RESUMEN
Genetic abnormalities in SCCHNs are frequent and may be useful for screening, follow-up and prognosis. A biopsy or resection generally is utilized to identify these alterations but analysis of scraped or exfoliated tumor cells has been proposed as simpler and more versatile. It is unknown how well genetic abnormalities in scrapes reflect those in the tumor. Therefore, we compared DNA alterations in tumor scrapes obtained prior to treatment with alterations in microdissected tumor biopsies. Eight primary squamous-cell carcinomas of the head and neck (SCCHNs) were examined at 14 loci to determine loss of heterozygosity (LOH) at sites on 3p, 9p, 11p, 11q and 17p and amplification of cyclin D1 (CCND1). All biopsies contained DNA alterations, but only 3/8 scrapes contained unequivocal abnormalities; 4/8 contained subtle alterations that could not have been definitively identified without comparison to the paired biopsies. Overall, 22 alterations were detected in the biopsies: 8/22 were found unequivocally in the scrapes; 7/22 were identifiable in scrapes only after the biopsy alterations were defined and 7/22 were absent from scrapes. One LOH in scrape, but not biopsy, DNA was found. Discrepancies between scrapes and tumors tended to increase if multiple tumor samples were examined. We conclude that DNA alterations can be detected in scrapes of SCCHNs but may inaccurately reflect the tumor's complex genetic abnormalities. This may be due to contamination of scrapes with normal cells or to genetic heterogeneity within the tumor not represented in the scrape. Although examining scrapes of SCCHNs is an attractive technique, its clinical utility may have limitations.
Asunto(s)
Biopsia , Carcinoma de Células Escamosas/genética , ADN de Neoplasias/genética , Pérdida de Heterocigocidad , Neoplasias Orofaríngeas/genética , Manejo de Especímenes/métodos , Ciclina D1/genética , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Valor Predictivo de las PruebasRESUMEN
Loss of heterozygosity (LOH) at chromosome 11q23 has been found in a variety of epithelial human neoplasms, suggesting that this region contains a tumor suppressor gene(s) important to tumorigenesis. We investigated whether LOH at 11q23 could be detected in squamous cell carcinoma of the head and neck (SCCHN), and whether loss at this site was associated with specific clinical parameters. Fifty-six matched blood and SCCHN tumor samples taken at the time of diagnosis were evaluated for LOH at three microsatellite markers at 11q23. Multiplex PCRs with [alpha-32P]dCTP labeling of the amplified DNA strands were performed. Clinical data were obtained from medical record review. LOH at 11q23 was found in 13 of 52 (25%) evaluable tumors. There was no association between LOH at 11q23 and amplification of the CCND1 (cyclin D1) oncogene or inactivation of the p53 gene, which had been determined previously. With a mean follow-up of 24 months, an association independent of tumor size or stage was found between LOH at 11q23 and recurrent disease (P = 0.04). Among subjects who received radiotherapy (RT) as a component of their treatment, LOH at 11q23 was associated with persistent or recurrent locoregional disease (P = 0.05). LOH at 11q23 occurs in a subset of SCCHN. It is associated with a higher likelihood of recurrent disease, perhaps related to resistance to RT. The specific gene(s) and mechanism(s) responsible remain to be identified. Until then, LOH at 11q23 might become a marker identifying patients likely to do poorly with conventional therapy.
Asunto(s)
Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 11 , Neoplasias de Cabeza y Cuello/genética , Pérdida de Heterocigocidad , Recurrencia Local de Neoplasia/genética , Carcinoma de Células Escamosas/radioterapia , Femenino , Estudios de Seguimiento , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Análisis de SupervivenciaRESUMEN
The authors have investigated whether genetic abnormalities in two genes, loss of heterozygosity (LOH) of p53 and amplification of the cyclin D1 gene, correlate with clinical outcome in 56 matched pairs of blood and tumor from patients with squamous cell carcinoma of the head and neck (SCCHN). Frequency of p53 LOH was 47.4%, of cyclin D1 amplification 33.9%, and of both abnormalities together 23.7%. p53 LOH was associated with T4 (P = 0.003) and stage IV (P = 0.015) tumors. Cyclin D1 amplification was associated with recurrences and/or metachronous tumors (P = 0.007). The total number of p53 and cyclin D1 abnormalities (scored as zero, one, and two) show a pattern that seems to be additive; the increase in the number of these abnormalities is associated with a proportional increase in the frequency of T4, stage IV, presence of recurrences and/or metachronous tumors, and possibly a proportional decrease in the disease-free interval in the sample. The association of the markers with recurrences and/or metachronous tumors persists if the tumor stage effect is mathematically removed. The combined analysis of the p53 and cyclin D1 abnormalities seems to be more informative than either of them individually and may have predictive value in SCCHN.
Asunto(s)
Carcinoma de Células Escamosas/genética , Amplificación de Genes , Genes bcl-1 , Genes p53 , Neoplasias de Cabeza y Cuello/genética , Pérdida de Heterocigocidad , Carcinoma de Células Escamosas/patología , Supervivencia sin Enfermedad , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Estadificación de Neoplasias , Neoplasias Primarias Secundarias , Pronóstico , Resultado del TratamientoRESUMEN
Polymorphisms in the tumor suppressor gene p53 were identified by Southern blots of denaturing gradient gels (melting polymorphisms). Five polymorphisms were identified in white blood cell DNA, with the frequency of heterozygotes varying from 4 to 27%. The strategy of hybridizing the same blots with different probes suggests that two of the polymorphisms are located in the region of exons 5-6 and one is in the region of exons 7-9. This study illustrates the use of this method for analysis of tumor suppressor genes and suggests future applications, such as for the assay of loss of heterozygosity. That assay is used to detect gene deletions in tumors, an important event in human tumorigenesis.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Genes p53/genética , Polimorfismo Genético/genética , Neoplasias de la Mama/genética , Sondas de ADN , ADN de Neoplasias/sangre , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Linfocitos/química , MutaciónRESUMEN
The treatment of locally advanced squamous cell carcinomas of the head and neck presents a challenge for oncologists. Radiation therapy alone fails to control many of these tumors. Chemotherapy added to radiation therapy has not clearly demonstrated an improvement in survival in the majority of trials reported to date. In this study, we have evaluated whether IFN-alpha-2a and/or 13-cis-retinoic acid (RA) enhance radiation cytotoxicity in a head and neck squamous cell carcinoma cell line (FaDu). Using a clonogenic cell survival assay, IFN-alpha-2a (1000 units/ml) or RA (1 microM) alone did not significantly enhance radiation cytotoxicity. The combination of the two agents, however, significantly increased the cytotoxicity of radiation against FaDu cells. The calculated survival fraction at 2 Gy was decreased from 0.649 with radiation alone to 0.477 when combined with the other two agents (P = 0.016), and the MID was decreased from 3.318 to 2.499 Gy (P = 0.028). A Phase I clinical trial to combine IFN-alpha-2a and/or RA in patients with unresectable head and neck cancer has been initiated.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Interferón-alfa/farmacología , Isotretinoína/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , División Celular/efectos de los fármacos , Humanos , Interferón alfa-2 , Proteínas Recombinantes , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiación , Ensayo de Tumor de Célula MadreRESUMEN
The paraoxonase polymorphism was analysed in the serum of 248 individuals from Saudi Arabia. The phenotyping method used was one based on the ratio of paraoxonase/arylesterase, described in earlier studies. The distribution of this ratio in the sample was trimodal and it provided a good resolution for the identification of three phenotypes: A-homozygotes with low activity; B-homozygotes with high activity; AB-the corresponding heterozygotes, with intermediate activity. Gene frequencies were 0.7296 for the allele A with low activity and 0.2704 for the allele B with high activity. These frequencies are close to those observed in Caucasian samples from North America and Europe. Phenotypic frequencies in the sample fit the Hardy-Weinberg equilibrium.
Asunto(s)
Monoéster Fosfórico Hidrolasas/genética , Polimorfismo Genético , Adulto , Arildialquilfosfatasa , Hidrolasas de Éster Carboxílico/sangre , Femenino , Frecuencia de los Genes , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Monoéster Fosfórico Hidrolasas/sangre , Arabia SauditaRESUMEN
The fluoride variant of human butyrylcholinesterase owes its name to the observation that it is resistant to inhibition by 0.050 mM sodium fluoride in the in vitro assay. Individuals who are heterozygous for the fluoride and atypical alleles experience about 30 min of apnea, rather than the usual 3-5 min, after receiving succinyldicholine. Earlier we reported that the atypical variant has a nucleotide substitution which changes Asp 70 to Gly. In the present work we have identified two different point mutations associated with the fluoride-resistant phenotype. Fluoride-1 has a nucleotide substitution which changes Thr 243 to Met (ACG to ATG). Fluoride-2 has a substitution which changes Gly 390 to Val (GGT to GTT). These results were obtained by DNA sequence analysis of the butyrylcholinesterase gene after amplification by PCR. The subjects for these analyses were 4 patients and 21 family members.
Asunto(s)
Butirilcolinesterasa/genética , Inhibidores de la Colinesterasa/farmacología , Mutación , Fluoruro de Sodio/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Butirilcolinesterasa/sangre , ADN/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Homología de Secuencia de AminoácidoRESUMEN
Genomic blots from man, monkey, cow, sheep, pig, rabbit, dog, rat, mouse, guinea pig, and chicken DNA were hybridized with probes derived from the four exons of the human butyrylcholinesterase gene (BCHE) (Arpagaus, M., Kott, M., Vatsis, K. P., Bartels, C. F., La Du, B. N., and Lockridge, O. (1990) Biochemistry 29, 124-131). Results showed that the BCHE gene was present in a single copy in the genome of all these vertebrates. The polymerase chain reaction was used to amplify genomic DNA from these animals with oligonucleotides derived from the human BCHE coding sequence. The amplified segment contained 423 bp of BCHE sequence including the active site serine of the enzyme (amino acid 198) and a component of the anionic site, aspartate 70. Amplification was successful for monkey, pig, cow, dog, sheep, and rabbit DNA, but unsuccessful for rat, guinea pig, mouse, and chicken DNA. Amplified segments were cloned in M13 and sequenced. The mouse sequence was obtained by sequencing a genomic clone. The highest identity of the human amino acid sequence was found with monkey (100%) and the lowest with mouse (91.5%). The sequence around the active site serine 198, Phe-Gly-Glu-Ser-Ala-Gly-Ala, was conserved in all eight animals as was the anionic site component, aspartate 70. A phylogenetic tree of mammalian butyrylcholinesterases was constructed using the partial BCHE sequences.
Asunto(s)
Evolución Biológica , Butirilcolinesterasa/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Butirilcolinesterasa/sangre , Bovinos , Sondas de ADN , Exones , Humanos , Datos de Secuencia Molecular , Filogenia , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , VertebradosRESUMEN
1. New information identifying nucleotide alterations of human butyrylcholinesterase allows the use of more specific nomenclature for the variants commonly known as atypical, fluoride, silent, and K variant. 2. In addition to suggesting a system of trivial names and abbreviations, we provide a list of formal names that follow the guidelines of the Committee for Human Gene Nomenclature. 3. It is suggested that formal names be included in publications whenever possible.
Asunto(s)
Butirilcolinesterasa/clasificación , Secuencia de Aminoácidos , Butirilcolinesterasa/genética , Inhibidores de la Colinesterasa/farmacología , ADN/genética , Genes , Variación Genética , Genotipo , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa , Fluoruro de Sodio/farmacología , Especificidad por Sustrato , Terminología como AsuntoRESUMEN
Our laboratory has recently shown that several variant forms of human butyrylcholinesterase, associated with unusual sensitivity to succinylcholine, are caused by specific mutations within the structural DNA coding for this enzyme. Atypical (dibucaine-resistant) butyrylcholinesterase is caused by a point mutation at nucleotide position 209(GAT-- greater than GGT), which changes aspartate 70 to glycine. One fluoride-resistant variant family has a point mutation at nucleotide 728(ACG-- greater than ATG), which changes threonine 243 to methionine. Another type of fluoride-resistant variant has a point mutation at nucleotide 1169(GGT-- greater than GTT), which changes glycine 390 to valine. One type of silent phenotype is due to a frame-shift mutation at nucleotide position 351(GGT-- greater than GGAG). A polymorphic site at nucleotide position 1615 (GCA/ACA), coding for Ala/Thr, accounts for the quantitative K-variant, which causes an approximate one-third reduction of activity, if Thr occupies that position at codon 539. Examples are given to illustrate the advantages of using a combination of the new DNA analytical techniques, including: the use of allele-specific probes, with the standard serum cholinesterase phenotyping methods. More accurate typing of patients with certain variants is now possible; pedigree analysis will be aided by the improved methodology.
Asunto(s)
Butirilcolinesterasa/genética , Alelos , Análisis Mutacional de ADN , ADN de Cadena Simple , Fluoruros/farmacología , Genotipo , Humanos , Sondas de Oligonucleótidos , FenotipoRESUMEN
A frameshift mutation that causes a silent phenotype for human serum cholinesterase was identified in the DNA of seven individuals of two unrelated families. The mutation, identified using the polymerase chain reaction, causes a shift in the reading frame from Gly 117, where GGT (Gly)----GGAG (Gly+ 1 base) to a new stop codon created at position 129. This alteration is upstream of the active site (Ser 198), and, if any protein were made, it would represent only 22% of the mature enzyme found in normal serum. Results of analysis of the enzymatic activities in serum agreed with the genotypes inferred from the nucleotide sequence. Rocket immunoelectrophoresis using alpha-naphthyl acetate to detect enzymatic activity showed an absence of cross-reactive material, as expected. One additional individual with a silent phenotype did not show the same frameshift mutation. This was not unexpected, since there must be considerable molecular heterogeneity involved in causes for the silent cholinesterase phenotype. This is the first report of a molecular mechanism underlying the silent phenotype for serum cholinesterase. The analytical approach used was similar to the one we recently employed to identify the mutation that causes the atypical cholinesterase variant.
Asunto(s)
Colinesterasas/genética , Glicina , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , Colinesterasas/sangre , ADN/sangre , ADN/genética , Femenino , Humanos , Leucocitos/enzimología , Masculino , Datos de Secuencia Molecular , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , SuccinilcolinaRESUMEN
A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. A polymorphic site near the C terminus of the coded region was detected, but neither allele at this locus segregated consistently with the atypical trait. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for all 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of 32P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. We conclude that the Asp-70----Gly mutation (acidic to neutral amino acid substitution) accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool.