Asunto(s)
COVID-19 , Mortalidad Hospitalaria , Hospitalización , Hospitales , Humanos , Pronóstico , Estudios Retrospectivos , SARS-CoV-2RESUMEN
Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR-based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%-91%) and a specificity of 88% (95% CI, 84%-92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas and 20 metastasizing primary melanomas revealed that DRTs had a marginal benefit as prognostic markers. DRTs have early potential to act as molecular biomarkers of melanoma on FFPE specimens pending validation, and DRTs may have applicability as prognostic markers in melanoma or other tumor types if new DRTs to relevant loci are developed.
Asunto(s)
Biomarcadores de Tumor/genética , Melanoma/diagnóstico , Neoplasias Cutáneas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Masculino , Melanoma/genética , Persona de Mediana Edad , Nevo/diagnóstico , Nevo/genética , Sensibilidad y Especificidad , Neoplasias Cutáneas/genética , Adulto JovenRESUMEN
A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.
Asunto(s)
Variaciones en el Número de Copia de ADN , Melanoma/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Sitios Genéticos , Humanos , Masculino , Melanoma/genética , Persona de Mediana Edad , Nevo/diagnóstico , Nevo/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
The purpose of this study was to evaluate whether paralogue ratio tests (PRT) using real-time PCR can accurately determine the DNA copy number (CN) using formalin fixed paraffin embedded (FFPE) tissue. Histopathology diagnostic archives are an enormous resource of FFPE tissue, but extracted DNA is of poor quality and may be unsuitable for CN assessment, thus representing a missed opportunity for studies of genetic association and somatic change in cancer in large cohorts of easily accessible samples. Assays with paralogues on chromosomes 18 and 20 (18|20 PRT) and chromosomes 13 and X (13|X PRT) were tested using archived FFPE pathology samples with known CN, including tonsil, placentae, and FFPE melanoma cell lines. The assay proved accurate over the dynamic range from 1:1 to 1:3 and gave precise results when repeated four times over several weeks. The precision of the assay was marginally reduced once the CT value for 10 ng of FFPE DNA increased above 30 cycles, reflecting importance of DNA quality. The assays distinguished changes in CN ratio with high sensitivity and specificity. The 13|X PRT could detect cells with distinct genotypes microdissected from within the same FFPE sample. Therefore, PRTs are suitable for analyzing CN in FFPE tissues.
