Biomarkers of TGF-β signaling pathway and prognosis of pancreatic cancer

Abstract

Background: Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

Method: We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

Results: The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

Conclusion: Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.

Figure 1. Typical Immunohistochemical staining pattern for TGF-βR2 and SMAD4 in pancreatic adenocarcinoma tumor tissues.

A: Positive nuclear expression of TGF-βR2 in a moderately differentiated ductal adenocarcinoma of the pancreas (Magnification: 10×40). B: Positive nuclear expression of SMAD4 in moderately differentiated ductal adenocarcinoma of the pancreas (Magnification: 10×40).

Figure 2. Overall survival of patients with various nuclear expression levels of TGF-βR2 and SMAD4.

Nuclear staining score 0–1 is defined as low for TGF-βR2 and a score 0 as low for SMAD4. Red line: TGF-βR2 is high and SMAD4 is low (HL); black line: both high (HH); green line: both are low (LL); purple line: TGF-βR2 is low and SMAD4 is high (LH). The median survival times are 7.8, 8.6, 11.3, and 15.6 months for the HL, HH, LL and LH groups, respectively. Log-rank test P values and results of Cox regression analysis are presented in Table 2. Thus, TGFβ-R2/SMAD4 ratio may be prognostic, with low values corresponding with an improved survival.

Figure 3. Plot of overall survival curve in patients with all patients (A), patients with localized (B), locally advanced (C) or metastatic tumors by SMAD4 SNP rs2704733 genotype.

Blue line: AA genotype; green line: AG/GG genotype. AA genotype was associated with an improved survival in the entire study population. On subgroup analysis, this survival difference was more relevant for advanced disease stage. P values by log-rank test are <0.0001, 0.369, 0.007, and 0.031 for panels A, B, C and D, respectively.