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Dührsen: Celgene: Honoraria, Research Funding.

Early thymic progenitors enter the thymus and are exposed to regional hypoxia while they develop in a step-wise manner to mature functional T-cells. Therefore, hypoxia might represent an important component of the highly specialized thymic microenvironment. On the molecular level the hypoxia-inducible factor pathway controls the cellular response to hypoxia. In this pathway, the von-Hippel-Lindau protein (pVHL) continuously mediates the destruction of the transcription factor hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. Under hypoxia HIF-1α degradation is inhibited leading to the activation of HIF-1α target genes.

No relevant conflicts of interest to declare.

Schultz-Fademrecht: Lead Discovery Center GmbH: Employment. Unger:Lead Discovery Center GmbH: Employment. Klebl:Lead Discovery Center GmbH: Employment. Choidas:Lead Discovery Center GmbH: Employment.

The transmembrane receptor NOTCH1 operates as a ligand-activated transcription factor controlling developmental processes, proliferation and apoptosis. In the context of cancer, activating NOTCH1 mutations are the most frequent oncogenic events in T-cell acute lymphoblastic leukemia and have been implicated in chronic lymphocytic leukemia (CLL) as well. The most prevalent CLL NOTCH1 mutation (N1ΔCT) leads to a truncation of the protein (p.P2515Rfs*4) and has been associated with impaired overall survival (OS). Here, we applied three different methods to study the N1ΔCT prevalence and subclone size in a cohort of n=275 CLL patients.Presence of the N1ΔCT mutation was analyzed using newly established restriction fragment length polymorphism (RFLP) and allele-specific PCR (AS-PCR) methodologies. A novel real-time PCR (qRT-PCR) assay was used to precisely quantify the N1ΔCT allele frequency. Presence of the N1ΔCT mutation was confirmed by conventional Sanger sequencing.Using RFLP analysis we detected the N1ΔCT mutation in n=17 CLL patients. In parallel, we used a more sensitive AS-PCR and identified n=12 additional N1ΔCT-mutated cases resulting in a total N1ΔCT mutation rate of 10.5% (n=29/275) in our cohort. The OS of RFLP-positive patients (RFLP+) was significantly shorter than the OS of N1ΔCT-unmutated patients (wt) (mean OS; RFLP+, 87 months vs. wt, 218 months; p=0.017). In contrast, OS of AS-PCR-positive cases (AS-PCR+) did not differ significantly from the OS of wt patients (mean OS; AS-PCR+, 175 months vs. wt, 218 months; p=0.42). These data prompted us to design a quantitative real-time PCR (qRT-PCR) assay, which is capable of precisely quantifying the size of the N1ΔCT-mutated subclones (allele frequency, %) in our CLL cohort. As expected, significantly different allele frequencies between RFLP+ (mean±SEM 27.1±3.4%), AS-PCR+ (3.7±0.6%) and wt patients (0.6±0.04%) were revealed by qRT-PCR (p<0.0001). In order to determine a methodology-independent cut-off which correlates with the clinical significance of the N1ΔCT mutation, we employed Receiver Operating Characteristics (ROC) analysis based on survival status and calculated a N1ΔCT allele frequency cut-off of 15.2% (AUC=0.71). Next, we determined N1ΔCT allele frequencies over time to investigate clone dynamics within individual patients (n=15 patients, mean observation period 87.4 months; range 5-186 months). Unexpectedly, the N1ΔCT allele frequencies remained relatively constant and none of the patients with N1ΔCT allele frequencies below 15.2% rose above this cut-off over time.Our data demonstrate that high abundance of a N1ΔCT-mutated CLL clone correlates with an aggressive disease course. In our CLL cohort a N1ΔCT allele frequency below 15% was of negligible clinical relevance. Thus, mere qualitative detection of a N1ΔCT mutation by PCR is not inevitably associated with shortened survival. Surprisingly, we did not observe that a minor N1ΔCT clone became dominant over time.No relevant conflicts of interest to declare.

The development of hematopoietic cells occurs in highly specialized microenvironments within bone marrow, thymus and spleen. Hematopoietic stem and progenitor cells are positioned at the lowest end of a bone marrow oxygen gradient, which implies a role for the hypoxic-response pathway in regulating hematopoiesis. In this pathway the von Hippel-Lindau protein (pVhl) is the central negative regulator and continuously mediates the proteasomal destruction of the hypoxia-inducible factor-1α (Hif-1α). Under hypoxic conditions Hif-1α destruction is inhibited and results in the expression of hypoxia-inducible genes.

The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their ability to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models.

The transmembrane receptor NOTCH1 operates as a ligand-activated transcription factor controlling developmental processes, proliferation and apoptosis. In the context of cancer, activating NOTCH1mutations are the most frequent oncogenic events in T-cell acute lymphoblastic leukemia and have been implicated in chronic lymphocytic leukemia (CLL) as well. The most prevalent CLL NOTCH1mutation (N1ΔCT) leads to a truncation of the protein (p.P2515Rfs*4) and has been associated with impaired overall survival (OS). Here, we applied three different methods to study the N1ΔCTprevalence and subclone size in a cohort of n=275 CLL patients.

May: InSCREENeX GmbH: Employment, Equity Ownership, Patents & Royalties.

Abstract 1491

Eickhoff: Lead Discovery Center GmbH (LDC): Employment. Baumann:Lead Discovery Center GmbH (LDC): Employment. Unger:Lead Discovery Center GmbH (LDC): Employment. Schultz-Fademrecht:Lead Discovery Center GmbH (LDC): Employment. Dürig:Roche: Speakers Bureau; GSK: Speakers Bureau; Santaris Pharma: Consultancy, Research Funding; Celgene: Research Funding.

Abstract 1789

No relevant conflicts of interest to declare.

Abstract 913