Application of Molecular Techniques in Diagnostic Pathology
Moderators: Dr. Ricardo Lloyd and Dr. George Kontogeorgos
Section 1 -
Development of In Situ Techniques and Recent Applications
Instutes of Pathology TUM
In situ hybridization (ISH) is a technique that allows direct analysis of both genes and gene
expression, through the localization of specific nucleic acid sequences to individual cells within a
morphologically preserved tissue. The hybridization of nucleic acid probes to their corresponding
targets can be used to detect structural changes within the genome (DNA-ISH), or changes in the
expression levels of specific transcripts (RNA-ISH) and complement immunohistochemistry studies of
protein expression. With the increasing availability of cDNA and cRNA probes to known human gene
sequences, hybridization studies can be performed to learn more about regulatory processes and gene
expression to diagnose specific diseases and to define the etiology of disease processes. Because this
detection can be performed on tissue sections, ISH provides additional morphologic information on the
spatial distribution and heterogeneity of gene expression in complex tissues. This offers ISH an
advantage over other molecular techniques in which homogenization precludes such evaluation.
Additionally this technology can be applied to most routine clinical samples, allowing for the
retrospective analysis of abnormalities in genes and gene expression in large numbers of routinely
prepared patient biopsy specimens. Although the basic principles of ISH have remained
unchanged, high-sensitivity detection, simultaneous assay of multiple species, and automated
data collection and analysis have advanced the field significantly. These powerful tools are
playing an increasingly important role in both clinical medicine and in translational research.
Development of ISH Techniques
Early in situ hybridizations depended on radioactive detection, followed in 1977 by the introduction
of fluorescently labeled antibodies that recognized specific DNA–RNA hybrids . A more
straightforward approach used the chemical coupling of a fluorochrome to a DNA or RNA probe for rapid and
direct visualization, known as fluorescence in situ hybridization . The coupling of a
fluorochrome to a DNA or RNA probe is often referred to as 'direct labeling'. By contrast, 'indirect
labeling' refers to the enzymatic or immunological detection of tags that have been incorporated into a
probe. The syntheses of modified nucleotide derivatives that contain a Biotin label, which could be
incorporated by polymerases into probes, were instrumental in the development of indirect labeling
techniques. During the last few years, several strategies have been developed to improve the sensitivity
of ISH by amplification of either target nucleic acid sequences prior to ISH or signal detection after
the hybridization is completed
. Non-isotopic detection methods are now used routinely for
both, DNA-ISH and RNA-ISH. Digoxigenin-, fluorescein-, alkaline phosphatase-, and biotin-labeled probes
are most commonly used. Application of some of these techniques has extended the utility of ISH in
diagnostic pathology and in research because of the ability to detect targets with low copy numbers of
DNA and RNA
Non-isotopically-labeled probes can also be readily applied to
ultrastructural RNA-ISH hybridization studies and provide excellent resolution with this technique.
Whereas the initial development of ISH involved expansion of the types of probe and number
of detectable targets, the outlook for future development of fluorescence techniques will
rather focus the expansion of the subjects of investigation as well as standardization
procedures. Clinical application of fluorescence imaging will require further advances in
mechanization that allow the probes to be delivered, imaged, and analyzed
automatically, thereby reducing operator-to-operator variability.
The recently developed tissue microarray (TMA) technology  is an ideal platform
for the introduction of high-throughput molecular profiling by ISH for tumor specimens at the
single cell level. To construct a TMA, small core biopsies are taken from representative
areas of paraffin-embedded tumor tissues and assembled in a single block. The numerous
advantages of this technology are obvious and have thus stimulated many constructors to evolve and
improve different technical approaches. With TMAs, multiple specimens can be simultaneously investigated
with different ISH techniques under identical laboratory conditions, resulting in a dramatic time and
cost reduction compared with conventional pathologic studies. Furthermore, this technology is less
exhausting for the finite original donor material, allowing for a significantly increased number of
assays per each case. In addition to pathological specimens such as tumor tissues, microarrays
may also contain corresponding normal tissue and internal controls. As the entire group of samples
is analyzed simultaneously in one experiment, enormous amounts of correlative information
about specific biomarkers at the DNA, RNA or protein level is provided. The next
challenge will be to apply multi-parameter ISH technology to these samples to correlate
putative genes and their products of prognostic value with specific morphological features
initially, and then extend studies to samples where the morphology is not sufficiently
Another important development is the increase in the number of differentially labeled probes that can
be hybridized and imaged. The discrimination of many more targets than the number of available,
spectrally resolvable fluorochromes can be achieved using combinatorial labeling or ratio
labeling . The organic fluorophores typically utilized for ISH have relatively broad emission
spectra and show significant photobleaching, attributes that limit their use in signal quantification.
The recent development of quantum dots (QDs) modified for use in biology provides an important new tool
for signal detection in ISH . QD fluorophores have several potential advantages. Their
photostability and narrow emission spectra make them eligible candidates for ISH to study the expression
of specific mRNA transcripts, furthermore facilitating the analyzing of multiple probes simultaneously.
QDs have been successfully used for immunolabeling of cellular molecules, such as HER2  and
other cellular targets. Not constricted by problems related to biocompatibility, QDs are now used for
in vivo cancer targeting and imaging, particularly when multi-photon imaging
is applied. QDs are also well suited for studies in correlative fluorescence and electron
The use of multi-photon microscopy approaches will also expand application of ISH imaging.
In multi-photon microscopy, a laser source fires short bursts of photons that are focused by
the microscope to arrive in pairs or triplets such that they summate to excite the
fluorophore of interest. Near-infrared excitation light is used, which penetrates biological
specimens more deeply and is far less toxic to live samples than visible light. This
scheme has already allowed the application of fluorescence imaging to many living
systems, including whole animals. Native fluorescent signatures that are present in tissues
due to normal physiology or pathophysiological processes can encode important clinical
information. Knowledge of how molecules interact in space and time is fundamental for
understanding cellular processes. A host of novel techniques have been developed for the visualization
of single target molecules in living cells, many based on ISH or immunocytochemistry. To extend the
applicability of ISH to living cells, special backbone-modified probes and specific conformations
(molecular beacons) have been designed. In the case of IC, conventional immunoreagents have been
fine-tuned with respect to size and affinity or replaced with new protein scaffolds based on ankyrin
repeat proteins. Other key advances include the use of proximity ligation to confirm vicinity binding
and the use of quantum dots, which have proven potential for cellular labeling.
Automation, Quantification and Analysis
A technical advance in ISH came with the development of machine automation, which could perform on
line all of the required incubations in a very precise and controlled fashion, freeing up technician time
from what had previously been a very labor intensive procedure. Automation allows for rapid and easy
optimization of variables such as probe concentration, hybridization temperature, reaction times, and
enzymatic permeabilization of tissue - all critical variables in successful ISH. Automation coupled with
improvements in detection of hybridization and the ready availability of nucleic acid probes has made ISH
more sensitive, consistent, reliable and feasible than ever before, with high-throughput capabilities.
There are several commercially available automated in situ hybridization machines that would greatly
improve the throughput as well as the reproducibility. These advances have highly expanded the
application of ISH in the study of both, the genome (DNA-ISH), or changes in the expression levels of
specific transcripts (RNA-ISH) in routine clinical samples as well as in basic science research.
By automation, the drawbacks of manual quantification and analysis of ISH can be circumvented.
Because fluorescent signal counting is tedious and often subjective, automated digital
algorithms for calculating signals are desirable. Objective criteria may be introduced, which
are followed without bias of the investigator or reduction of efficiency due to fatigue. The number of
cells detected may be increased without significantly increasing manual workload. Up to date, most
publications regarding automated ISH analysis report assessment of amplification of genes or enumeration
of centromeric probes. Some reports describe analyses of translocations applying locus specific probes.
An automated analysis follows the steps of manual FISH analysis. Cell nuclei must be correctly
recognized. Nuclear debris, large clusters of overlapping or touching nuclei, or unspecific background
staining must be excluded from further analysis, even if this exclusion results in the loss of a few
correctly acquired cells. Signals must be correctly detected. Since intra- and internuclear variation
regarding shape, size, and intensity of FISH signals is significant, it is not possible to differentiate
signals from artifacts, background noise, split, or partially overlapping signals in every case.
Therefore, it is crucial to exclude incorrectly recognized signals and cells with aberrant signal
patterns. Since nuclei are three-dimensional (3D) objects, measuring in 3D is vital to reduce the false
positive rate resulting from the overlap of signals in the 2D projection of the nuclei. New technology
provides a stack of images on multiple focal planes throughout a tissue sample.
Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in
ISH for Studying the Genome in Three Dimensions
In three-dimensional fluorescence in situ hybridization (3D-FISH) nuclei are fixed so that the spatial
relationships between chromosome territories are maintained. Sophisticated 3D image-acquisition
technology is used to collect a series of images throughout different sections of the nucleus, which
allows a detailed three-dimensional reconstruction. 3D-FISH has greatly benefited from improvements in
imaging technologies that use confocal lasers or sophisticated deconvolution algorithms. Interphase
FISH, both on fixed nuclei and in living cells, allows the study of the functional organization of the
genome and the dynamic interplay between the genome and its regulatory factors. Studies of chromosomes
within their natural tissue context using 3D-FISH have allowed the study of higher-order chromatin
architecture . In most tissues, the radial arrangement of chromosome territories has been
shown to correlate with gene density. However, in fibroblasts the distribution of chromosome territories
correlates more closely with chromosome size, indicating that there are tissue-specific differences in
three-dimensional genome organization. Three-dimensional studies are also providing increasing evidence
that chromatin location within the nucleus is an important constraint on gene activity. Recent 3D-FISH
studies indicated that portions of different chromosomes interact with each other, which implies that
related genes are brought together in the nucleus to coordinate their expression . However,
these technologies currently lack the resolution and speed that is required for the clinical diagnosis of
structural rearrangements and the instrumentation that is required is expensive. Nevertheless, analyses
of higher-order chromatin structure by 3D-FISH will be of importance in the future. New studies should
address distinct three-dimensional organizations in different tissue types, as higher-order chromatin
arrangements are likely to have fundamental implications for development and cell differentiation.
In situ hybridization, the method of choice for localization of specific nucleic acid
sequences in the native cellular or histological context, is a 20-year-old technology that has
developed continuously. Various methodologies and modifications have been
introduced to optimize the detection of DNA and RNA. Although the basic principles
of ISH have remained unchanged, high-sensitivity detection, simultaneous assay of
multiple species, and automated data collection and analysis have advanced the field
significantly. In the future, ISH techniques are likely to have significant further impact on
live-cell imaging and on medical diagnostics.
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