DETERMINING SPECIFIC THYROID TRANSCRIPTS
IN PERIPHERAL BLOOD:
A SINGLE CENTER STUDY EXPERIENCE
Makazlieva T, Eftimov A, Vaskova O, Tripunoski T,
Miladinova D, Risteski S, Jovanovic H, Jakovski Z
Tanja Makazlieva and Aleksandar Eftimov contributed equally to this study
and are considered first coauthors.
*Corresponding Author: Tanja Makazlieva, Ph.D., Institute of Pathophysiology and Nuclear Medicine,
Medical Faculty, Mother Teresa Street, No. 17, 1000, Skopje, Republic of Macedonia.
Mobile: +389-75-313-665. E-mail: tmakazlieva@medf.ukim.edu.mk or tmakazlieva@gmail.com
page: 13
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DISCUSSION
Since Ditkoff et al. [10] reported a possible usefulness
of the RT-PCR molecular technique in detection of
thyroid circulating cells in blood samples as an indicator of
metastatic TC, an increased number of studies on this issue
have been published in the past few years [17-19]. Besides
the possible use in follow-up of TC, some researchers
reported that evaluating TSHR-mRNA in patients with
indeterminate cytology, reports from fine needle aspiration
biopsies of thyroid nodules might improve cancer detection
and avoid unnecessary surgeries [20,21].
Using data from previous investigations into relative
quantification of gene expression in other malignancies,
we decided to perform a relative gene expression using
the 2–ΔΔCt method in evaluating relative expression of Tg
and TSHR genes in patients with TC compared with the
HC group. The analysis revealed an 8.57-fold higher level
in TCs patients than in HC individuals, and TCb patients
expressed TSHR by a 14.17-fold higher level than HC
individuals. The increase in fold change in expression was
higher for TCb with incomplete biochemical response than
in the TCs group, compared to HC subjects [11,14,15].
This finding may be due to dedifferentiation of tumor in the
TCs group of patients and low expression of the evaluated
transcripts for the target genes.
In our study, in almost all HC (except three cases)
expression of both Tg and TSHR genes was detected. Most
of the studies found the presence of Tg and TSHR expression
in blood of normal subjects [20-22]. This finding
can be explained due to the presence of transcripts for Tg
and TSHR from thyrocytes in peripheral blood of HC or
from ectopically transcribed Tg and TSHR, which may
indicate a low specificity of this method, but we detected
statistically significant lower expression in HC compared
to the TCs group.
Eszlinger et al. [22] analyzed the usefulness of quantification
of only Tg-mRNA in peripheral blood in the
follow-up of DTC patients. Their analysis differed from
ours because they did not find any statistically significant
difference in expression of Tg-mRNA in patients with
and without metastatic disease; hence, they concluded
that evaluation of Tg-mRNA was not a useful biomarker
in the follow-up of TC patients [22].
Ringel et al. [23] analyzed the possible reasons for
great variations in results between different researchers and
found several possible reasons, starting from the number
of cycles, selection of primers, selection of “housekeeping
genes” and difference in endpoint, or quantification of the
products in the assay or simply absence in standardization
of methodology [23]. Bojunga et al. [24] and Takano et al.
[25] in their studies didn’t find statistical significant difference
in expression levels between patients with and without
metastases. Bojunga et al. [24] reported different results
using “normal vs. high sensitivity PCR technique.” After 30
cycles of PCR, they detected Tg-mRNA in 9/13 patients with
known metastases, in half of the patients without metastases
and in 21/85 patients with benign disease. After using 40
cycles of PCR, Tg-mRNA expression was increased in 11/13
patients with metastases as well as in 61/85 patients with
benign disease and in 41/50 healthy controls [24].
Another important question regarding this methodology,
that should be discussed, is careful selection of
the primer pairs. Gupta et al. [13] in their research, used
several primer pairs of different exons. They found that
primer pairs for TSHR targeting 6 to 9 exons and 1 to 5
exons for Tg, were with specificity for thyroid tissue and
no reactivity in normal peripheral blood mononuclear cells
excluding possible illegitimate transcription [13,14]. In our
study, we used the same primer pairs as in the study of
Gupta et al. [13]. On the other hand, Savagner et al. [26]
also evaluated the possible effect of alternative splicing
using two different primer pairs from two non overlapping
regions. First in exons 10 and 11, in which no alternative
splicing was described, second in exons 6 and 7, in which
alternative splicing was found. Their study revealed higher
expression levels in controls depending on the TSH level,
even higher than in patients with metastatic disease, but
low levels in patients after thyroidectomy and ablation
and without signs of disease persistence. These authors
found that alternative variants of Tg-mRNA represented
approximately 30.0% of the measured value of Tg-mRNA
in controls and patients [25].
In our study, we noted significant difference in expressions
of both target genes between patients with structural
and biochemical incomplete response to treatment compared
to healthy individuals, and this finding was more
remarkable for expression of TSHR-mRNA. Our study
differs from the previous study in endpoint quantification
of the methodology. Savagner et al. [26] also used absolute quantification,
creating a standard curve from serial dilutions 102-105 copies
of Tg cDNA, generated from plasmids containing appropriate
cDNA inserted as a template and using a cutoff value
for TC patients’ values above 1 pg Tg-mRNA/μg RNA.
These authors found mean 10.6 ± 3.1 pg Tg-mRNA/μg total
RNA in normal healthy control individuals [26]. Chinnappa
et al. [14] analyzed the presence of TSHR-mRNA and TgmRNA
in 51 normal subjects, 67 patients with DTC, 27
patients with benign thyroid disease and eight patients with
DTC preoperatively. They used a similar methodology to
ours (38 cycles, GAPDH as reference gene and the same
primer pairs for Tg and TSHR), except for the method of
quantification. For the endpoint results, they used 2.0%
gel electrophoresis and visualization of the products with
ethidium bromide staining. The results of this study showed
absence of TSHR-mRNA and Tg-mRNA in blood samples
of normal subjects, which is contrary to what we found;
furthermore, they presented 97.0% sensitivity for TSHRmRNA
and 94.0% sensitivity for Tg-mRNA based on the
results obtained from patients with and without known
metastatic disease and from benign group [14].
In our study, we detected the presence of TSHRmRNA
and Tg-mRNA in 14/17 (82.4%) of HC and in all
TC patients, except in three patients with elevated sTg
levels, loco-regional persistence of the disease in one
patient and distant metastasis in two other patients. In
two patients, high levels of sTg >300 ng/mL were found,
as well as confirmed metastatic disease, in one patient
with diffuse pulmonary secondary deposits and several
131I therapies and in the other one with skeletal deposits.
Statistical analysis revealed significant difference in ΔCt
value for expression of Tg and TSHR genes between TCs
and TCb patients and TCr-excellent therapy responders
and HC. The greatest difference was detected between
the TCb and HC groups for TSHR, and lower, but statistically
significant difference between TCs and HC. This
finding may be due to the more aggressive variants of
tumors included in the TCs group and possible dedifferentiation
of the tumor. We also found that TSHR-mRNA
might be a more precise biomarker in follow-up of TC than
Tg-mRNA. Discrepancy in the results obtained in many
studies indicate that a larger multicenter study including
more subjects and standardization of the methodology
used, especially in quantification, is needed for understanding
the real significance of this method. Alternative
splicing, illegitimate transcription, empirically supposed
100.0% of amplification efficiency and dedifferentiation
of the tumor are also possible reasons for errors in the
detection of transcripts. The real need of new biomarkers
in the follow-up of thyroid carcinomas exists, especially
after introduction of the new recommendations for rationalization
of 131I ablation treatment in low risk DTC and
microcarcinomas, thus, sTg levels could not be used as a
reliable tumor marker in the follow-up of these patients.
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