Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Quantitative Assessment of Preanalytic Variables on Clinical Evaluation of PI3/AKT/mTOR Signaling Activity in Diffuse Glioma.

Author(s): Beccari S, Mohamed E, Voong V, Hilz S, Lafontaine M, Shai A, Lim Y, Martinez J, Switzman B, Yu RL, Lupo JM, Chang EF, Hervey-Jumper SL, Berger MS, Costello JF, Phillips JJ

Publication: Mod Pathol, 2024, Vol. 37, Page 100488

PubMed ID: 38588881 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study evaluated how cold ischemia time (20 min, 1 h, 6 h at room temperature), defined as the time between surgical excision and preservation, affects the levels of PI3K/AKT/mTOR biomarkers in FFPE human high-grade diffuse glioma specimens and compared biomarker levels between FFPE glioma specimens and case-matched specimens that were snap-frozen in liquid nitrogen then thawed in cold 10% neutral buffered formalin. Surgically resected high-grade diffuse glioma specimens from six patients (4 were an isocitrate dehydrogenase-wildtype glioblastoma (IDH-wildtype GBM), 1 was an IDG mutant astrocytoma, and 1 was an H3K27-altered diffuse midline glioma) were divided into four pieces (25-40 mg each) on gauze moistened with saline on petri dish on wet ice.  The four specimen pieces were snap frozen in liquid nitrogen immediately or held for 20 min, 1 h, or 6 h at room temperature before being fixed in 10% neutral buffered formalin for 2 h at room temperature followed by 24 h at 4°C. Formalin-fixed specimens were transferred to cold (4°C) 70% ethanol until dehydration, clearing, and paraffin embedding. Up to 9 tumor regions were sampled from IDH-mutant diffuse glioma tumors from eight patients during surgical resection to assess intratumoral heterogeneity; cold ischemia time of these samples was limited to ≤2 h. Levels of the PI3K/AKT/mTOR biomarkers phosphor- 4E binding protein 1 (4EBP1) and p-RPS6(S24/244) were determined by immunohistochemically staining 5 µm-thick FFPE sections using an autostainer and a confocal microscope.  Results were reported as the percentage of immunopositive tumor cells and phosphoprotein intensity per cell. 

   
   Orthotopic xenograft specimens were developed using cultured cells from the U87 human glioblastoma cell line that were transplanted in a murine model and were used to investigate potential changes in PI3K/AKT/mTOR biomarker levels with cold ischemia time (0, 20 min, 2 h, 6 h at room temperature) and with storage of slide-mounted FFPE sections for 3 or 5 months at room temperature, -20°C, and -80°C relative to controls that were sectioned ≤3 days of analysis. Xenografts of three mice per timepoint and temperature were used for analysis. Levels of PI3K/AKT/mTOR biomarkers were determined using the same workflow described above for human specimens.
    

   Summary of Findings:

   As cold ischemia time increased, the percentage of cells expressing p-4EBP1 decreased significantly in human GBM specimens, declining from expression in 100% of tumor cells after 20 min of cold ischemia on wet ice to approximately 40% and 50% after 1 h and 6 h at room temperature, respectively (p<0.05). The percentage of cells expressing p-RPS6 (S240/244) was not significantly affected by the cold ischemia times investigated (1 h and 6 h versus 20 min controls) in human GBM specimens. The authors noted an increase in the intensity of immunostaining at the periphery of the specimen (creating an “edge effect”) with progressive cold ischemia.  When case-matched specimens that were snap-frozen in liquid nitrogen and thawed in cold (4°C) 10% neutral buffered formalin were compared to FFPE specimens that we routinely processed within 20 min of collection, no significant differences in extent or the intensity of p-4EBP1 or pRPS6 (240-244) immunostaining were observed.  

   
   Intratumoral heterogeneity in p-RPS6 (S240/244) immunopositivity varied between patients, with differences in the percentage of immunopositive tumor cells ranging between 1.62% and 55.75%.  For a given patient, differences in the percentage of p-RPS6 (S240-244) immunopositive cells were a poor predictor of the distance between samples (R2=0.001).  The number of samples collected per patient was strongly correlated with the magnitude of the difference in pRPS6 (S240/244) staining between samples (R2=0.917), indicating that the extent of tumor sampling is important.  Although samples were limited to those with a cold ischemia time of ≤2 h, the percentage of cells that were immunopositive for p-RPS6 was very weakly correlated to cold ischemia time (R2=0.104; p=0.029). 

   
   When the stability of six phosphoprotein biomarkers was evaluated following cold ischemia times of 0, 20 min, 1 h, 2 h, and 6 h using the GBM orthotopic xenograft model system, significant declines in the percentage of cells expressing p-RPS6 (S240-244) and p-4EBP1 occurred after 2 h, and p-AKT after 6 h. However, when the phosphoprotein intensity per cell was quantified, all six biomarkers displayed an increase after only 20 minutes relative to immediately processed controls (P<0.05), with p-RPS6 (240/245), p-RPS6 (235/236), and p-mTOR exhibiting subsequent declines in specimens with ischemia ≥2 h. All six biomarkers also displayed more intense immunostaining at the periphery of the tumor (but not normal adjacent tissue) after a cold ischemia time of 2 h, which contributed to an increase in variability within a section. 

   
   When slide-mounted FFPE sections of GBM orthotopic xenografts were stored for ≤3 days to 5 months at room temperature, -20°C, or -80°C, changes in antigenicity were phosphoprotein-specific. Of the three phosphoproteins analyzed (p-4EBP1, p-AKT, and p-RPS6 (240/244)), the antigenicity of p-4EBP1 and p-AKT were stable in FFPE slides stored for up 3 months regardless of temperature, but slides stored for 5 months only displayed stable immunostaining for the two antigens when storage was at -80°C.  Conversely, RPS6 (S240/244) immunostaining displayed reductions in the percentage of positively stained cells after 3 months of storage at -80°C. 
    

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Mixed type

   Platform:

   Analyte	Technology Platform
   Protein	Immunofluorescence assay
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	20 min (on wet ice) 1 h (at room temperature) 6 h (at room temperature)
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Intratumoral sampling (exact positions not specified)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   Storage	Storage duration	≤3 d 3 months 5 months
   Storage	Storage temperature	Room temperature -20°C -80°C

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