Cláudia C. D. Nakabashi

A preoperative diagnostic test that distinguishes benign from malignant thyroid carcinoma based on gene expression

Accurate diagnosis of thyroid tumors is challenging. A particular problem is distinguishing between follicular thyroid carcinoma (FTC) and benign follicular thyroid adenoma (FTA), where histology of fine-needle aspirates is not conclusive. It is often necessary to remove healthy thyroid to rule out carcinoma. In order to find markers to improve diagnosis, we quantified gene transcript expression from FTC, FTA, and normal thyroid, revealing 73 differentially expressed transcripts (P < or = 0.0001). Using an independent set of 23 FTCs, FTAs, and matched normal thyroids, 17 genes with large expression differences were tested by real-time RT-PCR. Four genes (DDIT3, ARG2, ITM1, and C1orf24) differed between the two classes FTC and FTA, and a linear combination of expression levels distinguished FTC from FTA with an estimated predictive accuracy of 0.83. Furthermore, immunohistochemistry for DDIT3 and ARG2 showed consistent staining for carcinoma in an independent set 59 follicular tumors (estimated concordance, 0.76; 95% confidence interval, [0.59, 0.93]). A simple test based on a combination of these markers might improve preoperative diagnosis of thyroid nodules, allowing better treatment decisions and reducing long-term health costs.

Diagnosis of Suspicious Thyroid Nodules Using Four Protein Biomarkers

Purpose: Fine-needle aspiration (FNA) cytology, a standard method for thyroid nodule diagnosis, cannot distinguish between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC). Previously, using expression profiling, we found that a combination of transcript expression levels from DDIT3, ARG2, C1orf24, and ITM1 distinguished between FTA and FTC. The goal of this study was to determine if antibody markers used alone or in combination could accurately distinguish between a wider variety of benign and malignant thyroid lesions in fixed sections and FNA samples. Experimental Design: Immunohistochemistry was done on 27 FTA, 25 FTC, and 75 other benign and malignant thyroid tissue sections using custom antibodies for chromosome 1 open reading frame 24 (C1orf24) and integral membrane protein 1 (ITM1) and commercial antibodies for DNA damage–inducible transcript 3 (DDIT3) and arginase II (ARG2). FNA samples were also tested using the same antibodies. RNA expression was measured by quantitative PCR in 33 thyroid lesions. Results: C1orf24 and ITM1 antibodies had an estimated sensitivity of 1.00 for distinguishing FTA from FTC. For the expanded analysis of all lesions studied, ITM1 had an estimated sensitivity of 1.00 for detecting malignancy. Because all four cancer biomarkers did well, producing overlapping confidence intervals, not one best marker was distinguished. Transcript levels also reliably predicted malignancy, but immunohistochemistry had a higher sensitivity. Malignant cells were easily detected in FNA samples using these markers. Conclusions: We improved this diagnostic test by adding C1orf24 and ITM1 custom antibodies and showing use on a wider variety of thyroid pathology. We recommend that testing of all four cancer biomarkers now be advanced to larger trials. Use of one or more of these antibodies should improve diagnostic accuracy of suspicious thyroid nodules from both tissue sections and FNA samples.

One Month Is Sufficient for Urinary Iodine to Return to Its Baseline Value After the Use of Water-Soluble Iodinated Contrast Agents in Post-Thyroidectomy Patients Requiring Radioiodine Therapy

BACKGROUND There is a concern regarding the use of iodinated contrast agents (ICA) for chest and neck computed tomography (CT) to localize metastatases in patients with differentiated thyroid cancer (DTC). This is because the iodine in ICA can compete with (131)I and interfere with subsequent whole scans or radioactive iodine treatment. The required period for patients to eliminate the excess iodine is not clear. Therefore, knowing the period for iodine levels to return to baseline after the injection of ICA would permit a more reliable indication of CT for DTC patients. The most widely used marker to assess the plasmatic iodine pool is the urinary iodine (UI) concentration, which can be collected over a period of 24 hours (24U) or as a single-spot urinary sample (sU). As 24U collections are more difficult to perform, sU samples are preferable. It has not been established, however, if the measurement of iodine in sU is accurate for situations of excess iodine. METHODS We evaluated 25 patients with DTC who received ICA to perform chest or neck CT. They collected 24U and sU urinary samples before the CT scan and 1 week and 1, 2, and 3 months after the test. UI was quantified by a semiautomated colorimetric method. RESULTS Baseline median UI levels were 21.8 μg/dL for 24U and 26 μg/dL for sU. One week after ICA, UI median levels were very high for all patients, 800 μg/dL. One month after ICA, however, UI median levels returned to baseline in all patients, 19.0 μg/dL for 24U and 20 μg/dL for sU. Although the values of median UI obtained from sU and 24U samples were signicantly different, we observed a significant correlation between samples collected in 24U and sU in all evaluated periods. CONCLUSION One month is required for UI to return to its baseline value after the use of ICA and for patients (after total thyroidectomy and radioiodine therapy) to eliminate the excess of iodine. In addition, sU samples, although not statistically similar to 24U values, can be used as a good marker to evaluate patients suspected of contamination with iodine.

Diagnóstico de metástases de carcinoma papilífero de tiróide através da dosagem de tiroglobulina no líquido obtido da lavagem da agulha utilizada na punção aspirativa

The widespread use of neck ultrasonography (US) during the follow-up of patients with papillary thyroid carcinoma (PTC) has led to the discovery of small cervical lymph nodes (LN). Although US has a high sensitivity for diagnosing LN, fine needle aspiration biopsy (FNA) and measurement of thyroglobulin in fine needle aspirates (FNA-Tg) have proven to be invaluable tools. The aim of this study is to determine the sensitivity of the combined use of neck US, FNA biopsy and FNA-Tg for diagnosis of cervical lymph nodes. We have studied 32 patients with 44 LN detected by US, 19 classified as inflammatory and 25 as suspicious. 15 of those 25 suspicious LN had high FNA-Tg (13 of the 15 had positive cytology and 2 indeterminate). All of these 15 LN (11 patients) were proven to be PTC metastasis by histopathology. All 19 inflammatory LN and those 10/25 suspicious LN, had cytology negative for malignancy and undetectable FNA-Tg. We conclude that fine needle aspiration biopsy and FNA-Tg combined with neck US are essential for detecting positive cervical lymph nodes due to its high sensitivity and specificity and it should be considered the standard for investigating locally recurrent disease in patients with PTC.

Basal serum thyroglobulin measured by a second-generation assay is equivalent to stimulated thyroglobulin in identifying metastases in patients with differentiated thyroid cancer with low or intermediate risk of recurrence.

Background: Guidelines for the follow-up of differentiated thyroid cancer (DTC) recommend the measurement of TSH-stimulated thyroglobulin (s-Tg) instead of basal Tg on T4 therapy (b-Tg). However, these guidelines were established using first-generation Tg assays with a functional sensitivity (FS) of 0.5-1.0 ng/ml. Current more sensitive second-generation Tg assays (Tg2G; FS 0.05-0.10 ng/ml) have shown that low-risk DTC patients with undetectable b-Tg rarely have recurrences. Objectives: This study was undertaken to compare b-Tg using a chemiluminescent Tg2G assay (Tg2GICMA; FS 0.1 ng/ml) with s-Tg in DTC patients with an intermediate risk of recurrence. Methods: We evaluated 168 DTC patients with a low (n = 101) and intermediate (n = 67) risk of recurrence treated by total thyroidectomy (147 also treated with radioiodine), with a mean follow-up of 5 years. Results: b-Tg was undetectable with the Tg2GICMA in 142 of 168 patients. s-Tg was <2 ng/ml in 138 of these 142 patients, and only 3 of these 138 (2%) presented metastases on cervical ultrasound (US). Of the 4 of 142 patients with s-Tg >2 ng/ml, 1 had cervical metastases seen after radioiodine. Furthermore, 26 of 168 patients presented detectable b-Tg with the Tg2GICMA; 17 of these 26 patients also presented s-Tg >2 ng/ml. In 10 of these 17 patients, metastases were detected. Cervical US or b-Tg were positive in 14 of 15 patients with recurrent disease. Globally, the sensitivity and negative predictive value of the Tg2GICMA plus US were 93 and 99%, respectively. Conclusion: b-Tg measured with a Tg2GICMA and cervical US, used together, are equivalent to s-Tg in identifying metastases in patients with DTC with a low or intermediate risk of recurrence.

Development, characterization and clinical validation of new sensitive immunofluorometric assay for the measurement of serum thyroglobulin

OBJECTIVE In the last decade, data published stressed the role of highly-sensitive thyroglobulin (Tg) assays in the follow-up of differentiated thyroid carcinoma (DTC) patients. The present study describes a new, highly-sensitive Tg assay, compares it with an available commercial assay, and validates it in the follow-up of DTC patients. SUBJECTS AND METHODS The immunofluorometric high-sensitivity Tg assay is based on monoclonal and polyclonal antibodies produced at our laboratories. It was validated in 100 samples of 87 patients with DTC submitted to total thyroidectomy, 87% of whom also received radioiodine. For correlation, all samples were also tested using a commercial Tg assay (Beckman Access) with functional sensitivity (FS) of 0.1 ng/mL. RESULTS The new method showed FS of 0.3 ng/mL. The correlation between the two methods was good (r = 0.74; p < 0.0001). The diagnostic sensitivity was 88.9%, and it was increased to 100% when combined with neck US. CONCLUSION This new, high-sensitivity Tg assay presented a good correlation with Beckman Access assay and with the clinical outcome of the patients. The continuous availability of a validated assay is an additional advantage for long term follow-up of DTC patients.

Avaliação de fatores clínicos, laboratoriais e ultrassonográficos preditores de malignidade em nódulos tiroidianos

OBJECTIVE: To evaluate the risk of malignancy in thyroid nodules through clinical, laboratory, ultrasonographic and cytological aspects. PATIENTS AND METHODS: 741 nodules of 407 patients. RESULTS: The cytology was benign (60,5%), indeterminate (23,3%), malignant (8,3%) or nondiagnostic (7,6%). The prevalence of cancer in indeterminate citology was 18,5% (16% in follicular lesions, 44% in suspicious). The diagnosis of malignancy was 17,2% (n = 70). The frequency of cancer in women (15,2%) was lower than in men (27,9%). There was an inverse relation between age and cancer risk. There was no statistical significance in the prevalence of cancer according to number, size of nodules or TSH levels. Hypoechogenicity and microcalcifications on ultrasound were risk factors. CONCLUSION: The risk of malignancy was higher in men, hypoechoic nodules, with microcalcifications and was inversely related to age. The TSH level was not an independent factor predictive of malignancy.

Seven-year follow-up of a juvenile female with papillary thyroid carcinoma with poor outcome, BRAF mutation and loss of expression of iodine-metabolizing genes

BACKGROUND Recent studies reported that BRAF V600E mutation, the most prevalent genetic event found in papillary thyroid carcinoma, is an independent poor prognostic marker. Additionally, it correlates with a less differentiated tumor stage due to reduced expression of key genes involved in iodine metabolism. We previously described a patient with BRAF V600E mutation in primary tumor and a new mutation (V600E+K601del) in the matched-lymph node metastases. In the present study we report an unusual clinical behavior of PTC and correlate with BRAF mutational status and level of expression of TSHR and NIS. METHODS Quantitative PCR (qPCR) was used to evaluate the NIS and TSHR level of expression in matched papillary thyroid carcinoma and adjacent normal tissue. RESULTS In this study, we presented a seven-year follow up of a juvenile papillary thyroid carcinoma patient who had an aggressive tumor harboring BRAF mutation, and failed to conventional therapy. We found a markedly decrease of NIS and TSHR expression in primary PTC compared to adjacent normal thyroid tissue. CONCLUSION Our findings suggest that BRAF mutational status and decreased NIS and TSHR expression in this patient may reduce radioiodine uptake and lead to a negative response to radioiodine therapy.

Clinical impact of thyroglobulin (Tg) and Tg autoantibody (TgAb) measurements in needle washouts of neck lymph node biopsies in the management of patients with papillary thyroid carcinoma

Objectives The presence of thyroglobulin (Tg) in needle washouts of fine needle aspiration biopsy (Tg-FNAB) in neck lymph nodes (LNs) suspected of metastasis has become a cornerstone in the follow-up of patients with papillary thyroid carcinoma (PTC). However, there are limited data regarding the measurement of anti-Tg antibodies in these washouts (TgAb-FNAB), and it is not clear whether these antibodies interfere with the assessment of Tg-FNAB or whether there are other factors that would more consistently justify the finding of low Tg-FNAB in metastatic LNs. Materials and methods We investigated 232 FNAB samples obtained from suspicious neck LNs of 144 PTC patients. These samples were divided according to the patients serum TgAb status: sTgAb- (n = 203 samples) and sTgAb+ (n = 29). The TgAb-FNAB levels were measured using two different assays. Tg-FNAB was also measured using two assays when low levels (< 10 ng/mL) were identified in the first assay of the metastatic LNs from the sTgAb+ samples. Results The TgAb-FNAB results were negative in both assays in all samples. Low levels of Tg-FNAB were identified in 11/16 of the metastatic LNs of the sTgAb+ patients and 16/63 of the sTgAb- patients (p < 0.05) using assay 1. The measurement of the Tg-FNAB levels using assay 2 indicated additional metastases in 5 LNs of the sTgAb+ patients. Conclusions Factors other than the presence of TgAb-FNAB may contribute to the higher number of metastatic LNs with undetectable Tg-FNAB in the sTgAb+ group. In addition, the measurement of Tg-FNAB using different assays was useful to enhance the diagnosis of metastatic LNs, particularly when cytological and Tg-FNAB results are discordant.

Clinical utility of 18F-FDG PET/CT in the follow-up of a large cohort of patients with high-risk differentiated thyroid carcinoma

OBJECTIVE To evaluate the clinical utility of 18F-FDG PET/CT in patients with high-risk DTC. SUBJECTS AND METHODS Single-center retrospective study with 74 patients with high-risk differentiated thyroid cancer (DTC), classified in 4 groups. Group 1: patients with positive sTg or TgAb, subdivided in Group 1A: negative RxWBS and no foci of metastases identified at conventional image (n = 9); Group 1B: RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level (n = 13); Group 2: patients with histological findings of aggressive DTC variants (n = 21) and Group 3: patients with positive RxWBS (n = 31). RESULTS 18F-FDG PET/CT identified undifferentiated lesions and helped restage the disease in groups 1B and 2. The scan helped guide clinical judgment in 9/13 (69%) patients of group 1B, 10/21 (48%) patients of group 2 and 2/31 (6%) patients of group 3. There was no clinical benefit associated with group 1A. 18F-FDG PET/CT was associated with progressive disease. CONCLUSION 18F-FDG PET/CT is a useful tool in the follow-up of patients with high-risk DTC, mainly in the group of RxWBS not compatible with suspicious foci at conventional image or not proportional to sTg level and in those with aggressive DTC variants. Additionally, this study showed that 18F-FDG PET/CT was associated with progression and helped display undifferentiated lesions guiding clinical assessments regarding surgeries or expectant treatments.