Oncogenic TSHR Mutation in Papillary Thyroid Cancer

Abstract

Next-generation sequencing of papillary thyroid cancers has identified genomic alterations for which targeted therapy has proven effective. While thyroid stimulating hormone receptor (TSHR) mutations are relatively common in benign thyroid nodules, rare reports of TSHR mutations in malignant thyroid tumors are noted. Thyroid stimulating hormone (TSH) suppression is a standard of care for treatment of patients diagnosed with thyroid cancer, but for cancers harboring TSHR mutations, the effectiveness of this intervention is uncertain.

We present a case of a 49-year-old woman with a pT4aN1B papillary thyroid cancer who underwent radical thyroidectomy with bilateral neck dissection and RAI I-131 ablation, but poorly tolerated TSH suppression therapy due to tachycardia. Rising thyroglobulin levels and lymph node mapping showed recurrent/progressive disease in the thyroid bed. Genomic characterization of the tumor using a CLIA-certified next-generation sequencing assay identified an activating TSHR mutation, A623V. Clinical course and genomic profiling results were discussed in the setting of a multidisciplinary, molecular tumor board to generate therapeutic recommendations. As the TSHR A623V mutation may be associated with increased receptor sensitivity to TSH, further suppression of TSH with exogenous thyroid hormone was recommended. To achieve tolerability, beta-blocker was concurrently titrated with an increasing levothyroxine dose to control tachycardia while maximally suppressing TSH. Subsequent lymph node mapping showed no evidence of suspicious neck lymph nodes. Based on the results in this patient, aggressive therapeutic TSH suppression may represent an important therapeutic approach for patients with papillary thyroid cancer with TSH-sensitive TSHR mutations.

Introduction

Since 1980, the number of new cases of thyroid cancer more than doubled, resulting in its rank as the fifth most common malignancy in women in 2015. Thyroid cancer represents 6% of new cancer cases in women, and 60% of affected individuals are under age 55.1 Although most thyroid cancers are the well-differentiated, papillary, or follicular subtypes that carry a good prognosis, papillary thyroid cancers with poor prognostic features show the highest risk for recurrence.²Metastatic papillary thyroid cancer (PTC) can be managed effectively for prolonged periods with intermittent use of radioactive iodine (RAI) therapy, although the number of doses can be limiting. Once PTC becomes RAI-resistant or shows limited RAI accumulation, few effective treatments are available.

Thyroid stimulating hormone receptor (TSHR) and its endogenous ligand thyroid stimulating hormone (TSH) are pivotal factors in physiologic function and pathophysiology of benign and malignant thyroid diseases. Measurement of hormones involved in the thyroid axis function are used for screening and diagnosis of hyperthyroidism, hypothyroidism, and autonomously functioning nodules as well as for treatment and monitoring for thyroid cancer. TSHR is a G-protein coupled membrane receptor with an extracellular, leucine rich, ligand binding domain for TSH, 3 extracellular loops, and 3 intracellular loops linking the 7 helical structures comprising the transmembrane domain.³Constitutive activating mutations of TSHR, neuroblastoma RAS viral oncogene homolog (NRAS), and Guanine Nucleotide Binding Protein Alpha (GNAS) occur frequently in thyrotoxic disorders.^4-15Different frequencies of TSHR mutations ranging from 20% to 86% have been reported for benign thyroid diseases including toxic follicular adenomas, nodules, and multinodular goiter. TSHR mutations occur across all age ranges from infants to adults at similar frequencies, although the spectrum of mutations differs.^11,12Activating TSHR mutations have also been detected exclusively in hyper-functioning areas of the thyroid not presenting as discrete nodules, but not in nonfunctioning areas of the same gland.¹⁰The spectrum of mutations spanning the TSHR gene has been described, but the majority occurs in the transmembrane domain. In one family with familial non-autoimmune hyperthyroidism, a transmembrane region mutation, specifically TSHR L665F in helix 7, resulted in constitutive activation of TSHR and downstream activation of Gs proteins.¹⁶Such activation was thought to arise from structural changes in the receptor. Mutations in other genes such as NRAS and GNAS have also been reported for hyperfunctioning thyroid adenomas, though at variable frequencies.^5,12,16Esapa et al¹⁴evaluated a series of benign thyroid tumors, but only one GNAS R201C mutation in a Hurthle cell adenoma and one Gi2 alpha mutation R179C in a follicular adenoma were observed.

TSHR mutations are rare in malignant thyroid tumors, although individual cases have been reported including a hyper-functioning Hurthle cell carcinoma, an autonomously functioning follicular carcinoma, and papillary thyroid cancer in the setting of hyperthyroidism.^17-23Genes commonly mutated in papillary thyroid cancers include BRAF, NRAS, KRAS, and HRAS,^24-26suggesting that most of these cancers are driven by activation of the RAS-RAF-MEK signaling pathway. Moreover, the presence of mutations in the RAS-RAF-MEK pathway is associated with those papillary thyroid cancers having poor prognostic features.

Understanding the relationship between TSHR activating mutations, dependence on TSH, and development of thyroid cancer would aide in the therapeutic approach to these tumors. Herein, we describe a case of a papillary thyroid cancer found to contain the A623V activating mutation in TSHR and lacking mutations in either BRAF or NRAS. This mutation has been associated with increased sensitivity of receptor to TSH. Aggressive TSH suppression with aggressive management of symptoms resulted in clinical benefit and tumor regression.

Case Discussion

The patient was a 48-year-old woman with no family history of cancer and no known exposure to hazardous materials. She was in her usual state of health when a palpable thyroid nodule was noted on a routine physical exam. Laboratory studies showed a euthyroid state with TSH = 4.04 mIU/L (normal range 0.35-5.50m IU/L) and free T4 = 1.04 ng/dL (normal range 0.8- 1.8 ng/dL). Fine needle aspirations of a right upper thyroid nodule and a left jugular chain lymph node, identified on neck ultrasound, each showed malignant cells consistent with papillary carcinoma. She underwent thyroidectomy and right modified neck dissection. Pathology showed multifocal papillary thyroid carcinoma in the right lobe, isthmus and left lobe measuring up to 1.5 cm in greatest dimension with extension into bilateral skeletal muscle, multiple foci in adipose and fibro-connective tissue regional to the cricoid cartilage, and involvement of the bilateral re- current laryngeal nerves. No lymphovascular invasion was identified. Ten of a total of 41 lymph nodes demonstrated metastatic disease at multiple levels in the neck. Her disease was thus staged as pT4a N1b. Her postoperative course was complicated by vocal cord paralysis, extended ventilation, and MSSA tracheobronchitis resulting in a two-month recouperation with recovery of vocal cord function. A subsequent, additional left modified neck dissection showed metastatic papillary thyroid carcinoma in three of 35 lymph nodes. Unfortunately, this post-operative course was further complicated by a thoracic duct leak, chylous effusion, and bronchiolitis obliterans organizing pneumonia resulting in baseline hypoxemia and tachycardia. Replacement levothyroxine was started shortly after surgery, and 6 months post-operatively, TSH was 0.46 mIU/L.

Eight months after surgery, her TSH rose to 11.45 mIU/L and an I-123 scan with thyrogen stimulation showed a focal area of uptake at the anterior mid to lower neck with 24-hour radioiodine neck uptake value 0.8%. She completed thyroid ablation with 145 mCi I-131 one month later. A post-ablation scan 1 week later showed focal accumulation in the midline and neck, but no definite suspicious uptake outside of the neck. She was placed on levothyroxine 125mcg daily and TSH reached 0.63 mIU/L at 10 months after surgery, during which she experienced daily episodes of palpitations and tachycardia to greater than 110 beats per minute (bpm), dry skin, dry mouth, alopecia, and increased sensitivity to cold. Reducing the dose reduced the side effects but resulted in weight gain and increasing thyroglobulin from 1 ng/ mL to 8.2 ng/mL (normal range 1.5-38.5 ng/mL).

One year after initial diagnosis, she experienced an episode of palpitations, dizziness, headache, and shortness of breath. Cardiac evaluation was unremarkable with the exception of tachycardia. A chest X-ray showed a shadow within the right medial chest wall. TSH was 0.33 mIU/L and T4 was 9.2 (normal range 5.0-12.0 mcg/dL). A follow-up chest CT showed a resolving left lower lobe infiltrate and a new anterior superior mediastinal soft-tissue mass at the thoracic inlet presumed to be recurrence of the thyroid cancer. A PET/CT scan one month later revealed an asymmetric hypermetabolic focus with a maximum SUV of 6.5 situated posterolateral to the vocal cords on the right side. Relatively low-level FDG uptake was associated with the anterior soft tissue density mass reported on a prior CT scan, measuring 5.5 x 2.5 cm.

Comprehensive genomic profiling was performed on routine formalin-fixed, paraffin-embedded thyroid tissue from her thyroidectomy using the CLIA-certified FoundationOne platform.²⁷Targeted sequencing of the entire coding sequence was performed for 315 genes and introns of 28 genes involved in fusions at a depth of 500-1000X. Tumor genomic profiling from tissue from her thyroidectomy demonstrated two mutations, TSHR A623V and MDM4 S367L, but no evidence of BRAF or NRAS alterations. Her case was discussed at our institutional, multidisciplinary molecular tumor board. As the TSHR A623V mutation may result in activation in part through increased receptor sensitivity to TSH, the recommendation was to increase her levothyroxine to achieve maximal TSH suppression. Her multidisciplinary team concomitantly used beta-blockers, metoprolol 25 mg orally twice daily, and dropped her heart rate to 79 bpm, while increasing thyroid replacement. An additional RAI ablation was planned but the I—123 preliminary scan showed no RAI uptake in the previously positive area in the neck and only physiologic uptake in the salivary glands. Therefore, ablation was aborted, and shortly thereafter, her levothyroxine dose was increased from 125 μg to 137 μg. Following the titration of beta-blockers, her heart rate remained below 100 bpm and she experienced no further limitations or episodes of tachycardia and palpitations.

Evaluation 18 months post-diagnosis revealed resolution of tachycardia and a heart rate of 89 bpm. She denied any new symptoms and felt significantly better on the increased dose of levothyroxine (137 mcg daily). Her dose of metoprolol ER was escalated to 50 mg twice daily. TSH was 0.34 mIU/L. She underwent a neck ultrasound for lymph node mapping, which showed no abnormally enlarged or abnormal-appearing right-sided lymph nodes. A left-sided 15 x 6 x 9 mm lymph node in the superior jugular chain was more prominent than other lymph nodes but it appeared to have a hilum and showed normal fat echo. At 23 months post-diagnosis, her levothyroxine dose was adjusted again to 150 mcg daily and metoprolol to 75 mg twice daily with a TSH of <0.1 mIU/L and no tachycardia.

Discussion

We present a patient with a TSHR A623V-mutated, pT4aN1B papillary thyroid cancer who underwent a radical thyroidectomy with bilateral neck dissection and suffered severe post-operative pulmonary complications. Following clinical recovery, she received I-131 ablation. Symptomatic tachycardia limited TSH suppression and there was evidence of disease recurrence on suboptimal TSH suppression as shown by imaging. TSH suppression was recommended by the institutional molecular tumor board, given the potential for enhanced sensitivity of this TSHR-mutated thyroid cancer to endogenous TSH. Achieving side effect control with beta-blocker while increasing the levothyroxine dose for TSH suppression resulted in subjective and objective clinical benefit. Most TSHR mutated thyroid cancer in the literature has been associated with a hyper-functioning carcinoma or hyperthyroidism.17-22This patient was in a euthyroid state and had no histological evidence to suggest a hyper-functioning gland. However, the relationship between autonomously functioning thyroid cancer and presence of a TSHR mutation has not been systematically evaluated.

TSHR is characterized by a high level of constitutive activity in its wild-type state, but the physiologic relevance of this is not well understood.^28,40Castro et al⁴hypothesized that TSHR activating mutations result in higher levels of basal cAMP, which, in turn, lead to downstream pathway activation that promote the development of toxic follicular thyroid adenomas. Malignant thyroid tumors could also be hypothesized to arise with the same mechanism, although a second genomic alteration may be necessary for transformation. Given the relatively high frequency of TSHR mutations in non-malignant thyroid diseases but low frequency in thyroid cancer, this suggests that most papillary thyroid cancers do not arise from TSHR-mutated benign nodules.

Despite the recurrent observation of TSHR mutations in thyroid disease, debate still exists as to the relevance of TSHR mutations in the pathophysiology and clinical behavior of benign and malignant thyroid disorders. Although exon 10 is the most commonly interrogated exon for genomic evaluation of TSHR, somatic mutations affecting the third intracellular loop, sixth transmembrane segment, and second extracellular loop in addition to other receptor regions have also been described.³⁹The TSHR mutation observed in this papillary thyroid cancer occurs in the third intracellular loop of the transmembrane domain but has not been implicated as a contact residue responsible for oligomerization and contacts between transmembrane domains.^28,38Most TSHR activating mutations have been reported to be constitutively active.^{5-7,13,16,18,19,34,39}However, review of the literature suggests that only a limited number have unequivocal data supporting a link of constitutive activation.^41,42Moreover, the data do not clearly address whether mutated TSH receptors are truly ligand-independent as opposed to ligand &ldquo;hypersensitive.&rdquo; In fact, some TSHR mutations may confer constitutive activity but will either not respond to TSH or will respond similar to wild-type TSHR. In contrast, others may introduce structural changes leading to receptor stimulation at lower levels of TSH with or without change in basal TSHR activity.

The TSHR A623V, found in the papillary thyroid cancer of our patient, has been reported as occurring in well-differentiated thyroid cancer.^{17-19,21,22,34}This mutation has been shown to be oncogenic, as overexpression of TSHR A623V led to transformation of NIH3T3 cells.⁴³Intriguingly, although this mutated receptor was transforming by itself, suggesting constitutive activity, this increased activity may itself be dependent on some basal level of TSH found in normal serum conditions or due to intrinsically higher basal receptor activity. TSHR A623V transformation resulting in activation of the MAPK pathway validated findings by others that TSHR A623V showed higher basal activity as compared to wild-type TSHR, and also demonstrated greater dose-dependent, TSH-induced cAMP production as compared to wild-type TSHR.42 The latter supports the hypothesis that TSHR A623V displays ligand-dependent activity and may require some level of exogenous TSH for full activation.

Well-differentiated thyroid cancer retains dependence on TSH signaling for growth. Thus, TSH suppression therapy aimed at achieving TSH levels <0.1 mIU/L, if clinically feasible, have become a generally accepted guideline in the postoperative setting for intermediate and high-risk thyroid cancer. Adequate TSH suppression therapy often becomes suboptimal for patients such as the one reported here due to side effects. As the patient showed evidence of disease progression in the setting or suboptimal TSH suppression, this further raised the possibility that this TSHR A623V-mutated thyroid cancer may be hypersensitive to TSH. However, in this high-risk patient with recurring disease, we achieved suppression through concurrent management of tachycardia. Recently, Wang et al²⁹recognized that levothyroxine-induced TSH suppression to <0.05mIU/L and current recommended duration may cause potential harm even at normal TSH ranges. The comparison of patients with levothyroxine-induced TSH suppression and those not receiving levothyroxine in the setting of low-risk thyroid cancer showed no difference in tumor recurrence or disease-free survival over 6.5 years follow-up between these groups. Recognition of this finding resulted in a change in the 2015 ATA guidelines, whereby post-thyroidectomy TSH suppression is strongly recommended only for high-risk patients, but remains based only on &ldquo;moderate-quality evidence.&rdquo;³⁰While this patient meets criteria for high-risk disease from the outset, escalating thyroglobulin and RAI uptake, coupled with the presence of the TSHR mutation, further drove support for more aggressive TSH management. The long-term risk for osteoporosis was acknowledged for this patient, who we placed on both calcium and vitamin D supplementation.

Notable in this case of papillary thyroid cancer is the absence of other oncogenic mutations, commonly identified by other groups through next-generation sequencing, including BRAF V600E, NRAS, HRAS, and select fusions.^24,31-33For the 399 cases of papillary thyroid cancers reported in TCGA, only two mutations in TSHR, ie M453T and I640V, were reported.²⁴The papillary thyroid cancer with TSHR I640V demonstrated concomitant NRAS Q61R mutation, but neither showed an MDM4 mutation. Mutations affecting TSHR and proteins of the MAPK pathway do not appear to be mutually exclusive in thyroid cancer where comutations in both TSHR and NRAS or TSHR and KRAS have been reported.^19,20,24,34Cross et al²⁰speculated that the more aggressive tumor in their patient was a result of such a co-event. Occurrence of both BRAF and TSHR mutations in a single case has not yet been described; however, this may be due to other mechanisms affecting TSHR. For example, evidence from methylation-specific PCR suggests that epigenetic silencing of the TSHR promoter through hypermethylation occurs in roughly 73% of thyroid cancers harboring the BRAF V600E mutation, and that this correlated with elevated TSH levels.³⁵BRAF mutations have also been associated with poor prognostic features in papillary thyroid cancer and RAI resistance.^{25,26,31-33,36,37}Evaluation for associations between TSHR mutations and thyroid cancer prognosis or RAI refractory phenotype requires further evaluation.^17,20

In BRAF or RAS wild-type thyroid cancers, testing for TSHR mutation may be of greater importance for determining therapeutic actionability. However, data from this patient cannot rule out the potential benefit of testing for TSHR mutation in BRAF- and RAS-mutated papillary thyroid cancers as well. The probability of identifying a TSHR-mutated papillary thyroid cancer would be low as only 1% of papillary thyroid cancer in TCGA were TSHR-mutated.²⁴While most commercially-available tumor genomic profiling panels include BRAF and RAS, only a subset include TSHR in their panel and clinicians should consider reflex testing for TSHR, particularly in BRAF or RAS wild-type papillary thyroid cancer and in those patients with progression on suboptimal TSH suppression.

As a result of the absence of other actionable driver mutations in the RAS-RAF-MEK pathway in our case, recommendations were made for increased TSH suppression. Although the clinical significance of TSHR mutations in etiology of, and treatment of, hot thyroid nodules remains unresolved,⁴⁴this case demonstrates the association between targeting signaling through the TSH receptor and clinical response of a TSHR-mutated papillary thyroid cancer over a 6-month time period. Ideally, extended follow-up would be of interest for this patient as the longer-term benefit of TSH suppression in the setting of TSHR-mutated papillary thyroid cancer is not yet known. TSH-mediated signaling through a mutant TSHR is implicated as driving tumor growth in this case based on the temporal relationships between TSH suppression, thyroglobulin, and clinical benefit in the absence of other thyroid-directed therapies. This case raises important questions regarding more tailored use of TSH suppression therapy for specific thyroid cancers based on oncogenic drivers.

Clinical Pearls

1. Tumor genomic profiling of well-differentiated thyroid cancer identifies potentially actionable genomic alterations
2. Mutations in TSH receptor (TSHR) are found in a subset of malignant thyroid tumors and may be associated with non-hyperfunctioning gland
3. Maximal TSH suppression may be effective in some malignant thyroid tumors with TSHR mutations

Funding

Hugs for Brady, The Val Skinner Foundation, NIH P30CA072720. This research was supported by a generous gift to the Genetics Diagnostics to Cancer Treatment Program of the Rutgers Cancer Institute of New Jersey and RUCDR Infinite Biologics.

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