PROS AND CONS FOR FLUORESCENT IN SITU
HYBRIDIZATION, KARYOTYPING AND NEXT
GENERATION SEQUENCING FOR DIAGNOSIS AND
FOLLOW-UP OF MULTIPLE MYELOMA
Ikbal Atli E, Gurkan H, Onur Kirkizlar H, Atli E, Demir S, Yalcintepe S, Kalkan R, Demir AM
*Corresponding Author: Assistant Professor Emine Ikbal Atli, Department of Medical Genetics, Faculty
of Medicine, Trakya University, Balkan Campus, Highway D100, Edirne, Turkey 22030. Tel: +284-235-
76-41/23-30. Fax: +284-235-86-52. E-mail: eikbalatli@trakya.edu.tr
page: 59
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DISCUSSION
Genetic abnormalities hold an important place to
be able to risk stratification and the risk adapted treatment
strategies in MM [5,12]. Conventional cytogenetics,
FISH or microarray-based technologies are still golden
standard for identification of genetic abnormalities in MM
[10]. Even though conventional cytogenetic analysis is
an efficient approach, providing information about whole
chromosome status within a single experiment [13], but a
low mitotic index of plasma cells is the major problem of
MM cases which directly affects conventional cytogenetic
studies. In our study, conventional cytogenetic analysis
was successfully performed in 71.4% (25/35) of samples.
The rate of outcome with conventional cytogenetics in MM
patients varies between 29.5 and 64.0% in the literature
[14,15]. Our success rate was higher than the previously
described range even without using a specific stimulant
for plasma cells.
The identification of the cryptic translocations is an
important component of testing strategies in MM. Therefore,
FISH is a suitable technique for evaluation of these
diagnostic and prognostic cryptic translocations in MM.
Despite the advantages and disadvantages of all of these
techniques, we performed all of them in this study and we obtained different results from these techniques. Although
karyotyping is still the golden standard for hematological
malignancies, the results are not always beneficial for
clinical usage due to the limitations of the chromosomal
resolution. Fluorescent in situ hybridization is one of the
user-friendly and low-cost tests for prevention of resolution
limit of MM cases. High accuracy results are obtained
with frequent translocations (those accompanying the IgH
locus and others) and probes used specifically for deletion/
amplifications. However, FISH does not always allow low
clonal cell detection for minimal residual disease (MRD).
The other problem is that FISH probes are less sensitive
for detecting unbalanced translocations, and another technical
problem is adjusting of the cutoffs for various FISH
probes. In some laboratories, cutoffs are described on a
regular basis and adjust them as a >3 SD [13]. However, it
is not clear if these abnormalities have the same prognostic
significance when present at low levels at diagnosis. In both
instances, it is important to understand the limitations of
the FISH assay when interpreting and reporting the results.
Despite its pros and cons, FISH and karyotyping are still
the golden standard for diagnosis and follow-up of the disease.
Since 2016, several publications have been reported
related with NGS usage in MM. Published studies focused
on sequencing of the patients’ exome during the time of
diagnostic reading and relapse. These studies also confirm
the molecular heterogeneity of MM. The KRAS and NRAS
genes (approximately 25.0 and 20.0% of patients, respectively)
are the most frequently mutated genes in MM. The
prognostic information of these mutations is still limited.
Until now, detrimental prognostic value of mutations has
only been shown for TP53 mutations (observed in 6.0-8.0%
of patients) and other rare mutations (present in <3.0% of
the patients) may affect the outcome of the disorder [10].
Next generation sequence testing is suitable during the
low plasma cell percentage in the blood or bone marrow.
Next generation sequencing is very useful in the case of low
mitotic index, which cannot give permission for classical
methods to be able to identify the abnormalities. The major
limitation of NGS is not being used for covering broad
genomic regions and the ultra-deep sequencing of small
genomic regions, but it is applicable for detection of the MM-associated clonal rearrangements of the IgH genes that
are molecular markers for MRD evaluation. The complex
nature of the MM-related genes makes them a target for
the NGS approach. In this study, we identified pathological
variations in six cases that were reported normal using
conventional techniques. This showed us that the efficiency
of FISH and karyotype analyses are limited, and showed the
diagnostic and prognostic importance of NGS panels with
frequent variants in the myeloid series. Next generation
sequencing assays should be used in clinical practice due
to the sensitivity of 10–6 or because they are evaluated with
higher sensitivity and less expensive than other technologies
such as digital polymerase chain reaction (ddPCR) [6,10].
As a result, technological developments are important
in the classification of MM at the molecular level. It is inevitable
to perform both traditional cytogenetic and FISH
applications to reveal new recurrent specific chromosomal
abnormalities or other genetic abnormalities in MM patients.
Technologies such as NGS are an emergency tool
to determine the heterogeneous nature of the disease and
to determine the true clinical stage of the disease.
Acknowledgments. We would like to thank all the
patients who participated in this study.
Declaration of Interest. The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of this article.
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