The utility and limitation of single nucleotide polymorphism analysis on whole genome amplified mesenchymal tumour DNA in formalin fixed tumour samples.

Author(s): Liang CW, Lee YS, Marino-Enriquez A, Tsui K, Huang SH

Publication: Pathology, 2012, Vol. 44, Page 33-41

PubMed ID: 22157688 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 WGA and using FFPE rather than frozen gastrointestinal stromal tumor specimens for SNP and LOH analysis using Affymetrix NSP250k arrays. DNA was extracted with QIAamp with an increased proteinase K digestion time.

   Summary of Findings:

   After WGA of DNA from frozen specimens, 6% of calls did not match unamplified specimens. Using amplified DNA from FFPE specimens, 71% of probes were detected, but 18% of these did not match the call from the unamplified frozen specimen. Determining loss of heterozygosity (LOH) of 10-30 Mb regions was generally good using DNA from frozen specimens (amplified or not) or unamplified DNA from FFPE specimens, but use of amplified DNA from FFPE specimens resulted in up to 33% false positives. When smaller regions were considered, the false positive and negative rates using amplified DNA from frozen specimens or unamplified DNA from FFPE specimens increased as region size decreased, and the rates were highest when amplified DNA from FFPE specimens was used, followed by unamplified DNA from FFPE specimens. Copy number was correctly assigned 68% of the time using amplified DNA from frozen specimens, 59% of the time using unamplified DNA from FFPE specimens and 48% of the time using amplified DNA from FFPE specimens. Loss of detection and miscalls increased when the GC content was higher than 55%, particularly in frozen specimens, but high GC content increased accuracy of copy number determination. FFPE specimens also had increased loss of detection of probes with GC content below 35%. The authors report the two WGA kits performed similarly for SNP determination except when the GC content was below 35%, in which case, REPLI-g was preferable to GenomePlex, but that GenomePlex led to more accurate determination of copy number.

   Biospecimens

   • Tissue - Stomach
   • Tissue - Small Bowel
   • Tissue - Colorectal
   • Tissue - Esophagus

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic

   Platform:

   Analyte	Technology Platform
   DNA	Array CGH
   DNA	DNA microarray
   DNA	Whole genome amplification
   DNA	Electrophoresis
   DNA	SNP assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Whole genome amplification Specific	Nucleic acid amplification	REPL-g (Qiagen) Genomeplex (Sigma)
   Whole genome amplification Specific	Template/input amount	10 ng 30 ng 60 ng 100 ng 150 ng

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