Abstract #4458: Distinct clinical implications of allelic variations and aberrant expression of SMAD4 in colorectal cancer
Authors: Xu Jia, Venkat R Katkoor, Chandrakumar Shanmugam, Tom Callen, Ludwine Messiaen, Nirag C Jhala, James Posey, Upender Manne
Journal: Cancer Research (2009)

Background: SMAD4 is a candidate tumor suppressor gene, located at the 18q21.1 locus and mediates the transforming growth factor-\#946; (TGF-\#946;) signaling pathway. High frequency of SMAD4 allelic imbalance was reported in pancreatic and colorectal adenocarcinomas (CRCs). The current study has evaluated the prognostic value of its allelic imbalance and its phenotypic expression patterns in Stage II and Stage III CRCs. Materials & Methods: We retrieved 74 Stage II and 64 Stage III formalin-fixed paraffin-embedded archival tissues of CRCs and their corresponding normal (benign) tissues. The genomic DNA and RNA samples were extracted from these tissues. Genomic DNA samples were analyzed for the loss of heterozygosity (LOH) at 18q21 locus by assessing three microsatellite markers (D18S46, D18S363 & D18S474) which are close to or spanning the SMAD4 gene. The mRNA expression levels of SMAD4 were estimated by the real-time PCR method. All 138 tissues sections were analyzed for immunohistochemical (IHC) expression of SMAD4 using anti-human SMAD4 monoclonal antibody (Clone B-8; dilution 1:100; Santa Cruz). A tumor was considered as LOH when it demonstrates allelic loss in at least one of the three loci analyzed. Nuclear or Cytoplasmic staining pattern was noted based on its cellular localization. The status of LOH and the IHC staining patterns were correlated with patient overall survival using Kaplan-Meier method. Results: The LOH at any one of the three markers was detected in 48 Stage II (of 71 informative cases, 67%) and 33 Stage III (of 59 informative cases, 56%) CRCs. The CRCs which exhibited LOH had lower levels of mRNA (2.8 folds) when compared to CRCs without LOH. In Stage II cases, LOH was associated with short overall patient survival (log rank, p=0.017), but such an associated was not observed in patients with Stage III CRCs (log rank, p=0.518). Strong nuclear and cytoplasmic staining was noted in normal epithelial tissues. In both stage groups, LOH status or the mRNA levels were not correlated with the cellular localization of SMAD4. Although a similar frequency of decreased nuclear (28% & 37%) and cytoplasmic (19% vs. 25%) staining was observed in Stage II and Stage III CRCs, only decreased nuclear expression of SMAD4 was significantly associated with short survival of Stage III patients (log rank, p=0.025) but not in Stage II patients (log rank, p=0.931). Conclusions: These preliminary studies suggest that LOH of SMAD4 was a bad prognostic indicator of patients with Stage II CRCs and Stage II CRCs with SMAD4 allelic loss are behaving like Stage III tumors. It was interesting to note that lack of or decreased expression of nuclear SMAD4 was associated with short overall survival of patients with Stage IIII CRCs. These studies are supported by NCI/NIH grants, CA098932-01 & 2U54-CA118948.       Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4458.