RESUMO
Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.
Assuntos
Aneuploidia , Técnicas de Sonda Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Feminino , Formaldeído , Humanos , Masculino , Melanoma/química , Melanoma/genética , Melanoma/secundário , Técnicas de Sonda Molecular/estatística & dados numéricos , Nevo/química , Nevo/genética , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Inclusão em Parafina , Sensibilidade e Especificidade , Neoplasias Cutâneas/química , Neoplasias Cutâneas/genética , Fixação de TecidosRESUMO
In 2013 the European Medicine Agency (EMA) restricted the indication for anti-EGFR targeted therapy to metastatic colorectal cancer (mCRC) with a wild-type RAS gene, increasing the need for reliable RAS mutation testing. We evaluated the completeness and reproducibility of RAS-testing in the Netherlands. From 17 laboratories, tumor DNA of the first 10 CRC cases tested in 2014 in routine clinical practice was re-tested by a reference laboratory using a custom next generation sequencing panel. In total, 171 CRC cases were re-evaluated for hotspot mutations in KRAS, NRAS and BRAF. Most laboratories had introduced complete RAS-testing (65%) and BRAF-testing (71%) by January 2014. The most employed method for all hotspot regions was Sanger sequencing (range 35.7 - 49.2%). The reference laboratory detected all mutations that had been found in the participating laboratories (n = 92), plus 10 additional mutations. This concerned three RAS and seven BRAF mutations that were missed due to incomplete testing of the participating laboratory. Overall, the concordance of tests performed by both the reference and participating laboratory was 100% (163/163; κ-static 1.0) for RAS and 100% (144/144; κ-static 1.0) for BRAF. Our study shows that RAS and BRAF mutations can be reproducibly assessed using a variety of testing methods.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , GTP Fosfo-Hidrolases/genética , Testes Genéticos/métodos , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Sequência de Bases , Cetuximab/uso terapêutico , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação/genética , Países Baixos , Panitumumabe , Análise de Sequência de DNA/métodosRESUMO
To test the diagnostic usefulness of allelic imbalance (AI) analysis based on routinely paraffin-embedded tissue, a series of 55 benign Spitz naevi, Spitz tumours with uncertain malignant potential, and malignant Spitzoid melanomas was investigated. Laser microdissection was used to ensure representative sampling of lesional cells and to investigate AI in separate tumour areas of four melanomas. AI was found in 2/12 (17%) typical Spitz naevi, 3/9 (33%) atypical Spitz tumours, 12/17 (65%) atypical Spitz tumours suspicious for melanoma and 15/17 (88%) Spitzoid melanomas. Additional immunohistochemical staining for Ki-67 using the MIB-1 antibody revealed positive deeply situated lesional cells in 0/6 (0%) Spitz naevi, 1/8 (13%) atypical Spitz tumours, 5/14 (35%) atypical Spitz tumours suspicious for melanoma, and 7/14 (50%) Spitzoid melanomas, respectively. Two of the melanomas examined for AI in separate tumour areas showed intratumoural genetic heterogeneity. In view of the finding of AI and deeply situated Ki-67 positive cells not only in melanomas but also in Spitz tumours with uncertain malignant potential, these approaches appear to have no direct diagnostic applicability for the distinction between benign and malignant Spitz tumours. Further molecular studies will be required to determine whether Spitz tumours and Spitzoid melanomas are unrelated entities, or whether there is a true spectrum of tumour progression.