[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: There is an urgent need for biomarkers to identify malignant thyroid nodules from indeterminate follicular lesions. We have used a subtractive proteomic strategy to identify novel biomarkers by selecting ligands to goiter tissue from a 12-mer random peptide phage-displayed library using the BRASIL method (Biopanning and Rapid Analysis of Selective Interactive Ligands). After three rounds of selection, two highly reactive clones to the papillary thyroid tumor cell line NPA were further evaluated, and their specific binding to tumor proteins was confirmed using phage-ELISA. The antibody-like peptide CaT12 was tumor-specific, which was further tested by immunohistochemistry against TMAs (tissue microarrays) comprised of 775 human benign and malignant tissues, including 232 thyroid nodular lesions: 15 normal thyroid tissues, 53 nodular goiters (NG), 54 follicular adenomas (FA); 69 papillary thyroid carcinomas (PTC); and 41 follicular carcinomas (FC). CaT12 was able to identify PTC among thyroid nodular lesions with 91.2% sensitivity and 85.1% specificity, despite its non-specificity for thyroid tissues. Additionally, the CaT12 peptide helped characterize follicular lesions distinguishing the follicular variant of PTC (FVPTC) from FA with 91.9% accuracy; FVPTC from NG with 83.1% accuracy; FVPTC from the classic PTC with 57.7% accuracy; and FVPTC from FC with 88.7% accuracy. In conclusion, our strategy to select differentially expressed ligands to thyroid tissue was highly effective and resulted in a useful antibody-like biomarker that recognizes malignancy among thyroid nodules and may help distinguish follicular patterned lesions.
[show abstract][hide abstract] ABSTRACT: 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.
Clinical and Developmental Immunology 01/2013; 2013:846584. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 protein expression was assessed by immunhistochemistry and the gene expression 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 differentiated thyroid carcinoma cells.
Endocrine Related Cancer 11/2012; · 5.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Pathology - Research and Practice 07/2012; 208(8):489-92. · 1.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background: Immune responses against differentiated thyroid carcinomas (DTC) have long been recognized. Methods: In order to investigate the role of immune cell infiltration in the progression of DTC, we studied 398 patients - 253 with papillary and 13 with follicular thyroid cancers, as well as 132 with nonmalignant tissues. MUC1, NIS, ATM, PTEN, and CD56 protein expressions were assessed by immunohistochemistry; immune cell infiltration was identified using CD3, CD4, CD8, CD20, CD68, and FoxP3 immunohistochemical markers. In addition, we investigated infiltration of Th17 lymphocytes and myeloid derived suppressor cells (MDSCs). Findings: Furthermore, expression of MUC1, NIS, ATM, PTEN, CD56 and B7H1 was accompanied by infiltration of a mixture of different immune cells, suggesting this profile may be related to antitumor immune response. Tumors with high B7H1 expression were frequently enriched with MDSCs (p=0.03256), suggesting that a complex immune evasion mechanism is engaged in DTC microenvironment. Interpretation: Our data suggest that the immune response may be influenced by the status of tumor antigenicity in DTC. Immune cell infiltration helped characterize specific tumor histotypes.
Endocrine Related Cancer 03/2012; 19(3):L31-6. · 5.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: ATM is critical in response to ionizing radiation-induced DNA damage.
Variations in ATM are hypothesized to affect individual susceptibility to thyroid cancer. Our objective was to evaluate the association between ATM polymorphisms and thyroid cancer risk. DESIGN, PARTICIPANTS, AND METHODS: Six ATM single nucleotide polymorphisms (SNP) were genotyped in two independent case-control series including 592 patients with differentiated thyroid carcinoma (DTC) and 885 healthy individuals. An unconditional logistic regression model was applied to calculate odds ratios (OR) and 95% confidence intervals (CI) for each SNP with respect to risk of DTC and the combination effect of SNP on cancer risk.
The risk-allele frequencies of all the SNP were similar in the two case-control populations. Under a dominant model of inheritance, the G allele of ATM rs189037 exhibited a protective effect against DTC (adjusted OR = 0.8; 95% CI, 0.6-1.0; P = 0.04), and the G allele of rs1800057 was associated with increased risk of DTC (adjusted OR = 1.9; 95% CI, 1.1-3.1; P = 0.02). A protective haplotype (A-G-C-T-C-A) was associated with decreased risk of DTC in non-Hispanic whites (adjusted OR = 0.2; 95% CI, 0.0-0.8; P = 0.03). A significant dose-response relationship was observed between the total number of risk alleles of ATM and DTC risk (P = 0.01). Carriers of a combination of six to seven and eight to 10 risk alleles were at 30% (adjusted OR = 1.3; 95% CI, 1.0-1.7) and 50% (adjusted OR = 1.5; 95% CI, 1.1-2.1) increased risk of DTC, respectively.
Individual susceptibility to DTC may be attributable to polymorphisms of ATM, and the associations warrant confirmation in independent studies.
The Journal of clinical endocrinology and metabolism 03/2012; 97(6):1913-21. · 6.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: Forkhead box P3 (FoxP3) expression has been observed in human cancer cells but has not yet been reported in thyroid cells. We investigated the prognostic significance of both FoxP3 expression and intratumoral FoxP3(+) lymphocyte infiltration in differentiated thyroid carcinoma cells.
We constructed a tissue microarray with 385 thyroid tissues, including 266 malignant tissues (from 253 papillary thyroid carcinomas and 13 follicular carcinomas), 114 benign lesions, and 5 normal thyroid tissues.
We determined the expression of FoxP3 in both tumor cells and tumor-infiltrating lymphocytes using immunohistochemical techniques. Cellular expression of FoxP3 was evident in 71% of benign and 91.9% of malignant tissues. The nuclear and cytoplasmic expression patterns were quantified separately. A multivariate logistic regression analysis indicated that cytoplasmic FoxP3 expression is an independent risk factor for thyroid malignancy. Cytoplasmic FoxP3 staining was inversely correlated with patient age. Nuclear FoxP3 staining was more intense in younger patients and in tumors presenting with metastasis at diagnosis. FoxP3(+) lymphocytes were more frequent in tumors smaller than 2 cm, those without extrathyroidal invasion, and in patients with concurrent chronic lymphocytic thyroiditis.
We demonstrated FoxP3 expression in differentiated thyroid carcinoma cells and found evidence that this expression may exert an important influence on several features of tumor aggressiveness.
Clinics (São Paulo, Brazil) 01/2012; 67(5):483-8. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: We aimed to investigate the use of NIS mRNA and protein expression as a diagnostic and/or prognostic marker in patients with differentiated thyroid cancer (DTC).
This is a case-control study.
We studied 397 thyroid nodules tissue samples, including 224 papillary thyroid carcinomas (PTCs), 41 follicular carcinomas, 58 nodular goiters, 56 follicular adenomas and 18 normal tissues assembled in a tissue microarray.
NIS protein was identified using a monoclonal antibody that labelled only the follicular cell basolateral membrane of all 397 tissue samples. In addition, NIS mRNA was quantified in 145 DTC patients and 85 PTC cases were screened for BRAF(V600E) mutation.
We found low NIS mRNA expression and low or negative NIS protein expression in most DTC. NIS expression was lower in DTC patients over 45 years old and in tumours larger than 2 cm. There was a tendency for lower NIS expression in advanced stages and patients presenting recurrences. All 13 DTC patients who succumbed to the disease were NIS negative at immunohistochemistry and had very low mRNA expression. NIS expression was lower in PTC presenting BRAF(V600E) mutation. However, neither NIS immunohistochemical analysis nor NIS mRNA quantified expression could identify individuals with poor prognosis.
Our data suggest that NIS expression may help characterize patients' risk and individuals with a poor response to therapy, but is not useful as a diagnostic or prognostic marker, reinforcing the current concept that an appropriate management of DTC patient is the most important and modifiable prognostic factor.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: A 76-year-old man presented with small reddish, painless nodular lesions of the chest and on neck surgical scars. The patient had, 5 years before, a T3N1M0 classic papillary thyroid carcinoma that evolved with recurrent cervical lymph node metastasis despite 3 surgeries and a cumulative dose of 500 mCi of 131Iodine. Investigations included the lesion biopsy and Tg immunohistochemistry, which led to the diagnosis of rare skin metastasis from a well differentiated thyroid tumor. The cutaneous lesions also tested positive for sodium iodide symporter protein, with clear membrane immunohistochemical staining. However, the patient did not respond to an additional dose of 400 mCi of radioiodine, suggesting a nonfunctional sodium iodide symporter protein. The lesions rapidly spread to the chest wall and erythematous red-purple inflamed nodules became confluent over a 3-month period. The tumor invaded the trachea causing the patient's death.
The Endocrinologist 08/2009; 19(5):214-217. · 0.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: The glutathione-S-transferase (GST) gene family has an important role in the biotransformation and detoxification of different xenobiotics and endogenous carcinogens. GST profile has been associated to an increased risk for several types of tumors in different populations, but ethnic stratification makes data interpretation difficult. The Brazilian population represents a unique model in which the types and frequencies of GST gene polymorphisms are less influenced by ethnicity.
To evaluate the influence of GST profile in different age and gender groups regarding the risk of developing cancer and its relationship to smoking habit, the GSTT1, GSTM1, and GSTP1 genotypes of 785 Brazilian patients with cancer and 873 cancer-free controls paired on the basis of sex, age, ethnicity, diet and exercise routine, lifetime occupational history, smoking history, general health conditions, and previous diseases were compared.
A univariate logistic regression analysis demonstrated that age over 45 years (p=0.0417) and smoking (p=0.0015) were related to cancer. Multivariate analysis confirmed the importance of advanced age in susceptibility to cancer (p=0.0001). It was also observed that smoking significantly increased the risk of cancer among individuals over 45 years old (OR: 1.825, 95%CI: 1.241-2.682). However, no correlation between risk of cancer, smoking habit, age, or gender and any of the studied GST polymorphisms was found.
It is suggested that GST profile does not exert an important impact on the influence of tobacco smoking on cancer risk.
Medical science monitor: international medical journal of experimental and clinical research 02/2009; 15(1):CR10-5. · 1.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genetic polymorphisms in genes encoding for enzymes involved in the biotransformation of carcinogens have been shown to be relevant as risk for cancer and may be of considerable importance from a public health point of view. Considering that N-acetyltransferase 2 (NAT2) polymorphisms modulate the response to ionizing radiation, the strongest risk factor recognized to cause differentiated thyroid cancer (DTC) thus far, we sought to determine the influence of NAT2 detoxification system on thyroid cancer susceptibility.
We conducted a prospective case-control study, comparing 195 patients presenting with DTC that were previously genotyped for GSTT1, GSTM1, GSTP1, and CYP1A1, comprising 164 papillary carcinomas and 31 follicular carcinomas, with 196 control individuals paired for gender, age, ethnicity, diet routine, lifetime occupational history, smoking history, general health conditions, and previous diseases. We used PCR-RFLP assays and the combination of 6 variant alleles to define 18 NAT2 haplotypes that characterized slow, intermediate, or rapid phenotypes.
A multivariate logistic regression analysis identified the presence of *12A and the absence of *12B, *13, *14B, *14D, *6A, and *7A NAT2 haplotypes as risk factors for DTC. The inheritance of a rapid acetylation phenotype doubled the risk for a papillary carcinoma (odds ratio, 2.024; 95% confidence interval, 1.252-3.272). We found no relationship between genotypes and clinical, pathologic, or laboratory features of patients or between genotypes and outcome.
We showed that NAT2 genotypes and the NAT2 rapid acetylation phenotype are important susceptibility factors for DTC, suggesting that NAT2 detoxification system is involved in this tumor pathogenesis.
Clinical Cancer Research 02/2009; 15(1):406-12. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cigarette smoking is a well-recognized risk factor of Graves' disease and, particularly, Graves' ophthalmopathy. Hence, germline polymorphisms of detoxification genes and genes belonging to the major DNA repair-apoptosis pathways might have an important role in disease susceptibility. In addition, as some of these genes are regulated by thyroid hormones, they may affect the patients' outcomes. We aimed to assess the influence of the GST, CYP and TP53 gene polymorphisms in the risk of Graves' disease and its outcome.
Prospective case-control study.
A PCR-based strategy was used for GSTT1, GSTM1, GSTP1, CYP1A1 and TP53 codon 72 genotypes in a group of 400 Graves' disease patients, and to compare them to 574 control individuals with similar environmental exposure features.
GSTM1 and GSTT1 genotypes were equally distributed in cases and controls, respectively. However, GSTP1 (P < 0.0001), CYP1A1 (P < 0.0033) and Pro/ProTP53 (P < 0.0035) variants appeared more frequently in Graves' disease patients than in controls. A multivariate analysis indicated that cigarette smoking and inheritance of GSTP1, CYP1A1 and Pro/ProTP53 variants were important risk factors for Graves' disease, but only smoking appeared as an independent risk factor for Graves' ophthalmopathy. There was no association between clinical features, including ophthalmopathy or treatment outcome, and the studied genotypes.
We concluded that GSTP1, CYP1A1 and TP53, but not GSTT1 and GSTM1 germline polymorphisms, may be associated with smoking-related Graves' disease susceptibility and configure a risk profile for the disease. However, these polymorphisms do not influence the patients' response to treatment.
[show abstract][hide abstract] ABSTRACT: The large use of simple and effective diagnostic tools has significantly contributed to the increase in diagnosis of thyroid cancer over the past years. However, there is compelling evidence that most micropapillary carcinomas have an indolent behavior and may never evolve into clinical cancers. Therefore, there is an urgent need for new tools able to predict which thyroid cancers will remain silent, and which thyroid cancers will present an aggressive behavior. There are a number of well-established clinical predictors of malignancy and recent studies have suggested that some of the patients laboratory data and image methods may be useful. Molecular markers have also been increasingly tested and some of them appear to be very promising, such as BRAF, a few GST genes and p53 polymorphisms. In addition, modern tools, such as immunocytochemical markers, and the measure of the fractal nature of chromatin organization may increase the specificity of the pathological diagnosis of malignancy and help ascertain the prognosis. Guidelines designed to select nodules for further evaluation, as well as new methods aimed at distinguishing carcinomas of higher aggressiveness among the usually indolent thyroid tumors are an utmost necessity.
[show abstract][hide abstract] ABSTRACT: The identification of groups at high risk is fundamental to determine preventive strategies for skin cancer. Destructive reactive oxygen species produced by UVA or chemical carcinogens are metabolized by a series of enzymes. Polymorphisms of genes encoding for these enzymes may produce defective proteins with a diminished ability to detoxify a wide range of carcinogens.
To ascertain the influence and potential interactions of several polymorphisms of genes encoding four important antioxidant GST enzymes in the susceptibility to cancer among Brazilians.
We compared the genotypes of Glutathione S-Transferase mu, theta, pi and omega (GSTM1, GSTT1, GSTP1 and GSTO2) in a group of 102 patients with skin lesions and 124 controls.
Patients with Basal Cell Skin Carcinoma (BCC) presented the combined GSTM1-GSTT1+ genotype more frequently (49.1%) than controls (29.8%) (Fisher test; p=0.04), conferring a 2.273 (Odds Ratio; 95% CI=1.199-4.308) higher risk for BCC. We were not able to find any other association between genotypes or between any genotype and the patients' clinical features.
The GST profile may help identify Brazilian individuals at higher risk for BCC.
Revista medica de Chile 04/2007; 135(3):301-6. · 0.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: In contrast to most human malignancies, epidemiologic studies have frequently reported a reduced risk of differentiated thyroid cancer in tobacco consumers. Cytochrome P4501A1 (CYP1A1) gene variants may be related to an increased capacity to activate polycyclic aromatic hydrocarbons, producing highly reactive electrophilic intermediates that might damage DNA. Hence, the germline inheritance of a wild-type CYP1A1 gene may decrease the susceptibility for thyroid cancer. The present study was designed to investigate CYP1A1 (m1 and m2) role in thyroid tumorigenesis and its connection with GSTM1, GSTT1, GSTP1, GSTO1, and codon 72 of p53 genotypes. A total of 248 patients with thyroid nodules, including 67 benign goiters, 13 follicular adenomas, 136 papillary carcinomas, and 32 follicular carcinomas, and 277 controls with similar ethnic backgrounds were interviewed on their lifetime dietary and occupational histories, smoking habit, previous diseases, and other anamnestic data. DNA was extracted from a blood sample and submitted to PCR-restriction fragment length polymorphism assays. The wild-type CYP1A1m1 genotype was more frequent among papillary carcinoma patients (74.26%) than in the control population (62.45%; P=0.0147), reducing the risk for this type of cancer (odds ratio=0.564; 95% confidence interval=0.357-0.894). A multiple logistic regression analysis showed an inverse correlation between cigarette smoking (P=0.0385) and CYP1A1 germline inheritance (P=0.0237) with the susceptibility to papillary carcinomas. We were not able to find any correlation between smoking, clinical features, parameters of aggressiveness at diagnosis or during follow-up, and any of the GST or CYP genotypes considered separately or in different combinations. We suggest that CYP1A1 genotype might be associated with the reported reduced risk to papillary carcinomas among smokers.
Endocrine Related Cancer 01/2007; 13(4):1185-93. · 5.26 Impact Factor