Michela Palmieri

Gene expression profiles in primary ovarian serous papillary tumors and normal ovarian epithelium: identification of candidate molecular markers for ovarian cancer diagnosis and therapy.

With the goal of identifying genes with a differential pattern of expression between ovarian serous papillary carcinomas (OSPCs) and normal ovarian (NOVA) epithelium and using this knowledge for the development of novel diagnostic and therapeutic markers for ovarian cancer, we used oligonucleotide microarrays with probe sets complementary to 12,533 genes to analyze the gene expression profiles of 10 primary OSPC cell lines, 2 established OSPC cell lines (UCI‐101, UCI‐107) and 5 primary NOVA epithelial cultures. Unsupervised analysis of gene expression data identified 129 and 170 genes that exhibited >5‐fold upregulation and downregulation, respectively, in primary OSPC compared to NOVA. Genes overexpressed in established OSPC cell lines had little correlation with those overexpressed in primary OSPC, highlighting the divergence of gene expression that occurs as a result of long‐term in vitro growth. Hierarchical clustering of the expression data readily distinguished normal tissue from primary OSPC. Laminin, claudin 3, claudin 4, tumor‐associated calcium signal transducers 1 and 2 (TROP‐1/Ep‐CAM, TROP‐2), ladinin 1, S100A2, SERPIN2 (PAI‐2), CD24, lipocalin 2, osteopontin, kallikrein 6 (protease M), kallikrein 10, matriptase (TADG‐15) and stratifin were among the most highly overexpressed genes in OSPC compared to NOVA. Downregulated genes in OSPC included transforming growth factor‐β receptor III, platelet‐derived growth factor receptor α, SEMACAP3, ras homolog gene family member I (ARHI), thrombospondin 2 and disabled‐2/differentially expressed in ovarian carcinoma 2 (Dab2/DOC2). Differential expression of some of these genes, including claudin 3, claudin 4, TROP‐1 and CD24, was validated by quantitative RT‐PCR and flow cytometry on primary OSPC and NOVA. Immunohistochemical staining of formalin‐fixed, paraffin‐embedded tumor specimens from which primary OSPC cultures were derived further confirmed differential expression of CD24 and TROP‐1/Ep‐CAM markers on OSPC vs. NOVA. These results, obtained with highly purified primary cultures of ovarian cancer, highlight important molecular features of OSPC and may provide a foundation for the development of new type‐specific therapies against this disease.

Amplification of c-erbB2 oncogene : A major prognostic indicator in uterine serous papillary carcinoma

Overexpression of the epidermal growth factor type II receptor HER‐2/neu has been associated with resistance to chemotherapy and poor survival in several human tumors. In the current study, the authors have determined the frequency and clinical significance of HER‐2/neu gene amplification in uterine serous papillary endometrial carcinoma (USPC), a highly aggressive variant of endometrial carcinoma.

Gene expression fingerprint of uterine serous papillary carcinoma: identification of novel molecular markers for uterine serous cancer diagnosis and therapy

Uterine serous papillary cancer (USPC) represents a rare but highly aggressive variant of endometrial cancer, the most common gynecologic tumour in women. We used oligonucleotide microarrays that interrogate the expression of some 10 000 known genes to profile 10 highly purified primary USPC cultures and five normal endometrial cells (NEC). We report that unsupervised analysis of mRNA fingerprints readily distinguished USPC from normal endometrial epithelial cells and identified 139 and 390 genes that exhibited >5-fold upregulation and downregulation, respectively, in primary USPC when compared to NEC. Many of the genes upregulated in USPC were found to represent adhesion molecules, secreted proteins and oncogenes, such as L1 cell adhesion molecule, claudin-3 and claudin-4, kallikrein 6 (protease M) and kallikrein 10 (NES1), interleukin-6 and c-erbB2. Downregulated genes in USPC included SEMACAP3, ras homolog gene family, member I (ARHI), and differentially downregulated in ovarian carcinoma gene 1. Quantitative RT–PCR was used to validate differences in gene expression between USPC and NEC for several of these genes. Owing to its potential as a novel therapeutic marker, expression of the high-affinity epithelial receptor for Clostridium perfringens enterotoxin (CPE) claudin-4 was further validated through immunohistochemical analysis of formalin-fixed paraffin-embedded specimens from which the primary USPC cultures were obtained, as well as an independent set of archival USPC specimens. Finally, the sensitivity of primary USPC to the administration of scalar doses of CPE in vitro was also demonstrated. Our results highlight the novel molecular features of USPC and provide a foundation for the development of new type-specific therapies against this highly aggressive variant of endometrial cancer.

Human Papillomavirus Type 16 and 18 E7-Pulsed Dendritic Cell Vaccination of Stage IB or IIA Cervical Cancer Patients: a Phase I Escalating-Dose Trial

ABSTRACT The safety and immunogenicity of the human papillomavirus type 16 (HPV16) or HPV18 (HPV16/18) E7 antigen-pulsed mature dendritic cell (DC) vaccination were evaluated for patients with stage IB or IIA cervical cancer. Escalating doses of autologous DC (5, 10, and 15 × 106 cells for injection) were pulsed with recombinant HPV16/18 E7 antigens and keyhole limpet hemocyanin (KLH; an immunological tracer molecule) and delivered in five subcutaneous injections at 21-day intervals to 10 cervical cancer patients with no evidence of disease after they underwent radical surgery. Safety, toxicity, delayed-type hypersensitivity (DTH) reaction, and induction of serological and cellular immunity against HPV16/18 E7 and KLH were monitored. DC vaccination was well tolerated, and no significant toxicities were recorded. All patients developed CD4+ T-cell and antibody responses to DC vaccination, as detected by enzyme-linked immunosorbent spot (ELISpot) and enzyme-linked immunosorbent assays (ELISA), respectively, and 8 out of 10 patients demonstrated levels of E7-specific CD8+ T-cell counts, detected by ELISpot during or immediately after immunization, that were increased compared to prevaccination baseline levels. The vaccine dose did not predict the magnitude of the antibody or T-cell response or the time to detection of HPV16/18 E7-specific immunity. DTH responses to intradermal injections of HPV E7 antigen and KLH were detected for all patients after vaccination. We conclude that HPV E7-loaded DC vaccination is safe and immunogenic for stage IB or IIA cervical cancer patients. Phase II E7-pulsed DC-based vaccination trials with cervical cancer patients harboring a limited tumor burden, or who are at significant risk of tumor recurrence, are warranted.

Treatment of chemotherapy-resistant human ovarian cancer xenografts in C.B-17/SCID mice by intraperitoneal administration of Clostridium perfringens enterotoxin.

Ovarian cancer remains the most lethal gynecologic malignancy in the United States. Although many patients with advanced-stage disease initially respond to standard combinations of surgical and cytotoxic therapy, nearly 90% develop recurrence and inevitably die from the development of chemotherapy-resistant disease. The discovery of novel and effective therapy against chemotherapy-resistant/recurrent ovarian cancer remains a high priority. Using expression profiling, we and others have recently found claudin-3 and claudin-4 genes to be highly expressed in ovarian cancer. Because these tight junction proteins have been described as the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE), in this study we investigated the level of expression of claudin-3 and/or claudin-4 in chemotherapy-naive and chemotherapy-resistant primary human ovarian cancers as well as their sensitivity to CPE treatment in vitro. We report that 100% (17 of 17) of the primary ovarian tumors tested overexpress one or both CPE receptors by quantitative reverse transcription-PCR. All ovarian tumors showed a dose-dependent cytotoxic effect to CPE in vitro. Importantly, chemotherapy-resistant/recurrent ovarian tumors were found to express claudin-3 and claudin-4 genes at significantly higher levels when compared with chemotherapy-naive ovarian cancers. All primary ovarian tumors tested, regardless of their resistance to chemotherapeutic agents, died within 24 hours to the exposure to 3.3 microg/mL CPE in vitro. In addition, we have studied the in vivo efficacy of i.p. CPE therapy in SCID mouse xenografts in a highly relevant clinical model of chemotherapy-resistant freshly explanted human ovarian cancer (i.e., OVA-1). Multiple i.p. administration of sublethal doses of CPE every 3 days significantly inhibited tumor growth in 100% of mice harboring 1 week established OVA-1. Repeated i.p. doses of CPE also had a significant inhibitory effect on tumor progression with extended survival of animals harboring large ovarian tumor burdens (i.e., 4-week established OVA-1). Our findings suggest that CPE may have potential as a novel treatment for chemotherapy-resistant/recurrent ovarian cancer.

Overexpression of claudin-3 and claudin-4 receptors in uterine serous papillary carcinoma: novel targets for a type-specific therapy using Clostridium perfringens enterotoxin (CPE).

Uterine serous papillary carcinoma (USPC) represents a highly aggressive variant of endometrial cancer. Using gene expression profiling, we recently identified high expression of the claudin‐3 and claudin‐4 receptors in a limited set of USPC. These tight junction proteins represent the low‐ and high‐affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE) and are sufficient to mediate CPE binding and trigger subsequent toxin‐mediated cytolysis. The potential for targeting this pathway in the treatment of USPC was explored.

Discrimination between uterine serous papillary carcinomas and ovarian serous papillary tumours by gene expression profiling.

High-grade ovarian serous papillary cancer (OSPC) and uterine serous papillary carcinoma (USPC) represent two histologically similar malignancies characterised by markedly different biological behavior and response to chemotherapy. Understanding the molecular basis of these differences may significantly refine differential diagnosis and management, and may lead to the development of novel, more specific and more effective treatment modalities for OSPC and USPC. We used an oligonucleotide microarray with probe sets complementary to >10 000 human genes to determine whether patterns of gene expression may differentiate OSPC from USPC. Hierarchical cluster analysis of gene expression in OSPC and USPC identified 116 genes that exhibited >two-fold differences (P<0.05) and that readily distinguished OSPC from USPC. Plasminogen activator inhibitor (PAI-2) was the most highly overexpressed gene in OSPC when compared to USPC, while c-erbB2 was the most strikingly overexpressed gene in USPC when compared to OSPC. Overexpression of the c-erbB2 gene and its expression product (i.e., HER-2/neu receptor) was validated by quantitative RT–PCR as well as by flow cytometry on primary USPC and OSPC, respectively. Immunohistochemical staining of serous tumour samples from which primary OSPC and USPC cultures were derived as well as from an independent set of 20 clinical tissue samples (i.e., 10 OSPC and 10 USPC) further confirmed HER-2/neu as a novel molecular diagnostic and therapeutic marker for USPC. Gene expression fingerprints have the potential to predict the anatomical site of tumour origin and readily identify the biologically more aggressive USPC from OSPC. A therapeutic strategy targeting HER-2/neu may be beneficial in patients harbouring chemotherapy-resistant USPC.

The novel serine protease tumor-associated differentially expressed gene-15 (matriptase/MT-SP1) is highly overexpressed in cervical carcinoma.

Tumor‐associated differentially expressed gene–15 (TADG‐15/matriptase/MT‐SP1) is a novel transmembrane serine protease involved in numerous biologic processes, including activation of growth and angiogenic factors and degradation of extracellular matrix components. To assess the value of TADG‐15 as a possible marker for tumor detection and/or as a target for therapeutic intervention, the authors investigated the frequency of expression of TADG‐15 in human cervical tumors.

Human kallikrein 6: a new potential serum biomarker for uterine serous papillary cancer.

Purpose: The discovery of novel biomarkers might greatly contribute to improve clinical management and outcomes in uterine serous papillary carcinoma (USPC), a highly aggressive variant of endometrial cancer. Experimental Design: Human kallikrein 6 (hK6) gene expression levels were evaluated in 29 snap-frozen endometrial biopsies, including 13 USPC, 13 endometrioid carcinomas, and 3 normal endometrial cells by real-time PCR. Secretion of hK6 protein by 14 tumor cultures, including 3 USPC, 3 endometrioid carcinoma, 5 ovarian serous papillary carcinoma, and 3 cervical cancers, was measured using a sensitive ELISA. Finally, hK6 concentration in 79 serum and plasma samples from 22 healthy women, 20 women with benign diseases, 20 women with endometrioid carcinoma, and 17 USPC patients was studied. Results:hK6 gene expression levels were significantly higher in USPC when compared with endometrioid carcinoma (mean copy number by real-time PCR, 1,927 versus 239, USPC versus endometrioid carcinoma; P < 0.01). In vitro hK6 secretion was detected in all primary USPC cell lines tested (mean, 11.5 μg/L) and the secretion levels were similar to those found in primary ovarian serous papillary carcinoma cultures (mean, 9.6 μg/L). In contrast, no hK6 secretion was detectable in primary endometrioid carcinoma and cervical cancer cultures. hK6 serum and plasma concentrations (mean ± SE) among normal healthy females (2.7 ± 0.2 μg/L), patients with benign diseases (2.4 ± 0.2 μg/L), and patients with endometrioid carcinoma (2.6 ± 0.2 μg/L) were not significantly different. In contrast, serum and plasma hK6 values in USPC patients (6.1 ± 1.1) were significantly higher than those in the noncancer group (P = 0.006), benign group (P = 0.003), and endometrioid carcinoma patients (P = 0.005). Conclusions: hK6 is highly expressed in USPC and is released in the plasma and serum of USPC patients. hK6 may represent a novel biomarker for USPC for monitoring early disease recurrence and response to therapy.

Human papillomavirus type 16 (HPV-16) virus-like particle L1-specific CD8+ cytotoxic T lymphocytes (CTLs) are equally effective as E7-specific CD8+ CTLs in killing autologous HPV-16-positive tumor cells in cervical cancer patients: implications for L1 dendritic cell-based therapeutic vaccines.

ABSTRACT Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4+ and CD8+ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4+ and CD8+ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8+ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4+ and CD8+ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.