Helmut Deissler

Common Breast Cancer-Predisposition Alleles Are Associated with Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.

Expression Profiling of Mammary Carcinoma Cell Lines: Correlation of in vitro Invasiveness with Expression of CD24

Invasiveness and the capacity of tumor cells to form distant metastases are important cellular characteristics associated with a poor prognosis in breast cancer patients. In an approach to find genes that are potentially involved in these processes, RNA species showing different abundance in RNA pools from 12 invasive and 13 noninvasive mammary carcinoma-derived cell lines have been identified by hybridization to cDNA microarrays. CD24, keratin 19, keratin 8, GOB-4 and ezrin-radixin-moesin-binding phosphoprotein 50 were found to be preferentially expressed by noninvasive cells whereas vimentin was confirmed as a characteristic of invasive cells. Only differences in expression higher than 3-fold evident in three independent hybridization experiments were considered significant. For all cell lines, expression of mRNA coding for the adhesion molecule CD24, previously suggested to play an important role during tumor progression to more invasive phenotypes, has been quantified by real-time RT-PCR. Flow-cytometric analyses confirmed that CD24 mRNA reflects the amount of cell surface CD24 (Spearman R = 0.88, p = 10–6). CD24 mRNA was found to be absent or weakly expressed in 9/12 (75%) invasive cell lines compared to 3/13 (23%) noninvasive cell lines. The correlation between CD24 expression and invasiveness was calculated to be highly significant with χ2 = 6.74 and p = 0.0094. Future analyses of primary breast carcinomas are warranted to define the role of CD24 in future diagnostic and therapeutic approaches.

VEGF-induced effects on proliferation, migration and tight junctions are restored by ranibizumab (Lucentis) in microvascular retinal endothelial cells

Background: Because vascular endothelial growth factor (VEGF) signalling is deregulated in diabetic retinopathy, the potential therapeutic effects of VEGF inhibitors such as the human VEGF-specific antibody ranibizumab are currently being tested. A study was undertaken to determine whether VEGF-stimulated processes in retinal endothelial cells are reversed by ranibizumab. Methods: The influence of VEGF121 and VEGF165 on the proliferation and migration of immortalised bovine retinal endothelial cells (iBREC) was studied in the presence and absence of ranibizumab. In addition, the protein composition of tight junctions in the presence of VEGF and its inhibitor in iBREC was investigated. Results: While both isoforms stimulated proliferation of iBREC, only VEGF165 influenced cell migration. The addition of ranibizumab counteracted this stimulation without inhibition of the basal levels of migration and proliferation. Plasma membrane staining of the tight junction proteins occludin and claudin-1 disappeared in the presence of VEGF165; there was no effect on claudin-5 and ZO-1 was only weakly affected. The addition of ranibizumab restored plasma membrane localisation of occludin and claudin-1. For claudin-1, the variation in total protein expression corresponded with the observed effects of VEGF165 and ranibizumab. Conclusion: Ranibizumab reverses proliferation and cell migration stimulated by VEGF and delocalisation of tight junction proteins induced by VEGF165 in iBREC.

Germline mutations in the PALB2 gene are population specific and occur with low frequencies in familial breast cancer

The Partner and Localizer of BRCA2 (PALB2) protein has been linked to Fanconi anemia and breast cancer predisposition. Here we present data of a comprehensive mutation screening of the PALB2 gene in 818 familial cases of breast cancer from Germany. By analysing the entire coding region of PALB2, we found seven truncating mutations (six of them novel) in families tested negative for BRCA1/2‐mutations. In addition, two novel potentially disease causing missense mutations were found. Remarkably, only one mutation reported previously in other populations, was also identified in the German population. No PALB2 mutation carriers were identidfied in 450 unaffected controls. Thus, our observations indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the German population. As PALB2‐deficient tumors were shown to be sensitive to Poly(ADP‐ribose) Polymerase (PARP) inhibitors, our study has implications for newly developed, favourable treatment options in familial breast cancer.

Association of the Variants CASP8 D302H and CASP10 V410I with Breast and Ovarian Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Background: The genes caspase-8 (CASP8) and caspase-10 (CASP10) functionally cooperate and play a key role in the initiation of apoptosis. Suppression of apoptosis is one of the major mechanisms underlying the origin and progression of cancer. Previous case-control studies have indicated that the polymorphisms CASP8 D302H and CASP10 V410I are associated with a reduced risk of breast cancer in the general population. Methods: To evaluate whether the CASP8 D302H (CASP10 V410I) polymorphisms modify breast or ovarian cancer risk in BRCA1 and BRCA2 mutation carriers, we analyzed 7,353 (7,227) subjects of white European origin provided by 19 (18) study groups that participate in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). A weighted cohort approach was used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI). Results: The minor allele of CASP8 D302H was significantly associated with a reduced risk of breast cancer (per-allele HR, 0.85; 95% CI, 0.76-0.97; Ptrend = 0.011) and ovarian cancer (per-allele HR, 0.69; 95% CI, 0.53-0.89; Ptrend = 0.004) for BRCA1 but not for BRCA2 mutation carriers. The CASP10 V410I polymorphism was not associated with breast or ovarian cancer risk for BRCA1 or BRCA2 mutation carriers. Conclusions: CASP8 D302H decreases breast and ovarian cancer risk for BRCA1 mutation carriers but not for BRCA2 mutation carriers. Impact: The combined application of these and other recently identified genetic risk modifiers could in the future allow better individual risk calculation and could aid in the individualized counseling and decision making with respect to preventive options in BRCA1 mutation carriers. Cancer Epidemiol Biomarkers Prev; 19(11); 2859–68. ©2010 AACR.

New molecular targets of breast cancer therapy.

Background: The development of new chemotherapeutic agents and concepts of radiation therapy, administered as primary, adjuvant and palliative therapy, has led to new perspectives in breast cancer therapy. Apart from conventional chemotherapy, recently developed novel agents interfere with molecular mechanisms that are altered in cancer cells. Those targets are not necessarily breast cancer-specific. In this review we will focus on novel agents with potential or already proved benefit to breast cancer patients. Promising strategies include inhibition of growth factor receptors, blocking of tumor angiogenesis and signal transduction pathways, modulation of apoptosis, cancer vaccination, and inhibition of invasion and metastasis. Methods: Reports of relevant studies obtained from a search of MEDLINE and studies referenced in those reports were reviewed. Results: Apart from trastuzumab, other further developed compounds show promising results in clinical studies as a second generation of growth factor inhibitors. Different approaches in anti-angiogenetic therapy are under preclinical and clinical phase-II trials. Pro-apoptotic agents show synergistic effects with docetaxel in a clinical phase-I trial. Other compounds that target HSP 90, histone deacetylase and HMG-CoA reductase target atypical apoptotic pathways being lethal to tumor cells only but not to normal tissue, suggesting a tumor-specific way of action. MMP inhibitors have been demonstrating promising results in patients with refractory malignant pleural effusion in a phase-I trial. Several tyrosine kinase inhibitors currently under clinical investigation preliminarily show hopeful results in patients with advanced breast cancer. Furthermore, recent progress in defining the immunogenic epitopes of tumor antigens has rejuvenated the interest in cancer vaccines. Conclusion: Typical dose escalation studies leading to the highest clinically still tolerated dose do not appear to be equally appropriate for the estimation of efficiency of those compounds as for conventional cytotoxic regimes. Rather, escalation up to an amount of therapeutic agent that is sufficient for maximum target inhibition should be promoted, where classical measures of cytoreduction such as complete or partial remission are replaced both by time to progression and treatment failure as an appropriate measure of the efficacy of an agent.Hintergrund: Die Entwicklung neuer chemo- und strahlentherapeutischer Therapieansätze bietet neue Perspektiven in der systemischen Behandlung des Mammakarzinoms. Abgesehen von der klassischen Chemotherapie setzen diese Medikamente bei den in Tumorzellen veränderten subzellulären Mechanismen an. Wenngleich einige dieser neuen Therapieansätze nicht allein für das Mammakarzinom spezifisch sind, sondern auch in der Behandlung andere Tumorentitäten erprobt oder bereits eingesetzt werden, sollen in dieser Übersichtsarbeit des Umfangs wegen lediglich fünf für das Mammakarzinom relevante Gesichtspunkte besprochen werden: Wachstumsfaktoren und deren Rezeptoren, Invasion und Metastasierung, Angiogenese, Apoptose sowie die Immuntherapie. Methoden: Als Grundlage für diese Übersicht dienten in MEDLINE veröffentlichte Untersuchungen sowie darin zitierte Studien. Ergebnisse: Neben Trastuzumab zeigen neue, weiterentwickelte Substanzen bereits in klinischen Studien viel versprechende Ergebnisse als Inhibitoren von Wachstumsfaktoren. Verschiedene Ansätze der antiangiogenetischen Therapie werden in präklinischen Experimenten untersucht und teilweise in klinischen Studien erprobt. Proapoptotische Ansätze zeigen synergistische Effekte mit Docetaxel in der Therapie des metastasierten Mammakarzinoms. Andere Verbindungen gegen HSP 90, Histondeacetylase und HMG-CoA-Reduktase wirken in entarteten Zellen apoptotisch, nicht aber in Normalgewebe und scheinen somit eine gewissen Tumorspezifität zu besitzen. Die Identifizierung typischer Tumorantigene sowie neue Erkenntnisse immunologischer Mechanismen haben das Interesse der Immuntherapie des Mammakarzinom wiederbelebt. Schlussfolgerung: Typische dosisintensivierte Therapieregime bis zu einer von der Patientin gerade noch tolerierten Maximaldosis scheinen zur Beurteilung von Therapieeffekten solcher neu entwickelter Medikamente nicht geeignet zu sein. Vielmehr stellt die zur Hemmung des jeweiligen molekularen Ziels benötigte minimale Substanzkonzentration eine passende Messgröße dar. Klassische Einschätzungen des Therapieerfolgs anhand der Rate an kompletten oder partiellen Remissionen werden hier abgelöst werden von der Länge des Zeitintervalls bis zur Erkrankungsprogression oder bis zum Therapieversagen.

Low‐risk variants FGFR2, TNRC9 and LSP1 in German familial breast cancer patients

To validate common low‐risk variants predisposing for breast cancer (BC) in a large set of BRCA1/2 negative familial or genetically enriched cases from Germany, we genotyped 1,415 cases and 1,830 healthy women by MALDI‐TOF in 105 candidate SNPs. Significantly higher ORs than previously reported for heterozygous unselected cases were found for the minor allele in FGFR2 (OR = 1.43, 95% CI 1.30‐1.59, p‐value = 1.24 × 10−12) and for TNRC9 (OR = 1.33, 95% CI 1.19‐1.46, p‐value = 1.54 × 10−7). Most intriguing, however, were the ORs for homozygous carriers from high‐risk families for FGFR2 (OR = 2.05, 95% CI 1.68–2.51, LSP1 (OR = 0.49, 95% CI 0.28–0.86) and TNRC9 (OR = 1.62, 95% CI 1.27–2.07). Moreover, the additional validation of 99 CGEMS‐SNPs identified putative novel susceptibility alleles within the LSP1 gene (OR = 0.73, 95% CI 0.61–0.87, p‐value = 5.23 × 10−4). Finally, we provide evidence for the first time that a low‐risk variant located at 6q22.33 (rs6569479) is associated with estrogen receptor negative BC in familial cases (OR = 1.33, 95% CI 1.06–1.66; p‐value = 0.012). Our data confirm the impact of the previously identified susceptibility loci and provide preliminary evidence for novel susceptibility loci in familial BC cases and correlate them to specific histopathological subtypes defined by estrogen receptor status.

Tspan-1 is a tetraspanin preferentially expressed by mucinous and endometrioid subtypes of human ovarian carcinomas

In many human cancers, tumor progression was found to be associated with an altered expression of tetraspanins, a group of transmembrane adaptor proteins that are implicated in fundamental cellular processes. Although recognized as a characteristic of malignant cells of various origins, Tspan-1 has not yet been characterized in detail due to lack of specific antibodies. We describe the generation of Tspan-1-specific antibodies and immunohistochemical staining of different subtypes of ovarian carcinomas (n=72) that revealed significant differences in Tspan-1 expression that was pronounced in mucinous and endometrioid tumors. The observation that immunoreactivity was focused in intracellular vesicles often concentrated at the luminal sides of glandular structures further supported the assumption that Tspan-1 is involved in secretory pathways. In the group of serous ovarian carcinomas, pronounced expression of Tspan-1 was observed in FIGO stage III C-classified tumors of advanced stages. In summary, our results show that Tspan-1 is an important characteristic of malignant ovarian cancer cells and a potential therapeutic target.

Prediction of Nodal Involvement in Breast Cancer Based on Multiparametric Protein Analyses from Preoperative Core Needle Biopsies of the Primary Lesion

Purpose: Identification of molecular characteristics that are useful to define subgroups of patients fitting into differential treatment schemes is considered a most promising approach in cancer research. In this first study of such type, we therefore investigated the potential of multiplexed sandwich immunoassays to define protein expression profiles indicative of clinically relevant properties of malignant tumors. Experimental Design: Lysates prepared from large core needle biopsies of 113 invasive breast carcinomas were analyzed with bead-based miniaturized sandwich immunoassays specific for 54 preselected proteins. Results: Five protein concentrations [fibroblast growth factor-2 (FGF-2), Fas, Fas ligand, tissue inhibitor of metalloproteinase-1, and RANTES] were significantly different in the groups of patients with or without axillary lymph node metastasis. All 15 protein parameters that resulted in P values <0.2 and other diagnostic information [estrogen receptor (ER) status, tumor size, and histologic grading] were analyzed together by multivariate logistic regression. This yielded sets of five (FGF-2, Fas, Fas ligand, IP10, and PDGF-AB/BB) or six (ER staining intensity, FGF-2, Fas ligand, matrix metalloproteinase-13, PDGF-AB/BB, and IP10) parameters for which receiver-operator characteristic analyses revealed high sensitivities and specificities [area under curve (AUC) = 0.75 and AUC = 0.83] to predict the nodal status. A similar analysis including all identified parameters of potential value (15 proteins, ER staining intensity, T) without selection resulted in a receiver-operator characteristic curve with an AUC of 0.87. Conclusion: We clearly showed that this approach can be used to quantify numerous proteins from breast biopsies accurately in parallel and define sets of proteins whose combined analyses allow the prediction of nodal involvement with high specificity and sensitivity.

Actions of bevacizumab and ranibizumab on microvascular retinal endothelial cells: similarities and differences

Background Retinal endothelial cells are crucially involved in the genesis of diabetic retinopathy which is treated with vascular endothelial growth factor (VEGF) inhibitors. Of these, ranibizumab can completely restore VEGF-induced effects on immortalised bovine retinal endothelial cells (iBREC). In most experiments supporting diabetic retinopathy therapy with bevacizumab, only non-retinal EC or retinal pigment epithelial cells have been used. Also, bevacizumab but not ranibizumab can accumulate in retinal pigment epithelial cells. Objective To investigate the effects of bevacizumab on VEGF-induced changes of iBREC properties and potential uptake and accumulation of both inhibitors. Methods Uptake of VEGF inhibitors by iBREC with or without pretreatment with VEGF165 was visualised by immunofluorescence staining and western blot analyses. Measured transendothelial resistance (TER) of iBREC (±VEGF165) showed effects on permeability, indicated also by the western blot-determined tight junction protein claudin-1. The influence of bevacizumab on proliferation and migration of iBREC was studied in the presence and absence of VEGF165. Results Bevacizumab strongly inhibited VEGF-stimulated and basal migration, but was less efficient than ranibizumab in inhibiting VEGF-induced proliferation or restoring the VEGF-induced decrease of TER and claudin-1. This ability was completely lost after storage of bevacizumab for 4 weeks at 4°C. Ranibizumab and bevacizumab were detectable in whole cell extracts after treatment for at least 1 h; bevacizumab accumulated during prolonged treatment. Ranibizumab was found in the membrane/organelle fraction, whereas bevacizumab was associated with the cytoskeleton. Conclusion Both inhibitors had similar effects on retinal endothelial cells; however, some differences were recognised. Although barrier properties were not affected by internalised bevacizumab in vitro, potential adverse effects due to accumulation after repetitive intravitreal injections remain to be investigated.