RESUMO
KRAS genotyping is mandatory before anti-epidermal growth factor receptor monoclonal antibody therapy in metastatic colorectal cancer, which is the second leading cause of cancer-related death in the United States and in Europe. Thus, large-scale KRAS mutation screening is needed for efficient patient management and in the future metastatic colorectal cancer genotyping might also include the detection of the BRAF V600E mutation, which is a very strong negative prognostic factor in colorectal cancer. We report our experience of routine KRAS/BRAF mutation screening practice performed on 1130 formalin-fixed paraffin-embedded tumor samples from 992 colorectal cancer patients. DNA was extracted from macrodissected tumor areas highlighted by a pathologist, KRAS codons 12/13 and BRAF V600E mutations were assessed in a single SNaPshot® multiplex assay and each mutation was confirmed by an independent analysis. KRAS and BRAF mutations were, respectively, present in 41.8 and 6.5% of the tumor samples. If KRAS and BRAF mutations were mutually exclusive, four samples presented two concomitant KRAS mutations. Genotyping of paired primary tumors and metastases from 44 patients indicated that 5 patients (11.4%) presented discordant KRAS mutational status. KRAS genotype heterogeneity was also observed within primary tumor sites in seven cases. Non-reproducible KRAS artefactual mutations were detected in 53 samples (4.7%). We found that the prominent mechanism leading to these artefactual mutations was the fragmentation of DNA occurring during tissue processing. Routine KRAS genotyping performed on formalin-fixed paraffin-embedded tissues requires, therefore, the development of quality control scheme for molecular pathology, especially because of DNA damages induced by formalin fixation. The tumor heterogeneity observed in some patients indicates that it should be more appropriate to perform KRAS genotyping on metastases if sample is available.
Assuntos
Artefatos , Neoplasias Colorretais/genética , Técnicas de Genotipagem , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA/métodos , DNA de Neoplasias/isolamento & purificação , Formaldeído , Genótipo , Humanos , Metástase Neoplásica , Inclusão em Parafina , Mutação Puntual , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Fixação de TecidosRESUMO
SUMMARY: We describe a case of oligodendroglioma arising in an ovarian mature cystic teratoma associated with a loss of heterozygosity on the long arm of chromosomes 19 and 10. To our knowledge it is the second case reported in the literature at this site and the first one described in association with a characterized genetic alteration. The patient was 29 years old and presented with a history of 4 months of pelvic pain. Ultrasound examination showed a cystic mass arising in the left adnexa suggesting a teratoma. At laparotomy a cystic ovoid mass was found arising from the left adnexa, completely replacing the ovary. An ovariectomy was performed. Macroscopically a multilocular cyst containing hair, sebum, and a relatively well-defined solid zone of grayish-pink color strongly suggestive of a cerebral tissue, was observed. Microscopic analyses confirmed the teratomatous nature of the cyst. The solid area was composed of mature glial tissue in which was observed a proliferation of monotonous cells with round and homogenous nuclei, surrounded by a clear halo of cytoplasm ("honeycomb appearance") which immunohistochemically showed positivity for glial fibrilar acidic protein and for neurofilament protein. Ki-67 labeling index was about 3%. These findings were consistent with a low-grade oligodendroglioma arising in a mature ovarian cystic teratoma. Reverse transcription-polymerase chain reaction analysis showed a characterized loss of heterozygosity occurring in tumor DNA on chromosomes 10q and 19q13.