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Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project.

Author(s): Robbe P, Popitsch N, Knight SJL, Antoniou P, Becq J, He M, Kanapin A, Samsonova A, Vavoulis DV, Ross MT, Kingsbury Z, Cabes M, Ramos SDC, Page S, Dreau H, Ridout K, Jones LJ, Tuff-Lacey A, Henderson S, Mason J, Buffa FM, Verrill C, Maldonado-Perez D, Roxanis I, Collantes E, Browning L, Dhar S, Damato S, Davies S, Caulfield M, Bentley DR, Taylor JC, Turnbull C, Schuh A

Publication: Genet Med, 2018, Vol. 20, Page 1196-1205

PubMed ID: 29388947 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared DNA yield and integrity; NGS metrics; and detection of SNVs, InDels and copy number variations in matched FFPE and frozen tumor specimens. The effects of differences between bioinformatics pipelines used on SNV detection was also investigated. Matched tumor and peripheral blood specimens were collected from 52 cancer patients (10 breast, 12 colorectal, 7 endometrial, 4 prostatic, 14 renal, and 5 thoracic). A 5-mm punch of the tumor specimen was snap-frozen in liquid nitrogen and embedded in OCT. The remainder of the tumor was formalin-fixed in 10% formalin in sodium chloride or phosphate for an unspecified duration that was determined by specimen type and then paraffin-embedded. Frozen and FFPE sections were evaluated for cellularity, necrosis, and percentage viable cells. DNA was extracted from blood using QIAamp DNA Mini Kit from 30 mg frozen specimens using the All Prep DNA/RNA Mini Kit and from two 1-mm FFPE punches using truXTRAC FFPE DNA Kit with an overnight proteinase K digestion at 56˚C. DNA was quantified using Nanodrop and the Qubit dsDNA Broad Range Assay kit. Integrity was determined by electrophoresis and purity by A260/280 ratios. Quality was determined by the quantitative real-time PCR based FFPE QC PCR assay. Sequencing libraries were prepared using the TruSeq Nano DNA Library Prep and sequenced on a HiSeq2500 to 70X for tumor specimens and 30X for blood specimens.

   Summary of Findings:

   FFPE specimens had significantly lower yields of double-stranded DNA than matched frozen specimens (P=1.41 x 10^-6) and electrophoresis revealed the DNA was fragmented (<0.5-40 kb versus >40 kb). Further confirming the effect was due to degradation, the spectrophotometric yield and purity were comparable between frozen and FFPE specimens. FFPE specimens had lower median insert size (P=1.798 x 10^-9), read pass filter aligned ratios (P=3.618 x 10^-9), mean sequencing depth (77x versus 93x), and proportion of regions reaching a depth of 70X (0.351 versus 0.782), and a higher ratio of chimeric pairs (P=1.330 x 10^-8) than matched frozen specimens. Further, FFPE specimens had a higher AT dropout and lower GC dropout than matched frozen specimens (P=1.804 x 10^-9, both) and 1.1 to 5.8-fold higher standard deviation in the uniformity of coverage (P<2.2 x 10^-16).

   The number of SNVs and InDels detected depended on the variant detection method used. Mutect and Strelka had the highest SNV sensitivity (0.77 for Strelka and 0.76 for Mutect versus 0.49 for Shimmer and 0.51 for a Combination), but the positive predictive value (PPV) was highest for the combination or Mutect (0.89 for combination and 0.72 for Mutect versus 0.35 for Shimmer and 0.21 for Strelka). The number of C>T mutations in FFPE specimens was correlated with the increase in intragenic variants and was enriched in FFPE specimens, especially when analyzed by Strelka. The sensitivity for InDels was highest using Strelka (0.5 for Strelka, 0.28 for Shimmer, and 0.3 for the combination), but the PPV was highest using the combination or Shimmer (0.65 for combination, 0.62 for Shimmer, and 0.11 for Strelka).

   Overall, 6% of the variants were detected in both the FFPE and frozen specimens, 21% were only identified in frozen specimens, and 27% in just FFPE specimens with FFPE specimens having a greater proportion of C>A/G>T mutations and a lower proportion of C>T (24.2% versus 36%). The authors state the lower proportion of C>T mutations indicates that the mutations in FFPE specimens are not deamination artifacts. The overlap in SNV detection between FFPE and frozen specimens increased with increasing minimum allele frequency such that it was 71% in reliable areas of the genome and 63% overall when the minimal allele fraction was set to be 0.067 in one specimen. Further, percentage overlap in mutations was strongly correlated with the mutant allele fraction (r=0.731, P=1.09e^-9); modestly correlated with the number of C>A/G>T mutations (r=0.437, P=0.0013), proportion of reads aligned (r=0.492, P=0.000245), proportion of chimeric pairs (r=0.45, P=0.000934), median of reads covered at 70X (r=0.437, P=0.00134), and number of SNVs in the FFPE sample (r=0.458, P=0.000718); and weakly correlated with median insert size (r=0.368, P=0.00786). The authors consequently believe they are indicative of intra-tumoral variability. There was no increase in HighRisk InDels in FFPE specimens. Although 90% of the genes and InDels from the Cosmic cancer gene census were more frequently mutated in FFPE than frozen specimens, this was true for less than 1% of the functional mutations. Further, the sensitivities of frozen and FFPE specimens for mutations in Ion AmpliSeq Cancer Hotspot Panel v2 were 0.86 and 0.82, respectively. FFPE specimens had much more noise in the copy number intensity plots resulting in decreased reliability of copy number calls.

   The tumor content determined by the pathologists and the specimen purity determined by sequencing data were weakly correlated (r=0.347, P=0.00376). Among the tumor types, thoracic tumors had the most SNVs and the highest agreement in SNVs between FFPE and frozen specimens.

   Biospecimens

   • Tissue - Kidney
   • Tissue - Breast
   • Tissue - Colorectal
   • Bodily Fluid - Blood
   • Tissue - Prostate
   • Tissue - Ovary
   • Tissue - Adrenal Gland
   • Tissue - Uterus

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Electrophoresis
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry
   DNA	Spectrophotometry
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Spectrophotometry Specific	Technology platform	Qubit
   Next generation sequencing Specific	Data handling	Strelka Mutect Shimmer Combination

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2. Study Purpose

   This study investigated the effects of optimizing DNA extraction of NGS metrics and SNV detection in FFPE specimens and compared results to those obtained from frozen specimens. A 5-mm punch of each of five renal tumor specimens were snap-frozen in liquid nitrogen and embedded in OCT. The remainder of the tumor was formalin-fixed in 10% formalin in sodium chloride or phosphate for an unspecified duration that was determined by specimen type and then paraffin-embedded. DNA was extracted from 30 mg frozen specimens using the All Prep DNA/RNA Mini Kit and from two 1-mm FFPE punches using the truXTRAC FFPE DNA Kit or the QIAamp DNA FFPE Tissue kit with an overnight proteinase K digestion at 56˚C. The effects of reverse cross-linking temperature (90, 80, 65, and 56˚C), use of saline sodium citrate buffer, and duration (1 h versus 3 h) were investigated using pooled, extracted DNA from five patients. DNA was quantified using Nanodrop and the Qubit dsDNA Broad Range Assay kit. Integrity was determined by electrophoresis and purity by A260/280 ratios. Quality was determined by the quantitative real-time PCR based FFPE QC PCR assay. Sequencing libraries were prepared using the TruSeq Nano DNA Library Prep and sequenced on a HiSeq2500 to 70X for tumor specimens and 30X for blood specimens.

   Summary of Findings:

   All of the optimization protocols used for DNA extraction from FFPE specimens resulted in decreased or equivalent AT dropout and increased or equivalent global sequencing coverage, coverage uniformity, and overlap in SNV and InDel detection with that in frozen specimens than the non-optimized extraction method (QIAamp with reversal 90 ˚C for 1 hour or Covaris with reversal at 80˚C for 1 hour), with no clear preferred method. Further, the proportion of bases with >70X coverage was greater with all of the experimental protocols compared to the non-optimized protocol. The correlations in copy number changes were also greater using the optimization protocols than the original protocol. Using the optimized protocol, 98%, 86%, and 78% of tier 1, 2, and 3 mutations; respectively, were found in both FFPE and frozen specimens, 12 mutations were only found in the FFPE specimen and 6 were only found in the frozen specimen. Seven of the 12 only found in the FFPE specimen occurred at an allele frequency below 0.13.

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	truXTRAC FFPE DNA Kit QIAamp DNA FFPE Tissue kit
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Next generation sequencing Specific	Template modification	Reverse cross-linked at 90, 80, 65, or 56˚C Reverse cross-linked for 1 h or 3 h Reverse cross-linked with SSC Reverse cross-linked without SSC

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