Deregulated expression of superoxide dismutase-2 correlates with different stages of cervical neoplasia.

OBJECTIVE: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer. METHODS: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO). RESULTS: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining. CONCLUSIONS: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

Effect of a novel DNA vaccine on angiogenesis and tumor growth in vivo.

OBJECTIVE: To develop a DNA cancer vaccine that targets the vascular endothelial growth factor receptor. DESIGN: Mice were vaccinated intramuscularly with listeriolysin O-fetal liver kinase 1 (LLO-Flk1) or controls. Mice were also challenged subcutaneously with the tumor cell line TC-1. Tumor sizes were measured after vaccination. At the conclusion of the experiments, the tumors were harvested for immunohistochemical analysis and determination of hemoglobin content. SETTING: Research laboratory. SUBJECTS: Six- to 8-week-old C57BL/6 mice. INTERVENTION: Fifty micrograms of each vector was administered 3 times at weekly intervals. MAIN OUTCOME MEASURES: Tumor size, mean vessel density of tumors, hemoglobin content of tumor. RESULTS: Mice treated with the LLO-Flk1 vaccine experienced slower tumor growth relative to the other treatment groups. Complete tumor regression was observed in several cases. Immunohistochemical staining of tumors revealed fewer blood vessels in the mice vaccinated with the LLO-Flk1 vaccine relative to the other treatment groups. Finally, colorimetric assessment for hemoglobin suggested decreased vasculature in the tumor bed of these mice relative to the control groups. CONCLUSION: The novel DNA cancer vaccine LLO-Flk1 can slow tumor growth in vivo.

Analysis of human kallikrein 7 expression as a potential biomarker in cervical neoplasia.

Overexpression of kallikrein 7, a proteolytic enzyme important for epithelial cell shedding, may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion. In this study, we have evaluated hK7 (human kallikrein 7) protein levels by immunohistochemistry in 367 cervical histological samples including 35 cases of cervicitis, 31 low-grade cervical intraepithelial neoplasia, 51 high-grade cervical intraepithelial neoplasia (H-SIL), 197 squamous cervical carcinomas (SCC) and 53 cervical adenocarcinomas. We have observed that hK7 staining increased with the severity of cervical disease. Intense hK7 staining was found in 15.2% of cervicitis samples, in contrast to 55% of H-SIL and 68% of SCC. Moreover, 92.5% of adenocarcinomas also exhibited intense hK7 staining. Differences in the expression of hK7 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

Listeria monocytogenes delivery of HPV-16 major capsid protein L1 induces systemic and mucosal cell-mediated CD4+ and CD8+ T-cell responses after oral immunization.

Neutralizing antibodies are thought to be required at mucosal surfaces to prevent human papillomavirus (HPV) transmission. However, the potential for cell-mediated immunity in mediating protection against HPV infection has not been well explored. We generated recombinant Listeria monocytogenes (Lm) constructs that secrete listeriolysin O (LLO) fused with overlapping N-terminal (LLO-L1(1-258)) or C-terminal (LLO-L1(238-474)) fragments of HPV type 16 major capsid protein L1 (HPV-16-L1). Oral immunization of mice with either construct induced IFN-gamma-producing CD8+ and CD4+ T cells in the spleen and in the Peyer's patches with the C-terminal construct. Oral immunization with both constructs resulted in diminished viral titers in the cervix and uterus of mice after intravaginal challenge with vaccinia virus expressing HPV-16-L1.

The first clinical use of a live-attenuated Listeria monocytogenes vaccine: a Phase I safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix.

Invasive carcinoma of the cervix (ICC) is the second most common cancer in women worldwide. Lm-LLO-E7 vaccine is a live-attenuated Listeria monocytogenes (Lm) that secretes the HPV-16 E7 antigen fused to a non-hemolytic fragment of the Lm protein listeriolysin O (LLO). In this Phase I trial, the safety of Lm-LLO-E7 was assessed in 15 patients with previously treated metastatic, refractory or recurrent ICC. Patients received 1 of 3 dose levels of Lm-LLO-E7 (1 x 10(9)CFU, 3.3 x 10(9)CFU or 1 x 10(10)CFU) as an intravenous infusion, followed by a second dose 3 weeks later. All patients experienced a flu-like syndrome which responded to non-prescription symptomatic treatment. Severe (grade 3) adverse events related to Lm-LLO-E7 were reported in 6 patients (40%), but no grade 4 adverse events were observed. At the highest dose some patients had severe fever and dose limiting hypotension. By the end of the study protocol, 2 patients had died, 5 had progressed, 7 had stable disease and 1 qualified as a partial responder. This study shows for the first time that a live-attenuated Lm is safe to be administered to late stage ICC patients.

HPV variants and HLA polymorphisms: the role of variability on the risk of cervical cancer.

Human papillomavirus (HPV) infection is linked to the development of cervical cancer, and several cofactors contribute to the risk of disease. Research on the intratypic variability of HPVs has defined variants that are associated with persistent infections and are potentially more oncogenic, translating to a higher risk of malignant disease. The genetic variability of the host also plays a role in the risk of cervical cancer, especially genes controlling the immune response, such as HLA class I and II. These highly polymorphic genes are important risk determinants of HPV persistence and disease progression. The interaction between host and viral factors is complex and needs to be further investigated, paving the way to better define the patients at the highest risk of developing malignant diseases linked to HPV infection.

Multiple effector mechanisms induced by recombinant Listeria monocytogenes anticancer immunotherapeutics.

Listeria monocytogenes is a facultative intracellular gram-positive bacterium that naturally infects professional antigen presenting cells (APC) to target antigens to both class I and class II antigen processing pathways. This infection process results in the stimulation of strong innate and adaptive immune responses, which make it an ideal candidate for a vaccine vector to deliver heterologous antigens. This ability of L. monocytogenes has been exploited by several researchers over the past decade to specifically deliver tumor-associated antigens that are poorly immunogenic such as self-antigens. This review describes the preclinical studies that have elucidated the multiple immune responses elicited by this bacterium that direct its ability to influence tumor growth.

Construction and characterization of an attenuated Listeria monocytogenes strain for clinical use in cancer immunotherapy.

Listeria monocytogenes has been exploited previously as a vaccine vector for the delivery of heterologous proteins such as tumor-specific antigens for active cancer immunotherapy. However, for effective use of live vector in clinics, safety is a major concern. In the present study, we describe an irreversibly attenuated and highly immunogenic L. monocytogenes platform, the L. monocytogenes dal-, dat-, and actA-deleted strain that expresses the human prostate-specific antigen (PSA) using an antibiotic resistance marker-free plasmid (the dal dat DeltaactA 142 strain expressing PSA). Despite limited in vivo survival, the dal dat DeltaactA 142 strain was able to elicit efficient immune responses required for tumor clearance. Our results showed that immunization of mice with the dal dat DeltaactA 142 strain caused the regression of the tumors established by the prostate adenocarcinoma cell line expressing PSA. An evaluation of immunologic potency indicated that the dal dat DeltaactA 142 strain elicits a high frequency of PSA-specific immune responses. Interestingly, immunization with the dal dat DeltaactA 142 strain induced significant infiltration of PSA-specific T cells in the intratumoral milieu. Collectively, our data suggest that the dal dat DeltaactA 142 strain is a safe and potent vector for clinical use and that this platform may be further exploited as a potential candidate to express other single or multiple antigens for cancer immunotherapy.

Cancer immunotherapy targeting the high molecular weight melanoma-associated antigen protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature.

The high molecular weight melanoma-associated antigen (HMW-MAA), also known as melanoma chondroitin sulfate proteoglycan, has been used as a target for the immunotherapy of melanoma. This antigen is expressed on the cell surface and has a restricted distribution in normal tissues. Besides its expression in a broad range of transformed cells, this antigen is also found in pericytes, which are important for tumor angiogenesis. We generated a recombinant Listeria monocytogenes (Lm-LLO-HMW-MAA-C) that expresses and secretes a fragment of HMW-MAA (residues 2,160-2,258) fused to the first 441 residues of the listeriolysin O (LLO) protein. Immunization with Lm-LLO-HMW-MAA-C was able to impede the tumor growth of early established B16F10-HMW-MAA tumors in mice and both CD4(+) and CD8(+) T cells were required for therapeutic efficacy. Immune responses to a known HLA-A2 epitope present in the HMW-MAA(2160-2258) fragment was detected in the HLA-A2/K(b) transgenic mice immunized with Lm-LLO-HMW-MAA-C. Surprisingly, this vaccine also significantly impaired the in vivo growth of other tumorigenic cell lines, such as melanoma, renal carcinoma, and breast tumors, which were not engineered to express HMW-MAA. One hypothesis is that the vaccine could be targeting pericytes, which are important for tumor angiogenesis. In a breast tumor model, immunization with Lm-LLO-HMW-MAA-C caused CD8(+) T-cell infiltration in the tumor stroma and a significant decrease in the number of pericytes in the tumor blood vessels. In conclusion, a Lm-based vaccine against HMW-MAA can trigger cell-mediated immune responses to this antigen that can target not only tumor cells but also pericytes in the tumor vasculature.

Human papillomavirus type 16 variants in cervical cancer from an admixtured population in Brazil.

Several studies indicate that molecular variants of HPV-16 have different geographic distribution and risk associated with persistent infection and development of high-grade cervical lesions. In the present study, the frequency of HPV-16 variants was determined in 81 biopsies from women with cervical intraepithelial neoplasia grade III or invasive cervical cancer from the city of Belem, Northern Brazil. Host DNAs were also genotyped in order to analyze the ethnicity-related distribution of these variants. Nine different HPV-16 LCR variants belonging to four phylogenetic branches were identified. Among these, two new isolates were characterized. The most prevalent HPV-16 variant detected was the Asian-American B-2, followed by the European B-12 and the European prototype. Infections by multiple variants were observed in both invasive cervical cancer and cervical intraepithelial neoplasia grade III cases. The analysis of a specific polymorphism within the E6 viral gene was performed in a subset of 76 isolates. The E6-350G polymorphism was significantly more frequent in Asian-American variants. The HPV-16 variability detected followed the same pattern of the genetic ancestry observed in Northern Brazil, with European, Amerindian and African roots. Although African ancestry was higher among women infected by the prototype, no correlation between ethnical origin and HPV-16 variants was found. These results corroborate previous data showing a high frequency of Asian-American variants in cervical neoplasia among women with multiethnic origin.

Listeria-based vaccines for cancer treatment.

Listeria monocytogenes is a facultative intracellular bacterium that enters professional antigen-presenting cells by active phagocytosis. As a live bacterium, it induces antigen-presenting cell maturation and strong innate immunity which may assist in the immune response to poorly immunogenic antigens, such as tumor-associated antigens. Listeria produces virulence factors that allow it to escape from the phagolysosome and colonize the cytosol of the host cell. It is thus a potent vaccine vector for the presentation of passenger antigens to the major histocompatibility complex class I and II pathways of antigen processing and presentation. Recent progress in developing this bacterium as a vaccine vector for tumor-associated antigens is reviewed. In mouse models, recombinant Listeria carrying a number of such antigens has provided therapeutic immunity directed towards established tumors. Safety issues associated with live bacterial vaccine vectors and problems to be overcome in developing Listeria as a cancer immunotherapeutic for human use are also discussed.

Technology evaluation: HspE7 (Stressgen).

Stressgen is developing HspE7, a recombinant fusion protein comprising the human papillomavirus (HPV) E7 antigen and the heat shock protein Hsp65 from Mycobacterium bovis, as a potential therapy for conditions associated with HPV infection. This therapy is currently undergoing phase III clinical trials.

Infecções por Papilomavírus humano e neoplasia do colo uterino: efeito do polimorfismo dos genes HLA-DRB1 e DQB1 e respostas linfoproliferativas contra peptídeos virais / Infection for human Papillomavirus and lesions of the uterine cervix: effect of the polymorphism HLA-DRB1 and DQB1 genes and lymphoproliferative responses against virus peptide

Infecçäo persistente por tipos oncogênicos de papilomavírus humano (HPV) é considerada como o principal fator de risco para desenvolvimento de carcinoma invasivo do colo uterino (CCU) e de lesões intraepiteliais cervicais (SIL). Fatores genéticos do hospedeiro, como o polimorfismo de genes HLA (human leukocyte antigen), também têm sido implicados na suscetibilidade a estas patologias e à infecçäo por (HPV), como observado em diversos estudos caso-controle. Neste estudo investigou-se em uma coorte de mulheres (Ludwig-McGill cohort) se a variabilidade dos genes HLA-DRB1 e DQB1 influenciam na história natural das infecções por HPV e no risco de SIL. A tipificaçäo de DRB1 e DQB1 foi realizada em 620 amostras provenientes de um estudo epidemiológico prospectivo...