Elevated Preoperative TPO Ab Titers Decrease Risk for DTC in a Linear Fashion: A Retrospective Analysis of 1635 Cases.

CONTEXT: For some, chronic lymphocytic thyroiditis (Hashimoto thyroiditis) is an important risk factor for differentiated thyroid cancer (DTC). Surgical cohort studies even suggested a potential role for thyroid peroxidase antibodies (TPO-Abs) on that risk. OBJECTIVE: Our clinical observations argued against that possibility. We designed the present study to evaluate the relationship of TPO-Abs and DTC in a large patient population. METHODS: We recruited individuals who underwent thyroidectomies at 4 different clinical sites (USA: 1 clinic, 2000-2013, and Greece: 3 clinics, 2007-2021). We gathered data on TPO-Abs titers measured with commercially available chemiluminescence immunoassays, and reviewed patients' data including surgical pathology. TPO-Abs of 34 IU/mL or greater was deemed positive (TPO+) and TPO-Abs less than 34 IU/mL was deemed negative (TPO-). Odds ratios (OR) for DTC were calculated with the Fisher exact test and P less than .05 was deemed significant. RESULTS: We reviewed data from 8461 consecutive thyroid surgery cases. TPO-Abs titers were available for 1635 individuals: DTC n = 716 (43.8%), benign pathology n = 919 (56.2%), TPO+ n = 540 (33.0%), and TPO- n = 1095 (67.0%). DTC was found at a lower frequency in TPO+ (198/540, 36.7%) compared to TPO- (518/1095, 47.3%) patients, OR 0.64 (0.52-0.80; P < .0001). Rising TPO-Abs titers conferred protection against DTC in a linear fashion: TPO-Abs less than 10 IU/mL: 59.3%, TPO-Abs less than 34 IU/mL: 47.4%, TPO-Abs 34 to 100 IU/mL: 42.6%, TPO-Abs 100 to 500 IU/mL: 32.0%, TPO-Abs greater than 1000 IU/mL: 19.4%; P less than .0001. CONCLUSION: Higher TPO-Ab titers appear protective against DTC in our large multicenter cohort of patients who underwent thyroidectomies. Rising preoperative TPO-Abs titers conferred linearly increasing protection against DTC.

Thyroid Cancer Screening Using Tumor-Associated DN T Cells as Immunogenomic Markers.

Background: Thyroid nodules are an extremely common entity, and surgery is considered the ultimate diagnostic strategy in those with unclear malignant potential. Unfortunately, strategies aiming to predict the risk of malignancy have inadequate specificity. Our group recently found that the microenvironment of thyroid cancer is characterized by an enhanced immune invasion and activated immune response mediated by double-negative T lymphocytes (DN T) (CD3+CD4-CD8-), which are believed to enable or promote tumorigenesis. In the present work, we try to use the DN T cells' proportion in thyroid fine-needle aspiration (FNA) material as a predictor of the risk of malignancy. Methods: We recruited 127 patients and obtained ultrasound-guided FNA samples from subjects with cytology-positive or suspicious for malignancy and from those with benign nodular goiter associated with compressive symptoms (such as dysphagia, shortness of breath, or hoarseness), Hashimoto thyroiditis, and Graves' disease. Out of 127, we investigated 46 FNA samples of patients who underwent total thyroidectomy and for which postoperative histological diagnosis by the academic pathologists was available. We specifically measured the number of cells expressing CD3+CD4-CD8- (DN T) as a function of total CD3+ cells in FNA samples using flow cytometry. We correlated their FNA DN T-cell proportions with the pathological findings. Results: The DN T cells were significantly more abundant in lymphocytic infiltrates of thyroid cancer cases compared to benign nodule controls (p < 0.0001). When the DN T-cell population exceeded a threshold of 9.14%, of total CD3+ cells, the negative likelihood ratio of being cancer-free was 0.034 (96.6% sensitivity, 95% CI, 0.915-1.000, p < 0.0001). DN T cells at <9.14% were not found in any subject with benign disease (specificity 100%). The high specificity of the test is promising, since it abolishes a false-positive diagnosis and in turn unnecessary surgical procedures. Conclusion: The present study proposes DN T cells' proportion as a preoperative diagnostic signature for thyroid cancer that with integration of RNA transcriptomics can provide a simplified technology based on the PCR assay for the ease of operation.

Large, Slowly Growing, Benign Thyroid Nodules Frequently Coexist With Synchronous Thyroid Cancers.

CONTEXT: Thyroid nodules' size should not be the sole criterion for thyroidectomy; however, many patients undergo surgery for large or slowly growing nodules. OBJECTIVE: We evaluated risk for clinically significant thyroid cancer in patients with large or slowly growing nodules. METHODS: We reviewed data from 2 prospectively collected databases of patients undergoing thyroidectomies in tertiary referral centers in the USA and Greece over 14 consecutive years. We collected data on the preoperative surgical indication, FNA cytology, and surgical pathology. We included subjects operated solely for large or growing thyroid nodules, without any known or presumed thyroid cancer or high risk for malignancy, family history of thyroid cancer, or prior radiation exposure. RESULTS: We reviewed 5523 consecutive cases (USA: 2711; Greece: 2812). After excluding 3059 subjects, we included 2464 subjects in the present analysis. Overall, 533 thyroid cancers were identified (21.3%): 372 (69.8%) microcarcinomas (<1 cm) and 161 (30.2%) macrocarcinomas (≥1 cm). The histology was consistent with papillary cancer (n = 503), follicular cancer (n = 12), Hürthle cell cancer (n = 9), medullary cancer (n = 5), and mixed histology cancers n = 4. Only 47 (1.9%) of our subjects had any form of thyroid cancer in the nodule that originally led to surgery. The cancers were multifocal in 165 subjects; had extrathyroidal extension in 61, capsular invasion in 80, lymph node involvement in 35, and bone metastasis in 2 subjects. CONCLUSION: The risk of synchronous, clinically important thyroid cancers is small, but not null in patients with large or slow growing thyroid nodules. Therefore, more precise preoperative evaluation is needed to separate the patients who would clearly benefit from thyroid surgery from the vast majority of those who do not need to be operated.

Higher TSH Is Not Associated With Thyroid Cancer Risk in the Presence of Thyroid Autoimmunity.

BACKGROUND: Higher-but-within-normal thyrotropin (thyroid-stimulating hormone, TSH) is associated with higher risk for differentiated thyroid cancer (DTC) in surgical series. Our recent clinical observations suggest that this is not the case in the presence of autoimmune thyroid disease (AITD). We designed the present study to clarify this controversy. METHODS: We analyzed our prospectively collected database of patients referred for thyroid surgery at 2 tertiary care referral centers in Greece and the United States. We collected data for preoperative TSH, postoperative pathology, and thyroid peroxidase (TPO) antibodies titers. Subjects were subdivided into 2 groups, those with AITD (i.e., lymphocytic thyroiditis) and non-AITD. We excluded subjects with Graves disease, abnormal TSH (< 0.40 or > 4.50 mIU/mL), or recent use of levothyroxine. We compared the serum TSH among different groups using the Mann-Whitney test. RESULTS: A total of 3973 subjects were screened; 1357 met exclusion criteria. After all exclusions, data from 1731 non-AITD subjects and 329 AITD subjects were included in the analysis. AITD subjects had higher TSH than non-AITD subjects (2.09 vs 1.48; P < 0.0001). TSH values were higher in DTC compared with benign histology only in non-AITD subjects (1.65 vs 1.40; P < 0.0001). Progressively higher TSH was associated with higher incidence of DTC only in non-AITD subjects (P < 0.0001). In AITD subjects, TSH was similar between groups with or without DTC (2.02 vs 2.14; P = 0.21). CONCLUSIONS: TSH concentrations are not associated with the risk of developing DTC in the presence of thyroid autoimmunity, even though this seems to be the case for all other patients.

Incidentally Discovered Papillary Thyroid Microcarcinomas Are More Frequently Found in Patients with Chronic Lymphocytic Thyroiditis Than with Multinodular Goiter or Graves' Disease.

Background: Incidental finding of differentiated thyroid microcarcinomas (DTMc) in patients undergoing thyroid surgery for benign indications has become increasingly common. Even though carcinogenesis might relate to the background disease of the gland, the incidence of DTMc in the setting of various thyroid disorders remains unclear. We designed the present study to address this question. Materials and Methods: We reviewed data from two prospectively collected databases of consecutive patients undergoing thyroid surgery in two high-volume tertiary care referral centers, one in the United States (A) and the other one in Greece (B) over 18 years. We collected data on the preoperative surgical indication, fine-needle aspiration (FNA) cytology, and surgical pathology. We excluded subjects operated for thyroid cancer or with high risk for malignancy (FNA suspicious for thyroid cancer, follicular neoplasm, suspicious for follicular neoplasm, follicular lesion of undetermined significance/atypia of undetermined significance, or preoperative features of malignancy) and those with postsurgical pathology consistent with papillary thyroid cancer (PTC) ≥1 cm in largest diameter. We divided our subjects based on pathology data into those with chronic lymphocytic thyroiditis (CLT), Graves' disease (GD), or multinodular goiter (MNG). Results: We reviewed 6096 cases of thyroid surgery (A: 2711, B: 3385). We included 3909 subjects in the analysis. Overall, 569 (14.6%) PTC subjects were identified (A: 221/2003 [11%], B: 348/1906 [18.3%], odds ratios [OR] = 0.56, p < 0.0001). CLT was present in 617 subjects; PTC sonographic was present in 143 subjects (23.2%) (A: 79/404 [19.6%], B: 64/213 [30%], OR = 0.56, p = 0.003). GD was present in 359 subjects; PTC was present in 37 subjects (10.3%) (A: 12/197 [6.1%], B: 25/162 [15.4%], OR = 0.36, p = 0.004). MNG was present in 2933 subjects; PTC was present in 389 subjects (13.3%) (A: 130/1402 [9.3%], B: 259/1531 [16.9%], OR = 0.50, p < 0.0001). The incidence of PTC was significantly higher in CLT compared with MNG (OR = 1.75, p < 0.0001) or GD (OR = 2.25, p < 0.0001) but not in MNG compared with GD (OR = 1.29, p > 0.05). Conclusions: Incidentally discovered PTC are more commonly identified in surgical specimens from subjects with CLT compared with patients with MNG, while patients with GD present with a lower incidence compared with both groups. These data support previously published findings that euthyroid Hashimoto thyroiditis favors carcinogenesis, while GD may have a protective role.

The non-interventional approach to papillary thyroid microcarcinomas. An "active surveillance" dilemma.

BACKGROUND: Recent studies suggest that papillary-thyroid-microcarcinomas (PTMi) and follicular-variant-papillary-thyroid-cancers (FVPTC) are less aggressive overall. Our observations argue against. OBJECTIVES: To assess whether PTMi and FVPTC are indeed low-risk and could be safely followed without intervention. METHODS: We prospectively collected data of subjects with PTC on pathology post-thyroidectomy. Odds ratios (OR) were calculated with Fisher's exact test and differences between means were calculated using Mann Whitney's test. RESULTS: 696 met inclusion-criteria; 436 had macrocarcinomas (PTMa) and 260 had PTMi. PTMa were statistically significantly more likely to present multifocal [44.0% vs.28.1%], with extrathyroidal extension [22.1% vs.3.4%], lymph nodes involvement [25.5% vs.8.8%] and local invasion [3.1% vs.0.4%] (p < 0.05 for all), but not with distant metastasis [3.4% vs.1.3%, p > 0.05]. Therefore, PTMi measuring down to 0.01 cm, harbored aggressive features. We also identified 174 cases with FVPTC and 522 subjects with non-FVPTC. FVPTC had lower incidence of multifocality [40.1%, vs.60.9%], extrathyroidal extension [8.6% vs.17.4%] and lymphatic involvement [5.2% vs.24.0%], but not distant metastasis or local invasion [p > 0.05 for all]. Therefore, FVPTC measuring down to 0.5 cm, also harbored aggressive features. CONCLUSIONS: PTMi and FVPTC aggressive features are substantial enough to require careful evaluation, independent of their original tumor size before defaulting to just "active surveillance."

Nature of coexisting thyroid autoimmune disease determines success or failure of tumor immunity in thyroid cancer.

BACKGROUND: Thyroid cancer and thyroid autoimmunity are considered opposite extremes of immune-responses. However, several studies have suggested that thyroid cancer coexists with autoimmune thyroid diseases like Hashimoto Thyroiditis (HT) and Graves disease (GD). We have shown that the risk of developing thyroid cancer is higher in patients with a silent form of autoimmune thyroid disease -Euthyroid Hashimoto Thyroiditis-(EHT). METHODS: We analyzed data from 2633 consecutive patients with GD, HT, EHT and non-Autoimmune Thyroid Disease (Non-AITD) for the presence of Differentiated Thyroid Cancer (DTC). We further investigated the microenvironment, and cellular mechanism of protection from DTC in GD/EHT by ex-vivo aspirating infiltrates from thyroid samples. We also re-constituted in vitro the in-vivo microenvironment to mimic an in-vivo context. We isolated NK cells and differentiated macrophages into M1 and M2 phenotype from healthy human peripheral blood monocytes. RESULTS: DTC was less frequent/aggressive in GD as compared to EHT or Non-AITD. Intra-thyroidal immune-cell profiling revealed differential Natural Killer (NK) cell activity and macrophage polarization in the settings of GD versus EHT. In GD, NK-cells were activated, and macrophages showed M1-like phenotype whereas, in EHT, NK-cells were less active and macrophages displayed M2-like phenotype. Furthermore, in vitro co-cultures of NK-cells with differentiated macrophage subsets revealed that the presence of activated NK (NA) cells favors M1 macrophages, boosts macrophage action and amplifies the innate defense mechanisms. Moreover, co-culture of M2 macrophages with NA, increases the cytotoxicity of NK-cells and favors a pro-inflammatory microenvironment that reverts the anti-inflammatory M2 towards pro-inflammatory M1. CONCLUSION: Surveillance innate immune-cells like Natural Killer (NK) cells and macrophages are complementary to each other in their actions. We discovered here that activated NK-cells in the background of the thyroid autoimmune disease, GD, drive macrophage differentiation to the M1/killer phenotype which in turn is cytotoxic to cancer cells and down regulates the M2/repair phenotype. Understanding the molecular basis of macrophage-NK cell interface in Thyroid Cancer, ETH and GD will open new vistas for immunopathology and therapeutic intervention. Macrophages/innate immunity can be modulated from M2 to M1 phenotype to help treat thyroid cancer as naturally done by GD.

Hashimoto's thyroiditis pathology and risk for thyroid cancer.

BACKGROUND: Hashimoto's thyroiditis (HT) has been found to coexist with differentiated thyroid cancer (DTC) in surgical specimens, but an association between the two conditions has been discounted by the medical literature. Therefore, we performed this study to determine any potential relationship between HT and the risk of developing DTC. METHODS: We collected data for thyrotropin (TSH), thyroxine (T4), thyroid peroxidase antibody (TPO-Ab) titers, surgical pathology, and weight-based levothyroxine (LT4) replacement dose for patients who were referred for thyroid surgery. Patients with HT at final pathology were studied further. To estimate thyroid function, patients with preoperative hypothyroid HT (Hypo-HT) were divided into three equal groups based on their LT4 replacement: LT4-Low (<0.90 µg/kg), LT4-Mid (0.90-1.43 µg/kg), and LT4-High (>1.43 µg/kg). A group of preoperatively euthyroid (Euth-HT) patients but with HT by pathology was also studied. All subjects were also grouped based on their TPO-Ab titer in TPO-high (titer >1:1000) or TPO-low/negative (titer <1:1000 or undetectable) groups. The relationship of HT and DTC was studied extensively. RESULTS: Of 2811 subjects, 582 had HT on surgical pathology, 365 of whom were Euth-HT preoperatively. DTC was present in 47.9% of the Euth-HT, in 59.7% of LT4-Low, 29.8% of LT4-Mid, and 27.9% of LT4-High groups. The relative risk (RR) for DTC was significantly elevated for the Euth-HT and LT4-Low groups (p<0.001), but not for the LT4-Mid or LT4-High replacement dose groups. TPO-low/negative status conferred an increased RR in the Euth-HT and LT4-Low replacement dose groups (p<0.001 both), while TPO-high status decreased it in Euth-HT group (p<0.05) and made it nonsignificant in the LT4-Low group. CONCLUSIONS: HT pathology increases the risk for DTC only in euthyroid subjects and those with partially functional thyroid glands (LT4-Low) but not in fully hypothyroid HT (LT4-Mid and LT4-High). High TPO-Ab titers appear to protect against DTC in patients with HT.

Lymphocytic profiling in thyroid cancer provides clues for failure of tumor immunity.

Thyroid cancers are usually surrounded by a significant number of immune-reactive cells. Tumor-associated lymphocytes as well as background lymphocytic thyroiditis are frequently mentioned in pathology reports of patients who have undergone surgery for thyroid cancer. The nature of this lymphocytic reaction is not well understood. The fact that cancer can survive in this adverse microenvironment is indicative of immune regulation. We characterized the lymphocytic infiltration that accompanies thyroid cancer and compared it with that present in thyroid autoimmunity. We found that double-negative (DN) T cells were significantly more abundant in thyroid cancer than in thyroid autoimmunity. Although FOXP3(+) regulatory T cells were also present, DN T cells were the dominant cell type, associated with thyroid cancer. Furthermore, upon stimulation, the DN T cells associated with cancer remained unchanged, while the few (<5%) DN T cells associated with thyroid autoimmunity increased in numbers (>20%). CD25 expression on DN T cells remained unchanged after stimulation, which indicates that the increase in the absolute number of DN T cells in thyroid autoimmunity was at the expense of inactivation of single-positive T cells. We concluded that in the setting of thyroid cancer, DN T cells appear to suppress tumor immunity. In contrast, in thyroid autoimmunity, DN T cells were barely present and only increased at the expense of inactivated, single-positive T cells upon induction. Together, these findings indicate that thyroid cancer-associated DN T cells might regulate proliferation and effector function of T cells and thereby contribute to tumor tolerance and active avoidance of tumor immunity.

Eukaryotic translation initiation factor 5A inhibition alters physiopathology and immune responses in a "humanized" transgenic mouse model of type 1 diabetes.

Therapeutic options for treatment of type 1 diabetes (T1D) are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4(+) T cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7-treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D.

Diabetes-associated dry eye syndrome in a new humanized transgenic model of type 1 diabetes.

PURPOSE: Patients with Type 1 Diabetes (T1D) are at high risk of developing lacrimal gland dysfunction. We have developed a new model of human T1D using double-transgenic mice carrying HLA-DQ8 diabetes-susceptibility haplotype instead of mouse MHC-class II and expressing the human beta cell autoantigen Glutamic Acid Decarboxylase in pancreatic beta cells. We report here the development of dry eye syndrome (DES) after diabetes induction in our humanized transgenic model. METHODS: Double-transgenic mice were immunized with DNA encoding human GAD65, either naked or in adenoviral vectors, to induce T1D. Mice monitored for development of diabetes developed lacrimal gland dysfunction. RESULTS: Animals developed lacrimal gland disease (classically associated with diabetes in Non Obese Diabetic [NOD] mice and with T1D in humans) as they developed glucose intolerance and diabetes. Animals manifested obvious clinical signs of dry eye syndrome (DES), from corneal erosions to severe keratitis. Histological studies of peri-bulbar areas revealed lymphocytic infiltration of glandular structures. Indeed, infiltrative lesions were observed in lacrimal/Harderian glands within weeks following development of glucose intolerance. Lesions ranged from focal lymphocytic infiltration to complete acinar destruction. We observed a correlation between the severity of the pancreatic infiltration and the severity of the ocular disease. CONCLUSIONS: Our results demonstrate development of DES in association with antigen-specific insulitis and diabetes following immunization with clinically relevant human autoantigen concomitantly expressed in pancreatic beta cells of diabetes-susceptible mice. As in the NOD mouse model and as in human T1D, our animals developed diabetes-associated DES. This specific finding stresses the relevance of our model for studying these human diseases. We believe our model will facilitate studies to prevent/treat diabetes-associated DES as well as human diabetes.

Screening for parathyroid hormone resistance in patients with nonphenotypically evident pseudohypoparathyroidism.

OBJECTIVE: Hypocalcemia and hyperphosphatemia in the setting of elevated parathyroid hormone (PTH) and normal vitamin D metabolites, raises the possibility of PTH resistance. The idiopathic and inherited forms of PTH resistance are referred to as pseudohypoparathyroidism. Nonphenotypically evident pseudohypoparathyroidism can go undiagnosed for decades. We have designed a new test to diagnose PTH resistance and confirmed its clinical utility in the diagnosis of pseudohypoparathyroidism. METHODS: Our test consists of a subcutaneous injection of commercially available recombinant PTH and concomitant measurement of cyclic adenosine monophosphate in urine. We implemented the test in 2 patients with recalcitrant hypocalcemia and a healthy control subject. RESULTS: Our test unequivocally demonstrated PTH resistance in both patients. One of the patients had phenotypically evident pseudohypoparathyroidism type-1a hence, PTH resistance was suspected. The other patient with nonphenotypically evident disease, also showed PTH resistance and was later demonstrated to have pseudohypoparathyroidism type-1b at the genomic level and confirmed to be of familial type. CONCLUSION: Our results show for the first time the implementation of a simple new diagnostic tool designed to check for PTH resistance. This new test has already proven to be useful in few occasions at our institution. Larger populations, however, should be tested before implementation of such a test is considered a standard of care.