ALBERT

Description: Background: Cellular senescence has been recognized as a failsafe mechanism against hyperproliferation and might thus be induced by DNA replicative stress and oncogenic signaling, commonly termed oncogene-induced senescence (OIS). OIS has been described in several premalignant conditions such as colon adenomas and melanocytic nevi, with impaired OIS capabilities found in their malignant counterparts. Here, we analyze the possible impact of cellular senescence on malignant transformation in plasma cell disorders. Methods: Bone marrow and soft tissue biopsies from 125 patients with different stages of plasma cell disorders (16 monoclonal gammopathy of undetermined significance (MGUS), 32 smoldering multiple myeloma (SMM), 56 symptomatic multiple myeloma (MM), 21 extramedullary MM) as well as from 10 healthy donors were analyzed. Expression of OIS associated proteins p16INK4A, p21Cip1/Waf1, p27Kip1, phospho-Chk2, the DNA double-strand break marker γH2AX, as well as the proliferation marker Ki67 were assessed on plasma cells by immunohistochemistry. Additionally, double staining experiments for p21 and Ki67 were performed applying immunofluorescence confocal microscopy. Levels of protein expression were compared between different disease stages using the Kruskal-Wallis test. Results: A differential expression pattern was found for p21 in various stages of plasma cell disorders with peak expression of p21 in SMM compared to both healthy controls (p

Description: Introduction: The pathogenesis of multiple myeloma (MM) involves complex genetic and epigenetic alterations affecting the cellular signaling network, the detailed processes of which remain poorly understood. Recently available genomic data has revealed a diverse mutational landscape with relatively few recurrently mutated genes. Instead, there are clustered mutational hotspots within signaling pathways that have been described to be of pathogenetic relevance in MM. To date, however, only very limited data is available on the activation profile of signaling pathways in MM and most of these studies have been performed in vitro or on sorted patient cells, which may be not representative of primary cells within their native microenvironment. Patient samples and methods: In contrast to in vitro studies, we used an ex vivo immunohistochemistry-based technique to retrospectively analyze the activation status of the five most well-described signaling pathways in MM. Using key activation markers, we interrogated the RAS/RAF/MEK/ERK, JAK-STAT, canonical NF-kB, PI3K-AKT, and c-MYC signaling networks on bone marrow biopsies taken at the time of diagnosis from two independent patient cohorts. The training cohort included 148 newly diagnosed, symptomatic MM patients. The independent validation cohort was comprised of samples from 84 newly diagnosed, well documented MM patients who had been enrolled in the GMMG - HD3/4 clinical multicenter trials. All patients of the validation cohort had undergone upfront high-dose therapy and autologous stem cell transplantation. The activation pattern of each sample was independently scored by two pathologists relative to on-slide positive controls. Activation scores included signal intensity as well as the percentage of positive versus total tumor cells per sample to account for potential clonal heterogeneity. Principal component analysis (PCA) was applied to integrate signaling scores per pathway and sample. Associations of progression-free (PFS) and overall survival (OS) with pathway activation patterns were analyzed using the Cox regression model. Results: We first focused on activation signals that were present in the majority of myeloma cells per sample, potentially representing the major clone of the individual patient’s disease. Several clusters of activation could be distinguished with a NF-κB cluster being the most prominent (77% of cases), followed by samples with activated MEK/ERK signaling (19.6%) partially overlapping with PI3K-AKT activity (9.5%). An additional 5% of cases showed an activation of STAT3 and/or c-MYC in the majority of tumor cells. We next took all activation signals into account, including those present in less than 50% of myeloma cells per sample, potentially representing subclonal populations of tumor cells. Activation of a single pathway was found in 22%, two pathways in 30%, and more than 2 pathways in 47% of cases, while activation of any tested signaling event was absent in 2%. Several clusters of activation could be distinguished with a NF-kB cluster being the most prominent (82% of cases), followed by samples with activated MEK/ERK signaling (49%) partially overlapping with PI3K-AKT activity (41%). 29% of cases showed an activation of the JAK-STAT3 axis while c-MYC expression was detectable in 43% of the samples. Interestingly, the latter two signaling cascades tended to overlap with other activation clusters. A similar distribution of clusters of activation was found in the validation cohort. Importantly, association analyses with clinical data available for this cohort revealed a significantly shorter PFS (HR 4.59; p=0.038) for patients with STAT3 activation and a trend towards a shorter OS. Moreover, patients with c-MYC activation or those with more than one activated pathway had a significantly shorter overall survival (HR 9.54, p=0.019; and HR 3.77, p=0.003, respectively). In contrast, activation of NF-kB was associated with a more favorable outcome (HR 0.20, p=0.034) while MEK/ERK signaling appeared to confer a neutral prognosis. Conclusion: This study is the first comprehensive study of signaling pathways in multiple myeloma. Activation of signaling cascades differs substantially between patients and do not occur randomly but can be distinguished into defined clusters of activation. Most importantly, there appears to be a prognostic hierarchy of these clusters. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The pathogenesis of multiple myeloma (MM) involves complex genetic and epigenetic alterations, which ultimately affect oncogenic signaling networks. Analysis of pathway activation in primary MM cells within their native microenvironment will not only increase our understanding of MM cell biology but may open up new avenues towards tailored therapies that target individually activated signaling cascades. Methods: We established a (phospho-) immunohistochemistry (IHC)-assay to analyze key activation markers of oncogenic pathways and networks, namely RAS/RAF/MEK/ERK, PI3K/AKT, JAK/STAT3, canonical NFκB, and cMyc, in an exploratory cohort of 459 bone marrow (BM) biopsies from patients with smoldering MM (SMM), newly diagnosed symptomatic (NDMM) and refractory MM (RRMM). An independent validation cohort comprised BM samples from 462 NDMM patients who were uniformly treated within clinical trials. To integrate activation intensity and spread scores for each pathway and sample, non-linear principal component analysis (PCA) was applied. Results: Pathway profiling in progressing stages of plasma cell disorders showed the canonical NFκB pathway as nearly universally activated in SMM (97% of patients); signals for AKT, ERK, STAT3, and cMyc were only seen in subsets of SMM patients (≤33%), with STAT3 being the least activated pathway (6%). In NDMM and RRMM, NFκB activation significantly decreased to 90% and 82% of patients, respectively (p=0.02). In contrast, we observed a significant increase in activation of all other pathways and cMyc from SMM to NDMM and further to RRMM patients (p

Description: Activating mutations of the BRAF kinase (BRAF V600E) are found in virtually all cases of classical hairy-cell leukemia (HCL), suggesting disease-specific oncogene dependence. Case reports and early trial data demonstrate impressive activity of BRAF inhibitors, but optimal dosing and treatment duration remain unclear. Methods: We report on 21 patients with hairy cell leukemia across Europe (Heidelberg, Innsbruck, Nice, Munich, Cambridge, Erfurt, Freiburg, Luzern, Cologne, Leicester, London) treated with vemurafenib, a specific BRAF inhibitor outside of trials from 2011-2014. Centers provided clinical data and pathology specimen where available. Results: Presence of the BRAFV600E mutation was demonstrated in all patients. Median age of all patients included in this study was 64 (range 45-89) years. Patients received a median of 3 (range 0 to 12) prior treatment lines. Two patients were treated upfront (age, comorbidity). Median time between initial diagnosis and experimental treatment with vemurafenib was 8 (range 0-31) years. Vemurafenib was started at a dose of 240 mg bid in 18 patients and was continued at this dose in 14 patients. In four patients doses were escalated to 720 mg (n=1) and 960 mg (n=3), respectively. Three patients received 480 mg (n=2) bid or 960 mg (n=1) bid upfront. Median therapy duration was 90 (range 55-167) days, and 2 patients are still on therapy (day 85 and 275) at last follow up. All patient's blood counts improved meeting response criteria (Hb 〉 12g/dl, platelets 100.000/ µl, neutrophils 〉1000µl), except for a 89-year-old patient who did not improve with his hemoglobin due to renal anemia. Median time to neutrophil recovery (〉1000/ µl) was 39 (range 9-126) days, to platelet recovery (〉100.000 µl) 28 (range: 10-105) days and time to improvement of anemia (Hb 〉 12 g/dl) 67 (range: 10-105) days, respectively. Seven patients achieved a CR and 13 a PR. Patients who received more than 240 mg bid (n=8) did not have significant more CRs than patients who received 240 mg (n=14) (Fishers test p=0.16). CR did not translate into better EFS (HR 1.2, p=0.7, Figure 1). Immune histology staining was performed to assess p-ERK signaling. Median observation time was 12 months (range: 3-31 months) and median event free survival (retreatment or death) was 17 months for all patients (Figure 1). Survival at 12 months was 87%. Three of 21 patients died; one patient due to HCL disease progression after termination of vemurafenib, one patient developed an AML M6 and one patient died at the age of 89 years due to pneumonia off treatment in remission. Seven patients (28%) including the 2 patients treated upfront were retreated at relapse after a median of 10 months (range: 4-16 months) after stopping vemurafenib. Six patients were reexposed to vemurafenib and one received cladribine. All patients responded again to vemurafenib and two patients continue to receive ongoing treatment at 240mg bid and 480mg bid respectively, 18 months and 8.5 months from restarting therapy. Conclusion: Targeting a BRAF V600E can provide disease control in HCL. CR was achieved in 30% of patients and can be achieved with 240 mg bid. Relatively short EFS suggests that treatment duration and dosing regimen should be optimized and combination treatments involving BRAF inhibitors should be explored in refractory HCL patients. Moreover, dosing schedules and treatment duration may need to be individualized in patients with HCL carefully taking into account individual pharmacodynamics and response. Figure 1: EFS (re-treatment or death) of HCL patients after vemurafenib treatment. Patients with CR (solid line) and patients with PR (dashed line) have similar EFS. Figure 1:. EFS (re-treatment or death) of HCL patients after vemurafenib treatment. Patients with CR (solid line) and patients with PR (dashed line) have similar EFS. Disclosures Herold: Roche Pharma AG/Germany: Honoraria, Research Funding. Dearden:Roche, GSK, Gilead, Janssen, Napp: Honoraria.

Description: Key Points Low doses of the BRAF inhibitor vemurafenib are highly effective in refractory hairy cell leukemia. Abrogation of BRAF V600E–induced signaling was consistently seen with 240 mg of vemurafenib twice daily.