Bryan R. Haugen

Management of the patient with progressive radioiodine non‐responsive disease

Differentiated thyroid cancer accounts for a majority of the nearly 200,000 people in the United States with thyroid cancer. A significant minority of patients with thyroid cancer do not respond to conventional therapy (surgery, radioiodine, levothyroxine [LT4]). Current therapy for progressive, radioiodine non-responsive differentiated thyroid cancer includes surgery and external-beam irradiation (with or without low-dose weekly Adriamycin) for neck disease, and high-dose Adriamycin therapy for widely metastatic disease. Adriamycin therapy provides a 30% to 40% partial response of disease, but long-term cures are rare. Studies of combination chemotherapy do not show greater benefit than therapy with Adriamycin alone and carry understandably greater toxicity. Retinoic acid, octreotide, and tamoxifen therapies are currently being studied as future therapeutic possibilities. Chemotherapy may prove useful not only as a tumoricidal agent but as therapy for tumor re-differentiation in preparation for radioiodine therapy.

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

The molecular determinants governing cell-specific expression of the thyrotropin (TSH) β-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHβ promoter (−133 to −88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSHβ promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.

Towards improving the utility of fine‐needle aspiration biopsy for the diagnosis of thyroid tumours

Thyroid nodules are quite common with approximately 5% of the population, and approximately 10% of older individuals, having palpable thyroid nodules (Mazzaferri, 1993). Between 30 and 50% of people living in the United States and Europe have thyroid nodules noted on ultrasound examination. Fine-needle aspiration biopsy (FNAB) has become the mainstay of thyroid nodule evaluation. While the overall accuracy of FNAB is excellent, an indeterminate or suspicious biopsy can pose a diagnostic and management dilemma. Some of these issues are illustrated in the following patient vignettes.

The Use of a Combination of Galectin-3 and Thyroid Peroxidase for the Diagnosis and Prognosis of Thyroid Cancer

In this retrospective histologic study, galectin-3 had a sensitivity of 92% (22/24) for papillary thyroid carcinoma and 44% (4/9) for follicular thyroid carcinoma. Thyroid peroxidase (TPO) had a sensitivity of 50% (12/24) for papillary and 11% (1/11) for follicular carcinoma. The combination of galectin-3 and TPO had a sensitivity of 96% (23/24) for papillary and 44% (4/9) for follicular carcinoma. From a prognostic standpoint, of patients whose papillary carcinomas expressed both markers, all became free of disease. Of those whose papillary carcinomas expressed galectin-3 but not TPO, 57% (4/7) became free of disease, 29% (2/7) had persistent disease, and 14% (1/7) had progressive disease. This study confirms previous observations that galectin-3 alone is highly sensitive for papillary carcinoma but not adequately sensitive for follicular carcinoma. TPO alone is not adequately sensitive for the evaluation of any thyroid lesion. The combination of galectin-3 and TPO is complementary as a diagnostic and prognostic tool for patients with papillary carcinoma.

Programmed Death-1+ T Cells and Regulatory T Cells Are Enriched in Tumor-Involved Lymph Nodes and Associated with Aggressive Features in Papillary Thyroid Cancer

CONTEXTnRecurrent metastatic lymph node (LN) disease is common in patients with papillary thyroid cancer (PTC). Novel prognostic markers may be helpful in guiding a therapeutic approach. Our previous studies revealed that immune suppression is evident in PTC and associated with more severe disease.nnnOBJECTIVEnTo characterize the immune response to metastatic PTC, we assessed CD4(+) T cell polarization in LN. In addition, we investigated the role of programmed death-1 (PD-1) and T cell exhaustion.nnnDESIGNnUninvolved (UILN) and tumor-involved lymph nodes (TILN) were sampled ex vivo by fine-needle biopsy. T cell subsets were identified by flow cytometry. In parallel, archived TILN specimens were characterized by immunofluorescence.nnnSETTINGnThe study was conducted at the University of Colorado Hospital.nnnPATIENTSnData were collected on 94 LN from 19 patients with PTC undergoing neck dissection.nnnMAIN OUTCOMEnT cell subset frequencies were compared in UILN and TILN and assessed for correlation with recurrent disease and extranodal invasion.nnnRESULTSnRegulatory CD4(+) T cells (Treg) were enriched in TILN compared with UILN and further elevated in TILN from patients with recurrent disease. PD-1(+) T cells were present at high frequency in TILN and markedly enriched in TILN that showed evidence of extranodal invasion. In TILN, Treg frequency correlated with PD-1(+) T cell frequencies. Although PD-1(+) T cells produced interferon-γ, they failed to fully down-regulate CD27 and were not actively proliferating.nnnCONCLUSIONSnIncreased Treg and PD-1(+) T cell frequencies in LN may be indicative of aggressive recurrent PTC. Future prospective studies are necessary to determine the prognostic and therapeutic value of these findings in PTC.

Analysis of Pit-1 in regulating mouse TSH β promoter activity in thyrotropes

Abstract TSHβ gene expression is restricted to pituitary thyrotropes. Since Pit-1 is present in these cells, we characterized Pit-1 RNA and protein in thyrotropes, and tested its function in regulating TSHβ promoter activity. We demonstrate that both TtT-97 thyrotropic tumors and pituitaries contain four Pit-1 transcripts of 3.2, 2.6, 2.4, and 1.9 kb, respectively. Only two transcripts of 2.7 and 2.1 kb were detected in αTSH cells, a thyrotrope derived cell that no longer expresses TSHβ. Western analysis revealed Pit-1 protein in TtT-97 cells but not in αTSH cells. DNase I protection assays localized Pit-1 binding to three areas of the mouse TSHβ promoter. However, basal TSHβ promoter activity was minimally stimulated when αTSH cells or TtT-97 thyrotropes were co-transfected with mouse Pit-1 and a mTSHβ luciferase construct. These studies suggest that Pit-1 is not limiting for cell-specific expression of the TSHβ gene in thyrotrope-derived cells and implies that additional thyrotropic factors are likely required.

Determinants of thyrotrope-specific thyrotropin beta promoter activation. Cooperation of Pit-1 with another factor.

Thyrotropin (TSH) β is a subunit of TSH, the expression of which is limited to the thyrotrope cells of the anterior pituitary gland. We have utilized the thyrotrope-derived TtT-97 thyrotropic tumors to investigate tissue-specific expression of the TSHβ promoter. TSHβ promoter activity in thyrotropes is conferred by sequences between −270 and −80 of the 5′-flanking region. We have recently reported that the proximal region from −133 to −100 (P1) is required for promoter expression in thyrotropes. This region interacts with the pituitary-specific transcription factor Pit-1. While Pit-1 appears necessary for TSHβ promoter activity in thyrotropes, this transcription factor is not alone sufficient for promoter activity in pituitary-derived cells. In this report, we have generated a series of promoter mutations in the P1 region to identify additional protein-DNA interactions and determine their functional significance. We have found that Pit-1 interacts with the distal portion of the P1 region, and a second protein interacts with the proximal segment of this region. Each protein is able to independently interact with the TSHβ promoter, but neither alone can maintain promoter activity. Both proteins appear to be necessary for full promoter activity in thyrotropes. Southwestern analysis with the proximal segment of the P1 region (−117 to −88) reveals interaction with a 50-kDa protein. Interestingly, this protein is not found in the pituitary-derived GH3 cells and may represent a thyrotrope-specific transcription factor. Further characterization of this newly identified DNA-binding protein will further our understanding of the tissue-specific expression of the TSHβ gene.

Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer

BackgroundThyroid cancer is the most common endocrine malignancy, and many patients with metastatic differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) fail to respond to conventional therapies, resulting in morbidity and mortality. Additional therapeutic targets and treatment options are needed for these patients. We recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ATC and confers an aggressive phenotype when overexpressed in DTC cells.MethodsMicroarray analysis was used to identify downstream targets of PPARγ in ATC cells. Western blot analysis and immunohistochemistry (IHC) were used to assess thioredoxin interacting protein (TXNIP) expression in thyroid cancer cell lines and primary tumor specimens. Retroviral transduction was used to generate ATC cell lines that overexpress TXNIP, and assays that assess glucose uptake, viable cell proliferation, and invasion were used to characterize the in vitro properties of these cells. An orthotopic thyroid cancer mouse model was used to assess the effect of TXNIP overexpression in ATC cell lines in vivo.ResultsUsing microarray analysis, we show that TXNIP is highly upregulated when PPARγ is depleted from ATC cells. Using Western blot analysis and IHC, we show that DTC and ATC cells exhibit differential TXNIP expression patterns. DTC cell lines and patient tumors have high TXNIP expression in contrast to low or absent expression in ATC cell lines and tumors. Overexpression of TXNIP decreases the growth of HTh74 cells compared to vector controls and inhibits glucose uptake in the ATC cell lines HTh74 and T238. Importantly, TXNIP overexpression in T238 cells results in attenuated tumor growth and decreased metastasis in an orthotopic thyroid cancer mouse model.ConclusionsOur findings indicate that TXNIP functions as a tumor suppressor in thyroid cells, and its downregulation is likely important in the transition from differentiated to advanced thyroid cancer. These studies underscore the potential of TXNIP as a novel therapeutic target and prognostic indicator in advanced thyroid cancer.

PD-1+Tim-3+ CD8+ T Lymphocytes Display Varied Degrees of Functional Exhaustion in Patients with Regionally Metastatic Differentiated Thyroid Cancer

Severson and colleagues show that exhaustion of PD-1+ T cells in tumor-involved lymph nodes from patients with metastatic differentiated thyroid cancer was not complete. While PD-1+CD8+ T cells were variably dysfunctional in their ability to produce cytokines, their proliferative capacity was maintained, and PD-1+CD4+ T cells remained functional. Regional metastatic differentiated thyroid cancer (mDTC) provides a unique model in which to study the tumor–immune interface. These lymph node metastases persist for years, generally without progression to distant metastases. Although the immune system likely impedes disease progression, it is unsuccessful in eliminating disease. Our previous studies revealed that programmed death-1 (PD-1)+ T cells were enriched in tumor-involved lymph nodes (TILN). Tumor-associated leukocytes and tumor cells were collected from grossly involved lymph nodes from 12 patients to further characterize the phenotype and functional potential of mDTC-associated PD-1+ T cells. PD-1+CD4+ and PD-1+CD8+ T cells were enriched in 8 of 12 TILN samples. PD-1+ T cells coexpressed Tim-3 and CD69 and failed to downregulate CD27. CD8+ T cells, but not CD4+ T cells, from these samples were variably deficient in their ability to produce effector cytokines when compared with control TILNs that lacked resident PD-1+ T cells. PD-1+CD8+ T cells were capable of exocytosis but lacked intracellular perforin. Surprisingly, T-cell proliferative capacity was largely maintained in all samples. Thus, although PD-1 expression by mDTC-associated CD8+ T cells was associated with dysfunction, exhaustion was not complete. Notably, molecular markers of exhaustion did not translate to dysfunction in all samples or in CD4+ T cells. Regulatory T cells (Treg), PD-L1, and galectin-9 were commonly found in mDTC and likely contributed to the initiation of T-cell exhaustion and disease progression. Therapies that release the effects of PD-1 and Tim-3 and reduce the suppressive effects of Tregs may encourage tumor elimination in patients with mDTC. Cancer Immunol Res; 3(6); 620–30. ©2015 AACR.

A review of the potential uses for recombinant human TSH in patients with thyroid cancer and nodular goiter

Recombinant human TSH (rhTSH) has revolutionized the care of patients with differentiated thyroid cancer. Since its approval for clinical use in 2001 in Europe (1998 in the USA), rhTSH has greatly enhanced the surveillance of these patients by allowing the avoidance of hypothyroidism for TSH stimulation. Previously, a hypothyroid state was required for TSH stimulated diagnostic whole‐body radio‐iodine scans (DxWBS) and thyroglobulin (Tg) levels. Patients generally prefer rhTSH as a mechanism for TSH stimulation because symptoms of hypothyroidism can be completely avoided. Currently, rhTSH is only approved for diagnostic monitoring of differentiated thyroid cancer patients. There are many other potential uses for rhTSH, including facilitation of treatment of patients with thyroid cancer and nodular goiter. The diagnostic and therapeutic role of rhTSH in patients with differentiated thyroid cancer and nodular goiter will be discussed in this review.