Serum thyroglobulin (Tg) monitoring is a vital component of the

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1 41 Discordant Serum Thyroglobulin Results Generated by Two Classes of Assay in Patients with Thyroid Carcinoma Correlation with Clinical Outcome after 3 Years of Follow-Up David R. Weightman, Ph.D. 1 Ujjal K. Mallick, M.D. 2 John D. Fenwick, Ph.D. 3 Petros Perros, M.D. 1 1 Endocrine Unit, Freeman Hospital, Newcastle upon Tyne, United Kingdom. 2 Northern Centre for Cancer Treatment, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom. 3 Regional Medical Physics, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom. The authors thank Professor Michael Sheppard and Dr. Penny Clarke for permission to use the radioimmunoassay data, and Mrs Paula Simpson for invaluable help in accessing patients medical records. Address for reprints: Petros Perros, M.D., Endocrine Unit, Freeman Hospital, Newcastle upon Tyne NE7 7DN, United Kingdom; Fax: (011) ; Petros.Perros@ncl.ac.uk Received January 14, 2003; revision received March 19, 2003; accepted March 21, BACKGROUND. Serum thyroglobulin measurement is an integral part of monitoring patients with thyroid carcinoma, but analytic problems pose serious difficulties in the utility of this test. METHODS. Between 1997 and, serum samples from 83 patients with differentiated thyroid carcinoma were collected. Serum thyroglobulin was assayed by both radioimmunoassay and by an immunoradiometric assay. The disease status of patients with discordant serum thyroglobulin results was assessed in June Therefore, the predictive value of a single thyroglobulin measurement was assessed by evaluating the clinical status of patients 3 years later. RESULTS. Discordant serum thyroglobulin results were noted in 17 (20.4%) patients. Of the 17 patients with discordant results, 16 had adequate clinical followup data. Of these 16 patients, 11 patients had detectable levels of serum thyroglobulin by immunoradiometric assay (range, g/l) whereas levels were undetectable by radioimmunoassay ( 1 g/l). All 11 patients had evidence of metastases 3 years later. Two patients had undetectable serum thyroglobulin levels using the immunoradiometric assay ( 1 g/l), whereas they had detectable levels using radioimmunoassay (serum thyroglobulin g/l). The serum samples from both patients had normal recoveries and positive antithyroglobulin antibodies. Both patients developed metastases 3 years later. CONCLUSIONS. False-negative serum thyroglobulin results were significantly higher with the radioimmunoassay method compared with the immunoradiometric assay. The immunoradiometric assay is more reliable than the radioimmunoassay, particularly in patients who have no thyroglobulin antibodies. This finding is novel in that traditional immunoradiometric assay systems compared with radioimmunoassays usually have a higher incidence of false-negative results when assessed against clinical status. The immunoradiometric assay is subject to false-negative results in some patients with thyroglobulin antibodies, even when recovery experiments indicate the absence of interference. Thyroglobulin antibodies should be measured in all patients with differentiated thyroid carcinoma and if positive, results should be interpreted with extreme caution. Cancer 2003;98: American Cancer Society. KEYWORDS: thyroid, carcinoma, thyroglobulin, antibodies. Serum thyroglobulin (Tg) monitoring is a vital component of the assessment of patients with differentiated thyroid carcinoma (). 1 Often, clinical decisions (e.g., deciding whether patients should be subjected to further diagnostic procedures, surgery, and 2003 American Cancer Society DOI /cncr.11472

2 42 CANCER July 1, 2003 / Volume 98 / Number 1 radioiodine therapy) rely entirely on the serum Tg concentration. 2 Several assays are currently available. Because of the different methodologies, autoantibody interference, and other factors, discordance between assays is a common and serious problem. 3 7 Recent published data suggested that the threshold of serum Tg beyond which metastatic disease is likely is 2 g/l, which is close to the limit of detection of some of the available assays. 8,9 Comparisons of performance by different assays have utilized samples from patients who are known to be either free of disease or have metastases. 6,10 17 In clinical practice, physicians who manage patients with thyroid carcinoma are confronted frequently with patients whose disease status is unclear and for whom diagnostic tests may be contradictory. For example, a patient may have a negative radioiodine scan, but may have detectable serum Tg at low concentrations. 2 In this type of clinical scenario, it is crucial to perform a Tg assay. Before, requests for serum Tg from clinical oncologists in a local hospital were sent to an off-site laboratory that used an radioimmunoassay (RIA). Requests from the other two main hospitals were processed by the local laboratory, which utilized an immunoradiometric assay (). As patients with thyroid carcinoma often attended both the oncology and the surgical or endocrine clinics, serum Tg data were being generated by two different methods for the same patients. Discordant results between both assays were noted occasionally. This observation prompted the current study. Our goal was to evaluate prospectively these two commonly used assays by correlating serum Tg results with patients clinical status at the time of sampling and 3 years after the collection of samples. MATERIALS AND METHODS Study Design All requests for serum Tg received from hospitals in the city were processed by a single laboratory. Over a period of 4 months, all blood samples received by the laboratory for Tg measurement were included in the study. Serum Tg was assayed by the locally adopted. Aliquots of the same samples were sent to the University of Birmingham clinical biochemistry laboratory for measurement of Tg by RIA. During the 4-month study period, only one sample from each patient was received. Patients All patients had histologically proven. Patients disease status in June 2001 was recorded after evaluating their medical records. Disease stage was defined according to the ptnm classification. 18 All patients were receiving suppressive thyroxine therapy at the time of sampling, and all but one had undetectable levels of serum thyroid-stimulating hormone (TSH) by a third-generation assay. Assays The RIA was performed according to Black et al. 19 The lower limit of detection was 1 g/l. Intraassay and interassay variations were 2 6% and 8 15%, respectively, over the range 7 80 g/l (the reference range for normal adults was 1 35 g/l). The assay was standardized against a secondary human Tg preparation calibrated against CRM 457 [provided by Community Bureau of References of the Commission of the European Communities, Dr. Christus Profilis, Brussels, Belgium]. The methodology for the was described by Marquet et al. 20 (the kit was manufactured by Sanofi Diagnostics Pasteur, supplied by Biorad Laboratories, Harts, UK). The limit of detection (defined as the limit of confidence at 95%, i.e., 2 standard deviations for the zero point) was 1 g/l. Standard curves were produced with each assay run using human Tg standards supplied by the manufacturer (concentrations of standards were 0.2 g/l, 1.5 g/l, 5 g/l, 15 g/l, 50 g/l, 200 g/l, and 400 g/l). The human standards were calibrated against CRM 457 by the manufacturer of the kit. The intraassay and interassay variations were % and %, respectively, over the range g/l (the reference range for normal adults was 5 50 g/l). Recovery experiments were performed on all serum samples assayed by. A standard concentration of human Tg supplied by the manufacturer (30 g/l) was incubated overnight with the same volume of serum to be measured for Tg. Serum samples with and without added Tg were measured in the assay. The percentage recovery was calculated from the two Tg levels measured (percent recovery 2R /[P S] 100, where R the Tg value in the sample with added Tg; P the Tg value in the sample without added Tg; and S the concentration of added Tg). A percentage recovery between 80% and 120% indicated that there was no autoantibody interference (these data were supplied by the manufacturer in the kit insert). Anti-Tg antibodies were measured when an adequate volume of sample remained after the aliquots for serum Tg measurements (in 10 of the 17 discordant samples) were removed. A quantitative method employing an was used (TGAB Pasteur, Sanofi Diagnostics Pasteur, supplied by Biorad Laboratories). The standards were supplied by the kit manufacturer and were calibrated against the international reference preparation MRC 65/93. The intraassay and interassay coefficients of variation were % and

3 Discordant Serum Thyroglobulin Results/Weightman et al. 43 TABLE 1 False-Negative Tg Type of diagnosis Treatment before Tg recovery (%) Anti-TgAb (ku/l) Investigations and treatment 2001 Female 83 ptxn1m1 PTC 1996 Total thyroidectomy; RI 3000 MBq (for neck and mediastinal uptake); RI 5350 MBq (for neck and mediastinal uptake) Male 76 pt4n2m1 PTC 1996 Total thyroidectomy; RI 3200 MBq (for One negative challenge scan; neck lymph node metastases (histology); RIA Tg rose off thyroxine (TSH 10.1 mu/l, Tg 66 g/l) One negative challenge scan; metastases to the lung, spine, and mediastinum on CT scan with positive histology from mediastinum and spine; RI 5020 MBq CT: computed tomography; : differentiated thyroid carcinoma; : immunoradiometricassay; PTC: papillary thyroid carcinoma; RI: radioiodine; RIA: radioimmunoassay; Tg: thyroglobulin; TgAb: thyroglobulin antibody; TSH: thyroid-stimulating hormone % respectively. A concentration greater than 50 ku/l indicated the presence of antibodies (the reference range was derived from 165 normal subjects). Serum TSH levels were measured by the Immuno-1 method (Bayer Diagnostics, Berkshire, UK). Serum Tg results were considered concordant if they were undetectable or detectable by both methods. Discordance was defined as being present when serum Tg was greater than 1 g/l by one, but undetectable by the other assay. Statistical Analysis The statistical package SPSS version 11 was used to calculate the median and range and to perform the Spearman correlation analysis. RESULTS Concordance between the 2 assay methods for determining serum Tg levels was noted for 66 patients (Spearman correlation coefficient of 0.913; P 0.01). Of these, 56 patients had serum Tg levels below the limit of detection by both methods ( 1 g/l) and 10 patients had elevated serum Tg levels by both methods. Discordant Serum Thyroglobulin Assay Results False-negative immunoradiometric assay thyroglobulin Two patients had undetectable serum Tg with elevated RIA serum Tg (Table 1). Recovery experiments did not indicate any autoantibody interference. However, anti-tg antibodies were present in high concentration in serum samples from both patients. Imaging studies (radioiodine whole body scans) at the time of the abnormal result were positive in one and negative in the other patient (Table 1). Three years later, both patients had metastatic disease supported by both imaging and histologic evidence. False-negative radioimmunoassay thyroglobulin Eleven patients had detectable serum Tg levels (median, 3.6 g/l; range, g/l), but RIA serum Tg levels less than the limit of detection ( 1 g/l). Imaging studies were abnormal in three patients and normal in eight patients at the time when discordant Tg results were noted (Table 2). Ten of these patients had evidence of metastatic disease 3 years later. The remaining patient had faint uptake in the thyroid bed on the challenge scan, which subsequently disappeared. Further serum Tg measurements showed no detectable levels of serum Tg (Table 2). In this patient s case, the mildly elevated serum Tg may have been due to a thyroid remnant. Other discordant data One patient had a detectable (1.4 g/l) serum Tg concentration, whereas that concentration of serum Tg was undetectable by RIA. Three years later, the patient had received no further treatment, was clinically free of disease (Table 3), and had two negative challenge scans associated with an undetectable serum Tg concentration by. Two patients had a detectable level of RIA serum Tg with an undetectable level of Tg (Table 4). Both patients were clinically free of disease at the time of last follow-up 3 years later and both had had two negative challenge scans with undetectable serum Tg concentrations by. The Tg level was not remeasured by RIA in these three patients. Therefore, the possibility of persistent or recurrent disease (although unlikely in view of the low-

4 TABLE 2 False-Negative RIA Type of diagnosis Treatment before Tg recovery (%) Anti- TgAb (ku/ L) Investigations and treatment 2001 Female 23 pt4n2mx PTC 1997 Total thyroidectomy; RI 3340 MBq (for Female 79 ptxn1m1 FTC 1975 Total thyroidectomy; RI 3000 MBq (for ; RI 5000, 5000 MBq, (for mediastinal uptake) Female 37 pt4nxmx PTC 1995 Total thyroidectomy; RI 3000 MBq (for thyroid bed); RI 5000 MBq (for thyroid bed and mediastinal uptake) Female 21 pt3n1mx PTC (follicular variant) 1986 Total thyroidectomy; RI 3190 MBq (for thyroid bed and neck uptake); neck dissection; RI 4100 MBq (for neck uptake) Female 88 pt4n2mx PTC 1991 Total thyroidectomy; RI 3000 MBq (for Male 66 pt3nxmx FTC 1995 Partial thyroidectomy; RI 3000 MBq (for Two negative challenge scans; neck lymph node metastases (FNAB); Tg off T4 (TSH 0.05 mu/l Tg 5.2 g/l, TSH 64.1 mu/l, Tg 3.2 g/l) Positive challenge scan (mediastinal uptake); lung metastases on CT scan; Tg rises after thyroxine withdrawal (TSH 30 mu/l, Tg 190 g/l) Positive challenge scan (mediastinal metastases); RI 5000 MBq; Tg rose after thyroxine withdrawal (TSH 30 mu/l, Tg 60.1 g/l) One negative challenge scan; lung metastases on CT scan; Tg rises rises after thyroxine withdrawal (TSH mu/l, Tg 5.8 g/l) Neck lymph node metastases (histology) Positive challenge scan (; RI 3000, 3000, 5000 MBq (; lung metastases on CT scan; Tg rises after withdrawal (TSH 114 mu/l Tg 12.4 g/l) Female 81 PTC Neck lymph node metastases (FNAB) Male 75 pt4n1mx PTC 1995 Total thyroidectomy; RI 3000 MBq (for ; external beam irradiation to mediastinum Female 20 ptxn1mx PTC Total thyroidectomy; RI 3000 MBq (for Female 61 pt (multifocal) N1Mx PTC 1993 Total thyroidectomy; RI 3000 MBq (for Male 47 pt2nxmx FTC 1997 Total thyroidectomy; RI 3000 MBq (for Two negative challenge scans; mediastinal lymph nodes on CT scan; no data on Tg off T One negative challenge scan; Tg rose after T4 withdrawal (TSH mu/l), Tg 13 g/l, TSH 0.22 mu/l, Tg 1 g/l) Two negative challenge scans; Tg rose off T4 (TSH 20.9 mu/ L, Tg 10.3 g/l, TSH 0.05 mu/l, Tg g/l) Faint uptake in thyroid bed on challenge scan; negative subsequent challenge scan; Tg undetectable after thyroxine withdrawal (TSH 94.5 mu/l, Tg 1 g/l) CT: Computed tomography; : differentiated thyroid carcinoma; FNAB: fine-needle aspiration biopsy; FTC: follicular thyroid carcinoma; : immunoradiometric; PTC: papillary thyroid carcinoma; RI: radioiodine; RIA: radioimmunoassay; Tg: thyroglobulin; TgAb: thyroglobulin antibody; TSH: thyroid stimulating hormone.

5 Discordant Serum Thyroglobulin Results/Weightman et al. 45 TABLE 3 Positive, Negative RIA Tg Result Type of diagnosis Treatment before Tg recovery (%) Anti-TgAb (ku/l) Investigations and treatment 2001 Female 29 pt2n1mx PTC 1997 Total thyroidectomy; RI 3000 MBq (for Two negative challenge scans; Tg undetectable; no data of Tg off T4 : differentiated thyroid carcinoma; : immunoradiometricassay; PTC: papillary thyroid carcinoma; RI: radioiodine; RIA: radioimmunoassay; Tg: thyroglobulin; TgAb: thyroglobulin antibody; TSH: thyroid-stimulating hormone. TABLE 4 Positive RIA, Negative Tg Results Type of diagnosis Treatment before Tg recovery (%) Anti-TgAb (ku/l) Investigations and treatment 2001 Female 41 pt3n0mx PTC 1995 Total thyroidectomy; RI 3220 MBq (for ; RI 5440 MBq (for mediastinal uptake) Male 35 pt2n0mx PTC 1996 Total thyroidectomy; RI 3000 MBq (for Two negative challenge scans; Tg undetectable after thyroxine withdrawal Two negative challenge scans. : differentiated thyroid carcinoma; : immunoradiometricassay; PTC: papillary thyroid carcinoma; RI: radioiodine; RIA: radioimmunoassay; Tg: thyroglobulin; TgAb: thyroglobulin antibody; TSH: thyroid-stimulating hormone. risk status) cannot be excluded. The clinical status of these patients is therefore indeterminate. Repeat serum Tg data using both assays were performed only when specifically requested by clinicians. These data were available for 4 of the 16 patients with discordant results and the repeat samples were obtained 4 12 months after the original sample. One patient (row 1, Table 1) continued to have an undetectable serum Tg level by with low recovery (75.5) and an elevated serum Tg level by RIA (66 g/l). One patient with an elevated serum Tg concentration by (row 2, Table 2) had concordant results by both assays on subsequent testing (24.1 g/l by, 13 g/l by RIA). One patient with an elevated serum Tg concentration by and an undetectable concentration by RIA (row 3, Table 2) had similar results on repetition (2.6 g/l by, 1 g/l by RIA). Yet another patient (row 6, Table 2) who initially had a detectable serum Tg concentration by and an undetectable serum Tg concentration by RIA had concordant results on subsequent testing (serum Tg concentration by 5.8 g/l and 5.8 g/l by RIA). Serial measurements on at least two occasions in this very limited number of patients reduced the falsenegative rate of the RIA substantially. DISCUSSION Serum Tg monitoring is a valuable and sensitive tool in the follow-up of patients with (1,19). After total thyroidectomy and thyroid remnant ablation by radioiodine, serum Tg usually becomes undetectable, unless there is residual or metastatic disease. 21 Subsequently, an elevated serum Tg concentration is often the first sign of tumour recurrence. 22 Patients with elevated or rising serum Tg concentrations and negative challenge scans are not uncommon and some clinicians treat such cases with radioiodine. 2,23 In a significant proportion of these patients, the foci of abnormal radioiodine uptake can be seen in the postablation scan and the serum Tg concentration may decline after radioidine therapy. 1,23,24 Detection of even low concentrations of serum Tg (as low as 2 g/l) in patients who have been thyroidectomized and who have had radioiodine remnant ablation may indicate metastatic disease. 8,9 Undetectable serum Tg levels in the presence of metastatic disease is encountered occasionally. This may be caused by the nonsecretion of Tg by the tumor or by the secretion of molecular variants of Tg not detected by the antibody utilized in the particular assay, due to autoantibody

6 46 CANCER July 1, 2003 / Volume 98 / Number 1 interference or other technical reasons. 21 Anti-Tg antibodies are common in patients with 25 and they can interfere with assays leading to both the underestimation and overestimation of serum Tg levels. Immunoradiometric assays are more sensitive at low serum Tg concentrations and are less subject to anti-tg antibody interference than RIA. 1 For reliable detection of anti-tg antibody interference, a sensitive, quantitative assay is recommended. 25 For instance, hemagglutination assays are inadequate. A surrogate method for detecting interfering anti-tg antibodies is to perform recovery experiments. This study was undertaken because the same serum sample yielded discordant Tg results when measured by two assays. Our center s policy is to treat patients with with total thyroidectomy initially and then with radioiodine remnant ablation (3000 MBq). Challenge scans are performed 6 and 18 months after radioiodine ablation. Patients with positive scans receive further radioiodine therapy (5000 MBq) and are reassessed with challenge scans. 26 This study differs from others comparing Tg methods in that a single Tg measurement was correlated with the clinical status of patients after 3 years, during which time-independent data defining disease status (other than Tg measurements) were collected. In effect, we assessed prospectively the predictive value of a single Tg measurement using established and RIA methodologies. The most disturbing finding of this study is that false-negative serum Tg results by either assay are frequent (15.8% of a random sample of 82 patients). This is a more common problem with the RIA Tg assay (11 of 82 [13.4%]) than the (2 of 82 [2.4%]). This was an unexpected observation as many of the early RIAs were reported to show a positive interference by Tg antibodies, although occasional false-negative results were observed. 25 The RIA used in this study is well documented 20,22,27,28 and has been described as having minimal interference by Tg antibodies. 6 Nevertheless, the RIA demonstrated a higher number of false-negative results. The particular used in this study was designed to overcome the problems of autoantibody interference. This assay utilizes five monoclonal anti-tg antibodies specific for binding sites on the Tg molecule that are not believed to be autoantibody targets. 12,19,29 Theoretically, it allows a reliable assay of Tg, even in the presence of Tg antibodies. In at least two patients, the serum Tg assay failed to detect elevated serum Tg levels that were measurable by the RIA. Both patients had elevated serum anti-tg antibodies, yet recovery experiments did not indicate the presence of interference, demonstrating that recovery experiments are not always valid. Both patients had histologically proven metastatic disease 3 years later. We have demonstrated that when serum Tg is detectable by the method, this was predictive in all cases of future disease, even when serum Tg by RIA was undetectable or when 131 I scans were negative. In one patient, the serum Tg concentration was just detectable (1.4 g/l) by and was undetectable by RIA. This patient had low-risk. Two subsequent challenge scans were negative and other serum Tg measurements by revealed undetectable levels. Two patients had markedly elevated serum Tg levels by RIA (7.2 and 45 g/l), but undetectable serum Tg levels by. Although none of these three patients has shown other evidence of recurrent or persistent disease during 3 years of follow-up, their disease status remains unclear. The concordance rate improved for four patients for whom follow-up data on serum Tg levels were measured by both assays. This demonstrated that false-negative results can be overcome partially by repeated monitoring of serum Tg concentrations. Previous experience suggests that in the presence of anti-tg antibodies, immunometric assays underestimate and RIAs overestimate serum Tg concentrations. 21,25 Our data indicate that the reverse may also be true for RIA. A possible explanation for this may be poor specificity of the second antibody. Anti-Tg antibody interference is a serious problem in measuring serum Tg levels. Neither the recovery test nor measurement of anti-tg antibody can predict reliably interference in the assay. 6 Alternative methods that utilize reverse transcription-polymerase chain reaction techniques for detection of Tg mrna in peripheral blood are being developed and may overcome this difficulty. 30 False-negative serum Tg results are a serious potential analytic problem and multimonoclonal antibody s are no exception. The data from the current study illustrate the inadequacies of recovery experiments and concur with the view that they may be misleadingly reassuring. 31,32 The particular RIA used in this study was associated frequently with falsenegative results, with a higher incidence than that observed when using the, which is contrary to previous observations. 21,25 However, RIA in the presence of high anti-tg antibodies may be more reliable than. The direction of interference with RIA methods depends on several variables 33 and the above observation may not apply to other serum s. We suggest that the two assays may be complimentary. We concur with Spencer and Wang, 25 that in the presence of anti-tg antibodies an undetectable serum Tg result may be falsely reassuring and suggest

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