Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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DNA Methylation in Ovarian Tumors-a Comparison Between Fresh Tissue and FFPE Samples.

Author(s): Oliveira DVNP, Hentze J, O'Rourke CJ, Andersen JB, Høgdall C, Høgdall EV

Publication: Reprod Sci, 2021, Vol. , Page

PubMed ID: 33891290 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare methylation profiles generated using matched FFPE and frozen ovarian tumor specimens. The authors also investigated if a signature of differentially methylated loci in tumor compared to benign frozen specimens could be used to distinguish malignant and benign FFPE specimens. Tumor specimens were obtained from three patients with early-stage (stage I or II) high-grade serous carcinomas, three patients with late-stage (stage III or IV) high-grade serous carcinomas, one patient with a borderline ovarian tumor of low malignant potential, and one patient with an ovarian fibroma. All specimens were obtained by macroradical surgery prior to chemotherapy. Matched specimens were formalin-fixed and paraffin-embedded (processing detail not provided) and stored at room temperature or frozen and then stored at -80°C. All tissues were scored for tumor percentage by a pathologist and areas with high tumor content were selected for DNA extraction. DNA was extracted from a 2-mm core of FFPE tissue using the Maxwell RSC DNA FFPE Kit and from homogenized frozen tissue using the Maxwell RSC Tissue DNA Kit. DNA was further purified using QIAquick PCR Purification System and quantified using a Qubit fluorometer. DNA was bisulfite-converted using the EZ DNA Methylation Kit. Bisulfite-converted DNA from FFPE tissue was then repaired using the Infinium HD FFPE DNA Restore Kit and cleaned using the ZR-96 DNA Clean & Concentrator-5. Methylation was profiled using Infinium MethylationEPIC BeadChip Kit.

   Summary of Findings:

   A total of 838,438 probes were detected in all specimens and 27,421 failed detection (P>0.01) in one or more specimens. Of the probes not detected in one or more specimens, 87.4% were not detected in only an FFPE specimen, 2.72% were not detected in only a frozen specimen, and the remaining 9.92% were not detected in specimens of either type. The authors state the increased loss of detection shows that the quality of the FFPE specimen is below that of the frozen (P<0.0001). A very strong correlation was observed in expression of filtered probes (those known to be affected by common SNPs and those that cross-react with multiple loci in the genome) between frozen and FFPE specimens (r²=0.986, P=2.2×10⁻¹⁶). Hierarchical clustering based on the 2000 most variable probes clustered all frozen and FFPE pairs together and tumors separately from the benign specimens.  A total of 78 loci were hypermethylated and six hypomethylated in frozen malignant versus benign specimens. These 84 candidates were able to distinguish malignant from benign specimens in the matched FFPE specimens as well as in a previously published cohort of 99 high-grade serous adenocarcinoma and 12 normal fallopian tube specimens.

   Biospecimens

   • Tissue - Ovary

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Benign Malignant
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen

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