RESUMEN
PURPOSE: To evaluate the relationship between human papillomavirus (HPV) status and known prognostic makers for head and neck cancers including tumor hypoxia, epidermal growth factor receptor (EGFR) expression and intratumoral T-cell levels and to determine the prognostic impact of these markers by HPV status. METHODS AND MATERIALS: HPV status in 82 evaluable head and neck squamous cell carcinomas patients was determined by pyrosequencing and related to p16(INK4a) staining and treatment outcomes. It was correlated with tumor hypoxia (tumor pO(2) and carbonic anhydrase [CAIX] staining), EGFR status, and intratumoral lymphocyte expression (CD3 staining). RESULTS: Forty-four percent of evaluable tumors had strong HPV signal by pyrosequencing. There was a significant relationship between strong HPV signal and p16(INK4a) staining as well as oropharynx location. The strong HPV signal group fared significantly better than others, both in time to progression (TTP, p = 0.008) and overall survival (OS, p = 0.004) for all patients and for the oropharyngeal subset. Positive p16(INK4a) staining was associated with better TTP (p = 0.014) and OS (p = 0.00002). There was no relationship between HPV status and tumor pO(2) or CAIX staining. However, HPV status correlated inversely with EGFR reactivity (p = 0.0006) and directly with CD3(+) T-lymphocyte level (p = 0.03). Whereas CAIX and EGFR overexpression were negative prognostic factors regardless of HPV status, CD3(+) T-cell levels was prognostic only in HPV(-) tumors. CONCLUSION: HPV status was a prognostic factor for progression and survival. It correlated inversely with EGFR expression and directly with T-cell infiltration. The prognostic effect of CAIX and EGFR expression was not influenced by HPV status, whereas intratumoral T-cell levels was significant only for HPV(-) tumors.
Asunto(s)
Alphapapillomavirus/aislamiento & purificación , Carcinoma de Células Escamosas/virología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Receptores ErbB/análisis , Neoplasias de Cabeza y Cuello/virología , Infecciones por Papillomavirus/virología , Alphapapillomavirus/genética , Análisis de Varianza , Biomarcadores/análisis , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Hipoxia de la Célula , Femenino , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/aislamiento & purificación , Humanos , Inmunidad Celular , Linfocitos Infiltrantes de Tumor/citología , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/metabolismo , Reacción en Cadena de la Polimerasa , Pronóstico , Linfocitos T , Análisis de Matrices Tisulares/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: We investigated p16(INK4A) expression in branchial cleft cysts and its utility in distinguishing branchial cleft cysts from metastatic head and neck squamous cell carcinomas (SCCs) in fine-needle aspiration biopsies (FNABs). METHODS: A study set comprising 41 resections (15 SCC and 26 branchial cleft cysts) and a test set of 15 FNABs (11 SCC and 4 branchial cleft cysts) were analyzed with p16(INK4A) immunohistochemistry and human papillomavirus (HPV) polymerase chain reaction (PCR)/pyrosequencing. Cases with discrepant p16(INK4A) and PCR/pyrosequencing results were further evaluated with HPV in situ hybridization (ISH). SCCs were divided into keratinizing SCC and nonkeratinizing SCC groups and site of origin. RESULTS: Metastatic oropharyngeal nonkeratinizing SCC in the study set exhibited diffuse, strong p16(INK4A) (7 of 7) and HPV16 DNA positivity (6 of 6), while keratinizing SCC from the larynx and oral cavity was negative for p16(INK4A). p16(INK4A) reactivity in the branchial cleft cyst study set was characterized by focal, strong staining (6 of 21) involving the superficial squamous epithelium. HPV DNA was identified in 7 of 19 branchial cleft cyst study set cases by PCR/pyrosequencing, but these cases were negative by HPV ISH. In the test set, oropharyngeal nonkeratinizing SCC exhibited diffuse, strong p16(INK4A) (3 of 3) and HPV16 DNA (2 of 2), while metastatic keratinizing SCC was negative for p16(INK4A) and HPV DNA. All 4 FNABs of branchial cleft cysts were negative for p16(INK4A). Diffuse, strong p16(INK4A) correlated with oropharyngeal origin (P=.001) and nonkeratinizing morphology (P=.0001). CONCLUSIONS: Branchial cleft cysts can exhibit focal strong reactivity limited to the superficial squamous epithelium and glandular epithelium. Although p16(INK4A) immunohistochemistry may be helpful in distinguishing oropharyngeal nonkeratinizing SCC from branchial cleft cysts in FNAB specimens, it is not helpful in cases of keratinizing SCC because these cases are typically negative for p16(INK4A).
Asunto(s)
Branquioma/patología , Carcinoma de Células Escamosas/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Neoplasias de Cabeza y Cuello/patología , Orofaringe/patología , Adolescente , Anciano , Biopsia con Aguja Fina , Branquioma/metabolismo , Carcinoma de Células Escamosas/metabolismo , Quistes/metabolismo , Quistes/patología , Diagnóstico Diferencial , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Inmunohistoquímica , Queratinas/metabolismo , Masculino , Persona de Mediana Edad , Orofaringe/químicaRESUMEN
We describe the protocol through which we identify and characterize dynein subunit genes in the ciliated protozoan Tetrahymena thermophila. The gene(s) of interest is found by searching the Tetrahymena genome, and it is characterized in silico including the prediction of the open reading frame and identification of likely introns. The gene is then characterized experimentally, including the confirmation of the exon-intron organization of the gene and the measurement of the expression of the gene in nondeciliated and reciliating cells. In order to understand the function of the gene product, the gene is modified-for example, deleted, overexpressed, or epitope-tagged-using the straightforward gene replacement strategies available with Tetrahymena. The effect(s) of the dynein gene modification is evaluated by examining transformants for ciliary traits including cell motility, ciliogenesis, cell division, and the engulfment of particles through the oral apparatus. The multistepped protocol enables undergraduate students to engage in short- and long-term experiments. In our laboratory during the last 6 years, more than two dozen undergraduate students have used these methods to investigate dynein subunit genes.
Asunto(s)
Biología Computacional/métodos , Dineínas/genética , Genes Protozoarios/genética , Tetrahymena/genética , Animales , Bioensayo , Cilios/metabolismo , Dineínas/metabolismo , Regulación de la Expresión Génica , Marcación de Gen , Fenotipo , Filogenia , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Análisis de Secuencia de ADNRESUMEN
A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts.