| Part 1 Prognostic predictors for small hepatocellular carcinomaPurpose To Expore the predictive factors of SHCC.Patients and Methods The study cohort consisted 287 patients underwent hepatectomy for single SHCC. Clinical data (included age, gender, virus infection, serum alfa-fetoprotein level(AFP), capsule, tumor border, cirrhosis), histopathologic features (included differentiation, morphology subtype, microvascular invasion(MVI), tumor infiltrative lymphocytes (TIL), inflammatory injury grade and fibrosis stage of surrounding liver) were evaluated to identify prognostic factors influencing SHCC patients’ survival and MVI.Results There were 238 males (82.9%) and 49 (17.1%) females with median ages of 52 years. The median progression-free survival (PFS) and overall survival (OS) durations were both 54 months. About 94.0%(268/287) SHCC patients were infected by Hepatitis B. One hundred and two (36.4%) of the 280 (280/287) patients with pre-operation serum AFP level record had serum AFP level≥ 200ug/ml.One hundred and ninteen (66.1%) of the 180 (180/287) patients with post-operation serum AFP level record had AFP level ≥20ug/ml. Liver cirrhosis was present in 257 cases (89.5%). For 283 (283/287) patients with tumor border record,165 (58.3%) tumors were encapsulated and 118 (41.7%) patients were partially encapsulated. Two hundred and seventy two (97.8%) tumors had clear border. MVI was identified in 46 patients (16.0%).In univariate analysis, a significant correlation between the presence of MVI and shortened PFS was found (P=0.019). For patients with MVI, females (P=0.000, 0.003), patients with post-operation serum AFP level≥20ug/ml (P=0.011,0.008) or TIL ≥50/HPF (P=0.000,0.019), patients with clear tumor border (P=0.004, 0.043) and patients with Gl or G2 histological differentiation(P=0.013、0.012), MVI had a worse effect for the PFS and OS of these patients. MVI had a worse effect for the PFS of patients with cirrhosis (P=0.003) or aged< 50 years old (P=0.028).Conclusions1. MVI is an independent poorer prognostic factor for PFS of single SHCC patients.2. MVI had a worse effect for the PFS or OS of females, patients with post-operation serum AFP level≥ 20ug/ml or TIL≥50/HPF, patients with clear tumor border or G1/G2 histological differentiation. PFS was shorter for patients with cirrhosis and MVI or aged< 50y with MVI.Part 2 Differential expression of microRNAs between primary tumor and em bolus in fresh HCC samplesPurpose Select the differentially expressed microRNAs between HCC and corresponding embolus in fresh samples.Patients and Methods We collected the RNA of HCC and embolus tissues from 16 HCC patients. To detect the CT threshold of microRNAs, U47 is used as the reference gene. By using unpaired t test, we analyzed the differential expression of microRNAs in the two groups. Results 1. By analyzing the expression of 723 microRNAs in HCC and embolus samples,19 microRNAs was found statiatically differential expressed in two groups. Among them,15 microRNAs are reduced in embolus than HCC tissues, including hsa-miR-125a-3p (P=0.01), hsa-miR-575 (P=0.03), hsa-miR-320 (P=0.02), hsa-miR-122 (P=0.01), hsa-let-7c (P=0.03), hsa-miR-338-3p (P=0.02), hsa-miR-26a (P=0.02), hsa-miR-486-5p (P=0.00), hsa-miR-202 (P=0.04), hsa-miR-765 (/>=0.02), hsa-miR-125b-2* (P=0.01), hsa-miR-564 (P=0.03), hsa-miR-342-5p (P=0.03), hsa-miR-623 (P=0.03), hsa-miR-7-1* (P=0.01). Four microRNAs are expressed highly in embolus than in HCC tissues, including hsa-miR-21 (P=0.00), hsa-miR-551b (P=0.04), hsa-miR-27a (P=0.00), hsa-miR-876-3p (P=0.04).2. hsa-miR-125a-3p and hsa-miR-575 were chose as targeted genes to verify their expression in FFPE tissues.Conclusions1. Of the known 723 microRNAs,19 microRNAs are expressed significantly different in HCC and embolus samples. Among them,15 microRNAs are reduced in embolus than HCC tissues, including hsa-miR-125a-3p, hsa-miR-575, hsa-miR-320, hsa-miR-122, hsa-let-7c, hsa-miR-338-3p, hsa-miR-26a, hsa-miR-486-5p, hsa-miR-202, hsa-miR-139-3p, hsa-miR-765, hsa-miR-125b-2*, hsa-miR-564, hsa-miR-342-5p, hsa-miR-30b*, hsa-miR-623, hsa-miR-7-1*. Four microRNAs are highly expressed in embolus than in HCC tissues, including hsa-miR-21, hsa-miR-551b, hsa-miR-27a, hsa-miR-876-3p.2. hsa-miR-125a-3p and hsa-miR-575 were chose as targeted genes and verify their expression in FFPE tissues.Part 3 Correlation of targeted microRNAs and clinicopathological factors of HCC patientsPurpose Verify the expression of miR125A-3P and miR575 in paraffin tissues and analyze the correlation of targeted microRNAs with clinicopathological factors of HCC patients.Patients and Methods Microdissected HCC, MVI and Non-tumor tissues from 36 FFPE samples of HCC patients. Performing real-time RT-PCR, the correlation of differential expression of targeted genes were analyzed in the comparison of MVI,HCC and Non-tumor.Results 1. A total of 36 HCC patients with median age of 50 years, among them 31 are males,5 are females.33 cases were infected by HBV virus and 3 patients without hepatitis virus infection. Twenty-two patients had liver cirrhosis and 14 patients without cirrhosis. MVI was identified in 18 patients.2. Tissues were microdissected without contamination, RNA concentration was 4.1 422.1ng/ul.3. Among HCC and Non-tumor tissue, miR125A-3P and miR575 has no statistically correlation with gender, age, cirrhosis, tumor histological differentiation, MVI and TEL 4. In 18 patients with MVI, miR125A-3P showed statistically different between patients with TIL> 50/HPF or< 50/HPF in MVI and Non-tumor tissues.Conclusions1. For patients with MVI, miR125A-3P and miR575 are reduced in MVI than in HCC and Non-tumor, the expression of genes in Non-tumor is the highest.2. For patients without MVI, the expression of miR125A-3P and miR575 are lower in HCC than Non-tumor tissue.3. In patients with MVI and paired Non-tumor tissues, miR125A-3P showed statistically different between patients with TIL> 50/HPF or< 50/HPF.Part 4 Differential expression of target microRNAs in paraffin tissuesPurpose To compare the diverse expression of miR125A-3P and miR575 in HCC, MVI and Non-tumor tissues.Patients and Methods HCC, MVI and Non-tumor tissues were microdissected from 36 FFPE samples of HCC. Performing real-time RT-PCR, conducting t test, exploring the expression trends of targeted genes in MVI, HCC and Non-tumor.Results 1. Matching two of the three groups including MVI, HCC, Non-tumor in 18 patients with MVI, the expression of miR125A-3P in MVI tissue is lower than HCC and Non-tumor with P value 0.0053、0.0542, respectively. miR575 also displayed the same trend with miR125A-3P, with P value 0.121、0.2947, respectively. Both of the two genes are reduced in HCC than paired Non-tumor tissues.2. For HCC and Non-tumor tissues, miR125A-3P and miR575 is higher in patients with MVI than patients without MVI, miR125A-3P had P value statistically significant in two groups (P=0.0021,0.0336, respectively). miR575 didn’t show statistically different in two groups (P=0.1478,0.2977, respectively).Conclusions1. miR125A-3P is significantly different in MVI and HCC tissues for patients with MVI, miR575 didn’t displayed statistically different in the same groups. Neither two genes were statistically different in MVI and Non-tumor tissues for patients with MVI, nor in HCC and Non-tumor tissues for 36 HCC patients.2. In HCC and Non-tumor tissues, miR125A-3P and miR575 are higher in patients with MVI than patients without MVI and miR125A-3P was significantly different in the two groups. |
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