FoxP3 Expression in Papillary Thyroid Carcinoma: A Possible Resistance Biomarker to Iodine 131 Treatment

Abstract

Background:

The forkhead transcription factor FoxP3 plays an important role in regulatory T cell (Treg) functions. Tregs are critical in maintaining immunologic tolerance. It has been shown that vaccination against FoxP3-expressing cells is associated with enhancement of tumor immunity. Tregs appear to be increased in blood and in the tumor microenvironment of patients with different cancer types. Tumor cells themselves can express FoxP3. The present study investigates the possible role of FoxP3 expression in a series of human papillary thyroid cancers with a mean follow-up time of 15 years.

Methods:

One hundred five cases of papillary thyroid carcinoma (PTC) were investigated, and FoxP3 expression was evaluated in both tumor cells and tumor-associated infiltrates. For all patients, clinical/pathologic features were considered and the results analyzed by statistical tests.

Results:

Of the 105 PTC cases, 45 (43%) scored FoxP3-positive and 60 (57%) were negative. FoxP3 staining was localized predominantly in the cytoplasm of tumor cells. In some cases, both nuclear and cytoplasmic staining was seen in infiltrating cells. FoxP3 expression in tumor cells was correlated with the presence of extrathyroid invasion (p=0.04) and distant metastasis (p=0.04), but not with overall survival. Interestingly, FoxP3 expression in neoplastic cells was significantly associated with a resistance phenotype to radioiodine treatment (p=0.041).

Conclusions:

The data show an association of FoxP3 expression with features of PTC that seem to have a specific impact on radioiodine sensitivity.

Introduction

Forkhead box P3 (FoxP3) is a key marker of CD4⁺CD25⁺ regulatory T cells (Tregs) (1,2). The protein encoded by this gene is a member of the forkhead/winged-helix family of transcriptional regulators. Defects in this gene are associated with the immunodeficiency, polyendocrinopathy, enteropathy, and X-linked autoimmunity-immunodeficiency syndrome (2). Different isoforms have been identified. Briefly, FoxP3-expressing Tregs are important for maintaining immunological tolerance and overexpression of FoxP3 results in severe immunodeficiency (2). The mechanisms through which Tregs suppress immune reactions have been clarified in part (3). Genetic analysis of FoxP3-expressing T cells showed that FoxP3 can bind to approximately 700 different genes acting either as a transcriptional activator or repressor (4). In addition, FoxP3 inhibits immunological functions directly through cell-to-cell contact and indirectly through the secretion of anti-inflammatory mediators (5,6). High levels of Tregs were first reported in the peripheral blood of patients with different types of cancer (7). Subsequently, elevated percentages of Tregs have been reported in lymph nodes, ascites fluid, and tissues of patients with a variety of cancers (8). In the course of these studies, Zuo et al. (9) showed that epithelial cells of breast carcinoma and normal breast tissue were stained by FoxP3 antibody. Hinz et al. (10) detected FoxP3 expression in 60% of pancreatic adenocarcinoma specimens and in all the investigated pancreatic cancer cell lines. More recently, FoxP3-positive cells have been correlated with poor prognosis in a wide range of carcinomas (i.e., colon, ovarian, breast, gastric, thyroid, and melanoma) (7,11 –13). Similarly, accumulation of Tregs in local lymph nodes of patients with ovarian carcinoma has been associated with impaired prognosis (8). Currently, increased numbers of FoxP3⁺ CD4⁺ T cells in blood are considered a tumor progression marker in patients with pancreatic cancer (10,14). Malignant melanoma-initiating cells do enhance the numbers of circulating FoxP3⁺ Tregs (11). FoxP3 expression is also associated with decreased rates of survival in breast cancer (12). Merlo et al. (12) raised the possibility that expression of FoxP3 in epithelial breast cancer cells (i.e., in cells other than tumor-infiltrating lymphoid cells) could directly down-regulate T-cell functions. Though mechanisms through which Tregs regulate immune functions have been partially clarified (3), very little is known about how these cells may impact tumor progression in vivo. Using breast and colon carcinoma cells, Jung et al. (15) showed that FoxP3 expression is enhanced following up-regulation of p53, which is associated with DNA replication and repair (16). In ovarian cancer, a significant correlation was found between the number of FoxP3⁺ infiltrating Tregs and the expression of the CXCL12 and CXCR4 chemokines. Treatment with a selective CXCR4 antagonist was followed by tumor apoptosis and necrosis, reduction in intraperitoneal dissemination, and selective reduction of tumor-infiltrating Tregs (17). Finally, in animal models, it has been shown that immunization against FoxP3 and the high-affinity interleukin-2 receptor (IL-2Rα/CD25) does enhance tumor immunity (18).

A strong correlation has been demonstrated between papillary thyroid cancer (PTC) and inflammatory infiltration, particularly infiltration of lymphocytes, macrophages, and dendritic cells. It is also known that thyroid autoimmunity is associated with a higher than normal incidence of PTC (19 –22). Recent results of our group indicate that expression of CXCR4 together with the BRAF mutation status is associated with more aggressive PTC phenotypes and with an enhanced spread of tumor cells outside the thyroid capsule (23). In the present study, we investigated 105 consecutive PTC cases (mean follow-up time of 15 years) to assess the possible prognostic value of FoxP3 expression in epithelial thyroid cells.

Patients and Methods

Study group and clinicopathological features

The study included 105 patients who underwent total thyroidectomy at the Department of Surgery of the University of Pisa, Italy, from 1985 to 1992. A presurgical cytological diagnosis of PTC was available for all patients. The clinical follow-up of patients was performed at the Department of Endocrinology, University of Pisa. Institutional approval from the local Ethics Committee was obtained for this study. Surviving patients gave written informed consent to the use of their tissue specimens for present and future investigations.

Hematoxylin-eosin–stained sections of 105 patients from the archives of the section of Pathology of the University of Pisa were re-evaluated independently by two pathologists (CU, FB). A diagnostic concordance rate of 98% was achieved between the two investigators. Rare discordant cases were discussed with a third pathologist (AP). Tumors were reclassified according to the World Health Organization 2004 histopathological criteria (24). Histopathological re-evaluation at the time of diagnosis included, whenever possible, histotype, presence of capsule infiltrating tumor cells, infiltration of extrathyroidal tissue, and the presence and number of metastatic lymph nodes.

For the majority of patients (not for all) clinical/pathologic parameters were also re-evaluated (i.e., age, sex, tumor size, development of node metastasis, detection of distant metastases, number of doses and total dose [mCi] of radioiodine administered). Clinical/pathologic features are summarized in Table 1.

Table 1.

Clinical/Pathologic Features of Investigated Cases of Papillary Thyroid Carcinoma

Clinical/pathologic features	No. of cases/total (%)
Age (years)
<45	59/105 (56)
≥45	46/105 (44)
Sex
Male	23/105 (22)
Female	82/105 (78)
Tumor size (cm)
≤2	60/94 (64)
>2	34/94 (36)
Presence of tumor capsule
Yes	12/93 (13)
No	81/93 (87)
Multifocality
Yes	18/83 (22)
No	65/83 (78)
Bilaterality
Yes	5/77 (6)
No	72/77 (94)
Extrathyroidal invasion
Yes	26/94 (28)
No	68/94 (72)
Node metastasis
Yes	34/102 (33)
No	68/102 (67)
Distant metastasis
Yes	6/102 (6)
No	96/102 (94)
Clinical outcome
Cured	86/105 (82)
Not cured	19/105 (18)
Survival rate
Surviving	98/105 (93)
Not surviving	7/105 (7)
No. of ¹³¹I dose(s)
1	63/100 (63)
>1	37/100 (37)
Histotype of papillary thyroid carcinoma
Classic variant	77/105 (73)
Follicular variant	12/105 (11)
Solid variant	9/105 (9)
Tall-cell variant	4/105 (4)
Oxyphilic variant	2/105 (2)
Diffuse sclerosing variant	1/105 (1)

Clinical outcome

At the time of the study, 105 PTC patients had a mean follow-up period of 15 years (range 1 to 24 years; median±SD, 18±6.18). In agreement with current guidelines for the diagnosis and management of thyroid cancer (25,26), patients were considered cured when neck ultrasound plus whole-body scans gave negative results, and when serum thyroglobulin and anti-thyroglobulin antibodies were undetectable after thyroid hormone withdrawal or after thyrotropin stimulation. Patients who died of causes unrelated to PTC were considered cured or not cured according to the clinical status at the time of death.

At the end of the study, 86 of 105 patients (81.9%) were cured. In contrast, 19 of 105 patients (18.1%) had persistent disease. Of these, 7 of 19 (36.8%) died of metastatic PTC.

As a standard procedure, all patients were treated with one or more doses of ¹³¹I (30–100 mCi per dose) to ablate postsurgical thyroid remnants. When required, cumulative amounts up to 1120 mCi ¹³¹I were used.

Staining for FoxP3

Immunohistochemical analysis of FoxP3 expression was performed on formalin-fixed, paraffin-embedded tumor sections using mouse monoclonal anti-human FoxP3 antibody (ab22510; Abcam, Cambridge, United Kingdom). Sections were stained using the Ventana automated slide stainer (Ventana Medical Systems, Tucson, AZ).

Expression of FoxP3 in epithelial tumor cells and lymphocytes was evaluated independently by two investigators (CU and FB) who were blinded to clinical-pathologic data. Discrepancies between the two observers were discussed with a third pathologist (AP). Cytoplasmic staining of ≥25% tumor cells was considered positive. The possible association of FoxP3 expression with clinical/pathologic features, BRAF status, and survival data were investigated by statistical analysis.

Moreover the authors evaluated the presence of FoxP3 expression in T lymphocytes collateral to the tumor, scoring as positive or negative in 10 high power fields. The intensity of the staining was evaluated as weak and strong.

As a control thyroid tissue from 20 normal glands underwent surgery for head and neck tumors was stained. All the cases were negative for FoxP3 stain.

BRAF mutational analysis

BRAF analysis was conducted in 74 cases of 105. Excluded cases were inadequate for mutational analysis because these were old specimens (before 1990) that were fixed in an inappropriate fixative.

In the samples amenable to analysis, detection of a BRAF mutation was performed by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and direct DNA sequencing. PCR-SSCP screening of BRAF mutations was performed by amplifying exon 15 according to a standard procedure. DNA was used as a template in a 20 μL PCR mixture containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂ (pH 8.3), 0.2 mM dNTPs, 8 pmol of amplimers, and 1.25 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA). PCR primers for the BRAF exon 15 were as follows: 5′(F)-TCC TTT ACT TAC TAC ACC TCA GAT-3′ and 5′(R)-AGT GGA AAA ATA GCC TCA AT-3′. The amplicon size was 167 bp. Cycling conditions were as follows: initial denaturation (94°C, 5 minutes), then 35 cycles (denaturation, 94°C for 40 seconds; annealing, 55°C for 40 seconds; synthesis, 72°C for 40 seconds), followed by a final extension of 5 minutes at 72°C. All PCR products were visualized by electrophoresis in 2% agarose gels and purified using a PCR purification kit (Qiagen, Crawley, West Sussex, United Kingdom). Purified products were then diluted 1:1 with denaturing solution (1% xylene cyanol, 1% bromophenol blue, 0.1 mM EDTA, and 99% formamide), boiled for 5 minutes and immediately placed in ice to prevent the annealing of single-strand products. SSCP screening was carried out on the GenePhor Electrophoresis Unit using GeneGel Excel 12.5/24 (12.5% T, 2% C), according to manufacturer's instructions (GE Healthcare, Milano, Italy). Electrophoresis (600 V, 25 mA, 15 W) was performed at 18°C for 100 minutes. Gels were stained with PlusOne Silver Staining Kit (GE Healthcare), according to the manufacturer's instructions. Altered migration patterns in two or three independent PCR-SSCP runs were indicative of DNA mutations. Purified PCR products were then sequenced by a 3130 Genetic Analyzer (Applied Biosystem) automated sequencer using the Big Dye Terminator 3.1 (Applied Biosystem). DNA sequences were compared with the wild type exon 15 of the BRAF gene by using the Basic Alignment Search Tool (BLAST) software available at the National Center for Biotechnology Information.

Statistical methods

Overall survival (OS) was defined as the time elapsed from the date of surgery to the date of death or the date of the last follow-up. The disease-free survival (DFS) time was defined as the time elapsed from the date of surgery to the date of local recurrence or metastasis. The chi-square test was used to analyze the correlation of clinical and pathological data with FoxP3 expression. The population survival curve from a sample was analyzed with Kaplan–Meier analysis. Statistical significance was assessed using the log-rank test. The Statistica 6.0 software (StatSoft, Inc. Tulsa, OK) was used, and p values <0.05 were considered statistically significant.

Results

Expression of FoxP3 in PTC cells and infiltrating lymphoid cells

Among the tumor specimens that were immunostained for FoxP3, 45 of 105 (42.9%) scored positive, whereas 60 of 105 (57.1%) were negative. FoxP3 staining was localized predominantly in the cytoplasm of tumor cells. In a few specimens, both cytoplasmic and nuclear staining was observed. The intensity of staining was weak for all samples, with the exception of sporadic infiltrating cells. Of 105 tumor specimens immunostained for FOXP3, 29 (28%) were scored as positive in lymphocytes collateral to the tumors, and 76 (72%) as negative (Fig. 1A, 1B).

FIG. 1.

Expression of FoxP3 in epithelial cells of tall cell papillary thyroid carcinoma (A) and in infiltrating lymphocytes (B).

Expression of FoxP3 and clinical/pathologic data

As shown in Table 2, the correlation of FoxP3 expression with clinical/pathologic characteristics was significant only for the presence of extrathyroid invasion (p=0.04) and the presence of distant metastasis (p=0.04). No significant association was observed among FOXP3 expression in lymphocytes and clinico-pathological features analyzed.

Table 2.

Correlation of Clinical/Pathologic Features of Papillary Thyroid Carcinoma with Expression of FoxP3 in Tumor Cells

	No./total (%)
Clinical/pathologic features	FoxP3-positive	FoxP3-negative	p
Age
<45	23/45 (51)	36/60 (60)	NS
≥45	22/45 (49)	24/60 (40)
Sex
Male	6/45 (13)	17/60 (28)	NS
Female	39/45 (87)	43/60 (72)
Tumor size (cm)
≤2	25/39 (64)	35/55 (64)	NS
>2	14/39 (36)	20/55 (36)
Presence of tumor capsule
Yes	3/39 (8)	9/54 (17)	NS
No	36/39 (92)	45/54 (83)
Multifocality
Yes	8/38 (21)	10/45 (22)	NS
No	30/38 (79)	35/45 (78)
Bilaterality
Yes	3/35 (9)	2/42 (5)	NS
No	32/35 (91)	40/42 (95)
Extrathyroid invasion
Yes	15/39 (38)	11/55 (20)	0.04
No	24/39 (62)	44/55 (80)
Node metastasis
Yes	15/44 (34)	19/58 (33)	NS
No	29/44 (66)	39/58 (67)
Distant metastasis
Yes	5/44 (11)	1/58 (2)	0.04
No	39/44 (89)	57/58 (98)
Clinical outcome
Cured	36/45 (80)	50/60 (83)	NS
Not cured	9/45 (20)	10/60 (17)
Survival rate
Surviving	41/45 (91)	57/60 (95)	NS
Not surviving	4/45 (9)	3/60 (5)
No. of ¹³¹I dose(s)
1	26/45 (58)	37/55 (67)	NS
>1	19/45 (42)	18/55 (33)
Histotype of papillary thyroid carcinoma
Classic variant	37/45 (82)	40/60 (67)	NS
Follicular variant	2/45 (4)	10/60 (17)	NS
Solid variant	3/45 (7)	6/60 (10)	NS
Tall cell variant	2/45 (4)	2/60 (3)	NS
Oxyphilic variant	1/45 (2)	1/60 (2)	NS
Diffuse sclerosing variant	0 (0)	1/60 (2)	NS

NS, not significant.

No association was detected between FoxP3 expression and age, sex, tumor size, presence of tumor capsule, multifocality, lymph node metastasis, or histotype.

Comparing the survival patient cohort with the clinical/pathologic characteristics (Table 3), only one feature was significantly correlated with poor survival rates: presence of extrathyroid invasion (p=0.03). Other parameters were not significantly correlated.

Table 3.

Correlation of Clinical/Pathologic Features of Papillary Thyroid Carcinoma with Expression of FoxP3 in Tumor Cells in Survival Patient Cohort

	No./total (%)
Clinical/pathologic features	FoxP3-positive	FoxP3-negative	p
Age
<45	23/41 (56)	36/57 (63)	NS
≥45	18/41 (44)	21/57 (37)
Sex
Male	5/41 (12)	16/57 (28)	NS
Female	36/41 (88)	41/57 (72)
Tumor size
≤2	25/37 (68)	35/52 (67)	NS
>2	12/37 (32)	17/52 (33)
Presence of tumor capsule
Yes	3/37 (8)	9/51 (18)	NS
No	34/37 (92)	42/51 (81)
Multifocality
Yes	7/34 (21)	9/42 (21)	NS
No	27/34 (79)	33/42 (79)
Bilaterality
Yes	3/32 (9)	2/40 (5)	NS
No	29/32 (91)	38/40 (95)
Extrathyroid invasion
Yes	13/37 (35)	8/52 (15)	0.03
No	24/37 (65)	44/52 (85)
Node metastasis
Yes	13/40 (33)	16/55 (29)	NS
No	27/40 (68)	39/55 (71)
Distant metastasis
Yes	2/40 (11)	0/57 (0)	NS
No	38/40 (89)	55/57 (100)
Clinical outcome
Cured	32/41 (76)	50/57 (88)	NS
Not cured	9/41 (20)	7/57 (12)
No. of ¹³¹I dose(s)
1	24/41 (59)	34/52 (65)	NS
>1	17/41 (41)	18/52 (35)
Histotype of papillary thyroid carcinoma
Classic variant	35/41 (82)	37/57 (65)	NS
Follicular variant	2/41 (5)	10/57 (18)	NS
Solid variant	2/41 (5)	6/57 (11)	NS
Tall cell variant	1/41 (2)	2/57 (4)	NS
Oxyphilic variant	1/41 (2)	1/57 (2)	NS
Diffuse sclerosing variant	0 (0)	1/57 (2)	NS

Expression of FoxP3 and overall survival time and disease-free survival time

No correlation was found between expression of FoxP3 and OS or DFS. Similarly, FoxP3 positivity scores failed to correlate with OS or DFS. Moreover, no correlation was found between the FOXP3 expression in lymphocytes and OS and DFS or with radioactive iodine therapy.

Expression of FoxP3 and radioiodine therapy

As shown in Fig. 2A, expression of FoxP3 in PTC cells was significantly associated with the total dose of ¹³¹I that had been used for each case (p=0.041), not with the number of doses administered to each patient (p>0.1; Table 2). No significant correlation was detected between expression of FoxP3 in infiltrating lymphoid cells and the total dose of ¹³¹I used for each patient (Fig. 2B).

FIG. 2.

Expression of FoxP3 in papillary thyroid carcinoma cells was significantly associated with the cumulative dose of ¹³¹I that had been used for each case (A). No significant correlation was detected between expression of FoxP3 in infiltrating lymphoid cells and the total dose of ¹³¹I used for each case (B).

Expression of FoxP3 and BRAF status

Forty-four (62%) of the 72 cases analyzed were wild-type for BRAF and 27 (38%) harbored the V600E mutation. No correlation was found between expression and positivity of FoxP3 and BRAF status.

Discussion

In general, PTC has a good prognosis with high survival rates and a high cure rate. Only 10%–15% of cases are not cured by combined surgical and radioiodine treatments. The latter cases usually require more aggressive treatments that often fail to reach a definitive cure (27).

In this study, clinical/pathologic and prognostic features of a large series of cases were correlated with FoxP3 expression in tumor and infiltrating cells as a potential prognostic biomarker. Recently, FoxP3 expression, both in epithelial tumor cells and in collateral lymphocytes, has been correlated with the outcome of a variety of tumors. Hinz et al. (10) suggested that FoxP3 expression in epithelial pancreatic cancer cells could represent an example of molecular mimicry and could reveal a hidden mechanism of immune evasion by cancer cells. However, the function of FoxP3 in tumor cells could be extremely subtle since it has been reported that FoxP3 may down-regulate intracellular PDE3B (cyclic nucleotide phosphodiesterase 3B) in Tregs (28). PDE3B, in turn, is associated with the increased expression of metabolic fitness genes, better survival, and reduced apoptosis of tumor cells (29). Merlo et al. (12) reported that FoxP3 expression in breast cancer is associated with the survival rate, but not with local relapse. They hypothesized that expression of this marker was related to metastatic potential, not only to the suppression of the local immune response. Thus, FoxP3 was believed to influence the development of distant metastasis in breast cancer. In thyroid cancer, detection of infiltrating Tregs correlates with aggressive disease. Patients with tumor-associated lymphocytes (particularly FoxP3-positive cells in tumor-involved lymph nodes) exhibit disease progression and increased incidence of invasion and metastasis (30).

FoxP3 binds to the transcriptional start site of the chemokine receptors CXCR4 and CCR7 (31). CCR7 is a critical signal that determines T cell exit from peripheral tissue and is supposed to play a role in cancer invasion and metastasis (32). The CXCR4 receptor and its ligand CXCL12 are expressed widely in human cancers. Both are associated with disease progression at the levels of tumor cell proliferation, invasion, and angiogenesis. Righi et al. (33) showed that therapy with a selective CXCR4 antagonist in ovarian cancer resulted in increased tumor apoptosis and necrosis, reduction in intraperitoneal dissemination, and selective reduction of tumor-infiltrating FoxP3-positive T cells.

Recent evidence indicates that interactions between chemokine receptors and their ligands may also have critical roles in the development of PTC, including stimulating growth, progression, and metastasis. In particular, a strong association between CXCR4 expression, mutation of BRAF (serine/threonine-protein kinase B-Raf), and the degree of neoplastic infiltration has been reported (23). These data indicate that the CXCR4 expression associated with oncogenic activation may represent a determinant of the local aggressiveness of neoplastic cells. It has been hypothesized that, also in PTC, CXCR4 expression and BRAF mutation status may cooperate in conferring a particularly aggressive tumor phenotype (23). One possibility is the direct involvement of inflammatory cells (e.g., T lymphocytes and Tregs). Our data show that expression of FoxP3 can be detected not only in tumor-infiltrating T cells, but also in epithelial neoplastic cells. Expression of this marker appears associated with two negative prognostic factors (i.e., extrathyroidal tumor invasion and distant metastasis). More importantly, FoxP3 expression in neoplastic cells is associated with a resistance phenotype to radioiodine treatment. Here, it should be recalled that BRAF mutations are frequent in PTC and may be associated with loss of the sodium/iodide symporter (NIS). This event is associated with radioiodine-refractory disease (34).

In the investigated cohort, expression of FoxP3 was not associated with overall survival. The very nature of PTC may explain these seemingly contradictory data. In fact, PTC is characterized by a good prognosis with high survival rates of over 20 years (27). In our series of 105 patients, only 9% died of the disease.

In conclusion, the data show an association of FoxP3 expression with features of PTC that seem to have a specific impact on radioiodine sensitivity.

Footnotes

Acknowledgments

This study has been supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan, Italy); Istituto Toscano Tumori (Florence, Italy); Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR, Rome, Italy).

Author Disclosure Statement

The authors declare no conflicts of interest.

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