The Detection Of CTCs In Patients With Primary Hepatocellular Carcinoma And The Relationship Between CTCs And CD4+/CD8+、NLR、TTV、 Content Of HBV-DNA And Tumor Stage

Background and objectiveHepatocellular carcinoma(HCC) is derived from malignant of the liver,the incidence of the hepatocellular carcinoma account for the sixth of the global incidence of tumor,mortality of the hepatocellular carcinoma account for the third of global cancer related death.However,at present in our country primary liver cancer patients with new cases and the death of the patients caused by primary liver cancer are accounted for over fifty percent of the global new rate and mortality.The incidence of the hepatocellular carcinoma is still rising trend,but the overall survival in patients with primary liver cancer is still low.There are many causes that can lead to primary hepatocellular carcinoma, which the main cause is hepatitis virus infection, and primary hepatocellular carcinoma patients in our country caused by hepatitis virus infection, hepatitis B virus infection is the most common. There are many chronic hepatitis B patients in our country, At present, the number of primary hepatocellular carcinoma patients due to hepatitis B virus infection is is increasing, and the age of onset tends to be younger. In recent years, along with the continuous deepening study of HCC cell level and molecular level, we have more in-depth understanding and knowledge of HCC carcinogenesis, progression and metastasis of cytology and molecular mechanism 。At present, surgical resection of tumor is still the most effective treatment for patients with primary liver cancer, and is the only method possible to cure primary hepatocellular carcinoma。With the clinical diagnosis level constantly improve, the treatment scheme constantly updated and improvement and surgical technique continuous improvement and improve, the curative effect in primary liver cancer patients have obviously improved than before, and significantly extended primary hepatocellular carcinoma patients survival time. But because of the incidence of occult, no obvious symptoms at early stage and to visit already at an advanced stage so the mortality in patients with primary liver cancer rate is still high。According to the relevant literature report the 5-years survival rate after surgery is less than 50% in primary liver cancer patients, and the main causes of death in patients is postoperative tumor recurrence and metastasis.Therefore, postoperative tumor recurrence and metastasis is still the major problems and challenges that surgeons need to facing at present and future period of time.Thus, How to detection of tumor recurrence and metastasis after operation in early stageand take appropriate treatment to improve the prognosis of patients is very important.Circulating tumor cells (CTCs) refers to tumor cells which released into the peripheral blood by solid tumors or metastases lesions through spontaneously or due to operation of diagnosis and treatment, people called it as "disseminated tumor cells (DTC). In 1869 Australia scholar Ashworth found a kind of cell from the blood of patient who was die of metastasis carcinoma,which was similar to primary malignant tumor cells. But due to the technical level was limited and difficult to detect, circulating tumor cells did not get attention at that time, until the last century 90’s, the clinical value of circulating tumor cells has been paid close attention. With the development of science and technology and the improvement of detection technology, detecting circulating tumor cells in patients with tumor is becoming more and more common, and has been widely used in clinical such as diagnosis of malignant tumor, curative effect evaluation、monitoring of tumor recurrence and metastasis and individualized treatment. Through a variety of detection and identification technology, it is generally believed that in the peripheral blood circulation tumor cell has three different forms, namely mesenchmal type CTCs、epithelial type CTCs and hybrid type CTCs. With the in-depth study of the three different forms of circulating tumor cells,it is generally believed that mesenchmal type CTCs in malignant degree is far higher than that of epithelial type and hybrid type CTCs.If mesenchmal type CTCs are found in patients’ peripheral blood, while the risk of tumor progression, recurrence and metastasis significantly increase and with poor prognosis.At present, clinicians has begun to evaluate the prognosis of patients with primary liver cancer through detection of circulating tumor cells in peripheral blood of patients. Related study showed that if circulating tumor cells were found in the circulatory system,whose overall survival rate, postoperative survival rate, and the postoperative disease-free survival rate are significantly lower than those of patients with negative tumor cells in the circulatory system. Under normal circumstances, the tumor cells which released into the peripheral blood will be recognized and removed by the immune system of the body, but when the immune function is weak, part of the tumor cells will survive and this tumor cells will become the base of tumor recurrence and metastasis. At present, although the detection of circulating tumor cells more and more on primary hepatocellular carcinoma patients, but whether there is a link and whether there is a relationship between CTCs and clinical pathological factors and T cell immune function in primary hepatocellular carcinoma patients is not clear.In this study CanPatrol "method was used to detect the CTCs in peripheral blood of primary hepatocellular carcinoma patients, T cell subsets and CD4+/CD8+was detected by flow cytometry,meanwhile collecrted clinical pathological parameters,such as peripheral blood neutrophilic granulocyte count 、 lymphocytes count、quantitative hepatitis B virus、maximum tumor diameter and tumor stage o To investigate the relationship between CTCs and T cell immunity, NLR, TTV, HBV-DNA content and tumor stage, and provide d the basis for the curative effect evaluation and the choice of treatment of hepatocellular carcinoma patients.Method:1. Clinical data of 40 patients with primary liver cancer who underwent curative hepatectomy in Department of Hepatobiliary surgery, Nanfang Hospital Affiliated to Southern Medical University,from June 2014 to October 2015 were collected. There were 23 males and 17 females with age ranging from 21 to 71 years and the median age was 50 years. There were five inclusion criteria of the patients with primary liver cancer:(1)①atients should accordance with the clinical diagnosis of primary hepatocellular carcinoma before operation.Patients should have hepatitis B virus infection and the result of two pairs of semi-hepatitis confirmed HBsAg was positive. ②here were at least two kinds of imaging results accord with the imaging features of HCC,or at least one imaging results accord with the imaging features of HCC and tumor markers AFP in patients serum was higher than 400 ug/L. (2) The operation method of patients was radical resection and the following was the reference standard:In all of the preoperative examination no tumor metastasis were found,and the tumor should be resected completely.During the process must be strictly follow the principle of no tumor and no residual liver lesiona.The pathological examination results after the operation confirmed no residual tumor resection at the edgr of liver and excluded the positive margins patients. (3) More than two pathology experts confirmed that the tumor conform to HCC by looking at the sample after the operation; (4) All the selected patients should have not received radiofrequency ablation, hepatic artery chemoembolization, immune therapy, chemotherapy and local radiotherapy treatment. (5)A11 the selected patients were excluded from infectious diseases and blood system diseases etc. The clinical pathological parameters which need to record including age and sex of the patients, HBV-DNA content, the peripheral blood of neutral granulocyte and lymphocytes count, the maximum diameter of the tumor, the number of tumor and the tumor stage.2.Used CanPatrolTM detection technology which was developed by Guangzhou surexam company to test the type and count of CTCs in peripheral blood of the 40 cases of HCCAnalysising and comparing the difference of CTCs in groups of the clinical pathological parameters and analysising the relationship between CTCs and various clinical pathological parameters.3.Used the flow cytometry to check the number of CD4+ T cell and CD8+ T cell in peripheral blood of the 40 cases of HCC and calculated the ratio of CD4+ /CD8+,accorded the ratio of CD4+ /CD8+ to assess the T cell immune function. At the same time,compared the relationship and the correlational relationship between CTCs and T cell immune function.4. Accorded to the formula to calculate the ratio of the CD4+ /CD8 +, neutrophil to lymphocyte ratio(NLR) and total tumor volume (TTV) The computing method of tumor volume reference to the formula of spheroidal volume:tumor volume equal to 4/3×3.14 × r 3 (r=the maximum diameter of the tumor nodule which can be measured). Total tumor volume (TTV) was equal to the sum of the volume of the complete cytoreduction which can be measured in HCC patients, and the measured tumor radius was detceted by the iconography.Accorded to the number of lymphocytes and neutrophils which come from blood routine examination to calculated the NLR ratio, and accorded to the number of CD4+T cells and CD8+T cells calculated the ratio of CD4+ /CD8+. In this study,the ratio of CD4+ /CD8+,TTV, NLR was divided into high and low groups which was based on the average ratio of CD4+ /CD8+, the median of TTV and NLR. Accorded to the value of clinical test (1000 copies/ml), the content of HBV-DNA was divided into high and low groups. Accorded to HCC TNM staging (refer to the seventh edition of the AJCC Cancer Staging Manual) the patients were divided into the group of I, II stage, III, IV stage (stage III including III a, III b, IIIc; IV including IV a and IV b).5. Statistical treatment:Used software of SPSS 19 to statistical analysis the date.The results were expressed by the mean and standard deviation,The independent sanples T test was used to compared the different between groups and Spearman method was used to analysis the correlation.The P value less than 0.05 was considered have statistically significant.Results1.The CTCs could be detected in peripheral blood in patients with HCC, and there were three types of rumor cells in peripheral blood,they were epithelial type, mesenchymal type and mixed type; The CanPatrolTM detection technology which was developed by Guangzhou surexam company can be used for the detection of CTCS in peripheral blood of patients with primary liver cancer,and the accuracy was relatively high.2.Used the flow cytometry to detect T cell subsets in peripheral blood in patients with HCC, we found that the proportion of CD4+ T cells was lower,and the proportion of CD8+ and T cells was higher,the ratio of CD4+ /CD8+ was low,.This result indicated that the resistance mechanism disorders was existed in patients with primary liver cancer.The ratio of CD4+ /CD8+ was divided into high and low groups by taking the average value of the ratio of CD4+ /CD8+ as critical value, and compared the differences in the type and quantity of CTCs between the two groups. The results showed that in the higher group of the ratio of CD4+ /CD8+, interstitial type CTCs and total number of CTCs were lower than the low group, there were statistically significant differences between the two groups (P<0.05); But there were no statistically significant differences between the two groups in the number of the epithelial type CTCs and mixed type CTCs in the peripheral blood (P>0.05); And the results of correlational relationship showed that there was a negative correlation between the total number of CTCs in peripheral blood and T cells in patients. That was, if T cell immune function was worse, the total number of CTCs in the peripheral blood of patients was more,and the prognosis of patients was worse.3. According to the median of the total tumor volume (TTV) and neutrophil and lymphocyte ratio, there were divided into two groups, compared the differences of the CTCs’ type and quantity between the two groups, the results show that the total of CTCs、interstitial type CTCs and mixed type CTCs in high TTV group was significantly higher than that of low TTV group, there were significant differences between the two groups (P<0.05)And there was no significant difference between the two groups in the number of epithelial type CTCs (P>0.05); There were no significant differences between the high NLR group and low NLR group in the total number of CTCs, the number of interstitial type CTCs, mixed type CTCs and epithelial type CTCs in peripheral blood of HCC patinets (P>0.05); The results of the correlational relationship showed that there was a positive correlation between the total number of CTCs in peripheral blood and total tumor volume,while there was no obvious correlational relationship between the total number of CTCs in peripheral blood and neutrophil and lymphocyte ratio.4. Analyzed and compared the relationship about the type and quantity of the CTCs in the tumor staging between group Ⅰ and Ⅱ, Ⅲ and IV and group of high HBV-DNA content and low HBV-DNA content The results of the study show that the total of the CTCs, the number of interstitial type CTCs, mixed type CTCs in tumor staging of group I and II were less than group III and IV in patients peripheral blood, the differences between the two groups had statistical significance (P< 0.05), and differences between the two groups in the number of epithelial type CTCs had no statistical significance (P> 0.05).In the group of high HBV-DNA content and low HBV-DNA content, the differences in the total of CTCs, the number of interstitial type CTCs, mixed type CTCs and epithelial type in peripheral blood had no statistical significance (P> 0.05). The correlational analysis showed that there was a positive correlation between the total of CTCs in peripheral blood and tumor staging of patients, but no obvious correlational relationship with the content of the HBV-DNA.ConclusionTumor cells exists in peripheral blood of patients with hepatocellular carcinoma, and the CanPatrolTM detection technology which was developed by Guangzhou surexam company could be used for detecting CTCs of the primary hepatocellular carcinoma 。2. There are exist correlational relationship between the CTCs and CD4+ /CD8+ ratio (immune functional state of T cell), the TTV and the tumor staging, but no correlational relationship in the content of HBV-DAN and NLR.3. If T cell immune function were worse in patients, TTV was bigger and tumor staging was later, the number of CTCS,the number of interstitial type CTCs, mixed type CTCs which malignant degree considered to be higher will be more in peripheral blood of HCC patients. We can improve the prognosis of HCC patients by improving the patients’ immune function and reducing the tumor burden.