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Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.

Carrick DM, Mehaffey MG, Sachs MC, Altekruse S, Camalier C, Chuaqui R, Cozen W, Das B, Hernandez BY, Lih CJ, Lynch CF, Makhlouf H, McGregor P, McShane LM, Phillips Rohan J, Walsh WD, Williams PM, Gillanders EM, Mechanic LE, Schully SD - PLoS ONE (2015)

Bottom Line: While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined.Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining.This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Control and Population Sciences (DCCPS), National Cancer Institute, 9609 Medical Center Drive, Rockville, MD 20850, United States of America.

ABSTRACT
Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

No MeSH data available.


Related in: MedlinePlus

Workflow for the FFPE tissues.Three SEER RTRs selected cases that would likely meet the study criteria from their database. Cases were screened for histology/morphology, tumor nuclei content, and necrotic cell content to select cases meet the study criteria. The RTRs screened 30 or 31 cases; one RTR did not keep track of the number of cases they screened. Each RTR selected and sent 20 cases that met study criteria to the lab. One case was excluded from analyses due to incomplete information regarding storage time. 59 cases were considered for subsequent analyses. DNA was successfully extracted from 58 cases. WES was successful on 53 of the cases.
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pone.0127353.g001: Workflow for the FFPE tissues.Three SEER RTRs selected cases that would likely meet the study criteria from their database. Cases were screened for histology/morphology, tumor nuclei content, and necrotic cell content to select cases meet the study criteria. The RTRs screened 30 or 31 cases; one RTR did not keep track of the number of cases they screened. Each RTR selected and sent 20 cases that met study criteria to the lab. One case was excluded from analyses due to incomplete information regarding storage time. 59 cases were considered for subsequent analyses. DNA was successfully extracted from 58 cases. WES was successful on 53 of the cases.

Mentions: 59 FFPE tissue sections (distinct cases) were sent to the Molecular Characterization and Clinical Assay Development Laboratory (MoCha) at Frederick National Laboratory for Cancer Research (Fig 1). As specimens were retrospectively collected by the RTRs from multiple medical facilities and pathology labs within each of the three catchment areas, fixation times/conditions and storage conditions were unknown. Tissues were from high-grade serous ovarian adenocarcinomas (ICD-O-3 Topography code: C56.9; Morphology codes: 8441/3, 8460/3, 8461/3) and storage time ranged from 3 to 32 years (Table 1) based on decade when tissue was resected. This cancer type was selected because it is a rare, yet aggressive cancer and has high observed frequency of mutations in TP53 (90% of tumors, automated detection of TP53), which was used as a positive control [3]. This study was approved by institutional review boards at participating cancer registries (University of Southern California Health Sciences Campus, The University of Iowa and the University of Hawaii) and at the National Cancer Institute (NCI). The study was determined to be exempt from IRB review under 45 CFR 46.101(b)(4) for the use of coded or coded but unlinked tissue blocks from the SEER registries. No contact with subjects was made for this study.


Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.

Carrick DM, Mehaffey MG, Sachs MC, Altekruse S, Camalier C, Chuaqui R, Cozen W, Das B, Hernandez BY, Lih CJ, Lynch CF, Makhlouf H, McGregor P, McShane LM, Phillips Rohan J, Walsh WD, Williams PM, Gillanders EM, Mechanic LE, Schully SD - PLoS ONE (2015)

Workflow for the FFPE tissues.Three SEER RTRs selected cases that would likely meet the study criteria from their database. Cases were screened for histology/morphology, tumor nuclei content, and necrotic cell content to select cases meet the study criteria. The RTRs screened 30 or 31 cases; one RTR did not keep track of the number of cases they screened. Each RTR selected and sent 20 cases that met study criteria to the lab. One case was excluded from analyses due to incomplete information regarding storage time. 59 cases were considered for subsequent analyses. DNA was successfully extracted from 58 cases. WES was successful on 53 of the cases.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519244&req=5

pone.0127353.g001: Workflow for the FFPE tissues.Three SEER RTRs selected cases that would likely meet the study criteria from their database. Cases were screened for histology/morphology, tumor nuclei content, and necrotic cell content to select cases meet the study criteria. The RTRs screened 30 or 31 cases; one RTR did not keep track of the number of cases they screened. Each RTR selected and sent 20 cases that met study criteria to the lab. One case was excluded from analyses due to incomplete information regarding storage time. 59 cases were considered for subsequent analyses. DNA was successfully extracted from 58 cases. WES was successful on 53 of the cases.
Mentions: 59 FFPE tissue sections (distinct cases) were sent to the Molecular Characterization and Clinical Assay Development Laboratory (MoCha) at Frederick National Laboratory for Cancer Research (Fig 1). As specimens were retrospectively collected by the RTRs from multiple medical facilities and pathology labs within each of the three catchment areas, fixation times/conditions and storage conditions were unknown. Tissues were from high-grade serous ovarian adenocarcinomas (ICD-O-3 Topography code: C56.9; Morphology codes: 8441/3, 8460/3, 8461/3) and storage time ranged from 3 to 32 years (Table 1) based on decade when tissue was resected. This cancer type was selected because it is a rare, yet aggressive cancer and has high observed frequency of mutations in TP53 (90% of tumors, automated detection of TP53), which was used as a positive control [3]. This study was approved by institutional review boards at participating cancer registries (University of Southern California Health Sciences Campus, The University of Iowa and the University of Hawaii) and at the National Cancer Institute (NCI). The study was determined to be exempt from IRB review under 45 CFR 46.101(b)(4) for the use of coded or coded but unlinked tissue blocks from the SEER registries. No contact with subjects was made for this study.

Bottom Line: While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined.Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining.This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Control and Population Sciences (DCCPS), National Cancer Institute, 9609 Medical Center Drive, Rockville, MD 20850, United States of America.

ABSTRACT
Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

No MeSH data available.


Related in: MedlinePlus