Katarzyna Starska

The expression of SOCS1 and TLR4-NFkappaB pathway molecules in neoplastic cells as potential biomarker for the aggressive tumor phenotype in laryngeal carcinoma.

Suppressor of cytokine signaling 1 (SOCS1) is the key regulator of cytokine-mediated innate and adaptive immunity. One of the molecular mechanisms of SOCS1 is connected with inhibition of TLR4-NFkappaB pathway. The relationships among these molecules in laryngeal carcinoma are not exactly known. In this preliminary study we focused on their special activity and role in regulation of development and progression of laryngeal carcinoma. To investigate NFkappaB (p65 subunit) nuclear and cytoplasmic expression in 45 tumor samples of advanced laryngeal carcinoma IHC staining was performed. To determine the mRNA expression levels of TLR4, IRAK1, TRAF6 and SOCS1 in isolated neoplasm cells and non-cancerous adjacent mucosa epithelial cells RT-PCR was used. The invasiveness of laryngeal carcinomas was evaluated according to tumor front grading, TFG, which included tumor-related features (cytoplasmic differentiation, nuclear polymorphism, number of mitoses) and adjacent stroma-related characteristics of the peripheral edge of tumor infiltration (mode of infiltration, depth of invasion and plasmalymphocytic infiltration). The relationships between pT, pN status, the histological G grade, certain clinicopathological characteristics as well as postoperative observation time and the mRNA expression of the molecules mentioned earlier were investigated. Significant differences of TLR4-NFkappaB pathway molecules and SOCS1 mRNA expression in laryngeal tumor cells and normal adjacent mucosa cells as well as significant interconnections of TLR4, SOCS1 and NFkappaB(p65) in isolated tumor cells were obtained. This preliminary study demonstrated that the expression of SOCS1 and TLR4-NFkappaB pathway molecules had a strong association with the aggressiveness of laryngeal carcinoma. Positive relationships of TRAF6 in tumor margin cells with the histological grade and the mode of tumor invasion as well as the TFG total score were highlighted. Significant positive correlations were found between the TLR4 in tumor central cells and the TFG total score. Negative relationships of SOCS1 in tumor central cells with the histological grade were also noted. Significant positive correlations were found between the cytoplasmic NFkappaB(p65) and the mode of invasion as well as TFG total score. Our findings confirmed the importance of SOCS1 and TLR4-NFkappaB pathway molecules as potential biomarkers for assessment of the aggressive tumor phenotype in laryngeal carcinoma.

The role of tumor cells in the modification of T lymphocytes activity--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ cells in squamous cell laryngeal carcinoma. Part I.

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4(+) and CD8(+) lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69(+), CD71(+)) and late activation markers (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD3(+)CD4(+) and CD3(+)CD8(+) cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-γ in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69(+), CD71(+) and CD25(+) (high), CD26(+), HLA/DR(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-γ and IL-10 secretion with regard to CD4(+)CD69(+), CD8(+)CD69(+), CD4(+)CD71(+), CD8(+)CD71(+) antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4(+)HLA/DR(+) and CD8(+)HLA/DR(+) as well as between IL-10 concentration and CD4(+)HLA/DR(+) in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4(+) and CD8(+) lymphocytes in laryngeal carcinoma.

The − 5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer

Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the -5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region -5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the -5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that -5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer.

The effect of metallothionein 2A core promoter region single-nucleotide polymorphism on accumulation of toxic metals in sinonasal inverted papilloma tissues.

Metallothioneins (MTs) are intracellular thiol-rich heavy metal-binding proteins which join trace metal ions protecting cells against heavy metal toxicity and regulate metal distribution and donation to various enzymes and transcription factors. The goal of this study was to identify the -5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene, and to investigate its effect on allele-specific gene expression and Cd, Zn, Cu and Ni content in sinonasal inverted papilloma tissue (IP), with non-cancerous sinonasal mucosa (NCM) as a control. The MT2A promoter region -5 A/G SNP was identified by restriction fragment length polymorphism using 117 IP and 132 NCM. MT2A gene analysis was performed by quantitative real-time PCR. Metal levels were analyzed by flame atomic absorption spectrometry. The frequency of A allele carriage was 99.2% and 100% in IP and NCM, respectively. The G allele carriage was detected in 23.9% of IP and in 12.1% of the NCM samples. As a result, a significant association of -5 A/G SNP in MT2A gene with mRNA expression in both groups was determined. A significant association was identified between the -5 A/G SNP in the MT2A gene with mRNA expression in both groups. A highly significant association was detected between the rs28366003 genotype and Cd and Zn content in IP. Furthermore, significant differences were identified between A/A and A/G genotype with regard to the type of metal contaminant. The Spearman rank correlation results showed the MT2A gene expression and both Cd and Cu levels were negatively correlated. The results obtained in this study suggest that the -5 A/G SNP in the MT2A gene may have an effect on allele-specific gene expression and toxic metal accumulation in sinonasal inverted papilloma.

The expression of TLR pathway molecules in peripheral blood mononuclear cells and their relationship with tumor invasion and cytokine secretion in laryngeal carcinoma

PURPOSE The aim of this study was to analyze of TLRs mRNA expression in peripheral blood mononuclear cells as potential biomarkers of neoplastic lesions progress and to evaluate their role in the possible mechanisms responsible for the secretion of cytokines in laryngeal cancer. MATERIAL/METHODS The analysis of TLR2, TLR4, TRAF6, IRAK1 expression in isolated PBMCs by the reverse transcription PCR (RT-PCR) analysis as well as IL-6, IL-8 and TNF-α levels in supernatants of peripheral blood mononuclear cells from 55 patients with carcinoma of the larynx was performed by ELISA. The invasiveness of carcinoma was evaluated according to tumor front grading, TFG. RESULTS We noted that tumors with a well-defined borderline were characterized by significantly higher values of the average expression of TRAF6. Our research also confirmed that more aggressive carcinomas according to TFG, with a more dispersed type of invasion were characterized by significantly lower values of the average expression of IRAK1. Moreover, we observed that tumors with the invasion of cartilage were characterized by significantly lower values of the average expression of TLR4. In addition, the relationships of TLR2 with IL-6 and TNF-α level were highlighted. Significant interconnections were also found between the TLR4 and IL-8, TNF-α, IL-6 secretion after stimulation. The relationships of TRAF6 with IL-8 production after stimulation were noted. CONCLUSIONS Our findings confirmed the implication of the TLRs pathway molecules in proinflammatory cytokine secretions and their importance as encouraging potential indicators for assessment of the degree of aggressive tumor phenotype.

Kliniczno-histopatologiczna charakterystyka przypadków brodawczaka odwróconego jam nosa i zatok przynosowych leczonych w Klinice Laryngologii Onkologicznej UM w Łodzi w latach 2002–2006 – przegląd najnowszego piśmiennictwa

Summary Authors introduced diagnostic procedures, clinico-morphological features and treatment results of 16 cases of nasal cavity and paranasal sinuses inverted papillomas treated surgically on Department of Laryngological Oncology of Medical University of Lodz between 2002–2006. References review, definition, classification, clinical and histopathological criteria and treatment methods of Schneiderian papilloma were introduced in study.

Diagnostic impact of promoter methylation and E-cadherin gene and protein expression levels in laryngeal carcinoma

Aim of the study Inactivation of the tumor suppressor E-cadherin (CDH1) and its decreased expression is an important occurrence during carcinogenesis. Nevertheless, the relationship of CDH1 expression and the promoter methylation with laryngeal cancer cell aggressiveness is still unclear. The purpose of this study was to elucidate the gene and protein E-cadherin expression and the DNA methylation levels and to describe the correlations with morphological features in squamous cell laryngeal cancer. Material and methods The authors studied E-cadherin and the DNA methylation level in 86 cases to gain a further understanding of the clinicopathologic significance of analyzed parameters. The pathological evaluation included pTNM classification and the tumor front grading (TFG) criteria. Quantitative analysis of the amplified product in real time (qRT-PCR) for estimation of CDH1 mRNA was used. The methylation status was investigated by using methyl-specific polymerase chain reaction (MSP). The level of CDH1 protein expression by Western blot was determined. Results Downregulation of E-cadherin was found to be related to promoter methylation (p < 0.001). In tumors with the highest invasiveness according to TFG criteria the lowest E-cadherin gene and protein level in the study group was observed (p = 0.046 and p = 0.0002, respectively). In SCLC with muscle and cartilage invasion and disperse infiltration the lowest CDH1 gene and protein expression was noted (p = 0.0003 and p = 0.003 for deep invasion, p = 0.033 and p = 0.003 for multifocal infiltration, respectively). Conclusions The current findings suggest an association of E-cadherin tumor expression with progression of laryngeal cancer. CDH1 gene level may be an auxiliary molecular marker for advanced cases of laryngeal carcinoma; however, further studies are necessary.

Fibroblast growth factor receptor 1 and 3 expression is associated with regulatory PI3K/AKT kinase activity, as well as invasion and prognosis, in human laryngeal cancer

PurposeAberrant fibroblast growth factor receptor (FGFR) expression is thought to contribute to the development of many types of cancer. As yet, however, their impact on the course and prognosis of head and neck cancer remains to be determined. Here, we aimed to investigate the effects of expression of the FGFR family members FGFR1 and FGFR3, as well as their downstream PI3K/AKT signal-regulated kinases, on the aggressiveness and prognosis of laryngeal cancer.MethodsIn total 137 surgically removed squamous cell laryngeal cancer (SCLC) and 100 matched non-cancerous laryngeal mucosa (NCLM) samples were assessed for mRNA expression using quantitative real-time PCR. The corresponding proteins were analyzed by Western blotting. SLUG expression was assessed by immunohistochemistry. The expression data were subsequently related to tumor front grading (TFG), local/nodal recurrences, prognosis and overall survival.ResultsThe FGFR1, FGFR3 and PI3K/AKT kinase mRNA and protein levels were found to be significantly higher in the SCLC than the NCLM samples (p < 0.05). A high FGFR1 mRNA/protein expression level was found to be associated with an increased invasion rate, according to TFG scale and SLUG level, a high local/nodal recurrence rate and a poor prognosis (p < 0.05). Similarly, we found that a high FGFR3 mRNA/protein expression level was associated with a shorter survival time (p < 0.05). In addition, we found that high PI3K/AKT kinase mRNA/protein levels were associated with a high TFG (p < 0.05). We also found that FGFR1/3 mRNA and FGFR1 protein levels were inversely associated with overall survival (log-rank test: FGFR1 mRNA p = 0.03, FGFR3 mRNA p = 0.04, FGFR1 protein p = 0.03). Subsequent multivariate analyses revealed that high FGFR3 mRNA expression may serve as an independent poor prognostic factor (HR 2.32, 95% CI 1.03–6.59; p = 0.04). We also found that the p-PI3K regulatory kinase protein level was significantly associated with survival in the cohort studied (HR 1.78, 95% CI 0.64–8.53; p = 0.03).ConclusionsFrom our data we conclude that FGFR1 and FGFR3, as well as its downstream regulatory PI3K/AKT kinases, may serve as potential biomarkers for the invasiveness and prognosis of laryngeal cancer. The expression of FGFR1/3-PI3K/AKT regulatory pathway members may be instrumental for the identification of patients at risk for an unfavorable clinical outcome.

Gene/protein expression of CAPN1/2-CAST system members is associated with ERK1/2 kinases activity as well as progression and clinical outcome in human laryngeal cancer

Recent evidence indicates the involvement of calpains (CAPNs), a family of cysteine proteases, in cancer development and progression, as well as the insufficient response to cancer therapies. The contribution of CAPNs and regulatory calpastatin (CAST) and ERK1/2 kinases to aggressiveness, disease course, and outcome in laryngeal cancer remains elusive. This study was aimed to evaluate the CAPN1/2-CAST-ERK1/2 enzyme system mRNA/protein level and to investigate whether they can promote the dynamic of tumor growth and prognosis. The mRNA expression of marker genes was determined in 106 laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent mucosa (NCLM) controls using quantitative real-time PCR. The level of corresponding proteins was analyzed by Western Blot. SLUG expression, as indicator of pathological advancement was determined using IHC staining. Significant increases of CAPN1/2-CAST-ERK1/2 levels of mRNA/protein were noted in SCLC compared to NCLM (p < 0.05). As a result, a higher level of CAPN1 and ERK1 genes was related to larger tumor size, more aggressive and deeper growth according to TFG scale and SLUG level (p < 0.05). There were also relationships of CAPN1/2 and ERK1 with incidences of local/nodal recurrences (p < 0.05). An inverse association for CAPN1/2, CAST, and ERK1/2 transcripts was determined with regard to overall survival (p < 0.05). In addition, a higher CAPN1 and phospho-ERK1 protein level was related to higher grade and stage (p < 0.05) and was found to promote worse prognosis. This is the first study to show that activity of CAPN1/2- CAST-ERK1/2 axis may be an indicator of tumor phenotype and unfavorable outcome in SCLC.

Expression of CTLA-4 and Foxp3 in peripheral blood T cells of patients with squamous cell laryngeal carcinoma

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152) and Foxp3 (forkhead box P3) are receptors present on T cells which play a critical role in the down-regulation of antigen-activated immune responses. To evaluate the potential influences of CTLA-4 and Foxp3 on cancer invasiveness, a case-control study was conducted in 86 patients treated for squamous cell laryngeal carcinoma. The abundance of CTLA-4 and Foxp3 gene transcripts in the purified peripheral blood mononuclear cells (PBMCs) by quantitative real-time PCR (qRT-PCR) was determined. The analysis of proteins by Western blot was performed. The relationships between CTLA-4 and Foxp3 gene and protein expression as well as the aggressiveness of tumor determined on pT, type and depth of invasion were investigated. Our work revealed a significant dependence of mRNA CTLA-4 on tumor front grading (TFG) total score (p = 0.04) as well as CTLA-4 protein expression on pT (p = = 0.03) and type of invasion (p = 0.03). Advanced pT3-pT4 tumors with diffuse infiltration and > 14 TFG points were characterized by higher average values of CTLA-4 protein in PBMCs. Our data also demonstrated significant differences between Foxp3 protein levels in relation to pT (p = 0.04), depth of invasion (p = = 0.02) and type of invasion (p = 0.03). In tumors with the highest invasiveness identified by the pT3-pT4 status, deep invasion with involvement of cartilage and diffuse infiltration, the highest Foxp3 protein level was observed. In conclusion, these results suggest an impact of CTLA-4 and Foxp3 in determining proliferative and aggressive potential of laryngeal carcinoma, highlighting the significance of CTLA-4 and Foxp3 as potential predictive indicators.