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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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Size matching using FSM is a more critical determinant of array quality than other known variables.(A–H) Probe log2 ratio (signal intensity test DNA/signal intensity reference DNA) data plotted for a single chromosome (chr.13 or chr.1) from eight Agilent 1 M arrays (green;log2ratio<-0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). (A–C) Chromosome 13 plotted log2 ratios are representative profiles of three Agilent 1 M arrays of a single FFPE GBM specimen (GBM1) processed with the FSM ULS protocol (A), standard ULS protocol (B), or FSM ULS protocol after altered proteinase K digestion during DNA extraction (C) (plotted log2 ratio data for all chromosomes provided in Figure S2). (D–H) Chromosome 1 plotted log2 ratios are representative profiles of five Agilent 1 M arrays of a single FFPE GBM specimen (GBM2) processed using the FSM ULS protocol, with reduced DNA input in (E) and (F) (see Figure S3 and Figure S4 for detailed copy number analysis). Increased hybridization time (G) improved quality to a modest degree. Use of FFPE brain tissue as reference DNA (H) did not significantly improve results (dLRsd of 0.21 vs. 0.20 for standard reference).
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pone-0038881-g006: Size matching using FSM is a more critical determinant of array quality than other known variables.(A–H) Probe log2 ratio (signal intensity test DNA/signal intensity reference DNA) data plotted for a single chromosome (chr.13 or chr.1) from eight Agilent 1 M arrays (green;log2ratio<-0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). (A–C) Chromosome 13 plotted log2 ratios are representative profiles of three Agilent 1 M arrays of a single FFPE GBM specimen (GBM1) processed with the FSM ULS protocol (A), standard ULS protocol (B), or FSM ULS protocol after altered proteinase K digestion during DNA extraction (C) (plotted log2 ratio data for all chromosomes provided in Figure S2). (D–H) Chromosome 1 plotted log2 ratios are representative profiles of five Agilent 1 M arrays of a single FFPE GBM specimen (GBM2) processed using the FSM ULS protocol, with reduced DNA input in (E) and (F) (see Figure S3 and Figure S4 for detailed copy number analysis). Increased hybridization time (G) improved quality to a modest degree. Use of FFPE brain tissue as reference DNA (H) did not significantly improve results (dLRsd of 0.21 vs. 0.20 for standard reference).

Mentions: Having demonstrated the highly significant contributions of FSM analysis and matched DNA fragment sizes to aCGH quality, we sought to further assess the relative effects of fragment size compared to other previously reported variables such as Proteinase K digestion time, array hybridization time, and concentration and source of DNA in array hybridization reactions. DNA from a single FFPE tumor specimen, GBM1 (characterized by complex and highly aberrant copy number changes involving single-copy gains, single-copy losses, and regions of homozygous deletion on chromosome 13), was processed under multiple conditions and assayed with Agilent 1 M feature arrays. Comparison of Figure 6A and 6B supports our previous assertions regarding the significant improvement of data quality enabled by the FSM. Compared with data obtained following the FSM ULS protocol (Figure 6A) the standard ULS protocol yielded a higher dLRsd value (0.44) (Figure 6B) that precluded accurate detection of copy number aberrations (Figure S1).


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)

Size matching using FSM is a more critical determinant of array quality than other known variables.(A–H) Probe log2 ratio (signal intensity test DNA/signal intensity reference DNA) data plotted for a single chromosome (chr.13 or chr.1) from eight Agilent 1 M arrays (green;log2ratio<-0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). (A–C) Chromosome 13 plotted log2 ratios are representative profiles of three Agilent 1 M arrays of a single FFPE GBM specimen (GBM1) processed with the FSM ULS protocol (A), standard ULS protocol (B), or FSM ULS protocol after altered proteinase K digestion during DNA extraction (C) (plotted log2 ratio data for all chromosomes provided in Figure S2). (D–H) Chromosome 1 plotted log2 ratios are representative profiles of five Agilent 1 M arrays of a single FFPE GBM specimen (GBM2) processed using the FSM ULS protocol, with reduced DNA input in (E) and (F) (see Figure S3 and Figure S4 for detailed copy number analysis). Increased hybridization time (G) improved quality to a modest degree. Use of FFPE brain tissue as reference DNA (H) did not significantly improve results (dLRsd of 0.21 vs. 0.20 for standard reference).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376148&req=5

pone-0038881-g006: Size matching using FSM is a more critical determinant of array quality than other known variables.(A–H) Probe log2 ratio (signal intensity test DNA/signal intensity reference DNA) data plotted for a single chromosome (chr.13 or chr.1) from eight Agilent 1 M arrays (green;log2ratio<-0.3, black;-0.3≤log2ratio≤0.3, red;log2ratio>0.3). (A–C) Chromosome 13 plotted log2 ratios are representative profiles of three Agilent 1 M arrays of a single FFPE GBM specimen (GBM1) processed with the FSM ULS protocol (A), standard ULS protocol (B), or FSM ULS protocol after altered proteinase K digestion during DNA extraction (C) (plotted log2 ratio data for all chromosomes provided in Figure S2). (D–H) Chromosome 1 plotted log2 ratios are representative profiles of five Agilent 1 M arrays of a single FFPE GBM specimen (GBM2) processed using the FSM ULS protocol, with reduced DNA input in (E) and (F) (see Figure S3 and Figure S4 for detailed copy number analysis). Increased hybridization time (G) improved quality to a modest degree. Use of FFPE brain tissue as reference DNA (H) did not significantly improve results (dLRsd of 0.21 vs. 0.20 for standard reference).
Mentions: Having demonstrated the highly significant contributions of FSM analysis and matched DNA fragment sizes to aCGH quality, we sought to further assess the relative effects of fragment size compared to other previously reported variables such as Proteinase K digestion time, array hybridization time, and concentration and source of DNA in array hybridization reactions. DNA from a single FFPE tumor specimen, GBM1 (characterized by complex and highly aberrant copy number changes involving single-copy gains, single-copy losses, and regions of homozygous deletion on chromosome 13), was processed under multiple conditions and assayed with Agilent 1 M feature arrays. Comparison of Figure 6A and 6B supports our previous assertions regarding the significant improvement of data quality enabled by the FSM. Compared with data obtained following the FSM ULS protocol (Figure 6A) the standard ULS protocol yielded a higher dLRsd value (0.44) (Figure 6B) that precluded accurate detection of copy number aberrations (Figure S1).

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