Immunohistochemical expression of pi class glutathione S-transferase and alpha-fetoprotein in hepatocellular carcinoma and chronic liver disease

OBJECTIVE: To determine whether tumor marker pi glutathione transferase (GST-pi) is expressed in hepatocellular carcinoma (HCC) and other chronic liver diseases and to compare its expression with that of alpha-fetoprotein (AFP). STUDY DESIGN: Samples used were formalin-fixed, paraffin-embedded liver tissues: normal (n = 3), chronic hepatitis B (n = 15), cirrhosis (n = 15) and HCC (n = 30). The expression of AFP and GST-pi was detected by using immunohistochemistry with the peroxidase-antiperoxidase method. AFP immunoreactivity was based on the cytoplasm of the hepatocytes, while GST-pi immunoreactivity was based on the nuclei of hepatocytes. RESULTS: In normal liver tissues, AFP was not expressed. However, there was strong staining of GST-pi in bile duct epithelium cells and weak staining in hepatocytes. Our results showed higher AFP immunoreactivity in cases of HCC (36.7%) as compared to cirrhosis (6.7%) and hepatitis B (0%), whereas GST-pi immunoreactivity was lower in cases of HCC (53.3%) as compared to cases of cirrhosis (100.0%) and hepatitis B (93.3%). Percent sensitivity of AFP determination for HCC was 36.7% as compared to 53.3% for GST-pi, thus making GST-pi a more sensitive marker for detection of HCC. This study showed a significant relationship between the intensity and percentage of cells stained in hepatitis B, cirrhosis and HCC for GST-pi immunoreactivity (P < .001, .001 and .05, respectively) but not for AFP (P > .05). Statistical analysis showed that there was no significant relationship between expression of AFP and GST-pi in cirrhosis and HCC cases. Hepatitis B virus infection in HCC cases showed a positive rate of 46.7%, with AFP staining positively in 42.9% of tissues and GST-pi staining positively in 57.1% of tissues. CONCLUSION: AFP is a diagnostic but rather insensitive tissue marker for HCC. However, the absence of AFP in benign chronic liver disease makes this marker useful in differentiating between HCC and other chronic liver diseases, whereas GST-pi can be used as a diagnostic marker for HCC as well as in detecting other chronic liver diseases.     

Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.     

Pre-existing immunity to tyrosinase-related protein (TRP)-2, a new TRP-2 isoform, and the NY-ESO-1 melanoma antigen in a patient with a dramatic response to immunotherapy.

We have performed a detailed analysis of the recognition of melanoma Ags by the tumor-infiltrating lymphocytes (TIL) 1790, isolated from a patient who experienced a dramatic tumor regression following immunization with peptides from the gp100, MART-1, and tyrosinase Ags. This TIL was found to recognize HLA-A2-restricted CTL epitopes in tyrosinase-related protein (TRP)-2 (clone MR7) and NY-ESO-1 (clone M8). These epitopes were the same as the previously identified nonapeptide TRP-2: 180-188, and the overlapping NY-ESO-1 peptides, obtained by using lymphocytes from in vitro stimulation. We also cloned a previously unknown TRP-2 mRNA isoform (TRP-2-6b) that contained two novel exons alternatively spliced from the sixth intron between exons 6 and 7 of TRP-2 mRNA. The isoform encoded an HLA-A2-restricted antigenic epitope recognized by TIL clone MB4. An immunologic analysis of the patient's PBMC obtained before treatment showed the presence of high reactivity against NY-ESO-1 and both TRP-2 Ags, but not the Ags used for immunization. Because immune response against these Ags was less pronounced, it is possible that NY-ESO-1, TRP-2, and TRP-2-6b may be of importance in the generation of CTL-mediated tumor destruction and may have played a role in the dramatic tumor regression seen in this patient.     

A cluster of Ankyrin and Ankyrin-TPR repeat genes is associated with panicle branching diversity in rice

The number of grains per panicle is an important yield-related trait in cereals which depends in part on panicle branching complexity. One component of this complexity is the number of secondary branches per panicle. Previously, a GWAS site associated with secondary branch and spikelet numbers per panicle in rice was identified. Here we combined gene capture, bi-parental genetic population analysis, expression profiling and transgenic approaches in order to investigate the functional significance of a cluster of 6 ANK and ANK-TPR genes within the QTL. Four of the ANK and ANK-TPR genes present a differential expression associated with panicle secondary branch number in contrasted accessions. These differential expression patterns correlate in the different alleles of these genes with specific deletions of potential cis-regulatory sequences in their promoters. Two of these genes were confirmed through functional analysis as playing a role in the control of panicle architecture. Our findings indicate that secondary branching diversity in the rice panicle is governed in part by differentially expressed genes within this cluster encoding ANK and ANK-TPR domain proteins that may act as positive or negative regulators of panicle meristem’s identity transition from indeterminate to determinate state.     

Deleted in Liver Cancer 2 (DLC2) Was Dispensable for Development and Its Deficiency Did Not Aggravate Hepatocarcinogenesis

DLC2 (deleted in liver cancer 2), a Rho GTPase-activating protein, was previously shown to be underexpressed in human hepatocellular carcinoma and has tumor suppressor functions in cell culture models. We generated DLC2-deficient mice to investigate the tumor suppressor role of DLC2 in hepatocarcinogenesis and the function of DLC2 in vivo. In this study, we found that, unlike homologous DLC1, which is essential for embryonic development, DLC2 was dispensable for embryonic development and DLC2-deficient mice could survive to adulthood. We also did not observe a higher incidence of liver tumor formation or diethylnitrosamine (DEN)-induced hepatocarcinogenesis in DLC2-deficient mice. However, we observed that DLC2-deficient mice were smaller and had less adipose tissue than the wild type mice. These phenotypes were not due to reduction of cell size or defect in adipogenesis, as observed in the 190B RhoGAP-deficient mouse model. Together, these results suggest that deficiency in DLC2 alone does not enhance hepatocarcinogenesis.