Simultaneous assessment of DNA ploidy and biomarker expression in paraffin-embedded tissue sections.
OBJECTIVE
Simultaneous assessment of DNA ploidy and biomarker expression in paraffin-embedded tissue sections Aims: Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level.


RESULTS
Ploidy was measured in 22 paraffin-embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker gamma-H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections.


CONCLUSIONS
The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information.
Fleskens
Stijn J H M
SJ
Department of Otolaryngology-Head and Neck Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.
Takes
Robert P
RP
Otte-Höller
Irene
I
van Doesburg
Lisanne
L
Smeets
Annemieke
A
Speel
Ernst-Jan M
EJ
Slootweg
Pieter J
PJ
van der Laak
Jeroen A W M
JA
D000782:Aneuploidy; D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor / Q000032:analysis*; D002278:Carcinoma in Situ / Q000737:chemistry / Q000175:diagnosis / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000737:chemistry / Q000175:diagnosis / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D006657:Histones / Q000032:analysis; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D019394:Ki-67 Antigen / Q000032:analysis; D009062:Mouth Neoplasms / Q000737:chemistry* / Q000175:diagnosis / Q000235:genetics*; D016612:Paraffin Embedding; D011003:Ploidies; D011230:Precancerous Conditions / Q000737:chemistry* / Q000175:diagnosis / Q000235:genetics*; D012189:Retrospective Studies
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C468783:H2AX protein, human; D006657:Histones; D019394:Ki-67 Antigen; C440408:MIB-1 antibody