Clinical utility of the imunohistochemical co-expression of p53 and MDM2 in thyroid follicular lesions.

In order to investigate the possible correlation between p53 and MDM2 co-expression with clinicopathological features of differentiated thyroid cancer (DTC) and its use as diagnostic and/or prognostic markers, we used immunohistochemistry to evaluate 317 thyroid samples including 208 DTC and 94 benign nodules, in addition to 15 normal tissues. MDM2 and p53 expression were highly associated (r = 0.7161; p < 0.0001). The co-expression of p53-MDM2 was observed more frequently in malignant lesions (p < 0.0001) and helped characterize follicular patterned lesions distinguishing FVPTC from FA (p < 0.0001) and FVPTC from FTC (p < 0.0001). In addition, p53-MDM2 co-expression was associated with characteristics of less aggressiveness. It was more frequent in patients ≤45 years old (p = 0.0035), with unique tumors (p = 0.0095), tumors <2 cm (p < 0.0001), tumors without extrathyroid invasion (p = 0.0425), without metastasis at evolution (p = 0.0179), and in patients evolving free of disease after treatment (p = 0.0485). We suggest that p53-MDM2 co-expression profile analysis might help establishing diagnostic and determining prognostic of DTC patients.

Interleukin 10 expression is related to aggressiveness and poor prognosis of patients with thyroid cancer.

Most patients with thyroid cancer will evolve very well with current therapies. However, 10-30% of these patients will present recurrent disease and some of them will eventually die. IL-10 is an anti-inflammatory and immunosuppressive cytokine that can contribute to the immune escape of neoplastic cells. We aimed to investigate IL-10 as a molecular marker to improve the clinical management of patients with thyroid cancer. We retrospectively studied 162 patients with follicular cell-derived thyroid cancer who attended to our institution, including 63 classic papillary thyroid carcinomas, 46 follicular variant of papillary thyroid carcinomas, 11 poorly differentiated thyroid carcinomas and 42 follicular thyroid carcinomas. Patients were treated according to current guidelines and followed-up for 1-150 months. Additionally, we studied 96 samples of non-malignant tissues. We investigated the expression of IL-10 in tumor cells by semiquantitative and quantitative methods. Malignant tissues presented higher positivity (0.773 ± 0.140) than non-malignant samples (0.623 ± 0.190; p < 0.001). Tumors with extrathyroidal invasion at diagnosis presented higher levels of positivity for IL-10 (0.802 ± 0.125) than tumors without extrathyroidal invasion (0.731 ± 0.147; p = 0.004). We observed a positive correlation between tumor size and IL-10 positivity (correlation coefficient = 0.407; p < 0.001). Patients with IL-10 positivity above the median presented lower relapse-free survival rate compared to those patients whose tumors presented IL-10 positivity below the median. We suggest that a simple IL-10 IHC analysis could help selecting patients who would benefit from a more intensive approach.

CD8+ tumour-infiltrating lymphocytes and COX2 expression may predict relapse in differentiated thyroid cancer.

BACKGROUND/OBJECTIVE: There is an increasing rate of papillary thyroid carcinomas that may never progress to cause symptoms or death. Predicting outcome and determining tumour aggressiveness could help diminish the number of patients submitted to aggressive treatments. We aimed to evaluate whether markers of the immune system response and of tumour-associated inflammation could predict outcome of differentiated thyroid cancer (DTC) patients. DESIGN: Retrospective cohort study. PATIENTS: We studied 399 consecutive patients, including 325 papillary and 74 follicular thyroid carcinomas. MEASUREMENTS: Immune cell markers were evaluated using immunohistochemistry, including tumour-associated macrophages (CD68) and subsets of tumour-infiltrating lymphocytes (TIL), such as CD3, CD4, CD8, CD16, CD20, CD45RO, GRANZYME B, CD69 and CD25. We also investigated the expression of cyclooxygenase 2 (COX2) in tumour cells and the presence of concurrent lymphocytic infiltration characterizing chronic thyroiditis. RESULTS: Concurrent lymphocytic infiltration characterizing chronic thyroiditis was observed in 29% of the cases. Among all the immunological parameters evaluated, only the enrichment of CD8+ lymphocytes (P = 0·001) and expression of COX2 (P =0·01) were associated with recurrence. A multivariate model analysis identified CD8+ TIL/COX2 as independent risk factor for recurrence. A multivariate analysis using Cox's proportional-hazards model adjusted for the presence of concurrent chronic thyroiditis demonstrated that the presence of concurrent chronic thyroiditis had no effect on prognostic prediction mediated by CD8+ TIL and COX2. CONCLUSION: In conclusion, we suggest the use of a relatively simple pathology tool to help select cases that may benefit of a more aggressive approach sparing the majority of patients from unnecessary procedures.

P53 and expression of immunological markers may identify early stage thyroid tumors.

BACKGROUND: Besides its major role in cell proliferation, DNA repair, and apoptosis, functional p53 protein is involved in the induction of antitumor cytotoxic-T-cell activity against carcinoma cells. We aimed to investigate p53 and immune cell markers utility as diagnostic and prognostic markers of differentiated thyroid cancer (DTC). METHODS: ACIS-III system was used to evaluate p53 and immune cell markers including tumor-associated macrophages (TAM); CD68 and tumor-infiltrating lymphocytes (TIL) subsets such as CD3, CD4, CD8, and CD20 in 206 thyroid carcinomas, 105 benign nodules, and 18 normal tissues. Also, TP53 was sequenced in 78 out of 164 patients with papillary thyroid carcinoma. RESULTS: P53 expression was observed more frequently in malignant than in benign lesions (P < 0.0001) and helped discriminate follicular patterned lesions. In addition, p53 was more frequent in smaller (P = 0.0015), unique tumors (P = 0.0286), with thyroiditis (P = 0.0486) and without metastasis at diagnosis (P = 0.0201). TAM was more frequent in P53 negative tumors (P = 0.002). Infiltration of CD8+ TIL was found in 61.7% of P53 positive and 25.6% of P53 negative DTC (P < 0.001). CONCLUSIONS: We suggest that p53 and CD8+ TIL immune profile analysis might be useful in DTC.

Obesity and excess protein and carbohydrate consumption are risk factors for thyroid cancer.

Conflicting data concerning the association between obesity and differentiated thyroid cancer (DTC) may be attributed to the lack of records showing dietary intake and inadequate evaluation of nutrient composition. We evaluated 115 DTC patients carefully paired with 103 healthy control individuals by using a structured questionnaire, including a 24-h recordatory during 3 days, to investigate calorie intake and macronutrient (proteins, carbohydrates, and lipids) composition of the diet. We observed that excess weight (body mass index > 25 kg/m(2)) increased individual susceptibility to DTC [odds ratio (OR) = 3.787; 95% confidence interval (CI) = 1.115-6.814; P < 0.0001). This augmented risk was evident in women (OR = 1.925; 95% CI = 1.110-3.338; P = 0.0259) but not in men (P = 0.3498). Excess calorie intake was more frequent in patients with DTC than in controls (OR = 5.890; 95% CI = 3.124-11.103; P < 0.0001), and both excess protein (OR = 4.601; 95% CI = 1.634-12.954; P = 0.0039) and carbohydrate (OR = 4.905; 95% CI = 2.593-9.278; P < 0.0001) consumption were associated with an increased risk of DTC, contrarily to lipid/fiber intake and physical activity (P = 0.894 and 0.5932, respectively). In conclusion, our data indicate that overweight and risk of DTC are associated with higher protein and carbohydrate consumption than the rates recommended by the World Health Organization. The nutritional orientation should be part of preventive strategy targets designed to combat the increasing incidence of both obesity and DTC.

Serum interleukin measurement may help identify thyroid cancer patients with active disease.

BACKGROUND: Investigate the clinical utility of serum interleukin dosages of IL-2, IL-2R, IL-4, IL-6, IL-6R, IL-8, IL-10 and IL-12 in the diagnosis and characterization of patients with DTC. In particular, verify ILs utility in the identification of individuals who are evolving disease-free or with the active disease. METHODS: We evaluated 200 patients with malignant nodules (100 patients disease-free and 100 patients with recurrence/active disease); 60 benign nodules and 100 healthy controls, serum levels were assessed by ELISA. RESULTS: All ILs, but not IL-4, differentiated these three groups. We observed that IL-2, 2R and 10 serum concentrations were associated with thyroglobulin levels. Serum IL-2 was able to differentiate patients with active disease from the disease-free with a sensitivity of 98%, specificity of 58%, positive predictive value (PPV) of 70% and negative predictive value (NPV) of 97% (p=0.0007). IL-6R levels differentiated patients with active disease from the disease-free patients with 56% sensitivity, 63% specificity, PPV of 60% and NPV of 59% (p<0.0001). IL-8 values also distinguished patients with active disease from the disease-free ones with sensitivity of 50%, specificity of 76%, PPV of 68% and NPV of 60% (p=0.0025); using IL-12, we obtained a sensitivity value of 73%, specificity of 66%, PPV of 68% and NPV of 71% (p<0.0001). Furthermore, interleukin levels showed association with some tumor characteristics of aggressiveness. CONCLUSION: We suggest that the serum concentration of ILs may assist in the diagnosis and characterization of tumor malignancy helping identify patients with active disease who deserve closer medical attention.

Immunotherapy against endocrine malignancies: immune checkpoint inhibitors lead the way.

Immune checkpoint inhibitors are agents that act by inhibiting the mechanisms of immune escape displayed by various cancers. The success of immune checkpoint inhibitors against several tumors has promoted a new treatment strategy in clinical oncology, and this has encouraged physicians to increase the number of patients who receive the immune checkpoint therapy. In the present article, we review the main concepts regarding immune checkpoint mechanisms and how cancer disrupts them to undergo immune escape. In addition, we describe the most essential concepts related to immune checkpoint inhibitors. We critically review the literature on preclinical and clinical studies of the immune checkpoint inhibitors as a treatment option for thyroid cancer, ovarian carcinoma, pancreatic adenocarcinoma, adrenocortical carcinoma and neuroendocrine tumors. We present the challenges and the opportunities of using immune checkpoint inhibitors against these endocrine malignancies, highlighting the breakthroughs and pitfalls that have recently emerged.

Polymorphism in LEP and LEPR May Modify Leptin Levels and Represent Risk Factors for Thyroid Cancer.

Purpose. To understand the role of polymorphisms in the LEP (rs7799039 and rs2167270) and LEPR (rs1137101 and rs1137100) genes in DTC susceptibility and their effect on leptin levels. Methods. We studied 153 patients with DTC and 234 controls through TaqMan SNP Genotyping and ELISA, comparing these data to the clinicopathological data of patients with DTC. Results. Patients with AA genotype of rs7799039 had higher levels of serum leptin (9.22 ± 0.98 ng/mL) than those with AG genotype (10.07 ± 0.60 ng/mL; P = 0.005). Individuals with AG genotype of rs2167270 also produced higher serum leptin levels (10.05 ± 0.59 ng/mL) than the subjects with GG genotype (9.52 ± 0.79 ng/mL; P < 0.05). A multivariate logistic regression adjusted for gender, age, and BMI showed that the AG genotype of rs7799039 was an independent risk for DTC (OR, 11.689; P = 0.0183; 95% CI, 1.516-90.119). Similarly, AG and GG genotypes of rs1137101 increased the susceptibility to DTC (OR, 3.747; P = 0.027; 95% CI, 1.161-12.092 and OR, 5.437; P = 0.013; 95% CI, 1.426-20.729). Conclusions. We demonstrated that rs7799039 and rs2167270 polymorphisms modify the serum leptin concentrations in patients with DTC. Furthermore, polymorphisms rs7799039 and rs1137101 increase the risk of DTC development, although they do not correlate with tumor aggressiveness.

The role of the inflammatory microenvironment in thyroid carcinogenesis.

Immune responses against thyroid carcinomas have long been demonstrated and associations between inflammatory microenvironment and thyroid carcinomas repeatedly reported. This scenario has prompted scientists throughout the world to unveil how the inflammatory microenvironment is established in thyroid tumors and what is its influence on the outcome of patients with thyroid carcinoma. Many studies have reported the role of evasion from the immune system in tumor progression and reinforced the weakness of the innate immune response toward thyroid cancer spread in advanced stages. Translational studies have provided evidence that an increased density of tumor-associated macrophages in poorly differentiated thyroid carcinoma (DTC) is associated with an aggressive phenotype at diagnosis and decreased cancer-related survival, whereas well-DTC microenvironment enriched with macrophages is correlated with improved disease-free survival. It is possible that these different results are related to different microenvironments. Several studies have provided evidence that patients whose tumors are not infiltrated by lymphocytes present a high recurrence rate, suggesting that the presence of lymphocytes in the tumor microenvironment may favor the prognosis of patients with thyroid carcinoma. However, the effect of lymphocytes and other immune cells on patient outcome seems to result from complex interactions between the tumor and immune system, and the molecular pattern of cytokines and chemokines helps to explain the involvement of the immune system in thyroid tumor progression. The inflammatory microenvironment may help to characterize aggressive tumors and to identify patients who would benefit from a more invasive approach, probably sparing the vast majority of patients with an indolent disease from unnecessary procedures.

Obesity and thyroid cancer.

Many studies have provided observational data on the association of obesity and thyroid cancers, but only few of them propose mechanisms that would permit a better understanding of the causal molecular mechanisms of this association. Considering that there is an increasing incidence of both obesity and thyroid cancers, we need to summarize and link recent studies in order to characterize and understand the contribution of obesity-related factors that might affect thyroid cancer development and progression. Adipose tissue is involved in many vital processes, including insulin sensitivity, angiogenesis, regulation of energy balance, activation of the complement system, and responses such as inflammation. Although these processes have their own molecular pathways, they involve the same molecules through which obesity and adipose tissue might exert their roles in carcinogenesis, not only affecting MAPK and PI3K or even insulin pathways, but also recruiting local inflammatory responses that could result in disease formation and progression. This review describes five important issues that might explain the link between excessive weight and thyroid cancer: thyroid hormones, insulin resistance, adipokines, inflammation, and sexual hormones.

CD8+ TIL recruitment may revert the association of MAGE A3 with aggressive features in thyroid tumors.

BACKGROUND: We aimed to investigate a possible role of MAGE A3 and its associations with infiltrated immune cells in thyroid malignancy, analyzing their utility as a diagnostic and prognostic marker. MATERIALS AND METHODS: We studied 195 malignant tissues: 154 PTCs and 41 FTCs; 102 benign tissues: 51 follicular adenomas and 51 goiter and 17 normal thyroid tissues. MAGE A3 and immune cell markers (CD4 and CD8) were evaluated using immunohistochemistry and compared with clinical pathological features. RESULTS: The semiquantitative analysis and ACIS III analysis showed similar results. MAGE A3 was expressed in more malignant than in benign lesions (P < 0.0001), also helping to discriminate follicular-patterned lesions. It was also higher in tumors in which there was extrathyroidal invasion (P = 0.0206) and in patients with stage II disease (P = 0.0107). MAGE A3+ tumors were more likely to present CD8+ TIL (P = 0.0346), and these tumors were associated with less aggressive features, that is, extrathyroidal invasion and small size. There was a trend of MAGE A3+ CD8+ tumors to evolve free of disease. CONCLUSION: We demonstrated that MAGE A3 and CD8+ TIL infiltration may play an important role in malignant thyroid nodules, presenting an interesting perspective for new researches on DTC immunotherapy.

Clinical utility of KAP-1 expression in thyroid lesions.

Although there are evidences of the involvement of KAP-1 in other tumors, data on differentiated thyroid carcinomas (DTC) are still lacking. We aimed to evaluate KAP-1 clinical utility in the diagnosis and prognosis of DTC. We used both visual immunohistochemistry and a semiquantitative analysis to evaluate KAP-1 expression in 230 thyroid carcinomas and 131 noncancerous thyroid nodules. There were 43 follicular carcinomas (FC) and 187 papillary thyroid carcinomas (PTC), including 130 classic (CPTC), 4 tall cells (TCPTC), and 53 follicular variants (FVPTC). Patients were followed up for 53.8 ± 41 months. They were classified as free-of-disease (142 cases) or poor outcome (25 cases--10 deaths), according to their serum Tg levels and image evidences. KAP-1 was identified in 78 % PTC, 75 % TCPTC, 74 % FC, 72 % FVPTC, 55 % FA, 44 % hyperplasia, and 11 % normal thyroid tissues. A ROC analysis identified malignant nodules with 69 % sensitivity and 75 % specificity, using a cutoff of 73.19. In addition to distinguishing benign from malignant thyroid tissues (p < 0.0001), KAP-1 expression differentiated CPTC from nodular hyperplasia (p < 0.0001), CPTC from FA (p = 0.0028), FVPTC from hyperplasia (p = 0.0039), and FC from hyperplasia (p = 0.0025). Furthermore, KAP-1 was more expressed in larger tumors (>4 cm; p = 0.0038) and in individuals who presented recurrences/metastases (p = 0.0130). We suggest that KAP-1 may help diagnose thyroid nodules, characterize follicular-patterned thyroid lesions, and identify individuals with poor prognosis.

Differentiated thyroid carcinomas may elude the immune system by B7H1 upregulation.

B7H1 is consistently associated with inhibition of the immune system in many solid tumors. However, there is no report about its impact on differentiated thyroid carcinoma (DTC) presentation, aggressiveness, or evolution. Aiming to investigate the role of B7H1 in DTC and correlate this protein with other tumor-infiltrating immune cells, we studied 407 thyroid nodule tissue samples including 293 from DTC patients, all managed according to a same standard protocol. In addition, we obtained 5 normal and 114 benign thyroid lesions. Eighteen out of the 253 papillary thyroid carcinomas were paired with respective metastatic lymph node tissues. B7H1 (CD274) protein expression was assessed by immunohistochemistry and the gene expression was quantified by real-time PCR. Malignant tissues displayed a more intense B7H1 staining and higher mRNA levels than benign tissues (both P<0.0001). We observed a positive linear correlation between higher age at diagnosis and B7H1 mRNA levels (P=0.02896). Elevated levels of B7H1 protein were associated with the presence of CD4+, CD8+, CD20+, and FoxP3+ lymphocytes (all P<0.05); tumor-associated macrophages (P<0.0001); and the presence of myeloid-derived suppressor cells (P=0.03256). Stage II-IV patients presented higher B7H1 mRNA levels than stage I cases (P=0.03522). On the contrary, a decreased expression of B7H1 protein was observed in lymph node metastasis (P=0.0152). In conclusion, our data demonstrate that B7H1 expression is associated with features of aggressiveness, suggesting that this is an immune evasion mechanism of DTC cells.

mRNA BRAF expression helps to identify papillary thyroid carcinomas in thyroid nodules independently of the presence of BRAFV600E mutation.

Literature has consistently shown associations of BRAFV600E mutation with papillary thyroid cancer clinical features. However, the clinical utility of BRAF expression has not been clinically explored so far. We studied 67 thyroid nodules (32 benign nodules and 35 PTC cases). BRAF mRNA expression levels measured by a quantitative real-time PCR and a PCR-RFLP were used to identify BRAFV600E mutation. BRAF mRNA expression was significantly higher in malignant (198.2±373.9 AU) than in benign (4.1±6.9 AU) nodules (p<0.0001). BRAF expression identified malignancy with a sensitivity of 80.6%, specificity of 77.1%, positive predictive value of 75.8%, and negative predictive value of 81.8%. A cut-point of 4.712, identified by the ROC curve, was able to sort out malignant nodules with an accuracy of 78.8%. Although we did not find any correlation between the presence of BRAF V600E mutation and clinical or tumor features such as age (p=0.309), gender (p=0.5453), ethnicity (p=0.9820), tumor size (p=1.000), multifocality (p=0.2530) or mRNA levels (p=0.7510), the study power for BRAF expression and diagnosis (99%; FPRP=0.85) indicated that data is noteworthy despite the relative small number of patients investigated. We concluded that BRAF mRNA expression may help to identify PTC among thyroid nodules independently of the presence of BRAFV600E mutation.

Muc-1 expression may help characterize thyroid nodules but does not predict patients' outcome.

Our purpose was to evaluate MUC1 clinical utility in the diagnosis and prognosis of thyroid cancer patients. We studied the protein expression of MUC1 in 289 thyroid carcinomas and 121 noncancerous thyroid nodules. There were 41 follicular carcinomas (FC) and 248 papillary thyroid carcinomas (PTC) including 149 classic (CPTC), 20 tall cell (TCPTC) and 79 follicular variants (FVPTC). In addition, we used a quantitative real-time PCR (q-PCR) method to measure MUC1 mRNA expression levels in 108 carcinomas, 23 hyperplasias, and 19 FA. According to their serum Tg levels and other evidences of recurrence/metastasis, the patients were classified as free-of-disease (185 cases) or bad outcome (56 cases, 10 deaths). MUC1 protein was identified in 80.2% PTC; 48.8% FC; 68.3% FVPTC; 70% TCPTC; 21.8% FA; 30% hyperplasias and 6% normal thyroid tissues. MUC1 distinguished benign from malignant thyroid tissues (sensitivity = 89%; specificity = 53%). MUC1 also differentiated FC from FA (p = 0.0083). q-PCR mRNA expression of MUC1 also distinguished malignant from benign nodules (Mann-Whitney test, p < 0.0001). However, neither IHC nor mRNA MUC1 expression was associated with any clinical or pathological feature of aggressiveness or outcome. We suggest that MUC1 expression may help differentiate follicular patterned thyroid lesions.