Peter Dall

Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

PurposeAlternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo.Experimental designHMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival.ResultsIntravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone.ConclusionInjected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Limited relevance of the CHEK2 gene in hereditary breast cancer

To establish the importance of CHEK2 mutations for familial breast cancer incidence in the German population, we have screened all 14 of the coding exons in 516 families negative for mutations in both the BRCA1 and BRCA2 genes. We found 12 distinct variants in 30 unrelated patients (5.81%), including 5 that are novel and an additional 4 found for the first time in breast cancer. These aberrations were evaluated in 500 healthy women aged over 50 years and in the case of the 2 exon 10 mutations, 1100delC and 1214del4bp, in 1315 randomized healthy controls. According to our results, a statistically significant association for the exon 10 mutations was observed (p = 0.006). The prevalence of the 1100delC mutation in the German population, however, is significantly lower than those reported for other Caucasian populations both in familial breast cancer patients (1.6%) and controls (0.5%), and shows independent segregation with breast cancer in 2 of 4 families analyzed. The remaining 10 variants were more abundant in patients (21) compared to the controls (12) although the difference was not statistically significant. Interestingly, we found no increased breast cancer risk associated with the splice site mutation IVS2+1G→A or the most common missense mutation I157T, which account for more than half (12/21) of the variants observed in patients. The low prevalence and penetrance of the exon 10 deletion mutations together with no, or an uncertain elevation in risk for other CHEK2 mutations suggests a limited relevance for CHEK2 mutations in familial breast cancer. Further evaluation of the unique variants observed in breast cancer is required to determine if they may play a role in a polygenic model of familial breast cancer. Nevertheless, it seems premature to include CHEK2 screening in genetic testing.

Regression of cutaneous tumor lesions in patients intratumorally injected with a recombinant single-chain antibody-toxin targeted to ErbB2/HER2

ScFv(FRP5)-ETA is a recombinant single-chain antibody-toxin with binding specificity for ErbB2/HER2. Previously potent antitumoral activity of the molecule against ErbB2 overexpressing tumor cells was demonstrated in vitro and in animal models. Here we report on the first application of scFv(FRP5)-ETA in human cancer patients summarizing case reports collected in four different clinical centers. Eleven patients suffering from metastatic breast and colorectal cancers and from malignant melanoma were treated on a compassionate-use basis by intratumoral injection of scFv(FRP5)-ETA into cutaneous lesions once daily for 7–10 days. Total daily doses ranged from 60 to 900 µg, and total doses per treatment cycle ranged from 0.6 to 6.0 mg. Treatment caused injected tumors to shrink in six of the 10 cases evaluated (60%). Complete regression of injected tumor nodules was accomplished in four patients (40%) and partial reduction in tumor size in another two patients (20%). Adverse reactions were restricted to local symptoms such as pain and inflammation at injection sites which were fully reversible. Only in one patient treated at the highest daily doses systemic liver toxicity of grade 2 was observed and treatment was discontinued on day 7. No hematologic, renal, and/or cardiovascular toxicities were noted. Our results demonstrate that local therapy with scFv(FRP5)-ETA can be effective against ErbB2 expressing tumors justifying further clinical development of this reagent.

Growth retardation of tumors by adoptive transfer of cytotoxic T lymphocytes reprogrammed by CD44V6‐specific SCFV: ζ‐chimera

Variants of the CD44 protein family containing sequences encoded by variant exon 6 (v6) are involved in the metastatic spread of rat and human tumors. The rat‐specific antibody I.IASML, which recognizes a v6 epitope, interferes with metastatic dissemination of a rat pancreatic carcinoma. The single‐chain antigen‐binding fragment of this monoclonal antibody was fused to the ζ‐chain of the T‐cell receptor complex. The appropriate fusion gene was incorporated into a retroviral gene transfer vector. Murine cytotoxic T lymphocytes (CTLs) were infected, and cellular clones which express the single‐chain ζ‐chain fusion protein on their cell surface were selected. These CTLs are not MHC‐restricted in their CD44v6 recognition and exhibit in vitro lytic activity toward cells expressing CD44 variants comprising exon v6. Tumor cell xenografts grown in athymic nude mice are suppressed in their growth upon infusion of the genetically manipulated CTLs. Our data indicate that the CD44v6 epitope is an effective target for immune tumor therapy and demonstrate the efficacy of genetically engineered CTLs in targeting tumors expressing such epitopes.

Prognostic value of CD44 variant expression in primary breast cancer

CD44 is a family of cell surface transmembrane glycoproteins members which differ in the extracellular part by sequences derived by alternative splicing of 10 variant exons (v1– v10). CD44 proteins containing such variant sequences have been implicated in tumor metastasis formation. Here, we have evaluated the expression of CD44 variants by immuno‐histochemistry in primary breast cancer samples of 237 node‐negative and 230 node‐positive patients. For the analysis of samples derived from node‐negative patients, the exon‐specific antibodies used were DIII, vff7 and vff18 (v6), vff17 (v7/v8), fw11.24 (v9) and vff16 (v10). With the different antibodies which recognize v6 epitopes, the majority of tumors were positively stained (≥65% of the tumors) with varying intensities. Thirty‐nine percent of the tumors were positively stained with the antibody vff16, and approximately half of the tumors with the antibodies vff17 and fw11.24. The expression of CD44 v6 epitopes in tumors from node‐negative patients was associated with a favorable prognosis, both upon univariate and multivariate analysis. The expression of CD44 v7/8, v9 or v10 epitopes was not significantly related with relapse‐free survival. Samples from node‐positive patients were only examined with the antibodies vff7, vff17 and vff18. The staining with none of these antibodies was correlated with the length of relapse‐free survival of the patients. Our data suggest that, generally, the usefulness of knowledge of CD44 variant expression is of limited value for assessing the risk of relapse in patients with primary breast cancer. However, the expression of exon v6 of CD44 may be a marker to identify patients with a relatively favorable prognosis in node‐negative patients. Int. J. Cancer (Pred. Oncol.) 84:209–215, 1999.

Hormone Replacement Therapy after Treatment of Breast Cancer: Effects on Postmenopausal Symptoms, Bone Mineral Density and Recurrence Rates

Purpose: Breast cancer (BC) is the most frequent female carcinoma and the major cause of death in women aged 35–50 years. The total number of patients surviving BC and especially the morbidity rate of patients below the age of 55 years has increased significantly in the last several years. As a consequence, the number of BC patients suffering from the long-term effects of estrogen deficiency due to adjuvant treatment is increasing. At present, hormone replacement therapy (HRT) following BC treatment is applied individually and mainly depends on the severity of postmenopausal symptoms (PMS) experienced by these patients. Patients and Methods: In a retrospective study (total n = 185 BC patients, 64 with and 121 without HRT), the effect of HRT during or after adjuvant therapy [chemotherapy and/ or (anti-) hormonotherapy] has been investigated. The surveillance period was up to 60 months. Evaluated were HRT effects on (1) PMS measured by a comprehensive life quality questionnaire, (2) bone mineral density (BMD) measured by osteodensitometry and (3) morbidity as well as mortality rates. Results: Both groups did not differ with regard to tumor stage, lymph node involvement, metastasis, grading, and steroid hormone receptor status. A reduction in PMS was significant in women taking HRT (p < 0.001), especially in the subgroup of women ≤50 years (p < 0.0001). For both age groups, the median reduction in BMD (z-score) was less in women receiving HRT (≤50 years: without HRT –1.99 vs. with HRT –0.95, p < 0.05; >50 years: without HRT –2.29 vs. with HRT –1.19, p < 0.01). There were no statistically significant differences regarding morbidity and mortality (p = 0.29). Conclusion: In this study of BC patients, the use of HRT shows positive effects on PMS and BMD. There was no significant influence on morbidity or mortality. However, a reevaluation of HRT in the routine management of BC patients should await the results of prospective randomized trials.

Molecular mechanism of estrogen receptor (ER)α-specific, estradiol-dependent expression of the progesterone receptor (PR) B-isoform

The physiological effects of progesterone are mediated by the progesterone receptor (PR) isoforms PRA and PRB, transcribed from a single gene, under control of two distinct promoters. Both the isoforms display different, promoter- and cell line-specific transactivation properties. Upregulation of both isoforms in response to estradiol stimulation has been described, although the two promoters contain no classical estrogen response element (ERE). Therefore, we decided to investigate the regulation of PRB-expression through distinct estrogen receptor (ER)-isoforms: ERalpha and ERbeta We demonstrate, that in HeLa cells treated with E2, PRB promoter activity was enhanced (five-fold) by ERalpha, but not by ERbeta. ERbeta was also unable to stimulate activity of the PRB promoter in BT20 and Ishikawa cells, where ERalpha induced reporter activity by two-fold. Deletion of the AF1-but not AF2 domain from ERalpha resulted in loss of the transactivation potential in all cell lines tested. Furthermore, in BT20 cells deletion of the AF2 domain of ERalpha resulted in stronger transcriptional activation than that mediated through wild-type ERalpha. In SK-BR-3 cells both ERs repressed PRB promoter activity and this repression was enhanced by co-transfection of SRC1. However, strong estrogen-dependent stimulation was observed after deletion of AF2. We conclude that PRB expression is stimulated by ERalpha but not ERbeta in an unique, AF1-dependent but AF2-independent mechanism.

Expression of the hyaluronan receptor RHAMM in endometrial carcinomas suggests a role in tumour progression and metastasis.

Purpose. Interactions of hyaluronic acid (HA) with its binding protein RHAMM (receptor for HA-mediated motility) have been proposed as being important in promoting tumour progression and dissemination. This comparative study was designed to investigate the RHAMM expression patterns in endometrial carcinoma.Methods. We examined a series of 89 endometrial carcinomas and 15 normal endometrial tissues by immunohistochemistry, using a RHAMM-specific polyclonal antibody. Expression of RHAMM was assessed according to the pattern and intensity within (overall cytoplasm, center/periphery of tumours) and between the tumours. The staining results were compared to the corresponding clinical data (age, menopause status, histological staining, histological grading, lymph node status).Results. RHAMM-expression was detectable in 58% of the 89 tumours [Histological stage: pT1a (8/12); pT1b (16/37); pT1c (18/26); pT2 (6/9); pT3a (4/5)] and 13% (2/15) of the normal endometrial tissues. The positivity rates for RHAMM were 100% in patients with positive lymph nodes but only 50.7% in patients with negative lymph nodes (P<0-01). Additionally, the expression pattern showed a highly significant correlation (P<0.01) with the histological grade of the tumours [G1 (6/42), G2 (33/34), G3 (13/13)] and occurrence of lymph node metastases.Conclusions. Our results suggest that RHAMM expression may enhance and improve the invasion and metastasis of endometrial carcinomas.

Gene transfer to cervical cancer with fiber-modified adenoviruses

Successful adenoviral (Ad) vector–mediated strategies for cancer gene therapy mandate gene‐delivery systems that are capable of achieving efficient gene delivery in vivo. In many cancer types, in vivo gene‐transfer efficiency remains limited due to the low or highly variable expression of the primary Ad receptor, the coxsackie Ad receptor (CAR). In this study, we evaluated the expression of CAR on cervical cancer cells as well as CAR‐independent targeting strategies to integrins (Ad5.RGD), heparan sulfate proteoglycans (Ad5.pK7) or both (Ad5.RGD.pK7). We used a panel of established cervical cancer cell lines and primary cervical cancer cells isolated from patients to quantify the expression of CAR mRNA and to evaluate the gene‐transfer efficiency of fiber‐modified Ads. Of the fiber‐modified vectors, Ad5.pK7 and Ad5.RGD.pK7 displayed significantly enhanced gene‐transfer efficiency in vitro. Gene‐delivery efficiency in vivo was evaluated using an s.c. cervical cancer mouse model. Ad5.RGD.pK7 significantly improves tumor targeting in vivo, resulting in a significantly improved tumor/liver ratio in mice. Our results suggest that the double‐modified Ad5.RGD.pk7 vector enhances gene transfer to clinically relevant cervical cancer substrates, while the infectivity of nontarget cells in the mouse is not increased and comparable to Ad5. The fiber‐modified virus described here can help achieve higher clinical efficacy of cervical cancer gene therapy.

Characterisation of the murine gene encoding the intracellular hyaluronan receptor IHABP (RHAMM).

We have recently shown that the published cDNA sequence encoding the murine cell surface receptor for hyaluronan-mediated motility (RHAMM) in fact represents a partial sequence of the cDNA encoding a new intracellular hyaluronic acid binding protein (IHABP). Here we publish the genomic organisation, including 700bp sequences of the promoter region, of the IHABP gene. The IHABP gene consists of 18 exons and spans more than 25kb. Part of the IHABP gene is identical with the published data on RHAMM. The IHABP gene apparently possesses one promoter region with one major transcriptional start point. IHABP is ubiquitously expressed at the mRNA and the protein level in all murine tissues, suggesting that the function of this intracellular hyaluronan binding protein is not restricted to migrating cells.