Mesothelin as a target for cervical cancer therapy.

PURPOSE: The cell surface glycoprotein Mesothelin is overexpressed in several tumor entities and novel immune-based therapies are currently under the early clinical evaluation for the treatment of malignant pleura mesothelioma, ovarian cancer, and pancreatic cancer. Cervical cancer has not been recognized as a suitable target for Mesothelin-directed immune therapies so far. METHODS: To exploit a possible role of Mesothelin in cervical cancer treatment, we analysed Mesothelin expression in 79 cervical carcinomas and aligned expressions patterns with tumor growth parameters. A novel anti-Mesothelin drug conjugate (Anetumab Ravtansine) was applied for dose-efficiency studies in a Mesothelin positive tumor model for cervical cancer in Scid mice. RESULTS: In more than three-quarters (77%) of cervical adenocarcinomas, Mesothelin was expressed to high levels. Among squamous cell carcinomas of the cervix uteri expression levels were lower and expression patterns were less intense, but still ranged between 50-60% (57%). A significant correlation between Mesothelin expression levels and tumor grade, metastatic behaviour, and lymph- or hemangiosis was not found. The novel anti-Mesothelin-drug conjugate (Anetumab Ravtansine) showed a substantial dose-dependent therapeutic efficiency in a xenotransplant model for cervical cancer in SCID mice (hela cell tumors). Applying the ADC at a dose of 10 mg/kg twice weekly induced complete tumor regression in 88% of animals within 6 weeks. CONCLUSIONS: Mesothelin should be taken into account as a target in cervical cancer therapy and histological determination of Mesothelin expression should be considered in routine diagnostics of cervical carcinomas.

Validity of the colposcopic criteria inner border sign, ridge sign, and rag sign for detection of high-grade cervical intraepithelial neoplasia.

OBJECTIVE: To evaluate the association of three pathognomonic criteria, inner border, ridge sign, and rag sign with high-grade cervical intraepithelial neoplasia (CIN) using video exoscopy. METHODS: Retrospective evaluation of video recordings of 335 patients, referred for diagnostic colposcopy, who underwent cervical biopsies, and, if indicated loop excisions, was performed. The most severe histologic diagnosis was recorded. Sensitivity, specificity, positive, negative predictive value, and likelihood ratios for high-grade CIN were calculated. RESULTS: In 285 patients (85%), a single colposcopy directed biopsy was taken; 50 patients (15%) underwent two biopsies. One hundred sixty-two patients (48%) underwent subsequent magnification-guided loop excision. Sensitivity, specificity, positive predictive value, and negative predictive value of the inner border to detect high-grade CIN were 20%, 99%, 97.9%, and 34.8%, respectively. The positive likelihood ratio (LR+) was 20.3 and the negative likelihood ratio (LR-) was 0.81. Sensitivity, specificity, positive predictive value, and negative predictive value of the ridge sign to detect high-grade CIN were 52.5%, 96.4%, 96.8%, and 46.6%, respectively. The LR+ ratio was 13.2 and the LR- ratio was 0.49. Sensitivity, specificity, positive predictive value, and negative predictive value of the rag sign to detect high-grade CIN were 38.4%, 96%, 95.7%, and 40.2%, respectively. The LR+ ratio was 9.7 and the LR- ratio was 0.6. Only the ridge sign showed a correlation with young age. Presence of any two signs significantly increased the LR of the presence of high-grade CIN. CONCLUSION: The inner border, ridge sign, and the newly defined rag sign are objective, effective colposcopic signs and are significantly associated with high-grade CIN.

Evaluation of the VITOM in digital high-definition video exocolposcopy.

OBJECTIVE: : Our aim was to present our initial clinical experience using a novel exoscopically based colposcopy system (VITOM) for the evaluation of cervical, vulvar, and vaginal diseases. MATERIALS AND METHODS: : Women referred to the Charite Cervix Center, Charite University, Berlin, Germany, were included. Patients with abnormal Pap smear results, vulvar lesions, or a biopsy report of neoplasia of the lower genital tract were included into the study. The VITOM was used for colposcopic evaluation and directed biopsies. Colposcopic findings were reported according to the criteria of the Committee on Nomenclature of the International Federation of Cervical Pathology and Colposcopy. Histologic diagnosis was described as normal, low-grade lesion, high-grade lesion (including cervical intraepithelial neoplasia 2,3, vulvar intraepithelial neoplasia 2,3, vaginal intraepithelial neoplasia 2,3), or cancer. RESULTS: : We recruited 76 patients (54 with cervical, 4 with vaginal, and 18 with vulvar disease) to the prospective study. Four patients were pregnant. Of patients with cervical disease, 29% had a history of previous conization and 3.7% had a history of trachelectomy. The sensitivity, specificity, negative predictive value, and positive predictive value of the VITOM for cervical intraepithelial neoplasia 2, 3 were 90%, 77%, 90% and 77%, respectively. Concordance of exocolposcopic impressions and histologic results was higher in high-grade lesions (K = 0.68, 95% CI = 0.32-0.87, p < .001) than in low-grade lesions (K = 0.41, 95% CI = 0.1-0.41, p < .05). CONCLUSIONS: : Exocolposcopy with the VITOM is accurate and shows good correlation to histologic findings in high-grade disease of the lower genital tract. The potential advantages include patient and trainee involvement in examination, decision making, and documentation.

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.

Non-physiological overexpression of the low density lipoprotein receptor (LDLr) gene in the liver induces pathological intracellular lipid and cholesterol storage.

BACKGROUND: Gene therapy of familial hypercholesterolemia (FH) requires successful transfer and lifelong expression of a functional low density lipoprotein receptor (LDLr) gene in the liver. Most of the vector systems currently employed for gene therapy use promoter elements which do not modulate transgene expression in a physiological manner. METHODS: To study the in vivo effects of constitutive LDLr gene expression in the absence of interfering immunological reactions we established a new mouse model which combines homozygous LDLr deficiency and severe combined immune deficiency (SCID). RESULTS: Adenovirus-mediated transfer and expression of the LDLr gene under the control of a commonly used virus-derived promoter (minimal CMV promoter) leads to prolonged reduction of serum cholesterol levels in LDLr-deficient SCID mice. During the first 10 days after gene therapy serum cholesterol drops to about 10% of pretherapeutic values. Serum cholesterol persists on this level for 2 weeks and then slowly starts to rise again. Four months after vector application serum levels have reached about 40% of pretherapeutic values. However, as early as 5 days after gene transfer, the histological analysis of liver sections revealed the formation of crystalline lipid/cholesterol deposits in the cytosol of hepatocytes. During the following 8 weeks the amount of crystals increased in size and density. The intracellular storage of lipid and cholesterol reduced cell viability and induced an accelerated loss of therapeutic DNA from mice livers as was shown in a comparative expression study employing a transgene with a different metabolic function (human alpha 1-antitrypsin). CONCLUSIONS: The non-physiological constitutive overexpression of an LDL receptor gene induces an imbalance between the speed of LDL uptake and metabolism which leads to pathological accumulation of lipids and cholesterol in hepatocytes. To protect cells from negative effects of LDLr overexpression, future vector design should consider the use of physiologically controlled expression elements.

Hepatocarcinoma cells constitutively expressing adenoviral E1-genes provide a tumor model for intratumoral replication of E1-deficient adenoviruses.

The elimination of malignant tumors by intratumoral virus replication is a challenging therapeutic approach but is critically dependent on the speed and efficacy of intratumoral virus spread. The expression of oncolytic transgenes in the context of a replicating virus may help to enhance the therapeutic potency of this strategy. We have established a human hepatocarcinoma-derived cell line (Huh7-E1) which stably expresses adenoviral E1-genes. Tumors derived from these cells support replication of E1-deficient adenoviruses in SCID mice. This model can be used to evaluate E1-negative viruses encoding reporter genes or oncolytic transgenes in a replicating context. Most oncolytic viruses for human use could then be re-engineered as E1-postive viruses. Moreover, Huh7-E1 tumors release human alpha-1-antitrypsin (hAAT), which allows the monitoring of occult growing tumors (i.e. liver, peritoneum) by measuring serum hAAT levels.