Frederik Marme

Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay.

It has been hypothesized that carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, although the presence of CTCs is an indicator of poor prognosis in several carcinoma entities, the existence and phenotype of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. Here we developed a xenograft assay and used it to show that primary human luminal breast cancer CTCs contain MICs that give rise to bone, lung and liver metastases in mice. These MIC-containing CTC populations expressed EPCAM, CD44, CD47 and MET. In a small cohort of patients with metastases, the number of EPCAM+CD44+CD47+MET+ CTCs, but not of bulk EPCAM+ CTCs, correlated with lower overall survival and increased number of metastasic sites. These data describe functional circulating MICs and associated markers, which may aid the design of better tools to diagnose and treat metastatic breast cancer.

Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes

Exosomes are membrane vesicles that are released from many different cell types. Tumor derived-exosomes play a role in immune suppression. We hypothesized that in ovarian carcinoma patients exosomes initially produced at the local abdominal site may become systemic. We examined paired samples of ascites and blood from ovarian carcinoma patients for the presence of exosomes. We also studied the requirements for exosomal uptake by immune cells, the role of phosphatidyl-serine (PS) as uptake signal and the effect of exosome application on tumor growth. We used exosomes from ovarian carcinoma cell lines, malignant ascites and sera from ovarian carcinoma patients isolated by ultracentrifugation. PS-displayed by exosomes was detected by Anexin-V-FITC staining of latex beads adsorbed exosomes. For uptake experiments, labeled exosomes were exposed to cells in the presence or absence of cold Annexin-V as competitor. Uptake was examined by fluorescent microscopy and cytofluorographic analysis. Effects of exosomes on tumor growth were studied using SKOV3ip ovarian carcinoma cells in CD1 nu/nu mice. We found that malignant ascites-derived exosomes cargo tumor progression related proteins such as L1CAM, CD24, ADAM10, and EMMPRIN. We observed that exosomes become systemic via the blood stream. Uptake of ovarian carcinoma exosomes by NK cells was found to require PS at the exosomal surface but the presence of PS was not sufficient. Application of malignant ascites-derived exosomes to tumor bearing mice resulted in augmented tumor growth. Exosomes from the serum of tumor patients could be isolated from only one ml of blood and this analysis could serve for diagnostic purposes. We propose that tumor-derived exosomes could play a role in tumor progression.

Body Fluid Exosomes Promote Secretion of Inflammatory Cytokines in Monocytic Cells via Toll-like Receptor Signaling

Background: Exosomes, secreted from cells, have immunomodulatory capacities. Results: NFκB- and STAT3-mediated cytokine release is triggered by various types of ex vivo exosomes in a TLR-dependent fashion. Conclusion: Exosomes have inherent signaling capacities important for global inflammatory responses. Significance: Detailed knowledge about intercellular communication in cancer and inflammatory diseases is crucial for development of new therapeutic approaches. Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1β, TNF-α, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NFκB and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NFκB and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases.

CHEK2*1100delC Heterozygosity in Women With Breast Cancer Associated With Early Death, Breast Cancer–Specific Death, and Increased Risk of a Second Breast Cancer

PURPOSE We tested the hypotheses that CHEK2*1100delC heterozygosity is associated with increased risk of early death, breast cancer-specific death, and risk of a second breast cancer in women with a first breast cancer. PATIENTS AND METHODS From 22 studies participating in the Breast Cancer Association Consortium, 25,571 white women with invasive breast cancer were genotyped for CHEK2*1100delC and observed for up to 20 years (median, 6.6 years). We examined risk of early death and breast cancer-specific death by estrogen receptor status and risk of a second breast cancer after a first breast cancer in prospective studies. RESULTS CHEK2*1100delC heterozygosity was found in 459 patients (1.8%). In women with estrogen receptor-positive breast cancer, multifactorially adjusted hazard ratios for heterozygotes versus noncarriers were 1.43 (95% CI, 1.12 to 1.82; log-rank P = .004) for early death and 1.63 (95% CI, 1.24 to 2.15; log-rank P < .001) for breast cancer-specific death. In all women, hazard ratio for a second breast cancer was 2.77 (95% CI, 2.00 to 3.83; log-rank P < .001) increasing to 3.52 (95% CI, 2.35 to 5.27; log-rank P < .001) in women with estrogen receptor-positive first breast cancer only. CONCLUSION Among women with estrogen receptor-positive breast cancer, CHEK2*1100delC heterozygosity was associated with a 1.4-fold risk of early death, a 1.6-fold risk of breast cancer-specific death, and a 3.5-fold risk of a second breast cancer. This is one of the few examples of a genetic factor that influences long-term prognosis being documented in an extensive series of women with breast cancer.

Plasma microRNA panel for minimally invasive detection of breast cancer.

Over the last few years, circulating microRNAs (miRNAs) have emerged as promising novel and minimally invasive markers for various diseases, including cancer. We already showed that certain miRNAs are deregulated in the plasma of breast cancer patients when compared to healthy women. Herein we have further explored their potential to serve as breast cancer early detection markers in blood plasma. Circulating miR-127-3p, miR-376a and miR-652, selected as candidates from a miRNA array-based screening, were found to be associated with breast cancer for the first time (n = 417). Further we validated our previously reported circulating miRNAs (miR-148b, miR-376c, miR-409-3p and miR-801) in an independent cohort (n = 210) as elevated in the plasma of breast cancer patients compared to healthy women. We described, for the first time in breast cancer, an over-representation of deregulated miRNAs (miR-127-3p, miR-376a, miR-376c and miR-409-3p) originating from the chromosome 14q32 region. The inclusion of patients with benign breast tumors enabled the observation that miR-148b, miR-652 and miR-801 levels are even elevated in the plasma of women with benign tumors when compared to healthy controls. Furthermore, an analysis of samples stratified by cancer stage demonstrated that miR-127-3p, miR-148b, miR-409-3p, miR-652 and miR-801 can detect also stage I or stage II breast cancer thus making them attractive candidates for early detection. Finally, ROC curve analysis showed that a panel of these seven circulating miRNAs has substantial diagnostic potential with an AUC of 0.81 for the detection of benign and malignant breast tumors, which further increased to 0.86 in younger women (up to 50 years of age).

The role of genetic breast cancer susceptibility variants as prognostic factors

Recent genome-wide association studies identified 11 single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. We investigated these and 62 other SNPs for their prognostic relevance. Confirmed BC risk SNPs rs17468277 (CASP8), rs1982073 (TGFB1), rs2981582 (FGFR2), rs13281615 (8q24), rs3817198 (LSP1), rs889312 (MAP3K1), rs3803662 (TOX3), rs13387042 (2q35), rs4973768 (SLC4A7), rs6504950 (COX11) and rs10941679 (5p12) were genotyped for 25 853 BC patients with the available follow-up; 62 other SNPs, which have been suggested as BC risk SNPs by a GWAS or as candidate SNPs from individual studies, were genotyped for replication purposes in subsets of these patients. Cox proportional hazard models were used to test the association of these SNPs with overall survival (OS) and BC-specific survival (BCS). For the confirmed loci, we performed an accessory analysis of publicly available gene expression data and the prognosis in a different patient group. One of the 11 SNPs, rs3803662 (TOX3) and none of the 62 candidate/GWAS SNPs were associated with OS and/or BCS at P<0.01. The genotypic-specific survival for rs3803662 suggested a recessive mode of action [hazard ratio (HR) of rare homozygous carriers=1.21; 95% CI: 1.09-1.35, P=0.0002 and HR=1.29; 95% CI: 1.12-1.47, P=0.0003 for OS and BCS, respectively]. This association was seen similarly in all analyzed tumor subgroups defined by nodal status, tumor size, grade and estrogen receptor. Breast tumor expression of these genes was not associated with prognosis. With the exception of rs3803662 (TOX3), there was no evidence that any of the SNPs associated with BC susceptibility were associated with the BC survival. Survival may be influenced by a distinct set of germline variants from those influencing susceptibility.

Preeclampsia : increased expression of soluble ADAM 12

Preeclampsia is a multisystemic pregnancy-associated disease affecting about 3–7% of pregnancies worldwide and is still a principal cause of fetal and maternal morbidity and mortality. To identify potential markers, we have compared gene expression profiles from control and preeclamptic placental tissues taken at various age-matched gestational stages using complementary DNA microarray analysis. Besides previously identified preeclampsia-associated genes, novel differentially expressed transcripts were found. The soluble form of the disintegrin metalloprotease ADAM 12 (a disintegrin and metalloproteinase 12; meltrin-α) represented the most upregulated transcript. This was confirmed by in situ hybridization of sections of preeclamptic placentas and by serum protein analysis of preeclamptic pregnant women. Thus, ADAM 12 could serve as an early biomarker for preeclampsia that may be of predictive and/or functional significance.

Molecular and clinical dissection of CD24 antibody specificity by a comprehensive comparative analysis.

CD24 is a small, highly glycosylated cell surface protein that is linked to the membrane through a glycosyl-phosphatidylinositol anchor. It is overexpressed in many human carcinomas and its expression is linked to bad prognosis. Lately, lack or low expression of CD24 was used to identify tumor stem cells resulting in conflicting data on the usefulness of this marker. In many immunohistochemical studies, the mAb SN3b was used but the epitope and specificity of this antibody have never been thoroughly investigated. In other studies based mainly on cytofluorographic analysis, the mAb ML-5 was applied. In this study, we compared the epitope of mAb SN3b to the CD24 mAbs SWA-11 and ML-5 that both bind to the core protein of CD24. Using tissue microarrays and affinity-purified CD24 glycoforms, we observed only a partial overlap of SN3b and SWA11 reactivity. The mAb SN3b recognizes sialic acid most likely on O-linked glycans that can occur independently of the CD24 protein backbone. The SN3b epitope was not related to common sialylated cancer-associated glycan structures. Both SN3b epitope positive or negative CD24 glycoforms supported the binding of P-selectin and Siglec-5. In breast cancer, the SN3b reactivity was associated with bad prognosis, whereas SWA11 was not. In renal cell cancer, the SN3b epitope was completely absent but SWA11 reactivity was a prognostic factor. Our results shed new light on the tumorbiological role of CD24 and resolve discrepancies in the literature related to the use of different CD24 mAbs.

Outcome analysis of patients with primary breast cancer initially treated at a certified academic breast unit.

INTRODUCTION Evaluation of oncological outcome and prognostic factors of patients with primary breast cancer treated at a certified academic breast unit. PATIENTS AND METHODS We prospectively collected data of 3338 patients, diagnosed with primary breast cancer between 01.01.2003 and 31.12.2010 and treated at the Breast Unit Heidelberg, Germany, in order to analyze outcome in clinical practice. We evaluated local control rate (LCR), disease-free survival (DFS), distant disease-free survival (DDFS), observed overall survival (OS) and age-adjusted relative overall survival (ROS). In addition, the impact of known prognostic factors on these outcome variables was examined in univariate and multivariate analyses. RESULTS Of all patients, 368 (11.0%) had carcinoma in situ (CIS) and 197 (5.9%) had bilateral cancers. For the 2970 patients with invasive cancer, of which 49 patients (1.7%) had metastastic disease at time of diagnosis, DFS, LCR, DDFS, OS and ROS at 5 years were 79.8%, 84.7%, 81.2%, 86.3%, and 89.8%, respectively. In multivariate analysis age, pT category, nodal status, hormone receptor status and grading were identified as independent prognostic factors for OS. CONCLUSION Compared with recent population-based reports from Germany, more favourable patient characteristics and nominally higher survival was found among this large cohort of patients with primary breast cancer treated at a single certified breast unit.

Aesthetic and functional results after breast conserving surgery as correlates of quality of life measured by a German version of the Breast Cancer Treatment Outcome Scale (BCTOS).

This prospective cohort study was carried out to develop a German version of the Breast Cancer Treatment Outcome Scale (BCTOS) and to examine the relations of aesthetic and functional outcome after breast conserving surgery with quality of life (QoL). The study included 189 patients with one-sided, early stage breast cancer. A factor analysis indicated three internally consistent scales of the German BCTOS: Aesthetic Status, Functional Status and Breast Sensitivity Status. QoL was measured by the EORTC Quality of Life Questionnaire C30-BR23 (EORTC). All BCTOS scales were correlated with scales of the EORTC. Correlation magnitudes ranged from 0.24 to 0.67 (p < 0.001). A multiple regression analyses confirmed these results. The analysis of relevant covariates demonstrated that younger patients revealed poorer status on all BCTOS scales. Aesthetic and functional outcome seems to be closely related to quality of Life. The German BCTOS demonstrated to be a useful instrument.