Anlisis de la expresin de marcadores asociados a

Análisis de la expresión de marcadores asociados a linfocitos T reguladores y su correlación con variables clinicopatológicas y pronósticas en carcinomas de la cavidad oral Iryna Shukotska, Rafael Sirera, Luz García Alonso, Antoni Pla, Gracia Sarrión, Sandra Gallach, Alfonso Berrocal, Andrea Cabrera, José Vivente Bagán, Carlos Camps Hospital General Universitario Valencia SPAIN

Tumor infiltrating lymphocytes Stage IV ovarian cancer Zhang L, et al, N Engl J Med 2003

Regulatory T Lymphocytes (Treg) CTLA-4, Foxp-3, CD 4, CXCR 4, CD 127 low, CD 25 (IL-2 R)high IL-10, TGF- Weinberg RA, Cancer Biology, 2007

Intratumoral Balance of Treg Is Associated With Prognosis of Hepatocellular Carcinoma after Resection n=302 patients tissue microarrays Gao Q, J Clin Oncol 2007

Quantification of Regulatory T Cells Enables the Identification of High-Risk Breast Cancer Patients overall survival relapse-free survival ER-positive tumors ER-negative tumors IHC, n=222 patients stratified by the median number of Treg Bates GJ, J Clin Oncol 2007

HLA-G Biology l l Human non-classical MHC molecule Expressed in l Trophoblast l Supresses activity § § l l NKs CTLs Maternal tolerance to gentically different tissues Tumor cells l part of the strategies to scape immunosurveyance

IHC Expression of HLA-G Correlates with Poor Prognosis in Gastric Carcinoma IHC, n=160 patients HLA-G negative (—) HLA-G positive (- - - -) Yie S, Ann Surg Oncol 2007

HLA-G Expression: a Prognostic Indicator for Patients with CCR IHC, n=201 patients HLA-G negative (—) HLA-G positive (- --) Ye S, Mod Pathol 2007

Our Cohort l l l l In 36 frozen tissue from resected oral cancer patients 16 were male; Median age 64, 0 y [34 -92] Patients presented squamous carcinomas of the tongue (n=21) or other localization (n=15) Stages from I to IV (stage I=4; II=9; III=4; IV=5); 6 p metastatic disease 17 patients presented well-differentiated lesions, and the other 12 moderately-poor differentiated cells. Eight patients received post-surgery chemo and radiotherapy. It was possible to determine the expression of genes in 92% of the samples.

Nuestro estudio tanslacional 36 Pacientes cáncer oral resecable Extracción del m. RNA del tejido tumoral y sano Evaluación de la calidad e integridad del m. RNA CTLA-4, Foxp-3, IL-10, TGF- , CD 4, CD 8, CXCR 4 (CD 127), CD 25 (IL-2 R) y HLA-G Relación con variables clínicas Cuantificación relativa de la expresión génica RTq. PCR Transcripción Reversa m. RNA c. DNA Taq. Man® Gene Expression Assay (Applied Biosystems) High-Capacity c. DNA Reverse Transcription kit® (Applied Biosystems)

TILs and OS p= 0. 044 p= 0. 307

Correlation with tumor area Area <median Mediana Mínimo Máximo >median Mediana Mínimo Máximo p CTLA-4 FOXP 3 CD 4 CD 25 2, 66359 3, 04232 , 82571 , 103 , 325 , 032 19, 837 10, 845 2, 259 , 83433 1, 04936 , 24973 , 273 , 543 , 055 5, 975 4, 152 , 893 5, 577 , 029 , 038 , 022 , 013 2, 60388 , 104 6, 660 , 87239 , 067 Fox. P 3 and metastatic disease M Median n SD 0 1, 97262 25 1, 777964 1 4, 23079 6 3, 393249 Mann-Whitney, p=0. 046

Fox. P 3 and TTP TGF- and OS p= 0. 023 p= 0. 014 95% CI Fox. P 3 median lower 95% CI upper <per 75 >per 75 8, 700 5, 876 11, 524 TGFB median <per 75 38, 000 >per 75 7, 500 lower 3, 907 upper 11, 093

Treg phenotype Foxp 3 & TGF- p= 0. 032 95% CI median Both high other 13, 800 NR lower 5, 582 upper 22, 018

Treg phenotype (CD 25 high+CD 127 low) p= 0. 018 95% CI CD 25 high+CD 127 low other median lower upper 6, 100 4, 066 14, 070 NR

HLA-G 48% expressed HLA-G p= 0. 046 95% CI median expressed Not expressed lower upper 12, 899 17, 101 NR 15, 000

Conclusions l We have been able to evaluate the presence of tumor infiltrating Treg cell populations in OSCC using RT-PCR l The expression of CD 4 (Th) but not CD 8 (CTL), is a marker of good prognosis l Gene expression of markers associated with Treg function are higher in smaller, well differentiated tumors and metastatic disease l We have found that several molecules associated with Treg function act as prognostic markers l l Those patients with higher expression of Foxp 3 and TGF-b have poorer prognosis in TTP and OS We have shown HLA-G expression in about half of the samples, and its good prognostic value