Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine.
Author(s): Van Allen EM, Wagle N, Stojanov P, Perrin DL, Cibulskis K, Marlow S, Jane-Valbuena J, Friedrich DC, Kryukov G, Carter SL, McKenna A, Sivachenko A, Rosenberg M, Kiezun A, Voet D, Lawrence M, Lichtenstein LT, Gentry JG, Huang FW, Fostel J, Farlow D, Barbie D, Gandhi L, Lander ES, Gray SW, Joffe S, Janne P, Garber J, MacConaill L, Lindeman N, Rollins B, Kantoff P, Fisher SA, Gabriel S, Getz G, Garraway LA
Publication: Nat Med, 2014, Vol. 20, Page 682-8
PubMed ID: 24836576 PubMed Review Paper? No
Purpose of Paper
This paper compared next generation whole exome sequencing (WES) metrics, mutation detection, and copy number in matched FFPE and frozen specimens.
Conclusion of Paper
Although the coverage was 1.5-2-fold higher for FFPE than frozen specimens; sequencing metrics, detection rates for mutations, and exonic copy number were comparable in FFPE and frozen specimens. Importantly, WES data allowed for discovery of a novel KRAS mutation in a patient with lung cancer.
Studies
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Study Purpose
This study compared WES metrics, mutation detection, and copy number in matched FFPE and frozen specimens. Matched FFPE and frozen specimens from eleven patients with lung adenocarcinomas and the corresponding normal adjacent tissue were compared for mutation detection rates and copy number. For initial comparison of sequencing metrics, WES was performed on DNA from 99 unspecified FFPE specimens, 394 frozen blood specimens, and 367 unspecified frozen specimens. WES data from an additional 511 cases was analyzed to determine the suitability of this method for identification of therapeutically relevant mutations. FFPE sections or cores were deparaffinized using CitriSolv, lysed in proteinase K, and de-modified by incubation at 90°C before extraction with the QIAamp DNA FFPE Tissue Kit. The methods used for frozen and blood specimens were not specified. Library construction was performed with 10-100 ng of DNA using Illumina paired-end adapters and the KAPA Biosciences kit.
Summary of Findings:
Whole exome sequencing was successful with as little as 13.6 ng of DNA from frozen specimens or 16 ng of DNA from FFPE specimens, but greater than 80% of targeted bases and greater than 100X mean target coverage was obtainable with as little as 1 ng when extra sequencing was performed to account for duplication. Although FFPE and frozen specimens yielded a comparable percentage of target bases sequenced at 20X or more, percentage of selected bases, and percentage of zero-target bases; FFPE specimens were sequenced with 1.5-2-fold greater coverage than frozen specimens (75-100X versus 150-175X).
When only mutations detected in two copies in regions sequenced at sufficient depth in both frozen and FFPE specimens were considered, 91.4% of the mutations found in the FFPE specimens were validated in matched frozen specimens and 91% of mutations detected in frozen specimens were validated in FFPE specimens. When the data was down-sampled to reflect 90X coverage, 92.6% of variants identified in FFPE specimens were found in the matched frozen specimens and 91.5% of mutations found in the frozen specimens were found in the matched FFPE specimen. Exonic copy numbers were strongly correlated between matched FFPE and frozen specimens (R=0.79, P<0.0001)
Analysis of publicly-available data found that genes identified as tumor alterations relevant for genomics-driven therapy (TARGET) contained mutations in 80% of patients (408 of 511). Further, this method allowed for identification of a novel KRAS mutation in a FFPE specimen from a patient with lung cancer.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Normal Adjacent
- Neoplastic - Not specified
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Next generation sequencing Specific Nucleic acid amplification 90X coverage
90-175X coverage
Next generation sequencing Specific Template/input amount 1 ng DNA
10-100 ng DNA