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Relationship between formalin reagent and success rate of targeted sequencing analysis using formalin fixed paraffin embedded tissues.

Author(s): Amemiya K, Hirotsu Y, Oyama T, Omata M

Publication: Clin Chim Acta, 2019, Vol. 488, Page 129-134

PubMed ID: 30395866 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if the extent of DNA fragmentation is affected by the concentration (10%, 20%) and buffer used during formalin fixation and the duration of formalin fixation.  Surgically resected and biopsy liver specimens were dissected and fixed in 10 or 20% neural buffered formalin (NBF) or unbuffered formalin for 6 h to 7 d. Results were compared to controls that were snap-frozen in liquid nitrogen and then stored at -80°C. Tumor specimens were isolated via laser microdissection prior to DNA extraction with the QIAamp DNA FFPE kit and elution with QIAamp Mini Elute columns. DNA fragmentation was assessed by real-time PCR and the relative quantification of DNA via RQ values. RQ values reflect the ratio of a long (268 bp) to short (87 bp) amplicon, with a higher RQ value indicating better genomic DNA quality.

   Summary of Findings:

   Based on RQ values, all FFPE specimens yielded degraded DNA compared to snap-frozen liver specimens. When specimens were fixed for 24 h, DNA fragmentation was less severe when formalin was buffered compared to unbuffered and when a lower concentration of formalin (10% versus 20%)was used.  The highest RQ values were obtained from specimens fixed in 10% NBF followed by 20% NBF, 10% unbuffered formalin, and 20% unbuffered formalin. Long DNA fragments were amplifiable when liver specimens were fixed for 3 d or less, but fixation for 7 d resulted in severe DNA fragmentation; effects were dependent on formalin composition, with the least fragmentation observed among specimens fixed in 10% NBF, 10% unbuffered formalin, 20% NBF, followed by 20% unbuffered formalin.

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Time in fixative	6 h 12 h 1 d 3 d 7 d
   Real-time qPCR Specific	Length of gene fragment	87 bp 268 bp
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Formalin (unbuffered)
   Biospecimen Preservation	Concentration of fixative	10% 20%

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2. Study Purpose

   The purpose of this study was to determine if DNA fragmentation and NGS success differ between tumor specimens fixed in 10% NBF (180 specimens) and 20% unbuffered formalin (26 specimens). Specimens were fixed for 1, 3 or 7 d, and included both biopsies (53 specimens) and surgical resections (153 specimens). Tissue specimens were obtained from patients diagnosed with gastric cancer, lung cancer, hepatocellular carcinoma, metastatic lymph node, ovarian cancer, breast cancer, melanoma, peritoneum cancer, pancreatic cancer, endometrial cancer, and vaginal cancer. Tumor specimens were isolated via laser microdissection prior to DNA extraction with the QIAamp DNA FFPE kit and elution with QIAamp Mini Elute columns. DNA fragmentation was assessed by real-time PCR and the relative quantification of DNA via RQ values. RQ values reflect the ratio of a long (268 bp) to short (87 bp) amplicon, with a higher RQ value indicating better genomic DNA quality. NGS libraries were constructed with an Ion AmpliSeq Library Kit v 2.0. Targeted sequencing was performed with an Ion PGM system or an Ion Proton System, and data was analyzed with the Torrent Suite. NGS analysis was considered successful when sufficient mapped reads for a target region and coverage depth was obtained.

   Summary of Findings:

   While levels of the short DNA amplicon (87 bp) were comparable among specimens fixed for a mean of 2.5 d in 10% NBF or 20% unbuffered formalin, levels of the long DNA amplicon (268 bp) were significantly greater among specimens fixed in 10% NBF compared to those fixed in 20% unbuffered formalin (p=0.047); consequently, RQ values were significantly higher in specimens fixed in 10% NBF compared to 20% unbuffered formalin (0.38 vs 0.08; p<0.001).  NGS success rates were also substantially higher among specimens fixed in 10% NBF compared to 20% unbuffered formalin (98.3% vs 34.6%), but were similar among specimens fixed in 10% NBF for 1 to 7 d (range 95-100%). NGS success rates were comparable among biopsies and surgically resected specimens (98 vs 99%) that were fixed in 10% NBF, indicating that biopsies provide a sufficient amount of tissue for targeted NGS analysis. Successful targeted sequencing was possible for specimens fixed in 10% NBF regardless of tissue type.  A separate cohort of 860 specimens fixed in 10% NBF were used to confirm that FFPE specimens can be successfully used for NGS analysis.  Of those used for validation, five specimens failed to yield sufficient DNA for library construction, and nine specimens failed to produce NGS data of suitable quality resulting in a success rate of 98.4%.

   Biospecimens

   • Tissue - Liver
   • Tissue - Stomach
   • Tissue - Lung
   • Tissue - Lymph Node
   • Tissue - Ovary
   • Tissue - Vagina
   • Tissue - Skin
   • Tissue - Pancreas
   • Tissue - Uterus
   • Tissue - Other
   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Time in fixative	1 d 3 d 7 d
   Biospecimen Preservation	Concentration of fixative	10% 20%
   Biospecimen Acquisition	Method of tissue acquisition	Biopsy Surgical resection
   Real-time qPCR Specific	Length of gene fragment	87 bp 268 bp
   Biospecimen Acquisition	Biospecimen location	Stomach Lung Liver Lymph node Ovary Breast Skin Peritoneum Pancreas Uterus Vagina
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Formalin (unbuffered)

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