Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.     

Breast cancer micrometastases: Different interactions of carcinoma cells with normal and cancer patients' bone marrow stromata

The apparently dormant breast cancer micrometastases in haemopoietic marrow are correlated with distant metastatic carcinoma dissemination. We studied in vitro interactions of carcinoma cells with adjacent stromata, using connective tissue cell cultures from breast and bone marrow samples of normal donors, comparing them to the pericancerous breast tissue and bone marrows of 12 selected patients with invasive breast carcinomas. Cancer cells were detected by immunocytochemistry and RT-PCR in all the bone marrows and in most blood samples of the studied patients. We monitored the growth and interaction of carcinoma MCF-7 cells with the stromata. The normal breast stroma sustained typical massive cancer growth. The pericancerous breast stroma induced the invasive mesenchymal pattern of growth. Normal bone marrow stroma induced the same conversion and was highly adhesive, retaining the cells in the stroma, but carcinoma patients' bone marrow stromata underwent low adhesive interactions with cancer cells, releasing them potentially into the circulation. The semi-quantitative RT-PCR indicated an enhanced expression of the hepatocyte growth factor and its receptor c-met in breast and bone marrow stromata of cancer patients. The input of cancer cells into the normal bone marrow may induce modifications of the local microenvironment, favourable for growth and release of carcinoma cells into the systemic circulation, which correlate with the poor prognosis of patients with bone marrow micrometastases. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of N-(deoxyguanosin-8-yl)-4-azobiphenyl by (32)P-postlabeling analyses of DNA in human uroepithelial cells exposed to proximate metabolites of the environmental carcinogen 4-aminobiphenyl

DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), were analyzed by the (32)P-postlabeling method. Two adducts detected by (32)P-postlabeling were previously identified as the 3',5'-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955-961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295-301). In contrast to the dG-C8-ABP adduct, which was 3'-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-aminobiphenyl with deoxyguanosine-3'-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3'-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N(2)-yl)-4-azobiphenyl (dG-N==N-ABP). (32)P-Postlabeling analysis of the nuclease P1-enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-N==N-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA.     

Prognostic factors in laryngeal carcinoma: the role of apoptosis,p53, proliferation (Ki-67) and angiogenesis

Even though the roles of different known or suggested prognostic factors in laryngeal cancer have been studied in detail, clinical stage at time of diagnosis and anatomic subsite of the tumour remain the only practical predictors of clinical outcome and offer the only guidelines in the planning of treatment. In this study, the relative roles of known demographic and clinical prognostic factors, in addition to four histopathological factors, were evaluated in a sample of 100 laryngeal carcinoma patients with multivariate analysis using the Cox regression model. In addition to advanced stage (stage III-IV) (relative hazard of death (HR) 8.9, p=0.01) and supraglottic disease (HR 5.6, p=0.02), high apoptotic index (HR 11.1, p=0.05) was significantly associated with poor survival. Cell proliferation, p53 and angiogenesis did not significantly affect the prognosis. In the future, high degree of apoptosis could be used to identify patients with poor prognosis in laryngeal cancer.     

Overexpression of S100A4 is closely related to the aggressiveness of gastric cancer

Elevated levels of the calcium-binding protein S100A4 cause metastasis of benign rat mammary tumor cells. To investigate whether S100A4 plays an important role in the invasion and metastasis of gastric cancers, we examined the gene mutations in the coding regions and expression patterns of the S100A4 in gastric adenocarcinoma in Korea. Moderate to strong expression of S100A4 was found in 53 (68.8%) of the 77 gastric adenocarcinomas, whilst normal gastric epithelium either failed to stain or showed weak staining. Interestingly, S100A4 expression was more frequently observed in gastric cancer patients with advanced gastric cancer (p=0.039), positive lymph node metastasis (p=0.001), and peritoneal dissemination (p=0.022). No gene mutations were found in the analyzed genomic area in 77 gastric adenocarcinomas and 15 gastric cancer cell lines. We found one single nucleotide polymorphism without an amino acid change, A99G, in two cases. These data suggest that the overexpression of S100A4 may be closely related to the aggressiveness of gastric cancer in Korea.     

幽门螺杆菌cagA菌株感染对IL-8蛋白在胃癌组织中表达的影响

目的 探讨胃癌组织中HpcagA菌株感染对IL-8蛋白表达的影响。方法 采用流式细胞技术，对27例胃癌及相应的癌旁正常组织中IL-8蛋白的表达进行定量检测，用PCR法，对27例胃癌组织中HpcagA基因进行扩增。结果 27例胃癌组织中中，有25例（92%）可明显表达IL-8蛋白；而相应的癌旁正常组织中，基本没有IL-8蛋白的表达或仅有弱相应的癌旁正常组织中，基本没有IL-8蛋白的表达或仅有弱表达，HpcagA感染的胃癌组织中，IL-8的表达水平为64.27%，高于未感染HpcagA的胃癌组织（39.86%)。结论 IL-8在胃癌组织中的高表达与HpcagA感染有关。即HpcagA菌株感染可上调IL-8在胃癌组织中的表达水平。     

Immunological analysis of plasminogen activators from cultured human cancer cells

Summary Immunological similarities or differences between urokinase and plasminogen activators from 9 lines of cultured human caner cells with varying degrees of fibrinolytic activity were examined with antibodies against human urokinase.The antibodies completely inhibited the fibrinolytic activity of 4 lines of gastric cancer, 2 lines of lung cancer, 1 line of urinary bladder cancer and 1 line of renal cancer, indicating that the plasminogen activators from these cell lines were immunologically identical to urokinase. In 5 out of these cell lines, immunological identity was also confirmed by double diffusion analysis.The plasminogen activator from 1 line of lung cancer was found to be immunologically dissimilar to urokinase by a neutralization experiment and double diffusion analysis.These findings indicate that there are at least two immunologically distinguishable forms of plasminogen activators from human cancer cells.     

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

Association of common missense changes in ELAC2 (HPC2) with prostate cancer in a Japanese case–control series

 The recently identified prostate cancer susceptibility gene ELAC2 (HPC2) harbors two common missense variants, a serine to leucine substitution at residue 217 (Leu217) and an alanine to threonine substitution at residue 541 (Thr541). We genotyped the two variants in a Japanese cohort consisting of 350 prostate cancer patients 242 male population controls, and 114 male low-risk controls. Both missense alleles, Leu217 and Thr541, were carried at higher frequency in Japanese patients than in the controls (Leu217, P = 0.0012; Thr541, P = 0.0145), and the odds ratios associated with carrying these sequence variants were higher in Japanese than in Caucasians. Although the Leu217 and Thr541 variants of ELAC2 are less common in Japanese than in Caucasians, both variants confer significantly increased risk of prostate cancer in Japanese. Carriage of these variants was not associated with age at diagnosis, tumor stage, or tumor grade in these Japanese prostate cancer patients. The allele-specific pattern of risk observed in Japanese and familial Caucasian patients was qualitatively similar; however, the magnitude of that risk was considerably greater in Japanese than in Caucasians. Received: September 3, 2002 / Accepted: October 2, 2002