storage and use in pilot study assays. Abbreviations: GOG SDMC, Gynecologic Oncology Group Statistics and Data Management Center; UCMC, University of Chicago Medical Center. Sent backward rectangles and hexagons: open indicate new cut slides; filled in indicate aged (old cut slides).

Fig. 2.

Photomicrographs of comparative TOP2A, Ki67 and HER2 IHC staining of new cut (3 weeks old) and stored (old cut, >10 years old) slides sectioned from the same FFPE tumor blocks of endometrial cancers. (A & B) Example of endometrial cancer demonstrating no effect of slide storage on TOP2A IHC interpretation. Strong (3+) positive nuclear staining was observed in both sections. (C – F) Staining patterns of TOP2A (C & D) and Ki67 (E & F) illustrating complete loss of immunoreactivity in old cut slides of the same case. In new cut sections of this tumor, the percentages of positively stained cells were 50% for TOP2A and 30% for Ki67. (G & H) Tumor showing change in HER2 intensity staining from strongly positive (3+; G) to weak (1+; H). Stroma and inflammatory cells were negative internal controls for all biomarkers. Original magnification x200.

Fig. 3.

Graphical presentation of percentages of IHC positive cells for TOP2A (A & B) and Ki67 (C & D) and FISH HER2/CEP17 ratios (E & F) in new and old cut sections for each case of endometrial cancer. (A, C & E) Scatter Plots of identity: red lines mark agreement. (B, D & F) Bland-Altman Plots of difference. Solid grey lines mark the average difference and the limits of agreement specified as an average difference ± 1.96 x Standard Deviation. (A & C) Percentages of stained cells in new slides were mapped against percentages of stained cells in old slides from the same cases for TOP2A and Ki67, respectively. (B & D) Differences in percentages of cells stained positive between new and old sections were mapped against average values of percentages of positive cells from pairs of new and old sections for TOP2A (B) and Ki67 (D). In old slides, the difference from values in new slides ranged between 50% lower and 10% higher for TOP2A and between 0% and 35% lower for Ki67. Statistically significant loss of staining in stored slides by an estimated 12% for TOP2A and 10% for Ki67 was detected. (E) HER2/CEP17 ratios in new slides were mapped against HER2/CEP17 ratios in old slides from the same cases. (F) Differences in HER2/CEP17 ratios between new and old sections were mapped against average values of HER2/CEP17 ratios from pairs of new and old sections. In old slides, the variability from new slides ranged between 5.2 lower and 3.0 higher with an average estimated HER2/CEP17 ratio lover by 0.1 showing no difference from new sections.