Primary pulmonary hyalinizing clear cell carcinoma with fusions of both EWSR1::CREM and IRF2::NTRK3: report of a case with an aggressive behavior.

Primary pulmonary hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland-type tumor newly recognized in recent years, with approximately 21 cases reported to date in the English literature, which constitutes a challenge in pathology diagnosis, particularly in small biopsy specimens. Here, we present a case of pulmonary HCCC diagnosed by computed tomography-guided percutaneous lung biopsy in a 70-year-old man's right lower lung. Although the morphology and immunophenotype of the tumor suggested the diagnosis of mucoepidermoid carcinoma, fluorescence in situ hybridization failed to reveal the rearrangement of MAML2 gene, which is characteristic of mucoepidermoid carcinoma. Instead, further molecular genetic testing showed that the tumor harbored a rare EWSR1::CREM fusion combined with a previously unreported IRF2::NTRK3 fusion. Pulmonary HCCC is commonly regarded as a low-grade malignant tumor with an indolent course, but this case has a different biological behavior, presenting extensive dissemination and metastases at the time of diagnosis, which expands our understanding of the prognosis of this tumor. The patient has had five cycles of combination chemotherapy and has been alive with the tumor for eight months.

Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis.

BACKGROUND: Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified. METHODS: Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis. RESULTS: NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients. CONCLUSION: Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.

MR molecular imaging of tumors based on an optimal hTERT promoter tyrosinase expression system.

The early diagnosis and treatment of tumors is of vital significance to increase patient survival. Therefore, we constructed a lentiviral vector expressing tyrosinase (TYR) driven by an optimized human telomerase reverse transcriptase (hTERT) promoter or a cytomegalovirus(CMV) promoter in the hopes of performing noninvasive and real-time tumor-specific imaging. First, hTERT-TYR and CMV-TYR were constructed to infect cancer cell lines (telomerase-negative cell line: U2OS; telomerase-positive cell lines: SGC-7901, SW480 and HepG2). Subsequently, stable tyrosinase-expressing cell lines were sorted by flow cytometry out of these infected cancer cell lines. Then, the mRNA and protein levels of tyrosinase were analyzed. Thetyrosinase activity, melanin production and ferric ion adsorption were measured followed by an MR scan. Consequently the results showed that tyrosinase was only expressed in telomerase-positive tumor cells infected by hTERT-TYR, whereas tyrosinase was expressed in both telomerase-negative and telomerase-positive tumor cells infected by CMV-TYR. Finally, we performed in vivo tumor MR using a clinical 3T MR scanner and found increased signals at T1W1 from telomerase-positive cells infected by hTERT-TYR, which revealed that MR scanning could distinguish cells with hTERT -positive cells from hTERT-negative cells infected with the optimized lentivirus. The mechanism underlying this effect is that tyrosinase promotes melanin production and ferric ion adsorption only in hTERT-expressing cells. Taken together, these data show that this optimized hTERT promoter-driving tyrosinase expression system might be a useful diagnostic tool for the detection of tumors using MR imaging.

hTERT mediates gastric cancer metastasis partially through the indirect targeting of ITGB1 by microRNA-29a.

Human telomerase reverse transcriptase (hTERT) plays a key role in tumor invasion and metastasis, but the mechanism of its involvement in these processes is not clear. The purpose of this study is to investigate the possible molecular mechanism of hTERT in the promotion of gastric cancer (GC) metastasis. We found that the up-regulation of hTERT in gastric cancer cells could inhibit the expression of miR-29a and enhance the expression of Integrin ß1 (ITGB1). In addition, the invasive capacity of gastric cancer cells was also highly increased after hTERT overexpression. Our study also found that the restoration of miR-29a suppressed the expression of ITGB1 and inhibited GC cell metastasis both in vitro and in vivo. Taken together, our results suggested that hTERT may promote GC metastasis through the hTERT-miR-29a-ITGB1 regulatory pathway.

Effects of the total flavonoid extract of Xiaobuxin-Tang on depression-like behavior induced by lipopolysaccharide and proinflammatory cytokine levels in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaobuxin-Tang (XBXT), a traditional Chinese herbal decoction, has been used for the treatment of depressive disorders from ancient clinic. The aim of the study was to explore the involvement of inflammation or inflammatory markers in the antidepressant-like effects of XBXT-2. MATERIALS AND METHODS: Depression-like behavior was induced by lipopolysaccharide (LPS, 0.2mg/kg, i.p) in tail suspension test (TST) and forced swimming test (FST) in mice. The effects of the total flavonoids (XBXT-2) extracted from XBXT (25, 50, and 100mg/kg, p.o.) and duloxetine (DLX, 10mg/kg, p.o.) on the immobility time in TST and FST were determined 24h after LPS pretreatment. The locomotor activity was also determined to eliminate the false-positive activity. Additionally, in order to further evaluate the effect of XBXT-2 on inflammation, the levels of brain proinflammatory cytokines including IL-1ß and TNF-α were assessed by ELISA. RESULTS: The pretreatment with LPS significantly increased the immobility time in TST and FST in mice, as well as the brain levels of IL-1ß and TNF-α. XBXT-2 (25, 50, and 100mg/kg, p.o.) administration decreased the duration of immobility in TST and FST, and normalized the cytokines levels. The positive control DLX (10mg/kg, p.o.) exerted similar effects. Meanwhile, neither LPS pretreatment nor drugs treatment had any effect on mouse locomotor activity. CONCLUSIONS: These results suggest that inflammation and inflammatory cytokines may be involved in the antidepressant-like effects of XBXT-2.

Noninvasive and real-time monitoring of the therapeutic response of tumors in vivo with an optimized hTERT promoter.

BACKGROUND: Telomerase is commonly recognized as an effective anticancer target. The human telomerase reverse transcriptase (hTERT), the rate-limiting component of telomerase, is expressed in most malignant tumors, but it is not found in most normal somatic cells. Here, we report a real-time and noninvasive method to monitor tumor response to a lentivirus-based hTERT-conditional suicidal gene therapy. METHODS: In this study, we constructed a lentivirus system in which an optimized hTERT promoter was used to drive the expression of the cytosine deaminase (CD) gene, one of the suicide genes, and a green fluorescent protein (GFP) reporter gene (pLenti-CD/GFP). The lentivirus was used to infect telomerase-positive or telomerase-negative cell lines. In vitro and in vivo experiments were conducted to analyze the dynamic processes of exogenous gene expression noninvasively in cell culture and living animals in real time via optical imaging. RESULTS: The lentivirus was able to express the CD gene and GFP in telomerase-positive tumor cells and significantly decrease cell proliferation after the use of prodrug 5-flucytosine. However, it could not express GFP and CD in telomerase-negative cell lines, nor could it induce any suicidal effect in those cells. The in vivo study showed that telomerase-positive tumors can be visualized after intratumor injection of the lentivirus in tumor-bearing nude mice via an optical imaging system. Significant tumor growth suppression was observed in telomerase-positive tumors. CONCLUSIONS: Collectively, this technology provides a valuable, noninvasive method to evaluate the real-time therapeutic response of tumors in vivo.

Multimodal therapy for adult Wilms' tumour: an experience from one centre.

INTRODUCTION: Wilms' tumour (WT) is very rare in adults but very common in children. Treatment guidelines for adult patients with WT are still insufficient. Some study groups recommend that therapeutic protocols for adults with WT (AWT) should follow the guidelines that have been established for children. OBJECTIVE: To describe the clinical and pathological characteristics of AWT as well as the treatment protocols and outcomes for AWT at our treatment centre. MATERIAL AND METHODS: Seven patients (5 females and 2 males) were diagnosed with AWT in our hospital between 2002 and 2009. The tumours were staged and the patients were treated according to the paediatric regimen recommended by the National Wilms' Tumor Study Group. RESULTS: The median patient age at the time of diagnosis was 29 years (range, 16-37 years). Flank pain was the most common clinical presentation. One patient was in Stage I of disease development, two were in Stage II, two were in Stage III and two were in Stage IV. Anaplasia was present in 3 patients with Stage III or Stage IV disease. All of the patients but one underwent nephrectomy and 2 incomplete surgeries were performed. Seven patients received 2-drug or 3-drug chemotherapy (dactinomycin and vincristine and/or doxorubicin). Two patients with Stage III disease also received radiation therapy (a total dose of 3600 or 3960 cGy). Complete remission was achieved in 4 patients. Three patients (one with Stage III disease, 2 patients with Stage IV disease) died of their disease and those patients were all classified with an unfavourable histological type called anaplasia. With a median follow-up of 53.5 months (range, 40-102 months), the 3-year and 5-year overall survival rates were 57.1% (95% confidence interval, 20.4-93.8%). CONCLUSIONS: The results of this report suggest that histological anaplasia might be an adverse prognostic factor for AWT. Proper application of the diagnostic and therapeutic regimens established for children may improve the prognosis of adult patients with WT.

Inhibition of tankyrase 1 in human gastric cancer cells enhances telomere shortening by telomerase inhibitors.

Telomere stability is believed to be related to aging and tumorigenesis. Besides telomerase, telomere length is also regulated by several telomere-specific binding proteins. Tankyrase 1, a telomeric poly(ADP-ribose) polymerase (PARP), elongates telomere length by inhibiting TRF1 binding to telomeres. In order to study the synergistic action of tankyrase 1 and telomerase in the maintenance of telomere length in mammalian cells, we constructed anti-sense tankyrase 1 (aTNKS) eukaryotic expression vectors and then transfected them into the SGC-7901 human gastric cancer cell line, as well as SGC-7901 cells that had been transfected with antisense hTR (7901-ahTR) and antisense hTERT (7901-ahTERT) with DOTAP liposomes. The activity of telomerase, telomere length and telomerase-associated protein activities were measure by TRAP-ELISA, Southern blot and western blot analysis, respectively, in aTNKS transfected and untransfected cells. The results demonstrated that telomere length was significantly shorter in cells with concomitant tankyrase 1 and telomerase inhibition than by either tankyrase 1 or telomerase inhibition alone, in SGC-7901 cells. We also found that aTNKS had no effect on telomerase activity. These results reveal that inhibition of tankyrase 1 could shorten telomere length and play a synergistic role with telomerase inhibitors in telomere length shortening in the SGC-7901 gastric cancer cell line. Co-inhibition of tankyrase 1 and telomerase activity may be a rational strategy for telomere-directed gastric cancer therapeutics.

An optimized telomerase-specific lentivirus for optical imaging of tumors.

Advances in medical imaging techniques, such as ultrasound, computed tomography, magnetic resonance imaging, and positron emission tomography, have made great progress in detecting tumors. However, these imaging techniques are unable to differentiate malignant tumors from benign ones. Recently developed optical imaging of tumors in small animals provides a useful method to distinguish malignant tumors from their surrounding normal tissues. Human telomerase reverse transcriptase (hTERT) is normally inactivated in most somatic cells, whereas it is commonly reactivated in many cancer cells. In this study, we constructed a lentiviral vector that expresses green fluorescent protein (GFP) driven by an optimized hTERT promoter to create a noninvasive tumor-specific imaging methodology. The activity of this optimized hTERT promoter was found to be equal to the activity of SV40 and cytomegalovirus promoters. In vitro experiments showed that GFP was only expressed in telomerase-positive tumor cells infected with this lentivirus, whereas there was no GFP expression in telomerase-negative tumor cells or normal somatic cells. We also found that subcutaneous telomerase-positive tumors could be visualized 24 hours after an intratumoral injection with this lentivirus by using a charge-coupled device (CCD) camera. In contrast, telomerase-negative tumors could not be imaged after an intratumoral injection even for 30 days. These results suggest that infection with lentivirus containing this optimized hTERT promoter might be a useful diagnostic tool for the real-time visualization of macroscopically invisible tumor tissues using a highly sensitive CCD imaging system.

Cytotoxic T lymphocyte epitopes from human heparanase can elicit a potent anti-tumor immune response in mice.

Heparanase is expressed in almost all advanced tumors, and therefore it may serve as a potential target for tumor therapy. Our previous study has shown that heparanase can serve as a potential universal tumor-associated antigen (TAA) for the immunotherapy of advanced tumors. Further study demonstrated that the HLA-A*0201-restricted Cytotoxic T lymphocytes (CTL) epitopes Hpa525 (PAFSYSFFV), Hpa277 (KMLKSFLKA) and Hpa405 (WLSLLFKKL) from human heparanase could induce a potent anti-tumor immune response in vitro. The present study was designed to investigate whether the above peptides could induce immune responses in mice. Our results demonstrated that the effectors from heparanase peptide-immunized mice could effectively lyse various tumor cells that were heparanase positive and HLA-A*0201 matched. We also found that these peptide-specific CTLs did not lyse autologous lymphocytes that had low heparanase activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-gamma-producing T cells as compared to a negative peptide. These results suggest that Hpa525, Hpa277, and Hpa405 peptides are novel HLA-A*0201-restricted CTL epitopes capable of inducing heparanase-specific CTLs in mice. Because heparanase is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy of patients with advanced tumors.

hTERT promotes the invasion of telomerase-negative tumor cells in vitro.

Telomerase activation is a common feature of most types of human cancers. Although several studies have shown that activation of telomerase might participate in the progression of tumors, the mechanism by which telomerase activation causes the invasion and metastasis of tumors remains unclear. In this study, we transfected a vector containing the full-length cDNA of hTERT into a telomerase-negative osteosarcoma cell line U2OS (hTERT/U2OS). Vacant vector-transfected U2OS cells served as a control (EGFP/U2OS). We then compared the biological and vitodynamic changes in these transfected and untransfected U2OS cells. The hTERT protein was detected in hTERT/U2OS cells by Western blot analysis and immunochemistry assay. The telomere length in hTERT/U2OS cells was longer than that in EGFP/U2OS and untransfected U2OS cells. We also found using vacuum micropipette aspiration that hTERT transfection did not only promote the proliferation of hTERT-transfected U2OS cells but also increased the cellular adhesion capacity to the extracellular matrix. Transwell matrigel assay confirmed an increased invasion ability in hTERT/U2OS cells. These results strongly suggest that hTERT transfection promotes the invasion of telomerase-negative cells. Telomerase-mediated telomere maintenance enables these cells to achieve a fully malignant endpoint, including invasion and metastasis.

Induction of anti-tumour immunity by dendritic cells transduced with hTERT recombinant adenovirus in mice.

Dendritic cells (DCs) transfected with recombinant, replication-defective adenovirus (Ad) vectors encoding the human telomerase reverse transcriptase (hTERT) are potent inducers of cytotoxic T lymphocytes (CTLs) and anti-tumour immunity. However, previous studies have mostly been in vitro. In this study, we sought to determine whether DCs transfected with hTERT (DC/Ad-hTERT) could elicit a potent anti-tumour immunogenic response in vivo. We found that murine DCs transfected with recombinant adenovirus encoding the hTERT gene (DC/Ad-hTERT) induced hTERT-specific CTLs in vivo effectively, compared with Ad-LacZ-transduced DC (DC/Ad-LacZ) controls. These hTERT-specific CTLs lysed various tumour cell lines in an hTERT-specific and MHC-I molecule-restricted fashion. We also found that DC/Ad-hTERT could increase antigen-specific T-cell proliferation and augment the number of IFN-gamma secreting T-cells in mice. These data suggest that the DC/Ad-hTERT vaccine may induce anti-tumour immunity against tumour cells expressing hTERT in an MHC-I molecule-restricted fashion in vivo through the augmentation of the hTERT-specific CTL response. The DC/Ad-hTERT vaccine may thus be used as an efficient DC-based tumour vaccine in clinical applications.

HLA-A2-restricted cytotoxic T lymphocyte epitopes from human heparanase as novel targets for broad-spectrum tumor immunotherapy.

Peptide vaccination for cancer immunotherapy requires identification of peptide epitopes derived from antigenic proteins associated with tumors. Heparanase (Hpa) is broadly expressed in various advanced tumors and seems to be an attractive new tumor-associated antigen. The present study was designed to predict and identify HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in the protein of human Hpa. For this purpose, HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: 1) a computer-based epitope prediction from the amino acid sequence of human Hpa, 2) a peptide-binding assay to determine the affinity of the predicted protein with the HLA-A2 molecule, 3) stimulation of the primary T-cell response against the predicted peptides in vitro, and 4) testing of the induced CTLs toward different kinds of carcinoma cells expressing Hpa antigens and/or HLA-A2. The results demonstrated that, of the tested peptides, effectors induced by peptides of human Hpa containing residues 525-533 (PAFSYSFFV, Hpa525), 277-285 (KMLKSFLKA, Hpa277), and 405-413 (WLSLLFKKL, Hpa405) could effectively lyse various tumor cell lines that were Hpa-positive and HLA-A2-matched. We also found that these peptide-specific CTLs could not lyse autologous lymphocytes with low Hpa activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-gamma-producing T cells compared to a negative peptide. Our results suggest that Hpa525, Hpa277, and Hpa405 peptides are new HLA-A2-restricted CTL epitopes capable of inducing Hpa-specific CTLs in vitro. Because Hpa is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy for patients with advanced tumors.

H-2Kb-restricted CTL epitopes from mouse heparanase elicit an antitumor immune response in vivo.

The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancer-specific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumor-associated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K(b)-restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptide-binding assay to determine the affinity of the predicted epitopes with the H-2K(b) molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K(b), and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K(b) (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumor-bearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K(b)-restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies.

Dendritic cells reconstituted with a human heparanase gene induce potent cytotoxic T-cell responses against gastric tumor cells in vitro.

BACKGROUND AND AIMS: Dendritic cell-based tumor vaccination is a promising approach in the treatment of cancer. Strategies to modify dendritic cells (DCs) with tumor-associated antigens (TAAs) can elicit specific immune responses against tumors. Heparanase is overexpressed in gastric cancer, especially in invasive and metastatic cells, but is downregulated in differential normal tissue. Therefore, heparanase is a potential target in immunotherapy for patients with advanced gastric cancer who are not candidates for surgery. The present paper was designed to investigate the immune response of a heparanase gene-modified DC-based vaccine against gastric cancer cell lines in vitro. METHODS: DCs from peripheral blood mononuclear cells of healthy HLA-A2-positive donors were transfected with recombinant adenovirus containing the full-length cDNA of heparanase (rAd-Hpa) to generate heparanase gene-modified DC vaccine. T lymphocytes from the same donors were repeatedly activated by genetically modified DC vaccine to generate heparanase-specific cytotoxicity T lymphocytes (CTLs). CTL-mediated cell lysis of gastric cancer cells lines (KATO-III and SGC-7901) was analyzed in vitro by a standard (51)Cr releasing assay. IFN-gamma secretion was measured by ELISA in heparanase-specific CTLs cocultured with those gastric cancer cell lines. RESULTS: Our results showed that the expression of heparanase in DCs transfected with rAd-Hpa was significantly increased. Furthermore, DCs transfected with rAd-Hpa could induce heparanase-specific CTLs against HLA-matched and heparanase-positive gastric cancer cells in vitro, while there were no killing effects on autologous lymphocytes. Meanwhile, these rAd-Hpa-modified DCs could increase IFN-gamma secretion of effector cells when cocultured with KATO-III cells. CONCLUSIONS: These findings demonstrate for the first time that the transduction of DCs with rAd-Hpa can induce CTLs that specifically lyse heparanase-positive gastric cancer cells and increase IFN-gamma secretion in an MHC-restricted fashion. Heparanase gene-modified DC vaccine offers a great opportunity for immunotherapy in patients with advanced gastric cancer and possibly also with other malignancies.

In vitro anti-tumor immune response induced by dendritic cells transfected with hTERT recombinant adenovirus.

Transduction with recombinant, replication-defective adenoviral (Ad) vectors encoding a transgene is an efficient method for gene transfer into human dendritic cells (DC). Several studies have demonstrated that epitopes of the human telomerase reverse transcriptase gene (hTERT) can produce CTLs specific for malignant tumors. In this study, we constructed an hTERT recombinant adenovirus (rAd-hTERT) using DNA recombination. We found that human dendritic cells transduced with rAd-hTERT could effectively induce hTERT-specific cytotoxic T lymphocytes (CTLs) in vitro against various tumor cell lines, which were hTERT-positive and HLA-A2 matched. We also found that these hTERT-specific CTLs could not lyse autologous lymphocytes with low telomerase activity. Further studies revealed that rAd-hTERT transduced DCs could increase secretion of IFN-gamma by effector cells when they were co-cultured with hTERT-positive and HLA-A2 matched tumor cell lines. These data suggest that an hTERT vaccine can induce anti-tumor immunity against various tumor cells expressing hTERT in a HLA-A2-restricted fashion in vitro. The transduction of DCs with rAd-hTERT offers a great opportunity in cancer immunotherapy.

Identification of genes differentially expressed in human hepatocellular carcinoma by a modified suppression subtractive hybridization method.

To identify differentially expressed genes in human HCC in China, we applied a modified SSH method for cDNA subtraction. Such modification has made the method more effective for subtraction. We have obtained 36 and 24 differentially expressed cDNA fragments after modified SSH from 4 paired samples of human HCC and non-HCC tissues, respectively. Reverse Northern blotting analysis was performed to further identify the genes differentially expressed in the HCC and non-HCC tissue samples. There were 25 genes really overexpressed in HCC, and their corresponding encoding molecules may reflect the events of cell accelerated metabolism, proliferation, angiogenesis, anti-apoptosis, tumorigenesis (TLH107, TFH9) and the potential for metastasis. Of the 25 genes overexpressed in HCC, 5 were novel and their full-length cDNAs were cloned. These 5 novel genes are functionally associated with the occurrence and development of HCC according to the Database analysis. In the paired non-HCC tissues, there were 15 genes lowly or not expressed in HCC, and their encoding proteins function as tumor suppressors (TFA3, TFA11), acute-phase reactive proteins, and the blood plasma proteins that are mainly or exclusively synthesized in the liver. The distinct profiles of the differentially expressed genes in HCC and the paired non-HCC tissues have partially reflected the genetic alterations during HCC tumorigenesis. The novel HCC-specific gene TLH6 and the CT antigen encoding gene TLH107 may have diagnostic and therapeutic potentials in HCC and/or other solid cancers.