Keywords
papillary thyroid carcinoma - thyroglobulin antibodies - antitumor immunity - tetramer
analyses
PTC Papillary thyroid carcinoma
DTC Differentiated thyroid cancer
Tg Abs Anti-thyroglobulin-antibodies
HT Hashimoto’s thyroiditis
TPO Thyreoperoxidase
WBS Whole body scintigraphy
Introduction
Papillary thyroid cancer (PTC) is the most common malignant tumor of the thyroid
[1]. The etiology of PTC seems to be
multifactorial including genetic predispositions and environmental triggers [2]; moreover, comorbidities such as
simultaneously appearing autoimmune thyroiditis are discussed as risk factors [3]. PTC is characterized by a rather slow tumor
growth, a lymphatic spread without frequent distant metastases and an excellent
prognosis with a 10-year survival rate of more than 90% [4]. Interestingly, PTC shows an abundant
lymphocytic infiltration into the tumor site [5]. Around 30% of PTC patients additionally suffer from Hashimoto’s
thyroiditis (HT), also known as chronic lymphocytic thyroiditis, the most common
autoimmune disease of the thyroid. This frequent coincidence implies further
evidence of an important etiological link between PTC and immunological processes
in
the thyroid. In fact, the reported data about an improved prognosis of PTC patients
with present HT implies the question whether a simultaneously ongoing (auto)immune
reaction plays rather a protective role than a destructive one [6]
[7].
Due to these three characteristics, the excellent prognosis of this thyroid
malignancy, the local lymphocytic infiltration and the eventual benign association
with HT, the hypothesis of an existing antitumor-immunity affecting the PTC has been
postulated and remains a subject of discussion [8]
[9]
[10].
The role of anti-thyrogloblin antibodies (TgAbs) in this context is still a matter
of
debate. Stable or rising TgAb levels have long been associated with persistent or
progressive disease [11]
[12]
[13]
[14]
[15]. On the other hand, the question whether
TgAbs should turn out undetectable in order to establish DTC remission, is still
unclear. The latest guidelines of the American Thyroid Association (ATA) in the
management of PTC require the absence of TgAbs to define a patient as cured [16]. However, the observation that after total
thyroid ablation TgAbs required a long time (3 years) to disappear [17] and that a significant reduction of TgAb
levels after thyroidectomy correlated with a low risk of persistence or recurrence
of DTC prompted scholars to suggest decreasing TgAb levels over time as a favorable
prognostic factor [14]
[18]
[19]
[20]. However, other authors
recommended caution in using TgAbs as a prognostic marker of DTC [13].
The aim of our study was to correlate the presence of TgAbs with the clinical outcome
and to correlate these data with the cellular anti-tumor immunity in these patients.
To do this, tetramer analyses of a previously published paper [21] were reanalyzed and were correlated with
the present data. We show that (with the exception of one patient) TgAbs were only
seen in cured PTC patients. Tetramer positive cells were also higher in the group
of
TgAb positive patients compared to TgAb negative patients (however, in a very
limited number of patients).
Patients and Methods
Patients
The cohort included n=183 consecutive, unselected patients who had been treated
with total thyroidectomy (and lymphadenectomy when metastatic lymph nodes were
identified) because of PTC. Data were assessed retrospectively from chart
review. Patients were enrolled at the Department for Specific Endocrinology and
the Department of Nuclear Medicine at the University Hospital Duesseldorf form
2008 to October 2023 at the time of thyroid remnant ablation with
131I administered after l-thyroxine withdrawal, 2 to 5 weeks
after total thyroidectomy. Subsequent treatment consisted of
l-thyroxine at TSH-suppressive or replacement dose, 131I for
functioning metastatic lesions and surgery for metastatic lymph nodes. Median
follow-up (with interquartile range) was 97 months with a range of 3 to 320
months. PTC patients were defined as TgAb positive, if Tg antibodies could be
detected at least one time after initial therapy. The local Ethical Committee of
the Medical Faculty of the Heinrich-Heine-University Duesseldorf approved the
study (No. 2020–1146). In addition to the described patient cohort, we also
reanalyzed our already published patient cohort with known numbers of tumor
epitope-specific T cell determined by tetramer analyses [21]. We reanalyzed these data in the
context of Tg antibody positivity. These data were available in n=52 PTC
patients.
Laboratory measurements
Measurement of serum Tg and Tg antibodies, respectively, were performed by using
commercial assays. Tg was measured by different immunoassays such as Immulite
(Siemens Healthineers) and Cobas e801 (Roche Diagnostics, calibrated against the
Certified Reference Material CRM 457). Tg Abs were assessed by solid-phase
chemiluminescent immunoassays including Immulite (Siemens Healthineers) and
Cobas e801 (Roche Diagnostics).
Tetramer analysis and HLA typing was performed as described in our previous
publication [21]. These data were
reanalyzed. As previously described, the following tetramers have been chosen
for analyses: TPO1 [amino acids (AA) 857 to 865], LLlGGFAGL; TPO2 (AA 3 to 11),
ALAVLSVTL; TPO3 (AA 118 to 126), ALSEDLLSl; Tg1 (AA 2355 to 2363), GLLDQVAAL;
Tg2 (AA 2750 to 2758), GLREDLLSL; and Tg3 (AA 841 to 850), SLQDVPLAAL.
Follow-up
At the time of enrolment, all patients had Tg and TgAbs measured and underwent
neck ultrasound and whole-body scintigraphy (WBS). Laboratory tests and neck
ultrasound were next performed every 6–12 months. Central and bilateral neck
lymph node compartments and the superior mediastinum were evaluated at
ultrasound. Suspected lymphadenopathies or local recurrences were evaluated by
ultrasound-guided fine needle aspiration for cytological examination. Suspected
distant metastases were investigated by WBS, Computed Tomography and
18F-Fluorodeoxyglucose Positron Emission Tomography.
Remission of PTC was established on the following criteria: basal (on
levothyroxine) Tg was below the functional assay sensitivity of the used assays
and no evidence for structural thyroid disease. PTC was considered as persistent
when basal Tg was ≥2 ng/ml and/or presence of structural disease.
Statistical analyses
Prism software (PRISM 6, GraphPad Software, Inc., La Jolla, CA, USA) was used for
calculation of statistical significances and for graphical presentation: To
investigate clinical outcome of PTC patients chi2-test was used to
compare subgroups. p-Values<0.05 were considered as significant. Data from
tetramer analysis show no normal distribution; for this investigation we used
Mann–Whitney test.
Results
Frequency of Tg antibodies in PTC patients with and without signs of tumor
recurrence
During follow-up of in mean 97 months, most of the n=183 PTC patients had no
signs of tumor relapse. This was true for n=157 patients (85.8%) as the serum Tg
was below the assay’s functional sensitivities. In contrast, n=1 patient had Tg
serum levels above the detection limit (DL) and<1 ng/ml, n=2 patients Tg
serum levels ≥1 ng/ml and<2 ng/ml and n=23 patients had Tg serum levels
≥2 ng/ml ([Table 1]). Morphological signs
of tumor recurrence were seen in n=14 patients; all of these patients had serum
Tg levels ≥2 ng/ml. Importantly, with the exception of one patient, all TgAb
positive PTC patients (n=27) had no signs of tumor recurrence as the serum Tg
levels were below the assay’s functional sensitivities. Only one of the 27 TgAb
positive patients had serological (Tg ≥2 ng/ml) and morphological signs of tumor
recurrence. These differences did, however, not reach statistical
significance.
Table 1 Number of PTC patients depending on serum
thyroglobulin levels, anti-thyroglobulin antibodies, and
morphological detectable disease.
|
PTC patients (n=183)
|
Serum Tg (ng/ml)*
|
Number of patients
|
Number of patients with positive thyroglobulin antibodies
|
Number of patients with morphological detectable disease
|
|
<DL
|
157
|
26
|
0
|
|
>DL to<1
|
1
|
0
|
0
|
|
≥1 to<2
|
2
|
0
|
0
|
|
≥ 2
|
23
|
1
|
14
|
|
Sum
|
|
183
|
27
|
14
|
*Serum Tg at the end of the follow-up period. DL: Assays’
detection limit.
Prevalence of tumor epitope-specific T cells in PTC patients dependent on the
presence of thyroglobulin antibodies
Based on the aforementioned data, we reanalyzed our formerly published results
[21] in the context of Tg antibody
positivity. The sum of all Tg and/or TPO epitopes-specific T cells detected by
tetramer analyses were reanalyzed. Here, we could show that the number of TPO
epitope-specific T cells was higher in the group of TgAb positive patients
compared to those without Tg Abs (mean values: 0.28%±0.18 in Tg Ab negative
patients vs. 0.42%±0.13 in Tg Ab positive patients). An equal picture was seen
for Tg epitope-specific T cells (mean values: 0.25%±0.20 in Tg Ab negative
patients vs. 0.36%±0.14 in Tg Ab positive patients). Therefore, also the sum of
all tetramer positive T cells (TPO and Tg) revealed a similar picture: Tg Ab
positive 0.77%±0.21 versus Tg Ab negative 0.54%±0.38. These results did,
however, not reach statistical significance.
Discussion
The aim of our study was to correlate the presence of TgAbs with the clinical outcome
of PTC patients and to correlate these data with the cellular anti-tumor immunity
in
these patients. We could show that with the exception of one patient, all TgAb
positive patients had no signs of tumor recurrence. We also correlated these data
with the cellular immunity in PTC patients. To do this, tetramer analyses of our
previously published paper [21] were
reanalyzed and were correlated with the clinical outcome. Tetramer analyses were
available in 52 patients. The number of tetramer positive T cells were higher in the
group of PTC patients with TgAbs compared to PTC patients without TgAbs. These
differences did, however, not reach statistical significance due to the very limited
number of TgAb positive PTC patients tested. Still, these data suggest that the
appearance of TgAbs may be the result of the specific cellular immunity in PTC
patients.
TgAbs are a marker of autoimmune thyroid diseases [22]
[23], but may also be detected,
usually at low levels, in DTC and other non-autoimmune thyroid diseases [24]
[25]
as well as in few subjects with no thyroid disease [26]. In PTC, TgAbs arise due to an associated lymphocytic thyroiditis but
might also be induced by the stimulation of the immune surveillance elicited by the
tumor. In the follow-up of DTC the measurement of Tg, the marker of DTC, goes with
that of TgAbs, because TgAbs interfere with Tg measurement and are a surrogate
marker for persistent thyroid tissue. The impact of concomitant thyroid autoimmunity
on the course of DTC is debated. Some studies reported a favorable effect [5]
[27]
[28]
[29], whereas others observed a minor or no
effect on survival or recurrence risk [30]
[31]
[32]. Some studies related positive TgAbs after
near-total or total thyroidectomy to higher rates of persistent and recurrent DTC
[6]
[13]
[33]. At variance, a nationwide
US multicenter registry study reported no correlation between positive TgAbs and
disease-free and overall survival of DTC [34]
and another ruled out the influence of the TgAb status on the response to therapy
[35]. The additional observation that,
among DTC patients with positive TgAbs, those with a TgAb pattern typical of thyroid
autoimmunity had a less favorable prognosis supported the negative influence of
thyroid autoimmunity on the course of DTC [36]. On the other hand, a recent study suggested that positive TPOAbs are
associated with a lower risk of DTC recurrence [37]. It is worth noting that in many of these studies the
characterization of lymphocytic thyroiditis and its correlation with TgAbs were
inadequate or even lacking. All these aspects have been intensively discussed in a
recently published paper by Viola et al. [38].
Our data support the idea that the presence of TgAbs is connected to the cellular
antitumor immunity in these patients.
There are also some limitations of our study. First, there is the retrospective
design with analyses of patients who have been treated for PTC in the past. Second,
tetramer analyses for the detection of tumor epitope-specific T cells have been
performed many years after initial diagnosis. These data have than been combined and
reanalyzed. The phenomenon of a durable antitumor immunity over many years and
potentially life long is, however, known from other tumors as well. The potentially
better prognosis of these patients is in line with our data.
In summary, our study indicates an important role of Tg antibodies in PTC patients
and that the presence of TgAbs may correlate with the cellular immunity in PTC
patients. This, however, should also be reevaluated in a larger prospective
study.
