Estudio del polimorfismo de los genes KIR y NKG2D y de sus ligandos HLA clase I y MICA en pacientes con melanoma

Abstract

Los resultados y conclusiones del presente trabajo son los siguientes:

1. El genotipo KIR2DL3+/C1+ se asocia a protección frente al desarrollo de melanoma y de metástasis en ganglio centinela en pacientes diagnosticados de MES y MN.

2. La ausencia de KIR2DL3 (KIR2DL2 en homocigosis) en pacientes portadores de ligandos C1 podría representar un factor de riesgo para el desarrollo de melanoma nodular y para la progresión a la ulceración de la lesión tumoral.

3. El genotipo KIR2DL1+2DS1-C2C2 podría ser considerado como un factor de riesgo para el desarrollo de melanoma y de metástasis en ganglio centinela en individuos diagnosticados de MES.

4. El aumento de clones de células NK KIR2DS1+ observado en pacientes de melanoma sugiere un papel importante de dicha población celular en la respuesta inmunitaria frente al melanoma cutáneo.

5. Los SNPs de la región NKC estudiados individualmente no parece presentar asociación con el desarrollo y/o pronóstico del melanoma cutáneo.

6. El haplotipo NK-3 del bloque hb-2 de la región génica NKC parece estar asociado con un mayor riesgo de desarrollo de melanoma cutáneo.

7. La expresión aumentada del receptor NKG2D en células NK y linfocitos T CD8+ observada en los pacientes con melanoma cutáneo parece indicar un estado de activación de las mismas.

8. El alelo MICA*009 parece estar asociado con un mayor riesgo de desarrollo de melanoma cutáneo. No obstante, se requieren estudios con series más amplias para confirmar esta asociación. 9. El dimorfismo en posición 80 del gen MICA no parece estar asociado con el desarrollo del melanoma cutáneo.

10. La variante MICA-129Met está asociada con un mayor nivel de MICA soluble en plasma y una peor supervivencia de los pacientes de melanoma cutáneo. OBJECTIVES 1. To study the gene polymorphism of KIR receptors and their HLA class I ligands in patients with melanoma and in a control population. 2. To analyze the subtypes of NK cells and CD8 + T lymphocytes that express KIR receptors in patients with melanoma and in a control population. 3. To study the gene polymorphism of the NKG2D receptor and its MICA ligands in patients with melanoma and in a control population. 4. To analyze the expression of the NKG2D receptor in NK cells and CD8 + T lymphocytes of patients with melanoma and a control population. 5. To study the level of soluble MICA in plasma samples from melanoma patients at diagnosis and from a control population.

METHODOLOGY The KIR, HLA-A, B, C, and MICA genes typing was performed by SSO-PCR, reverse-SSO-PCR and SSP-PCR techniques, and the NKC gene region typing by an allelic discrimination assay with Taqman probes and by Sanger sequencing. These trials were carried out in 233 patients diagnosed with cutaneous melanoma and 200 healthy controls. The flow cytometry study to evaluate the expression of KIR receptors in NK cells and peripheral blood CD8 + T lymphocytes was performed in 35 patients diagnosed with melanoma and 24 healthy individuals, while the expression of NKG2D was evaluated in 48 patients with melanoma. and 37 healthy individuals. The determination of soluble MICA protein was carried out using a "sandwich" ELISA technique in the plasma of 30 patients diagnosed with cutaneous melanoma and 26 healthy individuals.

RESULTS AND CONCLUSIONS The results and conclusions of the present work were the following:

1. The KIR2DL3+/C1+ genotype is associated with protection against the development of melanoma and sentinel lymph node metastasis in patients diagnosed with SSM and NM.

2. The absence of KIR2DL3 (KIR2DL2 in homozygosis) in patients carrying C1 ligands could represent a risk factor for the development of nodular melanoma and for the progression to the ulceration of the tumor lesion.

3. The KIR2DL1+2DS1-C2C2 genotype could be considered as a risk factor for the development of melanoma and sentinel lymph node metastasis in individuals diagnosed with SSM.

4. The increase of KIR2DS1+ NK cell clones observed in melanoma patients suggests an important role of this cell population in the immune response against the cutaneous melanoma.

5. The NKC region SNPs studied individually does not seem to be associated with the development and/or prognosis of cutaneous melanoma.

6. The NK-3 haplotype of the hb-2 block of the NKC gene region seems to be associated with an increased risk of cutaneous melanoma development.

7. The increased expression of NKG2D receptor observed in NK cells and CD8+ T lymphocytes from patients with cutaneous melanoma seems to indicate an activation state of them.

8. The MICA*009 allele seems to be associated with an increased risk of cutaneous melanoma development. However, studies with larger series are needed to confirm this association. 9. The position 80 MICA gene dimorphism does not seem to be associated with the development of cutaneous melanoma.

10. The MICA-129Met variant is associated with a higher level of soluble MICA in plasma and a worse survival of cutaneous melanoma patients.