Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Frozen section histopathology and preanalytical factors affecting nucleic acid integrity in biobanked fresh-frozen human cancer tissues.

Author(s): Kim S, Kang J, Kim B, Kwak Y, Lee HS

Publication: J Pathol Transl Med, 2025, Vol. , Page

PubMed ID: 40936269 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of cold ischemia and frozen storage duration on DNA and RNA integrity by comparing unmatched colon adenocarcinoma (COAD), hepatocellular carcinoma (HCC), and renal cell carcinoma (RCC) specimens obtained after cold ischemia times ranging from <30 min to >120 min or frozen storage for 1-10 years. The authors also investigated potential correlations between DNA/RNA integrity and histopathologic parameters (tumor cell percentage, presence of normal tissue, extent of inflammatory cell infiltration, extent of stromal fibrosis, percentage necrosis or percentage extracellular mucin pool). A total of 105 tissue specimens were obtained during surgical resection from patients with colon adenocarcinoma (COAD), hepatocellular carcinoma (HCC), or renal cell carcinoma (RCC). The COAD specimens that were stored frozen for 1 year were collected immediately upon resection prior to pathology processing, but those stored for 3 years were collected pre-gross examination and were not washed, and those stored for >3 years were collected post-gross examination and after washing with water. All HCC specimens and partial nephrectomy specimens were collected in the operating room and delivered to the bank. RCC specimens collected during radical nephrectomy were collected during (specimens stored ≤ 5 years) or before (specimens stored >5 years) gross examination. Tissue specimen storage at 4°C prior to freezing (cold ischemia time) was ≤30 minutes (17 specimens), 31 to ≤60 minutes (18 specimens), 61 to ≤120 minutes (37 specimens), or >120 minutes (33 specimens). Specimens were then cut into 0.5 cm³ fragments and frozen by immersion in isopentane (temperature not specified) and stored in liquid nitrogen. Specimens were stored for 1 year (27 specimens), 3 years (25 specimens), 5 years (26 specimens) and 10 years (27 specimens).  After storage, each specimen was divided into two parts, and half was refrozen in optimal cutting temperature compound (OCT); RNA and DNA were extracted from the remaining half using the PureLink RNA Mini Kit and the QIAamp DSP DNA Mini Kit, respectively. RNA and DNA integrity were evaluated using Genomic ScreenTape Kits and an Agilent Tapestation 4200 instrument.  Sections (4 µm) were cut from OCT-embedded tissue and were H&E stained.  Pathologists graded the sections based on tumor cell percentage (0 if 0%, 1 if 1-30%, 2 if 31-60% and 3 if >60%), the presence of normal tissue (0 for no and 1 for yes), inflammatory cell infiltration (0 for no, 1 if scanty, 2 if moderate and 3 if marked), stromal fibrosis (0 for no, 1 if scanty, 2 if moderate and 3 if marked), percentage necrosis (0 if absent, 1 if <10%, 2 if 10-50% and 3 if >50%), and percentage extracellular mucin pool (0 if absent, 1 if <10%, 2 if 10-50% and 3 if >50%).

   Summary of Findings:

   Of the 101 tumor specimens analyzed, 61.76% (63 out of 102) had a DIN ≥ 7.0, with the highest DIN found in DNA extracted from HCC followed by RCC specimens and the lowest DIN observed in COAD specimens. The integrity of the DNA extracted from resected tumor specimens (assessed by median DIN) was not affected by the duration of frozen storage for any of the tumor types evaluated. The DIN was not significantly affected by the cold ischemia times evaluated (≤30 min, ≤60 min, ≤120 min, and >120 min).  DIN was not correlated with any of the histopathologic parameters scored (tumor cell percentage, presence of normal tissue, extent of inflammatory cell infiltration, extent of stromal fibrosis, percentage necrosis or percentage extracellular mucin pool).

   Of the 102 tumor specimens that underwent RNA extraction, 58.82% (60 out of 102)  had a RIN ≥ 7.0, with the highest RINs observed in RNA extracted from HCC specimens and the lowest in RNA extracted from COAD specimens. While RIN was not affected by the duration of frozen storage of HCC and RCC specimens, a non-significant trend toward decreased RIN with longer storage was noted among RNA from COAD specimens (median RIN=5.3 for specimens stored for 1 year versus median RIN of 5.0 for specimens stored for 10 years). Similarly, RIN was not significantly affected by the cold ischemia time evaluated (≤30 min, ≤60 min, ≤120 min, and >120 min), but there appeared to be a non-significant trend toward increased variability of RIN with longer cold ischemic time among COAD specimens. RIN was positively correlated with tumor cell percentage (r=0.411, P<0.001) and negatively correlated with the extent of inflammatory cell infiltration (r=-0.473, P<0.001), extent of stromal fibrosis (r=-0.391, P<0.001), percentage extracellular mucin pool (r=-0.255, P=0.010), and the presence of combined normal tissue (r=-0.223, P=0.024) but was not correlated with the percentage of necrotic tissue.  In a multivariable regression analysis, the extent of inflammatory cell infiltration and percentage of extracellular mucin pool were found to be independent negative predictors of RNA quality (P<0.05, both).

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Liver
   • Tissue - Kidney

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   Morphology	H-and-E microscopy
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Colon adenocarcinoma (COAD) Hepatocellular carcinoma (HCC) Renal cell carcinoma (RCC)
   Storage	Storage duration	1 year 3 years 5 years 10 years
   Biospecimen Acquisition	Cold ischemia time	≤30 min 31-60 min 61-120 min >120 min
   Biospecimen Acquisition	Biospecimen location	Renal cell carcinoma (RCC) Hepatocellular carcinoma Colon adenocarcinoma

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