RESUMEN
BACKGROUND: Sanger sequencing is one of several reliable methods in use to detect KRAS and BRAF mutations to facilitate clinical patient selection for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapy in unresectable metastatic colorectal adenocarcinoma (CRC). Most analyses are made on pretreatment biopsy or resection specimens. There is a scarcity of published studies on the suitability of cytological samples for KRAS testing in this setting. METHODS: DNA extraction was attempted on 11 search-retrieved paired cases of histological resections or excisions of CRC and their corresponding cytological samples (representing metastases) and tested for KRAS mutations in exon 2 and 3, as well as BRAF exon 15 mutations by Sanger sequencing. Only KRAS wild-type cases were subjected to BRAF analysis because this is the setting with true diagnostic value, as these mutations are mutually exclusive. RESULTS: Of the 11 paired cases analysed, only eight histology cases showed satisfactory DNA quality for sequencing. Thus, only eight of the corresponding cytology cases were analysed. Seven of the eight cases tested showed the same KRAS genotype on both the aspirated cytology specimen of metastatic carcinoma and the primary tumour (histological specimen), from which we derive an overall concordance rate of 87.5%. The single discordant case was likely to be a true difference as it was demonstrated again on repeat testing of both samples. No BRAF mutations were detected on the four KRAS wild-type cases. CONCLUSION: A range of cytological samples are suitable for KRAS and BRAF mutation testing, be it from previously stained preparations or cell blocks. These samples would be highly valuable in cases where cytological samples are the only material available for mutation testing.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Análisis Citogenético/métodos , Análisis Mutacional de ADN/métodos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Adenocarcinoma/patología , Adenocarcinoma/secundario , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Biopsia con Aguja Fina/métodos , Cetuximab , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/secundario , Receptores ErbB/genética , Receptores ErbB/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
OBJECTIVE: Studies have shown that c-kit mutation analysis of gastrointestinal stromal tumours (GISTs) obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) can be routinely performed. We validated c-kit exon 11 mutational analysis on cell block material obtained from fine needle aspiration cytology (FNAC) for diagnostic purposes and compared it with the same analysis in formalin-fixed paraffin-embedded full sections of the corresponding resection specimens. METHODS: c-kit mutation analysis was done on cell block material obtained from ten cases encountered in our department from 1999 to 2008 on which FNAC was attempted pre-operatively. The findings were compared with analysis on full paraffin section of the corresponding resected tumours in seven cases where patients opted for resection. c-kit exon 11 was examined via bidirectional nucleic acid sequencing. RESULTS: Our results showed 100% concordance for the presence and type of exon 11 mutation in the resected and aspirated tumours in all seven cases. These mutations had diagnostic value when compared with other neoplasms that are part of the cytomorphological differential diagnosis, such as leiomyosarcoma or gastric adenocarcinomas. CONCLUSION: Molecular cytopathology is a powerful tool that can complement morphology and immunohistochemical assessment of cytological material in routine practice for the diagnosis and prognostication of GISTs. We briefly discuss the advantages and limitations of the fine needle method of obtaining tissue for the diagnosis and prognostication of GISTs, and its current therapeutic strategies.
Asunto(s)
Análisis Mutacional de ADN , Exones/genética , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Proteínas Proto-Oncogénicas c-kit/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Biomarcadores de Tumor/genética , Biopsia con Aguja Fina , Femenino , Tumores del Estroma Gastrointestinal/diagnóstico , Tumores del Estroma Gastrointestinal/cirugía , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Reproducibilidad de los ResultadosRESUMEN
Peste des petits ruminants (PPR) is a viral disease of goats and sheep characterized by erosive stomatitis, enteritis, and pneumonia. The virus is a member of the family Paramyxoviridae and the genus Morbillivirus. The disease has high morbidity and mortality rates and has a substantial economic impact in developing countries. We have cloned and sequenced the cDNA of the nucleocapsid (N) gene of the Nigeria 75/1 strain of PPR virus (PPRV). A comparison of its nucleotide and deduced amino acid sequence with those of the N gene of the tissue culture-attenuated strain of PPRV was performed. A divergence of 8.9 and 5.0% was found at the nucleotide and amino acid level, respectively. A recombinant baculovirus that expresses the N protein in insect cells and larvae (Spodoptera frugiperda) was generated. The recombinant protein, characterized by Western blot analysis, was shown to have a molecular weight of 58 kDa and was recognized by anti-PPRV antibodies. The recombinant protein was used successfully as a coating antigen in an enzyme-linked immunosorbent assay for the serological diagnosis of PPRV.