Reiner Strick

Association between CYP2D6 polymorphisms and outcomes among women with early stage breast cancer treated with tamoxifen.

CONTEXT The growth inhibitory effect of tamoxifen, which is used for the treatment of hormone receptor-positive breast cancer, is mediated by its metabolites, 4-hydroxytamoxifen and endoxifen. The formation of active metabolites is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6) enzyme. OBJECTIVE To determine whether CYP2D6 variation is associated with clinical outcomes in women receiving adjuvant tamoxifen. DESIGN, SETTING, AND PATIENTS Retrospective analysis of German and US cohorts of patients treated with adjuvant tamoxifen for early stage breast cancer. The 1325 patients had diagnoses between 1986 and 2005 of stage I through III breast cancer and were mainly postmenopausal (95.4%). Last follow-up was in December 2008; inclusion criteria were hormone receptor positivity, no metastatic disease at diagnosis, adjuvant tamoxifen therapy, and no chemotherapy. DNA from tumor tissue or blood was genotyped for CYP2D6 variants associated with reduced (*10, *41) or absent (*3, *4, *5) enzyme activity. Women were classified as having an extensive (n=609), heterozygous extensive/intermediate (n=637), or poor (n=79) CYP2D6 metabolism. MAIN OUTCOME MEASURES Time to recurrence, event-free survival, disease-free survival, and overall survival. RESULTS Median follow-up was 6.3 years. At 9 years of follow-up, the recurrence rates were 14.9% for extensive metabolizers, 20.9% for heterozygous extensive/intermediate metabolizers, and 29.0% for poor metabolizers, and all-cause mortality rates were 16.7%, 18.0%, and 22.8%, respectively. Compared with extensive metabolizers, there was a significantly increased risk of recurrence for heterozygous extensive/intermediate metabolizers (time to recurrence adjusted hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.04-1.90) and for poor metabolizers (time to recurrence HR, 1.90; 95% CI, 1.10-3.28). Compared with extensive metabolizers, those with decreased CYP2D6 activity (heterozygous extensive/intermediate and poor metabolism) had worse event-free survival (HR, 1.33; 95% CI, 1.06-1.68) and disease-free survival (HR, 1.29; 95% CI, 1.03-1.61), but there was no significant difference in overall survival (HR, 1.15; 95% CI, 0.88-1.51). CONCLUSION Among women with breast cancer treated with tamoxifen, there was an association between CYP2D6 variation and clinical outcomes, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes and the presence of nonfunctional or reduced-function alleles with worse outcomes.

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

Cation–chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes

Mammalian interphase and mitotic cells were analyzed for their cation composition using a three-dimensional high resolution scanning ion microprobe. This instrument maps the distribution of bound and unbound cations by secondary ion mass spectrometry (SIMS). SIMS analysis of cryofractured interphase and mitotic cells revealed a cell cycle dynamics of Ca2+, Mg2+, Na+, and K+. Direct analytical images showed that all four, but no other cations, were detected on mitotic chromosomes. SIMS measurements of the total cation content for diploid chromosomes imply that one Ca2+ binds to every 12.5–20 nucleotides and one Mg2+ to every 20–30 nucleotides. Only Ca2+ was enriched at the chromosomal DNA axis and colocalized with topoisomerase IIα (Topo II) and scaffold protein II (ScII). Cells depleted of Ca2+ and Mg2+ showed partially decondensed chromosomes and a loss of Topo II and ScII, but not hCAP-C and histones. The Ca2+-induced inhibition of Topo II catalytic activity and direct binding of Ca2+ to Topo II by a fluorescent filter-binding assay supports a regulatory role of Ca2+ during mitosis in promoting solely the structural function of Topo II. Our study directly implicates Ca2+, Mg2+, Na+, and K+ in higher order chromosome structure through electrostatic neutralization and a functional interaction with nonhistone proteins.

Circulating micro-RNAs as potential blood-based markers for early stage breast cancer detection.

Introduction MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls. Methods We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718). Results Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202. Conclusions MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use.

Angiogenic growth factors in maternal and fetal serum in pregnancies complicated by intrauterine growth restriction

The present study was performed to compare serum concentrations of maternal and fetal angiogenic growth factors in IUGR (intrauterine growth restriction) and normal pregnancy at the time of delivery. VEGF (vascular endothelial growth factor), PlGF (placental growth factor), sFlt-1 (soluble fms-like tyrosine kinase 1), sKDR (soluble kinase domain receptor) and bFGF (basic fibroblast growth factor) were measured by ELISA in serum from a maternal peripheral vein, the umbilical vein and the umbilical arteries in 15 women with pregnancies complicated by IUGR and 16 controls (women with normal pregnancies). In IUGR, sFlt-1 was increased, and PlGF and sKDR were decreased, in both maternal serum and serum from the umbilical vein. Additionally, bFGF was increased in serum from the umbilical vein of women with pregnancies complicated by IUGR. No significant differences in growth factor concentrations between the groups were found in serum from the umbilical artery. In both groups, levels of VEGF were higher and levels of sFlt-1 were lower in serum from the umbilical vein and umbilical artery compared with maternal serum. PlGF levels were found to be lower in serum from the umbilical vein compared with maternal serum in both groups, whereas PlGF levels in serum from the umbilical artery were significantly lower only in the control group. These findings suggest an imbalance of angiogenic and anti-angiogenic factors in IUGR, with formation of an anti-angiogenic state in maternal and, to a lesser extent, umbilical vein blood. The placenta appears to play a central role in the release of sFlt-1 into maternal and umbilical blood. Umbilical artery blood was unaffected in IUGR, indicating that the fetus does not contribute to changes in angiogenic growth factor concentrations.

Estrogen and progesterone receptors: from molecular structures to clinical targets

Research involving estrogen and progesterone receptors (ER and PR) have greatly contributed to our understanding of cell signaling and transcriptional regulation. In addition to the classical ER and PR nuclear actions, new signaling pathways have recently been identified due to ER and PR association with cell membranes and signal transduction proteins. Bio-informatics has unveiled how ER and PR recognize their ligands, selective modulators and co-factors, which has helped to implement them as key targets in the treatment of benign and malignant tumors. Knowledge regarding ER and PR is vast and complex; therefore, this review will focus on their isoforms, signaling pathways, co-activators and co-repressors, which lead to target gene regulation. Moreover it will highlight ER and PR involvement in benign and malignant diseases as well as pharmacological substances influencing cell signaling and provide established and new structural insights into the mechanism of activation and inhibition of these receptors.

Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukemia

The translocation t(8;21)(q22;q22) is one of the most frequent chromosome translocations in acute myeloid leukemia (AML). AML1/RUNX1 at 21q22 is involved in t(8;21), t(3;21), and t(16;21) in de novo and therapy-related AML and myelodysplastic syndrome as well as in t(12;21) in childhood B cell acute lymphoblastic leukemia. Although DNA breakpoints in AML1 and ETO (at 8q22) cluster in a few introns, the mechanisms of DNA recombination resulting in t(8;21) are unknown. The correlation of specific chromatin structural elements, i.e., topoisomerase II (topo II) DNA cleavage sites, DNase I hypersensitive sites, and scaffold-associated regions, which have been implicated in chromosome recombination with genomic DNA breakpoints in AML1 and ETO in t(8;21) is unknown. The breakpoints in AML1 and ETO were clustered in the Kasumi 1 cell line and in 31 leukemia patients with t(8;21); all except one had de novo AML. Sequencing of the breakpoint junctions revealed no common DNA motif; however, deletions, duplications, microhomologies, and nontemplate DNA were found. Ten in vivo topo II DNA cleavage sites were mapped in AML1, including three in intron 5 and seven in intron 7a, and two were in intron 1b of ETO. All strong topo II sites colocalized with DNase I hypersensitive sites and thus represent open chromatin regions. These sites correlated with genomic DNA breakpoints in both AML1 and ETO, thus implicating them in the de novo 8;21 translocation.

Risk of estrogen receptor-positive and -negative breast cancer and single-nucleotide polymorphism 2q35-rs13387042

BACKGROUND A recent genome-wide association study identified single-nucleotide polymorphism (SNP) 2q35-rs13387042 as a marker of susceptibility to estrogen receptor (ER)-positive breast cancer. We attempted to confirm this association using the Breast Cancer Association Consortium. METHODS 2q35-rs13387042 SNP was genotyped for 31 510 women with invasive breast cancer, 1101 women with ductal carcinoma in situ, and 35 969 female control subjects from 25 studies. Odds ratios (ORs) were estimated by logistic regression, adjusted for study. Heterogeneity in odds ratios by each of age, ethnicity, and study was assessed by fitting interaction terms. Heterogeneity by each of invasiveness, family history, bilaterality, and hormone receptor status was assessed by subclassifying case patients and applying polytomous logistic regression. All statistical tests were two-sided. RESULTS We found strong evidence of association between rs13387042 and breast cancer in white women of European origin (per-allele OR = 1.12, 95% confidence interval [CI] = 1.09 to 1.15; P(trend) = 1.0 x 10(-19)). The odds ratio was lower than that previously reported (P = .02) and did not vary by age or ethnicity (all P > or = .2). However, it was higher when the analysis was restricted to case patients who were selected for a strong family history (P = .02). An association was observed for both ER-positive (OR = 1.14, 95% CI = 1.10 to 1.17; P = 10(-15)) and ER-negative disease (OR = 1.10, 95% CI = 1.04 to 1.15; P = .0003) and both progesterone receptor (PR)-positive (OR = 1.15, 95% CI = 1.11 to 1.19; P = 5 x 10(-14)) and PR-negative disease (OR = 1.10, 95% CI = 1.06 to 1.15; P = .00002). CONCLUSION The rs13387042 is associated with both ER-positive and ER-negative breast cancer in European women.

Recurrent aberrations identified by array-CGH in patients with Mayer-Rokitansky-Küster-Hauser syndrome

OBJECTIVE To identify genetic causes of Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. DESIGN Prospective laboratory study. SETTING University hospital. PATIENT(S) Fifty-six patients with MRKH syndrome. INTERVENTION(S) Identification of microdeletions and -duplications in a group of 48 MRKH patients by array-CGH. Results obtained by array-CGH were confirmed by RT-qPCR. Sequential analysis of two candidate genes LHX1 and HNF1B in a group of 56 MRKH patients. MAIN OUTCOME MEASURE(S) Identification of chromosomal regions and genes (recurrent and private) associated with MRKH syndrome. RESULT(S) We could delineate three definitively relevant regions (1q21.1, 17q12, and 22q11.21) and suggest that LHX1 und HNF1B are candidate genes for MRKH syndrome, because we identified recurrent deletions affecting these genes and a possible causative missense mutation in LHX1. CONCLUSION(S) Our findings suggest that different chromosomal regions are associated with MRKH syndrome.

High incidence of recurrent copy number variants in patients with isolated and syndromic Müllerian aplasia

Background Congenital malformations involving the Müllerian ducts are observed in around 5% of infertile women. Complete aplasia of the uterus, cervix, and upper vagina, also termed Müllerian aplasia or Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome, occurs with an incidence of around 1 in 4500 female births, and occurs in both isolated and syndromic forms. Previous reports have suggested that a proportion of cases, especially syndromic cases, are caused by variation in copy number at different genomic loci. Methods In order to obtain an overview of the contribution of copy number variation to both isolated and syndromic forms of Müllerian aplasia, copy number assays were performed in a series of 63 cases, of which 25 were syndromic and 38 isolated. Results A high incidence (9/63, 14%) of recurrent copy number variants in this cohort is reported here. These comprised four cases of microdeletion at 16p11.2, an autism susceptibility locus not previously associated with Müllerian aplasia, four cases of microdeletion at 17q12, and one case of a distal 22q11.2 microdeletion. Microdeletions at 16p11.2 and 17q12 were found in 4/38 (10.5%) cases with isolated Müllerian aplasia, and at 16p11.2, 17q12 and 22q11.2 (distal) in 5/25 cases (20%) with syndromic Müllerian aplasia. Conclusion The finding of microdeletion at 16p11.2 in 2/38 (5%) of isolated and 2/25 (8%) of syndromic cases suggests a significant contribution of this copy number variant alone to the pathogenesis of Müllerian aplasia. Overall, the high incidence of recurrent copy number variants in all forms of Müllerian aplasia has implications for the understanding of the aetiopathogenesis of the condition, and for genetic counselling in families affected by it.