Response of peripheral blood mononuclear cells to conditioned medium from cultured oral squamous cell carcinomas

The current study investigated the capacity for tumor factors secreted by head and neck squamous cell carcinoma (HNSCC) cell lines, KB, KB16, and HEP, to induce the secretion of various cytokines from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood samples collected from six healthy volunteers and these cells were incubated for 6, 24, 48, or 72 hours in the presence of 50 percent conditioned medium collected from cultured cell lines pretreated with, or without, stimulants such as phytohemagglutinin (PHA) or lipopolysaccharides (LPS). Aliquots of each supernatant were then assayed for levels of IFN-Γ, vascular endothelial growth factor (VEGF), TNF-α, and IL-4 using enzyme linked immunosorbent assays (ELISAs). Data collected were analyzed using Student's t-test, an ANOVA test followed by Tukey's test, and tests of Pearson's Correlation. PBMCs cultured with KB16-conditioned medium produced the highest levels of IFN-Γ. VEGF was also detected in conditioned media collected from all of the squamous cell carcinoma (SCC) cell lines used, and a significant difference in VEGF levels between control and KB- or KB16-conditioned media was observed. TNF-α was secreted by all PBMC groups within 6 hours of receiving conditioned media, and these levels increased up to the 24 hour timepoint, after which levels of TNF-α stabilized. In contrast, none of the supernatant samples contained detectable levels of IL-4. In combination, these data suggest that direct contact between fresh human PBMCs and conditioned media from tumor cells induces the secretion of TNF-α and VEGF by PBMCs, and this represents an initial angiogenic response.

Effect of suicide gene therapy in combination with immunotherapy on antitumour immune response & tumour regression in a xenograft mouse model for head & neck squamous cell carcinoma.

Background & objectives: Prodrug activation strategy as well as immunotherapy have been widely used for cancer gene therapy. In the present study, using a head and neck squamous cell carcinoma (HNSCC) xenograft nude mouse model, we have investigated whether the two therapies in combination could improve tumour cell kill. We also investigated induction of immune effector cells viz., NK (DX5+) and DC (CD11c+) in vivo, post-combination gene therapy. Methods: A retroviral vector producing cell line (PLTK47.1 VPC) carrying Herpes simplex virus thymidine kinase gene (HSVtk) was used for intratumoural injection into NT8e xenograft tumours followed by the prodrug ganciclovir (GCV). IL-2 plasmid DNA was injected intramuscularly. Immune cells were analyzed by flow-cytometry. Non parametric ANOVA was performed with Kruskal Wallis test. Results: IL-2 could induce proliferation of both NK cells (DX5+) and dendritic cells (CD11c+) in vivo. Apoptosis was higher in combination therapy group as compared to HSVtk/GCV alone or IL-2 alone and was mediated through caspase-3 dependent pathway. Significant reduction in tumour volume was seen in all 3 treatment arms as compared to controls. Interpretation & conclusions: Combination of suicide gene therapy and immunotherapy leads to successful tumour regression in a HNSCC xenograft mouse model. Immunotherapy could help in a systemic long lived anti-tumour immune response which would prove powerful for the treatment of metastatic cancers, and also for minimal residual disease. The results of this study may form the basis for Phase 1 clinical trials.

Field cancerization in head and neck squamous cell carcinoma: immunohistochemical expression of P53 and Ki67 proteins: clinicopathological study / Cancerização por campo em carcinoma de células escamosas: expressão imuno-histoquímica de proteínas p53 e Ki67: estudo clínico-patológico

OBJECTIVE: This study is aimed to evaluate the immunostaining influence of p53 and Ki67 proteins inareas of field cancerization of head and neck squamous cell carcinoma (HNSCC). We analyzed associationsof these proteins with clinicopathological parameters and the relation between their immunoexpression inHNSCC. MATERIAL AND METHOD: In a retrospecive analysis, 40 patients with HNSCC were selectedaccording to the recurrence of the disease, forming two groups: recurrent and non-recurrent HNSCC.Morphological gradations and imunnohistochemical analysis of p53 and Ki67 were performed in invasivefront and tumor adjacent epithelium. RESULTS: It was found significant associations between tumor recurrenceand p53 positivity in mucosa and invasive front. However, no association was found between p53immunostaining and the clinicopathological parameters. Ki67 was not related to any clinicopathologicalparameter either. The association between Ki67 and p53 expression was not significant. There was no significant inluence of recurrence in the clinicopathological parameters. Individuals with T1/T2 tumor size, non-recurrentand p53-negative in tumor adjacent epithelium presented better overall survival of HNSCC. CONCLUSION:p53 positivity in adjacent epithelium and invasive front of recurrent HNSCC is suggested in this study.

OBJETIVO: Avaliar a influência da imunoexpressão das proteínas p53 e Ki67 em áreas de campos de cancerização do carcinoma de células escamosas da cabeça e pescoço. Analisamos a associação dessas proteínas com parâmetros clínico-patológicos e a relação entre sua imunoexpressão.MATERIAL E MÉTODO: Em análise retrospectiva, 40 pacientes foram selecionados, de acordo com a recorrência da doença, formando dois grupos: com recorrência e sem recorrência da neoplasia. Gradações morfológicas e análises histoquímicas foram efetuadas na área de invasão e no epitélio adjacente ao tumor. RESULTADOS: Encontrou-se associações significativas entre a recorrência do tumor e positividade para p53 na mucosa e na área da lesão. Entretanto, não encontrou-se associação entre p53 e parâmetros clínico-patológicos. Ki67 não foi relacionada com qualquer parâmetro, igualmente. A associação entre expressão de Ki67 e p53 não foi significante e não houve influência significante de recorrência nos parâmetros clínico-patológicos. Indivíduos com tumores T1/T2, não recorrentes, e p53 negativos no epitélio adjacente ao tumor apresentaram melhor sobrevida à neoplasia. CONCLUSÃO: Positividade para p53 no epitélio adjacente e na área da neoplasia de carcinoma de células escamosas é sugerida por este estudo.

Prognostico valeu of intratumoral D57+T cells and microvessel density in head and neck squamous cell carcinoma / Valor prognóstico das células CD57+T e densidade dos microvasos no carcinoma de células escamosas de cabeça e pescoço

OBJECTIVES: This study aimed to examine the association of CD57+ T cells and MVD withclinical parameters and prognosis of HNSCC. MATERIAL AND METHOD: In a restrospectiveanalysis, 43 cases of primary HNSCC have been studied. We also analysed CD57 and CD31counting. T and N parameters was analized by Binary logistic regression analysis. Survival wasanalysed by Cox regression analysis. RESULTS: CD31 was not associated with anyclinicopathological parameters. CD57 immunoexpression was associated with locorregionalpresence. Cox regression test showed correlation of worse survival with locorregional metastasispresence. For binary logistic parameter, WHO Grade parameter was associated with smaller tumorsize and absent metastasis CD57+ T cells count was relationed with worse survival.CONCLUSION: There was no association between MVD and clinicopathological parameters.Locorregional metastasis presenting high CD57 positivity. No association was found betweenCD57 and the other clinicopathological parameters. Multivariate analysis showed that individualspresenting locorregional metastasis were associated with poor survival. CD57 count and WHOgrade were associated with larger tumor size.

OBJETIVO: Estudou-se a associação de células CD57+T e densidade microvascular comparâmetros clínicos e prognóstico do carcinoma de células escamosas de cabeça e pescoço(CCECP). MATERIAL E MÉTODO: Em análise retrospectiva, 43 casos de CCEP foramestudados. Analisamos também a contagem de CD57 e CD32. Os parâmetros T e N foramanalisados pela análise da regressão logística binária. A sobrevivência foi submetida a anális deregressão de Cox. RESULTADOS: CD31 não foi associada com nenhum parâmetroclinicopatológico. A imunoexpressão do CD57 associou-se com presença locorregional. O testede regressão de Cox demonstrou correlação entre pior sobrevivência com presença locorregionalde metástase. Para o parâmetro de logística binária, o parâmetro da OMS foi associado comtumores menores e ausência de metástases. A contagem das células CD57+ foi relacionada coma pior sobrevivência. CONCLUSÃO: Não houve associação entre microdensidade vascular eparâmetros clínico-patológicos. Metástases locorregionais apresentaram alta positividade paraCD57. Não foi encontrada associação entre CD57 e outros parâmetros clínico-patológicos. Análisemultivariada demonstrou que indivíduos apresentando metástases locorregionais apresentarampobre sobrevida. Contagem de CD57 e grau da OMS foram associados com tumores maiores.

Estudo do papel de células T reguladoras e moléculas co-estimulatórias ma regulação da resposta imune em doenças infecciosas e tumor de cabeça e pescoço / The role of the regulatory T cells and PD-1 molecules in the modulation of the immune response in chronic infections and head and neck tumors

O sistema imune serve como uma barreira contra os patógenos e ao crescimento anormal de células. Para impedir as respostas imunes excessivas ou indiscriminadas que podem comprometer a sobrevivência do organismo, diversos mecanismos regulatórios são ativados visando manter o delicado balanço entre início e término de uma resposta imune. As celular T reguladoras (Treg) parecem desempenhar papel central na regulação da resposta imune em infecções crônicas e durante o desenvolvimento de tumores. Outro mecanismo importante no controle da resposta imune é desempenhado por moléculas co-estimulatórias, dentre as quais estão CTLA-4 e PD-1, todas associadas à função das células T reguladoras. Um aspecto importante é q a sobrevida de tecido tumoral e de transplantes tem sido associada à função das células T reguladoras. Assim, buscamos definir o envolvimento de células T reguladoras e PD-1 na modulação da resposta imune L. braziliensis, ao fungo P. brasiliensis, à doença periodontal e ao tumor de cabeça e pescoço. Baseado nos resultados já publicados e em dados preliminares, as hipóteses são que: (a) a interação do parasita (ou célula tumoral) com o hospedeiro leva à migração de linfócitos T e efetores e células T reguladoras para o local da lesão; (b) a dinâmica do acúmulo dessas células em tais sítios determina a eficiência da eliminação do patógeno ou tumor. No caso das parasitoses, há o desenvolvimento de imunidade concomitante; (c) as células T regulam a resposta imune local de forma contato dependente e modulando a função de APC através da liberação de IL-10 e/ou TGF-β; (d) infecção e progressão tumoral levam à modulação da expressão de PD-1 nos leucócitos e seus ligantes nos órgãos; (e) a interação PD-PDL-1 regula a resposta imune local de forma a favorecer a persistência do patógeno e os mecanismos de escape tumoral.

The immune system serves as a barrier against pathogens and abnormal cellular growth. To avoid tissue and organ damage during immune response several regulatory mechanisms are activated to limit, terminate and attenuate T-cells response. Regulatory T cells (Treg) play a central role in the regulation of the immune response in chronic infections and tumor-specific immunity. Programmed death-1 (PD-1) is a transmembrane protein that acts as a negative regulator in effector T cells, modulating the delicate balance between effective antimicrobial immune defenses and immune-mediated tissue damage. However, recent data suggest that the PD-1:PD-L1 pathway can also block antitumor immune responses even when tumor antigens can be recognized. An important aspect it that the survival of tumor and transplant tissues has been associated with the function or regulatory T cells. Thus, we discuss the role of Treg cells and PD-1 molecules in the modulation of the immune response to L. braziliensis, P. brasiliensis, periodontal disease and head and neck tumors. Based on published results and preliminary data, the hypotheses are that: (a) the interaction of the parasite (or tumoral cells) with the host leads to the migration of effector T lymphocytes and Treg cells to the local; (b) the dynamics of cells accumulation in such sites determinate the elimination efficiency of tumors. In infectious disease, there is the development of concomitant immunity; (c) Treg cells regulate the local immune response, modulating the APC function through the release of IL-10 and/or TGF-β; (d) infection and tumor progression leads to the modulation of PD-1 expression in the leukocytes and their ligands in the tissue; (e) PD-PDL-1 interactions regulate the immune response and may mediate the persistence of pathogen and contribute to immune evasion by cancers.T.

Anergy testing in patients with head and neck cancer.

Patients with head and neck cancer were found to be deficient in were not clear [correction] Possible explanations include a change in T-lymphocyte numbers, particularly the helper/suppressor T-cell ratio, with the cause of this change still unknown. Tumor immunosuppressing factors and cancer-induced immunosuppression are proposed to be such causes. The deficiency of T cells resulted in an impaired cell-mediated immune response (CMIR), which lowered the host resistance, such facilitating the tumor to spread. As the CMIR can be evaluated by delayed hypersensitivity skin testing (= anergy screen), the objective of this study was to compare the CMIR function of patients with head and neck cancer to a non-cancer control group using this anergy screen. The study group consisted of 20 patients (17 males, 3 females, age range 10-76 years) with head and neck cancer, which were anti-HIV negative and had not received any therapy yet. The control group consisted of another 20 persons (17 males, 3 females, age range 21-72 years) without any cancer and who were also anti-HIV negative. Exclusion criteria were (1) eczema or skin disease in the area to be tested, (2) having received oral prednisolone within the last week and (3) an anti-HIV positive immune status. The antigens used in this study consisted of PPD (5 IU), tetanus toxoid (TT) (0.8 LF/ml and 1.6 LF/ml, Candida albicans (20 PNU/ ml and 200 PNU/ml), mumps-measles-rubella (MMR) vaccine (1:10 v/v and 1:5 v/v). The test was done by intradermal injection of 0.1 ml of each antigen. The anergy screen was considered positive when the test resulted in an erythema or induration larger than 5 mm at 72 hours after the injection. Complete anergy was diagnosed when there was no skin reaction at all, partial anergy when only 1 antigen tested positive and no anergy when there were positive skin reactions to two or more antigens. In the study group, 9 (45%) patients were diagnosed with complete anergy, 11 (55%) with partial anergy and none with no anergy, while in the control group, none were complete anergic, 3 (15%) were partially anergic and 17 (85%) had no anergy. There was a statistically significant difference (p < 0.01) between these two groups. In conclusion, patients with head and neck cancer seemed to have an impaired CMIR, with at least the partial anergy being statistical significantly different compared to the non-cancer group.

Undifferentiated head and neck tumors: the contribution of immunohistochemical technique to differential diagnosis

CONTEXTO: As neoplasias indiferenciadas em cabeça e pescoço e base do crânio não são raras. Ocorrem tanto em mucosas como em glândulas salivares, em partes moles e em linfonodos. O diagnóstico histopatológico preciso é fundamental na conduta terapêutica ideal e na caracterização do prognóstico de cada caso. OBJETIVOS: Avaliar a ocorrência destas neoplasias em nosso serviço, sua distribuição conforme o padrão celular, a idade do paciente e a localização do tumor, avaliando-se a freqüência dos casos em que o exame imunoistoquímico foi decisivo para o diagnóstico diferencial conclusivo. TIPO DE ESTUDO: Estudo de corte transversal. LOCAL: Hospital das Clínicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brasil. PARTICIPANTES: Foram estudadas 43 biópsias de neoplasias indiferenciadas diagnosticadas no ambulatório da Disciplina de Otorrinolaringologia, no período de 1990 a 1997. PROCEDIMENTOS: Aplicou-se um painel imunoistoquímico conforme o método complexo avidina-biotina-peroxidase (ABC), dependendo da idade dos pacientes, da localização do tumor e do padrão citoarquitetural das células neoplásicas. O laudo final foi emitido após nova análise conjunta com as lâminas coradas pela técnica da hematoxilina e eosina. VARIÁVEIS ESTUDADAS: Distribuição das neoplasias indiferenciadas de cabeça e pescoço, conforme o padrão celular, a idade do paciente e a localização do tumor. RESULTADOS: Os locais de ocorrência mais comuns foram os linfonodos, 20.9 por cento; faringe e pescoço, 16.3 por cento; seios paranasais, 14.0 por cento e cavidade nasal, 11.6 por cento. Estas neoplasias foram mais prevalentes na sétima década de vida (34.9 por cento), sendo duas vezes mais prevalentes em homens que em mulheres. O exame imunoistoquímico permitiu o diagnóstico conclusivo em 60.5 por cento dos tumores e o sugeriu em 20.9 por cento. Os padrões citoarquiteturais mais comuns foram: células redondas, 51.2 por cento; células epitelióides, 20.9 por cento; células fusiformes, 16.3 por cento; mixóides, 9,30 por cento e células pleomórficas, 2.3 por cento. CONCLUSÃO: Esses achados demonstram o papel fundamental do exame imunoistoquímico no diagnóstico conclusivo nestas neoplasias.

Prognostic significance of circulating immune complexes in malignant tumours of head and neck.

Circulating immune complexes (CIC) were estimated in 31 cases of head and neck malignancies by polyethylene glycol (PEG) precipitation and latex agglutination inhibition (LAI) techniques. Results were compared with 25 age and sex-matched control volunteers. Seropositivity for CIC by PEG precipitation test was 54.83% compared to 61.29% by LAI test. No positive case was detected in control group. Seropositivity for CIC by combination of both test results was 67.74%. In stage I cancer seropositivity for CIC by both techniques was 33.33%. In stage II it was 36.36% by PEG precipitation test and 45.45% by LAI test. In stage III it was 64.28% by PEG precipitation test and 71.42% by LAI test. In stage IV all cases were seropositive by both techniques. LAI test was more sensitive in CIC detection in cancer stages II and III. PEG precipitation test had the advantage of detecting quantitative CIC levels by PEG index. Follow-up in 10 postoperative cases revealed significant decline in CIC levels by both tests (p < 0.01) after 3 months of surgery; 90% patients became seronegative for CIC by PEG precipitation technique while 80% became seronegative by LAI technique. However, 15 patients were followed up after 3 1/2 years of surgery; 75% patients remained seronegative while 25% patients became seropositive by both tests and manifested clinical recurrences.

Immunological status of patients in carcinoma head and neck.

The cell mediated immune response in 45 patients of cancer, head and neck was evaluated by various parameters and compared with 20 control cases. (DNCB) Dinitrochlorobenzene cutaneous reactivity in vivo was compared with T lymphocytes, estimated as active rosette forming cells (ARFC) and Total rosette forming cells (TRFC) in peripheral blood. The ratio of TRFC/ARFC was studied in various histological subtypes of cancer head and neck. Lymphocytic infiltration around tumor mass was correlated with peripheral blood lymphocyte (PBL), ARFC and TRFC levels in blood. Significant difference was noted in the level of peripheral blood lymphocytes (PBL). TRFC and ARFC between control cases and cancer patients. There was a marked increase in all three parameters i.e. PBL, TRFC, ARFC (P less than 0.001) and a positive DNCB reaction in Adenocarcinoma of nasopharynx, thyroid and salivary gland and a significant decrease in Squamous cell Carcinoma of oral cavity and larynx (p less than 0.001) associated with impaired DNCB sensitivity. These parameters serve as an assessment of the degree of immune reactivity of the host to the malignant tumours.

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.