[DNA extraction using Chelex resin for oncogenic amplification analysis in head and neck tumours]. / Extracción de ADN con resina Chelex en el análisis de la amplificación oncogénica en carcinomas de cabeza y cuello.

DNA extraction from tissues can be the most laborious and complex step in amplifying DNA by PCR when phenol-choroform procedure is used. We compare this lengthy, slow and expensive extraction method with other two based in the use of Chelex-100 resin. This chelating resin has been applied for extracting DNA from different tissues to use with the PCR. These procedures are simple, rapid and do not require multiple steps. In this study we compared DNA extraction from 30 head and neck squamous cell carcinomas (HNSCC) using organic solvent precipitation, Chelex 100 resin with and without proteinase K pretreatment. The results show that proteinase K-Chelex 100 procedure is as efficient as the phenol-chloroform one.

Extracción de ADN con resina chelex en el análisis de la amplificación oncogénica en carcinomas de cabeza y cuello / DNA extraction using chelex resin for the oncogenic amplification analysis in nead and neck tumours

La extracción de ADN de tejido tumoral puede ser el proceso más laborioso y complejo en la amplificación de ADN mediante PCR cuando se utiliza el procedimiento con fenol-cloroformo. Comparamos este método de extracción extenso, lento y caro con otras dos técnicas basadas en el uso de resina Chelex-100. Esta resina quelante ha sido utilizada para la extracción de ADN de diferentes tejidos para su uso con la PCR. Estos procedimientos son simples, rápidos y no requieren múltiples pasos. En este trabajo comparamos la extracción de ADN procedente de 30 carcinomas epidermoides de cabeza y cuello utilizando la precipitación con solventes orgánicos, resina Chelex-100 con y sin procesamiento previo con proteinasa K. Los resultados evidencian que el procedimiento con proteinasa K y resina Chelex-100 es un método tan eficaz como la precipitación con fenol-cloroformo (AU)

DNA extraction from tissues can be the most laborious and complex step in amplifying DNA by PCR when phenol-choroform procedure is used. We compare this lengthy, slow and expensive extraction method with other two based in the use of Chelex-100 resin. This chelating resin has been applied for extracting DNA from different tissues to use with the PCR. These procedures are simple, rapid and do not require multiple steps. In this study we compared DNA extraction from 30 head and neck squamous cell carcinomas (HNSCC) using organic solvent precipitation, Chelex 100 resin with and without proteinase K pretreatment. The results show that proteinase K-Chelex 100 procedure is as efficient as the phenol-chloroform one (AU)

[Molecular changes in epidermoid carcinoma of the oropharynx]. / Alteraciones moleculares en los carcinomas epidermoides de la orofaringe.

In most of the studies about molecular alterations in squamous cell carcinomas of the head and neck there is not distinction between the different subsites of this area. The objective of this study is to describe the molecular alterations in squamous cell carcinomas of the oropharynx. Twenty-nine oropharyngeal carcinomas, with a minimum follow-up of 36 months, were studied. The molecular alterations analyzed were: the amplification of 11q13 region (in the 29 cases), and the MYC and ERBB1 oncogenes (in 22 cases); the integration of Human Papillomavirus (HPV) types 6b and 16 (in 22 cases); the loss of heterozygosity (LOH) of p53 and N-acetyltransferase-2 (NAT2) gene (in 12 and 13 informative cases, respectively); and the cellular DNA content (in 13 cases). The most frequent alterations found were the LOH at p53 (67%), and NAT2 (54%) locus, followed by 11q13 amplification (49%). ERBB1 amplification was found in 14% of the cases, and MYC amplification only in one (5%). Integration of the HPV was found in 23% of the cases. Nine (69%) of the 13 analyzed cases were aneuploid. The only alteration with a prognostic significance was 11q13 amplification that showed a tendency to be associated with a higher frequency of nodal metastases and tumor recurrence.

Alteraciones moleculares en los carcinomas epidermoides de la orofaringe / Molecular alterations in oropharyngeal squamous cell carninomas

En la mayoría de los estudios sobre alteraciones moleculares en los carcinomas epidermoides de cabeza y cuello no se realiza distinción entre las diferentes localizaciones de esta área. El objetivo de este estudio es describir las alteraciones moleculares propias de los carcinomas epidermoides de la orofaringe. Se estudian 29 carcinomas de orofaringe con un seguimiento mínimo de 36 meses.Las alteraciones moleculares analizadas fueron: la amplificación de la región 11q13 (en los 29 casos) y de los oncogenes MYC y ERBB1 (en 22 casos); la integración del virus del papiloma humano (HPV) tipos 6b y 16 (en 22 casos); las pérdidas de heterocigosidad de los genes p53 y NAT2 (en 12 y 13 casos informativos, respectivamente); y el contenido celular de ADN (en 13 casos). Las alteraciones más comunes fueron las pérdidas de heterocigosidad de los genes p53 (67 por ciento de los casos) y NAT2 (54 por ciento), seguidas de la amplificación de 11q13 (49 por ciento). La amplificación del ERBB1 se encontró en el 14 por ciento de los casos, y la del MYC en sólo un caso (5 por ciento). El 23 por ciento de los casos presentaban integración del HPV. De los 13 casos analizados 9 (69 por ciento) eran aneuploides. La única alteración con posible significado pronóstico fue la amplificación de 11q13, con tendencia a asociarse con una mayor frecuencia de metástasis ganglionares y de recidiva tumoral (AU)

In most of the studies about molecular alterations in squamous cell carcinomas of the head and neck there is not distinction between the different subsites of this area. The objective of this study is to describe the molecular alterations in squamous cell carcinomas of the oropharynx. Twenty-nine oropharyngeal carcinomas, with a minimum follow-up of 36 months, were studied. The molecular alterations analyzed were: the amplification of 11q13 region (in the 29 cases), and the MYC and ERBB1 oncogenes (in 22 cases); the integration of Human Papillomavirus (HPV) types 6b and 16 (in 22 cases); the loss of heterozygosity (LOH) of p53 and N-acetyltransferase-2 (NAT2) gene (in 12 and 13 informative cases, respectively); and the cellular DNA content (in 13 cases). The most frequent alterations found were the LOH at p53 (67%), and NAT2 (54%) locus, followed by 11q13 amplification (49%). ERBB1 amplification was found in 14% of the cases, and MYC amplification only in one (5%). Integration of the HPV was found in 23% of the cases. Nine (69%) of the 13 analyzed cases were aneuploid. The only alteration with a prognostic significance was 11q13 amplification that showed a tendency to be associated with a higher frequency of nodal metastases and tumor recurrence (AU)

Using polymerase chain reaction to human papillomavirus in oral and pharyngolaryngeal carcinomas.

PURPOSE: Increasingly, evidence has shown that human papillomavirus (HPV) plays a role in the induction of certain carcinomas. The presence of HPV sequences in 56 previously untreated oral and pharyngolaryngeal carcinomas was examined by the polymerase chain reaction (PCR). MATERIALS AND METHODS: After DNA extraction, samples underwent 40 replication cycles with specific oligonucleotide primers corresponding to sequences from the E6 open-reading frame of HPV-6b, HPV-16, and HPV-18. To determine the E6 genomic integration, positive samples were processed with specific primers for the corresponding HPV L1 genes. Genomic HPV DNA clones into PBR 322 was used as positive control. RESULTS: HPV E6 DNA of the 6b and 16 types was detected in 14 patients (25%). The L1 gene was not present. CONCLUSION: Detected HPV E6 DNA might be integrated into the cell genome in the positive cases as indicated by the absence of the L1 gene-coding for the viral capside. Histological and survival rates, were unrelated to the presence of HPV.

[Simultaneous screening of HPV-6b and 16 in pharyngolaryngeal cancer]. / Detección simultánea del HPV-6b y 16 en el cáncer faringolaríngeo.

It has been suggested that some human papilloma viruses (HPV) may play a causal role in cancer of the pharynx, larynx, and oral cavity, together with factors such as smoking, alcohol, toxins, and heredity. Using the polymerase chain reaction (PCR), we detected the two most common genotypes in pharyngolaryngeal cancer, HPV-6b and 16, in 15 patients from a series of 57 cases. One patient had both genotypes. The fact that this was the only positive case in which no other risk factors were present, particularly alcohol and smoking, suggests that the synergetic oncogenic action of both viruses could have played an important role in carcinogenesis.

[Amplification of oncogene c-erb-B1 and cellular DNA content in squamous cell carcinoma of the head and neck]. / Amplificación del oncogén c-erbB1 y contenido celular de ADN en los carcinomas epidermoides de cabeza y cuello.

Tumoral DNA content was studied by flow cytometry and PCR amplification of c-erbB1 in tissue samples from 31 patients with squamous cell carcinoma of the head and neck. Eighteen cases (58%) were aneuploid and 13 (42%) were diploid. Aneuploidy correlated with pharyngeal site and poorly differentiated tumors, but not with clinical stage or metastases. Six (19.3%) cases had c-erbB1 amplification, which correlated with tumor size, nodal metastasis, poor differentiation, and hypopharyngeal site. Only 20% of patients with amplification survived 30 months, compared with 64% of patients without amplification. None of the patients with aneuploidy and c-erbB1 amplification survived more than 15 months. To conclude, the measurement of cellular DNA content and c-erbB1 amplification seem to have prognostic value in squamous cell carcinoma of the head and neck.