Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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The impact of impaired tissue fixation in resected non-small-cell lung cancer on protein deterioration and DNA degradation.

Author(s): Butter R, Halfwerk H, Radonic T, Lissenberg-Witte B, Thunnissen E

Publication: Lung Cancer, 2023, Vol. 178, Page 108-115

PubMed ID: 36812759 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to assess the effects of under- and over-fixation of formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue on tissue quality (determined macroscopically and microscopically), immunohistochemical staining of 10 antigens, and DNA degradation.  Surgically resected lung tissue from 25 patients diagnosed with NSLC(9 with non-mucinous adenocarcinoma, 9 with squamous cell carcinoma, 2 with pleomorphic adenoma, 2 with carcinoid tumors, 1 with mucinous adenocarcinoma, and two with large cell carcinoma) were partially sliced along the axial plane every 1.5 cm, bandage mesh was placed between slices, and the tissue specimen was fixed in 10% neutral buffered formalin for 12-24 h. Cold ischemia time (time between resection and fixation) ranged between 0.5 to 21.5 h (mean 3.4 h).  Fixed specimens were sectioned again (0.5 cm thick sections) and examined macroscopically for adequate (specimen was a white/gray color throughout) or inadequate (specimen contained pink/red areas) fixation.  Specimens then underwent additional tissue processing and paraffin embedding (no additional details provided).  FFPE tissue sections were stained with hematoxylin and eosin for microscopic evaluation and by immunohistochemistry (IHC) for the following antigens: ALK, PD-L1, GAM2.5, CK7, c-Met, KER-MNF116, NapsinA, p40, ROS1, TTF1. For tissue slides stained by immunohistochemistry, areas containing the maximum and minimum staining intensity, necrotic zones, and evidence of adequate and inadequate fixation (detachment of the basement membrane) fixation were estimated.  To evaluate DNA degradation, adequately fixed and inadequately fixed regions were macroscopically dissected from 10 µm thick FFPE sections and DNA was isolated with the QIAamp DNA FFPE Tissue Kit, then assessed by multiplex PCR (100, 200, 300, 400 bp amplicons; gene not specified). The concentration of PCR amplicons was determined by electrophoresis with an Agilent Bioanalyzer. 

   Summary of Findings:

   Based on macroscopic evaluation of the fixed tissue segment before paraffin-embedding, 8 of the 25 specimens were adequately fixed (tissue was white or grey), 2 of the specimens were inadequately fixed (tissue was pink or red), and 15 specimens contained regions of both. Microscopic review of H&E-stained slides generated similar results, as 6 specimens were adequately fixed, 2 were inadequately fixed (detachment of the basement membrane), and 17 specimens contained regions of both. 
   Mean IHC scores of eight antigens were compared between adequately and inadequately fixed areas of the 25 tumors collected for the study; because specimens were excluded from analysis if immunopositive staining absent in both adequately and inadequately fixed tissue, a different number of specimens were analyzed for each antigen. Mean H-scores for both KER-MNF116 (256 versus 151, respectively; p<0.001) and p40 (293 versus 248, respectively; p=0.028) were significantly higher in regions that were adequately fixed and those that were inadequately fixed. Necrotic zones within a tumor had significantly lower mean H-scores than adequately fixed regions for CK7 (80 versus 186; p=0.044), KER-MNF116 (63 versus 256; p<0.001), and p40 (0 versus 293; p=0.002). 
   When mean H-scores were only examined in the subset of tumors that displayed both adequately and inadequately fixed regions, KER-MNF116 alone differed significantly, with a higher mean H-score in the adequately fixed region compared to the inadequately fixed region (249 versus 131; p=0.003). The minimum and maximum H-scores within a tumor differed significantly for all eight antigens evaluated (PD-L1, CAM5.2, CK7, c-MET, KER-MNF116, Napsin A, p40, TTF1; p≤0.008 for all).  Evidence of DNA fragmentation (the absence of 300 and 400 bp amplicons) was prominent in both adequately and inadequately fixed regions, and the concentration of PCR amplicons did not differ between the two region types for any of the amplicon lengths investigated.  Interactions between DNA degradation and cold ischemia time and fixation time were observed. Tumor specimens that had a cold ischemia time >16 h had a lower concentration of 300 (0 versus 0.13 ng/µl; p=0.004) and 400 bp (0 versus 0.0059 ng/µl; p=0.046) PCR amplicons than those with a delay of <6 h. Tumor specimens that were fixed in formalin for >24 h had a lower concentration of 300 (0.02 versus 0.14 ng/µl; p=0.024) and 400 bp (0 versus 0.0067 ng/µl; p=0.045) PCR amplicons than those that were fixed for <24 h. A potential influence of cold ischemia time and fixation time on immunohistochemical staining was not investigated.
    

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Benign
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Morphology	Macroscopic observation
   DNA	PCR
   Protein	Immunohistochemistry
   Morphology	H-and-E microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Time in fixative	<24 h >24 h
   Biospecimen Acquisition	Cold ischemia time	<6 h >16 h
   Immunohistochemistry Specific	Targeted peptide/protein	ALK PD-L1 GAM2.5 CK7 c-Met KER-MNF116 NapsinA p40 ROS1 TTF1
   PCR Specific	Length of gene fragment	100 bp 200 bp 300 bp 400 bp

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