96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

more effective than SAHA alone and possible for an individualized ESS
therapy.
SO-017
High-resolution 3D visualization and semi-automated characterization
of tumor angiogenesis
J . Ehling1 , B . Theek2 , F . Gremse2 , S . Baetke2 , R . Knüchel3 , F . Kiessling2 ,
T . Lammers2 1RWTH Aachen, Institute of Pathology & Institute of Biomedical Engineering,
Aachen, 2RWTH Aachen, Institute of Biomedical Engineering, Aachen,
3RWTH Aachen, Institute of Pathology, Aachen
Aims. The visualization and quantification of functional tumor blood
vessels is essential for assessing treatment responses to anti-angiogenic
therapies. In experiments using tumor xenografts, anti-angiogenic
effects are generally evaluated by immunohistochemistry (IHC). This
method has several limitations: for example, the 3D architecture and the
vessel functionality are not fully considered. To overcome these shortcomings,
we established a protocol based on ex vivo high-resolution microcomputed
tomography (µCT) for 3D visualization and characterization
of tumor angiogenesis.
Methods. Six different tumor models (A431, A549, Calu-6, DU145, MDA-
MB-231, MLS), differing in blood vessel density and maturation, were
analysed. After tumors reached a diameter of ~6 mm, mice were intracardially
perfused with the radiopaque contrast agent Microfil, which
polymerises intravascularly. Subsequently, the tumor was harvested and
scanned in a high-resolution µCT scanner (SkyScan, Belgium) with a
maximal spatial resolution of 3 µM. Tumor microvessels were visualized,
and vessel density, vessel size, number and order of branches, were analyzed
using a 3D rendering software (MeVisLab). Histological validation
was performed by CD31 and SMA staining.
Results. Vascular casting and high-resolution µCT analysis of the vasculature
in tumor models with large and highly mature vessels (CD31-positive
and SMA-positive), such as A549 or MLS, enabled the detection
of vessel branches up to the 7th order. Conversely, tumors primarily
containing small and immature vessels (CD31-positive and SMA-negative),
such as A431 or Calu-6, presented many randomly arranged small
vessels, without proper hierarchy and architecture. The rising order of
branches, the total number of branches and the distribution of branches
correlated very well with the SMA levels. In addition, a highly significant
correlation was observed between the vessel diameters measured by
high-resolution µCT and by histology.
Conclusions. An imaging protocol based on vascular casting and highresolution
µCT has been developed for the 3D visualization of the micromorphology
of tumor blood vessels. In addition, evidence is provided
showing that high-resolution 3D µCT imaging of vascular casts allows
for a semi-automated analysis of the micro-architecture of tumor vessels,
thereby making it an exquisite and highly useful tool for supplementing
IHC in translational focusing on tumor angiogenesis and antiangiogenic
treatment responses.
SO-018
Eyetracking experiments identify “fast and frugal” heuristics
during cancer grading
D . Bombari1 , B . Mora2 , S . Schaefer3 , F . Mast1 , H .-A . Lehr4 1 2 University of Berne, Cognitive Psychology, Bern, Switzerland, CHUV, Lausanne,
Institute of Pathology, Lausanne, Switzerland, 3Inselspital, Institute
of Pathology, Bern, Switzerland, 4CHUV, Lausanne, Institute of Pathology,
Lausanne, Switzerland
Aims. In prior studies on prostate carcinomas we found that during nuclear
grading, pathologists are heavily biased by the architectural growth
patter of the carcinomas (Fandel et al., J Pathol. 2008).
Methods. We asked 20 pathologists to assign nuclear grades to images
of high power fields of 40 prostate carcinomas projected on a computer
screen with an inbuilt eyetracking device. We wondered if pathologists
fixate on different nuclei when the same circular high power fields
(albeit turned and flipped to avoid recognition) were displayed before
background images of well-differentiated, tubule-rich or poorly differentiated,
solid carcinomas. Using Photoshop-based image analysis, we
analyzed nuclear size, chromasia, and roundness of those nuclei that the
pathologists fixated, and compared these morphometric data to a random
sample of nuclei contained within each high power field.
Results. (i) The selection of fixated nuclei largely followed the confirmation
bias induced by the tumor architecture. (ii) The selection of “matching”
nuclei explained only about one tenth of the manipulation of
nuclear grades induced by the tumor architecture, suggesting that it represents
nothing but an unconscious effort of our minds to vindicate the
gravitation of nuclear grades towards the tumor architecture. (iii) The
majority of pathologists based their subjective nuclear grade on a single
morphometric criterion (mostly nuclear size) and only few pathologists
considered more than one nuclear criterion during nuclear grading. This
observation has in the meantime been confirmed in a study on 44 pathologist
asked to grade nuclei in breast carcinomas.
Conclusions. (i) Counter the general expectation that pathologists rely in
their diagnosis solely (or mostly) on what they see through the microscope,
our experiments suggest that unconscious, poorly recognized biases
influence not only the interpretation of the histological image but also
the way we view the image (unwitting fixation on selected parts/aspects
of the image). (ii) While we eloquently teach medical students that nuclei
of aggressive tumors are enlarged, angulated, and hyper- as well as heterochromatic,
we personally ignore most of these different morphometric
criteria during our daily diagnostic routine and rather base nuclear
grades on a single criterion (mostly size). (iii) It may appear judicious to
recognize cognitive biases and heuristics as part of a consensuous error
culture in diagnostic anatomical pathology and in translational research
and to develop mechanisms to counteract/prevent ensuing blurs and/or
outright diagnostic errors.
This project was funded by a grant from the Fonds National Suisse (Proj .-N°
32000-120417) .
AG Gynäkopathologie und Mammapathologie I
SO-019
p16/Ki-67 dual-stained cytology using ThinPrep® liquid-based
cytology – sub-analyses of more than 9,000 PALMS participants
H . Griesser1 , F . Alameda2 , C . Bergeron3 , M . Labadie 4 , V . Maccalini5 , M . Sideri6 ,
R . Dachez7 , R . Ridder8 1 2 Center for Pathology and Cytodiagnostics, Koeln, Hospital del Mar, Barcelona,
Spain, 3Laboratoire Cerba, Cergy Pontoise, France, 4Laboratoire GRC,
Limonest, France, 5Ospedale Atri, Unita Gestionale Screening Regionale,
Atri, Italy, 6European Institute of Oncology, Milano, Italy, 7Institute Alfred
Fournier, Paris, France, 8mtm laboratories, Heidelberg
Aims. The PALMS trial (Primary ASC-US LSIL Marker Study) evaluated
the diagnostic performance of the novel p16/Ki-67 dual-stained cytology
testing in cervical cancer screening as well as in the triage of ASC-US or
LSIL Pap cytology results. The outcomes were compared to Pap cytology
and HPV testing in the screening setting, and to HPV testing in the triage
of ASC-US or LSIL. For both Pap cytology and dual-stained cytology
testing, liquid-based cytology and conventional cytology methods were
included in the PALMS trial.
Methods. We performed an analysis of the diagnostic performance of
dual-stained cytology, Pap cytology and HPV testing limited to the subcohort
of 9,231 women enrolled to the PALMS trial who had ThinPrep®
(Hologic) liquid-based cytology testing for both Pap and dual-stain.
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