Detection Of Circulating Tumor Cells In Peripheral Blood And Its Significance In Patients With Hepatocellular Carcinoma

Primary liver cancer(most of them are hepatocellular carcinoma, HCC) is one of the most frequent malignances worldwide, its incidence ranks the fifth most common cancer and is still increasing. And it has become the second cancer killer in China since 1990s. Although many efforts have been made to improve the prognosis and many progresses in clinical studies of HCC have been obtained in the past decades, the overall outcome of patients with HCC is still dismal with overall less than 5% of 5-year survival rate. So far, curative resection remains the major therapeutic method for HCC. However, only a definitive subset of patients has any chance of being cured, because of the great possibility of intrahepatic tumor recurrence after surgical operation. Metastasis is one of the major causes of tumor recurrence. Metastatic recurrence after operation has become one of the major obstacles to further prolonging survival. Therefore, there is a tremendous interest and urgency to search for predictors fro HCC metastasis. Many pathologic features, such as tumor size, number, cell differentiation, vascular invasion, and intrahepatic spreading, as well as tumor staging system, could provide some useful information in predicting the tumor recurrence and prognosis of patients, and triaging the patients who need and may benefit from adjuvant therapy. However, these features can not always provide exact enough information for the predictions of patients' outcome. Sometimes, the patients, even though they have the same TNM stages of disease, histopathological features of tumor, as well as treatment strategy (such as curative resection), have different clinical outcomes. This uncertainty in the prognostic prediction leads to overtreatment in patients with toxic agents that exert severe side effects. A better selection marker will be useful to predict which patients are in need of adjuvant therapies after successful surgical resection of the primary tumor, and in which patient the treatment should be avoided. The clinical relevance of tumor cells in peripheral blood has been studied for its use as a risk criterion for metastasis and for monitoring treatment results.Metastasis is the leading cause of cancer-related deaths. According to the traditional knowledge of metastasis, metastatic cells are rare and arise during advanced stage of tumor progression. However, our recent study on the gene expression profiling of HCC metastasis showed that metastatic programme is initiated in the primary tumour indicates that analysis strategies such as these can also be used to identify patients with pre-metastatic tumours, and also to predict the possibility of metastatic recurrence.The clinical relevance of tumor cells in peripheral blood has been studied for its use as a risk criterion for metastasis and for monitoring treatment results. Advance in immunocytochemistry and molecular assay made it possible to detect single cell in peripheral blood and marrow. Peripheral blood, however, is an ideal source for the monitoring of metastatic tumor cells, and many groups have demonstrated the presence of these circulating tumor cells(CTC) in patients with early-stage cancer without overt metastases.A new method of isolating CTCs by their larger size when compared with peripheral blood leukocytes was developed recently, this filtration assay is easy to perform, rapid and inexpensive with high sensitivity in detecting CTC. It can keep the cells undamaged and minimize the cell loss by avoiding the step of separating the single nuclear cells during immunomagnetic cell sorting. Therefore, we used this method to detect the CTC in peripheral blood of patients with HCC and evaluate its clinical significance. And also, the chromosomal abberations of CTC were analyzed using single-cell whole genomic amplification technique in combination with comparative genomic hybridization(CGH) method, to prove their malignant nature in the present study.PART 1Detection of circulating tumor cells in peripheral blood in patients with hepatocellular carcinomaObjective: To develop a new assay for the detection of circulating tumor cells(CTC) in peripheral blood samples of patients with HCC and to explore the effects of surgical resection on the number of CTC during operation.Methods: One modified filtration enrichment assay was established for this study. Filtrated HCC97-L cells were counted to evaluate the sensitivity of this method. Circulating cells were enumerated after filtration enrichment and immunostaining with anti-cytokeratin 8/18(CK8/18) in the peripheral blood samples from 75 patients with HCC, 15 cases of benign lesion and 10 cases of healthy control. In 18 patients with HCC, the blood samples were also collected during live transection.Results: The sensitivities of this assay in the 10 times repeated experiments ranged from 40% to 100% with an average of 68%. CTC were detected in 43 of 75(57.3%) preoperative blood samples from patients with HCC. No CTC was founded in the samples from patients with benign lesions and healthy control. In 18 patients with HCC, the blood samples were also collected during operation, the positive rate of CTC was 100%, which was much higher than that before operation(61%).Conclusions: CTC can be detected easily and rapidly in patients with HCC with filtration enrichment approach, liver resection may enhance the shedding of tumor cells into blood during surgery.PARTⅡChromosomal aberrations in single circulating tumor cells detected by comparative genomic hybridizationObjective: To detect the chromosomal aberrations of single CTC in patients with HCC and explore methods that may prove the malignancy of CTC.Methods: Thirty single cells that detected in partⅠin patients with early(14 cells) and advanced stage(16 cells) of HCC were isolated by laser capture microdissection(LCM) method. Genomic DNA of a single cell was amplified by single-cell whole genomic amplification method. Genomic alterations were analyzed by comparative genomic hybridization (CGH).Results: Twenty-nine of 30 single cells were successfully isolated by LCM method; Genomie DNA were successfully amplified by technique of single-cell whole genomie amplification in 27 out of the 29 single cells. Chromosomal aberrations were found in 22 of 27 single cells, while no significant chromosomal aberrations could be found in 4 out of 13 cells from patients with early stage of HCC, and 1 out of 14 cells in patients with advanced stage of HCC. The most frequent chromosomal aberrations include gains on chromosomes1q, 6p, 8q and losses on 1p, 4q, 8p, 13q, 16q, and 17p. Losses of 8p were found in 1 of 9 cells in patients with early stage and in 8 of 13 cells in patients with advanced stage of HCC. The difference between this two groups of patients was significant(P＜0.01).Conclusions: The amplified genomic DNA generated by single cell whole genomic amplification can be used for CGH. Most of CTC was found to have some degree of chromosomal aberrations, suggesting their malignant origin. The most frequent chromosomal aberrations include gains of 1q,6p,8q and losses of 1p,4q,8p,13q,16q,17p in CTC in patients with HCC. And 8p deletion was much more frequently found in the CTC from patients with advanced stage of HCC.PARTⅢClinical significances of circulating tumor cells in peripheral blood inpatients with hepatocellular carcinomaObjective: To evaluate the clinical significance and prognostic value of CTC in peripheral blood in patients with HCC.Methods: Circulating tumor cells were enumerated after filtration enrichment and immunostaining with anti-cytokeratin 8/18(CKS/18) in 75 patients with HCC, 15 cases of benign lesion and 10 cases of health control. Results: CTC were detected preoperatively in 28.6% of patients with early stage (stageⅠ～Ⅱ), and 82.5% of patients with advanced stage (stageⅢ～Ⅳ) of HCC. No CTC was founded in the control group. Comparing the CTC positive rates in patients with different clinicopathological features, the patients with multiple tumor nodules of HCC(83%) was much higher than those with single-nodular tumors(52.3%)(P＜0.05), patients with large size of tumor(＞5cm) (79%) was more than those with small tumor(＜＝5cm) (35%) (P＜0.01). There was no significant association between the CTC positive rates HBsAg, AFP serum level, liver cirrhosis, capsules of the tumor and Edmondson grade. CTC was associated with lower one-year FDS and OS in patients with HCC.Conclusions CTC can be detected easily in patients with HCC with filtration enrichment approach, even in some of patients with early stage of HCC. The positive rate of CTC increases with the advance of the tumor. CTC was associated with early recurrence of HCC after operation.