MHC-I upregulation safeguards neoplastic T cells in the skin against NK cell-mediated eradication in mycosis fungoides.

Cancer-associated immune dysfunction is a major challenge for effective therapies. The emergence of antibodies targeting tumor cell-surface antigens led to advancements in the treatment of hematopoietic malignancies, particularly blood cancers. Yet their impact is constrained against tumors of hematopoietic origin manifesting in the skin. In this study, we employ a clonality-supervised deep learning methodology to dissect key pathological features implicated in mycosis fungoides, the most common cutaneous T-cell lymphoma. Our investigations unveil the prominence of the IL-32ß-major histocompatibility complex (MHC)-I axis as a critical determinant in tumor T-cell immune evasion within the skin microenvironment. In patients' skin, we find MHC-I to detrimentally impact the functionality of natural killer (NK) cells, diminishing antibody-dependent cellular cytotoxicity and promoting resistance of tumor skin T-cells to cell-surface targeting therapies. Through murine experiments in female mice, we demonstrate that disruption of the MHC-I interaction with NK cell inhibitory Ly49 receptors restores NK cell anti-tumor activity and targeted T-cell lymphoma elimination in vivo. These findings underscore the significance of attenuating the MHC-I-dependent immunosuppressive networks within skin tumors. Overall, our study introduces a strategy to reinvigorate NK cell-mediated anti-tumor responses to overcome treatment resistance to existing cell-surface targeted therapies for skin lymphoma.

Boost of innate immunity cytokines as biomarkers of response to extracorporeal photopheresis in patients with leukaemic cutaneous T-cell lymphoma.

BACKGROUND: Extracorporeal photopheresis (ECP) has emerged as a systemic first-line immunomodulatory therapy in leukaemic cutaneous T-cell lymphoma (L-CTCL) and is now beginning to be utilized in other T-cell-mediated diseases. Although ECP has been used for nearly 30 years, its mechanisms of action are not sufficiently understood, and biomarkers for response are scarce. OBJECTIVES: We aimed to investigate the immunomodulatory effects of ECP on cytokine secretion patterns in patients with L-CTCL, to help elucidate its mechanism of action. METHODS: A total of 25 patients with L-CTCL and 15 healthy donors (HDs) were enrolled in this retrospective cohort study. Concentrations of 22 cytokines were simultaneously quantified by using multiplex bead-based immunoassays. Neoplastic cells in patients' blood were evaluated by flow cytometry. RESULTS: Firstly, we observed a distinct cytokine profile pattern difference between L-CTCLs and HDs. There was a significant loss of tumour necrosis factor (TNF)-α, and significant increase of interleukins (IL)-9, IL-12 and IL-13 in the sera of patients with L-CTCL compared with HDs. Secondly, patients with L-CTCL who received ECP were classified as treatment responders and nonresponders according to the quantitative reduction of malignant burden in their blood. We evaluated cytokine levels in culture supernatants from patients' peripheral blood mononuclear cells (PBMCs) at baseline and 27 weeks after ECP initiation. Strikingly, PBMCs purified from ECP responders released statistically higher concentrations of innate immune cytokines IL-1α, IL-1ß, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α in comparison with ECP nonresponders. In parallel, responders showed clearance of erythema, reduction of malignant clonal T cells in the blood, and a potent boost of relevant innate immune cytokines in individual patients with L-CTCL. CONCLUSIONS: Taken together, our results demonstrate that ECP stimulates the innate immune network, and facilitates redirection of the tumour-biased immunosuppressive microenvironment towards proactive antitumour immune responses. The alterations of IL-1α, IL-1ß, GM-CSF and TNF-α can be used as biomarkers of response to ECP in patients with L-CTCL.

Increased Chlormethine-Induced DNA Double-Stranded Breaks in Malignant T Cells from Mycosis Fungoides Skin Lesions.

Mycosis fungoides (MF) is a type of cutaneous T-cell lymphoma. Chlormethine (CL) is recommended as first-line therapy for MF, with a major purpose to kill tumor cells through DNA alkylation. To study the extent of treatment susceptibility and tumor specificity, we investigated the gene expression of different DNA repair pathways, DNA double-stranded breaks, and tumor cell proliferation of clonal TCR Vß+ tumor cell populations in cutaneous T-cell lymphoma skin cells on direct exposure to CL. Healthy human T cells were less susceptible to CL exposure than two T-lymphoma cell lines, resulting in higher proportions of viable cells. Interestingly, in T cells from MF lesions, we observed a downregulation of several important DNA repair pathways, even complete silencing of RAD51AP1, FANC1, and BRCA2 involved in homologous recombination repair. In the presence of CL, the double-stranded DNA breaks in malignant MF skin T cells increased significantly as well as the expression of the apoptotic gene CASP3. These data point toward an important effect of targeting CL on MF skin tumor T cells, which support CL use as an early cutaneous lymphoma treatment and can be of synergistic use, especially beneficial in the setting of combination skin-directed therapies for cutaneous T-cell lymphoma.

Post-COVID-19 psychosis: Cotard's syndrome and potentially high risk of harm and self-harm in a first-onset acute and transient psychotic disorder after resolution of COVID-19 pneumonia.

AIM: This report aims to illustrate the possibility of an acute onset of psychosis after COVID-19 infection in a patient without previous history of psychiatric disorders and to highlight the need for early screening and intervention in such cases. METHODS: Clinical presentation of a case, followed by clinical discussion and literature review of the effect of the new coronavirus SARS-CoV-2 and its impact on mental health in terms of neuropsychiatric conditions. RESULTS: We present a case of acute and transient psychotic disorder following complete recovery of COVID-19 bilateral pneumonia. The patient has no prior psychiatric history and presents with acute onset, disorganized behaviour, Cotard's delusion and a potentially high risk of psychotic homicide and suicide. CONCLUSION: Early intervention and treatment with antipsychotic medication are of crucial importance for the effective treatment and complete recovery of these patients.

Review of medicine utilization for Parkinson's disease management: the Bulgarian perspective.

BACKGROUND: Parkinson's disease (PD), which occurs in 1% of the population, is the second most common neurodegenerative disorder. Despite the broad spectrum of PD manifestations and high disease prevalence, there are insufficient data on medicine utilization and prescription strategies. The purpose of the current study was to analyze published data concerning treatment approaches and to compare them with Bulgarian therapeutic practice. DESIGN AND METHODS: We conducted a systematic review of the PubMed and Google Scholar databases, and we calculated medicine utilization in Bulgaria during 2018 and 2019 using the WHO methodology. RESULTS: The literature search identified a total of 311 publications, but only 12 met the inclusion criteria. Eleven studies pointed out that levodopa-containing medicine are the most frequently used, followed by dopamine agonists. The highest rate was found for levodopa-containing products and decarboxylase inhibitor (1.06 and 1.33 DDD/1000 inh/day), followed by anticholinergic Biperiden (0.494 and 0.455 DDD/1000 inh/day) during 2018 and 2019 in Bulgaria. CONCLUSION: Overall, the treatment approaches used in the last decade comply with guideline recommendations, despite variations in levodopa and dopamine agonist utilization. Even though new medicines have been approved for PD management, levodopa-containing products are still most often prescribed and used worldwide.

Oncolytic virotherapy-mediated anti-tumor response: a single-cell perspective.

Talimogene laherparepvec (T-VEC) is a genetically modified herpes simplex 1 virus (HSV-1) approved for cancer therapy. We investigate its effect on the clinical, histological, single-cell transcriptomic, and immune repertoire level using repeated fine-needle aspirates (FNAs) of injected and noninjected lesions in primary cutaneous B cell lymphoma (pCBCL). Thirteen patients received intralesional T-VEC, 11 of which demonstrate tumor response in the injected lesions. Using single-cell sequencing of the FNAs, we identify the malignant population and separate three pCBCL subtypes. Twenty-four hours after the injection, we detect HSV-1T-VEC transcripts in malignant and nonmalignant cells of the injected lesion but not of the noninjected lesion. Oncolytic virotherapy results in a rapid eradication of malignant cells. It also leads to interferon pathway activation and early influx of natural killer cells, monocytes, and dendritic cells. These events are followed by enrichment in cytotoxic T cells and a decrease of regulatory T cells in injected and noninjected lesions.

Blockade of programmed cell death protein 1 (PD-1) in Sézary syndrome reduces Th2 phenotype of non-tumoral T lymphocytes but may enhance tumor proliferation.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (L-CTCL) that arises from malignant clonally derived skin-homing CD4+ T cells. Based on advancements in our understanding of the mechanisms underlying L-CTCL, boosting the suppressed immune response emerges as a promising strategy in SS management. Immune checkpoint inhibitory molecules have already demonstrated efficacy in a wide spectrum of malignancies. Currently, agents targeting the programmed death-1 (PD-1) axis are under evaluation in L-CTCL. Here we investigated the expression of PD-1 and its ligands, PD-L1 and PD-L2 in blood and skin from patients with L-CTCL. We demonstrate that PD-1 expression is markedly increased on tumor T cells compared to non-tumor CD4+ T cells from SS patients and to CD4+ cells from healthy individuals. In contrast, PD-L1 shows decreased expression on tumor T cells, while PD-L2 expression is low without significant differences between these groups. Functional PD-1 blockade in vitro resulted in reduced Th2 phenotype of non-tumor T lymphocytes, but enhanced the proliferation of tumor T cells from SS patients. Our study sheds some light on the PD-1 axis in both peripheral blood and skin compartments in SS patients, which may be relevant for the treatment of L-CTCL with immune checkpoint inhibitor.

Pathogenesis and Therapy of Primary Cutaneous T-Cell Lymphoma: Collegium Internationale Allergologicum (CIA) Update 2020.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous disease group of unknown etiology with a complex immunological background. As CTCL arises from T cells that have a vital role in the antitumor response, their therapy is largely aimed at reversing the immunological mechanisms leading to or manifesting during this malignancy. Early disease stages can be controlled with skin-directed therapy in most CTCL cases. Still, advanced CTCL has a dismal prognosis and warrants systemic therapy. Despite considerable progress in understanding the pathophysiology of the disease and the numerous systemic treatment options available, long-term remission rates with conventional treatments alone are still low. Allogeneic hematopoietic stem cell transplantation is currently the only curative option for advanced CTCL, including mycosis fungoides and Sézary syndrome. The aims of this review is to summarize the recent findings on the immunology of this heterogeneous disease and to present the advances in its clinical management.

Selective inhibition of HDAC6 sensitizes cutaneous T-cell lymphoma to PI3K inhibitors.

Histone deacetylase (HDAC) inhibitors, approved for the treatment of cutaneous T-cell lymphoma (CTCL), are non-selective agents associated with an unsatisfactory response and considerable side-effects. Targeting single HDAC isoforms is considered to provide novel therapeutic options. HDAC6 is overexpressed in primary samples from patients with CTCL and preclinical studies using transgenic mice that spontaneously develop a CTCL-like disease, have suggested that combinations including HDAC6 inhibitors may be successful in the treatment of CTCL. PI3K inhibition is currently being tested in clinical trials for CTCL with promising results. Since HDAC6 is known to diminish the activity of Akt via its deacetylation, the aim of the present study was to evaluate the therapeutic potential of selective HDAC6 inhibitors in combination with PI3K inhibitors in CTCL. Through the genetic and pharmacological inhibition of HDAC6, it was demonstrated that combining HDAC6 with PI3K inhibition may be an attractive therapeutic option for patients with CTCL.

A novel proangiogenic B cell subset is increased in cancer and chronic inflammation.

B cells contribute to immune responses through the production of immunoglobulins, antigen presentation, and cytokine production. Several B cell subsets with distinct functions and polarized cytokine profiles have been reported. In this study, we used transcriptomics analysis of immortalized B cell clones to identify an IgG4+ B cell subset with a unique function. These B cells are characterized by simultaneous expression of proangiogenic cytokines including VEGF, CYR61, ADM, FGF2, PDGFA, and MDK. Consequently, supernatants from these clones efficiently promote endothelial cell tube formation. We identified CD49b and CD73 as surface markers identifying proangiogenic B cells. Circulating CD49b+CD73+ B cells showed significantly increased frequency in patients with melanoma and eosinophilic esophagitis (EoE), two diseases associated with angiogenesis. In addition, tissue-infiltrating IgG4+CD49b+CD73+ B cells expressing proangiogenic cytokines were detected in patients with EoE and melanoma. Our results demonstrate a previously unidentified proangiogenic B cell subset characterized by expression of CD49b, CD73, and proangiogenic cytokines.

The Potential Role of the Pharmacist in Supporting Patients with Depression - A Literature-Based Point of View.

OBJECTIVE: The current article is aimed at identifying the best practice for counseling around depression in community and outpatient pharmacies, resulting in a draft guideline, proposing key steps and an algorithm for integration of community pharmacists into care for patients with depression. METHODS: A literature review was performed followed by a detailed analysis, for the purpose of creation a short draft document used as a basis for creation of a guideline for pharmaceutical care for patients with depression. The technological scheme PRISMA flow diagram was applied. The paper is based on current knowledge, taking into consideration already published articles, guidelines, and recommendations about pharmaceutical care for patients with depression, giving a basis for further studies. RESULTS: This paper includes two main sections: 1) depression - a short description of the main symptoms, risk factors and pharmacotherapy guidelines available in Bulgaria important for the purposes of ensuring qualitative community-based pharmaceutical care; and 2) the pharmacists' role in providing high-quality care - the main aspects of pharmaceutical care for patients with depression with specific examples. CONCLUSION: The involvement of pharmacists in supporting depressive patients is crucial taking into account the specific characteristics of the pharmacological treatment: delayed onset of clinical results, risks in case of sudden pharmacotherapy abruption without physician consultation, multiple adverse drug reactions and drug-drug, drug-food and drug-alcohol interactions, etc. The current article could also be used as an initial document for creating a methodological guideline for providing pharmaceutical care services for patients with depression.

Costs and outcomes for individuals with psychosis prior to hospital admission and following discharge in Bulgaria.

PURPOSE: To examine the cost of psychotic disorders in Bulgaria prior to hospital admission and following discharge from two perspectives: healthcare and societal; and to evaluate the association between the costs and the patient's characteristics. METHODS: 96 individuals with psychosis experiencing psychotic exacerbation and their primary caregivers were evaluated upon the patients' hospital admission. The participants were followed up after 12 months. The costs were evaluated from healthcare and societal perspective using the Client's Sociodemographic and Service Receipt Inventory (CSSRI-EU). The psychopathology, functioning, quality of life and caregiver's burden were measured using standardized instruments. The mean differences in the costs and the associations with the clinical and socio-demographic characteristics of the patients were evaluated. RESULTS: The healthcare costs increase from EUR 120.66 (SD = 163.85) at baseline to EUR 177.54 (SD = 136.98) at follow-up. The total cost from societal perspective are up to sixfold higher than the healthcare costs at both assessments [EUR 717.41 (SD = 402.33) and 880.40 (SD = 1592.00), respectively] and do not change significantly. A major shift in the subtypes of costs, and significant associations of the costs with the socio-demographic and clinical characteristics, were found. CONCLUSIONS: Psychotic disorders and psychotic exacerbations have high societal costs. The underfunding of mental healthcare in Bulgaria is at the expense of high caregivers' and societal cost. The treatment of psychotic exacerbation is effective and investment in mental healthcare for the improvement of the psychopathology, social functioning, quality of life and the burden of informal care should be viewed as a sustainable investment.

ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions.

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a cell adhesion molecule of the immunoglobulin superfamily and has been implicated in diverse pathophysiological processes including T cell activation, leukocyte trafficking, and (lymph)angiogenesis. However, exploring the therapeutic potential of ALCAM blockade in immune-mediated inflammatory disorders has been difficult due to the lack of antibodies with blocking activity toward murine ALCAM. In this study, we identified and characterized a monoclonal antibody with high affinity and specificity for murine ALCAM. This antibody reduced in vitro T cell activation induced by antigen-presenting dendritic cells (DCs) as well as (trans)migration of murine DCs across lymphatic endothelial monolayers. Moreover, it reduced emigration of DCs from in vitro-cultured human skin biopsies. Similarly, antibody-based blockade of ALCAM reduced (lymph)angiogenic processes in vitro and decreased developmental lymphangiogenesis in vivo to levels observed in ALCAM-deficient mice. Since corneal allograft rejection is an important medical condition that also involves (lymph)angiogenesis, DC migration and T cell activation, we investigated the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection.

Divergent LAG-3 versus BTLA, TIGIT, and FCRL3 expression in Sézary syndrome.

In Sézary syndrome (SS) impaired T-cell function and cytokine profile lead to immune evasion. Immune checkpoints non-redundantly regulate immune responses and targeting them is promising. We evaluated the expression of BTLA, CTLA-4, FCRL3, LAG-3, and TIGIT in tumor and non-tumor SS T-cells.Compared to CD4+ T helper cells from ten healthy individuals, tumor cells of eight SS patients had a significant upregulation of BTLA (1.5-fold; p < .0001), FRCL3 (2.2-fold; p < .0028) and TIGIT (2.2-fold; p < .0003) expression. In contrast, we found a reduced expression of LAG-3+ cells in the blood of tumor patients (0.5-fold; p < .0014). Only weak alternations between tumor, non-tumor cells, and healthy controls were observed regarding CTLA-4 (0.5-fold; p < .2022). Our results show a diverse expression pattern of immune-regulatory molecules in SS patients. As these molecules are essential in the regulation of T-cell mediated tumor surveillance and defense, their specific targeting might be of clinical relevance.

PD-L1 expression is an independent predictor of favorable outcome in patients with localized esophageal adenocarcinoma.

Background. The outcome of patients with adenocarcinoma of the esophagogastric junction (AEG) remains poor. The programmed cell-death-protein-1 (PD-1), a co-inhibitory receptor primarily expressed by T-cells, represents a potential new therapeutic target. PD-1, PD-1 ligand 1 (PD-L1), and PD-L2 expression have all been described as prognostic factors in a variety of cancers. Their expression patterns in AEG, however, are poorly understood. We analyzed PD-L1, PD-L2 and PD-1 expression by tumor-infiltrating lymphocytes (TILs) and cancer-cells in tumor-biospecimens in AEG-patients. Methods. 168 patients who underwent esophagectomy because of AEG between 1992-2011 were included in this study. PD-L1, PD-L2 and PD-1 expression were evaluated by immunohistochemistry and correlated with various clinicopathological parameters, disease-free survival (DFS) and long-term overall survival (OS). Results. PD-L1 expression by cancer-cells (cancer-cell-PD-L1+) was found in 43.5% of patients whereas PD-L1 expression by TILs (TILs-PD-L1+) was observed in 69%. PD-L2 expression by cancer-cells and TILs was only found in 3.5% and 1.8%, respectively. Additionally, 77.4% of tumors contained PD-1+-cancer-cells and 81% PD-1+-TILs. Patients with increased expression of PD-1 by cancer-cells and TILs showed significantly reduced OS and DFS, as determined by univariate, but not multivariate analysis. Expression of PD-L1 by cancer-cells was found to be an independent predictor for improved DFS (p = 0.038) and OS (p = 0.042) in multivariate analysis. Conclusions. Cancer cells and TILs displayed PD-L1 expression in around 50% and PD-1 expression in around 80% of tumor-biospecimens obtained from AEG patients. Expression of PD-L1 is an independent predictor of favorable outcome in AEG, whereas PD-1 expression is associated with worse outcome and advanced tumor stage.

Aggressive Rare T-cell Lymphomas with Manifestation in the Skin: A Monocentric Cross-sectional Case Study.

Rare T- or NK-cell lymphomas with cutaneous manifestation may display a highly aggressive clinical course and major diagnostic/therapeutic challenges. This report describes our experiences with different lymphomas of this rare category and the therapeutic options used. This retrospective, descriptive, monocentric, cross-sectional case study, identified 4 rare aggressive T-/NK-cell lymphomas with manifestation in the skin, which were diagnosed in a tertiary care centre over a period of 4 years. Two patients had an Epstein-Barr virus-associated extranodal NK/T-cell lymphoma and 2 patients had a primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma. Concomitant extracutaneous involvement was observed in 2 of all 4 patients. Two patients had fulminant disease progression and resistance to chemotherapy. Two patients underwent allogeneic haematopoietic stem cell transplantation, which resulted in one complete remission and one partial remission. This report emphasizes the importance of an early diagnostic work-up and a prompt aggressive therapeutic approach.

PD1-positive tumor-infiltrating lymphocytes are associated with poor clinical outcome after pulmonary metastasectomy for colorectal cancer.

Pulmonary metastasectomy (PM) is routinely performed in colorectal cancer (CRC) patients with oligometastatic spreading to the lungs. Patients with an aggressive tumor phenotype should be excluded from PM, since its benefit is outweighed by early tumor recurrence and impaired prognosis. Expression of PD-1 and its ligands are prognostic factors in a variety of primary tumors. However, their impact on patients' outcome in the setting of PM for CRC has not been evaluated before. 53 CRC patients with pulmonary metastases receiving PM with curative intent were included in this study. Tissue samples of resected pulmonary metastases and available corresponding primary tumors were collected and assessed for PD-1, PD-L1 and PD-L2 expression by tumor-infiltrating lymphocytes (TILs) and tumor cells. Expression patterns were correlated with clinical outcome parameters. PD-1 and PD-L1 expression was commonly found in TILs and tumor cells. Expression levels significantly differed between metastases and primary tumors. High PD-1 expression by TILs was associated with impaired overall survival (low vs high expression (mean, 95% CI): 78 mo (60-96) vs 35 mo (25-44); p = 0.011). Additionally, the subgroup of patients, who experienced an upgrading in their TILs/PD1 status between primary and metastasis had a worse survival outcome compared with patients with the same grade or a downgrading (34 mo (26-42) vs 96 mo (72-120); p = 0.004). Thus, PD-1 expression by TILs is a strong prognostic marker in CRC patients with pulmonary spreading treated by PM. Moreover, this study provides a rationale for a therapeutic PD-1 pathway blockade in the treatment of CRC lung metastases. Future, large-scale studies are warranted to validate the findings of this single-center, retrospective analysis.

Expression of Programmed Cell Death Protein 1 by Tumor-Infiltrating Lymphocytes and Tumor Cells is Associated with Advanced Tumor Stage in Patients with Esophageal Adenocarcinoma.

BACKGROUND: Despite recent advances in the therapy for adenocarcinoma of the esophagogastric junction (AEG), overall prognosis remains poor. Programmed cell death protein 1 (PD1) is a co-inhibitory receptor primarily expressed by T-cells. Tumor cells can escape anticancer immune responses by triggering the PD1 pathway. Moreover, PD1 receptor engagement on cancer cells may trigger tumor-intrinsic growth signals. This study aimed to evaluate the potential clinical relevance of PD1 expression by tumor-infiltrating lymphocytes (TILs) and cancer cells in the AEG. METHODS: Patients with AEG who underwent esophagectomy from 1992 to 2011 were included in the study. Expression of PD1was evaluated by immunohistochemistry and correlated with long-term overall survival (OS), disease-free survival (DFS), and various clinicopathologic parameters. RESULTS: Tumor biospecimens from 168 patients were analyzed. In the analysis, 81% of the patients showed high tumoral frequencies (>5%) of PD1-expressing TILs (TIL-PD1+), and 77% of patient tumors harbored high levels (>5%) of PD1+ cancer cells (cancer-PD1+). Expression of PD1 by TILs and cancer cells correlated significantly (p < 0.05) with patients' tumor stage and lymph node involvement. Compared with the patients who had low tumoral frequencies of PD1+ TILs or cancer cells, the TIL-PD1+ and cancer-PD1+ patients demonstrated significantly reduced DFS in the univariate analysis (5-year DFS: 73.3 vs. 41.9%, log-rank 0.008 and 71.3 vs. 41.6%, p = 0.008, respectively). Additionally, the cancer-PD1+ patients showed significantly decreased OS in the univariate analysis compared with the cancer-PD1- patients (5-year OS: 68.8 vs. 43.5%; p = 0.047). However, these correlations did not reach significance in the multivariate analysis. CONCLUSIONS: The PD1 receptor is expressed by both TILs and cancer cells in AEG. High expression of PD1 is associated with advanced tumor stage and lymph node involvement, but not with survival.