A prognostic regulatory pathway in stage I epithelial ovarian cancer: new hints for the poor prognosis assessment.

BACKGROUND: Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. PATIENTS AND METHODS: We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. RESULTS AND CONCLUSIONS: Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, 'Activins/Inhibins' and 'Hedgehog' signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.

Profiling cancer gene mutations in longitudinal epithelial ovarian cancer biopsies by targeted next-generation sequencing: a retrospective study.

BACKGROUND: The majority of patients with stage III-IV epithelial ovarian cancer (EOC) relapse after initially responding to platinum-based chemotherapy, and develop resistance. The genomic features involved in drug resistance are unknown. To unravel some of these features, we investigated the mutational profile of genes involved in pathways related to drug sensitivity in a cohort of matched tumors obtained at first surgery (Ft-S) and second surgery (Sd-S). PATIENTS AND METHODS: Matched biopsies (33) taken at Ft-S and Sd-S were selected from the 'Pandora' tumor tissue collection. DNA libraries for 65 genes were generated using the TruSeq Custom Amplicon kit and sequenced on MiSeq (Illumina). Data were analyzed using a high-performance cluster computing platform (Cloud4CARE project) and independently validated. RESULTS: A total of 2270 somatic mutations were identified (89.85% base substitutions 8.19% indels, and 1.92% unknown). Homologous recombination (HR) genes and TP53 were mutated in the majority of Ft-S, while ATM, ATR, TOP2A and TOP2B were mutated in the entire dataset. Only 2% of mutations were conserved between matched Ft-S and Sd-S. Mutations detected at second surgery clustered patients in two groups characterized by different mutational profiles in genes associated with HR, PI3K, miRNA biogenesis and signal transduction. CONCLUSIONS: There was a low level of concordance between Ft-S and Sd-S in terms of mutations in genes involved in key processes of tumor growth and drug resistance. This result suggests the importance of future longitudinal analyses to improve the clinical management of relapsed EOC.

Mammaglobin B (SCGB2A1) is a novel tumour antigen highly differentially expressed in all major histological types of ovarian cancer: implications for ovarian cancer immunotherapy.

BACKGROUND: We studied the genetic fingerprints of ovarian cancer and validated the potential of Mammaglobin b (SCGB2A1), one of the top differentially expressed genes found in our analysis, as a novel ovarian tumour rejection antigen. METHODS: We profiled 70 ovarian carcinomas including 24 serous (OSPC), 15 clear-cell (CC), 24 endometrioid (EAC) and 7 poorly differentiated tumours, and 14 normal human ovarian surface epithelial (HOSE) control cell lines using the Human HG-U133 Plus 2.0 chip (Affymetrix). Quantitative real-time PCR and immunohistochemistry staining techniques were used to validate microarray data at RNA and protein levels for SCGB2A1. Full-length human-recombinant SCGB2A1 was used to pulse monocyte-derived dendritic cells (DCs) to stimulate autologous SCGB2A1-specific cytotoxic T-lymphocyte (CTL) responses against chemo-naive and chemo-resistant autologous ovarian tumours. RESULTS: Gene expression profiling identified SCGB2A1 as a top differentially expressed gene in all histological ovarian cancer types tested. The CD8+ CTL populations generated against SCGB2A1 were able to consistently induce lysis of autologous primary (chemo-naive) and metastatic/recurrent (chemo-resistant) target tumour cells expressing SCGB2A1, whereas autologous HLA-identical noncancerous cells were not lysed. Cytotoxicity against autologous tumour cells was significantly inhibited by anti-HLA-class I (W6/32) monoclonal antibody. Intracellular cytokine expression measured by flow cytometry showed a striking type 1 cytokine profile (i.e., high IFN-γ secretion) in SCGB2A1-specific CTLs. CONCLUSION: SCGB2A1 is a top differentially expressed gene in all major histological types of ovarian cancers and may represent a novel and attractive target for the immunotherapy of patients harbouring recurrent disease resistant to chemotherapy.

Diagnostic and prognostic impact of serum HE4 detection in endometrial carcinoma patients.

BACKGROUND: To date, no good marker for screening or disease monitoring of endometrial cancer (EC) is available. The aims of this study were to investigate HE4 gene, protein expression and serum HE4 (sHE4) levels in a panel of ECs and normal endometria (NEs) and to correlate sHE4 with patient clinicopathological characteristics and prognosis. METHODS: Using quantitative real-time PCR we tested 46 ECs and 20 NEs for HE4 gene expression. Protein expression was analysed by immunohistochemistry on tissue microarrays in 153 ECs and 33 NEs. Pre-operative serum samples from 138 EC and 76 NE patients were analysed with HE4-EIA assay. Association between sHE4 and patient clinicopathological characteristics or outcome was evaluated. RESULTS: Protein and HE4 gene were significantly upregulated in EC tissues and sera, compared with controls. High sHE4 levels were significantly associated with worse EC clinical characteristics. By univariate survival analysis, high sHE4 levels significantly correlated with decreased overall survival, progression-free survival and disease-free survival, retaining their independent prognostic value on the poorly differentiated EC cohort. CONCLUSION: We demonstrate, for the first time, that high sHE4 levels correlates with an aggressive EC phenotype and may constitute an independent prognostic factor for poorly differentiated-ECs. Determination of sHE4 could be clinically useful in identifying high-risk EC patients for a more aggressive adjuvant therapy.

Trefoil factor 3: a novel serum marker identified by gene expression profiling in high-grade endometrial carcinomas.

This study identifies the genetic fingerprint of poorly differentiated endometrioid endometrial carcinomas (G3-EEC) and analyses the potential utility of trefoil factor 3 (TFF3) as novel serum marker in G3-EEC. Affymetrix microarrays were used to identify the gene expression patterns of 19 snap-frozen G3-EEC and 15 normal endometrium (NE) biopsies. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry were used to validate TFF3 expression. Finally, TFF3 serum levels were determined by ELISA in 25 G3-EEC patients, 42 healthy controls, and in 13 endometrial hyperplasia patients. Hierarchical cluster analysis showed TFF3 as the top differentially expressed gene between 363 upregulated genes in G3-EEC, when compared with NE. Trefoil factor 3 gene expression levels analysed by qRT-PCR significantly correlated with Affymetrix results (P<0.001; rs=0.85). By immunohistochemistry, TFF3 protein was significatively more expressed in EEC compared with NE (P<0.01), with cytoplasmatic positivity in 79% G3-EEC and 18% NE. Patients harbouring G3-EECs had significantly higher TFF3 serum concentration by ELISA when compared with healthy patients (P<0.001) or patients harbouring endometrial hyperplasia (P=0.012). In conclusion, TFF3 is highly expressed at gene and protein level in G3-EEC. Further investigations on a wider set of samples are warranted to validate TFF3 as a novel serum marker for early detection and/or monitoring of G3-EEC patients.

Claudin-7 expression in human epithelial ovarian cancer.

Claudin-7 (CLDN-7) is a tight junction protein recently found highly differentially expressed in ovarian carcinoma. To evaluate its potential as a novel biomarker, in this study, we quantified and compared claudin-7 expression at messenger RNA and protein level in 110 patients harboring various histologic types of epithelial ovarian carcinomas (EOC). CLDN-7 transcript was found significantly overexpressed in both primary and metastatic EOCs compared to normal human ovarian surface epithelium cell lines (fold change = 111.4, P < 0.001) by reverse transcription-polymerase chain reaction. At the protein level, CLDN-7 expression was found significantly higher in tumors of primary and metastatic origin when compared to normal ovaries (P < 0.001), regardless of the histologic type, the grade of differentiation, and the pathologic stage of the disease (P = 0.12). Moreover, a strong immunoreactivity for CLDN-7 was detected in EOC cells present in ascites fluids, whereas ascites-derived inflammatory cells, histiocytes, and reactive mesothelial cells were negative. Finally, immunohistochemical expression of CLDN-7 was observed in several human normal epithelial control tissues analyzed. CLDN-7 is significantly overexpressed in all main histologic types of EOC and in single neoplastic cells disseminated in peritoneal cavity and pleural effusions, suggesting its potential role as novel diagnostic marker in ovarian cancer. Despite widespread expression of CLDN-7 in several human normal tissues, the high density of CLDN-7 molecules, their membranous localization on EOC cells, and their lack of expression on the celomic epithelium in the peritoneal cavity suggest that this target could be potentially suitable for antibody-mediated localized therapies of ovarian adenocarcinoma.

Mammaglobin B expression in human endometrial cancer.

Mammaglobin B (MGB-2) is an uteroglobin gene family member recently found highly differentially expressed in ovarian cancer by gene expression profiling. To evaluate its potential as a novel endometrial cancer biomarker, in this study we quantified and compared MGB-2 expression at messenger RNA and protein levels in endometrial tumors (endometrioid endometrial cancer [EEC]) with different grades of differentiation. MGB-2 expression was evaluated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) in fresh frozen biopsies and paraffin-embedded tissues derived from a total of 70 patients including 50 primary EEC and 20 normal endometria (NECs). High levels of MGB-2 gene expression were detected in 10 of 11 EEC G1 cases (91%), 16 of 17 EEC G2 cases (94%), and 6 of 22 EEC G3 cases (27%) by real-time PCR. In contrast, normal endometrial cells expressed low to negligible levels of MGB-2 by real-time PCR (P = 0.002 EEC vs NEC). Well- and moderately differentiated EECs overexpressed MGB-2 gene at significant higher levels when compared to NECs (P < 0.01). Pairwise differences between both G2 and G1 vs G3 cases for MGB-2 relative gene expression values were also statistically significant (G2 vs G3 P < 0.001, G1 vs G3 P = 0.016). MGB-2 protein expression was detected in 31 (86%) of 36 EEC and 0 of 5 atrophic NEC controls, while seven of eight (88%) of the proliferative/secretory/hyperplastic NECs focally expressed MGB-2 by IHC. MGB-2 is highly expressed in EEC, particularly in well- and moderately differentiated tumors, and may represent a novel molecular marker for EEC.

Expression of CD56 by human papillomavirus E7-specific CD8+ cytotoxic T lymphocytes correlates with increased intracellular perforin expression and enhanced cytotoxicity against HLA-A2-matched cervical tumor cells.

Human papillomavirus (HPV) infection represents the most important risk factor for developing cervical cancer. In this study, we examine the potential of full-length E7-pulsed autologous dendritic cells (DCs) to induce antigen-specific CTL responses from the peripheral blood of healthy individuals against HLA-A2-matched HPV-16 and HPV-18-positive tumor target cells in vitro. We show that DCs pulsed with E7 oncoprotein can consistently stimulate antigen-specific CTL responses that recognize and lyse HPV-16 or HPV-18-positive naturally infected cervical cancer cell lines. HPV-negative, EBV-transformed lymphoblastoid cell lines (LCLs) sharing the HLA haplotype of the target tumor cells, as well as autologous donor LCLs, were not significantly killed by E7-specific CTLs. Cytotoxicity against HLA-A2-matched HPV-16 and HPV-18 tumor target cells could be significantly inhibited by anti-HLA class I and by anti-HLA-A2 monoclonal antibodies. CD8+ CTLs expressed variable levels of CD56 and showed a strongly polarized Type 1 cytokine profile. Sorting of the CD8+ T cells on the basis of CD56 expression demonstrated that the most highly cytotoxic CTLs were CD56+ and expressed higher levels of perforin and IFN-gamma, compared with the CD8+/CD56- population. Taken together, these data demonstrate that full-length, E7-pulsed DCs can consistently induce E7-specific CD8+ CTL responses in healthy individuals that are able to kill naturally HPV-16 and HPV-18-infected cancer cells, and that CD56 expression defines a subset of CD8+ CTLs with high cytolytic activity against tumor cells.

Intrathecal cytotoxic T-cell immunotherapy for metastatic leptomeningeal melanoma.

A 49-year-old patient with primary, recurrent melanoma on the lower extremity developed metastatic leptomeningeal melanoma that did not respond to treatment with radiation therapy or intrathecal interleukin 2 (IL-2). Disease was characterized by neurological symptoms, including loss of hearing, loss of short-term memory, and gait disturbance. CD8+ CTLs were generated in vitro using autologous dendritic cells pulsed with peptides from the melanoma-associated antigens tyrosinase (145-156), Melan-A/MART-1 (26-35), and gp100/Pmel 17 (209-217). The CTLs exhibited up to 74% specific lysis against peptide-pulsed autologous EBV-transformed B cells, with Melan-A-specific CTLs yielding the greatest lytic activity. CD8+ CTLs possessed a type 1 cytokine profile, expressing tumor necrosis factor alpha and IFNgamma but not IL-4. Infusions of CTLs were supported with systemic low-dose IL-2 administration. 111In labeling and computerized gamma imaging were used to monitor the distribution of CTLs up to 48 h after infusion. Intra-arterial delivery via the right carotid artery was followed by redistribution of the CTLs to the lungs, liver, and spleen within 16 h. In contrast, delivery via an indwelling Ommaya reservoir resulted in prolonged retention of CTLs within the brain for at least 48 h after infusion. Marked but transient elevations in tumor necrosis factor alpha, IFN-gamma, and IL-6 in the cerebrospinal fluid were observed within 4 h of CTL infusion. There was no evidence of tumor progression throughout the treatment period, and clinically the patient showed some resolution of neurological symptoms.

Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus.

The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.

Effects of concurrent cisplatinum administration during radiotherapy vs. radiotherapy alone on the immune function of patients with cancer of the uterine cervix.

PURPOSE: To compare the effects of concurrent administration of cisplatinum (40 mg/m(2)/weekly) with radiation therapy (C-RT) to those induced by radiation therapy alone (RT) on the immune function of patients with locally advanced cervical cancer. METHODS AND MATERIALS: In 8 prospectively randomized patients (i.e., 4 receiving RT vs. 4 receiving C-RT), lymphocyte populations including CD3+, CD4+ and CD8+ T-cell subsets, B cells (CD19+) and natural killer cells (CD56+, CD16+, CD3-) were studied before, during, and after therapy. Expression of the activation marker CD25 on CD3+ T cells, intracellular levels of perforin in CD8+ and CD56+ cells, and interferon-gamma (IFN-gamma) and IL-2 in CD4+ and CD8+ T cells was also measured. Finally, lymphoblast transformation and natural killer (NK) cytotoxic activity were assessed. RESULTS: Both RT and C-RT significantly decreased the mean absolute number of all lymphocyte subsets compared to pretreatment levels (p > 0.001). However, no differences were detected in the characteristics or the magnitude of the lymphopenia induced by the two treatments. Both RT and C-RT increased similarly the percentages of CD25-positive lymphocytes (p > 0.001), and significantly decreased PHA-induced T-cell lymphoblast transformation (p > 0.001) and NK cytotoxic activity against K562 cells (p > 0.001). The percentage of perforin-positive and CD8+ T cells was not altered during either treatment, whereas the percentage of perforin-positive and CD56+ cells was significantly reduced during both treatments, and correlated with reduced cytotoxicity against K562 cells. The percentages of CD8+ IFN-gamma+ and CD4+ IFN-gamma+ T cells as well as that of CD8+ IL-2+ and CD4+ IL2+ T cells were not significantly altered by C-RT compared to RT alone. Finally, with both regimens, NK cells and B-cell numbers showed a more rapid recovery than T-cell numbers. CONCLUSION: Administration of concurrent cisplatinum to radiation may synergistically increase cytotoxic effects of radiation on tumor cells but does not alter the magnitude and the characteristics of radiation-induced immunosuppression.

Expression of surface antigens during the differentiation of human dendritic cells vs macrophages from blood monocytes in vitro.

High expression of MHC antigens and adhesion/costimulation molecules is considered as one of the major characteristics qualifying macrophages (M) and dendritic cells (DC) as professional antigen presenting cells. Since accessory activity of M is known to be weaker than that of DC but both M or DC can differentiate from blood monocytes (MO) depending on culture conditions (i.e. GM-CSF vs GM-CSF/IL-4), we investigated the kinetics of expression of MHC antigens and several adhesion/costimulation molecules during the differentiation of DC or M from blood MO. Blood MO cultured with GM-CSF consistently induced M that showed adherence to plastic and CD14 expression. In contrast, MO cultured with GM-CSF/IL-4 rapidly became nonadherent, acquired DC morphology and lost CD14 expression. M but not DC proliferated as demonstrated by [H3]thymidine incorporation. MHC Class I was highly expressed in both M and DC. In contrast, MHC Class II molecules were significantly higher on DC compared to M. CD80 was upregulated on both DC and M but only on a subset of cells. CD80 expression peaked at day 3 on M and declined thereafter, while on DC expression increased significantly until day 10. CD86 was upregulated on the majority of DC and M. However, while M maintained stable expression of CD86 after day 3, DC progressively upregulated CD86 throughout the culture period. CD1a expression was initially low in both cell types and peaked at day 3 in M declining thereafter, while expression remained stable on DC until day 10. ICAM-1 expression was significantly upregulated on M when compared to DC at day 3. However, on M, ICAM-1 expression became undetectable by day 5 while on DC it increased through day 10. Similarly, CD40 was transiently expressed on M until day 5, while on DC it continuously increased until day 10. Finally, in contrast to other antigens, LFA-3 was always more strongly expressed on M than DC at all culture periods. Taken together, these data suggest that M showed a rapid but transient upregulation in the expression of adhesion/costimulation molecules, suggesting that maximal accessory ability is reached by M at an earlier time point than DC. Significant differences in surface antigen expression DC vs M were recognizable for MHC class II, CD86, CD80, CD1a, CD40 and ICAM-1. Specifically, major differences occurred for MHC class II, CD86, CD40 and ICAM-1. Therefore, the higher accessory ability of DC compared to M in naive T cell priming may be related to qualitative and quantitative differences in expression of these immunologically important surface molecules.

Induction of ovarian tumor-specific CD8+ cytotoxic T lymphocytes by acid-eluted peptide-pulsed autologous dendritic cells.

OBJECTIVE: To evaluate the potential of dendritic cells pulsed with acid-eluted peptides derived from autologous ovarian cancer cells for eliciting a tumor-specific cytotoxic T cell response in women with advanced ovarian cancer. METHODS: CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous ovarian tumor peptide-pulsed dendritic cells were tested for their ability to induce an HLA class I-restricted cytotoxic T lymphocyte response against autologous tumor cells. To correlate cytotoxic activity by cytotoxic T lymphocytes with T cell phenotype, we used two-color flow cytometric analysis of surface markers and intracellular cytokine expression (interferon-gamma versus interleukin-4). RESULTS: CD8+ cytotoxic T lymphocyte responses against autologous ovarian tumor cells were elicited in three consecutive women who had advanced ovarian cancer. Although cytotoxic T lymphocyte populations from all women expressed strong cytolytic activity against autologous tumor cells, they did not lyse autologous lymphoblasts or Epstein-Barr virus-transformed cell lines, and they showed negligible cytotoxicity against the natural killer-sensitive cell line K-562. Cytotoxicity against the autologous tumor cells was significantly inhibited by anti-HLA class I (W6/32) and anti-HLA-A2 (BB7-2) monoclonal antibodies. CD8+ cytotoxic T lymphocytes expressed variable levels of CD56 and preferentially expressed interferon-gamma rather than interleukin-4. CONCLUSIONS: Peptide-pulsed dendritic cells induced specific CD8+ cytotoxic T lymphocytes that killed autologous tumor cells from women with advanced ovarian cancer. This finding might contribute to the development of active or adoptive immunotherapy for residual or resistant ovarian cancer after standard surgery and cytotoxic treatment.

Secretion of vascular endothelial growth factor in adenocarcinoma and squamous cell carcinoma of the uterine cervix.

OBJECTIVE: To determine whether major differences in vascular endothelial growth factor secretion exist between adenocarcinomas of the uterine cervix compared with squamous cell carcinomas. METHODS: The secretion of vascular endothelial growth factor by eight fresh cervical cancer cell preparations (four adenocarcinomas and four squamous cell carcinomas) and four established squamous cell lines was evaluated using a sensitive enzyme-linked immunosorbent assay in vitro. RESULTS: All cervical tumors secreted significant amounts of vascular endothelial growth factor, and no significant differences between fresh and established squamous cell lines were detectable. In contrast, a highly significant difference in vascular endothelial growth factor secretion was noted between fresh adenocarcinomas (mean = 2712, range between 1700 to 3500 pg/mL/10(5) cells/48 hours) when compared with fresh squamous (mean = 575, range between 200 to 950 pg/mL/10(5) cells/48 hours) or established squamous cervical carcinoma cell lines (mean = 712, range between 400 to 1000 pg/mL/10(5) cells/48 hours) (F-test, P< or =.001). CONCLUSION: These data strongly suggest that major differences in the secretion of vascular endothelial growth factor exist between squamous cell carcinoma and adenocarcinomas of the uterine cervix. Therefore, at least some of the differences in the natural biologic behavior of these two histologic types of cervical cancer, including the propensity for earlier lymphatic and hematogenous metastasis as well as the lower response to radiation treatment, could be related to major differences in the secretion of this powerful angiogenic and immunosuppressive cytokine.

Comparison of competitive and non-competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of hepatitis C virus (HCV) RNA.

A competitive reverse transcription (RT)-nested polymerase chain reaction (PCR) assay for HCV RNA was compared with the Roche Amplicor HCV Monitor assay, based on non-competitive, single step RT-PCR. A total of 83 serum samples were tested in parallel by both assays. All samples could be quantified by competitive RT-PCR (cPCR), whereas seven were negative by the non-competitive assay (ncPCR). The HCV RNA titre of the discordant samples assessed by cPCR was significantly lower than that of the remaining 76 (P < 0.001). Absolute HCV RNA titres were higher by cPCR than by ncPCR (P < 0.001), even if the results of the two methods were statistically correlated (P < 0.001). HCV RNA titre tested by cPCR was not significantly different between samples infected with genotype 1 or 2. However, values obtained by ncPCR were higher in samples with genotype 1 (P < 0.001). Furthermore, all seven discordant samples were infected with genotype 2. When both methods were used to measure serial dilutions of standard HCV RNA, we observed a bias to lower measurements with the ncPCR kit. This study shows a good correlation between the results of two PCR-based methods for the quantification HCV RNA. However, the degree of sensitivity and the absolute HCV RNA titre measured may vary according to the assay used.

Diagnosis of viral hepatitis with a nonisotopic hybridization assay.

The DNA enzyme immunoassay is an efficient method for the screening of PCR products derived from different hepatitis virus genomes, and allows to bypass both agarose gel electrophoresis and Southern blot hybridization with radioactively labeled probes. A wider application of this method will disclose new perspectives for the introduction of PCR in clinical laboratories.

The effects of irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical cancer.

PURPOSE: Studies were designed to analyse the effects of high doses of gamma-irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical carcinoma cell lines. MATERIALS AND METHODS: The expression of heat shock protein gp96 was evaluated at the transcriptional (Northern blot) and post-transcriptional levels (Western blot) in two human cervical carcinoma cell lines following exposure to high doses of gamma-irradiation. RESULTS: Doses of gamma-irradiation ranging from 25 to 100 Gy significantly and consistently increased the expression of heat shock protein gp96 on CaSki and HT-3 cervical cancer cells. The increase in the amount of protein was due to transcriptional up-regulation of this gene. Radiation doses unable to inhibit completely cell replication in the totality of tumour cells (i.e. 25 Gy), as well as higher (fully lethal) doses of irradiation (i.e. 50 to 100 Gy), were shown to up-regulate significantly the expression of heat shock protein gp96 mRNA in a dose-dependent manner. CONCLUSIONS: Recently, gp96 molecules have been implicated in the presentation of endogenous and viral antigens. A number of key elements in this pathway, including major histocompatibility complex (MHC) class I molecules as well as adhesion/co-stimulation molecules such as ICAM-1, are known to be sensitive to irradiation effects. The results show that radiation can also increase the expression of other immunologically important cell molecules such as a tumour rejection antigen (heat shock protein gp96) in human cervical cancer. Such findings may partially explain the increased immunogenicity of tumour cells following irradiation and further support a role for local radiation therapy as a powerful biologic response modifier.

Effects of retinoic acid on the expression of a tumor rejection antigen (heat shock protein gp96) in human cervical cancer.

Retinoids are a class of compounds structurally related to vitamin A which have been found to be active agents experimentally as well as clinically in the prevention and treatment of cervical cancer. Recent data have suggested that in addition to their key regulatory role during epithelial cell differentiation, they could also contribute to enhanced cellular and humoral immunity against tumor cells. Hsp gp96 molecules have recently been implicated in the presentation of tumor and viral antigens. A number of key elements in this pathway, including major histocompatibility complex (MHC) class I molecules as well as adhesion/co-stimulation molecules such as ICAM-1 have reported to be sensitive to retinoic acid up-regulation. In this study we analyzed at the transcriptional (Northern blot) and post-transcriptional levels (Western blot) the effects of retinoic acid on the expression of the tumor rejection antigen (heat shock protein gp96) in three human cervical carcinoma cell lines. Exposure of therapeutic doses of retinoic acid (i.e. 1 microM) significantly and consistently increased the expression of heat shock protein gp96 (Western blot analysis) on CaSki, SiHa and HT-3 cervical cancer cell lines. Northern blot analysis demonstrated that the increase in the amount of protein was due to the transcriptional upregulation of this gene. Taken together, our results show that retinoic acid can significantly increase the expression of yet another immunologically important cell molecule, the tumor rejection antigen heat shock protein gp96 in human cervical cancer. Such findings provide new information on the effects of retinoic acid on tumor cells and further support the role of retinoic acid as a powerful biologic response modifier.

Secretion of vascular endothelial growth factor in ovarian cancer.

Vascular endothelial growth factor (VEGF) is an important regulator of vascular endothelial cell function during vasculogenesis and tumor growth and is believed to play a major role in peritoneal fluid accumulation in ascites tumors. High VEGF production from primary tumors has been reported to correlate with increased metastatic spreading and worse prognosis compared to low VEGF secreting tumors. In addition, VEGF secretion has recently been proposed as one of the major factors responsible for defective immune function in cancer patients. In order to evaluate whether ovarian carcinomas actively secrete VEGF, in this study we have analyzed and quantified VEGF secretion in several fresh and established human ovarian carcinoma cell lines in vitro using a sensitive enzyme-linked immunosorbent assay (ELISA). In addition, VEGF levels were also evaluated in the ascitic fluids and plasma of six ovarian cancer patients. All fresh tumors secreted high levels of VEGF (mean = 5,046, range between 1,760 and 7,780 pg/ml/10(5) cells/48 hr) when compared to established ovarian carcinoma cell lines (mean = 493, range between 160 to 1,120 pg/ml/10(5) cells/48 hr) (p <0.02). Importantly, high grade malignancies were found to secrete larger amounts of VEGF (mean = 6,660 pg/ml) when compared to lower grade tumors (mean = 1,820 pg/ml) (p <0.01). Ascitic fluids from all patients were rich in VEGF (mean = 5,483, range between 1,300 and 11,200 pg/ml) and plasma levels of VEGF in ovarian cancer patients were significantly higher (mean = 408, range between 160 and 810 pg/ml) when compared with healthy individuals (mean = 46, range between 35 and 60 pg/ml) (p <0.01). Taken together, these data demonstrate that ovarian cancers secrete large amounts of VEGF in vitro and in vivo. This findings therefore suggest that this factor may play a crucial role in the genesis of ascitic fluid accumulation, angiogenesis and tumor induced immunosuppression in ovarian cancer patients. The design of anti-angiogenic treatment directed at blocking the action of VEGF may be a reasonable novel therapeutic approach in the treatment of ovarian cancer.

Development, characterization and distribution of adoptively transferred peripheral blood lymphocytes primed by human papillomavirus 18 E7--pulsed autologous dendritic cells in a patient with metastatic adenocarcinoma of the uterine cervix.

We describe a 27-year-old woman with systemic chemoresistant and radioresistant metastatic disease secondary to a recurrence of human papillomavirus (HPV) 18 infected cervical adenocarcinoma of the uterine cervix who received adoptive transfer of peripheral blood T cells stimulated with HPV 18 E7-pulsed autologous dendritic cells (DC). Extensive in vitro characterization of the DC-activated T cells derived from peripheral blood mononuclear cells (PBMC) included phenotypic analysis, cytotoxicity and intracellular cytokine production. High cytotoxicity activity was observed by CD8+T cells against autologous tumor cells, but not against NK-sensitive K562 cells, autologous Con-A lymphoblasts, or autologous Epstein-Barr virus-transformed lymphoblastoid cells. Blocking studies demonstrated that lytic activity was significantly inhibited by pretreatment of tumor targets with MAb specific for HLA class I as well as that of effector cells with anti-CD8, anti-LFA-1, but not anti CD3 MAb. Two-color flow cytometric analysis of the cytotoxic T cells revealed that a significant proportion of CD8+ cells was also CD56+. These double positive CTLs were thymically derived, as shown by expression of heterodimeric CD8 molecules (alpha/beta CD8) and were endowed with high cytotoxic activity against tumor cells. Analysis of intracellular cytokine expression showed that the striking majority of E7-pulsed DC activated CD8+ T cells strongly expressed IFN-gamma, TNF-alpha and IL-2 but not IL-4. The patient received two infusions of cytotoxic tumor-specific T cells at 2 week intervals, and in vivo distribution of the T cells was followed by 111 oxine labeling and serial gamma camera imaging. Persistent accumulation of radioactivity in the lungs, which harbored extensive metastatic disease, was detected up to 120 hrs after the infusion. Taken together, these results illustrate the potential of E7-specific and tumor-specific CTL-based immunotherapy for the treatment of patients with invasive cervical cancer.