Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine.

Author(s): Kim S, Park C, Ji Y, Kim DG, Bae H, van Vrancken M, Kim DH, Kim KM

Publication: J Mol Diagn, 2017, Vol. 19, Page 137-146

PubMed ID: 27840062 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of a delay to fixation, prolonged fixation (1-5 d), fixation in basic or acidic formaldehyde rather than neutral buffered formalin, and treatment of extracted DNA with UDG on NGS quality. The effects of formalin fixation duration were investigated using matched FFPE and fresh specimens collected from two patients during gastrectomy: one tumor specimen and one normal adjacent specimen from the first patient and a normal adjacent specimen from the second.  The FFPE specimens from the second patient was also used to investigate the effects of formalin pH, delayed fixation and treatment of extracted DNA with UDG.. Specimens were divided to investigate the effects of each pre-analytical factor. Specimens from the first patient were fixed for 1, 2, 3, 4 and 5 days in formalin ( pH 7), while the specimen from the second patient was fixed for 1, 2, 3, 4 and 5 days in formalin at a pH 5, 7 or 9. Additional specimens from the second patient were stored at room temperature (20-22˚C) in the absence of media for 24 h before fixation in formalin (pH 7) for 1-5 days. Details of paraffin-processing were not specified. DNA was extracted using the Qiagen DNA FFPE Tissue Kit and quantified by spectrophotometer and Qubit fluorometer. To investigate the impact of UDG pretreatment, DNA isolated from 4 archived FFPE blocks containing gastric adenocarcinoma specimens that were stored for > 10 years and a subset of specimens from the second patient (those fixed for 3 and 4 days in formalin (pH 7) and for 5 days in formalin (pH 9)) were volume reduced and treated with UDG for 2 h before sequencing. Next-generation sequencing was conducted using the Ion AmpliSeq Cancer Hotspot Panel version 2.

   Summary of Findings:

   Modest to strong correlations (r=0.60-0.73) were observed in mean target coverage between replicate DNA extractions from the four prospectively collected FFPE blocks and identical variant counts occurred in 50-76.2% of cases. When fresh specimens were compared the  tumor specimen procured from the  first patient had fewer SNVs with a Phred-scaled quality of >20 per read (>Q20) and higher coverage than the matched normal adjacent specimen; but when FFPE specimens were compared, the tumor specimen generally had lower coverage and the number of SNVs with a  >Q20displayed a biphasic response to fixation duration (Q20 was lower when specimens were fixed for 1 or 4 days, and higher when fixed for 3 or 5 days). The majority of FFPE specimens fixed (formalin, pH of 7) for 1 day  (2/3 specimens) had fewer SNVs with a >Q20 and reduced sequence coverage in comparison to case-matched fresh specimens. When conditions and durations of formalin fixation were examined, fixation for more than 2 days, a 24 h delay to fixation, or fixation in formalin with a pH of 5 generally resulted in fewer SNVs with a >Q20 and slightly lower than average coverage; however, effects were inconsistent and the differences in mean target coverage were less than those observed between replicate DNA extractions from the same block.  Further, a higher percentage of C:G>T:A nucleotide changes were observed  (i) among specimens fixed (for more than 3 days, (ii) among specimens fixed in formalin at a pH of 5 rather than 7 or 9, and (iii) among specimens subjected to a 24 h delay to fixation (formalin, pH 7). When filters for mapping quality, depth and strand bias were applied, many but not all of the low mapped quality values of C:G>T:A changes were removed. UDG treatment increased the total reads, the percentage of aligned reads, bases with a >Q20, and mean target coverage in prospectively collected FFPE specimens and 2 of the 4 archival FFPE specimens. In archival FFPE specimens, UDG treatment significantly affected allele frequency of C:G>T:A transitions, but the effect was nonsignificant in  prospectively collected FFPE specimens. UDG pretreatment did not appear to induce C:T>G:A changes in prospectively collected FFPE specimens. Further, while UDG pretreatment did remove C>T changes at the CpG dinucleotide site in 2 of the 4 archival specimens, the remaining 2 archival specimens and the prospectively collected specimens displayed a low number of C>T changes regardless of UDG treatment.

   Biospecimens

   • Tissue - Stomach

   Preservative Types

   • None (Fresh)
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Tumor Normal adjacent
   Biospecimen Preservation	Fixative pH	5 7 9
   Biospecimen Preservation	Time in fixative	0 days 1 day 2 days 3 days 4 days 5 days
   Next generation sequencing Specific	Template modification	UDG treated Not-treated
   Storage	Time at room temperature	0 h 24 h
   Storage	Storage duration	Archived >10 years Recently prepared
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Formaldehyde None (fresh)

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