A multiplex PCR predictor for aCGH success of FFPE samples.
Author(s): van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Hogervorst FB, Verhoef S, Nederlof PM
Publication: Br J Cancer, 2006, Vol. 94, Page 333-7
PubMed ID: 16333309 PubMed Review Paper? No
Purpose of Paper
This paper evaluated multiplex PCR as a method of assessing the suitability of DNA isolated from formalin-fixed paraffin-embedded (FFPE) specimens for analysis by array comparative genomic hybridization (aCGH). The effect of storage duration on successful amplification of a 157 bp amplicon and the effect of DpnII digestion on aCGH success was also examined.
Conclusion of Paper
The size distribution of extracted DNA and the maximum PCR amplicon size were highly variable between FFPE specimens. High quality aCGH data was obtained from almost all FFPE specimens with a maximum amplicon size of ≥200 bp by multiplex PCR, but was obtained in only a small percentage of FFPE specimens with a maximum amplicon size of 100 bp. In specimens with a maximum amplicon size of <200 bp, aCGH signal noise was too large to accurately detect gains and losses. Amplification of a 157 bp product was possible in only 82% of specimens archived more than 25 years, but was possible in >95% of specimens archived for less than 25 years. The authors report DpnII digestion had no effect on aCGH of FFPE specimens.
Studies
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Study Purpose
This study evaluated multiplex PCR as a method of assessing the suitability of DNA isolated from FFPE breast tumor specimens for analysis by aCGH. The study also sought to determine if amplification success was affected by storage duration, and if DpnII digestion was necessary for aCGH. Details of specimen procurement and FFPE processing were not provided, but the twelve specimens analyzed had been stored for 6-29 years before extraction. Ten 10 µm thick sections were deparaffinized in two changes of xylene, rehydrated in ethanol, stained with hematoxylin, and demodified by overnight incubation in sodium thiocyanate at 37˚C. Areas containing >70% tumor were scraped into a tube for proteinase K digestion at 55˚C for 44 h with additional enzyme added after 4, 20, and 28 h. DNA was then extracted using the QIAamp DNA extraction kit. DNA was assessed by electrophoresis and multiplex PCR amplification of 100, 200, 300, and 400 bp fragments of GAPDH. aCGH was conducted using custom arrays and DNA was obtained from the peripheral blood lymphocytes of six healthy women as a reference. The correlation between maximum amplicon size and aCGH success was investigated using DNA isolated from an additional 26 FFPE breast tumors for which aCGH had been performed previously and 93 FFPE breast tumors with prior multiplex PCR data. The effect of storage duration was investigated using an additional 1,345 archival FFPE specimens of unspecified type by amplification of an unspecified 157 bp amplicon.
Summary of Findings:
Most of the DNA extracted from FFPE specimens was shorter that 1 kb, but the size distribution of the DNA was highly variable between specimens. Multiplex PCR confirmed variability of DNA fragmentation, as the maximum amplicon size was 400 bp for 8% (1/12) of specimens, 300 bp for 50% (6/12) specimens, 100 bp for 25% (3/12) of specimens, and amplification failed for 6% (2/12) of specimens. Of these twelve specimens, aCGH failed in all five specimens with a maximum amplicon size of ≤ 100 bp but was successful in all seven specimens with a maximum amplicon size of >200 bp. When 26 FFPE specimens previously analyzed by aCGH were then assessed by multiplex PCR aCGH was successful in 100%l (21/21) of specimens with a maximum amplicon of ≥200 bp, 33% (1/3) of specimens with a maximum amplicon size of 100 bp, and was unsuccessful in the two specimens that failed to generate an amplicon. Similarly, in an additional 93 FFPE specimens, aCGH was successful in 100% (7/7) of specimens with a maximum amplicon size of ≥300 bp, 97% (38/39) of specimens with a maximum amplicon size of 200 bp, and 16% (6/37) of specimens with a maximum amplicon size of 100 bp. In specimens with a maximum amplicon size of <200 bp, signal noise was too large to detect copy number gains and losses. FFPE block storage duration did affect the successful generation of a 157 bp amplicon, as success rates ranged from 82% (202/246) of specimens fixed between 1970 and 1980 (25-35 years old), to 97% (666/682) of specimens fixed between 1980 and 1990, to 95% (397/418) of specimens fixed after 1990. The authors report DpnII digestion had no effect on aCGH of FFPE specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Array CGH DNA PCR DNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration <15 years
15-25 years
25-35 years
PCR Specific Targeted nucleic acid GAPDH
Array CGH Specific Technology platform Multiplex PCR
Array CGH Specific Template modification DpnII digested
Not DpnII digested
PCR Specific Length of gene fragment 300 bp
400 bp
100 bp
200 bp