ALBERT

Description: The homeobox transcription factor Cdx2 is one of the most frequent ectopically expressed proto-oncogenes in human AML and when retrovirally expressed causes AML in mice (Rawat et. al. PNAS 2004 and Blood 2008). As the leukemogenic potential of Cdx2 was dependent on its N-terminal transactivation domain, we now extended structure-function analyses by inactivating the evolutionarily conserved MAPK phosphorylation sites (S60, S99, S108, S156, S60-S99-108 (3S), and S60-S99-108-156 (4S)) in Cdx2. Peripheral blood analysis of the mice, transplanted with bone marrow cells retrovirally transduced with different Cdx2 serine mutants (n=5) after six weeks, revealed that all the Cdx2 serine mutants showed significant growth advantage over non-transduced carrier cells. However, assessment of engraftment after 16 weeks showed that mice transplanted with BM expressing the S99, the S108 mutants, the 3S and 4S failed to develop leukemic engraftment in contrast to wild type Cdx2, indicating that the S99 and S108 serine sites are critical for leukemic transformation. Furthermore, mice transplanted with BM expressing Cdx2 wild type (n=24), the S60 (n=3) or S156 mutant (n=5) developed AML after median latency of 120, 90 and 167 days post transplantation, respectively. In contrast S99, S108 and 3S mice developed AML after very long latency of 328 (5/5 mice), 341(3/5 mice) and 336 (3/5 mice) days post transplantation, respectively. Interestingly, 4S mice (n=5), did not develop any disease up to an observation time of 400 days, indicating that the transforming potential of Cdx2 was dependent on multiple N-terminal serines. As it was shown that the transcriptional activity of Cdx2 is dependent on the phosphorylation status of N-terminal serines in non-hematopoietic tissue we tested the phosphorylation status in hematopoietic cells: murine cells retrovirally transduced with Cdx2 and human AML cell lines positive for CDX2 expression showed a phosphorylated form of CDX2 and an activated Erk1/2 pathway, in contrast to AML cell lines negative for CDX2 expression. Based on this we tested whether inhibition of the MAPK pathway would impair the transforming potential of Cdx2. When Cdx2 transduced BM cells were incubated with MEK1/2 inhibitors (PD98059, U0126) for 14 days in liquid culture, viability of the cells was reduced by 78% (n=3, p

Description: Rationale Patients above 60 years of age account for up to one third of all patients with Hodgkin Lymphoma (HL). ABVD is considered standard of care for this patient cohort; however, both outcome and feasibility are poor, since tolerability of cytotoxic drugs is often markedly decreased. A major limitation is pulmonary toxicity due to bleomycin. We thus aimed at improving the ABVD regimen by replacing bleomycin with the immunomodulatory drug lenalidomide (Revlimid®, AVD-Rev), which has shown promising activity as single agent in HL. Methods We initiated the GHSG AVD-Rev dose finding trial (NCT01569204) for patients between 60 and 76 years of age, with first diagnosis of early unfavorable- or advanced-stage HL, good performance status (ECOG/WHO ≤2), and without evidence of severe organ dysfunction. Prophylactic anticoagulation (ASA or heparin) was mandatory. Depending on stage and response at interim staging, patients received four to eight cycles of AVD-Rev (standard-dose AVD on day 1 and 15 of a 28 days cycle and lenalidomide daily from day 1 to 21) followed by radiotherapy The daily lenalidomide dose for the first patient was 5 mg, and there were 8 possible dose levels ranging from 5 mg to 40 mg. Subsequently, all incoming information on dose limiting toxicities (DLT) during the first 4 cycles of therapy was used for dose level determination for the next patient using the EWOC (Escalation with Overdose Control) method. Critical adverse events including thromboembolism ≥CTC Grade II, hematological toxicity such as severe cytopenia (ANC〈 500/µl 〉7days with G-CSF support and thrombocytopenia below 25.000/µl ≥ 1 day), and resulting complications such as neutropenic fever and prolonged therapy delay were considered as dose limiting toxicities. Results 25 patients with a median age of 67 years (range 61-76) and with a CIRS-G comorbidity scoring of up to 7 points (n=2, range 0-7) were recruited and assigned to dose levels 5 mg (n=1), 10 mg (n=1), 15 mg (n=1), 20 mg (n=6), and 25 mg (n=16). Fifteen patients were male, 68% had advanced stage disease, and 80% had B-symptoms at diagnosis. After DLT evaluation of 20 patients, a pre-specified stopping criterion was reached and the recommended dose for a phase II trial was 25 mg. Dose delivery was high with a mean relative dose intensity of 91% (all dose levels, range: 63-104%, median: 97%), however at least one CTC Grade III-IV toxicity occurred in all 22 patients who were treated at dose levels 20 mg and 25 mg, and 16 of these patients had a CTC Grade IV toxicity. Dose limiting toxicities were observed in 2 of 6 (33%) and 8 of 16 (50%) patients at 20 mg and 25 mg, respectively, and were mainly hematologic but also included 3 thromboembolic events despite documented ASA prophylaxis. No DLT occurred in patients treated with

Description: The mechanisms underlying deregulation of HOX gene expression in AML are poorly understood. The ParaHox gene CDX2 was shown to act as positive upstream regulator of several HOX genes. In this study, constitutive expression of Cdx2 caused perturbation of leukemogenic Hox genes such as Hoxa10 and Hoxb8 in murine hematopoietic progenitors. Deletion of the N-terminal domain of Cdx2 abrogated its ability to perturb Hox gene expression and to cause acute myeloid leukemia (AML) in mice. In contrast inactivation of the putative Pbx interacting site of Cdx2 did not change the leukemogenic potential of the gene. In an analysis of 115 patients with AML, expression levels of CDX2 were closely correlated with deregulated HOX gene expression. Patients with normal karyotype showed a 14-fold higher expression of CDX2 and deregulated HOX gene expression compared with patients with chromosomal translocations such as t(8:21) or t(15;17). All patients with AML with normal karyotype tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression.

Description: Canonical Wnt signaling is critically involved in normal hematopoietic development and the self-renewal process of hematopoietic stem cells. Deregulation of this pathway has been linked to a large variety of cancers including leukemia. Lef-1, a key factor of the Wnt / beta-catenin signaling pathway, plays pivotal roles in lymphoid development, but little is known about the role of Lef-1 in myeloid hematopoiesis and leukemogenesis. We now show that Lef-1 is expressed in murine hematopoietic stem cells (‘LSK’, Sca+cKit+Lin−) and both myeloid and lymphoid subpopulations as well as in different human leukemias. Using a retroviral BM transplantation model, we demonstrate that ectopic expression of wild type Lef-1 (WT) and a constitutive active mutant of Lef-1 (CA) induces a severe disturbance of normal hematopoietic development: mice transplanted with bone marrow constitutively expressing Lef-1 had a significant increase in the number of circulating myeloid cells resulting in an inverted lymphoid-myeloid ratio in the peripheral blood (ratio: 0.28 (WT), 0.10 (CA) vs. 1.07 (GFP); p

Description: Background: Dose intensity is considered one of the prime determinants of antileukemic efficacy in induction treatment of acute myeloid leukemia (AML) - as demonstrated by the superior long-term results of double induction versus conventional induction. In an attempt to further pursue this historically successful strategy an ongoing phase II study of the AML-CG pilots the feasibility of the S-HAM regimen (HD-AraC 3g/m2/12h d1,2,8,9; Mitoxantrone 10mg/m2 d3,4,10,11) in first-line treatment of de-novo AML. In this regimen the interval between the two induction cycles is reduced from 17 days (double induction) to a minimum of 3 days (S-HAM) - thereby increasing dose-intensity more than 2-fold in the critical early phase of treatment. Results: In the past 18 months 99 patients have been recruited into the trial with a median age of 52 years (range 18 – 78). Of 93 patients evaluable for response the following results were achieved: CR 62%, CRp 24%, PL 6%, ED 8% - resulting in an overall response rate (ORR) of 86%. The early death rate (ED) of 8% and the toxicity profile compared favourably with a historical control group within the AML-CG 1999 study (subgroup: de-novo AML, age less than 60 years, HAM-HAM double induction) with an ED rate of 14% (ORR 68%, persistent leukemia (PL) 18%). The high antileukemic efficacy of S-HAM was also demonstrated by the fact that 88% of patients had a bone marrow blast count of 〈 10% one week after therapy as compared to less than 48% of patients of the HAM-HAM double induction group. If patients had an adequate blast clearance on day 18 pegylated G-CSF was applied every 10 days until neutrophil recovery. The median time to neutrophil recovery was 30 days after start of treatment with S-HAM which was substantially shorter than following either TAD-HAM or HAM-HAM double induction in the AML-CG 1999 trial (both with a median of 45 days). Since the S-HAM regimen has proven feasible at the present dose level a dose escalation was performed with an additional day of HD-AraC and Mitoxantrone in the first cycle of the sequential regimen. Conclusion: In the future the appropriate dose level of the S-HAM regimen will then constitute the experimental arm for a randomized comparison of a dose-intensified regimen S-HAM - combining a promising antileukemic activity with significantly reduced duration of critical neutropenia - versus standard double induction for patients younger than 60–70 years in the next generation of the AML-CG studies.

Description: Background: The prognosis of patients with acute myeloid leukemia (AML) has improved in recent years, partly due to the use of intensive double induction chemotherapy. Patients with adverse cytogenetic risk factors, especially those with a complex aberrant karyotype, however, still have a grave prognosis. Previous results from the German AMLCG study group have shown that younger patients with a complex karyotype may profit from double induction therapy containing one course of high dose AraC. It is unclear whether further intensification of induction chemotherapy may prolong survival. Methods: We investigated the outcomes of patients with unfavourable cytogenetics treated with either high-dose AraC and mitoxantrone (HAM) followed by thioguanine, conventional-dose AraC and daunorubicin (TAD) or with two courses of HAM in the prospectively randomized AMLCG-2000 trial. We included patients with unbalanced chromosomal aberrations (−5, −7, del(5q) or del(7q), or abnormalities involving 3q) and patients with a complex aberrant karyotype. Results: A total of 392 patients with unfavourable cytogenetics were analysed. Of those, 261 had de novo AML, 51 had secondary AML following a myelodysplastic syndrome (sAML) and 30 had therapy-related AML (tAML). The rate of complete remissions (CR) was significantly higher in patients with de novo AML compared with secondary or therapy-related AML (39 % vs. 20% vs. 23%, P=0.01). The overall survival (OS), however, was significantly shortened only in patients with tAML but not in those with sAML (median OS, 43 d (tAML) vs. 248 d (sAML) vs. 213 d (de novo AML), P=0.027). 233 patients had a complex aberrant karyotype. Although their CR rate was similar to patients with other unfavourable cytogenetics, OS was significantly worse (median, 169 vs. 327 days, P

Description: Introduction: The standard treatment for patients with relapsed or refractory (r/r) classical Hodgkin Lymphoma (cHL) is salvage chemotherapy followed by autologous stem cell transplantation (ASCT). The best outcome from the transplant is expected in patients who achieve complete remission (CR) after the salvage chemotherapy. Everolimus is a mammalian target of rapamycin (mTOR) inhibitor that has shown activity and an acceptable toxicity profile in patients with r/r cHL (Johnston PB et al. Am J Hematol 2010; Johnston PB et al. ISHL-9 2013). We therefore aim to improve the CR rate after salvage by an enforced salvage regimen consisting of everolimus plus time-intensified standard DHAP (Dexamethasone, High-Dose AraC [Cytarabine], Cisplatinum). Methods: We conducted a phase I trial of everolimus plus DHAP (ever-DHAP) for patients with r/r cHL eligible for ASCT to assess the recommended phase II dose (RPTD) of oral everolimus in this combination. Everolimus was administered for 14 days in each DHAP cycle starting from one day before DHAP. The second cycle of ever-DHAP was scheduled to start at day 14 of cycle one. Patients received two cycles of ever-DHAP. Everolimus dose levels of 2.5mg, 5mg, 7.5mg and 10mg were assessed in a modified 3+3 design. Generally, dose-limiting toxicity (DLT) was defined as CTCAE grade III/IV toxicity or unsuccessful stem cell mobilization. However, grade III/IV non-hematological toxicities known from DHAP were only counted as DLT from second occurrence in any patient onwards. Grade III/IV hematological toxicities were only counted as DLTs in case of prolonged time to recovery. Restaging by (PET)-CT was performed at day 21 of the second cycle given that the patient had recovered; PET was mandatory for all patients not achieving a CR in the CT scan. The rate of patients experiencing DLTs during 2 cycles of the combination therapy was the primary endpoint of the study. Secondary endpoints included adverse events, tumor related results of therapy or death, treatment administration, time to recovery after end of treatment and stem cell mobilization. Results: 14 patients were recruited between August 2012 and January 2014, all patients received at least one dose of everolimus. The median age was 33.5 years; six were female and eight were male. Eleven patients presented with stage III/IV disease. Overall, treatment was well tolerated. Two patients (both at the 7.5mg level) had a premature treatment termination and did not receive the 2nd cycle, one patient due to ototoxicity of grade I at investigator’s discretion and one patient due to neurotoxicity of grade III/IV. One additional patient (7.5mg cohort) suffered from ototoxicity of grade III/IV between end of treatment and restaging. Both grade III/IV toxicities were pre-defined as known from DHAP, thus they were not considered dose limiting at this first occurrence. No further non-hematological grade III/IV adverse events were reported. All patients but one experienced grade III/IV thrombocytopenia and leukopenia. No DLTs occurred and therefore 10mg of everolimus/day was chosen as RPTD. Duration of induction therapy including restaging after two cycles was 35-54 days. All patients who completed the 1st treatment cycle (n=13) had adequate stem cell mobilization (CD34+ cell count 2.9 - 31.1 x 106/kg; median 10 x 106/kg). So far one death occurred in a patient with pneumococcal sepsis one year after restaging. Responses according to CT scan in 12 patients who received two cycles of ever-DHAP were CR in 3 patients, CR unconfirmed (CRu) in 3 patients, partial remission (PR) in 5 patients and stable disease (SD) in 1 patient. This accounts for a CR/CRu rate of 50% (6/12) and an overall response rate of 92% (11/12). A PET scan was performed in 4 of 6 patients with CR/CRu; all had a negative PET (Deauville 1). In the 6 patients not achieving a CR/CRu 5 patients had a PET scan; 4 were PET positive (2 patients with Deauville 4 and 5 each) and 1 was PET negative (Deauville 1). Conclusions: Ever-DHAP with 10mg everolimus/day is safe and feasible in patients with r/r cHL. Based on the results of this phase I trial a randomized, placebo-controlled trial of ever-DHAP has been initiated and is currently recruiting. Disclosures von Tresckow: Takeda: Honoraria, Travel grants, Travel grants Other; Novartis: Honoraria, Research Funding. Off Label Use: Everolimus in relapsed or refractory Hodgkin Lymphoma. Topp:Affimed: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria. Engert:Millennium: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Affimed: Consultancy. Borchmann:Takeda: Honoraria, Research Funding, Travel grants Other.

Description: Interactions between leukemic cells and the bone marrow microenvironment are essential for the maintenance and progression of myeloid leukemias. G-protein coupled receptor 56 (Gpr56) is an adhesion molecule which collaborates with the extracellular matrix through interaction with collagen III binding and transglutaminase 2, and by this activating the Rho A response pathway. In the hematopoietic hierarchy, GPR56 expression is highest in stem cells and decreasing in expression with differentiation. It is a poor prognostic factor in cytogenetically normal acute myeloid leukemia (AML), and was identified as a member of a signature expressed in functionally validated human AML stem cells (Eppert et al., Nature Medicine 2011). To test its functional relevance for AML, we first analyzed the expression of GPR56 in primary patient samples (n= 74) with different genotypes from the AML dataset available in the TCGA database. Normal karyotype patients showed significantly higher expression of GPR56 (3.1 fold p

Description: The impact of a FLT3-internal tandem duplication (FLT3ITD) on prognosis of patients with acute myeloid leukemia (AML) is dependent on the ratio of mutated to wild-type allele. In 648 normal karyotype (NK) AML patients, we found a significant independent effect of the quantitative FLT3ITD mRNA level—measured as (FLT3ITD/wtFLT3)/(FLT3ITD/wtFLT3 + 1)—on outcome. Moreover, this effect was clearly seen in 329 patients with a mutated NPM1 gene (NPM1+), but not in 319 patients without a NPM1 mutation (wtNPM1). In a multivariate Cox regression model, the quantitative FLT3ITD mRNA level showed an independent prognostic impact on overall survival (OS) and relapse-free survival (RFS) only in the NPM1+ subgroup (OS: hazard ratio, 5.9; [95% confidence interval [CI]: 3.1-11.2]; RFS: hazard ratio, 7.5 [95% CI: 3.4-16.5]). The FLT3ITD mRNA level contributes to relapse risk stratification and might help to guide postremission therapy in NPM1-mutated AML.

Description: Efflux of Hoechst 33342 from normal hematopoietic cells identifies a “side population” (SP+) of negatively staining cells that, in the mouse, are largely CD34− and are enriched for primitive progenitors. To further characterize human SP+cells, blood or bone marrow from 16 patients with acute myeloid leukemia (AML) was analyzed for their presence, immunophenotype, and cytogenetic and functional properties, and for the relation between SP phenotype and multidrug resistance-1 (MDR-1) expression. The mean percentages of SP+ and MDR+ cells was 8.1% (range, 0.5%-29.9%) and 12.8% (range, 0%-54.8%), respectively, with no correlation between the 2 values. The percentages of SP+ cells that were CD34+CD38−, CD34+CD38+, or CD34− were 12% (range, 0.4%-50%), 25% (range, 0.5%-96%), and 63% (range, 4%-99%). Cytogenetically abnormal cells were always detected in the SP−CD34+CD38− and SP+CD34− fractions, and abnormal colonies (CFC), long-term culture-initiating cells (LTC-IC), and nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mouse leukemia–IC were detected in the former fraction. No progenitors were detected among SP+CD34− cells in any of these assays from 9 of 10 samples. In contrast, exclusively normal cells were detected in the SP+CD34+CD38−fraction from 9 of 15 samples, and CFC, LTC-IC, and multilineage engraftment in NOD/SCID mice from this subpopulation were also cytogenetically normal in 6 of 8, 6 of 7, and 2 of 2 cases studied, respectively. In contrast to murine studies, primitive progenitors are enriched among SP+CD34+CD38− cells from patients with AML. The molecular basis for Hoechst dye efflux is uncertain because it does not appear to be related to MDR-1 expression.