Standardised Ki-67 proliferation index assessment in early-stage laryngeal squamous cell carcinoma in relation to local control and survival after primary radiotherapy.

OBJECTIVES: Ambiguous results have been reported on the predictive value of the Ki-67 proliferation index (Ki-67 PI) regarding local control (LC) and survival after primary radiotherapy (RT) in early-stage laryngeal squamous cell cancer (LSCC). Small study size, heterogenic inclusion, variations in immunostaining and cut-off values are attributing factors. Our aim was to elucidate the predictive value of the Ki-67 PI for LC and disease-specific survival (DSS) using a well-defined series of T1-T2 LSCC, standardised automatic immunostaining and digital image analysis (DIA). METHODS: A consecutive and well-defined cohort of 208 patients with T1-T2 LSCC treated with primary RT was selected. The Ki-67 PI was determined using DIA. Mann-Whitney U-tests, logistic and Cox regression analyses were performed to assess associations between Ki-67 PI, clinicopathological variables, LC and DSS. RESULTS: In multivariate Cox regression analysis, poor tumour differentiation (HR 2.20; 95% CI 1.06-4.59, P = .04) and alcohol use (HR 2.84, 95% CI 1.20-6.71; P = .02) were independent predictors for LC. Lymph node positivity was an independent predictor for DSS (HR 3.16, 95% CI 1.16-8.64; P = .03). Ki-67 PI was not associated with LC (HR 1.59; 95% CI 0.89-2.81; P = .11) or DSS (HR 0.98; 95% CI 0.57-1.66; P = .97). In addition, continuous Ki-67 PI was not associated with LC (HR 2.03; 95% CI 0.37-11.14, P = .42) or DSS (HR 0.62; 95% CI 0.05-8.28; P = .72). CONCLUSION: The Ki-67 PI was not found to be a predictor for LC or DSS and therefore should not be incorporated in treatment-related decision-making for LSCC.

Assessment of the association between ACYP2 and laryngeal squamous cell carcinoma risk in Chinese males.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most prevalent malignant neoplasms of the upper respiratory tract. Studies have confirmed that an unstable chromosome constitution promotes the progress of laryngeal tumorigenesis, and ACYP2 has been confirmed as a telomere length-related gene. However, to date, the association between ACYP2 polymorphisms and LSCC susceptibility has not been investigated. METHODS: We performed this study to explore the effect of 11 single-nucleotide polymorphisms (SNPs) in ACYP2 on LSCC susceptibility in Chinese Han males. Unconditional logistic regression analysis adjusted for age was used to calculate the odds ratios and 95% confidence intervals. RESULTS: Based on allele and genotype models, our results showed that rs1682111 variant was significantly associated with a decreased LSCC susceptibility (p < 0.05). On the contrary, polymorphisms of rs10439478, rs11125529, rs12615793, rs843711, rs11896604, and rs17045754 were significantly associated with an increased LSCC risk (p < 0.05). The results of haplotype analysis indicated that haplotypes "TTCTCG" and "TTCTAA" in block 1 and "TG" in block 2 showed a risk factor for the development of LCSS (p = 0.009, p < 0.001, and p = 0.001, respectively). The results of Genotype-Tissue Expression analysis indicate that these significant SNPs were known to be associated with ACYP2 expression. CONCLUSION: Our data demonstrated that ACYP2 polymorphisms may exert effects on LSCC susceptibility in Chinese Han males.

Assessment of salivary total antioxidant capacity in patients with primary untreated head and neck squamous cell carcinoma with ORAC.

BACKGROUND: Oxidative DNA damage leads to abnormalities in endogenous cellular processes and constitutes a direct link between free radicals, antioxidants and squamous cell carcinoma (SCC). Oxygen radical absorbance capacity (ORAC) method is used to measure antioxidant capacity versus oxygen free radicals. There is no report on the application of ORAC to evaluate salivary antioxidant capacity in patients with head and neck SCC. This study investigated the relation between total antioxidant capacity of saliva, measured with ORAC method, and the incidence, location, size and histopathological grading of SCC. METHODS: Salivary antioxidant capacity was assessed in 30 male and female patients with histopathologically confirmed oral and laryngeal SCC. Histopathological grading of SCC was based on WHO classification. RESULTS: Mean ORAC in patients with SCC was higher than the mean value in the control group. Statistically significant difference was observed in the study group versus the control group. No significant differences were observed for ORAC values in relation to histopathological grading, tumour size and spread to lymph nodes. CONCLUSIONS: The obtained results can advocate local compensatory mechanism in saliva in response to increased oxidative stress associated with primary tumour. ORAC can become an additional factor in head and neck SCC prognosis, but further investigation is required.

The bioactive potential of red raspberry (Rubus idaeus L.) leaves in exhibiting cytotoxic and cytoprotective activity on human laryngeal carcinoma and colon adenocarcinoma.

In this article, the bioactive potential of red raspberry leaves, a by-product of this widely spread plant, mostly valued for its antioxidant-rich fruits, was determined. The polyphenolic profile and antioxidative properties of red raspberry leaf extract were determined and examined for potential biological activity. Cytotoxic effect, antioxidative/prooxidative effect, and effect on total glutathione concentration were determined in human laryngeal carcinoma (HEp2) and colon adenocarcinoma (SW 480) cell lines. SW 480 cells are more susceptible to raspberry leaf extract in comparison with HEp2 cells. The antioxidative nature of raspberry leaf extract was detected in HEp2 cells treated with hydrogen peroxide, as opposed to SW 480 cells, where raspberry leaf extract induced reactive oxygen species formation. Raspberry leaf extract increased total glutathione level in HEp2 cells. This effect was reinforced after 24 hours of recovery, indicating that induction was caused by products formed during cellular metabolism of compounds present in the extract. Comparison of the results obtained on these two cell lines indicates that cellular response to raspberry extract will depend on the type of the cells that are exposed to it. The results obtained confirmed the biological activity of red raspberry leaf polyphenols and showed that this traditional plant can supplement the daily intake of valuable natural antioxidants, which exhibit beneficial health effects.

Skp2, p27kip1 and EGFR assessment in head and neck squamous cell carcinoma: prognostic implications.

EGFR, p27kip1 and Skp2, have been implicated in human cancer development. We have studied these molecules in a search for molecular markers that may have prognostic value in head and neck squamous cell carcinomas (HNSCC). Tissue samples of 62 patients were collected and immunohistochemical analysis was carried out for p27kip1, Skp2 and EGFR protein evaluation. Western blot analysis of p27kip1 was performed. The p27kip1 expression is frequently down-regulated in HNSCC (44.4%, 20/45 cases). The immunohistochemical analysis showed p27kip1 cytoplasmic retention in 7/38 tumors. Strong Skp2 signals were detected at the invasive edge of the tumor in cells lacking p27kip1 staining. We found a high EGFR staining in 49% (23/47) of the cases. The staining tended to be more frequent in lymph node-positive cases. The dysplastic tissue exhibited no Skp2 immunoreactivity, whereas 51.06% (24/47) of invasive tumors expressed high levels. Of note is that Skp2 overexpression was the only factor that significantly correlated with a shorter overall survival in multivariate analysis (p=0.048). Our results suggest that Skp2 is a useful prognostic marker for HNSCC management.

Tumor risk assessment by means of immunocytochemical detection of early pre-malignant changes in buccal smears.

Increasing incidence of carcinomas in the upper aero-digestive tract, both in Germany and in other European countries requires development of new preventive strategies. The cure rate at advanced tumor stages remains poor in spite of a variety of available therapeutic methods. In the present study the quantitative assessment of a pre-malignant mucosa lesion within a field cancerization was performed by means of immunocytochemical methods. This may allow individuals with an increased risk of developing malignant disease to be identified. Cytosmears taken from healthy buccal mucosa of tumor patients (n=50) and from healthy probands (n=100) with different tobacco and alcohol consumption levels were examined with regard to identifying increased expression of the proliferation markers (PCNA, MIB1), of the tumor suppressor gene product p53 as well as the oncogene product cyclin D1. There was a significant difference in expression of investigated proliferation markers between tumor patients and healthy probands (p<0.0001). When comparing the rate of positively marked cell nuclei to cigarette pack years the marker cyclin D1 and MIB1 show an increased rate in the groups with high tobacco consumption as compared to the group with a low exposure (p>0.05). It could be possible to use the marker MIB1 and cyclin D1 to screen risk groups, since the relative morbidity risk (odds ratio) increases (by 45-62 times) if the threshold value of 4 positively marked cell nuclei is exceeded.

[Assessment of tissue inhibitor of metalloproteinases-2 (TIMP-2) in laryngeal cancer]. / Udzial tkankowego inhibitora metaloproteinaz-2 (TIMP-2) w raku krtani.

UNLABELLED: Metalloproteinases are the proteolytic enzymes that digest components of the extracellular matrix in many physiological and pathological conditions. Their activity is regulated by their natural inhibitors: non-specific alpha2-macroglobulin and specific tissue inhibitors of metalloproteinases (TIMP). There are four TIMPs. TIMP-2 is the only enzyme that is expressed in constitutive manner and has the ability to inhibit activity of all metalloproteinases. TIMP-2 has been identified in many carcinomas including cancers of lung, oral cavity, breast and colon. There was correlation between TIMP-2 expression and clinical course of the disease observed in most of the neoplasm. AIM: The aim of the research was to evaluate the expression of TIMP-2 in laryngeal cancer and to assess the prognostic significance of this factor. MATERIAL AND METHOD: 104 patients with laryngeal cancer, that underwent surgical treatment were included in the study. Only cases with at least a 5-year follow-up were included. Immunohistochemical studies were performed on formalin fixed, paraffin embedded sections by using monoclonal antibodies against TIMP-2 antigen and ABC detection system. RESULTS: TIMP-2 expression was cytoplasmatic, mainly in cancer cells, but also in some stromal cells. There was correlation between TIMP-2 expression and tumor size and grading observed. We didn't find any correlation between TIMP-2 and nodal metastases, recurrence and survival. CONCLUSIONS: Our results don't suggest that TIMP-2 expression may be used as a prognostic factor in patients with laryngeal cancer. Nevertheless there are more researches needed to explain the role of TIMPs in growth and progression of neoplastic tumors.

Carbon-11 tyrosine PET for visualization and protein synthesis rate assessment of laryngeal and hypopharyngeal carcinomas.

Accurate assessment of tumour extent and lymph node involvement in squamous cell carcinomas of the head and neck region is essential for therapy planning. Unfortunately, conventional diagnostic examination and imaging techniques, which monitor tumours on the basis of anatomical parameters, have drawbacks in clinical practice. The aim of this study was to investigate the feasibility of L-[1-(11)C]-tyrosine (TYR) positron emission tomography (PET) for visualisation of squamous cell carcinoma of the larynx and hypopharynx and quantification of tumour activity by assessment of protein synthesis rate (PSR). Dynamic TYR PET was performed on 31 patients with T1-T4 laryngeal or hypopharyngeal carcinoma before therapy. Plasma activity of TYR, (11)CO(2) and (11)C-protein levels were measured, and PSRs were calculated for primary malignancies. All 31 laryngeal and hypopharyngeal tumours were visualised as a hotspot (sensitivity 100%). The median PSR of the tumours (2.06 nmol ml(-1) min(-1); range 0.72-6.96) was significantly higher ( P<0.001) than that of non-tumour (background) tissue (0.51 nmol ml(-1) min(-1); range 0.22-0.89). L-[1-(11)C]-Tyrosine PET appears to be a potential method for visualisation of primary laryngeal and hypopharyngeal tumours. In vivo quantification of tumour activity by assessment of PSR is possible and may have a future role in the therapy planning and therapy evaluation of laryngeal and hypopharyngeal tumours.

Assessment of the ability of mononuclear blood cells to produce IFN-gamma in patients with laryngeal cancer.

The ability of mononuclear blood cells (PBMC), derived from patients with cancer of the larynx, to produce IFN-gamma in vitro was assessed in this paper. Thirty patients (27 male, 3 female) were qualified to the study. Their mean age was 65 (range: 41 to 78 years), tumour sizes found in the group were from T2 to T4, levels of pathologic malignancy G2 or G3. The percentage rates different blood cells phenotypes (CD3+, CD3+ HLADR+, CD4+, CD8+, CD14+ HLADR+, CD19+, CD56+) were evaluated by means of flow cytometry (Coulter EPICS XL). The control group consisted of 20 healthy blood donors. PBMC were derived by centrifugation of heparinized venous blood on Lymphoprep gradient according to Boyum method. Double cell cultures were performed for 24 hours with antibody anti-CD3 or recombinant interleukins 12 (rhIL-12) or 18 (rhIL-18). The statistic analysis was based on Student t and Mann-Whitney's test with significancy at p<0.05. A significant decrease in the production of IFN-gamma by PBMC in patients with laryngeal cancer after stimulation with antiCD3 (p=0.018), rhIL-12 (p=0.027), rhIL-18 (p=0.016) was found in comparison with the controls. The results suggest a decreased production of IFN-gamma in patients with cancer of the larynx.

[Assessment of usefulness of PCNA and protein p53 in subjects with laryngeal cancer with local recurrence]. / Ocena przydatnosci oznaczania markera PCNA i bialka P53 u chorych na raka krtani ze wznowa miejscowa.

The authors estimated PCNA and P53 in subjects with laryngeal cancer in whom local or nodal recurrences were observed. The study included 54 patients from Upper Silesia in age 37-79 years (mean 57 +/- 8.8). The mean value of the PCNA index in subjects with local recurrence (LR) was 24.2% +/- 12.1 while in subjects without LR 22.1% +/- 9.4 (p > 0.05). Additionally, 21 subjects were separated from the investigated group in whom no lymph node metastases were found during laryngectomy. Among these subjects in 16 LR was observed (PCNA index was 30.9% +/- 12.5) while in remaining 5 subjects, in whom LR did not develop, PCNA index was 21.7% +/- 11.2. Analysis of the P53 index in subjects with LR revealed significantly higher values (19.2% +/- 9.1) in comparison to cases without LR (13.2% +/- 6.3). Our study revealed usefulness of the P53 and PCNA as markers which could support the histological diagnostic process describing biology of the cancer cells. The demonstrated increase of PCNA and P53 index in patients with LR might be useful in prediction of LR.

[Assessment of utility of nm 23 antigen as a metastatic potential marker in laryngeal cancers: preliminary study]. / Ocena przydatnosci oznaczania antygenu nm 23 jako markera potencjalu przerzutowego raków krtani--doniesienie wstepne.

The aim of the study was the analysis of the utility of nm 23 protein in prediction of cervical lymph node metastases in patients with laryngeal cancer. A preliminary study was performed in 35 patients with laryngeal cancer with cervical lymph node metastases, which were confirmed by histopathologist. The control group consisted of 30 patients with laryngeal cancer without cervical lymph node metastases. In statistical analysis T and N were taken into account. In the investigated group with metastases the presence of positive immunostaining was found in 11% of cases while in the control group in 20%. The analysis of the presence of nm 23 protein revealed a weak usefulness of this marker as a factor which predicts the presence of the cervical metastases.

[Assessment of cell proliferation antigen Ki-67, protein p53 related to apoptosis and angiogenesis in laryngeal cancer]. / Ocena ekspresji antygenu proliferacji komórkowej Ki-67, bialka zwiazanego z apoptoza p-53 i intensywnosci angiogenezy w raku krtani.

Kinetics (growth fraction of tumour cell populations), death process of cancer cells (apoptosis and necrosis) and neovascularisation in tumour (angiogenesis) have influence on the growth of cancer. Sixty patients with laryngeal cancer treated in ENT Department of Medical Academy of Lodz were analysed. Proliferation activity of cancer cells was examined by means of selection appropriate antigen (Ki-67) characteristic for cell cycle utilising immunohistochemical techniques carried out on laryngeal cancer paraffin samples. Expression of selected protein connected with apoptosis (p-53) and intensity of angiogenesis were examine using selected antibody (anti-CD34) aimed against epithelial antigens. Above-mentioned markers were correlated with: stage of cancer progression, recurrences and metastasis of laryngeal cancer and follow-up of the patients. The morphological properties were examined as well. The researches on apoptosis, angiogenesis and proliferation of cancer cells can be used as prognostic factors for the patients with laryngeal cancer.

The influence of laser surgery on the assessment of "molecular margins" in carcinomas and mucous membranes of the upper aerodigestive tract.

BACKGROUND AND OBJECTIVE: A correlation has been shown between the occurrence of the p53 protein in margins of excised carcinomas and a higher incidence of recurrences. The presented study deals with thermic alterations in resection margins after laser surgery and their impact on p53 protein and LDH detection. STUDY DESIGN/MATERIAL AND METHODS: Healthy mucosa of the oropharynx and carcinoma samples were excised with scalpel, CO2 laser and Nd:YAG laser. Alterations of lactate dehydrogenase (LDH) and the p53 status of the samples were determined by immunohistochemistry and molecular biologic methods. RESULTS: Total activity of LDH was not altered by the different modes of excision. The expression of p53 could be detected in all mucosa specimen by ELISA. In laser-surgically excised tissue regularly a small zone adjacent to the cutting edge could be demonstrated where p53 could not be detected by immunohistochemistry. CONCLUSION: Immunohistochemical and molecular assessment of laser surgical margins is possible despite thermic artefacts. Laser surgery therefore does not minimize the prognostic value of resection margins of carcinomas of the upper aerodigestive tract.