Carmen Ferrándiz García

p16INK4a Gene Alterations Are Frequent in Lesions of Mycosis Fungoides

Mycosis fungoides is usually an indolent disease that, after a variable period of time in a stable phase, evolves into a tumoral form with aggressive behavior. The molecular events that mark this progression remain essentially unknown to date, and this prompted us to investigate the hypothetical role of p16(INK4a) silencing in mycosis fungoides progression. We analyzed the three most frequent mechanisms of inactivation of the p16(INK4a) gene (deletion, promoter hypermethylation, and mutation) in nine cases with patch/plaque and tumoral lesions of mycosis fungoides. The existence of alterations was investigated by microsatellite analysis, methylation-specific polymerase chain reaction, and direct sequencing. Alterations of the p16(INK4a) gene were found in four of nine of the plaque lesions (hypermethylation in three samples and allelic loss in one sample) and seven of nine in the tumor lesions (hypermethylation in five samples and allelic loss in three samples). No case presented point mutations. Although a higher incidence of p16(INK4a) hypermethylation was observed in the cases that failed to achieve a complete remission, the limited size of our series prompted us to evaluate this finding cautiously. The results of this study therefore showed a common genetic alteration that is found more frequently in tumoral lesions than it is in plaque lesions of mycosis fungoides. It also offers data that could suggest a relationship between p16(INK4a) hypermethylation and unfavorable clinical outcome. Broader studies are needed to confirm both relationships.

p16 INK4a Is Selectively Silenced in the Tumoral Progression of Mycosis Fungoides

Knowledge about the molecular mechanisms involved in the pathogenesis of tumoral progression in mycosis fungoides (MF) is still scarce. Because the 9p21 locus seems to be a good target for a detailed study in MF, this prompted us to compare the mechanisms of inactivation of the p16INK4a, p15INK4b, and p14ARF genes in aggressive and stable forms of MF, performing microsatellite analysis, methylation-specific polymerase chain reaction, direct sequencing, and p16INK4a protein expression by immunohistochemistry. Additionally, the p53 gene was also sequenced in tumoral lesions. Thirty-nine patients with stable MF were studied. Alterations in p16INK4a and p15INK4b genes were detected in 18% and 5% of the cases, respectively. None of the cases analyzed showed alterations of the p14ARF gene. In contrast with these findings, in the 11 patients with aggressive MF, alterations of the p16INK4a, p15INK4b, or p14ARF genes were found in 8 (73%), 3 (27%), and 2 (18%) cases, respectively. A significant proportion (4/11) of these alterations were already present in the p16INK4a gene in the initial plaque lesions in these aggressive forms of MF. Alterations in the p16INK4a gene, either methylation or loss of heterozygosity, were clearly more frequent than those in the p15INK4b and p14ARF genes. These p16INK4A alterations were confirmed using immunohistochemistry. None of the nine tumoral lesions analyzed showed mutations in exons 1-2 of the p16INK4a gene or in exons 5-8 of the p53 gene. These results seem to suggest that 9p21 alterations, and selectively p16INK4a silencing, could be a characteristic phenomenon in MF progression.

Posible implicación de las alteraciones moleculares de la vía de TNF en la tumorigénesis de la micosis fungoide. Descripción de un posible chip de diagnóstico molecular en micosis fungoide

Resumen — Introduccion . El diagnostico de la micosis fungoide es dificil, sobre todo en las fases iniciales, debido a su similitud morfologica con las dermatosis inflamatorias y la baja proporcion de celulas tumorales en el tejido. Pacientes, material y metodos Se utilizaron muestras de 29 pacientes con micosis fungoide con lesiones de mancha, placa o tumor; de 24 pacientes con micosis fungoide en estadios Ia, Ib o IIa provenientes de un ensayo clinico; de 11 dermatosis inflamatorias y de piel sana no fotoexpuesta. Se analizaron empleando un chip de ADN-c construido en nuestro laboratorio. Resultados Este estudio ha demostrado que, empleando estudios de micromatrices de ADN-c y normalizacion con muestras normales de la piel, es posible detectar una firma molecular que distingue este tipo de tumor de condiciones inflamatorias comunes de la piel. Se ha revelado una firma de 27 genes implicados en la tumorigenesis de micosis fungoide. Esta firma incluye la desregulacion de senalizacion de factor de necrosis tumoral (TNF) ademas de un lazo autocrino de TNF que promueve senalizacion antiapoptotica. Ademas, fue posible identificar un modelo predictivo de solo 6 genes que permite una distincion entre casos de micosis fungoide y casos de dermatosis inflamatorias con gran precision. Este modelo predijo correctamente el diagnostico del 97,3% de los casos en la serie original y en 97,0 % de los casos en una serie de validacion de 24 pacientes con micosis fungoide con estadios bajos. Conclusiones Hemos encontrado un grupo de 27 genes posiblemente implicados en la tumorigenesis de la micosis fungoide. Hemos identificado un posible chip de diagnostico de 6 genes.