Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples.

Author(s): Okojie J, O'Neal N, Burr M, Worley P, Packer I, Anderson D, Davis J, Kearns B, Fatema K, Dixon K, Barrott JJ

Publication: Curr Oncol, 2024, Vol. 31, Page 2441-2452

PubMed ID: 38785464 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield, purity and fragment size of DNA extracted from matched frozen and formalin-fixed, paraffin-embedded (FFPE) tumor specimens representing 12 different cancer types. The authors also investigated the potential effects of cold ischemia time and fixation duration on the yield and purity of isolated DNA. One tumor specimen per patient was collected from a total of 38 cancer patients (28 women and 10 men; 39-92 years of age) during surgical resection, resulting in one of each adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD),  uterine carcinosarcoma (UCS), and cervical squamous cell carcinoma and endocervical (CESC) specimen; two of each breast invasive carcinoma (BRCA) and sarcoma (SARC) specimen; three of each colon adenocarcinoma (COAD) and liver hepatocarcinoma (LIHC) specimen; four of each kidney renal cell carcinoma (KIRC) specimen; five uterine corpus endometrial carcinoma (UCEC) specimens, and thirteen ovarian serous cystadenocarcinoma specimens. Specimens were transported in Hypothermosol solution at 4°C and evaluated by a pathologist to ensure there was no severe necrosis, resulting in a mean cold ischemia time of 77.7 min (0-828 min). Half of each specimen was fixed in 10% formalin (mean of 15.7 h range 2-142 h) and paraffin-embedded before being stored at room temperature; the remaining half was snap-frozen and stored in liquid nitrogen.  Prior to DNA extraction, multiple (exact number not specified) FFPE scrolls (20 μm) were deparaffinized in xylene, rehydrated and weighed and frozen tissue was thawed (conditions not specified) and weighed. Tissues were lysed by incubation in Extracta DNA buffer containing Proteinase K at 55°C for 3 h and DNA was purified with the Zymo Research DNA Clean and Concentrator-5 Kit. DNA concentration and purity were evaluated by Nanodrop spectrometry. Fragment size profiles were evaluated using a 5300 Fragment Analyzer System and for fragments >6000 bp the Agilent Femto Pulse System. DNA Quality Numbers were assigned based on the percentage of fragments >300 bp with a score of 10 equating to 100% of fragments and 0 equating to 0% of fragments.

   Summary of Findings:

   The mean purity (ratio of absorbance at 260 to 280 nm) of the extracted DNA was comparable between FFPE and frozen specimens (1.76 versus 1.78); however, while seven FFPE specimens had DNA with 260/280 ratios >2.1, indicating potential protein contamination, none of the DNA from frozen specimens had ratios >2.1. The mean yield of DNA was much higher per mg of tissue for frozen specimens than deparaffinized and rehydrated FFPE tissue (222.1±35.2 ng/mg versus 52.8±18.5 ng/mg, P<0.001). Interestingly, while DNA yield from frozen specimens increased as tissue input increased, the same was not observed for FFPE tissue. The mean DNA Quality Number (DQN) was much higher for DNA extracted from frozen than FFPE specimens (9.8 versus 4.4, P<0.0005). Electropherograms of DNA from FFPE specimens also showed much lower DNA integrity, with only a single peak observed at approximately 300 bp rather than the peaks at 10,000 and 50,000 bp observed for DNA from frozen specimens. Area under the curve analysis of the electropherograms revealed that the DNA from frozen specimens had 8.9-fold more fragments that were >40,000 base pairs than DNA from FFPE specimens after normalization for DNA concentration (P<0.00005). DNA purity and DNA yield were not correlated with ischemia time or the duration of fixation.

   Biospecimens

   • Tissue - Adrenal Gland
   • Tissue - Bladder
   • Tissue - Prostate
   • Tissue - Breast
   • Tissue - Ovary
   • Tissue - Pancreas
   • Tissue - Uterus
   • Tissue - Liver
   • Tissue - Kidney
   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Sarcoma

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0-828 min
   Biospecimen Preservation	Time in fixative	2-142 h
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Biospecimen Aliquots and Components	Aliquot size/volume	10-19 mg tissue 20-34 mg tissue 35-50 mg tissue

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