Assessment of HPV 16, HPV 18, p16 expression in advanced stage laryngeal cancer patients and prognostic significance / Avaliação da expressão do HPV 16, HPV 18, e p16 em pacientes com câncer de laringe em estágio avançado e significado prognóstico

Abstract Introduction: Human papilloma virus is an etiological risk factor for a subset of head and neck squamous cell carcinomas. HPV has been proven to be a powerful prognostic biomarker for oropharyngeal cancer, but its role in the larynx has not been explored in depth. The developmental mechanisms of laryngeal carcinomas are quite complex and controlled by various factors. Smoking and alcohol are most important risk factors. Recent studies indicate that HPV infection also plays an important role in larynx carcinomas. HPV related laryngeal carcinomas especially occur at the supraglottic region of larynx. Objective: We aimed to determine the frequency of HPV/protein16 positivity in patients with laryngeal carcinoma and association of HPV and/or p16 positivity with variables such as age, sex, smoking habits, tumor localization, lymph node metastasis, recurrence and survival in advanced stage laryngeal carcinoma in our study. Methods: This retrospective study included 90 patients with advanced laryngeal carcinoma. The Control group was 10 normal larynx mucosa specimens. The presence of HPV was investigated polyclonally by polymerase chain reaction, and protein16 with immunohistochemical method. In HPV positive cases, the presence of HPV types 16, 18 were evaluated by polymerase chain reaction. Demographic features of patients were noted. Patient survival and association with HPV/protein16 was determined. Results: Polyclonal HPV positivity was detected in 11 (12.2%) of 90 cases. Out of these 11 cases, HPV 16 was positive in 6, HPV 18 in 4, and both HPV 16 and 18 were positive in 1. In 18 (20%) of the cases, p16 was positive. Six of the cases (6.6%) had both HPV and protein16 positivity. In cases where protein16 alone or HPV and protein16 were co-positive, alcohol use was less and the tumor was found more likely to be localized in the supraglottic area. These ratios were statistically significant. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant (p= 0.011). 55.6% of protein16 positive cases was located in the supraglottic region, 33.3% was glottic and 11.1% was transglottic. Although life expectancy over 5 years were numerically higher in HPV and protein16 positive cases, this was not found to be statistically significant. There was no statistically significant relationship between HPV positivity and mean age, differentiation, smoking and alcohol use, tumor progression, lymph node metastasis, localization, recurrence, cause of mortality and treatment methods in our study. The mean follow-up period of our patients was 6.7 years. Conclusion: The close relationship between HPV and oropharyngeal squamous cell carcinoma could not be shown in larynx malignancy in many studies, including our study. Our findings support a limited role of HPV in laryngeal carcinogenesis. Protein16 is not a reliable surrogate for HPV status in laryngeal cancers and is not a predictor of laryngeal cancer survival. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant. There is a need for more populated clinical trials, where neoplastic proliferation is better demonstrated and the accuracy of the results obtained is supported by different techniques.

Resumo Introdução: O papilomavírus humano é um fator de risco etiológico para um subconjunto de carcinoma espinocelular de cabeça e pescoço. Tem sido demonstrado que o HPV é um poderoso biomarcador prognóstico para o câncer de orofaringe, mas seu papel na laringe ainda não foi explorado em profundidade. Os mecanismos de desenvolvimento dos carcinomas de laringe são bastante complexos e controlados por vários fatores. Tabagismo e álcool são os fatores de risco mais importantes. Estudos recentes indicam que a infecção pelo HPV também desempenha um papel importante nos carcinomas da laringe. Os carcinomas laríngeos relacionados ao HPV ocorrem especialmente na região supraglótica. Objetivo: Nosso objetivo foi determinar a frequência da positividade para o HPV / proteína 16 em pacientes com carcinoma da laringe e a associação da positividade para o HPV e /ou proteína 16 com variáveis como idade, sexo, tabagismo, localização do tumor, metástase linfonodal, recidiva e sobrevivência de carcinoma da laringe em estágio avançado em nosso estudo. Método: Este estudo retrospectivo incluiu 90 pacientes com carcinoma laríngeo avançado. O grupo controle incluiu 10 amostras de mucosa laríngea normal. A presença de HPV foi inves-tigada por anticorpo policlonal através de reação de polimerase em cadeia e a proteína 16 por método imunohistoquímico. Nos casos positivos para o HPV, a presença dos tipos 16 e 18 do foi avaliada por reação de polimerase em cadeia. As características demográficas dos pacientes foram observadas. A sobrevida dos pacientes e a associação com HPV / proteína 16 foram determinadas. Resultados: A positividade com anticorpo policlonal do HPV foi detectada em 11 (12,2%) dos 90 casos. Desses 11 casos, o HPV 16 foi positivo em 6, o HPV 18 em 4 e o HPV 16 e 18 foram positivos em 1. Em 18 (20%) dos casos, a proteína 16 foi positiva. Seis dos casos (6,6%) apresentaram positividade para HPV e proteína16. Nos casos positivos apenas para a proteína 16 ou quando HPV e a proteína 16 foram co-positivos, a ingestão de álcool foi menor e o tumor apresentou maior probabilidade de estar localizado na área supraglótica. Essas proporções foram estatisticamente significantes. A localização supraglótica do tumor foi maior em casos positivos para proteína 16. A correlação entre positividade para proteína 16 e localização da área supraglótica foi estatisticamente significante (p = 0,011). Dos casos positivos para proteína 16, 55,6% foram supraglóticos, 33,3% glóticos e 11,1% transglóticos. Embora a expectativa de vida acima de 5 anos tenha sido numericamente maior nos casos positivos para HPV e proteína 16, isso não foi estatisticamente significante. Não houve relação estatisticamente significante entre positividade do HPV e média de idade, diferenciação, tabagismo e uso de álcool, progressão tumoral, metástase linfonodal, localização, recidiva, causa de mortalidade e métodos de tratamento em nosso estudo. O período médio de seguimento de nossos pacientes foi de 6,7 anos. Conclusão: A estreita relação entre HPV e carcinoma espinocelular orofaríngeo não pôde ser demonstrada na laringe em muitos estudos, inclusive no nosso estudo. Nossos achados confirmam um papel limitado do HPV na carcinogênese da laringe. A proteína 16 não é um substituto confiável para o status do HPV nos cânceres de laringe e não é preditor da sobrevida do câncer de laringe. A localização supraglótica do tumor foi maior em casos positivos para proteína16. A correlação entre positividade para proteína 16 e localização na área supraglótica foi determinada como estatisticamente significante. Há necessidade de ensaios clínicos com amostras maiores, nos quais a proliferação neoplásica seja melhor demonstrada e a precisão dos resultados obtidos seja apoiada por diferentes técnicas.

The evaluation of pretreatment neutrophil-lymphocyte ratio and derived neutrophil-lymphocyte ratio in patients with laryngeal neoplasms / Avaliação da relação neutrófilo-linfócito e relação neutrófilo-linfócito derivadano pré-tratamento em pacientes com neoplasias laríngeas

Abstract Introduction: Systemic inflammatory biomarkers are promising predictive and prognostic factors for solid cancers. The neutrophil-lymphocyte ratio and derived neutrophil-lymphocyte ratio are used to predict inflammation and used as biomarker in several malignancies. Objective: The purpose of this study was to demonstrate the diagnostic, predictive and prognostic role of neutrophil-lymphocyte ratio and derived neutrophil-lymphocyte ratio in patients with laryngeal neoplasms. Methods: A retrospective study was conducted on medical records involving 229 patients with benign, premalignant and malignant laryngeal neoplasms between 2002 and 2015. The diagnostic, predictive and prognostic role of neutrophil-lymphocyte ratio and derived neutrophil-lymphocyte ratio were evaluated using uni- and multivariate analysis. Results: The neutrophil-lymphocyte ratio and derived neutrophil-lymphocyte ratio were not statistically different between patients with benign, premalignant and malignant laryngeal neoplasms. Both neutrophil-lymphocyte ratio and derived neutrophil-lymphocyte ratio were predictive factors for stage, lymph node metastasis, and distant metastasis. Patients with high neutrophil-lymphocyte ratio value (≥4) had a poor prognosis when compared with patients with low neutrophil-lymphocyte ratio value (5 year, Overall Survival: 69.0% vs. 31.1%, p < 0.001; 5 year, disease free survival: 70.0% vs. 32.7%, p ˂ 0.001; 5 year, locoregional recurrence free survival: 69.7% vs. 32.0%, p < 0.001). Furthermore, neutrophil-lymphocyte ratio was an independent prognostic factor for 5 year: Overall survival (HR = 2.396; 95% CI 1.408-4.077; p = 0.001), Disease free survival (HR = 2.246; 95% CI 1.322-3.816; p = 0.006) and locoregional recurrence free survival (HR = 2.210; 95% CI 1.301-3.753; p = 0.003). Conclusion: Pretreatment neutrophil-lymphocyte ratio is a useful and reliable predictive and prognostic biomarker for patients with laryngeal carcinoma.

Resumo Introdução: Biomarcadores inflamatórios sistêmicos são fatores preditivos e prognósticos promissores para cânceres sólidos. A relação neutrófilo-linfócito e a relação neutrófilo-linfócito derivada são utilizadas para predizer a inflamação e como biomarcadores em várias malignidades. Objetivo: O objetivo deste estudo foi demonstrar o papel diagnóstico, preditivo e prognóstico da relação neutrófilo-linfócito e relação neutrófilo-linfócito derivada em pacientes com neoplasias laríngeas. Método: Foi realizado um estudo retrospectivo em prontuários médicos de 229 pacientes com neoplasias laríngeas benignas, pré-malignas e malignas entre 2002 e 2015. O papel diagnóstico, preditivo e prognóstico da relação neutrófilo-linfócito e relação neutrófilo-linfócito derivada foi avaliado por meio de análise uni- e multivariada. Resultados: A relação neutrófilo-linfócito e a relação neutrófilo-linfócito derivada não foram estatisticamente diferentes entre pacientes com neoplasias laríngeas benignas, pré-malignas e malignas. Ambas as relação neutrófilo-linfócito e relação neutrófilo-linfócito derivada foram fatores preditivos para o estágio, metástase linfonodal e metástase a distância. Pacientes com valor alto da relação neutrófilo-linfócito (≥ 4) apresentaram pior prognóstico quando comparados com pacientes com valor mais baixo da relação neutrófilo-linfócito (5 anos, Sobrevida Global: 69,0% vs. 31,1%, p < 0,001; 5 anos, sobrevida livre de doença: 70,0% vs. 32,7%, p < 0,001; 5 anos, sobrevida livre de recorrência loco-regional: 69,7% vs. 32,0%, p < 0,001). Além disso, a relação neutrófilo-linfócito foi um fator prognóstico independente para 5 anos: Sobrevida global (HR = 2,396; IC95% 1,408-4,077; p = 0,001), sobrevida livre de doença (HR = 2,246; IC95%: 1,322-3,816; p = 0,006) e sobrevida livre de recorrência loco-regional (HR = 2,210; IC95%: 1,301-3,753; p = 0,003). Conclusão: A relação neutrófilo-linfócito no pré-tratamento é um biomarcador preditivo e de prognóstico útil e confiável para pacientes com carcinoma de laringe.

The role of red cell distribution width in the locoregional recurrence of laryngeal cancer / O papel da amplitude de distribuição de eritrócitos na recorrência locorregional do câncer da laringe

Abstract Introduction: Although the red cell distribution width has been reported as a reliable predictor of prognosis in several types of cancer, to our knowledge few reports have focused on the prognostic value of red cell distribution width in laryngeal carcinoma. Objective: We aimed to explore whether the pretreatment red cell distribution width predicted recurrence in laryngeal cancer patients is a simple, reproducible, and inexpensive prognostic biomarker. Methods: All laryngeal cancer patients who underwent curative surgery (n = 132) over a 7 year study period were evaluated. Data on demographics, primary tumor site, T-stage, N-stage, histological features (differentiation; the presence of perineural/perivascular invasion), treatment group (total laryngectomy or partial laryngectomy) or adjuvant therapy (chemotherapy/radiotherapy); laboratory parameters (complete blood count, including the pre-operative red cell distribution width), and disease-free survival rates were retrospectively reviewed. All cases were divided into three groups by the red cell distribution width tertile [<13% (25th percentile) (n = 31), 13-14.4% (50th percentile) (n = 72), and >14.4% (75th percentile) (n = 29)]. Results: High-red cell distribution width group included more patients of advanced age, and more of those with recurrent and metastatic tumors (p = 0.005, 0.048, and 0.043, respectively). Individuals with red cell distribution width >14.4% (75th percentile) had lower disease free survival rates than did those with red cell distribution width <13% (25th percentile) (p = 0.014). Patients with red cell distribution width >14.4% at diagnosis were at a higher risk of locoregional recurrence (hazard ratio = 5.818, 95% confidence interval (95% CI) 1.25-26.97; p = 0.024) than patients with a normal red cell distribution width (<13%). Conclusion: We found that the pretreatment red cell distribution width was independently prognostic of disease free survival rate in patients with laryngeal cancer and may serve as a new, accurate, and reproducible means of identifying early-stage laryngeal cancer patients with poorer prognoses.

Resumo Introdução: Embora a amplitude de distribuição de eritrócitos tenha sido relatada como um preditor confiável de prognóstico em vários tipos de câncer, que seja de nosso conhecimento, poucos estudos se concentraram no valor prognóstico dessa medida no carcinoma laríngeo. Objetivo: Avaliar se a amplitude de distribuição de eritrócitos pré-tratamento prevê a recorrência em pacientes com câncer de laringe como um biomarcador prognóstico simples, reprodutível e não dispendioso. Método: Foram avaliados todos os pacientes com câncer de laringe submetidos à cirurgia curativa (n = 132) durante sete anos. Dados demográficos, local do tumor primário, estágio T, estágio N, características histológicas (diferenciação; presença de invasão perineural/perivascular), grupo de tratamento (laringectomia total ou laringectomia parcial) ou terapia adjuvante (quimioterapia/radioterapia); parâmetros laboratoriais (hemograma, inclusive a amplitude de distribuição de eritrócitos pré-operatório) e a sobrevida livre de doença foram revisados retrospectivamente. Todos os casos foram divididos em três grupos pelo tercil da amplitude de distribuição de eritrócitos [< 13% (percentil 25) (n = 31), 13%-14,4% (percentil 50) (n = 72) e > 14,4% (percentil 75) (n = 29)]. Resultados: O grupo com amplitude de distribuição de eritrócitos elevado incluiu um número maior de pacientes com idade avançada e mais pacientes com tumores recorrentes e metastáticos (p = 0,005, 0,048 e 0,043, respectivamente). Os indivíduos com a amplitude de distribuição de eritrócitos > 14,4% (percentil 75) apresentaram taxas de sobrevida livre de doença menores do que aqueles com a amplitude de distribuição de eritrócitos < 13% (percentil 25) (p = 0,014). Pacientes com a amplitude de distribuição de eritrócitos > 14,4% no diagnóstico apresentaram maior risco de recorrência locorregional [Hazard Ratio = 5,818, intervalo de confiança de 95% (IC 95%) 1,25-26,97; p = 0,024] do que pacientes com a amplitude de distribuição de eritrócitos normal (< 13%). Conclusão: Verificamos que a amplitude de distribuição de eritrócitos pré-tratamento foi um fator prognóstico independente de sobrevida livre de doença em pacientes com câncer de laringe e pode servir como um novo parâmetro, preciso e reprodutível, para identificar pacientes com câncer de laringe em estágio inicial com piores prognósticos.

The Expression of MicroRNA-155 in Plasma and Tissue Is Matched in Human Laryngeal Squamous Cell Carcinoma

PURPOSE: Tumor-associated microRNAs have been detected in cancer, though whether plasma microRNA-155 (miR-155) could be a potential biomarker for laryngeal squamous cell carcinoma (LSCC) prognosis is unclear. We aimed to determine how miR-155 can be used to predict the clinical characteristics of patients with LSCC and correctly diagnose them. MATERIALS AND METHODS: We collected tissue samples and peripheral blood samples before and after treatment from 280 LSCC cases and 560 controls. Real-time quantitative reverse transcription PCR was employed in this study to compare the relative expression of miR-155. RESULTS: A total of 280 LSCC patients and 560 age- and sex-matched controls were included in the study. The miR-155 level was more up-regulated in LSCC tissue than in the non-tumor tissues (13.6+/-2.4 vs. 3.1+/-0.80, p<0.001). Additionally, a significantly higher miR-155 level in plasma samples from LSCC patients than in those of the controls (8.9+/-1.25 vs. 1.8+/-0.8, p<0.001) was reported. Tissue miR-155 showed an area under the curve (AUC) of 0.933, with a sensitivity of 82.6% and a specificity of 89.2%. The AUC for plasma miR-155 was 0.757, with a sensitivity of 58.4% and a specificity of 69.5%. When early LSCC in TNM I stage was considered, tissue miR-155 showed an area under the curve of 0.804, with a sensitivity of 85.2% and a specificity of 87.3%. CONCLUSION: The expression of tissue and plasma miR-155 were significantly up-regulated in patients with LSCC. Our work will serve as a basis for further investigation, preferably large-scale validation in clinical trials.

Correlation between vascular endothelial growth factor expression and presence of lymph node metastasis in advanced squamous cell carcinoma of the larynx / Correlação entre expressão do fator de crescimento endotelial e presença de metástase linfática nos carcinomas epidermoides avançados de laringe

Introduction: Squamous cell carcinoma is the most common neoplasm of the larynx, and its evolution depends on tumor staging. Vascular endothelial growth factor is a marker of angiogenesis, and its expression may be related to increased tumor aggressiveness, as evidenced by the presence of cervical lymphatic metastases. Objectives: To evaluate the expression of the vascular endothelial growth factor marker in non-glottic advanced squamous cell carcinoma of the larynx (T3/T4) and correlate it with the presence of cervical lymph node metastases. Methods: Retrospective clinical study and immunohistochemical analysis of vascular endothelial growth factor through the German scale of immunoreactivity in products of non-glottic squamous cell carcinomas. Results: This study analyzed 15 cases of advanced non-glottic laryngeal tumors (T3/T4), four of which exhibited cervical lymphatic metastases. There was no correlation between vascular endothelial growth factor expression and the presence of cervical metastases. Conclusion: Although vascular endothelial growth factor was expressed in a few cases, there was no correlation with the spread of cervical lymph metastases. .

Introdução: O carcinoma de células escamosas é a neoplasia mais frequente da laringe e seu prognóstico depende do estadiamento. A progressão da doença está relacionada a fatores intrínsecos celulares do câncer, não conhecidos. O VEGF (vascular endothelial growth factor) é um marcador de angiogênese e sua expressão pode estar relacionada a uma maior agressividade tumoral, evidenciada pela presença de metástases linfáticas cervicais. Objetivos: Avaliar a expressão do marcador VEGF em carcinoma de células escamosas da laringe avançados (T3/T4), não glóticos e correlacionar quanto à presença de metástases linfáticas cervicais. Método: Estudo clínico retrospectivo de análise imunohistoquimica do VEGF através da escala Germânica de imunorreatividade em produtos de carcinomas epidermóides não glóticos. Resultados: Analisados 15 casos de tumores avançados de laringe (T3/T4) não glóticos, sendo sete com presença de metástases linfáticas cervicais. Não houve correlação entre a expressão do VEGF e a presença de metástases cervicais. Conclusão: O VEGF foi pouco expressado nos casos estudados e não foi observada sua correlação com a disseminação de metástase linfática cervical. .

Interferon-gamma and interleukin-10 production by mononuclear cells from patients with advanced head and neck cancer

OBJECTIVE: This study aims to evaluate the production of interferon-gamma and interleukin-10 by stimulated peripheral blood mononuclear cells isolated from patients with supraglottic laryngeal cancer before and after surgical treatment. METHODS: Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cultures of peripheral blood mononuclear cells isolated during the preoperative and late postoperative periods were stimulated with concanavalin A and Bacille Calmette-Guerin, and the supernatant concentrations of interferon-gamma and interleukin-10 were measured. RESULTS: For non-stimulated cultures, the interferon-gamma levels produced by the preoperative period and the late postoperative period cultures were lower than the levels produced by the control group cultures. The interferon-gamma levels after stimulation with concanavalin A were higher in the late postoperative period cultures than in the preoperative evaluation cultures. Stimulation with Bacille Calmette-Guerin led to the production of similar levels of interferon-gamma and interleukin-10 by all cultures; thus, stimulation increased the levels of interferon-gamma produced by both the preoperative and postoperative cultures relative to the levels produced by the corresponding unstimulated cultures. CONCLUSION: Patients with advanced supraglottic laryngeal cancer exhibit an in vitro deficiency in interferongamma secretion by mononuclear cells. Stimulated cells seem to recover this function during the postoperative period.

Association of vitamin A, vitamin C and zinc with laryngeal cancer.

BACKGROUND: The incidence of the cancers of the oral cavity, pharynx, esophagus and larynx in different population groups of India is amongst the highest reported in Asian countries. There is evidence that high dietary carotenoids and vitamin C may possibly decrease the risk of laryngeal cancer. Limited data is available from India on the association between these micronutrients and the risk of laryngeal cancer. AIMS: To assess the levels of vitamin A, vitamin C and zinc in laryngeal cancer patients and healthy controls. SETTING AND DESIGN: A hospital based case- control study. MATERIAL AND METHODS: One hundred and fifty five laryngeal cancer patients and a control group of 155 healthy individuals constituted the study population. Individuals in the control group were individually matched with the patients for their age +/- 2 years, sex and place of residence. Venous blood was drawn from the cases and controls and estimations of vitamin A, zinc and vitamin C was done utilizing the standard procedures. STATISTICAL ANALYSIS USED: Paired 't' test to compare the mean serum levels of vitamin A and zinc and plasma vitamin C between laryngeal cancer patients and controls. Univariate logistic regression analysis to calculate the odds ratios and the confidence intervals. RESULTS: The mean serum vitamin A, zinc and plasma vitamin C levels were significantly lower in laryngeal cancer patients as compared to the controls. CONCLUSIONS: The findings of the present study indicated a strong association of these micronutrients with laryngeal cancer in the Indian population.