Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen.

Author(s): van Seijen M, Brcic L, Gonzales AN, Sansano I, Bendek M, Brcic I, Lissenberg-Witte B, Korkmaz HI, Geiger T, Kammler R, Stahel R, Thunnissen E

Publication: Virchows Arch, 2019, Vol. 475, Page 191-199

PubMed ID: 31264038 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if the duration of a room temperature delay to fixation (0, 1, 6, 24, 48, 96 h) and time in fixative (18 h, 24 h, 2, d, 4, 7 d) affect tissue quality and immunohistochemical staining of cancer biomarkers in FFPE non-small cell lung carcinoma (NSCLC) specimens. In total, surgically resected tumor specimens (>4 cm) from 20 cases of NSCLC were used in the study.  Ten samples (0.5 x 0.5 x 0.3 cm) from each case were collected and subjected to different delays to fixation at room temperature (0, 1, 6, 24, 48, 96 h) and then fixed in 10% neutral buffered formalin (NBF) in PBS for 18 or 24 h, or fixed immediately (exact time not provided) in 10% NBF at room temperature for 18 h, 24 h (control), 2, d, 4, 7 d. Separate tissue microarrays (TMAs) were constructed for the DTF and TIF experiments; each TMA consisted of two 1 mm cores per time point. Additional details relating to tissue processing and TMA construction were not provided.  Slide-mounted FFPE sections (thickness not specified) were stained by immunohistochemistry (IHC) for the following antigens: CK AE1/AE3, CK 7, CAM 5.2, KER MNF-116, CK 5/6, p40, p80, p63, TTF-1, BRAFV600E, C-MET, ROS1, ALK, EGFR, Napsin A, D2-40, PD-L1, synaptophysin, chromogranin A, CD 56. IHC staining of each core for each antigen (with the exception of PD-L1) was assessed by five pathologists for the intensity of immunopositive staining and was assigned a score (scale: 0 to 7) that considered loss of cores, the intensity of staining, tissue quality, and the absence of tumor or normal tissue in the core (0=TMA core lost, 1=negative staining, 2= weak positive staining, 3=moderate positive staining, 4=strong positive staining, 5=poor tissue quality, 6=tumor not present, 7=normal tissue not present).  PD-L1 IHC staining was based on the percentage of immunopositive staining in the cell membrane (<1%, 1-5%, 5-10%, 10-25%, 25-50%, >50%).  DTF and TIF timecourses were compared to standardly processed specimens (fixed immediately in 10% NBF for 24 h) by statistical analysis, specifically, McNemar and Wilcoxon rank tests, and statistical significance was set at p<0.05.

   Summary of Findings:

   Of the 400 TMA cores evaluated, tumor tissue was present in 84% and normal adjacent tissue was present in 67%; sections of TMA cores detached from the slide during IHC for 27% of specimens in the TIF timecourse and 35% of specimens in the DTF timecourse. The percentage of sections of TMA cores that remained on the slide after IHC staining was lower for NSCLC specimens from the DTF timecourse than matched NSCLC specimens that underwent standard fixation (fixed immediately in 10% NBF for 24 h) in normal adjacent tissue for CK7 (p<0.01), C-MET (p<0.05), and chromogranin A (p<0.05); and in tumor tissue for CAM 5.2, p40, p63, C-MET (p<0.001 for all) and CK7, TTF-1, BRAFV600E, ROS1, PD-L1, and chromogranin A (p<0.05 for all). The percentage of sections of TMA cores that were retained after IHC staining did not differ when NSCLC specimens from the TIF timecourse were compared to matched specimens that underwent standard fixation. When all antigens were considered together, poor tissue quality (score 5, differentiation of normal and tumor specimens was difficult) was more prevalent among specimens that experienced a DTF of 1 h (tumor only, p<0.001), 24 h (tumor and normal adjacent, p<0.001 for both), 48 h (tumor only, p<0.001), and 96 h (tumor and normal adjacent, p<0.001 for both) than specimens that were processed immediately. When each antigen was considered individually, a score denoting poor tissue quality (score 5) was more prevalent among NSCLC specimens from the DTF timecourse than those that underwent standard processing for keratin MNF116 (in cores containing normal adjacent tissue, p<0.05), AE1/AE3 (in cores containing tumor, p<0.05), synaptophysin (in cores containing normal adjacent or tumor tissue, p<0.01 for both), and CD56 (in cores containing normal adjacent or tumor, p<0.01 for both). Differences in the number of specimens with poor tissue quality were not observed among NSCLC specimens from the TIF timecourse and controls that underwent standard fixation.  Relative to specimens processed under standard conditions, NSCLC specimens from the DTF timecourse displayed significant decreases in the intensity of immunopositive staining for D2-40 (in normal adjacent only, p<0.05), PD-L1 (in normal adjacent only, p<0.01), Ker MNF116 (in tumor only, p<0.05), TTF-1 (in tumor only, p<0.05), C-MET (in normal adjacent and tumor, p<0.05 for both), and Napsin A (in normal adjacent and tumor, p<0.05 for both). The intensity of immunopositive staining did not differ between NSCLC specimens that experienced standard (24 h) or prolonged fixation (≥48 h) for any of the antigens investigated.  For PD-L1 staining, the percentage of tumor cells with positive membrane staining was also significantly lower in NSCLC specimens from the DTF timecourse compared to those that experienced standard processing and fixation, with noticeable reductions in staining intensity after a 1 h DTF.  The intensity of PD-L1 staining did not differ between NSCLC specimens from the TIF timecourse and those that experienced standard fixation.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Tissue microarray
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 h 1 h 6 h 24 h 48 h 96 h
   Immunohistochemistry Specific	Targeted peptide/protein	CK AE1/AE3 CK 7 CAM 5.2 KER MNF-116 CK 5/6 p40 p80 p63 TTF-1 BRAFV600E C-MET ROS1 ALK EGFR Napsin A D2-40 PD-L1 synaptophysin chromogranin A CD 56
   Biospecimen Preservation	Time in fixative	18 h 24 h 2 d 4 d 7 d

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