Analysis of 4 single-nucleotide polymorphisms in relation to cervical dysplasia and cancer development using a high-throughput ligation-detection reaction procedure.

BACKGROUND: Host genetic characteristics and environmental factors may correlate with risk for cervical cancer development. Here we describe a retrospective screening study for single nucleotide polymorphisms (SNPs) in genetic markers TP53, MTHFR, CYP1A1, and CYP2E1 in 749 patients. METHODS: A multiplex ligation-dependent polymerase chain reaction approach was applied. We used archived material from human papillomavirus tests and correlated SNP genotypes to the corresponding clinical data. Semantic integration was used to identify and evaluate the clinical status from electronic health records. RESULTS: An association with cervical cancer and high-grade dysplasia was found for the rare homozygous CC genotype (rs4646903) in CYP1A1 (odds ratio [OR], 8.862). Odds ratios were also significantly elevated for heterozygous MTHFR CT genotype (rs1801133; OR, 1.457). No significant association was found in TP53 (rs1042522) and CYP2E1 (rs3813867). In addition, we found smokers at higher risk (OR, 2.688) and identified pregnancies as a significant risk factor (OR, 1.54). CONCLUSIONS: Our protocol enables a feasible way for further retrospective large sample size evaluation of potential genetic markers. This study revealed genetic associations of a rare SNP genotype with cervical dysplasia in one of the largest patient sample to date that warrants further investigation.

Epidermal growth factor receptor expression affects the efficacy of the combined application of saponin and a targeted toxin on human cervical carcinoma cells.

Cervical cancer is the second most common cancer in women worldwide. Targeting the epidermal growth factor receptor (EGFR) is a very promising approach since it is overexpressed in about 90% of cervical tumors. Here, we quantified the toxic effect of SE, a targeted toxin consisting of epidermal growth factor (EGF) as targeting moiety and the plant toxin saporin-3, on 3 common human cervical carcinoma cell lines (HeLa, CaSki and SiHa) and recently established lines (PHCC1 and PHCC2) from 2 different individuals. A human melanocytic and a mouse cell line served as negative control. Additionally, we combined SE with saponinum album, a saponin composite from Gypsophila paniculata, which exhibited synergistic properties in previous studies. The cell lines, except for SiHa cells, revealed high sensitivity to SE with 50% cell survival in the range of 5-24.5 nM. The combination with saponin resulted in a remarkable enhancement of cytotoxicity with enhancement factors ranging from 9,000-fold to 2,500,000-fold. The cytotoxicity of SE was clearly target receptor specific since free EGF blocks the effect and saporin-3 alone was considerably less toxic. For all cervical carcinoma cell lines, we evinced a clear correlation between EGFR expression and SE sensitivity. Our data indicate a potential use of targeted toxins for the treatment of cervical cancer. In particular, the combination with saponins is a promising approach since efficacy is drastically improved.

Creation and characterization of a xenograft model for human cervical cancer.

OBJECTIVE: Most of primary human cancer tissues show effective engraftment and proliferation after transplantation onto Scid mice. However xenotransplantation of vital specimens of cervical carcinoma has not been successful in the past, also the generation of cell lines from primary cervical cancer has hardly ever been possible. The lack of appropriate xenograft models impedes the search for improved specific therapeutic agents. METHODS: We explored the efficiency of different techniques for tumor transplantation and describe the first protocol to enable reliable and efficient engraftment of human cervical cancer in Scid beige mice. To demonstrate the value of this tumor model, we explored the therapeutic potency of a novel immunotoxin (SA2E). SA2E is a chimeric protein constructed by fusing the human epidermal growth factor and the plant protein toxin saporin. RESULTS: About 70% of transplanted tumors exhibited potent proliferation, and multiple retransplantation was possible in 40%. Local treatment with the immunotoxin SA2E had a dose dependent therapeutic effect and achieved a tumor volume reduction of up to 60%. CONCLUSIONS: Reliable engraftment and high reproducibility make this novel xenograft model an attractive test system to identify new therapeutic agents for cervical cancer.

Regulatory (FOXP3+) T cells as target for immune therapy of cervical intraepithelial neoplasia and cervical cancer.

Regulatory (FOXP3+) T cells (Tregs) comprise a subpopulation of CD4+ T cells that suppress autoreactive immune cells, thereby protecting organs and tissues from autoimmunity. Tregs have also been detected in human malignancies and their depletion or inactivation substantially improves cellular antitumor immunity in preclinical studies. Novel therapeutic strategies for cervical cancer and precancerous cervical intraepithelial neoplasia (CIN) focus on immune-modulatory and cancer vaccination approaches. In this context, the frequency of Tregs in cervical cancer and precancerous CIN could influence therapeutic strategies. We determined the frequency of infiltrating CD4+ and CD8+ T cells as well as FOXP3+ Tregs in high-grade CIN lesions (CIN III) and cervical carcinoma compared to colon carcinoma, skin melanoma, and bronchial carcinoma. We show that human papilloma virus-derived lesions have a significantly higher number of infiltrating lymphocytes and FOXP3+ Tregs compared to three other common tumor entities. In addition we explored the therapeutic effect of agonistic anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein antibodies that, by single systemic application, inactivate Tregs and induce strong intratumoral invasion of CD8+ T cells and complete tumor eradication in 70% of treated animals. The large number of Tregs in human papilloma virus-derived lesions suggests a pivotal role of Tregs for counteracting the host immune response. We therefore regard CIN and cervical cancer as prime targets for new immune-based non-invasive therapies.

Combining T-cell vaccination and application of agonistic anti-GITR mAb (DTA-1) induces complete eradication of HPV oncogene expressing tumors in mice.

We generated an adenovirus-based T-cell vaccine (Ad-p14) that reliably elicits T-cell responses to human papillomavirus (HPV) oncogenes of the 2 most common high-risk HPV serotypes. The artificial gene used to create the vaccine comprising 415 aa (1248 bp) was cloned by fusing 14 polymerase chain reaction fragments of HPV16 and HPV18 E6 and E7 oncogenes devoid of sequences with transforming potential. Although ensuring maximal biologic safety, the construct includes approximately 70% of the relevant T-cell epitopes. In a tumor model for cervical cancer (C3), therapeutic vaccination led to complete eradication in 100% of the mice. In a second model (TC1), it induced initial tumor mass reduction, but 90% of the animals showed delayed tumor progression. To further improve the therapeutic effect, vaccination was combined with systemic application of imiquimod, anti-CD4, alpha-interferon, or anti-GITR. Although adding alpha-interferon improved the therapeutic potential of Ad-p14 by 40%, the combination with anti-GITR resulted in complete and permanent eradication of all TC1 tumors. Ad-p14 has clinical potential for treating HPV-induced lesions, and the added effect of immune response modifiers stresses the importance of combined protocols for immunotherapy of malignant tumors.

A flow cytometry-based assay to assess minute frequencies of CD8+ T cells by their cytolytic function.

Limited sample size and low sensitivity of currently used functional assays challenge direct analysis of cytotoxic CD8+ T lymphocyte activity to quantify antigen-specific immunity after infection or vaccination. Our flow cytometry-based assay reproducibly detects at least three epitope-specific CD8+ T lymphocytes by their cytolytic function. As exemplified for viral epitopes restricted to the human leukocyte antigen (HLA)-A2, the HLA-A2+ human somatic cell hybrid T2 provided an about 10-fold more sensitive readout as compared to autologous B-lymphoblastoid cells or the human erythroleukemia cell line K562 transfected to express HLA-A2 when used as target cells. We named our assay VITAL-FR assay, referring to Hermans et al. (2004) and indicating the modification of using Far Red (FR) dye instead of CMTMR. Under optimal conditions the VITAL-FR assay proved 30 times more sensitive than the 51chromium-release assay to assess epitope-specific target cell lysis. The high overall sensitivity of the VITAL-FR assay basically depended on the negligible spectral overlap of the emission of a stable Far Red fluorescent reporter with the green tracer for target cell labelling. It also profited from long co-incubation of effector and target cells of up to 72, from prior in-vitro culture increasing the frequency of epitope-specific CD8+ T cells and from generic, easily accessible standardized target cells that were used with only 10(3) specific and 10(3) control target cells per individual experimental reaction. Our functional approach with the VITAL-FR assay therefore ideally suits for monitoring CD8+ T cell-mediated cytotoxicity in e.g. vaccination studies with known MHC-restricted immunogenic peptides in scientific and diagnostic applications.