The Role of Advanced Computing

Techniques in FISH Analysis

Introduction
Originally used as a gene mapping tool for the Human Genome Project, Fluorescent in situ Hybridization (FISH) has been
around since the 1980s. As a highly specific technique that can pinpoint genetic sequences as well as chromosomal
abnormalities, FISH is widely used as a diagnostic procedure to identify cytogenetic abnormalities that can cause cancer,
neurodevelopmental disorders, aneuploidy and so on.

With the advent of advanced computing techniques, FISH has the potential to transform the field of clinical diagnostics
through computational modelling and image pattern analysis.

Fluorescent in situ Hybridization (FISH)

FISH uses a fluorescent probe1 to bind to a specific, complementary sequence of DNA. A fluorescent microscope can then
be used to visualize the hybrid probe-target sequence as it stands out in high contrast from its surrounding sequences
[ CITATION OCo08 \l 1033 ].
Owing to its specificity, FISH is the perfect intermediate between nucleotide sequence analysis and chromosomal studies.
As a result, it is used for detecting aberrations on a genetic and chromosomal level, such as fusion genes that are formed due
to chromosomal rearrangement between non-homologous chromosomes.

This is best exemplified by the BCR-ABL

oncogene that causes chronic myeloid
leukemia: a segment of chromosome 9
containing the ABL proto-oncogene2 fuses with
the BCR gene3 on chromosome 22. The
resulting fusion chromosome is known as the
Philadelphia chromosome and can be
easily viewed when 2 differently colored
fluorescent probes bind to the BCR and
ABL gene sequences alongside each other[
CITATION OCo08 \l 1033 ].
FISH removes the need for cell culturing, Interphase FISH detecting normal nuclei (A) and BCR/ABL fusion containing a
mainstay of the conventional metaphase nuclei (B) shown by the yellow fluorescence (mixing red and green colors
karyotypic analysis. Consequently, FISH creates yellow). Taken from Dewald et al. (1998).
analysis can be performed during the
interphase stage of the cell cycle which makes it suitable to be used to scan for cancers with low cell proliferation rates,
such as haemopoietic malignancies[ CITATION Lyn01 \l 1033 ]. A systematic review by Ratan et al. (2017) states that
FISH was found to be more accurate and reliable for the diagnosis of oncological and clinical disorders than other molecular
profiling methods.

Artificial Intelligence (AI)

AI is an interdisciplinary field that combines economics, mathematics, philosophy, computer science and statistics to
identify associations within data in a way that resembles human learning.

1
A single-stranded DNA molecule made up of fluorophore-bound nucleotides.
2
A gene that regulates normal cell growth and as such, can cause cancer (uncontrolled cell growth) when mutated.
3
A promotor i.e. a specific DNA sequence that initiates gene expression.
An AI accomplishes its intended goal using a set of instructions called algorithms. AI algorithms can process and
subsequently learn from data in a number of ways. If the system is trained on a dataset that has a known correct output, this
is called supervised learning; if it is trained on data alone without human intervention, it is known as unsupervised learning
[ CITATION Nic19 \l 1033 ].
Another method of ‘teaching’ the AI system is through reinforcement learning where the system figures out how to make a
sequence of decisions that ultimately result in accomplishing a goal in a completely unknown environment. Reinforcement
occurs through a system of rewards and penalties that encourage the AI algorithm to learn through a succession of trial and
error.

A set of AI algorithms working together are known as models. Machine Learning (ML) models involve a wide array of
algorithms that apply statistical techniques to a large dataset with unique data points. A ML system is designed to
automatically improve its analytical ability with every analysis. Another model is known as deep learning and it mimics the
way a human neuron processes information to identify and then amplify recognizable patterns in a large dataset. This is
called a neural network and it involves processing data through a network of interconnected nodes that provide a final
result[ CITATION Pan18 \l 1033 ].

A combination of these models and techniques has allowed AI algorithms to accurately predict outcomes when given
enough quantifiable data – sometimes with nearly human accuracy. This is invaluable in multiple fields, especially clinical
diagnostics and medical analytics.

Big Data
Big Data is a term used to describe immense datasets with multiple sources that often keeps adding data in real time.
Conventional databases and data processing softwares are unable to handle the ever-expanding volume of Big Data
[ CITATION IBM \l 1033 ]. Consequently, novel and advanced analytical methods are needed to identify patterns,
comprehend trends and predict outcomes. These include machine learning, deep learning, predictive analytics, natural
language processing and data mining models that involve linear regression, naïve Bayes, kNN[ CITATION WuX08 \l
1033 ][ CITATION XWu14 \l 1033 ].

Role of AI and Big Data in FISH Analytics

Optimization of FISH Assays
Although FISH is a powerful technique for aberration detection, FISH assays can take a lot of time. This is in part due to the
slow diffusion rate of the probes into the cell(s) being analyzed as well as the time taken by the probe to hybridize to its
DNA target sequence. Measuring the actual hybridization rate of the probes in situ can lead to more efficient probe designs
that will, in turn, shorten the total time taken by the FISH assay. Computational models that calculate FISH probe
hybridization kinetics have been proposed but they are unable to predict the optimum hybridization conditions for a specific
probe sequence and length.

However, Ostromohov et al. (2018) recently presented a procedure that can measure the real time hybridization rate for any
probe and target sequence by constantly delivering probes to analyzed cells and siphoning away unhybridized probes
immediately. With this newfound ability to quantify hybridization conditions such as probe concentration, sequence, length
and types; volume exclusion and destabilizing agents content; ionic strength and rate-enhancing agents, more accurate
computational models can be developed that will ultimately lead to more efficient probe designs and optimized FISH
assays[CITATION Nad18 \l 1033 ].

Another pertinent development is the emergence of thermodynamic models that increasingly predict highly accurate
temperatures for the denaturation of the DNA double helix (known as melting), a critical value for molecular techniques like
FISH, Polymerase Chain Reaction (PCR), microarrays etc. For FISH however, multiple chemical reagents are used, and
different parameters need to be considered than needed for PCR or other molecular biology techniques. So it becomes
imperative to modify existing DNA/RNA based thermodynamic models. Melting temperature is also not always the optimal
hybridization temperature, which is why there is a great of deal of customization that is still required to optimize current
thermodynamic models for FISH assays [ CITATION Fon15 \l 1033 ].
Automated FISH Analysis
Biological pattern recognition combines machine learning with optical biological assays or biomedical imaging to provide
reliable outcomes. The eventual goal is to reach consistently accurate high-throughput imaging analysis on par with or even
exceeding human accuracy. This can be accomplished through both supervised machine learning where the system is
trained on examples or unsupervised learning where the system learns to cluster relevant data using pre-set rules
[ CITATION Sha10 \l 1033 ][ CITATION Che18 \l 1033 ].
Automated cytogenetic analysis techniques have been in use since 1980’s, mainly for karyotyping. A system enhanced
images, segmented and aligned chromosomes and compared feature vectors in the images with stored feature vectors of
standard chromosomes. This allowed the automatic diagnosis of abnormal chromosomal patterns and distributions
[ CITATION Bri07 \l 1033 ].
Modern image processing and pattern recognition techniques have a come a long way from those rudimentary beginnings
and have evolved into a variety of applications. Lerner et al. (2001) have used a neural network classifier to optimize and
sharpen FISH images by improving discrimination between the actual fluorescence of the hybridized probes and the
reflected or background fluorescence scattered by the 3-dimensional cell debris [ CITATION Ler01 \l 1033 ]. Nandi et al.
(2009) have automated nuclei segmentation, the first (and usually the longest) step of analyzing tissue images. They used a
hybrid, data-driven segmentation algorithm in concert with intelligent, supervised pattern classification system which learns
by collating features of manually segmented nuclei as well as the outcomes of multiple classifiers [ CITATION Nan09 \l
1033 ]. Perhaps the most promising results is the successful application of deep learning (through a convolutional neural
network) for high-throughput image processing of DNA FISH signals by Gudla et al (2017). The workflow, dubbed
‘SpotLearn’ has proven accuracy even for images with low signal and high noise ratio [CITATION Gud17 \l 1033 ].

These developments in advanced computing techniques applied to biological pattern recognition are important for not only
clinical diagnosis, but also risk prediction. Wang et al. (2010) reported a 92-98% agreement between a trained
cytogeneticist and automated FISH analysis system for early stage cervical cancer diagnosis [ CITATION Wan10 \l 1033 ].
A more recent study that used deep learning to identify HER-2 gene amplification in breast cancer reported accuracy
validated by 3 independent pathologist and a set of 57 reference images [ CITATION Zak19 \l 1033 ]. While further work
on pattern recognition and image processing still needs to be done, the future of automated FISH analytics has proven to be
promising.

References

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[2] L. Kearney, "The impact of the new FISH technologies on the cytogenetics of haematological malignancies," British
Journal of Haematology, pp. 648-658, 2001.

[3] J. H. C. H. &. B. M. Nichols, "Machine learning: applications of artificial intelligence to imaging and diagnosis,"
Biophysical Reviews, p. 111–118 , 2019.

[4] T. S. P. &. A. R. Panch, "Artificial intelligence, machine learning and health systems.," Journal of global health,
2018.

[5] IBM, "Big data analytics," [Online]. Available: https://www.ibm.com/analytics/hadoop/big-data-analytics.

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[7] X. Z. G. W. a. W. D. X. Wu, "Data mining with big data," IEEE Transactions on Knowledge and Data Engineering,
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Hybridization (FISH) Kinetics," Analytical Chemistry, pp. 11470-11477, 2018.

[9] G. N. W. J. A. N. Fontenete S, "Prediction of melting temperatures in fluorescence in situ hybridization (FISH)

procedures using thermodynamic models," Criticial Reviews Biotechnology, pp. 566-577, 2015.

[10] L. Shamir, J. D. Delaney, N. Orlov, D. Eckley, Mark and I. G. Goldberg, "Pattern Recognition Software and
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[11] C. J. H. T. M. e. a. Chen, "Unsupervised Learning and Pattern Recognition of Biological Data Structures with Density
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[12] A. P. a. R. G. Britto, "A Review of Cytogenetics and its Automation," Journal of Medical Sciences, pp. 1-18, 2007.

[13] B. &. C. W. &. D. S. &. H. M. &. B. C. Lerner, "Automatic signal classification in fluorescence in situ hybridization
images.," Cytometry, 2001.

[14] G. P. M. K. M. T. L. S. Nandy K, "Automatic nuclei segmentation and spatial FISH analysis for cancer detection.,"
Conf Proc IEEE Eng Med Biol Soc., 2009.

[15] P. R. N. K. P. G. &. M. T. Gudla, "SpotLearn: Convolutional Neural Network for Detection of Fluorescence In Situ
Hybridization (FISH) Signals in High-Throughput Imaging Approaches," Cold Spring Harbor Symposia on
Quantitaive Biology, 2017.

[16] Z. B. Z. R. e. a. Wang X, "Automated analysis of fluorescent in situ hybridization (FISH) labeled genetic biomarkers
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[17] F. d. B. W. W. M. e. a. Zakrzewski, "Automated detection of the HER2 gene amplification status in Fluorescence in
situ hybridization images for the diagnostics of cancer tissues.," Sci Rep , 2019.