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DNA fragmentation simulation method (FSM) and fragment size matching improve aCGH performance of FFPE tissues.

Craig JM, Vena N, Ramkissoon S, Idbaih A, Fouse SD, Ozek M, Sav A, Hill DA, Margraf LR, Eberhart CG, Kieran MW, Norden AD, Wen PY, Loda M, Santagata S, Ligon KL, Ligon AH - PLoS ONE (2012)

Bottom Line: While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations.Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues.Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT
Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

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DNA fragmentation and thermodegradation are unpredictably variable.(A) Gel electrophoresis image of DNA extracted from 22 FFPE tissue specimens stored in paraffin from one to 13 years. (B) Mode fragment size of samples in (A) plotted by age of paraffin block, linear regression of data indicated by dashed line. (C) Gel electrophoresis image of DNA from six FFPE specimens intact prior to labeling (i), after ULS labeling only (0), or after ULS labeling plus 1 min heat fragmentation (1). (D) Mode fragment size of lanes marked 0 and 1 plotted for the six FFPE samples from the gel shown in (C). (E) Gel electrophoresis image of DNA from three frozen specimens with i, 0, and 1 indicating same conditions as in (C), and another samples after ULS labeling conditions plus 2 min heat fragmentation (2). (F) Plot of mode fragment size for lanes marked 0, 1, and 2 plotted for the three frozen samples in (E).
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pone-0038881-g003: DNA fragmentation and thermodegradation are unpredictably variable.(A) Gel electrophoresis image of DNA extracted from 22 FFPE tissue specimens stored in paraffin from one to 13 years. (B) Mode fragment size of samples in (A) plotted by age of paraffin block, linear regression of data indicated by dashed line. (C) Gel electrophoresis image of DNA from six FFPE specimens intact prior to labeling (i), after ULS labeling only (0), or after ULS labeling plus 1 min heat fragmentation (1). (D) Mode fragment size of lanes marked 0 and 1 plotted for the six FFPE samples from the gel shown in (C). (E) Gel electrophoresis image of DNA from three frozen specimens with i, 0, and 1 indicating same conditions as in (C), and another samples after ULS labeling conditions plus 2 min heat fragmentation (2). (F) Plot of mode fragment size for lanes marked 0, 1, and 2 plotted for the three frozen samples in (E).

Mentions: Utilizing our DNA extraction protocol with over 100 FFPE brain tumor specimens (block ages ranging from one to 15 years, all estimated to contain >50% tumor tissue) obtained from six different institutions, 100% of samples yielded DNA with average fragment sizes greater than 400 bp. Indeed, for most samples the fragment sizes were well above this size threshold and in agreement with general size ranges reported in other studies ([9], [19]). Agarose gel electrophoresis of 22 DNA extracts from FFPE tissue blocks ranging in age from one to 13 years confirmed this observation (Figure 3A). In fact, plotting the mode fragment size of each smear against block age reveals a statistically significant relationship (r = −0.77, p<0.0001) between advanced age and decreased fragment size (Figure 3B). Despite this relationship, our results support the conclusion that the initial (post-extraction) degradation of FFPE-derived DNA does not preclude obtaining fragment distributions within the optimal range (Figure 2), even among DNA samples isolated from archival specimens over ten years old.


DNA fragmentation simulation method (FSM) and fragment size matching improve aCGH performance of FFPE tissues.

Craig JM, Vena N, Ramkissoon S, Idbaih A, Fouse SD, Ozek M, Sav A, Hill DA, Margraf LR, Eberhart CG, Kieran MW, Norden AD, Wen PY, Loda M, Santagata S, Ligon KL, Ligon AH - PLoS ONE (2012)

DNA fragmentation and thermodegradation are unpredictably variable.(A) Gel electrophoresis image of DNA extracted from 22 FFPE tissue specimens stored in paraffin from one to 13 years. (B) Mode fragment size of samples in (A) plotted by age of paraffin block, linear regression of data indicated by dashed line. (C) Gel electrophoresis image of DNA from six FFPE specimens intact prior to labeling (i), after ULS labeling only (0), or after ULS labeling plus 1 min heat fragmentation (1). (D) Mode fragment size of lanes marked 0 and 1 plotted for the six FFPE samples from the gel shown in (C). (E) Gel electrophoresis image of DNA from three frozen specimens with i, 0, and 1 indicating same conditions as in (C), and another samples after ULS labeling conditions plus 2 min heat fragmentation (2). (F) Plot of mode fragment size for lanes marked 0, 1, and 2 plotted for the three frozen samples in (E).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376148&req=5

pone-0038881-g003: DNA fragmentation and thermodegradation are unpredictably variable.(A) Gel electrophoresis image of DNA extracted from 22 FFPE tissue specimens stored in paraffin from one to 13 years. (B) Mode fragment size of samples in (A) plotted by age of paraffin block, linear regression of data indicated by dashed line. (C) Gel electrophoresis image of DNA from six FFPE specimens intact prior to labeling (i), after ULS labeling only (0), or after ULS labeling plus 1 min heat fragmentation (1). (D) Mode fragment size of lanes marked 0 and 1 plotted for the six FFPE samples from the gel shown in (C). (E) Gel electrophoresis image of DNA from three frozen specimens with i, 0, and 1 indicating same conditions as in (C), and another samples after ULS labeling conditions plus 2 min heat fragmentation (2). (F) Plot of mode fragment size for lanes marked 0, 1, and 2 plotted for the three frozen samples in (E).
Mentions: Utilizing our DNA extraction protocol with over 100 FFPE brain tumor specimens (block ages ranging from one to 15 years, all estimated to contain >50% tumor tissue) obtained from six different institutions, 100% of samples yielded DNA with average fragment sizes greater than 400 bp. Indeed, for most samples the fragment sizes were well above this size threshold and in agreement with general size ranges reported in other studies ([9], [19]). Agarose gel electrophoresis of 22 DNA extracts from FFPE tissue blocks ranging in age from one to 13 years confirmed this observation (Figure 3A). In fact, plotting the mode fragment size of each smear against block age reveals a statistically significant relationship (r = −0.77, p<0.0001) between advanced age and decreased fragment size (Figure 3B). Despite this relationship, our results support the conclusion that the initial (post-extraction) degradation of FFPE-derived DNA does not preclude obtaining fragment distributions within the optimal range (Figure 2), even among DNA samples isolated from archival specimens over ten years old.

Bottom Line: While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations.Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues.Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.

ABSTRACT
Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

Show MeSH
Related in: MedlinePlus