The SMAD4 gene is located on chromosome 18q21 and encodes the SMAD4 protein belonging to the SMAD family of transcription factor proteins, which act as mediators of TGF-β signal transduction. The TGF-β/SMAD4 signaling pathway controls signal transduction from cell membrane to nucleus, and is responsible for a wide range of cellular processes such as proliferation, differentiation, apoptosis, migration, as well as cancer initiation and progression. Alterations in the SMAD4 gene was primarily discovered in pancreatic cancer (duct adenocarcinoma) but occur in a variety of cancers such as colorectal cancer, gastric cancer, prostate cancer, melanomas, head and neck cancers and many others, though with higher frequencies in gastrointestinal tract cancers. Loss of SMAD4 expression in tumors has also been shown to affect cancer progression and therapy, such as reduced response to adjuvant chemotherapy. Tonsil is recommendable as external control for SMAD4: The vast majority of basal squamous epithelial cells in the surface epithelium must show a strong, predominantly nuclear staining reaction, whereas superficial squamous epithelial cells typically should be negative or only faintly demonstrated. Virtually all other cells e.g. lymphocytes must show an at least moderate, predominantly nuclear but also cytoplasmic staining reaction. In addition, a tumor (pancreas or colon) with loss of SMAD4 due to inactivation of the gene could be included, in which stromal cells (internal tissue control) should display the required staining reaction - at least moderate intensity. However, for SMAD4 it has to be emphasized that internal positive tissue controls being e.g. normal stromal cells adjacent to the neoplastic cells are preferred to external controls. An observed intact nuclear expression of SMAD4 proteins in the internal normal cells together with loss of SMAD4 proteins in the neoplastic cells is diagnostic essential.