Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue.
Author(s): Mathieson W, Mommaerts K, Trouet JM, Mathay C, Guan P, Carithers LJ, Rohrer D, Valley DR, Blanski A, Jewell S, Moore HM, Betsou F
Publication: J Histochem Cytochem, 2019, Vol. 67, Page 159-168
PubMed ID: 30562131 PubMed Review Paper? No
Purpose of Paper
The aim of this paper was to identify mRNA markers of cold ischemia time that could distinguish formalin-fixed, paraffin-embedded (FFPE) tissues that experienced suboptimal delays prior to fixation in three different tumor types. A total of 23 mRNA transcripts were evaluated by qRT-PCR in colon, kidney and ovarian tumor specimens that had been subjected to a cold ischemia time of 1, 2, 3, and 12 h at room temperature prior to formalin fixation and paraffin embedding.
Conclusion of Paper
Of the 23 mRNA evaluated, 21 transcripts were successfully amplified for all FFPE tumor specimens and cold ischemia times evaluated. The greatest sources of variability in gene expression were identified to be between patients (50.9%), cold ischemia times (18.1%), and collection sites (11.4%), with only 0.5% of variability attributed to tissue type. Hydroxymethylbilane synthese (HMBS) and RNA polymerase II subunit A (POLR2A) were determined to be the most stable housekeeping genes during cold ischemia, although level of erythropoietin receptor (EPOR), BRCA1 associated RING domain I (BARD1), MER proto-oncogene, tyrosine kinase (MERTK), myosin, heavy chain 10, non-muscle (MYH10), Jun proto-oncogene, AP-1 transcription factor subunit (JUN), and exocyst complex component 7 (EXOC7) were not significantly affected by cold ischemia. The least stable transcripts in tumor specimens subjected to a cold ischemia time of 1-12 h included Fos proto-oncogene, AP-1 transcription factor subunit (FOS), BTG anti-proliferation factor 2 (BTG2), MDM4, p53 regulator (MDM4), protein kinase cAMP-activated catalytic subunit alpha (PRKACA), bete-2-microglobulin (B2M), Kruppel like factor 6 (KLF6), early growth response 1 (EGR1), dual specificity phosphatase 1 (DUSP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all had a CV >5%). The majority of transcripts increased as ischemia time progressed, although the levels of some transcripts decreased with progressive ischemia time in a some patients (2 to 32 patients). Based on these findings, the authors developed a Cold Ischemia Score, calculated as (Ct PRKACA - Ct POLR2A) + (Ct FOS - Ct POLR2A) + (Ct EGR1 - Ct POLR2A), that was capable of distinguishing FFPE specimens that experienced a cold ischemia time of 1 or 3 h versus 12 h, but not among cold ischemia times ≤ 3 h.
Studies
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Study Purpose
The purpose of this study was to identify mRNA markers of cold ischemia time in three different tumor types. Specimens were collected and processed as part of the National Cancer Institute's Biospecimen Pre-analytical Variables (BPV) Program in collaboration with the Integrated Biobank of Luxembourg (IBBL). Tumors were collected from a total of 40 consenting patients from four different collection sites and included 18 kidney, 12 colon, and 10 ovarian specimens. For each tumor specimen, equally sized aliquots were subjected to a cold ischemia time of 1, 2, 3, or 12 h at room temperature in a humidified chamber, fixed in 10% neutral buffered formalin for 10 to 12 h, and processed for paraffin-embedding. FFPE blocks were sectioned at a central facility within 3-18 months, and RNA was extracted from a single 10 µm-thick FFPE section per block the same day using the QIAsymphony RNA kit and associated automated robot, and included the optional DNase digestion. Isolated RNA was concentrated with a column prior to quantification by spectrophotometry (OD 260 nm). RNA was reverse transcribed using random primers and analyzed by qRT-PCR using the TaqMan system by operators that were blind to the cold ischemia of individual samples. Levels of 23 transcripts identified by the authors from the existing literature were evaluated by qRT-PCR and both the ΔCt and ΔΔCt methods; 19 transcripts were evaluated for their potential as cold ischemia markers and 4 as potential housekeeping genes. Statistical significance was determined by ANOVA and Tukey post-hoc tests. Specimen composition requirements for tumor content (>50% by surface area) and necrosis (<20%) were evaluated for each FFPE block as well as the QC FFPE block by a pathologist. Multiple sections were analyzed from each specimen and cold ischemia timepoint such that qRT-PCR results were reflective of different depths of the tumor block.
Summary of Findings:
Although mRNA transcripts SLC25A25 and GUCY2C only produced results in 11 of the 40 patients evaluated, qRT-PCR analysis of the remaining 21 transcripts was successful for all FFPE specimens and cold ischemia timepoints. Sources of variability in gene expression were identified by sum-squared analysis, and 50.9% was attributed to between patient variability, 18.1% to cold ischemia time, 11.4% to collection site, 0.5% to tissue type, and 19.1% to unnamed sources. Given the low percentage of variability attributed to different tissue types, data was pooled for subsequent analysis. HMBS and POLR2A were determined to be the most stable housekeeping genes during cold ischemia, although EPOR, BARD1, MERTK, MYH10, JUN, and EXOC7 were also identified as stable (CV<4% for all). The least stable transcripts in tumor specimens subjected to a cold ischemia time of 1-12 h included FOS, BTG2, MDM4, PRKACA, B2<, KLF6, EGR1, DUSP1, and GAPDH (all had a CV >5%). The majority of transcripts increased as ischemia time progressed using the ΔCt method, with 76-95% of patients having a significant difference depending on the gene when 1 and 12 h timepoints were compared. When 2 and 3 h cold ischemia timepoints were compared 57-73% of patients displayed an increase in mRNA level depending on the gene A reduction in mRNA levels as ischemia time progressed was less common, as only 4 patients displayed a reduction in levels of ≥15 genes, 2 patients displayed a reduction in 4-6 genes, and 32 patients displayed a reduction in ≤3 genes when 1 and 12 h ischemia timepoints were compared; no commonalities were identified among these patients. The authors developed a Cold Ischemia Score, calculated as (Ct PRKACA - Ct POLR2A) + (Ct FOS - Ct POLR2A) + (Ct EGR1 - Ct POLR2A), that was capable of distinguishing FFPE specimens that experienced a cold ischemia time of 1 or 3 h versus 12 h, but not among cold ischemia times ≤ 3 h. The optimal cutoff for Cold Ischemia Score was determined to be 2.9 for the dataset, which yielded a sensitivity of 62% and a specificity of 84%. The authors report similar results when Cold Ischemia Score was calculated using the ΔΔCt method.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 1 h
2 h
3 h
12 h
Real-time qRT-PCR Specific Data handling ΔCt
ΔΔCt
Real-time qRT-PCR Specific Targeted nucleic acid EPOR
BARD1
MERTK
MYH10
JUN
EXOC7
HMBS
POLR2A
FOS
BTG2
MDM4
PRKACA
B2M
KLF6
EGR1
DUSP1
GAPDH