Discussion On Clinical And Pathological Significance Of Identifying Tumor Clonal Origin From Hepatocellular Carcinoma Patients Who Received Liver Transplantation

【Objective】 To discuss the clinical and pathological significance of identifying tumor clonal origin from hepatocellular carcinoma patients who received liver transplantation.【Method】We collected pathological data, preoperative serum AFP levels and postoperative follow-up data of pluri-nodular hepatocellular carcinoma patients who received liver transplantation in our hospital during Aug 2005 and Aug 2010 and were diagnosed of conforming with standard of UCSF. Those whose information was not completely collected were excluded and the number of remained patients was summed up to 60. 60 cases’ paraffin embedded blocks of non-tumor tissue and tumor tissue were sliced up into five and a-10μm-thick sections, and then were reserved after xylene-ethanol dewaxing. The tumor tissue and non-tumor tissue in dewaxing sections were precisely obtained under the microdissection microscope; the DNA was extracted using DNA extraction kit. We selected 12 high-frequency microsatellite losses of heterozygosity(LOH) sites and detected the LOH state of tumor tissue by the mean of SSCP-PCR after compounding primer, and then determined the pattern of tumor clonal origin according to each patient’s LOH state of different microsatellite loci. SPSS20.0 statistical software was used to analyzing the correlation between the pattern of tumor clone origin and disease-free survival rate, the pathological characteristics and the level of serum AFP in patients who received liver transplantation. We collected 14 cases of hepatocellular carcinoma(D-HCC) patients who were treated with liver transplantation in our hospital during Aug 2004 and Nov 2010. 2 block tumor tissues and 1 block un-tumor tissues were collected from tumor stoves spacing more than 3 cm within left and right lobes of pathological liver and un-tumor region respectively, all samples were sliced up into five and a-10μm-thick sections and were reserved after xylene-ethanol dewaxing. The tumor tissue and non-tumor tissue in dewaxing sections were precisely obtained under the microdissection microscope; the DNA was extracted using DNA extraction kit. We selected 12 high-frequency microsatellite losses of heterozygosity(LOH) sites and detected the LOH state of tumor tissue by the mean of SSCP-PCR after compounding primer, and then determined the pattern of tumor clonal origin according to each patients’ LOH state of different microsatellite loci. Preoperative serum AFP concentration, postoperative disease liver pathological data and postoperative follow-up data were collected to summarize the clinical and pathological features of 14 cases of D-HCC patients.【Results】A total of 60 patients were qualified 53 males and 7 females. Their age ranged from 38 to 71, with average 53. Their background diseases included 45 cases of HBV cirrhosis, 11 cases of HCV cirrhosis, 3 cases of alcoholic cirrhosis and 1 case of cryptogenic cirrhosis. There were two tumor nodules in 45 patients and three tumor nodules in 15 patients. 135 tumor nodules were collected from 60 cases of HCC patients with multiple nodules, with diameters ranging from 0.8cm to 3.8cm. The microsatellite LOH detections showed the tumor clone origin were IM type, IM+MO type, MO type and unidentified type, with the percentage of 33.33%(20/60), 8.33%(5/60), 55%(33/60)and 3.33%(2/60)respectively, two patients whose tumor clone origin could not be unidentified were removed and the remaining 58 patients had IM at 34.48%(20/58), MO at 56.90%(33/58) and IM+MO at 8.62%(5/58). 3-years cumulative disease-free survival, microscopic tumor thrombus,tumor incidence of low differentiation rate and AFP concentration in IM group, MO group and IM+MO group were 50.00% and 78.79% and 40%,100% and 18.18% and 100%, 80% and 51.52% and 80%, 226.80μg/L(2.78μg/L-3000.00μg/L) and 24.59μg/L(1.16μg/L-531.30μg/L) and 122.58μg/L(16.20μg/L-1055.00μg/L) respectively. The area under the ROC curve for preoperative serum AFP concentration of IM and MO groups was 0.792, corresponding 95% confidence interval was 0.659-0.926. The best determination boundary value was 122.30μg/L, the sensitiveness was 0.750 and the specificity was 0.818. Statistical analysis showed that three-year tumor-free survival ratio in IM group was obviously lower than that in MO group(P <0.05); the occurrence of microscopic tumor thrombus and preoperative serum AFP concentration were higher in IM group than that in MO group(P<0.05); tissue differentiation degree in IM group was obviously lower than that in MO group(P<0.05) too; there was no significance between IM+MO group and IM group on the three-year tumor-free survival ratio, microscopic tumor thrombus and tissue differentiation degree(P>0.05). 14 cases of patients with D-HCC LOH test results showed that 11 cases with the clonal origin of type IM, 3 patients with tumor clone origin type of MO and IM at the same time. Preoperative serum AFP concentration of 14 patients was 0.53μg/L- 427.04μg/L, with 9 patient’s serum AFP level>20.00μg/L and 4 patient’s serum AFP level>200.00μg/L. The preoperative imaging inspection showed the image that similar to cirrhosis, accompanied with their livers and spleens swelled to some extent, but no space occupying lesions was discovered. The tumors spread throughout the livers, with the diameters ranging from 0.1 to 1.0 centimeters and the number more than 100, just like the cirrhosis nodules. The pathological grading of tumors were grade I and II, the tumor thrombus can be detected through microscope. in all D-HCC patients. The tumor-free survival period after liver transplantation was ranging from 4.5 to 37.4 months and in an average of 13.5±6.7 months.【Conclusion】1.There were two main types of tumor clonal origin of pluri-nodular hepatocellular carcinoma: MO type and IM type; The three-year tumor-free survival ratio after liver transplantation was obviously higher in HCC with MO type than that with IM type, the prognosis was similar between IM+MO type and IM type. The tumor clonal origin may be act as one of important reference indexes to predict tumor recurrence after liver transplantation in patients with pluri-nodular hepatocellular carcinoma.2. It is helpful to forecast the tumor recurrence ratio after transplantation by combining tumor clonal origin with pathological characteristics and preoperative serum AFP.3. The main type of D-HCC clonal origin is IM type and it mostly indicates extensive intrahepatic metastasis; the occurrence of D-HCC is occult and with low sensitivity on clinical characteristics and imaging inspections, needle biopsy could be carried out when it is difficult to be distinguished.