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Hammersen: Mundipharma: travel support Other. Off Label Use: Bendamustine in the treatment of agressive Non-Hodgkin Lymphoma. La Rosee:Mundipharma: Honoraria, travel support Other.

Hemophagocytic Lymphohistiocytosis (HLH) is a rare and potentially life-threatening disorder in adults. Patients (pts) with fever of unknown origin, lymphadenopathy, deteriorating performance status, encephalopathy and elevated systemic inflammatory parameters frequently show no clear cut diagnostic criteria specific for HLH. Among adults, late onset of inherited HLH is possible, but acquired HLH triggered by infection, neoplasia or autoimmune disease is more frequent than in pediatrics. Due to the variability of clinical presentation, large-scale unreporting can be assumed. We therefore commenced a German registry for HLH pts, aiming to sensitize clinicians and to establish an HLH network as starting point for research and clinical guideline activities.We performed a retrospective multicenter case study in Germany. Diagnostic criteria were derived from the pediatric revised HLH-04 protocol. Treatment and outcome parameters were evaluated.In 29 adult pts registered so far, median age was 46 years (18–81), 13 pts were female. HLH in 10 pts (34%) was malignancy-associated (aggressive B-cell lymphoma, n=6; B-CLL n=1; AML n=2; melanoma n=1), infection-associated (IHLH) in 13 pts (45%), 2 pts (7%) had connective tissue disease (CTD), in 1 pt HLH developed after therapeutic plasma exchange for TTP and in 3 pts (10%) the cause could not be determined. The main pathogen in IHLH pts was EBV (9/29, 31%) followed by other viruses (CMV n=1; HSV n=1; VZV n=1; Parvovirus B19 n=1). Of the 13 IHLH pts, 4 pts showed an EBV-reactivation post-allogeneic stem cell transplantation (SCT) for acute leukemia (n=3) and severe aplastic anemia (n=1) and 1 pt a systemic VZV infection after autologous peripheral blood SCT for aggressive B-cell NHL. 2 pts showed an EBV- and a lymphoma-triggered HLH respectively during late pregnancy. In 2 pts with EBV-associated HLH sequencing revealed mutations in XIAP and XLP-1 genes. The median time from onset of symptoms to diagnosis was 14 days (range 1–60 days). Most pts received polyvalent immunoglobulin (20/29, 69%) and HLH-04 based therapy, including steroids (14/29, 48%), cyclosporine (6/29, 21%), and etoposide (10/29, 34%). 5 pts (17%) received rituximab and 3 pts (10%) R-CHOP for lymphoma. 2 pts each (7%) received alemtuzumab and cyclophosphamide. One patient underwent allogeneic SCT but died. With a median follow up of 413 days (range, 54–1204 days), 12 pts (41.3%) survived. 2 pts were lost to follow-up. Median survival after diagnosis was 151 days (range, 14–1204+ days).Adult pts with HLH in our cohort showed a heterogeneous clinical syndrome most frequently triggered by EBV. HLH represents a final common pathway of an aberrantly activated immune system responding to a variety of triggers. If not recognized and treated early, HLH in adults has a very poor prognosis. Clinical registries need to include adult HLH pts in order to facilitate basic and clinical research to improve outcome of this frequently fatal immune response syndrome.No relevant conflicts of interest to declare.

The BCR-ABL oncoprotein promotes growth and survival by activating Ras-dependent MAPK-signalling, which can be blocked by the Abl-kinase inhibitor imatinib, the current standard treatment for chronic myelogenous leukemia (CML). However, paradoxical MAPK-activation is observed in CD34+ progenitor cells of CML patients when exposed to imatinib in the presence of growth factors. Cytokine-dependent MAPK-activation may underlie primary resistance of the leukemic stem cell to imatinib. Second generation BCR-ABL inhibitors have been developed, but their potential to modulate the MAPK has not been evaluated. Here we demonstrate differential MAPK-modulating activity of currently available ABL-inhibitors and report a cell line model mimicking paradoxical, cytokine dependent MAPK-activation in response to BCR-ABL-inhibition. CD34+ enriched mononuclear cells (MNC) derived from CML patients prior to treatment (n=5) were cultured in serum-free media in the presence of standard growth factor mix (GF). Overnight exposure of single agent or combined inhibitors (nilotinib, dasatinib), and subsequent analysis of MAPK-activation was performed. BCR-ABL-transformed Baf3 and 32D cells were grown in the presence of increasing concentrations of tyrosine kinase inhibitors up to 24 hrs. Cultures were kept either with or without Interleukin 3 (IL3). Signaling was studied after SDS-page of whole cell lysates and subsequent Western blot analysis. Inhibitor isodoses were determined using tetrazolium based proliferation assays. Results: CD34+ CML MNC show significant and dose dependent activation of MAPK1/2 in response to nilotinib ([fold change of control]; 0.2μM: 2.7±1.1, p<0.05; 1.0μM: 3.2±1.2, p<0.05). Co-treatment with imatinib tends to enhance the MAPK-activation seen with nilotinib alone. In contrast, exposure of patient cells (n=5) to dasatinib results in a significant MAPK-inhibition (12nM: 0.4±0.3, p<0.05; 60nM: 0.5±0.3, p<0.05). However, combined application of nilotinib with dasatinib overrides the MAPK-inhibitory activity of dasatinib (dasatinib: 0.4±0.3, p<0.05 vs dasatinib+nilotinib: 2.3±2; n.s.). The myeloid 32Dp210 cells when grown in the presence of IL3 reflect the results seen in primary cells with activation of MAPK1/2 in the presence of nilotinib. Time course experiments show peak-activation at 6hrs after start of treatment (0.04μM: 2.8±1.6, n.s.; 0.2μM: 2.8±1.9; n.s.). Without IL3, MAPK’s are significantly inhibited confirming the cytokine-dependence of MAPK-activation (p<0.05). This contrasts data obtained with dasatinib, which leads to MAPK-inhibition even in the presence of IL3 (12nM: 0.5±0.2, n.s., 60nM: 0.5±0.1, n.s.). In lymphoid Bafp210, nilotinib and dasatinib both effectively inhibit MAPK1/2 even in the presence of IL3, demonstrating cell context dependent MAPK-modulation in response to Abl-kinase-inhibitors. Conclusions: Nilotinib induces significant MAPK1/2-activation in CD34+ CML progenitors similar to previously reported results with imatinib. Effective MAPK-inhibition in CD34+ cells by dasatinib may underlie a possible higher stem cell activity of dasatinib. Reversal of MAPK-inhibition in combination experiments indicates that nilotinib acts through mechanisms that cannot be influenced by dasatinib. 32Dp210 cells may prove useful to study the exact mechanisms of paradoxical MAPK-activation.

The Abl-kinase-inhibitor imatinib is current standard treatment in chronic myelogenous leukemia (CML). Resistance to imatinib in most cases is governed by BCR-ABL mutations. Mutation T315I prevents binding of imatinib through steric hindrance. Patients with advanced Ph+ leukemia carrying T315I-mutant BCR-ABL rapidly progress on continuous imatinib therapy. The growth advantage of T315I-expressing cells may be solely explained by selection. However, the rapid onset of progression led us to ask, whether an additional imatinib-dependent mechanism may be accounted for this phenomenon. In imatinib responding cells, BCR-ABL-inhibition leads to effective blockade of BCR-ABL-depending singalling cascades such as the Ras/MAPK-pathway. However, paradoxical MAPK-activation in response to imatinib has been reported as a possible cytokine dependent resistance regulating factor. Using lymphoid hematopoietic cell lines (Baf3), we studied the effect of imatinib and the second generation inhibitor dasatinib on MAPK1/2 in interleukin 3 (IL3) dependent Baf3 cells. Baf3 cells transformed to IL3-independence by unmutated (p210) or mutant BCR-ABL (p210T315I) were treated with Abl-inhibitors in the presence or absence of additional IL3. Cellular BCR-ABL- and MAPK-activity were studied by Western blot analysis. Proliferation assays were used to study the functional significance of imatinib-induced signalling alterations in p210T315I -expressing cells. Imatinib at 5μM for 24hrs leads to significant MAPK1/2 inhibition in Bafp210wt cells in the absence of additional IL3 ([fold change vs untreated control] MAPK1/2: 0.07±0.06, p<0.05). In contrast, IL3 prevents imatinib from interfering with MAPK-signalling as observed in the presence of IL3 (MAPK1/2: 0.88±0.37, ns). Prevention of MAPK-inhibition correlates with rescue of Bafp210wt cells treated with imatinib in the presence of IL3 in viability count proliferation assays. Surprisingly, imatinib treatment in Bafp210T315I cells induced significant (p<0.05) and dose dependent activation of MAPK1/2 (1μM: 1.2±0.3; 5μM: 1.4±0.3) in the absence of IL3. This was not seen in cultures where IL3 was added. We further tested equipotent doses of the combined Src/Abl-inhibitor dasatinib in Bafp210T315I cells. Contrary to imatinib, dasatinib does not lead to MAPK-activation in Bafp210T315I cells (2.5nM: 0.9±0.8 n.s.; 12nM: 0.9±0.6 n.s.). Asking whether imatinib-induced MAPK-activation in p210T315I expressing cells would translate into a growth or survival advantage of these cells, proliferation studies with imatinib alone or combined chemotherapy were performed. Whereas imatinib alone did not consistently modulate cell growth, combined treatment studies with imatinib (5μM) and increasing concentrations of the chemotherapeutic agent decitabine demonstrated a slight but consistent growth advantage of combined vs decitabine only treated cells. Conclusions: MAPK-activation in imatinib-treated p210T315I -expressing cells indicates off-target activity of imatinib leading to activation of growth and survival promoting signalling intermediates. The combined Src/Abl-inhibitor dasatinib differs from imatinib in that it does not alter cellular MAPK-activity in Baf p210T315I cells. Different target profiles may explain this differential inhibitor effect. Antagonistic antiproliferative activity of imatinib/decitabine points to a possible functional link of imatinib-induced MAPK-activation leading to increased survival of imatinib-resistant cells treated with imatinib.