Doing more with less: fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)1-11
Number of pages11
JournalVirchows Archiv
DOIs
Publication statusAccepted/In press - Jan 27 2016

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Fluorescence In Situ Hybridization
Genes
Neoplasms
Mutation
Hematoxylin
Eosine Yellowish-(YS)
Peroxidase
Alkaline Phosphatase
Immunohistochemistry
Technology

Keywords

  • Direct sequencing
  • Fish
  • Immunohistochemistry
  • Next generation sequencing

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Cell Biology
  • Molecular Biology

Cite this

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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.",
keywords = "Direct sequencing, Fish, Immunohistochemistry, Next generation sequencing",
author = "Giuseppe Pelosi and Federica Perrone and Elena Tamborini and Alessandra Fabbri and Testi, {Maria Adele} and Adele Busico and Giulio Settanni and Benedetta Picciani and Enrica Bovio and Angelica Sonzogni and Barbara Valeri and Marina Garassino and {de Braud}, Filippo and Ugo Pastorino",
year = "2016",
month = "1",
day = "27",
doi = "10.1007/s00428-016-1906-0",
language = "English",
pages = "1--11",
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T1 - Doing more with less

T2 - fluorescence in situ hybridization and gene sequencing assays can be reliably performed on archival stained tumor tissue sections

AU - Pelosi, Giuseppe

AU - Perrone, Federica

AU - Tamborini, Elena

AU - Fabbri, Alessandra

AU - Testi, Maria Adele

AU - Busico, Adele

AU - Settanni, Giulio

AU - Picciani, Benedetta

AU - Bovio, Enrica

AU - Sonzogni, Angelica

AU - Valeri, Barbara

AU - Garassino, Marina

AU - de Braud, Filippo

AU - Pastorino, Ugo

PY - 2016/1/27

Y1 - 2016/1/27

N2 - 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.

AB - 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.

KW - Direct sequencing

KW - Fish

KW - Immunohistochemistry

KW - Next generation sequencing

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U2 - 10.1007/s00428-016-1906-0

DO - 10.1007/s00428-016-1906-0

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JO - Virchows Archiv - A Pathological Anatomy and Histopathology

JF - Virchows Archiv - A Pathological Anatomy and Histopathology

SN - 0945-6317

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