paraffin-embedded tissue sections
A.J.H.M. Fleskens
R.P. Takes
I. Otte-Höller
L. van Doesburg
A. Smeets
E.J.M. Speel
P.J. Slootweg
J.A.W.M. van der Laak
Simultaneous assessment of DNA ploidy and biomarker expression in paraffin-embedded tissue sections
abstraCt
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.
methods and 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 prolifer- ation 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 γ-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.
IntroduCtIon
Chromosomal instability or microsatellite instability may be important causes of genomic alterations leading to carcinoma. DNA aneuploidy, an aberrant chromosome number, has been suggested as a useful marker for neoplastic progression of premalignant lesions at different localizations, including oesopha- gus, skin, head and neck, and colon.1–9 DNA ploidy status may prove especially
informative when combined with phenotypic information describing expression levels of proteins related to, for example, cell cycle progression or DNA damage response. For instance, the combined analysis of DNA ploidy and expression of different biomarkers is capable of estimating the malignant potential of Barrett’s oesophagus10 and colonic cancer.11
Ploidy and protein expression may be assessed simultaneously at the individual cell level using flow cytometry. Alternatively, image cytometry enables DNA ploidy assessment by microscopy, allowing for subsequent visual inspection of nuclei of interest.12 Cytometric ploidy analysis of (pre)malignancies is mostly
performed on intact nuclei, extracted from thick (50 μm) tissue sections (Hedley procedure13). Both techniques mentioned above suffer from the fact that
the original tissue context is lost, rendering correlation with histopathology impossible.14 This may, for example, result in overlooking small aneuploid cell
populations, because of dilution of the data by the abundant presence of normal euploid cells.15 Also, when dealing with small tissue fragments from
biopsy specimens, insufficient material may be available for isolation of nuclei.16
In contrast, image cytometric measurement enables analysis of protein expres- sion in situ in immunohistochemically stained tissue sections. Although possible, ploidy analysis in such relatively thin sections is cumbersome. On one hand, measurement in thin tissue sections (i.e. 2–5 μm) is hampered by reduced precision because of truncation of nuclei (Figure 1).17 As a result, sensitivity
for detecting aneuploid cells is compromised.18,19 On the other hand, use of
thick sections (i.e. >10 μm) may lead to biased sampling with preference for smaller nuclei and nuclei in less densely populated areas of the tissue because of nuclear overlap.20,21 Mathematical correction models, described to overcome
the effect of nuclear truncation,18,20 have only limited applicability in practical
situations.22 It has been shown that ploidy analysis of 7-μm sections without
integrated optical density (IOD) correction is more sensitive in detecting aneuploid subpopulations compared with analysis of 5-μm sections with IOD correction.23 Other studies have shown that IOD correction may even be
harmful to the data.22,24 Sections of thickness ≥7 μm do not require IOD
92 93
The present paper aims to describe an analysis model that offers improved sensitivity for detecting aneuploid cells in tissue sections, based on image cytometric DNA ploidy measurement. This improvement is realized by com- bining a stoichiometric DNA dye with MIB-1 (a monoclonal antibody directed against the nuclear Ki67 antigen) proliferation marker,25 making the distinction
possible between euploid cells that have entered the cell cycle (i.e. MIB-1+) and resting (G0) aneuploid cells (i.e. MIB-1)). This analysis may be combined with other relevant markers at the individual cell level. Because of such multiplexing, highly specific prognostic information can be derived from selectively sampled parts of a (pre)malignancy, probably facilitating tailored treatment in the near future. Also, the study of basic development and progression of malignancies is facilitated using this new approach. The method also allows assessment of the spatial distribution of aneuploid cells.
Figure 1. schematic representation of the effect of nuclear truncation caused by sectioning. shown is an example nucleus (height in the section 7 μm) at three different section thicknesses (2, 7 and 12 μm). the top of the figure gives an impression of the amount of nuclear overlap. the effect of truncation increases with decreasing section thickness, whereas the amount of nuclear overlap decreases with decreasing section thickness. sections below approximately 7 μm require correction of the measured integrated optical density values. Biased sampling because of larger amounts of nuclear overlap will occur when section thickness exceeds a certain value.
materIaLs and methods
patIent seLeCtIon
Twenty-two specimens from oral biopsies were retrospectively obtained from the archives of the Department of Pathology at the Radboud University Nijmegen Medical Centre (Nijmegen, the Netherlands). All specimens were routinely fixed in 4% buffered formalin and paraffin embedded. Standard 4 μm-thick
haematoxylin and eosin-stained sections were used for classification of the lesions.
The 22 tissue specimens in this study consisted of one specimen without abnormalities, five specimens showing hyperkeratosis, one mild dysplasia, six moderate dysplasias, six severe dysplasias, one carcinoma in situ (CIS) and two squamous cell carcinomas, according to World Health Organization criteria.26 From each tissue block, one 50-μm section was cut for isolation
of nuclei and subsequent cytospin preparation. Additionally, sections were cut for DNA ploidy assessment (thickness 6–7 μm) and fluorescence in situ hybridization (FISH) analysis (4 μm), as described below.