ALBERT

Beschreibung: The transcription factor, CCAAT enhancer binding protein alpha (C/EBPα), is crucial for granulopoiesis and is deregulated by various mechanisms in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported in 10% of human patients with AML. Even though the C/EBPα mutants are known to display distinct biologic function during leukemogenesis, the molecular basis for this subtype of AML remains elusive. We have recently showed the significance of deregulation of C/EBPα-regulated microRNA (miR) in AML. In this study, we report that miR-34a is a novel target of C/EBPα in granulopoiesis. During granulopoiesis, miR-34a targets E2F3 and blocks myeloid cell proliferation. Analysis of AML samples with CEBPA mutations revealed a lower expression of miR-34a and elevated levels of E2F3 as well as E2F1, a transcriptional target of E2F3. Manipulation of miR-34a reprograms granulocytic differentiation of AML blast cells with CEBPA mutations. These results define miR-34a as a novel therapeutic target in AML with CEBPA mutations.

Beschreibung: Introduction: Residual minimal disease (MRD) in hematological malignancies has become a valid tool to predict clinical relapse after allogeneic stem cell transplantation (ASCT). Methods and Patients: We screened 154 patients with myelofibrosis who underwent ASCT for molecular residual disease by qPCR or next-generation sequencing (NGS) for JAK2V617F, MPLW515L and MPLW515K or Calreticulin (L367fs*46, and K385fs*47) mutations in peripheral blood (PB) on days +100 and +180 after transplantation. Out of 154 pts, 103 were JAK2V617F (n=101), 31 Calreticulin (CALR), and 4 MPL mutated, while 13 pts were triple negative. 136 pts could be followed after ASCT with one molecular marker. The median age of the pts was 58 years (range, 32-75 y). Patients had either primary myelofibrosis (n= 90), post ET/PV myelofibrosis (n=40), myelofibrosis in acceleration or were transformed to AML (n=6). Conditioning mainly relied on a busulfan-based reduced-intensity regimen. Donor were HLA-identical sibling (n=26), matched unrelated (n=71) or mismatched unrelated (n=39). JAK2V617F, MPL, and CALR mutations were measured by usage of Taqman PCR or in case of CALR Type 2 mutation by digital PCR on day +100 and day +180 from PB as described elsewhere. Results: After a median follow up of 78 months (range, 49-101 months) the 5-year estimated overall survival was 60% (95% CI: 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. On days +100 and +180 after transplantation in 27% and 12% of the patients the underlying mutation was still detectable in peripheral blood. The percentage of complete clearance was higher in CALR-mutated patients (96%) in comparison to MPL (75%) and JAKV2617F (70%) mutated pts. Whereas there was a trend for better survival for CALR-mutated patients in comparison to JAK2-mutated patients (71% vs 57%; p=0.48), once a patient achieved molecular remission post-transplant the risk of relapse remained low independently of the underlying mutation. Patients who were alive and without relapse at days +100 or +180 but with still detectable mutation in PB had a significantly higher risk of relapse than those who were molecular negative (62% vs 10%, p

Beschreibung: Despite the significant improvement in outcomes for myeloma patients (pts) in the more recent years (yrs), the disease remains incurable. Because of high morbidity and mortality the role of allo-SCT in treatment of myeloma remains controversial. Registry data from the European Society of Blood and Marrow Transplantation (EBMT) suggest an increasing use of allo-SCT as salvage therapy after failure to auto-SCT. In present single center study we provide data on the use of allo-SCT focusing on pts with first relapse/progressive disease after autografting. A total of 89 pts (males, 62%) with median age of 53y (24-73) relapsed after an autograft (single, 64%; tandem, 36%) and received an allo-SCT during 2000 - 2015 yrs at University of Hamburg were included. The majority of the pts experienced advanced disease (stage III, 80%) and short remission duration after auto-SCT (≤24 mo, 81%) with median of 15 mo (2-91). A total of 76% of pts received a reinduction therapy prior to allo-SCT, whereas 24% did not. Twelve pts (14%) had high risk cytogenetics (defined as detection of del17p and/or t(4;14) by FISH). Twelve pts experienced extramedullary relapses. At time of transplantation pts were in CR (10%), vgPR (22%), PR (27%), SD (16%) or PD (25%). Allo-SCTs were performed mostly with peripheral stem cell (92%) and from unrelated (matched, 40%; mismatched, 32%) donors. Myeloablative conditioning was used in 70% of pts (busulfan-based, 61%; treosulfan-based, 9%). A total of 28 pts (31%) received a maintenance therapy (lenalidomide, 79%) starting at median 6 mo (4-8) post-transplant. Furthermore, 19 pts (24%) received DLIs (positive immunofixation, n=15; prophylaxis, n=4) within a median of 10 mo (4-27). The cumulative incidence (CI) of acute GvHD (grade II-IV) at d100 was 43% (32-55%). The CI of chronic GvHD at 5y post-transplant was 51% (39-63%; mild, n=19, moderate, n=12, severe, n=5). Of the 19 pts receiving DLIs, 5 (26%) developed GvHD (grade II-IV). Six (40%) of 15 pts responded (CR). The CI of TRM at d100 and at 5y were 8% (4-16%) and 19% (12-28%), respectively. We observed improved TRM in pts who didn't receive radiation before allo-SCT (p=0,03). The CI of relapses at 5y was 61% (49-72%). We observed a trend to lower relapse rate of 29% in pts with longer remission duration (〉24 mo, p=0,07). The 5y probability of EFS was 28% (19-39%). The median EFS was 29 mo (22-36). In univariate analysis, we observed decreased survival probability for pts with high risk cytogenetics (p=0,026), remission duration ≤24 mo (p=0,008) and non-responders to salvage therapy (p=0,037). In multivariate analysis the negative impact of remission duration ≤24 mo (HR 6,9; 1,8-25,6; p=0,004) and failure to salvage therapy (HR 3,3; 1,2-9,0; p=0,021) were confirmed. In landmark analysis at d100 we observed improved survival for responding pts (p

Beschreibung: Background: The recent discovery of the somatically acquired calreticulin mutation in about 30% of the myelofibrosis patients provided a new diagnostic marker, which can also be used as marker of minimal residual disease (MRD) following curative treatment with allogeneic stem cell transplantation (ASCT). More than 80% of CALR mutations are of two types: type-1 variants result from a 52-bp deletion and produce the protein change p.L367fs*46, and type-2 variants are caused by a 5-bp insertion and produce the protein change p.K385fs*47. Whereas Sanger as well as next-generation sequencing (NGS) readily allow detection of CALR mutations in newly diagnosed patients, their applicability for MRD detection is limited. Real-time quantitative PCR (qPCR) has therefore been suggested as a potential alternative. In order to combine the increased sensitivity of qPCR with the excellent accuracy of digital PCR (dPCR) we developed a duplex dPCR assay detecting the CALR type-2 mutation in combination with its wild-type allele. Methods and Patients: To address sensitivity and reliability of the novel dPCR assay we first tested it on a new, UKE-1 derived cell line harboring one copy of the CALR type-2 mutation (UKE-1CALR2), which was established by lentiviral gene transfer and single-cell sorting. We generated serial dilutions of UKE-1CALR2 in buffy-coat (BC) cells, isolated DNA and submitted it to dPCR. Using 120 ng EcoRI-restricted genomic DNA we detected up to one UKE-1CALR2 in 10,000 BC cells (0.01%) indicating excellent sensitivity. As expected the detection limit could be further increased by applying the p.K385fs*47-specific dPCR as a singleplex assay. Results: Using our new technique we next performed MRD analysis in CALR+ patients who underwent allogeneic stem cell transplantation and compared results with respective qPCR data. Out of 143 patients with myelofibrosis who underwent allogeneic SCT 92 were JAK2V617 positive, 4 MPL positive and 35 CALR positive. Out of these 35 patients 21 harbored the CALR type-1 and eight the CALR type-2 mutation. In seven out of those eight patients both qPCR and digital PCR could be applied for MRD monitoring after SCT. In 3/7 patient qPCR as well as dPCR were negative on day +20, +100 and +180 after transplantation, respectively. In 2 patients dPCR remained positive at days +100 and +180 days before turning negative, whereas qPCR was already negative. In one patient dPCR remained positive 6 months after SCT, whereas qPCR was negative since day +80. None of the above patients had experienced clinical relapse. In contrast, there was one patient who relapsed 28 months after transplantation. That patient was MRD-negative by qPCR until one months before relapse, whereas dPCR, which initially also became negative after SCT (day +180), converted to positivity already 1 year after transplantation and was steadily increasing until clinical relapse. Conclusion: Our data indicates that the new CALR type-2-mutation specific dPCR assay combines excellent accuracy with high sensitivity thus allowing the monitoring of deep molecular remission and the early detection of MRD in relapsing patients with myelofibrosis after stem cell transplantation * BF and AB contributed equally Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 1203 Poster Board I-225 HHCT using reduced intensity conditioning (RIC) and immunomagnetic CD3/CD19 graft depletion is of low toxicity and enables fast engraftment even with grafts of low CD34 content. Immune reconstitution may be improved compared to graft CD34 selection due to the cotransplantation of facilitating cells, CD34- progenitors, dendritic cells and natural killer (NK)-cells. A prospective multicenter phase II study of HHCT using CD3/CD19-depleted grafts after RIC with fludarabine (150-200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day -5 to +14) was initiated. No post grafting immunosuppression was applied if the graft contained 500 granulocytes/μL (range, 9-50) and 11 days to 〉20.000 platelets/μL (range, 7-38). Incidence of grade II-IV acute GVHD and chronic GvHD (cGvHD) was 47% and 15%, respectively. Non-relapse-mortality on day 100 was 25% and 44% for the whole study period. Current overall survival (OS) is 18 of 60 patients (30%) with median follow-up of 525 days (range, 21-1542) of patients alive resulting in a Kaplan-Meier estimate 1-year OS of 41% and 2-year OS of 24%. Kaplan-Meier estimate 1-year OS was 40 % in AML, 38 % in ALL and 67% in NHL patients. No positive impact of KIR-mismatch on survival even in the subgroup of patients with AML was observed. Patients with cGVHD had a trend toward better survival with 56% vs. 39% (p=0.09). 14 patients died because of infections. Detailed immune reconstitution was analyzed in 24 patients. NK-cell engraftment was fast with median of 248 CD16+56+CD3- cells/μl (range, 1-886) on day 20. Increased natural cytotoxicity receptors (NKP30, NKP44, NKP46) and NKG2A, but decreased NKG2D and KIR-expression was observed on NK-cells in the first 3 months. T-cells regenerated delayed with median of 191 CD3+ cells/μl (range, 38-799) on day 100. A slow reconstitution of CD8+ and CD4+ T-cells with median of 66 CD8+ (range, 8-170) vs. 70 CD4+ cells/μl (range, 12-301) on day 100 and 157 (range, 32-379) vs. 181 cells/μl (range, 19-980) on day 400 was observed. The subset of memory T-cells regenerated faster compared to naïve T-cells with median of 25 CD4+45RA+ (range, 4-109) vs. 80 CD4+45RO+ cells/μl (range, 0 to 255) and 46 (range 7-191) vs. 164 cells/μl (range, 66-323) on days 80 and 250, respectively. T-cell repertoire was skewed with oligoclonal T-cell expansion to day 100 and normalization after day 200. B-cell reconstitution was slow with median of 8 (range, 0-407) CD19+20+ cells/μl on day 100. 6 of these 24 patients received donor-lymphocyte-infusions for relapse or mixed chimerism resulting in acceleration of immune recovery in T- and NK-cells. In conclusion, HHCT using RIC and CD3/CD19 depleted grafts is of low toxicity and allows fast engraftment even with low doses of CD34 cells. Overall survival seems promising in a high risk patient cohort. Recovery of NK cells occurs early and fast. KIR receptor expression remains low in the first 3 months. T- and B-cell reconstitution is delayed but seems faster compared to published data after CD34 selection. To evaluate the treatment protocol earlier during the course of disease a new study has just been initiated. Disclosures: Off Label Use: The use of Fludarabine, Thiotepa, Melphalan and OKT-3 in the conditioning is off-label-use.

Beschreibung: Introduction Primary or post-ET/post-PV myelofibrosis is one of the Philadelphia chromosome-negative chronic myeloproliferative neoplasia characterized by significantly reduced overall survival. More recently several mutated genes have been detected, which allow, in addition to clinical factors, to identify patients with a significantly shorter overall survival and a higher risk of transformation into acute leukemia. Allogeneic stem cell transplantation is still the only curative treatment option for patients with myelofibrosis, and due to the inherent risk of the treatment procedure careful selection of the patients is required. Molecular genetics may help to select patients for allogeneic stem cell transplantation. Patients and methods To determine the impact of mutated genes in patients with myelofibrosis who underwent allogeneic stem cell transplantation we analyzed samples from 169 patients with a median age of 58 years (r: 18 - 75) who received allogeneic stem cell transplantation either from related (n = 36) or unrelated (n = 133) donor. Stem cell source were more often peripheral blood stem cells (n = 165) than bone marrow (n = 4). The intensity of conditioning was mainly reduced intensity (n = 166), rather than myeloablative conditioning (n = 3). Patients suffered from primary myelofibrosis (n = 110), post-ET/PV myelofibrosis (n = 46), while 13 patients were in acceleration or had transformed into acute myeloid leukemia. According to dynamic IPSS (DIPSS) (n = 165) the patients were either low (n = 7), intermediate-1 (n = 35), intermediat-2 (n = 91), or high risk (n = 32). Regarding molecular genetics we found JAK2V617F mutations in 62%, calreticulin (CALR) mutations in 20%, MPL mutations in 4%, U2AF1 in 7%, SRSF2 in 10%, SF3B1 in 4%, ASXL1 in 29%, IDH1 in 2%, IDH2 in 3%, CBL in 1%, DNMT3A in 4%, TET2 in 10%, EZH2 in 4%, while none of the patients showed mutations in ETV6 and PTPN11. Overall, only in 11 patients no mutation could be detected. One mutation could be detected in 41%, 2 mutations in 30%, 3 mutations in 11%, 4 mutations in 5%. Results During follow-up 39 patients experienced relapse and 46 patients experienced non-relapse mortality. From the non-molecular factors regarding disease-free survival in univariate analysis age 〈 58 (p 〈 0.01), intermediate-1 and low risk according to DIPPS (p = 0.002), HLA-matched vs. mismatched (p = 0.04) were significant factors for improved disease-free survival. Regarding molecular markers improved disease-free survival was seen for patients with mutations in CALR (p = 0.005), while negative impact on disease-free survival was seen for mutations in U2AF1 (p = 0.035), ASXL1 (p = 0.05), IDH2 (p = 0.006), DNMT3A (p = 0.029). No significant difference could be seen for patients with EZH2, IDH1, SRSF2, and SF3B1 mutations. There was no difference in disease-free survival for patients without any mutation vs. 1, and more than 1 mutation (p = 0.12). Regarding the previously described unfavorable mutations ASXL1, SRSF2, EZH2, IDH1, and IDH2, we found 40 patients who had at least 1 of these unfavorable mutations, 11 had 2 of these mutation, and 1 had 3 of these unfavorable mutations. However, the estimated 5-year disease-free survival did not differ significantly between patients without any of these unfavorable mutations, with 1 or with 2 of them (47 vs. 40 vs. 41%, p = 0.5). Conclusions These results suggest that some molecular marker such as ASXL1, U2AF1, IDH2 and DNMT3A negatively influence DFS in myelofibrosis after allogeneic stem cell transplantation in a univariate analysis. In contrast, the poor prognosis of the recently described unfavorable mutated genes SRSF2, EZH2, and IDH1 was not observed and may therefore be overcome by allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 4467 Background: Relapse incidence (RI) and non-relapse mortality (NRM) are competing risks limiting overall survival (OS) after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML). Disease and transplant specific factors predicting relapse like measurement of minimal residual disease (MRD) and chimerism analysis are widely used to aid prophylactic and preemptive treatment decisions. Prediction of NRM mostly relies on pretransplant features. Although most transplant centers routinely perform bone marrow (BM) cytomorphology after SCT for AML, the impact of factors beyond blast count is not well studied. Study Design: We analyzed frequencies and prognostic impact of dysplasia and cellularity upon BM cytomorphology of 112 patients (60 m/52 f, median age 53 [range 17–72] years) with AML at 1st manifestation/ relapse at day 30 (d30) and day 100 (d100) after SCT. Using peripheral blood as main graft source (n=106), donors were unrelated in 87 cases, related in 25. Conditioning was reduced (RIC, n=72) or myeloablative (MAC, n=40). All patients received G-CSF from day 5 until stable engraftment was achieved. Dysplasia was assessed following WHO criteria with different thresholds (10%, 20%, 50%) to define a hematopoietic lineage as dysplastic. We performed a correlation of dysplasia and age-adapted cellularity with outcome measures, calculating RI and NRM as competing risks. Only patients who achieved blast clearance on d30 after SCT were included in the study. Patients who developed hematological relapse between d30 and d100 were only evaluated for d30. At d30 (d100), BM aspirates from 75 (65) patients were available for morphologic evaluation. Result: Dysplasia was a frequent event both at d30 and d100, with ≥10% dysplastic features in granulopoiesis in 25.0% of cases at d30 (31% d100), in erythropoiesis in 34.6% of cases at d30 (43.6% d100) and in megakaryopoiesis in 47.7% of cases at d30 (63.5% d100). Overall, cellularity at d30 was increased in 17.3% (d 100: 6.5%), reduced in 37.3% (d100: 38.7), and normal in 45.3% (d100: 54.8%). No significant correlation with CMV reactivation or with the type of immunosuppression (cyclosporine/ methotrexate versus cyclosporine/ mycophenolic acid) was noted. Cumulative incidences of 2-year-RI and 2-year-NRM were 34% (95% CI, 24%-44%) and 17% (95% CI, 9%-25%). Dysplasia both at d30 and d100 did not correlate with OS or RI. Yet, a statistically significant correlation of normal overall cellularity at d30 with less relapses (RI 20.6%) when compared with reduced overall cellularity (RI 32.1%) or increased overall cellularity (RI 76.9%; p=0.001) was observed. Estimated 2-year-OS was 59% in pts with normal overall cellularity versus 31.4% (reduced) and 44.0% (increased), respectively (p=0.009). The same results, favoring normal cellularity, were observed for each lineage (granulopoiesis, erythropoiesis, megakaryopoiesis). Conversely, increased overall cellularity at d30 correlated with lower NRM (8.3%) when compared to normal (NRM 23.7%) and reduced overall cellularity (NRM 39.6%, p=0.031). Thus, whereas reduced overall cellularity at d30 correlated both with higher RI and higher NRM, the impact of increased cellularity on survival was less clear. The analysis of subdistributive hazards in the competing risk factor model revealed a cumulative RI of 62% (95%CI 35%-89%, HR 6.68, p=0.00014) for increased cellularity, making it the most potent hazard in this analysis. Presence of an informative sample was of prognostic value, too (2-year-OS/ NRM 54.7%/ 80.4% for “evaluable” versus 20%/ 36.9% for “not evaluable” due to low cellularity, p

Beschreibung: The monoclonal antibody Rituximab has been successfully used to treat steroidrefractory chronic graft versus host disease (cGvH) after allogeneic stem cell transplantation (SCT). We hypothesized that Rituximab might reduce the incidence of aGvH. We found that patients conditioned with a regimen containing Rituximab experienced significantly less aGvH when compared to a regimen without Rituximab (23.1% versus 66.7%, p = 0.032). Furthermore did we find that the recovery of CD3+CD4+ lymphocytes was delayed following administration of Rituximab. As expected, patients were devoid of CD19+CD20+ B lymphocytes after administration of Rituximab; interestingly, both the recovery of CD3+CD8+ lymphocytes and CD16+CD56+ NK cells did not differ between patients receiving Rituximab and Rituximab-naíve patients. Furthermore, our data revealed a significant correlation of the transplants CD34+ HSC content with the occurrence of aGvH (p = 0.031) but only a trend towards a correlation of CD3+ cells with the occurrence of aGvH (p = 0.274). Taken together, our data establish a mechanism of how Rituximab might reduce the incidence of aGvH next to depleting host and perhaps donor antigen presenting cells. It remains to be elucidated whether this effect translates to a graft versus tumor effect and to survival as well.