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C. Hernando



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    MO16 - Prognostic and Predictive Biomarkers IV (ID 97)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO16.02 - Tumor and Stroma Treg markers in resectable NSCLC (ID 2753)

      16:20 - 16:25  |  Author(s): C. Hernando

      • Abstract
      • Presentation
      • Slides

      Background
      Immunosuppressive regulatory T lymphocytes (Tregs) have been proved to play a critical role in immune tolerance to tumor. In this study we have analyzed several markers related to Tregs, in both tumor and stroma areas in patients with resectable NSCLC.

      Methods
      Tumor FFPE samples from 135 early-stage NSCLC patients were used in this retrospective study. The most representative areas of tumor cells and tumor stroma of each sample were carefully micro-dissected. RTqPCR using hydrolysis probes was performed to determine the expression of Treg markers such as: CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNF-a as well as CD4 and CD8. Relative gene expression was assessed using GAPDH and CDKN1B as endogenous controls and results were normalized against a human cDNA as a reference. FOXP3 protein expression was assessed by immunohistochemistry, in 80 of the 135 patients included in this study. The absolute number of FOXP3-positive lymphocytes was determined in both tumor and stroma areas by averaging the cell counts in 10 fields (400X). All statistical analyses were considered significant at p< 0.05.

      Results
      Gene expression analyses revealed an over-expression of CD25 (5.40X and 7.95X, respectively) and down-expression of CD127 (0.28X and 0.37X, respectively) in both, tumor and stroma. There was a tendency toward higher expression of FOXP3 (1.67X and 2.01X, respectively) and CTLA-4 (1.92X and 1.76X, respectively) as well. Paired Wilcoxon test showed significant gene expression differences between tumor and stroma in FOXP3 (p=0.006), CD25 (p<0.0001), CD4 (p<0.0001), CD8 (p=0.028), IL-10 (p<0.0001) and TGFB-1 (p<0.0001). Survival analyses revealed that patients with a “Treg profile” (↑CD25/↓CD127) had a reduced overall survival (OS), whilst those patients with higher levels of the ratio FOXP3 stroma/tumor had worse time to progression (TTP) (Table 1). Spearman test revealed a significant association between stromal FOXP3 expression levels and the number of FOXP3-positive lymphocytes (by IHC) in stroma, p=0.006. Moreover, chi-square test showed that patients with squamous cell carcinoma histology presented a higher number of FOXP3-positive lymphocytes than those patients with adenocarcinoma, p= 0.035. Table 1: OS for “Treg profile” and TTP for Ratio FOXP3 Stroma/Tumor

      OS
      Median (months) 95% CI p
      Others 74.33 65.96 - 82.69 0.003
      "Treg profile" 29.90 4.91 - 6.54
      TTP
      Median (months) 95% CI p
      ↓ Ratio FOXP3 Stroma/Tumor NR -- 0.040
      ↑ Ratio FOXP3 Stroma/Tumor 32.50 16.25- 48.74

      Conclusion
      Gene expression of Treg markers in tumor microenvironment seem to play an important prognostic role in early-stage NSCLC patients. Furthermore, preliminary IHC analysis indicated a correlation between mRNA and protein levels for FOXP3 in NSCLC patients. Supported in part, by grants PS09/01149, RD06/0020/1024 and RD12/0036/0025 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III (ISCIII).

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-030 - KRAS mutations in resectable NSCLC patients. Prognostic implications. (ID 2273)

      09:30 - 09:30  |  Author(s): C. Hernando

      • Abstract

      Background
      Development of Non-Small Cell Lung Cancer (NSCLC) requires multiple genetic and epigenetic alterations, with some differences according to etiology and histology. The most frequently mutated genes in these tumors are EGFR and KRAS (present mostly in adenocarcinomas), however, the prognostic value of KRAS mutations in NSCLC is still controversial.

      Methods
      Fresh tumor tissue samples (n=150) were obtained from resectable NSCLC patients. DNA was extracted by standard methods based in TriZol® and analyzed for KRAS mutational status by RTqPCR with ARMS technology and Scorpions probes. Non-parametric methods were used fos statistical analysis. Progression free survival (PFS) and overall survival (OS) were evaluated by Kaplan-Meier method (log-rank test). A p value ≤ 0.05 was considered statistically significant.

      Results
      Baseline characteristics of the patients were: median age, 64 years [26-82]; 86.0% male; 71.3% ECOG-PS 0; 40% adenocarcinomas (ADC). KRAS mutations were detected in 10.7% of the tumors (n= 150). Table 1 summarizes the mutations found in our cohort. In the subgroup of ADC + ADC-SCC samples, mutant KRAS represents 20% of the tumors. Considering only the never-smoker group of patients, 31.6% of the samples were mutated for KRAS. Our results showed that patients with KRAS mutated tumors had significantly shorter PFS than patients with wild type KRAS (11.633 vs 45.833 months, respectively, p= 0.043) and a trend to a shorter OS (23.067 vs 66.967 months, respectively, p= 0.074). Table 1: Distribution of KRAS mutations in our cohort

      n %
      Wild Type 134 89.3
      12SER 1 0.7
      12CYS 5 3.3
      12ASP 7 4.7
      12VAL 3 2.0
      TOTAL 150 100.0

      Conclusion
      KRAS gene mutation is a poor prognostic factor for PFS in our cohort of resectable NSCLC; therefore, the determination of the mutational status of KRAS gene might be implemented routinely in clinical practice. This work was supported in part, by a grant [RD06/0020/1024 and RD12/0036/0025] from Red Temática de Investigación Cooperativa en Cáncer, RTICC, and Instituto de Salud Carlos III (ISCIII).