RESUMEN
Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.
Asunto(s)
Cromograninas/genética , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico/métodos , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Quísticas, Mucinosas y Serosas/genética , Quiste Pancreático/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Anciano , Anciano de 80 o más Años , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico/normas , Femenino , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Quísticas, Mucinosas y Serosas/diagnóstico por imagen , Neoplasias Quísticas, Mucinosas y Serosas/patología , Quiste Pancreático/diagnóstico por imagen , Quiste Pancreático/patología , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/patología , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normasRESUMEN
B-cell chronic lymphocytic leukemia (B-CLL) consists of at least 2 subtypes with either somatically mutated or unmutated immunoglobulin heavy-chain variable region (IgVH) genes. A prognostic significance of the infiltration pattern in bone marrow trephine biopsy has been described before. The combined pattern analysis and detection of 70-kd zeta-associated protein (ZAP-70) expression in formalin-fixed, paraffin-embedded bone marrow trephines has not been investigated so far. To evaluate the relationship between ZAP-70 expression, mutation status, and the infiltration pattern in B-CLL, we analyzed bone marrow trephine biopsies from B-CLL patients (n = 35). The expression of ZAP-70 was related to the infiltration type: in all samples with diffuse infiltration pattern, the leukemic cells showed ZAP-70 staining, whereas leukemic cells in a nodular infiltration pattern were negative. By contrast, the mixed-pattern type showed a variable ZAP-70 expression. Besides definitely negative or positive ZAP-70 expression, a few samples showed a faint ZAP-70 staining, and the classification into the positive or negative group was difficult. In addition, the infiltration type was related to the mutation status in a subset of samples: mutation of the IgVH gene was restricted to the nondiffuse infiltration pattern and was not found in cases with diffuse infiltration of bone marrow. The expression of ZAP-70, detected by an immunohistochemical assay and also by real-time quantitative reverse transcriptase-polymerase chain reaction assigned 83% of the chronic lymphocytic leukemia cases to the suspected immunoglobulin mutation subtype. In 2 patients with ZAP-70 expression, a mutated IgVH status was found. These cases exhibited a mixed pattern of infiltration. We conclude that the pure nodular type of marrow infiltration in B-CLL is associated with IgH hypermutation and ZAP-70 negativity, whereas the predominantly diffuse type of infiltration reveals unmutated IgH genes with ZAP-70 overexpression. The mixed type of infiltration is displayed by mutated as well as unmutated cases with a varying pattern of ZAP-70 expression.