title: Use of formalin-fixed paraffin-embedded samples for gene expression studies in breast cancer patients.
abstract: To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Musella
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Callari
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Di Buduo
authors.affiliation: Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Scuro
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Dugo
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Miodini
authors.affiliation: Department of Medical Oncology, Ospedale San Raffaele, Milan, Italy.
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Bianchini
authors.affiliation: Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Biagio
authors.initials: B
authors.lastname: Paolini
authors.affiliation: Department of Medical Oncology, Ospedale San Raffaele, Milan, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Gianni
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Daidone
authors.affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Vera
authors.initials: V
authors.lastname: Cappelletti
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods; D010865:Pilot Projects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
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