Hans Georg Bender

Multistep carcinogenesis of breast cancer and tumour heterogeneity.

Abstractu2002Breast cancer emerges by a multistep process which can be broadly equated to transformation of normal cells via the steps of hyperplasia, premalignant change and in situ carcinoma. The elucidation of molecular interdependencies, which lead to development of primary breast cancer, its progression, and its formation of metastases is the main focus for new strategies targetted at prevention and treatment. Cytogenetic and molecular genetic analysis of breast cancer samples demonstrates that tumour development involves the accumulation of various genetic alterations including amplification of oncogenes and mutation or loss of tumour suppressor genes. Amplification of certain oncogenes with concomitant overexpression of the oncoprotein seems to be specific for certain histological types. Loss of normal tumour suppressor protein function can occur through sequential gene mutation events (somatic alteration) or through a single mutational event of a remaining normal copy, when a germline mutation is present. The second event is usually chromosome loss, mitotic recombination, or partial chromosome deletion. Chromosome loci 16q and 17p harbour tumour suppressor genes, which seem to be pathognomonic for the development or progression of a specific histological subtype. There are an overwhelming number of abnormalities that have been identified at the molecular level which fit the model of multistep carcinogenesis of breast cancer. When the functions of all of these genes are known and how they participate in malignant progression, we will have the tools for a more rational approach to diagnosis, prevention and treatment. This review deals only with the factors that are involved in the conversion of a normal breast cell into a malignant cell rather than those required for invasion and metastases. A key critical long-term step in the molecular analysis of breast cancer will be to link the specific molecular damage with the effects of environmental carcinogens.

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.

Effect of human papillomavirus vaccines on vulvar, vaginal, and anal intraepithelial lesions and vulvar cancer

OBJECTIVE: Human papillomavirus (HPV) is a necessary cause for cervical cancer, and it has been associated with vulvar and vaginal cancer and vulvar (VIN) and vaginal (VaIN) and anal (AIN) intraepithelial neoplasia. We assessed the prevalence of HPV (and the types) to estimate the possible effect of a HPV vaccine on lower genital tract disease prevention. METHODS: Two hundred fifty-eight samples of VIN, VaIN, AIN, and vulvar cancer from 241 women were included in the study. The diagnosis of surgical samples was made using published histomorphologic criteria. The DNA was extracted for HPV detection and typed using polymerase chain reaction and sequencing. RESULTS: The analyses were performed on 210 intraepithelial neoplasia samples (VIN2/3, VaIN2/3, AIN2/3) and 48 vulvar carcinoma samples. Human papillomavirus DNA was detected in 92%, 91%, 89%, and 60% of the VIN, VaIN, AIN, and vulvar carcinoma samples, respectively. High-risk HPV types 16 or 18 were detected in 76%, 64%, 81%, and 42% of the VIN2/3, VaIN2/3, AIN, and vulvar carcinoma samples. Women with HPV-positive samples were younger than those with HPV-negative samples (46 years compared with 55 years and 51 years compared with 61 years, for the VIN2/3 and vulvar carcinoma samples, respectively). Human papillomavirus–positive vulvar carcinoma was more frequent in women aged younger than 56 years (77%), than in those aged 56 years or older (41%). CONCLUSION: Based on the data obtained in this study, widely-implemented prophylactic HPV vaccination could make an important contribution to the reduction of the risk for cervical cancer and could also prevent about half the vulvar carcinomas in younger women and about two thirds of the intraepithelial lesions in the lower genital tract. LEVEL OF EVIDENCE: II-3

New aspects of vulvar cancer: Changes in localization and age of onset

OBJECTIVEnTo characterize the changes in incidence, age of disease onset, tumor site and patients characteristics in women with invasive vulvar cancer in a German University Hospital unit over a 28-year period.nnnMETHODSnThe clinical records for women treated for invasive vulvar cancer from 01/1980 until 06/2007 were analyzed. We performed a retrospective analysis for three 9-year periods: 1/1980 to 02/1989; 3/1989 to 04/1998 and 05/1998 to 06/2007. For each cohort, the number of cases treated, age of disease onset, tumor site and further characteristics were extracted and statistically evaluated.nnnRESULTSnA total of 224 patients with vulvar cancer were identified between 1/1980 and 6/2007. The number and mean age changed significantly over time: between 1/1980 and 02/1989 53 women with a mean age of 65.6 years were treated for invasive vulvar cancer, between 03/1989 and 04/1998 this number increased to 69 women with a mean age of 63.9 years and in the last period, 102 women with a mean age of 57.0 years were treated for vulvar cancer. The total increase was 192%. In the first period 11% of the women were aged 50 years or less compared with over 41% in the third period (p=0.001). Two-third of the tumors women aged<50 years were HPV-positive. Significant changes in the tumor site were observed; from labial position to the region between clitoris and urethra: 37% in the last period compared with 19% in the first period (p>0.05).nnnCONCLUSIONSnAlthough in the literature the incidence of invasive cancer has been reported to be stable or only minimally increased, the results of this study show that the number of patients presenting with invasive vulvar cancer has doubled within the last three decades at one university hospital unit in Germany, with a nearly 4-time increase in younger patients (+372%) due to HPV high risk infection. The tumor localization changed significantly from the labia to the area between the clitoris and urethra. Assuming that these limited data reflect the general trend in the incidence of HPV-induced vulvar cancer, widely-implemented prophylactic quadrivalent HPV vaccination, which has been proven to be highly effective against anogenital disease, could make an important contribution to the reduction of the risk of vulvar carcinomas in younger women.

Clinical impact of detection of loss of heterozygosity of BRCA1 and BRCA2 markers in sporadic breast cancer

The development of familial and sporadic breast cancer is based on genetic alterations of tumour-suppressor genes, for which loss of heterozygosity (LOH) is one mechanism of gene inactivation. To investigate LOH of BRCA1 (17q21) and BRCA2 (13-q12-13) in sporadic breast cancer, polymerase chain reaction (PCR)-based fluorescent DNA technology for detection of microsatellite polymorphisms was applied. A total of 137 breast cancer and 15 benign breast specimens with matched normal tissue were examined. Fluorescent-labelled PCR products were analysed in an automated DNA sequencer (ALFTM Pharmacia). Losses at both loci were correlated with different histological types, age, tumour size, lymph node status, grading and steroid hormone receptor expression, [SHR: oestrogen receptor (ER), progesterone receptor (PgR)]. For BRCA1 (D17S855, THRA1, D17S579) losses could be detected in invasive ductal carcinoma (IDC; n = 108) in 32-38%, invasive lobular carcinoma (ILC; n = 19) in 21-42% depending on the marker applied, but not in benign breast tumours (n = 15). Losses of BRCA1 markers correlated with larger tumour size, higher grade, and PgR expression. For BRCA2 (D13S260, D13S267, D13S171) losses could be detected in 108 IDCs in 30-38%, in 19 ILCs in 17-39% depending on the marker applied, but not in benign breast tumours. Losses of BRCA2 markers correlated only with higher grade. Microsatellite analyses combined with detection of fluorescent-labelled PCR products by an automated laser DNA sequencer can be used for routine determination of LOH. In sporadic breast cancer, LOH of BRCA1 of BRCA2 does not add decisive prognostic value as stated for familial breast cancer.

Comparison of rapidly cycled tandem high-dose chemotherapy plus peripheral-blood stem-cell support versus dose-dense conventional chemotherapy for adjuvant treatment of high-risk breast cancer: results of a multicentre phase III trial

BACKGROUNDnBreast cancer with extensive axillary-lymph-node involvement has a poor prognosis after conventional treatment. In trials with historical controls, high-dose chemotherapy produced improved outcomes. We compared an intensive double-cycle high-dose chemotherapy regimen with an accelerated conventionally dosed regimen in high-risk breast cancer in a multicentre trial.nnnMETHODSnPatients with at least nine positive nodes were randomly assigned either two courses of accelerated (2-week intervals, with filgrastim support), conventionally dosed epirubicin and cyclophosphamide followed by two courses of high-dose chemotherapy (epirubicin, cyclophosphamide, and thiotepa supported by peripheral-blood progenitors) or four identical cycles of epirubicin and cyclophosphamide followed by three cycles of accelerated cyclophosphamide, methotrexate, and fluorouracil. The primary endpoint was event-free survival. Analyses were done both by intention to treat and per protocol.nnnFINDINGSn403 patients were enrolled; 201 were assigned high-dose chemotherapy and 202 conventional treatment. The mean number of positive nodes was 17.6, and median follow-up was 48.6 months. 4-year event-free survival (intention-to-treat analysis) was 60% (95% CI 53-67) in the high-dose chemotherapy group and 44% (37-52) in the control group (p=0.00069). The corresponding overall survival was 75% (69-82) versus 70% (64-77; p=0.02). There were no treatment-related deaths.nnnINTERPRETATIONnOur finding of significant improvements in both event-free and overall survival for high-dose chemotherapy compared with a dose-dense conventional regimen contrasts with the results of other studies. The discrepancy might be due partly to design differences (tandem, brief induction) between our regimen and those studied in other trials. This approach merits further study.

Patterns of allelic loss on chromosome 17 in sporadic breast carcinomas detected by fluorescent-labeled microsatellite analysis.

Loss of heterozygosity (LOH) on chromosome 17 is a frequent genetic alteration in breast cancer. To assess whether the location of potential tumor suppressor genes is compatible with the LOH pattern in individual tumors, we analyzed allele loss on chromosome 17 in 121 invasive ductal breast carcinomas and 16 benign breast tumors with 14 polymorphic microsatellite markers (4 on 17p and 10 on 17q). Fluorescent polymerase chain reaction (PCR) for typing microsatellites coupled with DNA fragment analysis in an automated DNA sequencer was applied. Frequencies of LOH varied from 29.4% (D17S1322) to 57.4% (TP53‐Alu). No LOH could be detected in benign breast tumors. In 54 tumors the deletion patterns were consistent with the complete loss of 17p (n = 28), 17q (n = 9), or the whole chromosome 17 (n = 17). Five smallest regions of overlap (SROs) were identified in tumors with interstitial deletion patterns. On 17p, two foci were detected affecting the TP53 locus and the hypermethylated in cancer I (HICI) region (17p13.3). On 17q, SROI was localized between markers THRAI and D17S855, centromeric to the breast/ovarian cancer gene BRCAI; SRO2 was flanked by markers AFM234 and NMEI, and SRO3 was centered between markers MPO and GH. Associations between LOH and histopathological characteristics were determined. Significant correlations were found between higher grade and loss of the TP53 gene (marker TP53, P = 0.019), loss of the BRCAI region (P < 0.009), LOH of marker AFM155 (P = 0.003) and marker NMEI (P = 0.026). For positive estrogen receptor status, only LOH of the THRAI marker correlated significantly, whereas highly significant correlations were determined between positive progesterone receptor and markers centromeric to the BRCAI region D17S250 (P = 0.00002), THRAI (P = 0.0006), and the intragenic BRCAI markers [D17S1322 (P = 0.021), D17S855 (P = 0.029)]. Results presented in this study identify five independent regions of chromosome 17 which are likely to contain potential tumor suppressor genes involved in the carcinogenesis of sporadic breast cancer. Genes Chromosom. Cancer 18:181–192, 1997.

Comprehensive analysis of 130 multicentric intraepithelial female lower genital tract lesions by HPV typing and p16 expression profile

PurposeHPV associated cervical transformation is characterized by well-defined steps, including persistent HPV infection and deregulated oncogene expression. Recent studies have suggested that a number of lower genital tract lesions are clonally related to cervical lesions. In the current study, HPV infections and oncogene expression were assessed in a large series of patients with multicentric lower genital tract disease to analyze the transformation steps in extracervical disease.MethodsOne hundred and thirty biopsies of 52 women treated for multicentric synchronous or metachronous lower genital tract intraepithelial neoplasias were collected. Up to seven multicentric specimens taken from one patient were studied with a maximum follow up of 20xa0years. HPV typing and p16ink4a immunostaining was performed.ResultsHPV DNA was present in 121 of 130 specimens (93%). HPV16 was frequently found in VIN, VaIN and AIN (73, 60 and 77%, respectively), whereas only 37% of CIN were HPV16 positive. Infections with identical HPV types in multicentric lesions were diagnosed in 46% of the HPV positive patients. p16INK4a expression was negative in the nine HPV negative lesions whereas about 90% of the high grade lesions showed diffuse p16 staining.ConclusionOur findings indicate that multicentric lower genital tract disease evolves through different pathways. Some cases were related to a high susceptibility towards HPV infections, while others exhibited features of clonal propagation of transformed cervical cell clones. The clinical management of the latter group is particularly challenging, because malignant cell clones can persist over a long time course.

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.