Non-invasive detection of c-myc p64, c-myc p67 and c-erbb-2 in colorectal cancer.

OBJECTIVE: Colorectal cancer is a major cause of cancer mortality in the industrialized nations in the West. Because mortality is closely related to the stage of the disease at the time of diagnosis, detection at an early stage is likely to result in improved recovery rates. Since current diagnostic procedures such as colonoscopy are invasive and the fecal occult blood test (FOBT) lacks sensitivity and specificity for the detection of early lesions, the development of non-invasive methods based on molecular markers of neoplasia can lead to earlier diagnosis and more favorable outcomes for patients with colorectal cancer. Recent advances in the technology for isolating colonocytes from stool (SCSR (somatic cell sampling and recovery)) provide a non-invasive tool for the study of biomarkers expressed in colorectal cancer. The aim of this study was to detect mRNA expression of three biomarkers: (c-erbb-2 and two forms of c-myc: p64 and p67) in fecal colonocytes and to evaluate its use in diagnosing colorectal cancer. MATERIAL AND METHODS: Colonocytes (SCSR cells) were isolated from stools from 30 subjects: 15 colorectal cancer patients and 15 normal controls. One cancer patient was excluded from the final data analysis because the tumor was a gastrointestinal lymphoma. Each sample yielded two fractions: a pellet and an interphase. Expression of c-myc p64, c-myc p67 and c-erbb-2 mRNA was evaluated in each of the fractions by reverse transcriptase-polymerase chain reaction (RT-PCR). A marker was considered positive upon detecting an amplicon of the expected size in agarose gel electrophoresis. RESULTS: c-myc p64 mRNA expression was observed in both fractions in 78.5% of colorectal cancer patients, compared with 13.3% in the control group (p=0.009). For c-myc p67, 78.6% of the colorectal cancer patients showed mRNA expression in both fractions in comparison with only 13.3% of the controls (p=0.003). C-erbb-2 showed no significant difference in mRNA expression between colorectal cancer and controls. When the data were analyzed for co-expression of c-myc p64 and c-myc p67, in both pellet and interphase, sensitivity was 64% and specificity was 100%. CONCLUSIONS: Fecal colonocytes isolated by somatic cell sampling and recovery (SCSR) technology could be used for the non-invasive assessment of the expression of biomarkers of colon cancer such as c-myc p64, c-myc p67 and c-erbb-2. The expression of c-myc p64 and c-myc p67 in colonocytes showed a significant association with colorectal cancer and may be helpful as a biomarker for the non-invasive detection of colorectal cancer.

Raf-1 activation suppresses neuroendocrine marker and hormone levels in human gastrointestinal carcinoid cells.

Gastrointestinal carcinoid cells secrete multiple neuroendocrine markers and hormones including 5-HT and chromogranin A. The intracellular signaling pathways that regulate production of bioactive molecules are not completely understood. Our aim was to determine whether activation of the raf-1/MEK/MAPK signal transduction pathway in carcinoid cells could modulate production of neuroendocrine markers and hormones. Human pancreatic carcinoid cells (BON) were stably transduced with an estrogen-inducible raf-1 construct creating BON-raf cells. Activation of raf-1 in BON-raf cells led to a marked induction of phosphorylated MEK and ERK1/2 within 48 h. Importantly, raf-1 activation resulted in morphological changes accompanied by a marked decrease in neuroendocrine secretory granules by electronmicroscopy. Moreover, induction of raf-1 in BON-raf cells led to significant reductions in 5-HT, chromogranin A, and synaptophysin levels. Furthermore, treatment of BON-raf cells with MEK inhibitors PD-98059 and U-0126 blocked raf-1-mediated morphological changes and hormone suppression but not ERK1/2 phosphorylation. These results show that raf-1 induction suppresses neuroendocrine marker and hormone production in human gastrointestinal carcinoid cells via a pathway dependent on MEK activation.

Coprocytobiology: on the nature of cellular elements from stools in the pathophysiology of colonic disease.

The gastrointestinal epithelium is known to undergo constant and rapid renewal resulting in millions of cells being shed into the fecal stream every day. The conventional wisdom was that these cells disintegrate upon exfoliation and will not survive the transit through the intestinal tract. In 1990, we (P.N.) made the discovery that a significant number of these cells remain intact and viable and that they can be isolated. The implications of this important discovery became apparent when we demonstrated that these cells are exclusively of colonic origin, are anatomically representative of the entire colon, and can be used for clinical investigations of disease processes. The term coprocytobiology (CCB) was coined to encompass the broad range of applications of this new technology. The somatic cell sampling and recovery (SCSR) process involves the isolation of exfoliated colonocytes from a small sample of stool ( approximately 1 g) collected and transported in a unique medium at ambient temperature, providing cells for the detection of a number of biomarkers of disease propensity. These exfoliated colonocytes express cytokeratins indicating epithelial lineage as well as colon-specific antigen. Over the years, the study of exfoliated colonocytes has provided striking new insights into the biology of colon cancer and inflammatory bowel disease, including detection of p53 gene mutations, reverse transcriptase polymerase chain reaction amplification, and identification of CD44 splice variants, neoplasia-associated specific binding of plant lectins, and expression of COX-2, the inducible form of cyclooxygenase. The functional diversity of cells isolated by SCSR is revealed by the demonstration of cell surface markers such as secretory component, IgA, and IgG on the one hand and the amplification and cloning of the human insulin receptor and the expression of the multidrug resistance gene mdr-1 on the other hand. This review portrays the immense potential of CCB as a powerful tool for investigating the pathophysiology of disease, identifying genetic variants in pharmacogenetics, assessment of mucosal immunity, and several other applications that use somatic cells.