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The age of paraffin block influences biomarker levels in archival breast cancer samples.

Author(s): Chen H, Fang QQ, Wang B

Publication: Oncol Lett, 2020, Vol. 20, Page 525-532

PubMed ID: 32565978 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study evaluated how the duration of formalin-fixed, paraffin-embedded (FFPE) block storage (1-10 y) affected the extent and intensity of estrogen receptor (ER) and progesterone receptor (PR) immunohistochemical staining and the intensity of fluorescence in situ hybridization (FISH) signal for human epidermal growth factor receptor 2 (HER2) and chromosome enumeration probe 17 (CEP17) in core needle biopsy (CNB) and surgically resected breast cancer tumor specimens.  Non-case matched CNB (44 specimens) and surgically resected (46 specimens) breast tumor specimens were procured from 100 patients diagnosed with breast cancer (median age: 56.7 y) who had not received neoadjuvant chemotherapy; all specimens were collected on a weekday to maintain consistency in fixation and processing and were fixed in 10% neutral buffered formalin for 6-24 h at room temperature. FFPE blocks stored for 1, 3, 5, 7, and 10 y were evaluated to determine the effects of FFPE block storage on IHC and ISH results; 4-10 CNB and 7-10 mastectomy specimens were analyzed for each FFPE block storage timepoint by immunohistochemistry and FISH.  For immunohistochemistry, ER and PR results were compared to the results of the original test, 1-10 y prior. ER and PR expression was evaluated by immunohistochemical staining of 4 µm thick slide-mounted FFPE sections; sections were deparaffinization with xylene, endogenous peroxidase activity was quenched with 3% H2O2, and non-specific binding was blocked with normal goat serum, then incubated with the primary antibody and counterstained with hematoxylin. The intensity and extent of immunopositive staining was represented by Q-score (range: 0-7), a sum of the intensity score (0=no staining of any nuclei; 1= weak staining; 2= moderate staining; 3= strong clear positive staining) and distribution score (0= 0%; 1= 1–25%;2= 26–50%;3= 51–75%; 4= >75%).  For FISH, prior test results for HER2 and CEP17 were not available, so the results of older blocks were compared to recently collected blocks.  For FISH analysis, FFPE sections (4 µm) were deparaffinized in xylene incubated in protease solution (1ug/ml) at 37°C, hybridized with a dual color HER2/CEP17 probe, and counterstained with 4',6-diamidino-2-phenylindole (DAPI). Thirty invasive tumor nuclei that were randomly selected were evaluated for signal intensity (0= no visible signal; 1= weak; 2= signal visible but not intense; 3= intense signal).

   Summary of Findings:

   Effects of FFPE storage duration on immunohistochemistry staining were dependent upon specimen collection method (biopsy versus surgical resection).  CNB stored as FFPE blocks for 10 years had a significantly lower Q-score for ER (3.17±1.33 versus 6.17±0.75, P<0.05) and PR (1.50±1.22 versus 4.50±1.38, P<0.05) than the original analysis. Differences in ER and PR Q-scores among the other storage timepoints for CNB specimens and all timepoints for mastectomy specimens were comparatively small and not significant.

   The intensity of the FISH signal for HER2 was adversely affected by FFPE block storage in CNB specimens, as no signal was observed among specimens that were stored for 7 (8 specimens) and 10 years (4 specimens), respectively, and significant reductions in the intensity of the HER-2 FISH signal was observed when CNB blocks stored for3 years (1.78±0.44) or 5 years (1.44±0.73) were compared to those stored for 1 year (2.60±0.70, P<0.05 for both). The intensity of HER-2 signal in mastectomy specimens stored as FFPE blocks for 7 (1.00±0.71) and 10 y (0.86±0.69) was also significantly lower than those stored for 5 y (2.00±0.58), 3 y (2.30±0.48), or 1 y (2.33±0.87)(P<0.05 for all), although blocks containing mastectomy specimens that were stored for 1-3 y did not differ from one another.

   A FISH signal for CEP17 was not generated in CNB-containing FFPE blocks stored for 7 or 10 y (4 and 8 specimens, respectively) and mastectomy-containing FFPE blocks stored for 10 y (7 specimens). CNB-containing FFPE blocks stored for 1 year (2.60±0.70) had FISH CTEP signal that was significantly more intense than those stored for 3 y (1.78±0.44) or 5 y (1.22±0.67) (P<0.05).  In mastectomy FFPE blocks, significant differences in CTEP signal were limited to comparisons between blocks stored for 7 y (0.22±0.44) and those stored for 1 y (2.00±0.87), 3 y (2.10±0.32), or 5 y (1.86±0.38) (P<0.05 for all).

   When FISH signal intensity was compared between CNB and mastectomy specimens, significant differences were limited to HER2 when FFPE blocks stored for 3 years were compared (1.78±0.44 versus 2.30±0.48) and CEP17 when FFPE blocks stored for 5 y were compared (1.22±0.67 versus 1.86±0.38) (P<0.05 for all).

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   DNA	In situ hybridization

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1 y 3 y 5 y 7 y 10 y
   In situ hybridization Specific	Targeted nucleic acid	HER-2 CEP17
   Immunohistochemistry Specific	Targeted peptide/protein	ER PR

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