| [Background] Primary liver cancer is a highly malignant, early invasive andmetastatic tumor. Early diagnosis is the key to reducing mortality. Alpha-fetoprotein(AFP) is a classic marker for hepatocellular carcinoma (HCC) screening anddiagnosis, but the diagnostic value of alpha-fetoprotein, especially for earlyhepatocellular carcinoma, has been disputed. Serum markers for early diagnosis ofHCC have their own advantages and limitations, including glypican-3(GPC3),alpha-fetoprotein heterogeneity3(AFP-L3), des-γ-carboxy prothrombin (DCP), Golgiprotein73(GP73) and transforming growth factor beta2(TGF-β2). The diagnosticvalue of these serum markers alone or panel remains unclear for HCCs with serumAFP <200ng/ml. Glypican-3(GPC3) is useful not only as a novel tumor marker, butalso as a target antigen for immunotherapy. The purpose of this study is to furtherclarify the advantage of serum GPC3alone or with other serum markers in thediagnosis of HCC and preliminary elaborated GPC3peptide-specific T cell responsesin patients with HCC or cirrhosis and its related factors. Thus, this study may providea theoretical basis for the early diagnosis and treatment of HCC.1. The diagnostic values of serum marker alone or panels inpatients with hepatocellular carcinoma[Materials and Methods] The study included99patients with HCC,47cirrhosis patients,25patients with active hepatitis and25healthy individuals. Theconcentrations of serum GPC3, GP73and TGF-β2were detected by the ELISA assay.The levels of serum AFP, AFP-L3and DCP were measured by theimmunofluorescence liquid phase binding assay. [Results]1.1Healthy individuals as a control group, the analysis of the diagnosticvalue of a single serum marker in HCC.ROC analysis was performed for each marker to determine its diagnosticaccuracy. DCP and AFP have the best efficacy with an area under the ROC curve(AUC) of0.85, which is higher than that of AFP-L30.8, GPC30.8, GP730.8andTGF-β20.78. The cut-off value was AFP>200ng/ml, AFP-L3>15%,DCP>110mAU/ml, GPC3>1.5ng/ml, GP73>2.6IU/ml and TGF-β2>1300pg/ml,respectively. The sensitivity of serum DCP, GPC3and AFP-L3were66%,64%and57%, the specificity was94%, the odds ratio (OR, positive likelihood ratio andnegative likelihood ratio) were30.4,26.8and20.8.As a single HCC diagnostic marker with the same specificity (94%), thediagnostic value of serum DCP, GPC3or AFP-L3was significantly higher than that ofserum AFP (which sensitivity and OR were52%and17).1.2Cirrhosis patients as a control group, the analysis of the diagnostic valueof serum markers in HCC.The odds ratio of serum DCP>110mAU/ml was13.06, the highest value in thesix markers. The positive predictive value (PPV) and negative predictive value (NPV)were87.23%and65.66%. The odds ratio of serum GPC3>1.5ng/ml was11.88. ItsPPV and NPV were89.36%and58.59%. The positive predictive value of serumAFP>200ng/ml was91.49%, but the OR value (8.96) is lower than the OR value ofGPC3and DCP. The PPV, NPV and OR of serum AFP-L3, GP73and TGF-β2werelow. Two-marker combination analysis: The odds ratio of serum AFP+DCP was22.57,the highest value in the11two-marker combinations. The PPV and NPV were91.86%and66.67%. The odds ratio of serum GPC3+DCP was18.82. Its PPV and NPV were89.74%and68.89%. There were no advantages in the PPV, NPV and OR of other9two-marker combinations than that of single marker. Three-marker combinationanalysis: The odds ratio of serum AFP+GPC3+DCP was32.73, the highest value inthe7three-marker combinations. Its PPV and NPV were89.11%and80%. The OR,PPV and NPV of other6three-marker combinations and two-marker combinations were similar.Cirrhosis patients as a control group, the diagnostic values of serum markerswere (AFP+GPC3+DCP)>(AFP+DCP)>(GPC3+DCP)>DCP>GPC3.1.3Cirrhosis patients as a control group, the diagnostic values of serummarker alone or panels in HCCs with AFP≤200ng/mlThe PPV and NPV of serum DCP>110mAU/ml were85.37%and69.49%, thesensitivity and specificity were66.04%and87.23%. The PPV and NPV of serumGPC3>1.5ng/ml were86.11%and64.62%, the sensitivity and specificity were57.41%and89.36%. The PPV, NPV, sensitivity and specificity of serum AFP-L3,GP73and TGF-β2were lower than that serum DCP or GPC3. The PPV and NPV ofserum GPC3+DCP were81.48%and75.61%, the sensitivity and specificity were81.48%and75.61%. No PPV and NPV of other combinations were higher than thatof serum GPC3+DCP.The diagnostic values of serum markers in HCCs with≤A2F0P0ng/ml were(AFP+GPC3+DCP)>(AFP+DCP)>(GPC3+DCP)>DCP>GPC3.1.4The level of serum TGF-β2in patients with HBV-related cirrhosis was679.3±114.8pg/ml and was significant lower than that in HCCs (1546±193.2pg/ml) orpatients with alcoholic and HBV-related cirrhosis (905.2±96.20pg/ml)(p<0.01andp<0.05, respectively). The results suggested that HBV-related cirrhosis patients withconcomitant alcoholic liver injury may lead to increase the levels of serum TGF-β2,thereby increasing the risk of HCC. The levels of serum TGF-β2in HCCs with serumGPC3>1.5ng/ml were similar with that in HCCs with serum GP≤C13.5ng/ml. Theresults suggested that serum GPC3and TGF-β2correlation in hepatocellularcarcinoma is not clear and further research was needed.[Conclusions]1.5The serum DCP or GPC3are good diagnostic markers of HCC and can beused as indicators of HCC screening and early diagnosis in patients with cirrhosis.The serum AFP+GPC3+DCP is the best indicator of HCC screening and earlydiagnosis.1.6The serum GPC3or DCP are beneficial to the early diagnosis of HCC patients with AFP≤200ng/ml. The serum GPC3+DCP is the best indicator of HCCearly diagnosis in patients with AFP≤200ng/ml.2. GPC3peptide-specific T-cell response and its related factor[Materials and Methods] The study included25patients with HBV-relatedHCC,22patients with HBV-related cirrhosis and25healthy control subjects. In thepresence or absence of anti-CD152antibodies, the GPC3peptide-specific T cellsresponse rates and the intensity of the reaction in PBMCs were detected by theELISPOT assay. T lymphocyte subsets were detected by the FC assay. Theconcentrations of serum CD152were detected by the ELISA assay.[Results]2.1GPC3peptide-induced specific T cells responses:Specific T cells response rates induced by GPC3298-306and GPC3144-152were33.3%and26.7%, the intensity of the reaction were106SFU/106PBMC and203SFU/106PBMC, which were higher than that induced by GPC3155-163and GPC344-52.In the case of co-stimulatory with anti-CD152antibodies and GPC3-peptide, thereaction rates were increased (19.5%vs.16.1%), but the intensity of the reaction wasreduced (58.75±13.7SFU/106PBMC vs.109.1±31.9SFU/106PBMC).The results showed that GPC3might be used as targets for immunotherapy, butthere was a large difference of immunogenic in different GPC3-peptide. Theimmunotherapy effect may be decreased when CD152antigen was closed.2.2Peripheral blood T lymphocyte subsets:Higher frequencies of CD3+, CD3+CD4+, CD4+CD25+FoxP3+,CD8+CD57+CD27+T cells and lower levels of CD4+CD28+CD27+, CD3+CD8+T cellswere observed in patients with HCC and cirrhosis than that control groups. TheCD8+CD28-CD27+T cells in cirrhosis groups were detectable at the reducedfrequencies. The CD8+GPC3-Tetramer+T cells were no significant difference in HCC,cirrhosis and control groups.The immune disorders in patients with HCC and cirrhosis exhibited increasedproportion of Treg cells and reduced level of cytotoxic T cells. The results showed that a higher proportion of cytotoxic T cells in the late stage of differentiation. Thereduced frequencies of CD8+CD28-CD27+T cells might be related with the increasedintensity of the GPC3-peptide-induced specific T cells responses.2.3Correlation in the serum GPC3, CD152and T cell subsets：Higher frequencies of CD4+CD28+CD27+T cells and lower levels ofCD8+GPC3-Tetramer+T cells were observed in cirrhosis patients withGPC3>1.5ng/ml. Higher frequencies of CD8+and CD8+GPC3-Tetramer+T cells andlower levels of CD8+CD28+/27-T cells were observed in HCCs with GPC3>1.5ng/ml.The concentrations of serum GPC3were negatively correlated with thefrequencies of CD8+GPC3-tetramer+T cells in cirrhosis and were positively correlatedwith that in HCC. The increased CD4+CD28-and CD8+CD28-T cells in patientswith HCC and cirrhosis might be not due to T cell activation by occupation of the Tcell receptor, because it was not accompanied by an increase in CD152expression.[Conclusions]2.4The results show that GPC3may be used as targets for immunotherapy. TheGPC3peptide-specific T cells exist in the peripheral blood of patients with HCC orcirrhosis, low levels of GPC3-specific T cells may exist in some of the healthypopulation. In ELISPOT assay, anti-CD152antibody may help to increase thedetection rates of GPC3peptide-specific T cells, but the role of CD152in the GPC3peptide-specific T cell responses requires further study.2.5The Features of the immune cells in HCC and cirrhosis are higher proportionof Treg and CD4+T cells, lower levels of CD8+T cells and higher frequencies ofCD28-T cells. It suggests that the changes of T-cell subsets in cirrhosis patients mayincrease the risk of liver cancer.2.6The high levels of serum GPC3in cirrhosis and HCC may be have differenteffects on the changes of T-cell subsets; further research is needed to understand itsmechanism and significance. |
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