Genome-wide, high-resolution detection of copy number, loss of heterozygosity, and genotypes from formalin-fixed, paraffin-embedded tumor tissue using microarrays.

Author(s): Jacobs S, Thompson ER, Nannya Y, Yamamoto G, Pillai R, Ogawa S, Bailey DK, Campbell IG

Publication: Cancer Res, 2007, Vol. 67, Page 2544-51

PubMed ID: 17363572 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of DNA extraction method on DNA yield from FFPE ovarian tumors.

   Summary of Findings:

   The highest yield and the largest RAPD-PCR products (800 bp) were obtained using phenol-chloroform extraction or a modified QIAgen protocol. In contrast, use of the Promega, Invitrogen, and Gentra Systems kits produced less consistent results and failed to produce an 800 bp RAPD-PCR product. The DNA yield from the amplification step of the Mapping 50K Xba Assay was 19.2 ug from phenol-chloroform extracted tissue and 21.4 ug with the Qiagen kit.

   Biospecimens

   • Tissue - Ovary

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	RAPD PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	MagneSil Genomic Fixed Tissue System (Promega) ChargeSwitch gDNA Micro Tissue kit (Invitrogen) PureGene (Gentra Systems) Modified protocol with DNeasy Tissue kit (Qiagen) Phenol-chloroform extraction

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2. Study Purpose

   The purpose of this study was to determine the effects of preservation method and array type on genotype and LOH detection by microarray. Ovarian, breast and colorectal specimens were stored frozen for 3-12 years prior to analysis or formalin fixation.

   Summary of Findings:

   The amplicon size from FFPE tissue was <700 bp which was smaller than that from fresh tissue. Using FFPE specimens, the highest call rate (86.30) was obtained after hybridization to the 10K v2 Xba array and the lowest (31.86) using the 50K Xba array. Hybridization of FFPE specimens to the 250K Sty and 250K Nsp arrays resulted in call rates of 75.17 and 79.84, respectively. In contrast, from matched fresh tumor specimens, the call rates ranged from 90.07 (50K Xba) to 93.99 (250K Nsp). The reduced call rates in FFPE specimens were consistent with the decreased amplification success from larger fragments. The concordance of calls was 93.6% between FFPE and frozen specimens, but concordance increased to 97.4% when SNPs located on fragments >700 bp were excluded. Consistent LOH data between FFPE and frozen specimens was obtained when SNPs located on fragments >700 bp were excluded. When the 10K, 100K and 500k mapping arrays were compared, call rates were inferior using the 100k array. The authors report that while call rates declined with storage of FFPE tissue, the PCR based analysis was more predictive of successful genotype and LOH determination than storage duration.

   Biospecimens

   • Tissue - Ovary
   • Tissue - Colorectal
   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen
   • None (Fresh)

   Diagnoses:

   • Neoplastic
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh) Snap frozen
   Storage	Storage duration	1-17 years
   DNA microarray Specific	Type of array	10K v2 Xba 50K Xba 250K Nsp 250K Sty 500 K

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