Reporting tumor molecular heterogeneity in histopathological diagnosis.
Author(s): Mafficini A, Amato E, Fassan M, Simbolo M, Antonello D, Vicentini C, Scardoni M, Bersani S, Gottardi M, Rusev B, Malpeli G, Corbo V, Barbi S, Sikora KO, Lawlor RT, Tortora G, Scarpa A
Publication: PLoS One, 2014, Vol. 9, Page e104979
PubMed ID: 25127237 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare next-generation sequencing (NGS) results between frozen and formalin-fixed paraffin-embedded (FFPE) specimens and to determine if mutation rates match immunohistochemical (IHC) staining results.
Conclusion of Paper
The number of reads and the mutational frequency were comparable between matched FFPE and frozen specimens. For FFPE specimens, the frequency of mutations was very strongly correlated with the dilution of the template DNA, and mutation rates matched immunostaining results.
Studies
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Study Purpose
The purpose of this study was to compare NGS results between frozen and FFPE specimens and to determine if mutation rates match IHC staining results. DNA was extracted from 30 FFPE upper gastrointestinal and hepatobiliopancreatic tumors and 5 matched FFPE and frozen pancreatic solid pseudopapillary tumors. The QIAamp DNA FFPE Tissue Kit was used for extraction from FFPE specimens and the QIAamp DNA Mini kit was used for extraction from frozen specimens. NGS was used to identify mutations in 46 cancer-associated genes and results were verified by Sanger sequencing.
Summary of Findings:
A similar number of sequences were obtained using the 5 matched FFPE and frozen specimens. Further, the frequency of the beta-catenin (CTTNB1) mutation was ~50% of the percentage of tumor cells and generally was the same between matched FFPE and frozen specimens. Overall mutation frequencies as low as 1% and 2% were found for frozen and FFPE specimens, respectively. Further, the frequency of detection of mutations in DNA from a hepatic carcinosarcoma was very strongly correlated with the dilution of the DNA (r=-0.917-0.997). Overall, an average of 1800x coverage was achieved using FFPE specimens, and the expected mutations in cancer-associated genes were identified. Importantly, some tumors showed multiple mutations with differing frequencies indicating tumor heterogeneity. Finally, the tumor protein p53 (TP53) mutation rates matched the proportion of p53 IHC stained cells in both Intrahepatic cholangiocarcinoma (ICC) cases examined, and SMAD family member 4 (SMAD4) mutation rates matched the proportion of SMAD4 loss of staining in the single Ampulla of Vater carcinoma (AVC) case examined.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Next generation sequencing DNA DNA sequencing Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Next generation sequencing Specific Template/input amount 100% DNA
95% DNA
90% DNA
75% DNA
50% DNA
25% DNA
10% DNA
0% DNA
Next generation sequencing Specific Technology platform IHC