Gene-expression analysis of single cells-nested polymerase chain reaction after laser microdissection

AIM: The structural and functional characteristics of cells are dependent on the specific gene expression profile. The ability to study and compare gene expression at the cellular level will therefore provide valuable insights into cell physiology and pathophysiology.METHODS: Individual cells were isolated from frozen colon tissue sections using laser microdissection. DNA as well as RNA were extracted, and total RNA was reversely transcribed to complementary DNA (cDNA). Both DNA and cDNA were analyzed by nested polymerase chain reaction (PCR). The quality of isolated DNA and RNA was satisfactory.RESULTS: Single cells were successfully microdissected using an ultraviolet laser micromanipulator. Nested PCR amplification products of DNA and cDNA of single cells could clearly be visualized by agarose gel electrophoresis.CONCLUSION: The combined use of laser microdissection and nested-PCR provides an opportunity to analyze gene expression in single cells. This method allows the analysis and identification of specific genes which are in volved inphysiological and pathophysiological processes in a complex of variable cell phenotypes.

Prediction of Response to Multimodality Treatment inEsophageal Cancer

Patients with locally advanced esophageal cancer have a dismal prognosis when treated exclu- sively by surgery. This fact prompted many investigators to apply neoadjuvant treatment strategies in an e?ort to improve survival. Results from phase III randomized trials are encouraging however, they revealed 五笔字型计算机汉字输入技术 that only patients with major histopathological response will bene?t from treatment. Therefore, predic- tive molecular markers indicating response or non-response to neoadjuvant treatment would be extremely helpful in selecting patients for current and future treatment protocols. In this paper we review the role of the molecular markers ERCC1 (excision repair cross-complementing 1 gene) and c-erbB-2 (synonym: HER2/neu) in predicting response to radiochemotherapy and outcome for patients with locally advanced resectable esophageal cancers (cT2-4, Nx, M0). The results are promising and it appears that we might expect to unequivocally identify with ERCC1 and c-erbB-2 respectively, approximately up to one third of patients who ful?l the criteria for neoadjuvant treatment for locally advanced esophageal cancer but will not bene?t from our treatment protocol. Integration of such markers in the clinical setting might prevent a substantial number of patients from expensive, non-e?ective and potentially harmful therapies, and could lead to a more individualized type of combined multimodality treatment in the near future.

CORRELATION BETWEEN THE NUMBER OF MITOTIC FIGURES AND THE PERCENTAGE OF Ki67－POSITIVE CELLS IN NON－HODGKIN’S LYMPHOMAS

Proliferative activity in 106 cases of non-Hodgkin’s lymphoma was estimated using the monoclonal antibody Ki67 and by counting the number of mitotic figures. The percentage of Ki67-positive cells was compared with the median number of mitotic figures per square millimeter. Both Ki67 positivity and the number of mitotic figures were found to be greater in high grade than in low grade lymphomas, although there was an overlap between the two grades of malignancy. A close correlation was found between the number of mitotic figures and the percentage of Ki67-positive cells not only in all lymphoma typo taken together (r_s= 0. 834 . P<0. 001). but also in B-cell lymphoma (r_s= 0. 8 1 P< 0. 001) and T-cell lymphoma (r_s = 0. 764. P<0. 00 1) taken seprately. Thus, both methods are useful for the estimation of proliferative activity. but each method has its advantages and disadvantages.

Neoadjuvant Treatment for Esophageal Cancer

Because the con?icting data currently available from the performed randomized trials it is very di?cult to provide strict guidelines for the treatment of patients with locoregional advanced esophageal cancers. Surgery however, remains the standard of care for potentially resectable disease. Preoperative chemotherapy is still controversial with two large randomized trials resulting in two di?erent conclusions regarding the survival bene?t. Preoperative chemoradiation is also controversial since only one randomized trial showed a clear survival bene?t however, the patients treated with surgery alone in this trial had an unusually poor outcome. And the study by Urba et al was not powered enough to show a clear survival bene?t for patients treated with neoadjuvant chemoradiation. The results of three metaanalysis of these randomized studies show lower rate of resection, higher rate of R0-resection, more often postoperative mortality and better prognosis for patients with neoadjuvant radiochemotherapy. As a consequence one may consider o?ering neoadjuvant chemotherapy or neoadjuvant radiochemotherapy to patients with locally- advanced disease under the premise that patients have a good performance status and understand the controversies about this therapeutic option. Larger trials with su?cient power to clearly detect survival bene?ts for patients treated with neoadjuvant chemotherapy or radiochemotherapy are necessary before this therapeutic option will be the standard of care.

A study on expression of hMLH1, hMSH2 and PCNA in colorectal adenomas and malignant change type in adenomas

object To explore the effect of hMLHland hMSH2 in carcinogenesis and progression of colorectal carcinoma,and their influences on proliferation in colorectal adenoma and malignant change in adenomas, Methods we investigated the expression of mismatch repair (MMR） gene hMLH1, hMSH2 and proliferation cell nuclear antigen (PCNA)by immunohistochemistry in 63 cases colorectal adenomas,20cases malignant change in adenomas and 20ceses colorectal carcinomas (CBCs). Results The positive rate of hMLH1 and hMSH2 in colorectal adenomas,malignant change in adenomas and CBCs were significantly lower than that in normal mucosa. With increasing dysplasia in adenomas, the expression rate of hMLH1 and hMSH2 protein decreased gradually; The PCNA label index of tumors with overexpression hMSH2 were significantly higher than that of tumors with negative hMSH2 in colorectal malignant change type in adenomas and CRCs. PCNA lable index of tumors with overexpression hMSH2 were significant higher than that of tumors with negative hMSH2 in adenomas with grade Ⅱ, Ⅲ dysplasia. The changes of PCNA label index in colorectal adenomas, malignant change type in adenomas and CBCs were not association with the expression of hMLH1. Conclusion It indicated that the MMB genes mutation and abnormal function of DNA mismatch repair maybe participate in carcinogenesis of CBCs. It is might be an early event in carcinogenesis of CBCs. It suggested that the reduction of proliferating activities in colorectal neoplasms might be related to mutation and defect of MMB gene.