pH-dependent vs. constant release of mesalazine in the treatment of ulcerative colitis: Do drug delivery concepts determine therapeutic efficacy? (Review).

Inflammatory bowel diseases (IBD) have developed to become a major global health problem. Ulcerative colitis (UC) is one of two main types of IBD, and >90% of patients suffering from mild or moderate forms of UC are treated with mesalazine, a well-tolerated and cost-effective drug. To allow oral administration, the drug has to be protected from resorption before it can reach the affected sites in the colon. The drug is therefore released from most currently used medications either constantly slow (time-dependent) or triggered by an increased pH during gastrointestinal transition. Both variants are widely used in clinical practice and it is surprising that they have not yet been compared directly in a large clinical study. In this overview, the evidence that may suggest preferential use of one type of mesalazine formulation over the other in general or for defined subgroups of patients is summarized and evaluated. Data from in vitro modelling of drug release and measurements of drug concentrations in colonic mucosa suggest that in many cases, constant release and pH-dependent formulations are of similar therapeutic efficiency; however, pH-triggered release may be superior in patients with proctitis-type UC or sites of inflammation in the proximal colon. Additionally, patients with a long gastric residence time, slow small intestinal transition, disease-related diarrhea or sensitivity to systemic adverse effects may benefit more from pH-dependent release formulations. In general, medications based on both concepts show similar efficacies, but the pH-dependent release formulations seem to be more robust in the treatment of a not further classified group of patients with UC. Future comparative clinical studies are required to clearly define the subgroups of patients that should be treated preferably with constant or pH-dependent release formulations of mesalazine.

Sitagliptin and the Blood-Retina Barrier: Effects on Retinal Endothelial Cells Manifested Only after Prolonged Exposure.

Inhibitors of dipeptidyl peptidase-4 (DPP-4) are widely used to treat diabetes mellitus, but data concerning their effects on the barrier stability of retinal endothelial cells (REC) in vivo and in vitro are inconsistent. Therefore, we studied whether the barrier properties of immortalized endothelial cells of the bovine retina (iBREC) were affected by the inhibitors of DPP-4 sitagliptin (10-1000 nM) and diprotin A (1-25 µM). Their effects were also investigated in the presence of VEGF-A165 because diabetic patients often develop macular edema caused by VEGF-A-induced permeability of REC. To detect even transient or subtle changes of paracellular and transcellular flow as well as adhesion of the cells to the extracellular matrix, we continuously monitored the cell index (CI) of confluent iBREC grown on gold electrodes. Initially, the CI remained stable but started to decline significantly and persistently at 40 h or 55 h after addition of sitagliptin or diprotin A, respectively. Both inhibitors did not modulate, prevent, or revert the persistent VEGF-A165-induced reduction of the CI. Interestingly, sitagliptin and diprotin A increased the expression of the tight-junction protein claudin-1 which is an important component of a functional barrier formed by iBREC. In contrast, expressions of CD29-a subunit of the fibronectin receptor-or of the tetraspanin CD9 were lower after extended treatment with the DPP-4 inhibitors; less of the CD9 was seen at the plasma membrane after prolonged exposure to sitagliptin. Because both associated proteins are important for adhesion of iBREC to the extracellular matrix, the observed low CI might be caused by weakened attachment of the cells. From our results, we conclude that extended inhibition of DPP-4 destabilizes the barrier formed by microvascular REC and that DPP-4 inhibitors like sitagliptin do not counteract or enhance a VEGF-A165-induced barrier dysfunction as frequently observed in DME.

A low-frequency haplotype spanning SLX4/FANCP constitutes a new risk locus for early-onset breast cancer (<60 years) and is associated with reduced DNA repair capacity.

Only a fraction of breast cancer (BC) cases can be yet explained by mutations in genes or genomic variants discovered in linkage, genome-wide association and sequencing studies. The known genes entailing medium or high risk for BC are strongly enriched for a function in DNA double strand repair. Thus, aiming at identifying low frequency variants conferring an intermediate risk, we here investigated 17 variants (MAF: 0.01-0.1) in 10 candidate genes involved in DNA repair or cell cycle control. In an exploration cohort of 437 cases and 1189 controls, we show the variant rs3810813 in the SLX4/FANCP gene to be significantly associated with both BC (≤60 years; OR = 2.6(1.6-3.9), p = 1.6E-05) and decreased DNA repair capacity (≤60 years; beta = 37.8(17.9-57.8), p = 5.3E-4). BC association was confirmed in a verification cohort (N = 2441). Both associations were absent from cases diagnosed >60 years and stronger the earlier the diagnosis. By imputation we show that rs3810813 tags a haplotype with 5 additional variants with the same allele frequency (R2 > 0.9), and a pattern of association very similar for both phenotypes (cases <60 years, p < 0.001, the Bonferroni threshold derived from unlinked variants in the region). In young cases (≤60 years) carrying the risk haplotype, micronucleus test results are predictive for BC (AUC > 0.9). Our findings propose a risk variant with high penetrance on the haplotype spanning SLX4/FANCP to be functionally associated to BC predisposition via decreased repair capacity and suggest this variant is carried by a fraction of these haplotypes that is enriched in early onset BC cases.

Safety of monoclonal antibodies and related therapeutic proteins for the treatment of neovascular macular degeneration: addressing outstanding issues.

INTRODUCTION: The vascular endothelial growth factor (VEGF) inhibitors most widely used to treat neovascular age-dependent macular degeneration (nAMD) are different proteins with structural features potentially relevant to adverse effects (AEs). Two of these are also established in cancer therapy (with higher dosages and AEs). The importance of ocular AE and extraocular activities is still a subject of controversy and ongoing research. AREAS COVERED: Potential risks of intraocular VEGF inhibition based on prospective studies, in vitro investigations, pharmacokinetics, and hints from anti-cancer treatment. EXPERT OPINION: nAMD is a frequently observed chronic clinical condition severely affecting the visual function of elderly persons. Intravitreal injection of VEGF-inactivating proteins is highly effective to prevent loss of vision. Anti-VEGF therapy is well tolerated, and low rates of ocular and systemic AEs in smaller trials suggest a very high benefit/risk ratio. The proteins established in nAMD therapy show similar efficacies. In the controversy over the off-label use of bevacizumab purely on grounds of much lower cost, the small, but potentially relevant differences between the available drugs are easily either dramatized (by pharmaceutical companies) or trivialized (by health insurances) and even political interference is involved. Facing the lack of a convincing body of evidence regarding safety, further long-term study results seem necessary.

VEGF but not PlGF disturbs the barrier of retinal endothelial cells.

Elevated permeability of retinal endothelial cells (REC), as observed in diabetic retinopathy (DR), is induced by extended exposure to ≥25 ng/ml vascular endothelial growth factor A165 (VEGF165) for up to 3 d and this effect is more pronounced when equimolar amounts of basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-1) are present. Down-regulation of the tight-junction protein claudin-1 and its loss from the plasma membrane is associated with induced higher permeability, whereas other tight-junction proteins (e.g. claudin-3, claudin-5, ZO-1) show only subtle changes in our experimental setting. Using immortalized bovine REC (iBREC) as a well-established model, we investigated effects of other members of the VEGF family, i.e. VEGF121, placental growth factor (PlGF-1 and PlGF-2) and viral VEGF-E which activate different sets of VEGF receptors, on barrier function after extended treatment: iBREC were incubated with 1-100 ng/ml of the growth factors for up to 2 days before barrier function was assessed by measuring transendothelial resistance (TER). Presence of TJ-proteins was determined by western blot analyses and immunofluorescence staining. Similar experiments were performed to evaluate whether the primary actions of PlGF-1, PlGF-2 or VEGF121 are modulated by bFGF or IGF-1 when all growth factors (each at 25 ng/ml, but 10 ng/ml IGF-1) act simultaneously at equimolar concentrations. We also studied the potential normalization of the barrier disturbed with combinations of growth factors by addition of the VEGF-specific Fab fragment ranibizumab or the recombinant protein aflibercept which binds VEGF and PlGF. Whereas 1 ng/ml VEGF-E were sufficient to impair the iBREC barrier, a higher concentration of 100 ng/ml VEGF121 was needed to reduce TER and expression of claudin-1 over 2 days. By PlGF-1 or PlGF-2, the barrier was not affected even at the highest concentration tested (100 ng/ml) and these factors also did not modulate the effect of VEGF165. The weak barrier derangement caused by VEGF121 was slightly enhanced by bFGF and IGF-1. After induction of the barrier breakdown with various combinations of all growth factors included in the study, normal TER and claudin-1 expression was re-established by ranibizumab. Both VEGF inhibitors ranibizumab and aflibercept similarly reinstated lost claudin-1, even when applied at a small fraction of the clinically relevant concentrations. These results show that VEGF-A, but not PlGF impairs the barrier function of iBREC and that the longer isoform VEGF165 is more potent than VEGF121. To induce barrier dysfunction in iBREC, activation of VEGF receptor 2 - probably in concert with neuropilin-1 - seems to be sufficient because VEGF-E and VEGF165, but not PlGF-1/-2 reduced TER or claudin-1 expression.

Identification of alternative transcripts of rat CD9 expressed by tumorigenic neural cell lines and in normal tissues.

CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology (5 transmembrane domains) and a deviating spectrum of functions can be expected.

Ranibizumab efficiently blocks migration but not proliferation induced by growth factor combinations including VEGF in retinal endothelial cells.

BACKGROUND: Proliferation and migration of retinal endothelial cells (REC) are associated with the development of proliferative diabetic retinopathy. REC proliferation is stimulated by isoforms of vascular endothelial growth factor-A (i.e., VEGF121 and VEGF165), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1) of which VEGF165 also enhances migration of REC. Effects induced by VEGF-A can be blocked with ranibizumab, a VEGF-binding Fab fragment used in therapy of diabetic macular edema. In this study, we investigated potential angiogenic effects of placental growth factors (PlGF-1, PlGF-2) as other members of the VEGF family and whether the primary action of VEGF165 is modulated in the presence of bFGF, IGF-1 and PlGF-1/-2. We also studied how effects of growth factor combinations can be attenuated with ranibizumab. METHODS: Effects of single growth factors or their combinations on proliferation and migration of immortalized bovine retinal endothelial cells (iBREC) were studied with or without ranibizumab or the inhibitor of VEGF receptors KRN951. RESULTS: Proliferation of iBREC was significantly stimulated by 1-100 ng/ml PlGF-1 or PlGF-2, but additive effects were not observed with various combinations of the tested growth factors. Ranibizumab neutralized VEGF's effect on proliferation but was not effective when the other growth factors were used in combination with VEGF. bFGF and IGF-1 but not PlGF-1 or PlGF-2 stimulated iBREC migration as single agents, and they further enhanced VEGF-induced migration. The effects of such growth factor combinations including VEGF on migration were efficiently blocked by targeting only VEGF with ranibizumab. Migration induced by VEGF plus bFGF and IGF-1 was also almost completely inhibited by KRN951 interfering with VEGF receptor signalling. CONCLUSIONS: Migration but not proliferation of iBREC induced by combinations of bFGF, IGF-1, PlGF-1 or PlGF-2 together with VEGF is efficiently suppressed by ranibizumab. VEGF-mediated signalling through VEGFR2 seems to control REC migration dominantly in the presence of other growth factors.

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 VEGF(165) was visualised by immunofluorescence staining and western blot analyses. Measured transendothelial resistance (TER) of iBREC (±VEGF(165)) 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 VEGF(165). 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.

Medium-sized deletion in the BRCA1 gene: Limitations of Sanger sequencing and MLPA analyses.

We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification. This showed that a considerable fraction of medium-sized deletions are undetectable by currently used standard methods of mutation analyses. We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions. Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.

Association of death receptor 4 variant (683A > C) with ovarian cancer risk in BRCA1 mutation carriers.

Dysregulation of apoptosis plays an important role in carcinogenesis. Therefore, apoptosis-associated genes like the death receptor 4 (DR4, TRAIL-R1) are interesting candidates for modifying the penetrance of breast and ovarian cancer in carriers of BRCA1 and BRCA2 mutations. The DR-4 haplotype 626C-683C [626C > G, Thr209Arg (rs4871857) and 683A > C, Glu228Ala (rs17088993)] has recently been linked to an increased risk of breast cancer. To evaluate whether DR4 626C > G or DR4 683A > C modifies the risk of breast or ovarian cancer in carriers of BRCA1 and BRCA2 mutations, we undertook a national multicenter study including data of 840 carriers of breast cancer gene (BRCA) mutations. DNA samples were collected from 12 German research centers between 1996 and 2005 and were genotyped by the Taqman allelic discrimination assay. The association between genotypes and incidence of breast or ovarian cancer data was evaluated using a Cox proportional hazards regression model. We found evidence for a significant association of DR4 683A > C with a higher risk for ovarian cancer in carriers of BRCA1 mutations [n = 557, hazard ratio 1.78 (1.24-2.55), p = 0.009]. Our results thus indicate that the DR4 683A > C variant modifies the risk of ovarian cancer in carriers of BRCA1 mutations.

Medium-sized deletion in the BRCA1 gene: limitations of Sanger sequencing and MLPA analyses

We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification. This showed that a considerable fraction of medium-sized deletions are undetectable by currently used standard methods of mutation analyses. We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions. Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.

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 analyzing 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 identified 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, favorable treatment options in familial breast cancer.

Molecular indicators of non-sentinel node status in breast cancer determined in preoperative biopsies by multiplexed sandwich immunoassays.

PURPOSE: Purpose of this study was to determine the accuracy of prediction of non-sentinel lymph node (NSLN) involvement in sentinel node (SLN)-positive breast cancer patients based on protein concentrations measured in lysates from initially taken breast biopsies. METHODS: Data on protein expression, previously generated by multiplexed bead-based immunoassays, were analysed by multivariate logistic regression to define parameter sets of value to predict NSLN involvement. Receiver-operator characteristics (ROCs) were calculated as indicators of diagnostic significance. RESULTS: Analyses of data from all patients (n = 99) resulted in parameter sets that allowed direct prediction of the NSLN status with a ROC area under the curve (AUC) of 0.83. The clinically most relevant prediction of NSLN status in SLN-positive patients (n = 37) based on only seven parameters (including TIMP-2 as the most relevant single value) was possible with high accuracy indicated by an AUC of 0.89. CONCLUSIONS: Parallel assessment of protein concentrations in breast biopsies is a highly promising approach to predict nodal involvement and even the NSLN status in SLN-negative breast cancer patients. Such diagnostic information could substantially reduce the number of completion axillary lymph node dissections in clinical practice.

Inhibition of vascular endothelial growth factor (VEGF) is sufficient to completely restore barrier malfunction induced by growth factors in microvascular retinal endothelial cells.

BACKGROUND: Deregulated expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-1 (IGF-1) is associated with the pathogenesis of diabetic retinopathy. The VEGF(165)-induced increase in permeability of retinal endothelial cells (REC), probably resulting in diabetic macular oedema (DME), could be completely restored by the VEGF-binding Fab fragment ranibizumab in vitro. We investigated whether bFGF and IGF-1 as single factors or in combination with VEGF(165) influence permeability and tight junctions in immortalised bovine REC (iBREC) and if these effects could be restored by inhibition of VEGF. METHODS: As a measure of changes in cellular permeability, transendothelial electrical resistance (TER) was monitored during long-term treatment of iBREC with growth factors in the absence or presence of ranibizumab or KRN951 (an inhibitor of VEGF receptors). Expression of claudin-1, as an indicator of functional tight junctions, was assessed by western blot analysis. RESULTS: Whereas VEGF(165) decreased TER and expression of claudin-1 in a concentration-dependent manner, long-term treatment of iBREC with 10-100 ng/ml bFGF or/and IGF-1 did not. Changes in claudin-1 expression or TER, induced by 25 ng/ml VEGF(165), were slightly enhanced by bFGF and/or IGF-1 and were accompanied by a slightly increased secretion of VEGF. Complete reversion of these effects was achieved by prolonged treatment with ranibizumab and partly by exposure to KRN951. CONCLUSION: Our findings indicate that VEGF(165), but not IGF-1 or bFGF, is mainly responsible for changes in cellular permeability observed in REC. This supports VEGF targeting as a therapeutic concept for DME.

Evidence for SMAD3 as a modifier of breast cancer risk in BRCA2 mutation carriers.

INTRODUCTION: Current attempts to identify genetic modifiers of BRCA1 and BRCA2 associated risk have focused on a candidate gene approach, based on knowledge of gene functions, or the development of large genome-wide association studies. In this study, we evaluated 24 SNPs tagged to 14 candidate genes derived through a novel approach that analysed gene expression differences to prioritise candidate modifier genes for association studies. METHODS: We successfully genotyped 24 SNPs in a cohort of up to 4,724 BRCA1 and 2,693 BRCA2 female mutation carriers from 15 study groups and assessed whether these variants were associated with risk of breast cancer in BRCA1 and BRCA2 mutation carriers. RESULTS: SNPs in five of the 14 candidate genes showed evidence of association with breast cancer risk for BRCA1 or BRCA2 carriers (P < 0.05). Notably, the minor alleles of two SNPs (rs7166081 and rs3825977) in high linkage disequilibrium (r² = 0.77), located at the SMAD3 locus (15q22), were each associated with increased breast cancer risk for BRCA2 mutation carriers (relative risk = 1.25, 95% confidence interval = 1.07 to 1.45, P(trend) = 0.004; and relative risk = 1.20, 95% confidence interval = 1.03 to 1.40, P(trend) = 0.018). CONCLUSIONS: This study provides evidence that the SMAD3 gene, which encodes a key regulatory protein in the transforming growth factor beta signalling pathway and is known to interact directly with BRCA2, may contribute to increased risk of breast cancer in BRCA2 mutation carriers. This finding suggests that genes with expression associated with BRCA1 and BRCA2 mutation status are enriched for the presence of common genetic modifiers of breast cancer risk in these populations.

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; P(trend) = 0.011) and ovarian cancer (per-allele HR, 0.69; 95% CI, 0.53-0.89; P(trend) = 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.

Multiplexed immunoassays for the analysis of breast cancer biopsies.

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated.

Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene.

Germline mutations in a number of genes involved in the recombinational repair of DNA double-strand breaks are associated with predisposition to breast and ovarian cancer. RAD51C is essential for homologous recombination repair, and a biallelic missense mutation can cause a Fanconi anemia-like phenotype. In index cases from 1,100 German families with gynecological malignancies, we identified six monoallelic pathogenic mutations in RAD51C that confer an increased risk for breast and ovarian cancer. These include two frameshift-causing insertions, two splice-site mutations and two nonfunctional missense mutations. The mutations were found exclusively within 480 pedigrees with the occurrence of both breast and ovarian tumors (BC/OC; 1.3%) and not in 620 pedigrees with breast cancer only or in 2,912 healthy German controls. These results provide the first unambiguous evidence of highly penetrant mutations associated with human cancer in a RAD51 paralog and support the 'common disease, rare allele' hypothesis.

B-cell-specific elements enhance sustained gene expression mediated by self-replicating extrachromosomal vectors.

Retroviral vectors have been considered the most promising vehicles for the transfer of therapeutic genes into cells of the hematopoietic system. In clinical studies, however, cases of leukemoid clonogenic expansion of the transduced cells in vivo caused by semirandom integration of the foreign DNA in the genome have changed the focus to other types of vectors. In addition to their superior safety profile, a higher packaging capacity might be an advantage of non-viral vectors in certain applications. Prolonged transgene expression of non-viral vectors can be achieved by inclusion of Epstein-Barr virus (EBV)-derived elements mediating episomal replication and retention. Furthermore, a variety of cis acting elements have been explored in an attempt to enhance gene transfer efficiency. Our study confirmed that prolonged transgene expression can be achieved in B-lymphoid cells with EBV-derived vectors containing the EBV latent gene EBNA-1 and the EBV latent origin of replication oriP. In addition, we demonstrated that the inclusion of enhancer elements of the immunoglobulin κ light chain (Ei and E3') associated with its matrix attachment region (MAR) resulted in a 10-fold increase in transgene expression in B-lymphoid cells. It can be concluded that these elements are generally useful modules for improving the efficiencies of non-viral vectors in the B-lymphoid lineage.

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 x 10(-12)) and for TNRC9 (OR = 1.33, 95% CI 1.19-1.46, p-value = 1.54 x 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 x 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.