Martin J. Cannon

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.

Endemic Burkitt's lymphoma: a polymicrobial disease?

Endemic Burkitts lymphoma is the most common childhood cancer in equatorial Africa. Two ubiquitous human pathogens are thought to be responsible for the aetiology of this disease: Epstein–Barr virus and Plasmodium falciparum malaria. New data suggest how these two pathogens might interact to result in disease and provide insights into the emerging concepts of polymicrobial disease pathogenesis.

Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8+ Cytotoxic T Lymphocytes

Interleukin-10 (IL-10) is widely known as an immunosuppressive cytokine by virtue of its ability to inhibit macrophage-dependent antigen presentation, T-cell proliferation, and Th1 cytokine secretion. However, several studies have challenged the perception of IL-10 solely as an immunosuppressive cytokine. As part of an investigation on potentiation of the cytotoxic activity of human papillomavirus E7-specific CD8(+) cytotoxic T lymphocytes (CTL) for adoptive transfusions to cervical cancer patients, we found that IL-10 in combination with IL-2, unlike several other combinations, including IL-2 with IL-12, gamma interferon (IFN-gamma), tumor necrosis factor alpha, and transforming growth factor beta, was able to consistently increase cytotoxicity. This augmentation in cytotoxic activity correlated with a significant increase in the cytoplasmic accumulation of perforin as detected by fluorescence-activated cell sorter. Surface expression of both the alpha and beta chains of the CD8 heterodimeric coreceptor and CD56 molecules was increased by exposure of CTL to IL-10. More importantly, we found that administration of IL-10 in combination with IL-2 after antigen stimulation consistently increased the intracellular expression of Th1 cytokines (i.e., IFN-gamma and IL-2) compared to results for control CD8(+) T cells cultured in IL-2 alone. In kinetic studies, proliferation, intracellular perforin levels, cytotoxic activity, and IFN-gamma expression were consistently elevated in CTL cultures containing IL-10 compared to control cultures, both at early and late time points following stimulation. In contrast, intracellular IL-2 expression was consistently increased only at early time points following stimulation with autologous tumor cells or solid-phase anti-CD3 antibody. Taken together, these data support the use of IL-10 in combination with IL-2 for the in vitro expansion and potentiation of tumor-specific CTL for clinical use in the therapy of cancer.

Cytotoxic T cell specificity for respiratory syncytial virus proteins: fusion protein is an important target antigen

We examined the specificity of BALB/c cytotoxic T (Tc) cells for respiratory syncytial virus (RSV) components, using recombinant vaccinia viruses (VV) coding for several individual RSV proteins. We found that immunization with the different VVs yielded the following Tc memory cell populations: high levels of RSV-specific Tc cells were induced with the fusion protein VV, but low levels were induced with VV coding for the RSV nucleoprotein. Tc cell recognition of attachment glycoprotein, part of the matrix molecule or 1A internal protein was poor. While high levels of fusion protein-specific Tc cells were induced by the fusion protein VV, they showed poor cross-reactivity between the A2 and 8/60 RSV strains compared with Tc cells primed by RSV infection.

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.

Vaccination with HPV-18 E7–Pulsed Dendritic Cells in a Patient with Metastatic Cervical Cancer

To the Editor: The management of disseminated carcinoma of the cervix that is no longer amenable to control with surgery or radiation therapy has not improved significantly with the advent of moder...

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.

Dendritic Cells and Macrophages Are Productively Infected by Poliovirus

ABSTRACT Expression of the poliovirus receptor (PVR) on cells is a major host determinant of infection by poliovirus. Previously, the only immune cell type known to express PVR was the blood-derived monocyte, which is susceptible to infection at very low frequency. We demonstrate that professional antigen-presenting cells—macrophages and dendritic cells, generated upon differentiation of monocytes—retain expression of PVR and are highly susceptible to infection by type 1 Mahoney strain of poliovirus. Maximal cell-associated titers of virus are obtained within 6 to 8 h postinfection, and cell death and lysis occurs within 24 h postinfection. Similar kinetics are observed in cells infected with the Sabin 1 vaccine strain. Although protein synthesis and receptor-mediated endocytosis are inhibited upon poliovirus infection of these critical antigen-presenting cells, we demonstrate for the first time that functional presentation of antigen occurs in these infected cells via the HLA class II pathway.