GRIDINA et al.638
BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024
In many tumors, chromosomal rearrangements
not only accompany the process of tumor develop-
ment, but act as the main cause (driver) of cell onco-
logical transformation. One of the examples is recipro-
cal translocations observed in Burkitt’s lymphoma, in
which the translocation of the MYC gene from chromo-
some 8 to chromosome 14 under the influence of the
immunoglobulin heavy chain enhancer results in dys-
regulation of its expression [1]. If breakpoints occur
within the genes, this can lead to the gene fusions re-
sulting in the formation of chimeric proteins. Such fu-
sion proteins often involve transcription factors (ERG,
MYB) or protein kinases (ABL1, ALK, BRAF, EGFR, JAK2,
RET) that play a pivotal role in the oncogenic process.
The TMPRSS2-ERG gene fusion, which is prevalent in
a majority of prostate adenocarcinomas and approxi-
mately 20% of high-grade prostate intraepithelial neo-
plasias, illustrates this mechanism. TMPRSS2 is a serine
protease regulated by the androgen-dependent promot-
er and its fusion with the ERG oncogene results in the
ERG overexpression, a key event in the prostate cancer
pathogenesis [2]. Similar mechanisms involving ERG
fusion with other partners, such as NDRG1, EWS, and
FUS, have been implicated in other cancer types[3-5].
Gene fusion, a hallmark of various cancers, can
dysregulate gene expression and alter the function of
the encoded protein. Thus, if gene fusion results in the
truncation of one of the fusion partners, this can lead
to its overexpression due to the loss of negative reg-
ulatory elements (e.g., binding sites for microRNA) or
domains determining the protein lifespan. A notable
example is the MYB-NFIB gene fusion in adenoid cys-
tic carcinoma, resulting from the t(6;9) translocation
[6]. In this fusion, the chimeric transcript partially or
completely loses a region encoding the C-terminal reg-
ulatory domain of MYB containing the sites for protein
post-translational modification, as well as a non-cod-
ing sequence essential for the binding of microRNAs.
Consequently, the absence of these regulatory ele-
ments in the MYB portion of the fusion protein leads
to the upregulation of MYB expression and prolonged
protein lifespan [7].
Gene fusions can also lead to the production of
chimeric proteins with significantly altered functional
domains. In the norm, the FGFR3 receptor tyrosine ki-
nase is activated through the homo/heterodimerization
in the presence of fibroblast growth factor (FGF) as a
ligand [8]. The translocation between chromosomes
4 and 7 results in the FGFR3 fusion with BAIAP2L1.
Theresulting chimeric protein possesses the ability for
constitutive, ligand-independent homodimerization.
This aberrant dimerization is facilitated by the BAR
domains of BAIAP2L1, resulting in the FGFR3 kinase
activation and potent oncogenic activity [9].
For the diagnostic purposes and long-term stor-
age, tumor samples are preserved as formalin-fixed
paraffin-embedded (FFPE) tissue blocks through for-
malin fixation and subsequent embedding in paraffin.
FFPE blocks have many advantages, including stability
at room temperature, extended shelf life, and compat-
ibility with immunohistochemical analysis. However,
such fixation and storage of samples can lead to the
degradation of nucleic acids and appearance of arti-
facts, which requires optimization of molecular anal-
ysis methods [10]. Furthermore, the degradation and
modification of nucleic acids in FFPE samples compli-
cate the use of RNA sequencing for the detection of
biomarkers [11].
Routine methods for identification of chromosom-
al rearrangements in tumor tissues include FISH (flu-
orescence in situ hybridization), immunohistochemical
analysis, and RT-PCR. These approaches have obvious
limitations in the detection of novel or complex chro-
mosome rearrangements. Recent advances in high-
throughput sequencing have revolutionized clinical
genetics. Whole-genome sequencing (WGS) and whole-
exome sequencing (WES) using the short-read technol-
ogy have excelled in identifying SNVs and unbalanced
chromosomal rearrangements, but their accuracy in
repetitive genome regions is limited. Detection of bal-
anced rearrangements using WGS and WES depends
on the presence of chimeric reads encompassing the
rearrangement breakpoints and therefore requires a
high sequencing depth. Long-read sequencing methods
(PacBio and Oxford Nanopore) are effective for detect-
ing balanced chromosomal rearrangements, but their
efficiency diminishes when analyzing FFPE samples
due to the DNA degradation. Balanced chromosomal
rearrangements often trigger carcinogenesis through
two mechanisms: gene fusion and disruption of gene
expression resulting from alterations in the gene reg-
ulatory environment. Consequently, RNA sequencing
has emerged as an important tool for analyzing tumor
samples [12-14]. However, this technique demands a
high RNA quality, which is challenging when RNA is
isolated from FFPE samples [11-16]. Degraded RNA
fragments may lack crucial information on the fusion
sites. Moreover, RNA-seq technology faces sensitivity
issues in the case of low expression of fusion tran-
scripts [13] and fusions with non-coding regions [17]
and requires significant sequencing depth (20-30 mil-
lion paired-end reads) or targeted gene enrichment
[11]. The Hi-C (high-throughput chromatin conforma-
tion capture) method has been increasingly used in
recent years as an alternative approach for detect-
ing various types of chromosomal rearrangements.
Theadvantage of the method is its ability to detect bal-
anced rearrangements at a lower sequencing depth.
This efficiency is partly due to the fact that Hi-C does
not rely solely on the reads containing the breakpoint.
Instead, it analyzes changes in the chromatin contact
frequency within broad genomic regions and, therefore,