Deep clonal profiling of formalin fixed paraffin embedded clinical samples.

Author(s): Holley T, Lenkiewicz E, Evers L, Tembe W, Ruiz C, Gsponer JR, Rentsch CA, Bubendorf L, Stapleton M, Amorese D, Legendre C, Cunliffe HE, McCullough AE, Pockaj B, Craig D, Carpten J, Von Hoff D, Iacobuzio-Donahue C, Barrett MT

Publication: PLoS One, 2012, Vol. 7, Page e50586

PubMed ID: 23226320 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 formalin fixation and paraffin embedding and WGA on analysis of copy number and SNV by aCGH and NGS in flow cytometry-sorted cells. Details of formalin fixation were not specified but were stated to be standard. FFPE specimens were rehydrated and digested, frozen and DAPI stained prior to flow cytometry. Matched specimens were frozen in liquid nitrogen and stored at -80 degrees C. DNA was extracted from sorted FFPE nuclei using a modified QIAamp micro kit protocol, after which, DNA was amplified with Ovation WGA FFPE.

   Summary of Findings:

   The diploid and aneuploid nuclei histograms from FFPE specimens were wider than from matched frozen specimens indicating more DNA fragmentation. However, when 50,000 sorted FFPE nuclei were used rather than 10,000 or 25,000 cells, there was a robust aCGH signal, allowing for detection of loss of tumor necrosis factor alpha-induced protein 8 (TNFAI8). 19 of the top 20 ranked amplicons from FFPE specimens had >0.9 overlap with the same interval in the matched frozen specimen. All FFPE tumor specimens had clinically relevant changes in copy number. Amplification with the Ovation WGA FFPE kit led to increased signal but also led to increased non-specific amplification and decreased resolution. However, the Adrenomedullin 2 (ADM2) aCGH intervals were comparable between frozen specimens, amplified and unamplified FFPE specimens. The genome profiles of frozen, amplified and unamplified FFPE specimens by next generation sequencing were identical, as determined by the ADM2 interval. 22 mutations identified in a cell line were generally detected in all specimen types, but 2 of the mutations were not called in the fresh frozen or unamplified FFPE specimens. The authors hypothesize this was possibly due to allelic drift in the cell line or non-tumor cells in the sorted specimens.

   Biospecimens

   • Tissue - Pancreas
   • Tissue - Ovary
   • Tissue - Bladder
   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Array CGH
   DNA	Whole genome amplification
   DNA	Next generation sequencing

   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	Amplified Unamplified
   Array CGH Specific	Template/input amount	10,000 nuclei 25,000 nuclei 50,000 nuclei

   View more