title: Doing more with less: fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections.
abstract: Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF, EGFR, KRAS, C-KIT, and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3'-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen). For FISH analysis, the concordance rate between the original diagnosis and that obtained on H&E- or P-IHC-stained tissue sections (AP-IHC was not on record for this set of patients) was 95% (18 out of 19 tumor sections). Only one tumor sample, diffusely positive for MLH1, did not yield any nuclear hybridization signal. For sequencing analysis, the concordance rate was 100% on negative P-IHC and positive AP-IHC-stained sections, regardless of the subcellular localization of the reaction product. Mutations were detected in only 52% of cases expressing nuclear/nuclear-cytoplasmic markers, regardless of the sequencing technology used (p = 0.0002). In conclusion, stained sections may be a valuable resource for FISH or sequencing analysis, but on cases expressing nuclear markers sequencing results need to be interpreted cautiously.
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy. giuseppe.pelosi@unimi.it.|Department of Biomedical and Clinical Sciences "Luigi Sacco", Università degli Studi, Milan, Italy. giuseppe.pelosi@unimi.it.
authors.forename: Giuseppe
authors.identifier: http://orcid.org/0000-0003-4725-4692
authors.initials: G
authors.lastname: Pelosi
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Perrone
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Tamborini
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Fabbri
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Maria Adele
authors.initials: MA
authors.lastname: Testi
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Busico
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Giulio
authors.initials: G
authors.lastname: Settanni
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Benedetta
authors.initials: B
authors.lastname: Picciani
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Bovio
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Angelica
authors.initials: A
authors.lastname: Sonzogni
authors.affiliation: Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Giacomo Venezian, 1, 20133, Milano, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Valeri
authors.affiliation: Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Garassino
authors.affiliation: Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: De Braud
authors.affiliation: Division of Thoracic Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Ugo
authors.initials: U
authors.lastname: Pastorino
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D005240:Feasibility Studies; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000235:genetics*; D013194:Staining and Labeling; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor
keywords: Direct sequencing; Fish; Immunohistochemistry; Next generation sequencing
references.citation: J Thorac Oncol. 2015 Nov;10(11):1560-9
references.pmid: 26317919
references.citation: DNA Cell Biol. 2010 Oct;29(10):639-46
references.pmid: 20590475
references.citation: Histochemistry. 1991;96(3):215-28
references.pmid: 1717413
references.citation: Anticancer Res. 2012 Apr;32(4):1463-73
references.pmid: 22493387
references.citation: J Phys Chem B. 2013 Nov 21;117(46):14345-50
references.pmid: 24164302
references.citation: Cytopathology. 2015 Apr;26(2):99-105
references.pmid: 24831172
references.citation: PLoS One. 2013 Nov 13;8(11):e80478
references.pmid: 24236184
references.citation: PLoS One. 2015 Mar 19;10(3):e0120232
references.pmid: 25789737
references.citation: Acta Cytol. 2000 Nov-Dec;44(6):981-6
references.pmid: 11127756
references.citation: Lung Cancer. 2012 Sep;77(3):507-14
references.pmid: 22705117
references.citation: Am J Surg Pathol. 2010 Aug;34(8):1180-5
references.pmid: 20661016
references.citation: J Histochem Cytochem. 1979 Jan;27(1):44-8
references.pmid: 438504
references.citation: Transl Lung Cancer Res. 2014 Oct;3(5):291-300
references.pmid: 25806314
references.citation: J Clin Endocrinol Metab. 2015 Jul;100(7):2743-50
references.pmid: 25965083
references.citation: Biotech Histochem. 2009 Aug;84(4):159-77
references.pmid: 19579146
references.citation: Int J Surg Pathol. 2013 Jun;21(3):229-39
references.pmid: 23486764
references.citation: Acta Cytol. 1974 Jul-Aug;18(4):300-11
references.pmid: 4135333
references.citation: Arch Pathol Lab Med. 2013 May;137(5):668-84
references.pmid: 22970842
references.citation: Cancer Cytopathol. 2012 Feb 25;120(1):52-61
references.pmid: 21793228
references.citation: J Thorac Oncol. 2012 Feb;7(2):281-90
references.pmid: 22071786
references.citation: Ann Oncol. 2014 Sep;25(9):1681-90
references.pmid: 24718890
references.citation: N Engl J Med. 2006 Feb 2;354(5):526-8; author reply 526-8
references.pmid: 16452569
references.citation: Clin Cancer Res. 2007 Apr 15;13(8):2369-77
references.pmid: 17438095
references.citation: Int J Gynecol Cancer. 2005 Jan-Feb;15(1):70-80
references.pmid: 15670300
references.citation: Histochemistry. 1986;85(5):353-64
references.pmid: 2430916
references.citation: Am Soc Clin Oncol Educ Book. 2015;:e141-7
references.pmid: 25993165
references.citation: Arch Pathol Lab Med. 2013 May;137(5):685-705
references.pmid: 22913371
references.citation: Cancer Cytopathol. 2015 Jan;123(1):30-9
references.pmid: 25186473
references.citation: Lancet Oncol. 2011 Feb;12(2):175-80
references.pmid: 21277552
references.citation: Clin Cancer Res. 2010 Oct 15;16(20):4909-11
references.pmid: 20926402