Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Impact of the quality of resected thyroid cancer tissue sample on next-generation sequencing testing.

Author(s): Hatanaka KC, Nakamura K, Katoh R, Ito K, Hirokawa M, Miyauchi A, Matsuno Y, Kano S, Okada Y, Mori J, Ito YM, Hatanaka Y

Publication: Pathol Int, 2024, Vol. 74, Page 77-86

PubMed ID: 38226479 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to assess the impact of the concentration (10 vs 15%) and duration of formalin fixation (≤48 h, 48-72 h), and the storage duration of formalin-fixed, paraffin-embedded (FFPE) blocks (<1 to 10 y) on DNA and RNA integrity markers and next-generation sequencing (NGS) success rates for activating rearranged during transfection (RET) alterations of proto-oncogenes in thyroid cancer specimens. The relationship between DNA and RNA integrity and NGS success was also investigated. In total, 203 surgically resected medullary and papillary thyroid tumor specimens collected at three different hospitals were used for the study; the distribution of specimens was such that each fixation or storage parameter included 10 or more specimens. FFPE blocks had a tumor cell content ≥30% and a tumor area of ≥10 mm x 10 mm. DNA and RNA were isolated at a central laboratory from ten 5 µm-thick FFPE sections using the AllPrep DNA/RNA Mini Kit by Qiagen. RET fusions and RET mutations were detected in RNA and DNA samples, respectively, by NGS analysis using an Oncomine Dx Target Test (ODxTT) on a ODxTT multi-CDx system; NGS success rate was calculated based on the percentage of analyzed samples that returned validated results.  DNA integrity number (DIN, range: 1-10), RNA integrity number (RIN, range 1-10), and DV200 value (the percentage of RNA fragments longer than 200 bp) were determined with a 4200 TapeStation instrument.  Nucleic acid integrity was also quantified by quantitative PCR (qPCR)-generated double delta quantification cycle (ddCq) values using an Agilent NGS FFPE QC Kit for DNA and delta Cq (ΔCq) values using an RNA QC Assay for RNA.

   Summary of Findings:

   Of the 203 FFPE thyroid tumor samples evaluated in the study, one sample had nucleic acid yields that were insufficient for analysis.  When the remaining 202 FFPE thyroid tumor samples were considered together (regardless of time in fixative or formalin concentration), the overall NGS success rate was 90% for DNA and 93% for RNA.  NGS success rate was dependent on FFPE block age, as FFPE blocks stored for <3 y tended to have higher NGS success rates (RNA: 100%; DNA: 98-100%) than those stored for 3 to <5 y (RNA: 88-91%; DNA: 83-86%); the trend was progressive, with NGS success rates of 78% and 67% for RNA and DNA, respectively, in FFPE blocks stored for 9 to <10 y. When FFPE block age was limited to <3 y, FFPE thyroid tumor specimens that were fixed in 15% formalin generally had lower DNA NGS success rates than those fixed in 10% formalin (≥95% versus 70-90%, respectively), although the two cohorts also differed in the duration of formalin fixation (10% formalin for ≤48 h, 15% formalin for >48- 72 h). However, when 24 FFPE specimens with the same time in fixative (>48- 72 h) and FFPE block age (3 to <4 y) were compared, DNA and RNA NGS success rates were higher in specimens that were fixed in 15% formalin than 10% formalin (RNA: 90 versus 67%; DNA: 80 versus 67%). FFPE block storage also adversely affected the DNA and RNA integrity markers evaluated. DIN began to decrease and the quantitative PCR-based ddCq value began to increase after 3 years of storage compared to those from blocks stored for <1 y. The RNA marker DV200 began to decrease and the ΔCq value began to increase after 2 years of storage compared to blocks stored for <1 y, but only slight differences were observed in RIN among different FFPE block ages. In contrast, the two DNA integrity markers and three RNA integrity markers were not affected by the concentration of formalin or the duration of fixation. For DNA, formalin concentration, time in fixative, FFPE block age, DIN, and ddCq were all identified as significant predictors of NGS success with the ODxTT assay. For RNA, formalin concentration, FFPE block age, RIN, DV200, and ΔCq were identified as significant predictors of NGS success with the ODxTT assay.  Multivariate regression analysis identified ddCq and ΔCq as significant contributors to DNA and RNA NGS success rates, respectively.  When all nucleic acid quality metrics were excluded from the multivariate logistic regression model, FFPE block age was identified as a “significant contributor to RNA NGS success rates”.  The authors identified quality metric thresholds for NGS success for DNA (ddCq <1.72; DIN ≥3.00) and RNA (ΔCq <10.85; DV200 ≥63.21).

   Biospecimens

   • Tissue - Thyroid Gland

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   DNA	Next generation sequencing
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Real-time qPCR
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Concentration of fixative	10% Formalin 15% Formalin
   Biospecimen Preservation	Time in fixative	≤48 h 48-72 h
   Storage	Storage duration	<1 y 1 to <2 y 2 to <3 y 3 to <4 y 4 to <5 y 5 to <6 y 6 to <7 y 7 to <8 y 9 to <10 y
   Real-time qPCR Specific	Quality metrics	DIN ddCq
   Real-time qRT-PCR Specific	Quality metrics	RIN DV200 ΔCq

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