title: Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples.
abstract: Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.
authors.affiliation: Translational Genomics Research Institute, 445 N. 5th Street, Phoenix, AZ 85004, USA. ghostetter@tgen.org
authors.forename: Galen
authors.initials: G
authors.lastname: Hostetter
authors.forename: Su Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Stephanie
authors.initials: S
authors.lastname: Savage
authors.forename: Gerald C
authors.initials: GC
authors.lastname: Gooden
authors.forename: Michael
authors.initials: M
authors.lastname: Barrett
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Lalitamba
authors.initials: L
authors.lastname: Alla
authors.forename: April
authors.initials: A
authors.lastname: Watanabe
authors.forename: Janine
authors.initials: J
authors.lastname: Einspahr
authors.forename: Anil
authors.initials: A
authors.lastname: Prasad
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Nickoloff
authors.forename: John
authors.initials: J
authors.lastname: Carpten
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Trent
authors.forename: David
authors.initials: D
authors.lastname: Alberts
authors.forename: Michael
authors.initials: M
authors.lastname: Bittner
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics; D055028:Comparative Genomic Hybridization / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D053938:DNA Fragmentation; D003851:Deoxyribonucleases; D005091:Exons; D005404:Fixatives; D005557:Formaldehyde; D018628:Gene Dosage; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding; D012878:Skin Neoplasms / Q000235:genetics
chemical_list: D005404:Fixatives; D005557:Formaldehyde; D004247:DNA; D003851:Deoxyribonucleases