TSH-FREE THYROXINE DISCORDANCE IN AN ATHYREOTIC PATIENT DURING IPILUMINAB AND NIVOLUMINAB THERAPY

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1 Case Report TSH-FREE THYROXINE DISCORDANCE IN AN ATHYREOTIC PATIENT DURING IPILUMINAB AND NIVOLUMINAB THERAPY Jacqueline Jonklaas, MD ABSTRACT Objective: The objective of this case report is to illustrate the clinical confusion resulting from discordance between serum thyrotropin and free thyroxine measurements in an athyreotic patient receiving immunotherapy with ipiluminab and nivoluminab. Methods: The case of a 50-year-old athyreotic man with metastatic melanoma is described. The patient was discontinued from his levothyroxine replacement by his primary care physician based on an elevated free thyroxine concentration of 3.87 ng/dl. Shortly afterwards, the patient was started on immunotherapy with ipiluminab and nivoluminab. After a subtherapeutic levothyroxine dose of 0.68 µg/kg/day was initiated concurrently with his immunotherapy, the patient continued to have a high free thyroxine level but additionally developed an elevated serum thyrotropin value of 33 miu/l. He also became symptomatically hypothyroid over a period of 4 months. Resumption of a full weight-based dose resulted in normalization of the patient s thyrotropin and free triiodothyronine, but there was persistence of the free thyroxine elevation. Results: The patient s immunotherapy was associated with development of marked discordance between his free thyroxine values measured using a luminescent oxygen Submitted for publication August 19, 2015 Accepted for publication November 16, 2015 From the Division of Endocrinology, Georgetown University, Washington, DC. Address correspondence to Dr. Jacqueline Jonklaas, Division of Endocrinology, Georgetown University, Suite 230, Building D, 4000 Reservoir Road, NW, Washington, DC jonklaaj@georgetown.edu. DOI: /EP15975.CR To purchase reprints of this article, please visit: Copyright 2016 AACE. channeling immunoassay and his serum thyrotropin. This discordance resulted in clinical confusion that lead to the patient s levothyroxine being severely underdosed for his athyreotic state. It subsequently became clear, based on mass spectrometry methods and use of an alternate electrochemiluminescent assay to measure free thyroxine concentrations, that the patient s serum thyrotropin concentration accurately indicated his clinical situation and that he required an increase in his levothyroxine dose. Conclusion: Heightened awareness of the potential for multiple situations to cause inaccurate assessment of free thyroid hormone concentrations using different immunoassays will allow for interpretation of anomalous thyroid function results with such confounding factors in mind. With this knowledge, prevention of this patient s prolonged period of iatrogenic hypothyroidism would have been possible. (AACE Clinical Case Rep. 2016;2:e296-e301) Abbreviations: BMI = body mass index; BW = body weight; FT3 = free triiodothyronine; FT4 = free thyroxine; LT4 = levothyroxine; T4 = tetraiodothyronine; T3 = triiodothyronine; TSH = thyrotropin INTRODUCTION Ipiluminab and nivoluminab are immune modulators with efficacy for treating metastatic melanoma by virtue of blocking cytotoxic T lymphocyte associated antigen 4 interaction with its ligands and blocking the programmed death receptor 1, respectively (1,2). Ipiluminab, in particular, has been noted to have multiple endocrine-related side effects (3-6). The most documented is hypophysitis, with resultant secondary adrenal insufficiency (3-6), and less often, secondary hypothyroidism and hypogonadism (3,7). Thyroiditis (5) and adrenalitis (7) have also been e296 AACE CLINICAL CASE REPORTS Vol 2 No. 4 Autumn 2016 Copyright 2016 AACE

2 Copyright 2016 AACE TSH-Free Thyroxine Discordance, AACE Clinical Case Rep. 2016;2(No. 4) e297 reported. Development of discordance between measurement of thyroid hormones and thyrotropin (TSH) during immune modulator therapy has not been described previously. We describe a patient whose treatment with ipiluminab and nivoluminab coincided with a new discrepancy between the free thyroxine (FT4) value measured using a luminescent oxygen channeling immunoassay using the Siemens Vista platform and his serum TSH, while the FT4 values measured by two other methods (an electrochemiluminescence immunoassay using the Elecsys 2010 Modular Analytics system and tandem mass spectrometry) remained concordant. The clinical confusion that this caused is discussed. CASE REPORT A 50-year-old man presented for treatment of metastatic melanoma. He had been diagnosed with stage IIA melanoma 3 years prior. He was then determined to have metastatic disease after a computed tomography scan performed for evaluation of flank pain showed lung and liver lesions. Stage I papillary thyroid cancer had been diagnosed 15 years earlier. Following this thyroidectomy, he received radioactive iodine therapy using an activity of 30 mci and was prescribed a suppressive dose of levothyroxine (LT4) of 175 µg. His body weight (BW) at this time was 99 kg, and his body mass index (BMI) was 28.9 kg/m 2. A whole-body radioiodine scan 1 year later did not show any iodine-avid tissue. He had not continued follow-up with his endocrinologist, but his primary physician had continued his 175-µg LT4 dose with TSH values of 0.01 to 0.1 miu/l being achieved and also had documented adherence to his LT4 regimen. When presenting to his primary care physician in October 2014 prior to treatment for his melanoma, the patient described multiple symptoms of hyperthyroidism. He described himself as bouncing off the walls. His BW at his time was 110 kg, and his BMI was 32.1 kg/ m 2. The patient had no contact with animals in his job or personal life. Based on his low serum TSH value of 0.1 miu/l (normal, 0.4 to 4.5 miu/l) and high FT4 value of 3.87 ng/dl (normal, 0.76 to 1.46 ng/dl) (see Table 1), he was taken off LT4 by his primary physician. He consulted with an oncologist, and in November 2014, he was started on combination therapy with ipiluminab and nivoluminab. He was also restarted on a lower doses of LT4 of 75 µg. He was not taking any supplements such as biotin. For the next several months, he continued on checkpoint inhibitor therapy, although he was switched to nivoluminab as a single therapy in February From November through February he was symptomatically hypothyroid, with complaints of constipation, weight gain, and fatigue. By February 2015, his TSH had risen progressively to 33.1 miu/l, although his FT4 remained elevated at 1.74 ng/ dl (normal, 0.76 to 1.46 ng/dl). The monitoring of his thyroid function is shown in Table 1, with all testing being performed at the main hospital laboratory with a luminescent oxygen channeling immunoassay using the Siemans Dimension Vista Analyzer. The FT4 assay used a biotinylated anti-t4 mouse monoclonal antibody. The free triiodothyronine (FT3) assay used a biotinylated anti-t3 sheep monoclonal antibody. In March 2015, he was referred to an endocrinologist because of his elevated serum TSH value of 33.2 miu/l that had persisted despite an elevated FT4 value of 2.16 ng/dl (normal, 0.76 to 1.46 ng/dl). At this time, his FT3 concentration was below the reference interval at 1.52 pg/ ml (normal, 2.18 to 3.98 pg/ml) (see Table 1). His physical examination upon referral showed multiple stigmata of thyroid hormone deficiency, including cool, dry skin and delayed relaxation of his deep tendon reflexes. His total protein was normal at 6.8 mg/dl, albumin was normal at 3.9 g/dl, and thyroxine-binding globulin was normal at 16 µg/ml. Additional thyroid evaluation performed at outside laboratories showed low FT4, FT3, total T4, and total T3 values using their standard methodology. Specifically, FT4 measured by tandem mass spectrometry was low at 0.55 ng/dl (normal, 0.8 to 1.7 ng/dl), FT4 measured by electrochemiluminescent immunoassay using a polyclonal sheep anti-t4 antibody and the Elecsys 2010 Modular Analytics system was low at 0.7 ng/dl (normal, 0.82 to 1.77 ng/ dl), and FT3, also measured by electrochemiluminescent immunoassay using a monoclonal sheep anti-t3 antibody and the Elecsys 2010 Modular Analytics system, was low at 1.2 pg/ml (normal, 2.0 to 4.4 pg/ml) (see Table 2). The patient was restarted on 175-µg LT4, with a prompt normalization of his serum TSH to 1.48 miu/l. Despite the continued normal TSH ranging between 0.44 and 1.48 miu/l, the pattern of the patient s free thyroid hormones measured at the main hospital laboratory persistently showed an elevated FT4 of between 2.78 and 3.66 ng/ dl (normal, 0.76 to 1.46 ng/dl) but a normal FT3 concentration of between 2.40 and 3.11 pg/ml (normal, 2.18 to 3.98 pg/ml). The patient felt well without symptoms of hypothyroidism. Testing for T4 antibodies, T3 antibodies, and human anti-mouse antibodies were all negative (see Table 2). Additional thyroid testing at the outside laboratories could not be repeated because of insurance restrictions. DISCUSSION This patient was not recognized as being hypothyroid by his nonendocrinology health care team because his FT4 was reported as being high when measured by the institutional luminescent oxygen channeling immunoassay, at a time his serum TSH was also high. His FT4 values measured by an alternate electrochemiluminescent assay and by tandem mass spectrometry were, however, low. Prior to beginning his immune modulator therapy he was considerably overtreated with LT4 and was symptom-

3 e298 TSH-Free Thyroxine Discordance, AACE Clinical Case Rep. 2016;2(No. 4) Copyright 2016 AACE Date TSH (normal range miu/l) Table 1 Serial Thyroid Test Results From Main Hospital Laboratory FT4 a (normal range ng/dl) FT3 b (normal range pg/ml) FT4/FT3 ratio Ipi/ Nivo Nivo 9/ / / LT4 dose µg Symptoms 9/1/ hyperthyroid 10/22/ off hyperthyroid 11/17/ X 75 hypothyroid 11/21/ X 75 hypothyroid 11/25/ X 75 hypothyroid 12/9/ X 75 hypothyroid 12/17/ X 75 hypothyroid 12/30/ X 75 hypothyroid 1/6/ X 75 hypothyroid 1/27/ X 75 hypothyroid 2/6/ X 75 hypothyroid 2/17/ X 75 hypothyroid 3/3/ X 75 hypothyroid 3/17/ X 75 hypothyroid 3/31/ X 175 euthyroid 4/14/ X 175 euthyroid 4/28/ X 175 euthyroid 5/12/ X 175 euthyroid 5/26/ X 163 euthyroid 6/9/ X 163 euthyroid 6/23/ X 163 euthyroid 7/7/ X 163 euthyroid 7/21/ X 163 euthyroid Abbreviations: FT3 = free triiodothyronine; FT4 = free tetraiodothyronine; Ipi = ipiluminab; LT4 = levothyroxine; Nivo = nivoluminab; TSH = thyrotropin. Free thyroid hormones were measured by the main hospital laboratory employing a luminescent oxygen channeling immunoassay using the Siemens Dimension Vista Analyzer from 2012 onwards. Regular font indicates concordance between TSH and FT4 prior to immunotherapy. Bold indicates discordance between TSH and FT4 after immunotherapy. a The FT4 assay used a biotinylated anti-t4 mouse monoclonal antibody. b The FT3 assay used a biotinylated anti-t3 sheep monoclonal antibody. atically hyperthyroid. His October 2014 laboratory results were consistent, with a low TSH and a high FT4 value. Once treated with immunotherapy in the form of ipiluminab and nivoluminab, the patient s FT4 and TSH values became discordant: he had elevated FT4 values accompanying his high TSH values. However, his TSH and FT3 levels were concordant: high TSH values were accompanied by low FT3 values (see Table 1 and Fig. 1). Moreover, the elevated TSH and low FT3 levels were consistent with his predicted state of underreplacement based on the subtherapeutic LT4 dose of 75 µg (BW-based dose = 0.68 µg/kg/day) and his symptoms of hypothyroidism. Upon resumption of a 1.6-µg/kg/day dose of LT4, the pattern of his thyroid analytes changed and his TSH and FT3 became normal, but his FT4 became further elevated. Subsequent additional thyroid testing at different laboratories revealed

4 Copyright 2016 AACE TSH-Free Thyroxine Discordance, AACE Clinical Case Rep. 2016;2(No. 4) e299 Thyroid analyte Date 3/26/15 FT4 by dialysis/tandem mass spectrometry (ng/dl) Table 2 Additional Thyroid Testing Patient value Reference interval FT4 a direct (ng/dl) FT3 b (pg/ml) Total T4 (µg/dl) Total T3 (ng/dl) TSH (miu/l) Thyroxine binding globulin (µg/ml) Albumin (g/dl) FT4/FT3 ratio 0.58 T4/T3 ratio Date 10/14/15 T4 antibodies negative negative T3 antibodies negative negative Human anti-mouse antibodies 13 <74 Laboratory Esoterix (Endocrine Sciences Laboratory), Calabasas Hills, CA LabCorp, Burlington, NC The free thyroid hormones were measured using an electrochemiluminescence immunoassay using the Elecsys 2010 Modular Analytics system Quest Diagnostics Nichols Institute, San Juan Capistrano, CA Abbreviations: FT3 = free triiodothyronine; FT4 = free tetraiodothyronine; LT4 = levothyroxine; T3 = triiodothyronine; T4 = tetraiodothyronine; TSH = thyrotropin. a The FT4 assay used a polyclonal sheep anti-t4 antibody. b The FT3 assay used a monoclonal sheep anti-t3 antibody. normal FT4 values by an alternate immunoassay (electrochemiluminescent assay) and by tandem mass spectrometry at the same time as the institutional immunoassay detected a high FT4 value (see Table 2). There are at least four theoretical explanations for this confusing series of thyroid function tests: (1) antibody interference with TSH measurement; (2) inhibition of the type 2 iodinase by nivoluminab; (3) antibody interference with FT4 measurements; and (4) confounding factors associated with FT4 assessment in the sample specific to the luminescent oxygen channeling immunoassay. This assay has been shown to have the same dependence on protein binding concentrations as other immunoassays (8) and, moreover, the patient did not have any binding protein abnormalities. Antibody interference with TSH measurements has been described (9) but is least likely, as at all times the patient s serum TSH was entirely consistent with his clinical picture. The pattern of these thyroid function tests shown in Table 1 is consistent with ipiluminab or nivoluminab acting as a type 2 deiodinase inhibitor. This action would explain the finding that the patient persistently had a much higher FT4 level than FT3 level: either high FT4 and low FT3, or even higher FT4 and normal FT3. In fact, while taking immunotherapy, he appeared to need a high FT4 concentration in order to achieve a normal FT3 concentration. An effect of these agents to inhibit the type 2 deiodinase has not been described previously. However, such an action on deiodinases is reminiscent of that of sorafenib, in which accelerated activity of the type 3 deiodinase has been proposed (10). An effect on the type 3 deiodinase does not seem likely to explain the effect of ipiluminab or nivoluminab, as this would be expected to be associated with a low FT4 concentration, which was not seen in this patient (Table 1). However, once the additional thyroid testing shown in Table 2 is taken into account, this proposed mechanism of an effect on deiodinases no longer becomes tenable. The values of all 4 immunoassays (T4, FT4, T3, FT3) were low according to the outside commercial immunoassay. The FT4 value measured by tandem mass spectrometry after a separation by dialysis further confirmed that the FT4 was truly low. Mass spectrometry is a method that is known to be resistant to problems such as extremes of protein binding and heterophilic antibodies (11). Thus, it becomes most likely that while being treated with ipiluminab and nivoluminab, the patient s FT4 is being incorrectly assessed in the main hospital laboratory. A possible reason for this overestimation of FT4 values could be interfering antibodies (9,12-14). The proposed antibody or antibodies would be interfering with FT4 assessment measured by the in-house assay. These antibodies would block the fixed number of

5 e300 TSH-Free Thyroxine Discordance, AACE Clinical Case Rep. 2016;2(No. 4) Copyright 2016 AACE assay anti-t4 binding sites, so that a greater amount of tracer is displaced, and the FT4 appears elevated (15). Such antibodies could be due to an immune-related side effect of ipiluminab or nivoluminab. The major possible sources of antibody interference in thyroid hormone immunoassays are heterophilic antibodies (including rheumatoid factor and human anti-mouse antibodies), and thyroid hormone autoantibodies (16). Specimens containing rheumatoid factor, for example, are known to have increased variation in FT4 and FT3 assays across platforms (17). Heterophilic antibodies, although more frequently affecting TSH assays, also affect thyroid hormone assays, and may even interfere with both T4 and T3 assays (18). T4 and T3 autoantibodies may be present in 1 to 8% of a healthy population (19) but may be more frequent in individuals with an underlying thyroid disorder (16,20). When interfering antibodies are present and falsely elevate FT4 values, the interference can be removed by treatment of the serum with polyethylene glycol (18,21) or by adding excess nonimmune immunoglobulin from the same species as the reagent antibody (16). If an interfering antibody related to immunotherapy is the responsible agent, there are several interesting considerations. The first is that neither T4 antibodies nor human anti-mouse antibodies appeared to be the culprit in this case, leaving open the possibility of an unidentified heterophilic antibody. Secondly, the erroneous FT4 readings were specific to the luminescent oxygen channeling immunoassay. Interestingly, this is the only assay that used mouse monoclonal antibodies, and again the patient tested negative for these. In addition, the FT4 and FT3 levels appear to be proportional to each other (both increasing as the LT4 dose is increased) (see Fig. 1). If the FT4 values are falsely high, one might expect the value to be consistently high and not show association with other thyroid hormone values. The final interesting issue is that the patient had 2 very high FT4 values prior to receiving immunotherapy, which he first received in November This raises the issue of whether any putative interfering antibodies could have preceded the initiation of immunotherapy and perhaps be related to the melanoma or other causes. The only factor that argues against pre-existing antibodies is that in September-October 2014, the patient was symptomatically hyperthyroid and his low TSH was entirely congruent with his elevated FT4 levels. Another potential confounding factor that has been noted to affect thyroid analyte assessment is consumption of large doses of biotin. This causes falsely elevated thyroid hormone readings in competitive assays employing biotin-streptavidin affinity in their design (22). However, this does not appear to be the explanation in this case, as the patient was not taking biotin. In addition, such interference might be expected to cause high readings in both the FT4 and FT3 assays, which were both biotin-based, rather than false elevation of only the FT4 assay. CONCLUSION Fig. 1. Relationship between serum thyrotropin and free thyroid hormone levels over time. In summary, interfering antibodies are a possible, albeit unconfirmed, mechanism accounting for the patient s FT4 being higher than his clinical situation suggests. An alternative mechanism could be unacknowledged biotin ingestion with a falsely high reading in only a biotin-based FT4 assay but not a biotin-based FT3 assay. Nevertheless,

6 Copyright 2016 AACE TSH-Free Thyroxine Discordance, AACE Clinical Case Rep. 2016;2(No. 4) e301 regardless of the mechanism(s), the serum TSH appeared to truly predict this patient s thyroid status and clearly should have been the analyte upon which clinical decisionmaking was based. The patient would have been spared a 4-month period of hypothyroidism if his LT4 had been reduced rather than discontinued in response to the FT4 value of 3.87 ng/dl. In another reported case (14), falsely elevated FT4 values led to unnecessary carbimazole treatment, whereas in this particular case the consequence was failure to recognize the patient s evolving hypothyroidism. The fact that the patient is currently euthyroid (based on his TSH) while taking 175 µg LT4, whereas he was overreplaced on this dose prior to his immunotherapy, is perhaps partially explained by his intervening weight gain of 11 kg. DISCLOSURE The author has no multiplicity of interest to disclose. REFERENCES 1. Ascierto PA, Marincola FM, Atkins MB. What s new in melanoma? Combination! J Transl Med. 2015;13: Pennock GK, Chow LQ. The evolving role of immune checkpoint inhibitors in cancer treatment. Oncologist. 2015;20: Ryder M, Callahan M, Postow MA, Wolchok J, Fagin JA. Endocrine-related adverse events following ipilimumab in patients with advanced melanoma: a comprehensive retrospective review from a single institution. Endocr Relat Cancer. 2014;21: Min L, Vaidya A, Becker C. Association of ipilimumab therapy for advanced melanoma with secondary adrenal insufficiency: a case series. Endocr Pract. 2012;18: Corsello SM, Barnabei A, Marchetti P, De Vecchis L, Salvatori R, Torino F. Endocrine side effects induced by immune checkpoint inhibitors. J Clin Endocrinol Metab. 2013;98: Weber JS, Kähler KC, Hauschild A. Management of immune-related adverse events and kinetics of response with ipilimumab. J Clin Oncol. 2012;30: Min L, Ibrahim N. Ipilimumab-induced autoimmune adrenalitis. Lancet Diabetes Endocrinol. 2013;1:e Monneret D, Guergour D, Vergnaud S, Laporte F, Faure P, Gauchez AS. Evaluation of LOCI technologybased thyroid blood tests on the Dimension Vista analyzer. Clin Biochem. 2013;46: Klee GG. Interferences in hormone immunoassays. Clin Lab Med. 2004;24: Abdulrahman RM, Verloop H, Hoftijzer H, et al. Sorafenib-induced hypothyroidism is associated with increased type 3 deiodination. J Clin Endocrinol Metab. 2010;95: van Deventer HE, Soldin SJ. The expanding role of tandem mass spectrometry in optimizing diagnosis and treatment of thyroid disease. Adv Clin Chem. 2013;61: Bolstad N, Warren DJ, Nustad K. Heterophilic antibody interference in immunometric assays. Best Pract Res Clin Endocrinol Metab. 2013;27: Koulouri O, Moran C, Halsall D, Chatterjee K, Gurnell M. Pitfalls in the measurement and interpretation of thyroid function tests. Best Pract Res Clin Endocrinol Metab. 2013;27: Ghosh S, Howlett M, Boag D, Malik I, Collier A. Interference in free thyroxine immunoassay. Eur J Intern Med. 2008;19: Tate J, Ward G. Interferences in immunoassay. Clin Biochem Rev. 2004;25: Després N, Grant AM. Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem. 1998;44: Martel J, Després N, Ahnadi CE, et al. Comparative multicentre study of a panel of thyroid tests using different automated immunoassay platforms and specimens at high risk of antibody interference. Clin Chem Lab Med. 2000;38: Fiad TM, Duffy J, McKenna TJ. Multiple spuriously abnormal thyroid function indices due to heterophilic antibodies. Clin Endocrinol (Oxf). 1994;41: Sakata S, Matsuda M, Ogawa T, et al. Prevalence of thyroid hormone autoantibodies in healthy subjects. Clin Endocrinol (Oxf). 1994;41: Sakata S, Nakamura S, Miura K. Autoantibodies against thyroid hormones or iodothyronine. Implications in diagnosis, thyroid function, treatment, and pathogenesis. Ann Intern Med. 1985;103: Sakata S, Komaki T, Ogawa T, et al. Evaluation of thyroid function in patients with thyroid hormone autoantibodies. Clin Chim Acta. 1993;219: Barbesino G. Misdiagnosis of Graves disease with apparent severe hyperthyroidism in a patient taking biotin megadoses. Thyroid. 2016;26: