Thyroglobulin Value Predict Iodine-123 Imaging Result in Differentiated Thyroid Cancer Patients

Simple Summary This study was prompted to assess the diagnostic performance of 123I-Dx-WBS-SPECT/CT in the identification of incomplete structural response in the early follow-up of DTC patients and derived optimized basal-Tg thresholds as a yardstick for scintigraphic imaging. In this light, we reviewed the records of 124 low or intermediate-risk DTC patients who underwent thyroid surgery followed by radioiodine therapy. The response to initial treatments was evaluated 6–12 months after radioiodine therapy. According to 2015 ATA criteria, 87, 19 and 18 patients were classified to have excellent response (ER), indeterminate/incomplete biochemical response (BIndR/BIR) or structural incomplete response (SIR), respectively. Among 37 patients with less than ER, 18 had a positive 123I-Dx-WBS-SPECT/CT. Interestingly, metastatic disease noted at 123I-Dx-WBS-SPECT/CT mainly involved lymph nodes of the central compartment with a corresponding negative ultrasound of the neck. The optimized basal-Tg cut-off was settled at 0.39 ng/mL by ROC curve analysis (AUC = 0.852) aiming to discriminate patients with positive or negative 123I-Dx-WBS-SPECT/CT, respectively. Basal-Tg exceeding this cutoff level independently predicts a positive 123I-Dx-WBS-SPECT/CT. In conclusion, 123I-WBS-SPECT/CT identified lymph node metastases in 14/37 patients with less than ER and negative neck ultrasound, thus modifying the management of such patients. The diagnostic performance of 123I-Dx-WBS-SPECT/CT imaging significantly increases in patients with basal-Tg levels ≥ 0.39 ng/mL. Abstract Background: In differentiated thyroid cancer (DTC) patients, the response to initial treatments is evaluated 6–12 months after radioiodine therapy (RIT) according to the 2015 American Thyroid Association (2015 ATA) criteria. In selected patients, diagnostic 131-radioiodine whole-body scintigraphy (Dx-WBS) is recommended. We evaluated the diagnostic performance of 123I-Dx-WBS-SPECT/CT imaging in detecting incomplete structural responses in the early follow-up of DTC patients and, additionally, derived optimized basal-Tg value as a yardstick for scintigraphic imaging. Methods: We reviewed the records of 124 low or intermediate-risk DTC patients with negative anti-thyroglobulin antibody. All patients had undergone (near)-total-thyroidectomy followed by RIT. The response to initial treatments was evaluated 6–12 months after RIT. Results: According to the 2015 ATA criteria, 87, 19 and 18 DTC patients were classified to have excellent response (ER), indeterminate/incomplete biochemical response (BIndR/BIR) or structural incomplete response (SIR), respectively. Among patients with less than ER, 18 had a positive 123I-Dx-WBS-SPECT/CT. Metastatic disease at 123I-Dx-WBS-SPECT/CT mainly involved lymph nodes within the central compartment, and corresponding neck ultrasound examinations were negative. The ROC curve analysis was performed to define the best basal-Tg cut-off (i.e., 0.39 ng/mL; AUC = 0.852) able to discriminate patients with and without positive 123I-Dx-WBS-SPECT/CT, respectively. The overall sensitivity, specificity, accuracy, PPV and NPV were 77.8%, 89.6%, 87.9%, 56.0% and 95.9%, respectively. Basal-Tg cut-off was an independent risk factor for having a positive 123I-Dx-WBS-SPECT/CT. Conclusion: 123I-Dx-WBS-SPECT/CT identified lymph node metastases in 14/37 patients with less than ER and a negative neck ultrasound, thus modifying the management of such patients. The diagnostic performance of 123I-Dx-WBS-SPECT/CT significantly increased in patients with basal-Tg values ≥ 0.39 ng/mL.


Introduction
Thyroid cancer represents the most frequent endocrine malignancy [1][2][3][4], with differentiated thyroid cancer (DTC) being the most common subtype (80% and more of all thyroid cancers). The incidence of DTC has been increasing in the last decades with an overall increased annual incidence of about 3%, mainly represented by papillary histotype, small tumors and female patients [5][6][7][8][9][10]. Thyroidectomy followed by risk-adapted 131 I therapy (RIT) and levothyroxine (LT4) therapy are the standard of care leading to excellent response in more than 80% of patients. Notwithstanding, long-term follow-up is recommended after primary treatment since the risk of persistent or recurrent disease is not negligible, especially among intermediate to high-risk patients [5,[11][12][13][14]. According to the 2015 ATA guidelines [15], the response to initial treatments is evaluated at 6-12 months by using basal and/or stimulated serum thyroglobulin (Tg) and neck-ultrasound (nUS). The use of diagnostic whole-body scintigraphy (Dx-WBS) with iodine radioisotopes ( 123 I or 131 I) is suggested in selected DTC patients [5,15,16]. Additional single photon emission computed tomography/computed tomography (SPECT/CT) is recommended in addition to planar imaging whenever possible [16]. Indeed, SPECT/CT is able to: (i) detect a higher number of radioiodine-avid foci than planar imaging; (ii) improve anatomic/topographic localization of radioiodine-avid foci; and (iii) distinguish pathological from aspecific uptakes, respectively. As per consequence, the diagnostic performance of radioiodine imaging significantly increased after the introduction of SPECT/CT in clinical practice, especially in detecting small metastatic lymph nodes within the central compartment [5,[15][16][17][18]. The diagnostic performance of radioiodine imaging improves in patients with higher Tg levels (either basal and/or stimulated). Thus, the present study was prompted to assess the diagnostic performance of 123 I-Dx-WBS-SPECT/CT in detecting structural incomplete response (SIR) at early DTC follow-up. In addition, we assessed the relationship between basal/stimulated Tg levels and 123 I-Dx-WBS-SPECT/CT results to derive optimized Tg cut-off(s) to select patients for 123 I-Dx-WBS-SPECT/CT, thus reducing the number of useless studies.

Patients
We retrospectively reviewed the records of 124 consecutive DTC patients [F = 94, M = 30; female to male ratio (F/M) = 3.1:1; mean age ± SD = 47.1 ± 14.1, median age = 46, range = 18- The study was conducted in accordance with the Declaration of Helsinki. In addition, the protocol was approved by the Ethics Committee of the "G. Martino" University Hospital (Project identification code 19/17). All subjects gave informed consent for their inclusion prior to enrollment in the study.

Methods
All patients had undergone (near)-total-thyroidectomy [(n)-TT] followed by RIT within 3 months. Before RIT (i.e., postoperative period), all patients were assessed by a neck ultrasound (nUS) and laboratory test [1 month after surgery] before performing RIT. Afterward, patients underwent RIT after rhTSH administration according to standard protocol (i.e., intra-muscle injection of 0.9 mg rhTSH daily for 2 consecutive days). Administered 131 I activities ranged from 2220 to 3700 MBq for ablative purpose and 3700 to 5550 MBq for adjuvant purpose, respectively. A post-therapy whole-body scan with single photon emission tomography-computed tomography (PT-WBS-SPECT/CT) was obtained in all patients (5 to 7 days after RIT), as already described [19,20]. Then, all patients were revaluated 3 months after 131 I therapy by a clinical examination and laboratory test (TSH, FT4, Tg and TgAb), while a laboratory test and additional nUS and 123 I-Dx-WBS-SPECT/CT were obtained about 9-12 months later (i.e., response to initial treatments, see specific paragraph). 123 I-Dx-WBS-SPECT/CT examinations were performed to assess the response to initial treatment in patients fulfilling the 2015 ATA criteria for scintigraphic reassessment, including those with positive PT-WBS-SPECT/CT and/or isthmus topography of malignancy, as already reported [16,21]. Additional diagnostic [e.g., fine needle cytology (FNC) and Tg measurement in the aspirate washout] or therapeutic approaches (e.g., lymph node dissection or 131 I therapy) were performed according to the patient's response to initial treatment and the judgement of the attending physician.
The response to initial treatments was assessed 8-12 months after 131 I therapy by: Finally, all patients were required to drink at least 1.5 L of water and take laxative drugs 1 day before the examination in order to achieve a better target/background ratio.
According to the 2015 ATA guidelines (15), responses to treatment were classified in three categories: (I) excellent response (ER) [i.e., stimulated Tg < 1 ng/mL, and no loco-regional and/or distant metastases at morphological and/or functional imaging; (II) biochemical indeterminate (BIndR) or incomplete (BIR) response [i.e., negative imaging and stimulated Tg between 1 and 10 ng/mL or >10 ng/mL, respectively]; and (III) structural incomplete response (SIR) [evidence of structural disease independently from Tg and TgAb results].

Statistical Analysis
Continuous data were expressed as mean and standard deviation and categorical variables as numbers ratio and relative percentage. The non-parametric approach was used since most of the numerical variables were not normally distributed, as verified by the Kolmogorov-Smirnov test. In order to assess the existence of significant differences between groups of patients (ER vs. less than ER), the Mann-Whitney test was applied with references to numerical parameters [basal Tg (day 1), early and late stimulated Tg (day 3 and day 5, respectively) and the Chi-Square test for categorical variables. The Spearman correlation test was applied in order to assess the possible correlation between the Tg values (day 1, day 3 and day 5) and 123 I-Dx-WBS-SPECT/CT results, respectively. The Receiver Operating Characteristic (ROC) curve was plotted in order to set the optimal Tg cut-off for day 1, day 3 and day 5 to predict a positive 123 I-Dx-WBS-SPECT/CT result; the area under the curve (AUC) was calculated with a relative 95% Confidence Interval and the significance. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and diagnostic accuracy (DA) were evaluated according to cut-offs resulting through ROC analysis (0.39, 0.38 and 0.71 for day 1, day 3 and day 5, respectively). Finally, univariate and multivariate logistic regression models were estimated in order to identify significantly independent predictive factors for positive 123 I-Dx-WBS-SPECT/CT. The covariates were gender, age, histotype, histological variant, size, malignant nodule topography and basal Tg (day 1) cut-off. Statistical analyses were performed using SPSS 27.0 for Window package. A p-value lower than 0.05 was considered to be statistically significant.
Demographic, clinical and pathological data of the patients with ER, BIndR/BIR and SIR are reported in Tables 2-5.      Interestingly, among patients of the Group B, no one developed structural disease during the subsequent follow-up. Patients with BIndR did not undergo any treatment (i.e., wait and see strategy) and basal Tg decreased and became undetectable over time with corresponding negative nUS examinations. Conversely, as per local protocols, BIR patients underwent an additional 131 I therapy course, and relative PT-WBS-SPECT/CT examinations showed radioiodine-avid lymph nodes located in the central compartment. At follow-up evaluation, one of these patients showed an excellent response after retreatment while the remaining one was down-staged from BIR to BIndR status, respectively. It is noteworthy that 123I-Dx-WBS-SPECT/CT was positive (i.e., abnormal foci) in all SIR patients with sensitivity, specificity, positive predictive value (PPV), NPV and accuracy of 48.6%, 100%, 100%, 82.1% and 84.6%, respectively. Metastatic disease detected at 123I-Dx-WBS-SPECT/CT involved local (i.e., VI, VII Robbins' level), regional (i.e., II-V Robbins' level) and loco-regional (i.e., VI, IV Robbins' level) lymph nodes in 13, four and one patients, respectively. Interestingly, lymph node metastases were located within the tracheoesophageal space in 9 out of 14 patients with local metastatic disease. The overall mean size of the metastatic lymph nodes was 11.1 ± 3.7 mm (median = 11, range = 6-18 mm). Notably, 123 I-planar imaging detected functional disease in 6 out of 18 patients (sensitivity 33%), confirming the significantly higher sensitivity of SPECT/CT imaging (p = 0.006). Notably, nUS was negative in 14 out of 18 patients. In particular, nUS did not detect any local lymph node metastases while pathological lymph nodes were detected in four out of five patients with regional (n = 3) or loco-regional (n = 1) metastatic disease. Accordingly, overall nUS sensitivity, specificity, PPV, NPV and accuracy were 10.8%, 100%, 100%, 72.5% and 73.4%, respectively. However, excluding patients with local lymph node metastases (n = 14), nUS diagnostic performance in detecting regional or loco-regional lymph node metastasis dramatically increased [sensitivity, specificity, positive predictive value (PPV), NPV, accuracy 80%, 100%, 100%, 98.9% and 73.4%, respectively). Regional or loco-regional lymph node metastasis was confirmed in four out of five patients by FNC with Tg washout measurement, and all SIR patients underwent functional lymphadenectomy according to local standard of care (also taking into account that in 9 out of 18 patients, the size of the metastatic lymph node was >10 mm), while no patients accepted a "wait and see" approach. No patients suffered by early or late side effects/complications due to surgical approach while lymph node metastases were confirmed at histopathological analysis in all SIR patients.
Finally, in univariate and multivariate logistic regression analysis, the basal Tg cut-off emerged as the only independent predictor of positive 123 I-Dx-WBS-SPECT/CT results regardless of age, gender, histotype, histological variant, tumor size, malignant nodule topography or risk classification, according to 2015 ATA (Table 7). Interestingly, the risk of having a positive 123 I-Dx-WBS-SPECT/CT result was 36% higher in DTC patients with basal Tg ≥ 0.39 ng/mL than others (Odds ratio = 3.60) (Figure 2). Interestingly, the risk of having a positive 123 I-Dx-WBS-SPECT/CT result was 36% higher in DTC patients with basal Tg ≥ 0.39 ng/mL than others (Odds ratio = 3.60) ( Figure  2). No pathological radioiodine uptake was noted. On the contrary, axial SPECT-CT images (C-F) showed abnormal radioiodine uptake in a small para-tracheal lymph-node (red arrows) of the right central compartment (VI Robbins' level). At the time of 123I-Dx-WBS-SPECT/CT study, nUs did not show any lymph-node with suspected/pathological features. Basal-Tg was 0.9 ng/ml while early and late-stimulated-Tg levels were <10 ng/mL (2.7 and 7.7 ng/ml, respectively).
The patient under-went functional lymphadenectomy of the right central compartment and histophatological analysis confirmed the presence of micrometastasis in 4 out of 13 removed lymph-nodes.

Discussion
Thyroid surgery followed by postoperative risk-adapted RIT therapy is the standard of care in many DTC patients, leading to an excellent response in more than 80% of patients. The response to initial treatment is commonly assessed 6-12 months after RIT by using a laboratory test (i.e., basal and stimulated Tg measurements) and nUS. In addition, the use of 131−123 I-Dx-WBS is also recommended in selected DTC patients (e.g., high risk cancers, positive TgAb, poorly informative post-therapy whole-body scintigraphy) according to the 2015 ATA guidelines [15]. More recently, its use has also been suggested in lower risk DTC patients having a malignant nodule located in the isthmus [19,21].
All in all, the use of diagnostic whole-body scintigraphy has markedly decreased in No pathological radioiodine uptake was noted. On the contrary, axial SPECT-CT images (C-F) showed abnormal radioiodine uptake in a small para-tracheal lymph-node (red arrows) of the right central compartment (VI Robbins' level). At the time of 123I-Dx-WBS-SPECT/CT study, nUs did not show any lymph-node with suspected/pathological features. Basal-Tg was 0.9 ng/mL while early and late-stimulated-Tg levels were <10 ng/mL (2.7 and 7.7 ng/mL, respectively). The patient underwent functional lymphadenectomy of the right central compartment and histophatological analysis confirmed the presence of micrometastasis in 4 out of 13 removed lymph-nodes.

Discussion
Thyroid surgery followed by postoperative risk-adapted RIT therapy is the standard of care in many DTC patients, leading to an excellent response in more than 80% of patients. The response to initial treatment is commonly assessed 6-12 months after RIT by using a laboratory test (i.e., basal and stimulated Tg measurements) and nUS. In addition, the use of 131−123 I-Dx-WBS is also recommended in selected DTC patients (e.g., high risk cancers, positive TgAb, poorly informative post-therapy whole-body scintigraphy) according to the 2015 ATA guidelines [15]. More recently, its use has also been suggested in lower risk DTC patients having a malignant nodule located in the isthmus [19,21].
All in all, the use of diagnostic whole-body scintigraphy has markedly decreased in DTC management over the last decades, likely due to suboptimal diagnostic performances in terms of both sensitivity and detection rate. In addition, the so-called stunning effect due to diagnostic 131 I activity administration further contributed to the reduced use of diagnostic whole-body scintigraphy, since it may reduce the effectiveness of subsequent RIT cycles, as reported by some authors, while others have contradicted this hypothesis [15,[22][23][24][25]. Notably, the improved sensitivity of both Tg assays and nUS also contributed to the decreased use of diagnostic whole-body scintigraphy in clinical practice [12,13,25]. All in all, a consensus on the use of diagnostic whole-body scintigraphy after initial RIT has not been reached until now [26][27][28].
Our present results are well in line with previous ones and showed a significantly better diagnostic performance of hybrid over planar imaging (p = 0.006). Indeed, 123 I planar images were positive in 6 out of 37 patients with less than ER (15.4%) and in 6 out of 18 patients with SIR (33%), respectively.
Moreover, hybrid imaging overperformed nUS in detecting lymph node metastasis within the central neck compartment. Indeed, this is not surprising due to the well-known anatomical limitations of the ultrasound exploration of such a district. As per consequence, it can be difficult for well-trained ultra sonographers to detect lymph node metastasis (especially small sized metastatic disease).
In our cohort of DTC patients with less than ER to initial treatments, 123 I-Dx-WBS-SPECT/CT changed response classification from BIndR (n = 10) or BIR (n = 4) (better prognosis) to SIR (worse prognosis) in 37.8% of patients with increased Tg and negative nUS. Accordingly, a change in patients' management was prompted.
On the other hand, negative 123 I-Dx-WBS-SPECT/CT results occurred in 19 out of 37 DTC patients with BIndR (n = 17) or BIR (n = 2). In such patients, nUS was negative as well. It should be noted that many patients in the BIndR range will spontaneously reach and maintain undetectable basal Tg levels during follow-up, thus making a Tg follow-up the most reasonable approach in such cases [15].
The most relevant result of our present study has been to set an optimized basal Tg cutoff at 0.39 ng/mL able to provide high diagnostic performance and retain an independent prognostic role, regardless of other routinely considered risk factors such as age, gender, histotype, histological variant, tumor size, malignant nodule topography and risk classification according to 2015 ATA. In addition, the risk of having a positive 123I-Dx-WBS-SPECT/CT result in DTC patients with basal Tg ≥ 0.39 ng/mL was 36% higher than in other ones (Odds ratio = 3.60).
Even if the Tg interpretation criteria are adapted to the methods employed in clinical practice due to the high inter-assay Tg variability, our results are well in line with previous studies where highly sensitive assays were adopted. Interestingly, no structural recurrences were detected over time in patients with basal hsTg values below~0.3-0.4 µg/L in newly available highly sensitive assays, including the method adopted in our study, and basal hsTg emerged as the only independent predictor of cancer relapse in multivariate analysis, conferring hazard ratios for reduced disease-free survival of 67.94 and 81.61, depending on the assay employed [33,34].
As per consequence, the basal-Tg cut-off value could be useful to address the use of 123 I-Dx-WBS-SPECT/CT in DTC patients with basal-Tg values ≥ 0.39 ng/mL and rule out such procedure in patients with lower basal-Tg values (NPV 95.9%). Some potential limitations of our study must be also addressed. First, considering the retrospective design, a selection bias cannot be completely excluded. However, the study is not interventional, clinical practice in our centers is homogeneous and strict inclusion criteria were adopted, making a significant effect unlikely. Second, the follow-up length might not be considered sufficient in our patients; nevertheless, some recurrences could turn out positive during lifelong follow-up. Indeed, most relapses occur during early follow-up and significant bias is unlikely. Third, nUS were performed in different services by different operators and this may have reduced the overall accuracy. On the other hand, it should be noted that all ultrasonographers were well trained in DTC patients' management, and used high standing US machines. Moreover, many metastatic lymph nodes were located in anatomical regions difficult to approach by nUS, such as the central neck compartment.
Finally, the differences between different Tg assays in terms of analytical and clinical performance should be carefully accounted for to correctly use Tg assays in clinical practice [13]. Accordingly, cut-off limits reported in our study should be considered with caution, and their transferability to local settings carefully evaluated.

Conclusions
123 I-Dx-WBS is a useful diagnostic tool to integrate Tg measurement and nUS in assessing the response to initial treatments in DTC patients. Planar imaging should be completed by SPECT/CT imaging whenever possible to improve the diagnostic performance of the method, especially in detecting small sized lymph node metastases within anatomical regions (i.e., the central compartment) difficult to explore by nUS. In our series, 123 I-Dx-WBS-SPECT/CT changed the response assessment (from BIndR/BIR to SIR) and, consequently, the clinical management of more than 35% DTC patients with less than ER to initial treatments. The accuracy of 123 I-Dx-WBS-SPECT/CT increases when patients are selected based on a basal serum Tg level above 0.39 ng/mL. Thus, the use of basal Tg values is suggested to increase the diagnostic accuracy of 123 I-Dx-WBS-SPECT/CT and reduce the number of useless radioiodine imaging.