The clinical value of thyroglobulin antibodies and Hashimoto's thyroiditis in the follow-up of patients

48 Pages·2014·1.63 MB·English

MASTER THESIS

The clinical value of thyroglobulin antibodies and Hashimoto’s thyroiditis in
the follow-up of patients with differentiated thyroid carcinoma

Author:
J.M. Klopstra
Student 2048590

E-mail: [email protected]

Supervisors:
Prof. dr. T.P. Links, internist-endocrinologist
Drs. M.S. Klein Hesselink, PhD-student

Location:
University Medical Center Groningen
Department of Endocrinology

Period:
September 16th, 2013 – March 4th, 2014
ABSTRACT

The clinical value of thyroglobulin antibodies and Hashimoto’s thyroiditis in the follow-
up of patients with differentiated thyroid carcinoma

INTRODUCTION. Differentiated thyroid carcinoma (DTC) is the most common endocrine
malignancy. Thyroglobulin (Tg) is the tumor marker of DTC, however, thyroglobulin
antibodies (TgAbs) decrease the reliability of Tg measurements. For TgAb measurements, a
luminescence immunoassay (TgAb-ILMA) and a radioimmunoassay (TgAb-RIA) are used in
our institute. In literature, Hashimoto’s thyroiditis (HT) is associated with both DTC and
TgAbs. HT is often associated with a more favorable disease outcome, although the exact
causality and clinical association between these associations is unknown. We aim to find out
which TgAb assay is clinically the most reliable. Furthermore, we aim to create more insight
in the clinical outcome of TgAb positive and negative DTC patients, either with or without
HT.
METHODS. All DTC patients still in follow-up in our institute between March 21st, 2006
and September 2nd, 2013 were included. Their medical files were consulted for patient-related
data, laboratory values of Tg and TgAbs, cumulative radioiodine doses, pathological data and
final disease state. Research questions involving TgAbs only used a subgroup of patients who
were diagnosed with DTC after March 21st, 2006, the date of introduction of the TgAb assays
in our institute. Patients without available data were excluded from analysis.
RESULTS. 621 DTC patients were included in this study, 236 of them were included in the
subgroup for TgAb analysis. 54 of these 236 (22.9%) patients were TgAb-ILMA positive, 43
(18.2%) were TgAb-RIA positive and 37 (15.7%) were positive in both assays. Only 6
patients (2.5%) were TgAb-RIA positive and TgAb-ILMA negative. They had small tumors,
were all in remission and did not show any recurrence. 578/621 DTC patients had available
pathological data of the thyroidectomy. HT was found in 87/578 patients (15.1%). The
presence of HT was associated with female gender (89.7% vs. 70.1%; p <0.001), younger age
(42.3 vs. 46.8 years; p = 0.023), less distant metastases (1.1% vs. 9.4%; p = 0.009), lower
cumulative radioiodine doses (p = 0.015) and a more favorable disease outcome (p = 0.001),
compared to the absence of HT. From the subgroup for TgAb analysis, 225/236 patients had
both available pathological data and reliable TgAb values. Comparison between TgAb
positive patients with HT (23/225; 10.2%) and TgAb positive patients without HT (13/225;
5.8%) revealed a younger age (47.9 years vs. 61.1 years; p = 0.011) and no T4-tumors
(extensive local tumor growth) (0.0% vs. 26.7%; p = 0.007) in the first group. Besides, the
number of patients in remission was higher (65.2% vs. 53.3%) and persistent disease was
lower (4.3% vs. 26.7%) (p = 0.049).
CONCLUSIONS. TgAb-ILMA is more suitable for detecting TgAbs in DTC patients than
TgAb-RIA; abolishing the latter would not lead to missing patients with extensive disease.
HT-patients have a more favorable disease state and outcome, compared to non-HT patients.
TgAb positivity with HT is associated with a more favorable disease state and outcome than
without HT.
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SAMENVATTING
De klinische waarde van thyreoglobuline-antilichamen en de ziekte van Hashimoto bij
de follow-up van patiënten met gedifferentieerd schildkliercarcinoom

INTRODUCTIE. Het gedifferentieerd schildkliercarcinoom (DTC) is de meest
voorkomende endocriene maligniteit. Thyreoglobuline (Tg) is de tumormerkstof van DTC,
echter, de betrouwbaarheid van Tg-metingen wordt gehinderd door de aanwezigheid van
thyreoglobuline-antilichamen (TgAbs). In onze kliniek worden zowel een luminescentie-
immunoassay (TgAb-ILMA) als een radio-immunoassay (TgAb-RIA) gebruikt voor TgAb-
bepalingen. In de literatuur wordt de ziekte van Hashimoto (HT) vaak geassocieerd met
zowel DTC als TgAbs, hoewel het exacte causale en klinische verband tussen de genoemde
associaties nog altijd onbekend is. Het doel van deze studie is vast te stellen welke TgAb-
assay het meest geschikt om in onze kliniek te gebruiken. Verder proberen we meer inzicht te
krijgen in de klinische uitkomst van zowel TgAb-positieve als TgAb-negatieve DTC
patiënten, met of zonder HT.

METHODE. Alle DTC-patiënten die in onze kliniek nog in follow-up waren tussen 21 maart
2006 en 2 september 2013, werden geïncludeerd. Hun medische dossiers werden
geraadpleegd voor patiëntgerelateerde data, laboratoriumwaarden van Tg en TgAbs,
cumulatieve dosis radioactief jodium, pathologische gegevens en de uiteindelijke
ziektestatus. Onderzoeksvragen aangaande TgAbs werden alleen beantwoord aan de hand
van een subgroep van patiënten die met DTC werden gediagnosticeerd ná 21 maart 2006, de
introductiedatum van de TgAb-assays. Patiënten van wie geen gegevens te verkrijgen waren,
werden geëxcludeerd uit de studie.

RESULTATEN. 621 DTC-patiënten werden geïncludeerd, 236 van hen waren beschikbaar
voor de TgAb-analyse. Het aantal TgAb-RIA positieve/TgAb-ILMA negatieve patiënten was
gering (n = 6; 2,5%). Deze 6 patiënten hadden allemaal relatief kleine tumoren, waren
allemaal in remissie en toonden geen recidief. Van 578/621 DTC-patiënten was het
pathologieverslag van de thyreoïdectomie beschikbaar. HT werd bij 87/578 patiënten (15,1%)
vastgesteld. De aanwezigheid van HT was in vergelijking met de afwezigheid van HT
geassocieerd met vrouwelijk geslacht (89,7% versus 70,1%; p <0,001), lagere leeftijd (42,3
versus 46,8 jaar; p = 0,023), minder afstandsmetastasen (1,1% versus 9,4%; p = 0,009),
lagere cumulatieve dosis radioactief jodium (p = 0,015) en een betere ziekte-uitkomst (p =
0,001). In de subgroep voor TgAb-analyse waren van 225/236 patiënten zowel het
pathologieverslag als geschikte TgAb-waarden aanwezig. De vergelijking tussen TgAb-
positieve patiënten met HT (23/225; 10,2%) en TgAb-positieve patiënten zonder HT (13/225;
5,8%) laat een lagere leeftijd (47,9 jaar versus 61,1; p = 0,011) en geen T4-tumoren (lokaal
extensieve tumorgroei) (0,0% versus 26,7%; p = 0,007) zien in de eerstgenoemde groep.
Bovendien was het aantal patiënten in remissie hoger (65,2% versus 53,3%) en dat met
persisterende ziekte lager (4,3% versus 26,7%) (p = 0,049).

CONCLUSIES. Voor TgAb-metingen in DTC-patiënten is TgAb-ILMA geschikter dan
TgAb-RIA; afschaffen van de TgAb-RIA-assay leidt niet tot het missen van patiënten met
uitgebreide ziekte. HT-patiënten hebben een gunstiger ziektestatus en –uitkomst, vergeleken
met patiënten zonder HT. TgAb-positiviteit met HT wordt geassocieerd met een gunstiger
ziektestatus en –uitkomst dan TgAb-positiviteit zonder HT.
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TABLE OF CONTENTS
ABSTRACT 2

SAMENVATTING 3

1. INTRODUCTION 5
1.1 Epidemiology 5
1.2 Diagnosis and treatment 5
1.3 Follow-up: thyroglobulin 6
1.4 Follow-up: thyroglobulin antibodies 7
1.5 Thyroglobulin antibody assays 7
1.6 Clinical relevance of thyroglobulin antibodies 8
1.7 Hashimoto’s thyroiditis 8
1.8 Relevance of the study 9
1.9 Aim of the study 9

2. PATIENTS AND METHODS 11
2.1 Study population 11
2.2 Data collection and specification 11
2.3 Laboratory methods 13
2.4 Ethics 14
2.5 Statistical analysis 14

3. RESULTS 15
3.1 Patient characteristics 15
3.2 Additional value of using both TgAb-assays (aim 1) 15
3.3 Characteristics of TgAb-positive patients vs. TgAb-negative patients (aim 2) 18
3.4 Hashimoto’s thyroiditis and disease outcome (aim 3) 19
3.5 TgAb-positivity and Hashimoto’s thyroiditis combined (aim 4) 21

4. DISCUSSION 24
4.1 Conclusions 28

5. ACKNOWLEDGEMENTS 30

6. REFERENCES 31

7. APPENDICES 37
Appendix I: Institutional protocol 37
Appendix II: TNM-classification of thyroid carcinoma (7th edition) 40
Appendix III: Stage grouping classification (American Joint Committee on Cancer) 41
Appendix IV: Flowchart of subdivision and exclusion 42
Appendix V: Characteristics of TgAb-ILMA positive/TgAb-RIA negative and TgAb-
ILMA negative/TgAb-RIA positive patients 43
Appendix VI: Characteristics of TgAb-positive patients who received additional
radioiodine therapy based on their TgAbs only 44
Appendix VII: Characteristics of TgAb-positive patients without HT and TgAb-positive
patients with HT 46
Appendix VIII: List of abbreviations and cut-off values 48
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1. INTRODUCTION

1.1 EPIDEMIOLOGY
Thyroid carcinoma covers about 3.6% of all malignant diseases and 96% of all malignant
endocrine tumors (1). Of these, differentiated thyroid carcinoma (DTC) is the most common,
accounting for at least 94% (2). Overall, the 10-year relative survival of patients with DTC is
excellent (93% for papillary carcinoma and 85% for follicular carcinoma) (3).The a priori
chance of palpating a malignant thyroid nodule in the general practitioner’s office is
estimated at maximally 5% (4).
DTC originates from the follicular epithelial cells in the thyroid gland. DTC is subdivided
into two main groups: papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma
(FTC). PTC occurs three times more frequently than FTC (4). Another histological subtype is
the medullary thyroid carcinoma, which accounts for 5% of all thyroid cancers. This tumor
secretes calcitonin, originates from the C cells in the thyroid, and can be found in rare
familiar neuro-endocrine tumor syndromes (2). The undifferentiated or anaplastic thyroid
carcinoma is the most malignant type of thyroid carcinoma is the. This subtype accounts for
1% of all thyroid cancers and has the worst prognosis (1,5).
Thyroid cancer is a relatively rare disease with a total annual incidence of 4.0/100,000 (6).
According to the Dutch cancer registry, the annual incidence in 2011 was 2.0/100,000 for
men and 4.5/100,000 for women (611 new patients in total) (5). Thyroid cancer can occur in
people of all ages and both genders, however, the peak incidence in men (65 years of age)
occurs about 15-20 years later than in women (50 years of age) (7). This is probably due to
the fact that women are more likely to present with benign nodular thyroid disease than men,
and are therefore controlled by a physician more regularly, whereas men may less timely seek
medical care (8). A large retrospective study by Hughes et al. showed that the age group
diagnosed in the period between 1973 and 2006 with PTC has shifted from patients in their
30s to patients in the 40-50-year-old age group. After 1999, PTC became more common in
patients older than 45 years (9).
The incidence of thyroid cancer has increased over the past three decades (10,11), with an
accelerated increase since the year 2000 (12). Increased incidence rates have been found
among individuals in higher income quintiles. This also applies for living in cities, compared
to rural areas (11). The overall assumption is that the increased incidence is due to the
increased detection of subclinical disease, i.e. small papillary cancers, and not due to an
increase in the true occurrence of thyroid cancer (13,14). Still, many environmental and
genetic factors, possibly influencing the incidence, have to be investigated (15).
1.2 DIAGNOSIS AND TREATMENT
Thyroid cancer usually presents with a nodule. Thyroid ultrasonography is used to assess the
size of the nodule and to guide fine-needle-aspiration cytology (FNAC) (16). When
suspecting a malignant thyroid tumor, FNAC could be performed (17). However, the final
diagnosis is made by histological examination after surgery (18). The cytological difference
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between a follicular adenoma and a follicular carcinoma cannot be made by FNAC, so a
histological examination is always needed when a follicular neoplasm is present (17).
A PTC is usually an unencapsulated tumor with papillary and follicular structures, which
shows typical nuclear characteristics. The tumor is multifocal in 20-80% of patients. It may
spread to the regional lymph nodes or, rarely, hematogenically to the lungs. FTC does not
show the typical nuclear characteristics of a PTC and it usually possesses a capsule. An
oncocytic or Hürthle-cell carcinoma is recognized as a subtype of FTC. Lymph node
metastases are rare in FTC, but distant metastases may occur in the lungs and bones (16).
The first step in the treatment of DTC is a total thyroidectomy. Prior to this, an ultrasound
examination of the neck is recommended, in order to investigate potential lymph node
metastases (19). A total thyroidectomy is associated with the lowest incidence of local and
regional recurrence (20). Patients with clinically or histologically proven lymph node
metastases are treated with an additional lymph node dissection. A prophylactic central
compartment neck dissection for patients with PTC is controversial (21). An important
feature of postsurgical DTC treatment is an ablation therapy with radioactive iodine (131I). 131I
is an example of targeted radiation therapy for PTC and FTC, because iodine is specifically
taken up by follicular thyroid (tumor) cells (4,22). If residual tumor and/or metastases are
present after initial 131I-treatment, patients undergo repeated cycles of 131I treatment. A whole
body scan (WBS) 3-7 days after administration of 131I evaluates the possible presence of
residual tumor, and lymph node or even distant metastases (21).
During follow-up, patients are treated lifelong with levothyroxine (T4) to prevent
hypothyroidism. Another important purpose of this treatment is suppression of thyrotropin
(TSH) levels, since TSH is believed to enhance follicular (tumor) cell growth and metabolism
(23). About 21% of DTC patients are diagnosed with a recurrence, of which roughly 80%
occur in the lymph nodes and 20% in other sites of the body (24). Although rare, recurrences
can occur up to 40 years after initial therapy (25).
1.3 FOLLOW-UP: THYROGLOBULIN
In the follow-up of DTC, thyroglobulin (Tg) in the serum is the only tumor marker. It is a
large glycoprotein that normally acts as storage for T4 and T3 (26). It is produced by both
normal and malignant differentiated thyroid follicular cells (27). An increased serum Tg level
after total thyroidectomy and ablation therapy is highly suspicious for having residual thyroid
(tumor) tissue or metastases. Subsequently, a rising serum Tg value is suspicious for a local
or distant recurrence (2,4,6,7,16,18,19,22,23,27). It is, however, known that 25% of the
patients require 18 months or more after thyroidectomy to show undetectable Tg values, even
though they are cured (28). Recently, it has been shown that Tg can be used as an early
prognostic marker for predicting remission up to 18-24 months after initial treatment (27). An
important detection method for Tg is the immunoradiometric (Tg-IRMA) assay, which is
standardized on the European reference standard and has a functional sensitivity of < 1.0
ng/mL (29,30).

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1.4 FOLLOW-UP: THYROGLOBULIN ANTIBODIES
An important issue concerning the reliability of Tg measurements is the presence of
thyroglobulin antibodies (TgAbs) (31-33). TgAbs are able to bind with serum Tg, by which
they lower the concentration of free circulating Tg. This may lead to a misinterpretation of
the serum Tg value, i.e. unidirectional underestimation of the value by Tg-I(R)MA assays and
either under- or overestimation by Tg-radioimmunoassays (RIAs) (34-36). In the presence of
TgAbs, Tg cannot be used as a reliable tumor marker for disease activity, because of the
possible falsely low value. Subsequently, patients may be labeled disease free based on low
or undetectable serum Tg values, whilst having a persistent or even recurrent disease.
Increased Tg values may indicate interference by other molecules that bind similarly like Tg
in the Tg-RIA detection method, thereby making the Tg value unusable. Besides that, widely
different relationships between Tg and TgAbs have been described, indicating TgAb-Tg
epitope heterogeneity in DTC sera (31,34,36). It has recently been suggested that TgAbs of
an individual patient contain a specific IgG pattern that binds to an equally specific site in the
Tg assay (37). Currently, every Tg assay is hindered by the influence of TgAbs (31,34,36,38).
Recently, it has therefore been recommended to assess the presence of TgAbs quantitatively
in concurrence with every Tg measurement in DTC patients (32).
1.5 THYROGLOBULIN ANTIBODY ASSAYS
Roughly, two methods for TgAb detection exist: a direct antibody measurement and a
recovery test (31,32). The latter indirectly measures TgAb interference, by measuring the Tg
concentration before and after adding Tg to a serum sample (31). However, opinions on
recovery tests vary highly and therefore Tg recovery tests are currently not recommended for
the measurement of TgAbs (31,32,39). The currently used direct TgAb assays are basically
the same as those used for detecting Tg. They consist of a competitive polyclonal TgAb-RIA,
which has a long incubation time, and the non-competitive TgAb-IMA, with monoclonal
antibodies and a short incubation time (31,40). Subclasses of both assay types are highly
discordant, thereby indicating heterogeneity between different TgAbs from different patients.
Other factors that may explain the discordance are methodology differences and differences
in the structure of Tg molecules between patients suffering from an autoimmune thyroid
disease and patients who do not (35,40).
The frequency of TgAb interference is related to the quantitative TgAb level. However, the
ratio of the Tg values measured by IMA and RIA assays (Tg-IMA/Tg-RIA ratio), varies
widely between patients, confirming qualitative differences in TgAb activity at patient level.
The format of the Tg assay, the TgAb concentration and the TgAb epitope specificity all
influence the qualitative effects of TgAb positivity (34). Currently, both a TgAb-RIA assay
from Brahms Laboratories and a luminescence immunoassay (TgAb-ILMA) from Abbott
Laboratories are used in the UMCG. Both methods are considered to be complementary to
each other, i.e. TgAb positive patients may be detected by only one method and not by both
(35,36). However, our institutional laboratory calculated that results by both TgAb-RIA and
TgAb-ILMA are only correlated in sera with co-existing Tg concentrations below 988
ng/mL. Results of TgAb-RIA are false positive in sera with Tg values above this level,
7
probably due to Tg interference, suggesting competition of endogenous Tg with the Tg tracer.
TgAb-ILMA does not show this characteristic.
1.6 CLINICAL RELEVANCE OF THYROGLOBULIN ANTIBODIES
The prevalence of TgAbs in patients with DTC is above twice as high as in the normal
population (31,36,39,41). However, our group previously reported a prevalence of TgAbs in
DTC patients of 13%, whereas other studies reported widely various prevalences (up to 36%)
(36,39,42). Furthermore, the prevalence is higher in women than in men, in whites versus
blacks or Hispanics and in PTC versus other thyroid carcinoma subtypes (43).
TgAbs may be suitable for predicting the health outcome in DTC patients. The presence of
TgAbs may be indicative for developing a (recurrent) thyroid malignancy (44-48). This
phenomenon is found in TgAb positive patients of whom the TgAbs were measured 6-12
months after ablation therapy (44,48). TgAbs might be a predictive factor independent of co-
existent autoimmune thyroiditis (46). Moreover, a change in serum TgAb concentration
during follow-up might as well be predictive for recurrent disease (44,47). However, it may
take up to three years after thyroidectomy and ablation therapy for TgAbs to disappear (33).
Although there is no complete consensus about the predictive value of TgAbs, the common
assumption is that TgAbs respond to the mass of thyroid (tumor) tissue in the body (33,41).
Therefore, more tumor tissue may be present if TgAb values are higher, indicating that TgAb
positivity is associated with a less favorable disease state and outcome. Also, the slope of the
TgAb values is recommended to be used as a surrogate tumor marker, rather than a single
serum Tg level and/or TgAb level measurement at the time of diagnosis (31,32,44).
1.7 HASHIMOTO’S THYROIDITIS
DTC is not the only entity in which TgAbs are present: TgAbs are strongly associated with
the presence of autoimmune thyroid diseases (AITD) like Graves’ disease and Hashimoto’s
thyroiditis (HT) (49,50). HT is the most common cause of hypothyroidism and goiter in non-
iodine deficient areas. It consists of a chronic thyroiditis, which is histopathologically
characterized by a diffuse lymphocytic infiltration with germinal center formation and
thyroid follicle obliteration. Often, there is epithelial destruction (51). The degree of
lymphocytic infiltration correlates with the amount of thyroid autoantibodies (33,51). A
prevalence of up to 46.2% of TgAb positivity in HT patients has been described, measured in
six different assays (40,50). The other way around, up to 52.4% of TgAb positive patients
show HT on histology (50).
Expectably, because of the strong link between TgAbs and both HT and DTC, the association
between these diseases is subject of discussion (52,53). Jankovic et al. showed a mean
prevalence of DTC with co-existing HT of 27.6% in their literature review (52). Several
meta-analyses showed that HT is closely related to DTC, especially PTC (34,54-58). Other
studies did not find this association (46,50,59).
It is important to distinguish between focal lymphocytic infiltration around the tumor or
diffuse lymphocytic infiltration, the latter being HT. A chronic infiltration may induce
malignant transformation in epithelial cells. Conversely, a lymphocytic infiltration may attack
8
the tumor, thereby hampering its growth. Therefore, it remains unclear whether HT is either a
stimulating factor for the development of DTC or the result of an autoimmune response
against the tumor or whether it is an epiphenomenon next to DTC (52,54). A more favorable
disease outcome has been found in DTC patients when an AITD is present, e.g. lower
recurrence rates, smaller primary tumors, lower rate of lymph node involvement et cetera
(34,54,56,60-64). However, this association is not fully confirmed (65,66).
Taking the above mentioned knowledge into account, we aim to assess the role of HT
presence in the prognosis of DTC, compared to the non-HT DTC population. Additionally,
because of the strong link between TgAbs and both DTC and HT, we also aim to assess the
role of HT presence in the prognosis of DTC, based on TgAb positivity and negativity. For
the conflicting indication that HT is associated with a more favorable outcome and TgAb
positivity with an unfavorable outcome in DTC, we aim to characterize the disease
characteristics and outcome.
1.8 RELEVANCE OF THE STUDY
Summarizing, Tg is the tumor marker used in the follow-up of DTC patients. However,
serum Tg values can be hampered by TgAbs, of which the measurement is challenging. The
value of a co-existing HT in DTC patients as a risk factor also needs to be confirmed.
No certainty exists in literature which assay is the most suitable for detecting TgAbs.
Furthermore, no clear answer exists on the association between HT and PTC, and the role of
TgAbs in both diseases. This study aims to provide useful additional knowledge for
optimizing the follow-up of DTC patients, i.e. if the influence of TgAbs and HT on DTC
disease outcome is known, follow-up protocols may be corrected for these features, thereby
supplying patient-tuned health care in the follow-up of DTC.
1.9 AIM OF THE STUDY
The aim of the study is to optimize the follow-up of DTC patients. More detailed, the
following aims are set:
1. To assess the additional value of using both TgAb assays which are used in our clinic
(TgAb-RIA and TgAb-ILMA).
2. To compare the patient characteristics, disease characteristics and disease outcome of
TgAb positive patients with TgAb negative patients.
3. To assess the prognosis of DTC patients with and without histologically confirmed HT.
4. To assess the role of TgAb positivity in the disease outcome in DTC patients with HT or
without HT.
The corresponding hypotheses are:
1. TgAb-ILMA is more suitable for detecting persistent/recurrent disease in DTC patients
than TgAb-RIA, thereby abandoning the additional value.
2. Patients who are TgAb positive show a less favorable disease outcome compared to
patients who are TgAb negative.
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3. The prognosis of DTC patients in the presence of HT is more favorable than in non-HT
patients.
4. TgAb positive DTC patients without HT show less favorable disease characteristics and
outcome than TgAb positive DTC patients with HT.
10