Fabian Birzele

Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R2 = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients

Purpose: CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform expression impacts antitumoral responses in vivo to treatment with RG7356, a humanized anti-CD44 antibody inhibiting CD44–HA interaction. Experimental Design: CD44 isoform expression on various tumor cell lines was analyzed by RNASeq while data on patients with different tumor types were obtained from the publicly available TCGA RNASeq dataset as well as a phase I clinical study (NCT01358903). We analyzed the link between HA production and CD44 isoform expression as well as the consequences of blocking the CD44-mediated cell adhesion to HA using RG7356. The correlation between CD44 isoform expression and antitumor response to RG7356 treatment was investigated in the corresponding murine xenograft in vivo models as well as in a subset of patients treated with RG7356 from a recently completed phase I clinical trial. Results: CD44 isoform expression, in particular expression of CD44s, is associated with HA production and predicts response to treatment with RG7356 in tumor xenograft models. Furthermore, patient data suggest that CD44 isoform status is a potential predictive biomarker for clinical response to treatment with RG7356. Conclusions: We provide new insights into the close interplay between CD44 and HA and a potential biomarker to enrich patient responses to RG7356 in the clinic. Clin Cancer Res; 21(12); 2753–62. ©2015 AACR.

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8–12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Molecular targets and pathways involved in liver metastasis of colorectal cancer

We here summarize the current view of molecular mechanisms involved in the dissemination process of colorectal cancer cells to the liver as deduced from preclinical models. We focus on molecular aspects of the current understanding of the biology of liver metastases formation and survival, both being crucial for identification and validation of possible therapeutic targets and review the latest findings elucidating some features of the liver as a metastatic niche. In more detail, we outline the role of proteases and of major pathways such asc-MET signaling and its modulation by factors such as MACC1 and TIMP1, as well as Notch and TGFβ signaling. The relevance of these signalling pathways during tumor-stroma interactions in this context will be addressed. In addition, the functional role and validation of targets such as PRL3, Trop-2, L1CAM, S100A4, S100P, CD133, LIPC, and APOBEC3G are summarized.

Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models

BackgroundVenetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML.MethodsThe effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments.ResultsCombination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies.ConclusionsOur findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Significance Diphthamide is a conserved modification on eukaryotic translation elongation factor 2 (eEF2). Analyses of genetically defined diphthamide-deficient cell lines indicate that this modification determines not only sensitivity of cells to the ADP-ribosylating toxins Pseudomonas exotoxin A and diphtheria toxin, but it also modulates nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and TNF receptor signaling pathways. The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1–DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

Acute tumour response to a bispecific Ang-2-VEGF-A antibody: insights from multiparametric MRI and gene expression profiling

Background:To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A).Methods:Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg−1 dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 106 mm2 s−1), vascular perfusion/permeability (Ktrans, min−1) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed.Results:Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour Ktrans (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis.Conclusions:Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.

MDM2 antagonist clinical response association with a gene expression signature in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is uniquely sensitive to p53 activation 1, 2 as ≈90% of patients carry wild-type TP53 and frequent MDM2 overexpression.3 MDM2 blocks p53 transactivation and targets p53 for ubiquitin-dependent degradation.4, 5 Nutlins have been characterized as potent and selective small-molecule MDM2 antagonists.1, 6–8 RG7112 was the first such MDM2 antagonist to undergo clinical assessment in solid tumors and leukaemia trials.1, 2, 9 As not all patients with functional p53 will respond to MDM2 antagonists, diagnostic tools may identify patients likely to respond.

Manananggal - a novel viewer for alternative splicing events

BackgroundAlternative splicing is an important cellular mechanism that can be analyzed by RNA sequencing. However, identification of splicing events in an automated fashion is error-prone. Thus, further validation is required to select reliable instances of alternative splicing events (ASEs). There are only few tools specifically designed for interactive inspection of ASEs and available visualization approaches can be significantly improved.ResultsHere, we present Manananggal, an application specifically designed for the identification of splicing events in next generation sequencing data. Manananggal includes a web application for visual inspection and a command line tool that allows for ASE detection. We compare the sashimi plots available in the IGV Viewer, the DEXSeq splicing plots and SpliceSeq to the Manananggal interface and discuss the advantages and drawbacks of these tools. We show that sashimi plots (such as those used by the IGV Viewer and SpliceSeq) offer a practical solution for simple ASEs, but also indicate short-comings for highly complex genes.ConclusionManananggal is an interactive web application that offers functions specifically tailored to the identification of alternative splicing events that other tools are lacking. The ability to select a subset of isoforms allows an easier interpretation of complex alternative splicing events. In contrast to SpliceSeq and the DEXSeq splicing plot, Manananggal does not obscure the gene structure by showing full transcript models that makes it easier to determine which isoforms are expressed and which are not.