Abstract: Recently, overlapping molecular phenotypes of hematopoietic and neuropoietic cells were described in mice. Here, we examined primary human CD34+ hematopoietic stem and progenitor cells applying specialized cDNA arrays, real-time reverse-transcriptase–polymerase chain reaction (RT-PCR), and fluorescent-activated cell sorter (FACS) analysis focusing on genes involved in neurobiologic functions. We found expression of vesicle fusion and motility genes, ligand- and voltage-gated ion channels, receptor kinases and phosphatases, and, most interestingly, mRNA as well as protein expression of G protein–coupled receptors of neuromediators (corticotropin-releasing hormone 1 [CRH 1] and CRH 2 receptors, orexin/hypocretin 1 and 2 receptors, GABAB receptor, adenosine A2B receptor, opioid κ1 and μ1 receptors, and 5-HT 1F receptor). As shown by 2-color immunofluorescence, the protein expression of these receptors was higher in the more immature CD38dim than in the CD38bright subset within the CD34+ population, and completely absent in fully differentiated blood cells, suggesting that those receptors play a role in developmentally early CD34+ stem and progenitor cells. The intracellular concentration of cyclic adenosine monophosphate (cAMP) in CD34+ cells was diminished significantly upon stimulation of either CRH or orexin receptors, indicating that those are functionally active and coupled to inhibitory G proteins in human hematopoietic cells. In conclusion, these findings suggest a molecular interrelation of neuronal and hematopoietic signaling mechanisms in humans.

Abstract: Patients with acute myeloid leukemia ( AML ) carrying FLT 3‐ ITD mutations ( FLT 3‐ ITD +) who relapse after allogeneic transplantation (allo‐ SCT ) have a very dismal prognosis with the currently available treatment options. Methods We treated eight patients with FLT 3‐ ITD + AML who had relapsed in median 91 d (range, 28–249) following allo‐ SCT with a combination of the multikinase inhibitor sorafenib and the DNA methyltransferase inhibitor azacitidine (Aza). Results Patients received a median of five cycles of Aza (range, 2–9) and sorafenib with a median daily dosage of 750 mg (range 400–800) for 129 d (range, 61–221). Six of eight patients received donor lymphocyte infusions ( DLI ) with a median number of two DLI per patient (range, 1–4). Following this treatment, four patients (50%) achieved a complete remission and three of them a complete molecular remission. Median duration of CR was 182 d (range, 158–406), and two patients remain in ongoing remission for 406 and 168 d. Median overall survival was 322 d (range, 108–574 d) with three patients being currently alive. Conclusion Taken together, the combination of sorafenib, Aza, and DLI shows promising efficacy and deserves further evaluation in larger patient groups.

Abstract: Treatment of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) remains a great challenge. Aiming to evaluate the combination of venetoclax and hypomethylating agents (HMAClax) for the treatment of relapse of myeloid malignancies after alloHSCT, we retrospectively collected data from 32 patients treated at 11 German centers. Venetoclax was applied with azacitidine ( n = 13) or decitabine ( n = 19); 11 patients received DLI in addition. HMAClax was the first salvage therapy in 8 patients. The median number of cycles per patient was 2 (1–19). All but 1 patient had grade 3/4 neutropenia. Hospital admission for grade 3/4 infections was necessary in 23 patients (72%); 5 of these were fatal. In 30 evaluable patients, overall response rate (ORR) was 47% (14/30, 3 CR MRD neg , 5 CR, 2 CRi, 1 MLFS, 3 PR). ORR was 86% in first salvage patients versus 35% in later salvage patients ( p = 0.03). In 6 patients with molecular relapse (MR), ORR was 67% versus 42% in patients with hematological relapse (HR) ( n = 24, p = n.s.). After a median follow-up of 8.4 months, 25 patients (78%) had died and 7 were alive. Estimated median overall survival was 3.7 months. Median survival of patients with HMAClax for first versus later salvage therapy was 5.7 and 3.4 months ( p = n.s.) and for patients with MR (not reached) compared to HR (3.4 months, p = 0.024). This retrospective case series shows that venetoclax is utilized in various different combinations, schedules, and doses. Toxicity is substantial and patients who receive venetoclax/HMA combinations for MR or as first salvage therapy derive the greatest benefit.

Abstract: Acute myeloid leukemia (AML) is a phenotypically and prognostically heterogeneous hematopoietic stem cell disease that may be cured in eligible patients with intensive chemotherapy and/or allogeneic stem cell transplantation (allo-SCT). Tremendous advances in sequencing technologies have revealed a large amount of molecular information which has markedly improved our understanding of the underlying pathophysiology and enables a better classification and risk estimation. Furthermore, with the approval of the FMS-like tyrosine kinase 3 (FLT3) inhibitor Midostaurin a first targeted therapy has been introduced into the first-line therapy of younger patients with FLT3-mutated AML and several other small molecules targeting molecular alterations such as isocitrate dehydrogenase (IDH) mutations or the anti-apoptotic b-cell lymphoma 2 (BCL-2) protein are currently under investigation. Despite these advances, many patients will have to undergo allo-SCT during the course of disease and depending on disease and risk status up to half of them will finally relapse after transplant. Here we review the current knowledge about the molecular landscape of AML and how this can be employed to prevent, detect and treat relapse of AML after allo-SCT.

Abstract: Acute myeloid leukemia (AML) is characterized by an expansion of leukemic cells and a simultaneous reduction of normal hematopoietic precursors in the bone marrow (BM) resulting in hematopoietic insufficiency, but the underlying mechanisms are poorly understood in humans. Assuming that leukemic cells functionally inhibit healthy CD34+ hematopoietic stem and progenitor cells (HSPC) via humoral factors, we exposed healthy BM-derived CD34+ HSPC to cell-free supernatants derived from AML cell lines as well as from 24 newly diagnosed AML patients. Exposure to AML-derived supernatants significantly inhibited proliferation, cell cycling, colony formation, and differentiation of healthy CD34+ HSPC. RNA sequencing of healthy CD34+ HSPC after exposure to leukemic conditions revealed a specific signature of genes related to proliferation, cell-cycle regulation, and differentiation, thereby reflecting their functional inhibition on a molecular level. Experiments with paired patient samples showed that these inhibitory effects are markedly related to the immunomagnetically enriched CD34+ leukemic cell population. Using PCR, ELISA, and RNA sequencing, we detected overexpression of TGFβ1 in leukemic cells on the transcriptional and protein level and, correspondingly, a molecular signature related to TGFβ1 signaling in healthy CD34+ HSPC. This inhibitory effect of TGFβ1 on healthy hematopoiesis was functionally corrobated and could be pharmacologically reverted by SD208, an inhibitor of TGFβ receptor 1 signaling. Overall, these data indicate that leukemic cells induce functional inhibition of healthy CD34+ HSPC, at least in part, through TGFβ1, suggesting that blockage of this pathway may improve hematopoiesis in AML.

Abstract: The current classification system for Myelodysplastic Syndromes lumps all therapy-related (tMDS) into one subgroup assuming all tMDS had the same poor prognosis. We have put together a database including 2032 patients with a diagnosis of tMDS from several different IWG centers and the MDS clinical research consortium. With the idea of developing an individual scoring system for tMDS, we decided to start by optimizing the cytogenetic part of the IPSSR. First, we did an extensive review of karyotypes. Finally, 1245 patients had complete data and correct ISCN formula to be used for score development. We could show regarding karyotypes there are very limited differences between primary and tMDS. Mainly the distribution of risk groups differs with complex occurring more (37%) and normal karyotypes occurring less frequent, although still accounting for 30%. There are few exceptions that are relatively special for tMDS, like translocations including 11q23. A few karyotypes are less frequent; therefore, we could not evaluate the value of IPSS-R cytogenetics for all karyotypes. However, if we apply IPSS-R cytogenetics to our patient cohort, we can separate 5 different risk groups as in pMDS. We tested the performance of the score by using the Dxy. As main endpoint we chose transformation-free survival giving better information about the severity of the disease compared to the single endpoints survival and AML transformation that where calculated for completeness as well. The Dxy for the IPSS-R cytogenetic part is 0.31 for transformation-free survival. This indicates an effective prognostic performance although not as good as in pMDS. Several attempts were done to develop a tMDS specific cytogenetic score. The best draft scoring component achieves a Dxy of 0.33. Counting the number of aberrations achieves a score of 0.30. If normal clone present or not is added, the performance of this very simple model is improved with a Dxy of 0.32. As we could show, all these different approaches lead to a comparable performance. One can argue that still regarding a few karyotypes the prognostic impact is slightly different between p and tMDS (e.g. +8). On the other hand, the most practical approach seems to be to adopt the original cytogenetic part of the IPSS-R for further score development since clinicians do not need to use different scoring systems for different MDS subtypes. While the final analyses for the development of a tMDS specific risk score are currently under way, extensive calculations regarding the performance of different scores like WHO- (Dxy 0.24), FAB-classification (Dxy 0.19), WPSS-R (Dxy 0.35), IPSS-R (Dxy 0.37), and IPSS-R+age (Dxy 0.36), show all these systems can separate different risk groups within our cohort. However, these results also show an inferior performance of the scoring systems in t compared to pMDS. There are multiple possible reasons for this. The most important seem to be tMDS patients are often not cured from the primary disease and its disease specific risk of death should ideally be considered. Unfortunately, we don't have that data. And second, we included treated as well as untreated patients. It seems not to be feasible otherwise since the selection bias for old unfit patients would be unacceptable. We could show already in pMDS that the score performances are considerably worse if we analyze treated patients and the score performance in our cohort is better if limited to untreated patients. To conclude, we can say existing classification and scoring systems work in tMDS and can separate groups with clearly different risk for death and transformation. Although we could not develop a tMDS specific cytogenetic score this could be seen positively since it underlines tMDS do not seem to be much different regarding disease specific risk. This should initiate a discussion of a revision of the WHO-classification and encourage clinicians to use the existing tools for risk assessment and treatment decisions. A simple solution could be to use the WHO classification for pMDS and precede each subgroup with a t, like tMDS-SLD, and so on. Such an approach would be of importance for patients falsely classified as tMDS. After all this classification is done according to anamnestic information only and sporadic cases cannot be excluded. Until now, in the first analyzes performed with the final tMDS-database, we did not find any indication that risk factors established in pMDS would lose or change their meaning in tMDS. Figure. Figure. Disclosures Komrokji: Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. List:Celgene: Research Funding. Roboz:Orsenix: Consultancy; Eisai: Consultancy; Novartis: Consultancy; Celltrion: Consultancy; Astex Pharmaceuticals: Consultancy; Argenx: Consultancy; Janssen Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Argenx: Consultancy; Janssen Pharmaceuticals: Consultancy; Pfizer: Consultancy; Cellectis: Research Funding; Daiichi Sankyo: Consultancy; Sandoz: Consultancy; Otsuka: Consultancy; Daiichi Sankyo: Consultancy; Eisai: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy; Novartis: Consultancy; Celltrion: Consultancy; Celgene Corporation: Consultancy; Cellectis: Research Funding; Orsenix: Consultancy; Aphivena Therapeutics: Consultancy; Otsuka: Consultancy; Jazz Pharmaceuticals: Consultancy; Sandoz: Consultancy; Roche/Genentech: Consultancy; Aphivena Therapeutics: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Bayer: Consultancy; Astex Pharmaceuticals: Consultancy; Celgene Corporation: Consultancy; AbbVie: Consultancy. Döhner:Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Pfizer: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Valent:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Platzbecker:Celgene: Research Funding. Lübbert:TEVA: Other: Study drug; Celgene: Other: Travel Support; Cheplapharm: Other: Study drug; Janssen: Honoraria, Research Funding. Díez-Campelo:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Stauder:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva: Research Funding. Germing:Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Abstract: Abstract 2898 Multiple myeloma (MM) patients often suffer from hematopoietic impairment already at the time of diagnosis with anemia as the prevailing symptom. Given the overt affection of the bone marrow in MM patients by the invasion of malignant plasma cells, we hypothesized that hematopoietic insufficiency in these patients may originate from a functional impairment of hematopoietic stem and progenitor cells. Quantitative analysis of BM CD34+ HSPC cell subsets from MM patients and age-matched healthy donors showed a significant decline of all HSPC subsets including hematopoietic stem cells, common myeloid and lymphoid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in MM patients. The greatest diminution was observed in megakaryocyte-erythrocyte progenitors (MEP) which were 4.9-fold reduced in comparison to healthy donors. Transcriptional analyses of CD34+ HSPC subsets revealed a significant deregulation of signaling pathways that was particularly striking for TGF beta signaling and suggested increased activation of this signaling pathway. Immunhistochemical staining of phosphorylated smad2, the downstream mediator of TGF receptor I kinase activation, in bone marrow sections and immunoblotting of purified CD34+ HSPC of MM patients confirmed the overactivation of TGF beta signaling. On a functional level, we observed significantly reduced long-term self-renewal and clonogenic growth, particularly of the erythroid precursors BFU-E and CFU-E, in CD34+ HSPC of MM patients which could be restored by inhibition of TGF beta signaling. Proliferation and cell cycle analyses revealed a significantly decreased proliferation activity in CD34+ HSPC and, particularly, MEP. Again, this was reversible after inhibition of TGF beta signaling. In addition, the transcriptional analyses showed disturbance of pathways involved in the adhesion and migration of HSPC and the gene encoding for the principal hyaluronan receptor CD44 throughout the HSPC subsets. This was corroborated by immunofluorescence imaging of CD44 on HSPC subsets showing a marked downregulation in the patients' cells. In line, the adhesion of CD34+ HSPC subsets to hyaluronan and their migration towards SDF-1 was significantly inhibited. Subsequent xenotransplantation of CD34+ HSPC from MM patients and healthy donors into myeloma-free recipients revealed even increased long-term engraftment of CD34+ HSPC obtained from MM patients and normal differentiation capacities suggesting that the observed functional alterations in fact depend on the MM-related bone marrow microenvironment. Our data show that hematopoietic impairment in patients with multiple myeloma originates, at least in part, from functional alterations of hematopoietic stem and progenitor cells. These alterations seem to depend on the disease-related changes of the bone marrow microenvironment. Currently, experiments are underway to elucidate in more detail the role of the microenvironment and the responsible structures for the impairment of HSPC in MM patients. These data will be presented. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy.