Studies On β-Catenin Mutation And Aberrant Expression In Sporadic Colorectal Cancers And Raman Spectra Of Single Optically Trapped Sporadic Colorectal Cancer Cells

Objective:(1)To observe β -Catenin gene aberrant expression and mutation in sporadic colorectal cancers(SCRC) and explore its clinicopathological correlation. (2)To detect microsatellite instability (MSI) status of SCRCs and elucidate the relationship between MSI status and β-Catenin gene mutation. (3) Quantitative detection of β-Catenin mRNA in SCRCs for analyzing its role on β -Catenin aberrant expression and clinicopathological significance. (4) Measuring Raman spectra of single SCRC cells in order to establish diagnostic model for identifying SCRC cells and analyze biochemical changes.Methods: (1) Immunohistochemical study for β -Catenin aberrant expression was carried out in 90 SCRC samples and 22 sporadic colorectal adenoma samples. (2) PCR amplification and direct DNA sequencing were used to identify β-Catenin mutation in the same SCRC and sporadic colorectal adenoma samples stated above. (3) PCR amplification and capillary electrophoresis were employed to detect Bat25 and Bat26 microsatellite loci. (4) The concentration of β -Catenin mRNA in 81 SCRC samples and 28 adjacent normal colorectal mucosa was determined using TaqMan real-time quantitative PCR. (5) Single-cell Raman spectra of SCRC cells from 10 patients were obtained by using laser tweezers Raman spectroscopy (LTRS) and analyzed by principal component analysis and logistic regression to establish a diagnosis algorithm.Results: (1) In normal colorectal mucosa, β-Catenin was detected in the cell membranes of epithelial cells and was not detected in the cytoplasm and nuclei. In SCRCs and sporadic colorectal adenomas , β -Catenin stainwas found in the cytoplasm and nuclei and membrane stain was reduced with different extent. The cytoplasmic and nuclear aberrant expression rate of β-Catenin was higher in SCRC(88. 9% and 41.1% respectively) than that in sporadic adenomas(59. 1% and 18. 2% respectively) ( P<0. 05) . Theβ -Catenin cytoplasmic aberrant expression rate in ulcerative and infiltrating type, well and moderately differentiated SCRCs was significantly higher than that in polypoid type, poorly differentiated SCRCs respectively. The nuclear aberrant expression rate in ulcerative and infiltrating type of SCRC(50. 0%) was higher than that in polypoid type of SCRC(26. 5%) ( P<0. 05) and in the invasive front of SCRCs the nuclear aberrant expression was frequently observed. (2) β-Catenin gene mutation was identified in one out of 90 SCRCs, whereas none of 22 sporadic colorectal adenomas was identified with β-Catenin gene mutation. The only mutation was at condon 41 (ACC→GCC), changing a threonine to alanine.(3)8 out of 90 SCRC cases showed high microsatellite instability(MSI-H), one of which had β-Catenin gene mutation, whereas there was no β -Catenin gene mutation detected in SCRC samples with microsatellite stability(MSS). ( 4 ) The β-Catenin mRNA level in SCRCs (2. 5267 ± 2. 2839) was lower than that in adjacent normal colorectal mucosa(5. 0029 ± 3. 3264), P < 0. 05. In addition, The β-Catenin mRNA level in lymph node-positive cases and ulcerative and infiltrating growth types was significantly lower (1.8266+1.2883, 2.2023 ± 2.0351) , when compared with that in lymph node-negative cases and polypoid growth type respectively (3. 3592 ± 2. 8805, 3.1083 ± 2.6103), P < 0. 05. No significant difference of β-Catenin mRNA level was found between β -Catenin cytoplasmic or nuclear aberrant expression cases and those without β -Catenin cytoplasmic or nuclear aberrant accumulation. (5) A diagnosis algorithm was established using principal component analysis and logistic regression, based on a calibration set comprising of 320 Raman spectra from 8 patients. It was further validated by Raman spectra data from 2 new patients with sensitivity and specificity 82.5% and 92.5%, respectively. Spectra analysis showed that the level of nucleic acid and protein in SCRC cells was higher than in normal cells, whereas the level of lipids was lower in SCRC cells.Conclusions:1. In SCRCs, the cytoplasmic and nuclear aberrant expression rate of β -Catenin was 88. 9% and 41. 1% respectively and the aberrant expression rate was only related to tumourigenic stage, growth type and differentiation of SCRCs. The nuclear aberrant expression was frequently observed in the invasive front of SCRCs, which might be related to invasive behavior of SCRC.2. In SCRCs, the β-Catenin mutation is rare, suggesting that β -Catenin mutation is not the main cause of β -Catenin cytoplasmic and nuclear aberrant expression. β-Catenin mutation may be prone to occur in SCRC samples with MSI-H phenotype.3. In SCRCs, the β-Catenin mRNA level was significantly lower than that in adjacent normal colorectal mucosa, which perhaps results from negative feedback mechanism of biologic cells. The β -Catenin mRNA level in SCRCs was not in association with β-Catenin cytoplasmic and nuclear aberrant expression.4. The decreased β-Catenin mRNA level was significantly associated with the presence of lymph node metastases and ulcerative and infiltrating growth types, indicating that the decreased β-Catenin mRNA level may regard as one of markers for evaluating the biological behaviors of SCRCs. 5.Single cell Raman spectra diagnosis algorithm can be used to distinguish SCRC cells with normal epithelial cells sensitively, with potential application in the clinical diagnosis of epithelial cancers . In addition , single cell Raman spectra can provide a wealth of biochemical information about SCRC cells.6 . Laser tweezers Raman spectroscopy (LTRS) is a nondestructive, reagentless and objective method, deserving generalization in cytologic diagnosis and research.