Alamandine treatment prevents LPS-induced acute renal and systemic dysfunction with multi-organ injury in rats via inhibiting iNOS expression.

Sepsis is defined as the dysregulated immune response leading to multi-organ dysfunction and injury. Sepsis-induced acute kidney injury is a significant contributor to morbidity and mortality. Alamandine (ALA) is a novel endogenous peptide of the renin-angiotensin-aldosterone system. It is known for its anti-inflammatory and anti-apoptotic effects, but its functional and vascular effects on sepsis remain unclear. We aimed to investigate the effects of ALA, as a pre- and post-treatment agent, on lipopolysaccharide (LPS)-induced systemic and renal dysfunction and injury in the LPS-induced endotoxemia model in rats via functional, hemodynamic, vascular, molecular, biochemical, and histopathological evaluation. 10 mg/kg intraperitoneal LPS injection caused both hepatic and renal injury, decreased blood flow in several organs, and renal dysfunction at 20 h in Sprague-Dawley rats. Our results showed that ALA treatment ameliorated systemic and renal inflammation, reduced inflammatory cytokines, prevented the enhancement of the mortality rate, reversed vascular dysfunction, corrected decreased blood flows in several organs, and reduced renal and hepatic injury via inhibiting iNOS (inducible nitric oxide synthase) and caspase expressions in the kidney. In addition, expressions of different ALA-related receptors showed alterations in this model, and ALA treatment reversed these alterations. These data suggest that ALA's systemic and renal protective effects are achieved through its anti-inflammatory, anti-pyroptotic, and anti-apoptotic effects on hemodynamic and vascular functions via reduced iNOS expression.

Association of Human Leukocyte Antigen Genotypes with Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine-Induced Subacute Thyroiditis.

Background: Despite mass vaccination, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine-induced subacute thyroiditis (SAT) is rarely seen as a complication. The reason why some individuals are susceptible to developing vaccine-induced SAT is not known. SAT develops in genetically predisposed individuals who carry specific human leukocyte antigen (HLA) haplotypes. It is unknown whether specific HLA alleles are associated with SARS-CoV-2 vaccine-induced SAT. Objective: This study compared the HLA profiles of patients with SARS-CoV-2 vaccine-induced SAT to controls, to assess whether there is an association between specific HLA genotypes and development of SAT. The relationship between HLA genotypes and the clinical course of SARS-CoV-2 vaccine-induced SAT was also evaluated. Methods: A case-control study was conducted in a Turkish tertiary care center. Fourteen patients with SARS-CoV-2 vaccine-induced SAT and 100 healthy controls were included. HLA-A, HLA-B, HLA-C, HLA-DQB1, and HLA-DRB1 frequencies were analyzed by next-generation sequencing. Results: The frequencies of HLA-B*35 and HLA-C*04 alleles were significantly higher in SARS-CoV-2 vaccine-induced SAT cohort when compared with controls (HLA-B*35: 13 [93%] vs. 40 [40%], p < 0.001; HLA-C*04: 13 [93%] vs. 43 [43%], p < 0.001, respectively). More severe thyrotoxicosis was seen in patients having HLA-B*35 and HLA-C*04 homozygous alleles (free thyroxine: 4.47 ng/dL [3.77-5.18] vs. 1.41 ng/dL [1.22-2.63], p = 0.048). Inflammation tended to be more severe in homozygous patients (C-reactive protein: 28.2 mg/dL [13.6-42.9] vs. 4.8 [1.2-10.5], p = 0.07). Conclusions: The frequencies of HLA-B*35 and HLA-C*04 alleles were higher in SARS-CoV-2 vaccine-induced SAT compared with controls. Homozygosity for HLA-B*35 and HLA-C*04 was associated with thyrotoxicosis and a greater inflammatory reaction. Our findings should be confirmed in studies of other populations.

Prognostic role of serum cytokines and soluble HLA-G levels in children with leukemia who undergo allogeneic stem cell transplantation.

This is the first study to have investigated the prognostic role of cytokines and soluble human leukocyte antigen-G (sHLA-G) levels in pediatric leukemia patients who have undergone allogeneic stem cell transplantation (allo-SCT). Forty-one patients with acute leukemia (n = 28, acute lymphoblastic leukemia (ALL) and n = 13, acute myeloblastic leukemia) were recruited. Patients were examined at diagnosis (n = 26), in the pre-transplantation period (PreTx) (n = 26), on the day of transplantation (Tx0) (n = 41), and on post-transplantation Days 14 (PostTx14) (n = 41) and 28 (PostTx28) (n = 41). Serum levels of pro-inflammatory cytokines (interleukin [IL]-1, IL-2, IL-6, Tumor necrosis factor [TNF]-α), anti-inflammatory cytokines (IL-4, IL-10), and sHLA-G were measured by Enzyme-Linked ImmunoSorbent Assay. Median levels of all cytokines tested and sHLA-G were significantly higher at diagnosis and at the post-transplant time points than at PreTx (all p < 0.05). At the time of diagnosis (specifically ALL) and at PostTx14, elevated IL-4, IL-10, and/or sHLA-G were associated with higher post-transplant relapse rates (all p < 0.05). Elevated IL-2 and TNF-α at Tx0 were associated with lower survival rates (both p < 0.05). Levels of serum cytokines and sHLA-G may be useful predictors of survival and relapse in pediatric leukemia patients who undergo allo-SCT.

Interleukin-21 and Interleukin-32 gene expression levels and their relationship with clinicopathological parameters in colorectal cancer.

BACKGROUND AND AIMS: The immune cells in tumor microenvironment release chemokines and cytokines which determine the immune phenotype of the tumor and play an important role in the prognosis. Present study evaluates the gene expression levels of IL-21 and IL-32 and their relations to clinicopathologic parameters in colorectal cancer. PATIENTS AND METHODS: 31(17F) patients with colorectal cancer were included. Samples were obtained from normal and tumor tissues. After RNA isolation, IL-21 and IL-32 gene expression levels were measured. Immunohistochemistry was also carried out for CD4+, CD8+ and NKcells to measure cell density. The relations between expression levels, immune cell density and differentiation, stage, presence of vascular, perineural invasion and lymph node metastasis(MLN) were investigated. RESULTS: IL-32 gene expression levels were increased in tumor tissues. IL-21 levels were found to be decreased in 50% of the patients. IL-32 levels were also increased with the stage however, it was decreased significantly with the increased number of the MLN. On the other hand, expression levels of IL-21 increased significantly with the presence of vascular invasion. CD4+ density was decreased with increased T-stage, vascular invasion whereas CD8+ density decreased only with the vascular invasion. CONCLUSIONS: IL-32 expressed by tumor microenvironment reveals that expression increased to control tumor growth, but levels are decreased with the increased number of MLNs which might be due to decreased CD4+ cell density. Changes on IL-21 and IL-32 together with the changes on immune cell density, indicate their role in tumor growth and invasion in colon cancer. KEY WORDS: Colorectal Cancer, Cytokines, Immune Cell Density, Interleukin-21, Interleukin-32, Tumor Microenvironment.

Evaluation of HLA class I and HLA class II allele profile and its relationship with clinical features in patients with alopecia areata: a case-control study.

BACKGROUND: Alopecia areata (AA) is an autoimmune disease where autoimmune dysregulations along with genetic susceptibility are hypothesized to play a role in pathogenesis. OBJECTIVE: The aim of this study in to evaluate HLA-A, HLA-B, HLA-C, HLA-DQB1, and HLA-DRB1 profile and its relationship with clinical features in AA patients. MATERIALS AND METHODS: Ninety-eight patients with AA and 100 healthy controls were included in the study. HLA-A, HLA-B, HLA-C, HLA-DQB1, and HLA-DRB1 frequencies were analyzed using polymerase chain reaction-sequence specific primers (PCR-SSP). RESULTS: HLA-B*39 and HLA-HLA-DRB1*15 allele frequencies were increased (p = .022 and p = .023, respectively), HLA-A*11 and HLA-B*35 frequencies were decreased (p = .006 and p = .014, respectively) in AA patients. HLA-B*13 and HLA-DRB1*11 were associated with poor prognostic factors. A class I allele, HLA-B*13 was associated with recurrence (p = .023) and presence of nevus flammeus (p = .022), while the class II allele HLA-DRB1*11 was associated with widespread hair loss (diffuse or universal alopecia) (p = .026), presence of ophiasis (p = .049) and juvenile onset (p = .018). CONCLUSION: Belonging to two different classes of HLA family, HLA-B*13 and HLA-DRB1*11 alleles identified separate set of risk factors. In addition to increasing the risk of AA, HLA alleles may affect the prognosis of the disease.

Spondyloarthritides: Theories and beyond.

Spondyloarthritides (SpA) are a family of interrelated rheumatic disorders with a typical disease onset ranging from childhood to middle age. If left untreated, they lead to a severe decrease in patients' quality of life. A succesfull treatment strategy starts with an accurate diagnosis which is achieved through careful analysis of medical symptoms. Classification criterias are used to this process and are updated on a regular basis. Although there is a lack of definite knowledge on the disease etiology of SpA, several studies have paved the way for understanding plausible risk factors and developing treatment strategies. The significant increase of HLA-B27 positivity in SpA patients makes it a strong candidate as a predisposing factor and several theories have been proposed to explain HLA-B27 driven disease progression. However, the presence of HLA-B27 negative patients underlines the presence of additional risk factors. The current treatment options for SpAs are Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), TNF inhibitors (TNFis), Disease-Modifying Anti-Rheumatic Drugs (DMARDs) and physiotherapy yet there are ongoing clinical trials. Anti IL17 drugs and targeted synthetic DMARDs such as JAK inhibitors are also emerging as treatment alternatives. This review discusses the current diagnosis criteria, treatment options and gives an overview of the previous findings and theories to clarify the possible contributors to SpA pathogenesis with a focus on Ankylosing Spondylitis (AS) and enthesitis-related arthritis (ERA).

D2 vs D2 Plus Para-aortic Lymph Node Dissection for Advanced Gastric Cancer.

OBJECTIVES: Gastric cancer is a common malignancy worldwide. Effective treatment by interdisciplinary cooperation is important, and surgery still plays an important role. MATERIAL AND METHODS: In a ten-year period, 355 patients were diagnosed to have gastric cancer. One hundred and sixty-two patients with a median (range) age of 58 (23-83) years were eligible for the study. There were 107 patients in D2 and 55 patients in D2 lymphadenectomy plus para-aortic lymph node (PALN) dissection group. The two groups were compared in terms of complications, morbidity, mortality and long-term survival. RESULTS: Length of stay was 12 (8-34) days for D2 and 14 (8-42) days for D2 plus PALND. Total number of operative mortality was 8/162 (5%), and it was not different between the groups. Twenty patients (18%) had complications in D2 group and 9 (17%) patients in D2 plus PALND group. Overall survival was also similar between the groups, but patients with T3-T4 tumors, patients with stage IIIA and IIIB disease had better survival with D2 plus PALN dissection. We found that the depth of invasion, PLN, ratio (PLN/TLN), stage and LND were all prognostic variables. CONCLUSION: This study showed that D2 plus PALN dissection for advanced gastric cancer can be performed as safely as a standard D2 dissection by experienced surgeons without increasing postoperative morbidity and mortality. D2 plus PALN dissection should be preferred in the advanced stage of the disease (IIIA-IIIB) as it increases the rate of survival.

The relationship between non-segmental Vitiligo, HLA genotype and oxidative stress.

BACKGROUND: Vitiligo is an autoimmune disease characterised by acquired loss of melanocytes. Although the pathogenesis of vitiligo remains unknown, oxidative stress and autoimmune dysregulations are considered to play a role. OBJECTIVE: The aim of this study was to evaluate the HLA profile and total antioxidant capacity (TAC) and their relationship to clinical characteristic of vitiligo patients. METHODS: Ninety-one vitiligo patients and 100 healthy controls were included in the study. We analysed HLA allele frequencies using sequence-specific oligonucleotide Prob (SSOP) method. Serum total antioxidant capacity (TAC) levels were measured and compared between vitiligo patients and controls. RESULTS: HLA-A*02 allele frequency was increased (OR = 1.6, CI = 1.12-2.24, P = .009), HLA-A*11 (OR = 0.46, CI = 0.32-0.91, P = .019) and HLA-DRB1*01 (OR = 0.39, CI = 0.16-0.92, P = .029) frequencies were decreased in vitiligo patients. HLA-A*02 allele especially increased the risk of late onset (Vitiligo onset >30 years of age) vitiligo (OR:3.67, 95% CI: 1.63-8.26, P = .002). Serum TAC levels were similar between vitiligo patients and healthy controls but TAC levels were significantly lower in patients who did not have an HLA-DRB1*01 allele (1.52 vs 1.61, P = .033). CONCLUSION: Our study showed that HLA-A*02 increases, HLA-A*11 and HLA-DRB1*01 decreases vitiligo susceptibility in Turkish patients as well as a possible relationship between HLA and TAC.

The Effect of Bariatric Surgery on Exocrine Pancreatic Function.

INTRODUCTION: After bariatric surgery (BS), patients might suffer from nutrient maldigestion, malabsorption, and vitamin deficiencies. In this study, our aim was to assess pancreatic functions after BS using fecal elastase-1 assay (FE-1). MATERIAL AND METHODS: Sixty patients (21M) undergoing BS and 20 (6M) healthy controls were included into the study. Stool samples were collected 1 year after surgery. Ten patients from one anastomosis gastric bypass (OAGB) and single anastomosis duodenal switch (SADS) groups with the lowest value of FE-1 and GIQLI scores were given pancreatic enzyme replacement therapy (PERT). After PERT, FE-1, excess weight loss (EWL), BMI, GIQLI scores, and vitamin D levels were measured. RESULTS: Vitamin D levels were detected as 19.04 (9-46.5) pg/ml, 15.1 (8.4-23.6) pg/ml, 17.8 (5-30) pg/ml, and 21.79 (11-40.3) pg/ml after sleeve gastrectomy (SG), OAGB, SADS, and control groups, respectively (p = 0.04). GIQLI scores in the first year were found to have increased in all patients (p = 0.02). FE-1 levels were found as 642.35 (566.3-711.4) µg/g, 378.52 (183.5-561.1) µg/g, 458.88 (252.5-593, 5) µg/g, and 518.2 (351.6-691) µg/g for the SG, OAGB, SADS, and control groups, respectively. There was a strong inverse correlation between EWL and FE-1 levels at the end of the first year (Spearman's rho = - 0.688, p = 0.003). After having performed PERT for patients with the lowest FE-1 levels, the levels increased to 683.39 (615.5-720) µg/g in the OAGB and 691.5 (643.1-720) µg/g in the SADS groups (p = 0.011). CONCLUSION: FE-1 measurements demonstrated that many patients suffer from malabsorption after OAGB or SADS, whereas functions remain normal after SG. PERT corrects pancreatic functions without affecting weight loss and also contributes to the normal serum level of vitamin D.

Association of class II human leukocyte antigen (HLA) alleles with pulmonary sarcoidosis.

Background and objectives: Sarcoidosis is a systemic inflammatory disease of unknown etiology that involves any part of the body, mainly the lungs and thoracic lymph nodes. The clinical presentation is heterogeneous based on the degree and extent of organ involvement. The existence of variable clinical presentations and treatment responses suggest an important role of genetic predisposition. In genetic studies, sarcoidosis was found to be associated with several genes, but the strongest link was with HLA region. The aim of this study was to investigate the association of HLA class II alleles with the extent and course of disease in Turkish patients with sarcoidosis. Methods: The study included 103 patients with sarcoidosis and 100 unrelated healthy controls. HLA-DRB1 and HLA-DQB1 typing was performed by using Polymerase Chain Reaction-Sequence Specific Priming (PCR-SSP) method at low resolution level. Results: HLA-DRB1* and -DQB1* analysis revealed that while the frequency of HLA-DRB1*01 was significantly higher in the control group, HLA-DRB1*13 and -DQB1*06 alleles were more frequent in the sarcoidosis patients. When the patients were grouped based on clinical outcome as remitters and non-remitters, HLA-DRB1*10 allele was only detected in the remitters, whereas the frequency of HLA-DQB1*06 allele was significantly higher in non-remitters. Conclusions: This study supported the association of HLA alleles with sarcoidosis. In a considerably high number of patients with Turkish origin, the frequency of HLA-DRB1*13, -DRB1*10 and HLA-DQB1*06 alleles was significantly associated with increased risk and clinical outcome. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 143-149).

Netrin-1 is associated with macrophage infiltration and polarization in human epicardial adipose tissue in coronary artery disease.

BACKGROUND: Inflammatory activity originating from the epicardial adipose tissue (EAT) may have a role in coronary artery disease (CAD) pathogenesis. The relationship between macrophage infiltration, polarization in the EAT, and netrin-1 gene expression was investigated. METHODS: Macrophage infiltration and polarization were examined by immunohistochemical methods and expression levels of netrin-1, Unc5b, and cytokines related with M1-macrophage subtype (IL-12 and IL-18) were determined by quantitative polymerase chain reaction in subcutaneous and epicardial adipose tissue obtained from patients undergoing coronary artery bypass grafting and non-coronary cardiac surgery. RESULTS: CAD patients had higher CD68+ (p=0.005) and CD11c+ (p<0.001) macrophage count in EAT when compared to the controls. CD11c+/CD206+ macrophage ratio, which reflects dominancy of M1-macrophage phenotype, was significantly increased in EAT of CAD patients when compared to that of the controls (p=0.008). CAD patients had significantly higher netrin-1, Unc5b, and IL-18 gene expression in the EAT when compared to the control group (p<0.001, p<0.001, and p=0.006 respectively). Increased macrophage infiltration and polarization were associated with higher netrin-1, Unc5b, and IL-12 gene expression in EAT (p<0.05). CONCLUSIONS: Findings suggest a link between enhanced netrin-1 expression in EAT and macrophage infiltration and polarization in patients with CAD.

STEAP4 and HIF-1α gene expressions in visceral and subcutaneous adipose tissue of the morbidly obese patients.

AIM AND BACKGROUND: Obesity is a multifactorial disease in which environmental and genetic factors play an integrated role. Determining such target genes will help to elucidate the mechanisms underlying complex diseases such as obesity and diabetes which are usually seen together. Present study investigates the expression levels of STEAP4 and HIF-1α in visceral and subcutaneous adipose tissue. PATIENTS AND METHODS: 30(6M) morbidly obese patients undergoing bariatric surgery were included in the study. The patients were grouped according to the BMI as Group I (BMI <50kg/m(2)) and Group II (BMI ≥50kg/m(2)). Samples from visceral (omentum) and subcutaneous adipose tissues were obtained from each patient and real-time PCR (qPCR) was carried out for STEAP4 and HIF-1α gene expressions. Correlations between expression levels and clinical parameters were analyzed. RESULTS: Mean age of the patients recruited to the study was 37.4 (18-64) years. Mean BMI was 46 (36-60) kg/m(2). STEAP4 expression in visceral adipose tissue was significantly higher than subcutaneous tissue. Visceral STEAP4 expression was also found to be reduced with increased BMI. It was also lower in patients with HbA1C over 6. Furthermore, expression of subcutaneous and visceral HIF-1α was significantly higher in Group II. There was a significant correlation between BMI, glycosylated hemoglobin, STEAP4 and HIF-1α gene expression. CONCLUSIONS: Obesity and related disease are linked with the fact that there is a low grade inflammation in the adipose tissue of the obese individuals. Counter-regulatory processes such as STEAP4 protein family are overwhelmed by the proinflammatory stimuli. HIF-1α expression is increased due to tissue hypoxia and pro-inflammatory stimuli in the obese individuals, which results in increased visceral STEAP4 expressions.

Current adjuvant therapeutic approaches for pancreatic cancer.

Pancreatic cancer continues to be the fourth leading cause of death despite advancements in surgical and adjuvant therapeutic approaches. In the present review, the current cytotoxic therapeutic approaches and advanced targeted therapies are objectively discussed with consideration to the current literature.

Importance of serum SELDI-TOF-MS analysis in the diagnosis of early lung cancer.

BACKGROUND: Different methods of diagnosis have been found to be inefficient in terms of screening and early diagnosis of lung cancer. Cancer cells produce proteins whose serum levels may be elevated during the early stages of cancer development. Therefore, those proteins may be recognized as potential cancer markers. The aim of this study was to differentiate healthy individuals and lung cancer cases by analyzing their serum protein profiles and evaluate the efficacy of this method in the early diagnosis of lung cancer. MATERIALS AND METHODS: 170 patients with lung cancer, 53 under high risk of lung cancer, and 47 healthy people were included in our study. Proteomic analysis of the samples was performed with the SELDI-TOF-MS approach. RESULTS: The most discriminatory peak of the high risk group was 8141. When tree classification analysis was performed between lung cancer and the healthy control group, 11547 was determined as the most discriminatory peak, with a sensitivity of 85.5%, a specificity of 89.4%, a positive predictive value (PPV) of 96.7% and a negative predictive value (NPV) of 62.7%. CONCLUSIONS: We determined three different protein peaks 11480, 11547 and 11679 were only present in the lung cancer group. The 8141 peak was found in the high-risk group, but not in the lung cancer and control groups. These peaks may prove to be markers of lung cancer which suggests that they may be used in the early diagnosis of lung cancer.

Response letter regarding the interpretation of gene expression data.

This is a response letter to Verna E's comments regarding our previous manuscript published last year in the World Journal of Gastroenterology entitled "Relationship between LYVE-1, VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer", which evaluated the relationship between these expression levels and clinicopathological parameters (Ozmen F et al, World J Gastroenterology 2011; 17: 3220-3228). The mean values for lymphatic vessel endothelial hyaluronan receptor-1, CD44 and vascular endothelial growth factor receptor-3 expression (represented as 2(-ΔΔCt)) were 1.13, 1.24 and 1.17, respectively, suggesting an increase in gene expression in tumor tissue compared to normal tissue. Despite the increase in gene expression in the cancer tissues (2(-ΔΔCt) > 1), only some of the results reached statistical significance, which was thoroughly discussed in our paper. In the present letter, we report that his comments are flawed and result in confusion. Therefore, we herein provide more explanation regarding gene expression in gastric cancer. We hope that this letter will address Verna E's misunderstandings.

The roles of epigenetic modifications of proapoptotic BID and BIM genes in imatinib-resistant chronic myeloid leukemia cells.

In chronic myeloid leukemia (CML), epigenetic modifications such as promoter hypermethylation and inactive histone modification are known mechanisms of drug resistance. In our study, we investigated the roles of promoter hypermethylation of BIM and BID genes and H3K27me3 histone modification on imatinib resistance. We detected higher expression levels of BIM and BID genes and lower expression levels of EZH2, EED2, SIRT1, and SUZ12 genes in imatinib-resistant K562/IMA-3 cells compared to imatinib-non-resistant K562 cells. While we determined the EZH2 and DNMT enzymes as bounded to the promoter of the BIM gene, we did not detect hypermethylation of this promoter. We also found the H3K27me3 histone modification promoter of BIM and BID genes in both cell lines. In conclusion, our results support the notion that DNA promoter methylation may be formed independently from EZH2-H3K27me3 and pro-apoptotic BIM and BID genes are not methyllated in the imatinib resistance of CML cells.

Relationship between LYVE-1, VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer.

AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their levels and clinicopathological parameters in gastric cancer. METHODS: Tissue samples were obtained from 33 patients (8 females) with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS: LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic features such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the distribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION: LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.