L-Asparaginase Ex Vivo Activity Distorts Plasmatic Asparagine Depletion Assessment

Abstract L-asparaginase (L-ASP) is a well-established and important agent having demonstrated significant clinical efficacy in the treatment of acute lymphoblastic leukemia (ALL) Many cancerous cells are deficient in asparagine synthetase (ASNS), requiring endogenous plasma asparagine (ASN) for proliferation. Without this essential amino acid, the malignant cells undergo apoptosis. Plasma levels of ASN have been considered a relevant surrogate biological parameter to assess L-ASP efficacy. The aim is to maintain ASN levels below a threshold varying from 0.1 to 3µM. However, the measurement of ASN in presence of L-ASP is problematic, considering that 1 International Unit (IU) of L-ASP is able to cleave 1µmol of ASN per minute at 37°C. As the normal value of plasma ASN is around 50µM in humans, it requires about 30 seconds for 100 IU/L of L-ASP in the blood stream to fully deplete L-ASN, also considering there is no de novo ASN production., Plasma ASN is provided by normal cells expressing ASNS and by food intake. Once blood is drawn, no endogenous source of ASN is present and in the presence of active L-ASP a rapid ASN ex vivodepletion is expected to occur in the sampling tube. L-ASP activity can be reduced by low temperature (iced water) and/or by lowering the pH through addition of sulfosalicylic acid (Pieters et al, Blood, 2008). It is recommended to cool down the sample for 15 minutes on iced water immediately after the patient’s blood is drawn, and to centrifuge at 1000g for 15 minutes at +4°C to recover the plasma. A solution of 10% sulfosalicylic acid is added to the plasma 1:4 (v/v) before centrifugation at 3000g for 5 minutes to extract the proteins, including L-ASP. Here we report our investigation on the impact of the duration between blood draw and immersion in “iced water” and the impact of the sulfosalicylic acid concentration when “free” E. coliL-ASP (L-ASP) or L-ASP encapsulated in erythrocytes (L-ASP/RBC) is present. L-ASP or L-ASP/RBC was added to human blood, in concentrations comparable to patients receiving L-ASP treatment (0-200 IU/L). The sampling process used in clinical practice was strictly applied to the sample, except that different times (5 sec to 30 min) at room temperature (RT) between the addition of L-ASP (considered as the moment of the blood draw) and the step of cooling were tested. Two quantities (100µL and 500µL) of sulfosalicylic acid were also tested. ASN was quantified in the samples after the completion of the whole process to assess whether variations in these parameters could impact the measurement. When 20 IU/L of L-ASP was present in the sample, ASN was depleted by 75% after a 5-sec delay at RT, and below the limit of quantification (BLLQ=2µM) after 10 min. When reducing sulfosalicylic acid to a suboptimal quantity (100µL), complete depletion (BLLQ) of ASN was obtained after 5 sec at RT with 20 IU/L of L-ASP. These results confirmed the necessity of the acidic deproteinization of the samples but also revealed a significant ex vivo activity of L-ASP leading to overestimation of ASN depletion in plasma. Considering L-ASP/RBC, 25% ASN depletion was observed at 200IU/L after 3 min. at RT, and ex vivocomplete depletion was reached with 2000IU/L and a 30-minute delay at RT. In conclusion, considering current methods, due to inability to control ex vivo L-ASP metabolism, it is practically impossible to have a measurement of plasmatic ASN which reflects the in vivo reality. Indeed, in the presence of low concentrations of free L-ASP, a very rapid depletion of ASN is observed ex vivo. In the time needed to cool down and centrifuge samples further “artificial” depletion occurs rapidly and the patient can be erroneously considered ASN depleted. However, the comparison of the phamacodynamics between “free” asparaginases may be still considered valuable because the error is probably equal in both sides. On the contrary, “artificial” depletion is much less when the L-ASP is encapsulated in RBCs (thanks to the delay of asparagine to pass through the RBC membrane), leading to a potential bias to compare L-ASP or L-ASP/RBC. Finally, L-ASP activity is believed to be a more relevant marker than the measurement of ASN depletion. This is consistent with the approach taken by regulatory agencies in recent years, favoring the duration of L-ASP activity over 100IU/L as endpoint over depletion endpoints. Disclosures Berlier: ERYTECH: Employment, Equity Ownership. Godfrin:ERYTECH Pharma: Employment, Equity Ownership.

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IDH Mutations Can Be Detected In 28.7% of All Normal Karyotype AML and Have Unfavourable Impact on the NPM1+/FLT3-ITD- Genotype

Blood ◽

10.1182/blood.v116.21.102.102 ◽

2010 ◽

Vol 116 (21) ◽

pp. 102-102

Author(s):

Susanne Schnittger ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

De Novo ◽

Hot Spot ◽

Normal Karyotype ◽

Prognostic Impact ◽

Employment Equity ◽

Therapeutic Decisions ◽

Idh Mutations ◽

Equity Ownership ◽

De Novo Aml ◽

The Impact

Abstract Abstract 102 Introduction: Mutations in IDH1 and IHD2 have recently been shown to play an important role in AML. As they code for enzymes from the citric acid cycle mutations within these genes from the mechanistical point of view are a totally new kind of mutation associated with AML. In IDH1 one mutational hot spot (amino acid R132) and in IDH2 two hotspots (R140 and R172) have been reported. We aimed at further delineating the impact of IDH1 and IDH2 mutations in AML and analyzed the interaction with other mutations in normal karyotype (NK) AML. Methods: 526 AML patients were selected according to normal karyotype and availability of mutational status for FLT3-ITD, NPM1 and MLL-PTD. Further mutation analyses were available in subgroups of the cohort (FLT3-TKD: n=318, CEBPA: n=369, RUNX1: n=174, NRAS: n=220). Female/male ratio was 283/243 and age ranged from 20.0–90.1 years (median, 66.9 years). 435 had de novo AML (82.6%), 71 AML following MDS (s-AML,13.5%) and 20 AML after previous treatment of other malignancies (t-AML, 3.8%). The respective base exchanges in R132, R140, and R172 were analysed by a melting curve assay with subsequent sequencing of the positive samples. Results: Overall, in 151 pts (28.7%) IDH mutations (IDHmut) were detected. In detail, 68 mutations (12.9% of all cases) were detected in IDH1 (R131C: n=35, R131L: n=17, R131H: n=7, R131G: n=6, R131S: n=3) and 83 mutations (15.8%) in IDH2 (R140Q: n=72, R140L: n=2, R140W: n=1, N141G: n=1, R174K: n=7). IDH1mut and IDH2mut were mutually exclusive in this cohort. IDH1mut were more frequent in females (18.2% vs 8.6 % in males, p=0.001), whereas there was no sex difference for IDH2. According to history IDH1 was equally distributed in de novo AML, s-AML and t-AML whereas IDH2 was more frequent in de novo compared to s- and t-AML (19.6% vs. 7.6 vs 11.8%, p=0.048). According to FAB the most prevalent subtype was FAB M1 with IDHmut in 23.2% compared to 9.8% in all other FAB (in detail: IDH1: 44.8% vs. 23.9%, IDH2: 27.0% vs. 15.1%; p<0.001, for both). IDH1 was underrepresented in M4 (4.9% vs. 15.0 % in all other subtypes, p=0.004), whereas the distribution of IHD2 was not different in M4 vs. all others. The immunophenotype (n= 297) of IDHmut cases tended to be more immature and featured a lower expression of monocytic markers. The analyzed 78 IDHmut cases, as compared to 219 IDHwt cases, showed a significantly higher expression of MPO and CD117 while CD116, CD11b, CD14, CD15, CD36. CD56, CD64, CD65 and CD7 were lower expressed. Age, WBC count, and platelet count were not different between IDH1, IDH2 and IDHwt cases. IDH mutations are not mutually exclusive of other mutations. However, the frequency of CEBPAmut in IDHmut compared to IDHwt was decreased (7.7% vs. 13.7, p=0.001) (IDH1: 0% vs 11.7%, p=0.022 and IDH2: 7.7% vs 13.4%, p=0.053). MLL-PTD was more frequent in IDHmut vs. IDHwt (44.7 vs. 5.8%, p=0.039), however, this is restricted to IDH1mut vs. IDH1wt (26.3 vs. 6.3%, p=0.018). RUNX1mut are distributed equally in IDH2mut and IDH2wt (20.0% vs 27.3%) but are underrepresented in IDH1mut compared to IDH1wt (2.2% vs. 28.7%, p=0.068). FLT3-ITDs are equally distributed between IDHmut and IDHwt, however, those IDH1mut with FLT3-ITD have lower FLT3-ITD/FLT3wt ratios compared to FLT3-ITD+ IDH1wt cases (mean: 0.16 vs. 0.72; p=0.005). All other mutations were distributed equally in IDHmut compared to IDHwt. For survival analysis only cases with de novo AML <65 years were included (n=164, IDHmut: n=37, n=, IDHwt: 127). In the total analysis there was no effect on overall survival or event free survival (EFS). However there was a trend for shorter EFS of the IDHmut vs. IDHwt (median: 439 days vs. not reached, p=0.080) in cases with NPM1+/FLT3-ITD- genotype. For IDH2 there was a significant adverse effect in the NPM1+/FLT3-ITD- group (median EFS: 397 vs. 679 days, p=0.045). Summary: IDH mutations belong to the most frequent mutations in NK AML and can occur together with all other known mutations. There is a high preponderance for the FAB M1 subtype and a more immature immunophenotype for both IDH mutations and a strong female preponderance for IDH1. In addition, an adverse prognostic impact of IDH mutations was shown for the NPM1+/FLT3-ITD- genotype. Further analyses should focus on the definition of the role and place of IDH mutations for therapeutic decisions in patients with AML. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Gene Amplifications Are Rare Events in AML and MDS and Are Associated with Complex Karyotype, TP53 Deletions and Very Poor Survival

Blood ◽

10.1182/blood.v118.21.2524.2524 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2524-2524

Author(s):

Claudia Haferlach ◽

Vera Grossmann ◽

Melanie Zenger ◽

Alexander Kohlmann ◽

Wolfgang Kern ◽

...

Keyword(s):

Gene Amplification ◽

De Novo ◽

Chromosome Instability ◽

Snp Array ◽

Complex Karyotype ◽

Employment Equity ◽

Double Minutes ◽

Hoxd Cluster ◽

Equity Ownership ◽

The Impact

Abstract Abstract 2524 Background: Gene amplifications are usually defined as the presence of more than 6 copies of a gene per cell. These supernumerary copies are located either extrachromosomally in double minutes (small acentric chromosome structures) or intrachromosomally in homogeneously staining regions. Such gene amplifications are rare but recurrent phenomenons in AML and MDS. So far, only small case studies have been reported. Aims: 1) to determine the frequency of gene amplifications in a large AML and MDS cohort, 2) to characterize the amplified regions and accompanying abnormalities, 3) to analyze the impact of specific amplifications on outcome. Patients and Methods: Out of 4,248 AML and 3,689 MDS studied by chromosome banding analysis (CBA) we identified 105 AML patients (2.5%) with gene amplifications (80/3,478 (2.3%) de novo AML, 7/478 (1.5%) s-AML, 18/292 (6.2%) t-AML) and 46 (1.2%) MDS. All cases with gene amplification were studied by 24-color FISH in addition to CBA in order to characterize the amplified regions and the accompanying abnormalities in detail. Further, interphase (IP)-FISH was performed with probes for TP73, HOXD cluster, EVI1, CMYC, JAK2, NUP214, MLL, ZNF4, GLTSCR1, ERG, RUNX1, BCR and CLRF, if 24-color FISH suggested amplification of these genes. In a subcohort of 12 patients genomic arrays (Human CGH Whole-Genome Array, NimbleGen, Madison, WI; Genome-Wide Human SNP Array 6.0, Affymetrix, Santa Clara, CA) were performed to characterize the amplified region in more detail. Results: In 28/151 pts (18.5%) the amplification was located in double minutes and in the remaining 123 cases intrachromosomally (81.5%). The following regions were found to be amplified: 1p (n=1, containing TP73), 2q (n=1, containing HOXD cluster), 3q (n=1, containing EVI1), 7p (n=1), 8q (n=29, containing CMYC in 28/29 pts), 9p (n=2, containing JAK2), 9q (n=1, containing NUP214), 11q (n=81, containing MLL in 80/81 cases), 13q (n=2), chromosome 19 (n=10, containing ZNF4 in 5 cases and GLTSCR1 in 2 cases), 21q (n=19, containing ERG in 16 and RUNX1 in 6 cases), 22q (n=2, containing BCR) and Xp (n=1, containing CLRF). In median, 8 accompanying chromosomal aberrations per cases were observed (range 0–21). 124/151 (82.1%) cases had a complex aberrant karyotype, defined as 4 or more abnormalities. However, in 2 cases the double minutes were the sole abnormalities. Gene amplifications were not observed in patients with disease defining aberration like t(8;21), inv(16), t(15;17) or those carrying NPM1 or CEPBA mutations (mutation status available in 89 and 37 patients, respectively). However, 2 cases with t(6;11)(q27;q23)/MLL-AF6 harbored an amplification of CMYC. In 88 cases the copy number status of TP53 was determined by IP-FISH. A TP53 deletion was detected in 49 (55.7%) pts. Interestingly, 14/16 (87.5%) cases with double minutes compared to 35/72 (48.6%) patients with intrachromosomal gene amplifications showed a TP53 deletion (p=0.004). Only 3 chromosomal regions were amplified in double minutes: 8q24/CMYC (n=14), 11q23/MLL (n=12) and 13q (n=2). In 6 cases with 8q amplification, 2 cases with 11q amplification and 4 cases with 21q amplification genomic arrays were performed. While the amplified region was quite homogeneous in cases with 8q amplification and contained in all cases the CMYC gene, amplified regions on 11q and 21q were heterogeneous and amplified regions were interspersed with regions of deletions. Interestingly, MLL and CBL were amplified in all analyzed cases with 11q23 amplification. In all analyzed cases with 21q22 amplification ERG was located within the amplified region while RUNX1 was amplified in 3/4 cases and deleted in the remaining case. In AML, overall survival was short in cases with gene amplification (median OS 11.3 months) and was particularly short in cases accompanied by complex karyotype (6.3 mo vs 18.6 mo in cases with non-complex karyotype, p=0.049). Conclusions: 1) MLL is the most frequently amplified gene in AML and MDS. 2) Gene amplifications occur predominantly in complex aberrant karyotypes. 3) Prognosis is poor in this subset of cases, and even more dismal if these amplifications are accompanied by complex karyotype. 4) The association of gene amplifications, complex karyotypes and TP53 deletions suggests that the unfavorable prognosis is due to chromosome instability facilitating the occurrence of additional genetic aberrations triggering resistance to chemotherapy. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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ASXL1 exon 12 Mutations Are Frequent in AML with Intermediate Risk Karyotype and Are Independently Associated with An Extremely Poor Outcome

Blood ◽

10.1182/blood.v118.21.416.416 ◽

2011 ◽

Vol 118 (21) ◽

pp. 416-416

Author(s):

Susanne Schnittger ◽

Christiane Eder ◽

Tamara Alpermann ◽

Annette Fasan ◽

Vera Grossmann ◽

...

Keyword(s):

De Novo ◽

Strong Association ◽

Multivariable Analysis ◽

Adverse Impact ◽

Prognostic Impact ◽

Intermediate Risk ◽

Employment Equity ◽

Mutational Status ◽

Equity Ownership ◽

The Impact

Abstract Abstract 416 Introduction: ASXL1 mutations have recently been described in a number of different myeloid malignancies. Data on frequency, association with other markers and outcome in AML are rare. Aim: The aim of this study was to evaluate ASXL1 mutations (ASXL1mut) in AML with intermediate risk karyotype for frequency, association with other mutations and impact on outcome. Methods: We analyzed 476 cases with intermediate risk de novo AML for ASXL1 mutations by direct Sanger sequencing of exon 12. Other mutations were analyzed as described previously and were available in part of the patients (NPM1: n=474, FLT3-ITD: n=473, FLT3-TKD: n=407, MLL-PTD: n=474, CEBPA: n=447, RUNX1: n=150, WT1: n=384, IDH1: n=464 and IDH2: n=444, TET2: n=109, NRAS: n=191; KRAS: n=110, DNMT3A: n=83). 397 cases had a normal karyotype (NK) and 79 had intermediate risk aberrant cytogenetics (according to MRC). Female/male ratio was 221/255 and age ranged from 18.5–100.4 y (median: 66.4). Results: Overall, in 70/476 patients (14.7%) ASXL1mut were detected. In detail, the most frequent mutation was p.G646WfsX12 (n=36) followed by p.E635RfsX15 (n=9), and p.Y591X (n=2). The remaining 21 mutations were non-recurrent consisting of 2 frameshift, 13 nonsense and 6 missense mutations. All mutations were detected with a mutation/wildtype load of 40–50% and none of the cases had more than one ASXL1mut. ASXL1mut were more frequent in males than in females (56/255, 22.0% vs 14/221, 6.3%, p=0.001) and were associated with higher median age (72.4 yrs vs 64.1 yrs, p<0.001). In detail, in the cohort > 65 yrs 21.7% (n=55/254) and in those <65 yrs only 6.8% (n=15/222) were ASXL1mut (p<0.001). With respect to morphology ASXL1mut were more frequent in AML without maturation than in all others (37.5% vs 14.3%, p=0.022). In 242 cases immunophenotyping data was available and cases with ASXL1mut (n=34) had a higher expression of CD13 (mean±SD, 55±23% vs. 43±25%, p=0.012), CD34 (46±32% vs. 24±26%, p<0.001), CD133 (29±27% vs. 16±23%, p=0.006) and HLA-DR (42±25% vs. 30±24%, p=0.009) as well as a lower expression of CD33 (66±21% vs. 77±21%, p=0.005) and thus had a more immature immunophenotype as compared to ASXL1wt. There was no association with leukocyte or platelet counts. With regard to cytogenetics ASXL1mut were more frequent in those with aberrant karyotype than in NK (20/79, 25.3% vs 50/397, 12.6%, p=0.008). Generally, ASXL1mut were observed together with all other molecular mutations but there was a strong correlation to RUNX1mut (n=18/43, 41.9% vs 19/107, 17.8% in RUNX1wt, p=0.003) and a negative correlation with NPM1mut (n=9/274; 3.3% vs. n=61/200, 30.5% in NPM1wt, p<0.001) and DNMT3Amut (1/26, 3.8% vs. 19/55 in DNMT3A, 34.5%, p=0.002). Patients with ASXL1mut had a shorter overall survival (OS) (median: 11.2 vs 38.8 months, p<0.001) and event free survival (EFS) (median: 9.0 vs 23.9 months, p<0.001). In detail, this adverse impact could be shown for both NK (OS: median: 10.9 vs 38.3 months, p<0.001; EFS: 9.8 vs. 26.5 months, p<0.001) and intermediate risk aberrant cytogenetics (OS: median: 8.6 vs 38.8 months, p<0.001; EFS: 5.3 vs 21.5 months, p=0.011), separately. Although the ASXL1mut were much more frequent in the elderly and compared to the ASXL1wt had a shorter OS (median: 7.0 vs 16.3 months, p=0.002) an adverse effect on survival could also be shown in the cohort <65 yrs (median OS: 11.6 vs 47.3 months, p<0.001 and median EFS: 9.3 vs 34.5 months, p<0.001). Because of the high coincidence of the two mutations the impact of ASXL1mut in dependence of RUNX1 status was analyzed. In the RUNX1mut (n=43) the ASXL1mut (n=18) still had an adverse impact on EFS (median: 5.3 vs 15.6 months, p=0.010) and a trend for shorter OS (10.7 vs. 20.5 months, p=0.079). In a multivariable analysis ASXL1 is an unfavourable factor for OS independent of age and RUNX1 mutational status (p=0.026, RR: 2.0). Conclusions:ASXL1 mutations belong to the most frequent mutations in intermediate risk AML. There is a strong association with male sex, high age, immature phenotype and RUNX1mut. Still, ASXL1mut retained its independent very poor prognostic impact. Although the number of known molecular markers in AML is continuously increasing and selection of the most import markers for diagnostic work-up seems challenging this data indicates that ASXL1 is one of the most prominent candidates. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Ex Vivo Activity of Immunotherapeutic Approaches Targeting CD38 Against Daratumumab-Resistant Multiple Myeloma Patient Samples

Blood ◽

10.1182/blood-2019-127893 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1848-1848

Author(s):

Maria Karvouni ◽

Heyue Zhou ◽

Arnika Kathleen Wagner ◽

Qiangzhong Ma ◽

Alamdar H. Baloch ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Therapeutic Potential ◽

Ex Vivo ◽

Phase 1 ◽

Employment Equity ◽

Multiple Myeloma Patient ◽

Myeloma Cells ◽

Car T ◽

Equity Ownership

Background: Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. The identification of CD38, a transmembrane glycoprotein overexpressed on MM cells, led to the development of target-specific therapeutics such as the FDA approved monoclonal antibody (mAb) Daratumumab (DARA). Although a valuable treatment option for refractory/relapsed (R/R) MM patients, DARA has a limited response rate of below 50%, which highlights the clinical need for novel therapeutics. Aims: Aiming to further exploit the therapeutic potential of CD38 in the MM setting, immunotherapies based on the novel anti-CD38 mAb CD38A2 were tested. Methods: For the first approach, the CD38A2 mAb -that binds to a unique, distinct from DARA's, CD38 epitope- was conjugated with either the alkylating agent Duomycin (ADC-136) or the microtubulin binder Duostatin (ADC-129). The ADCs were compared to DARA, in cultures of primary MM cells from patients refractory to DARA treatment. In a second approach, a chimeric antigen receptor (CAR) consisting of the CD38A2 scFv and the intracellular domains of CD28 and CD3ζ was used to transduce primary T and NK cells from R/R MM patients. The functionality of the CAR-T and CAR-NK cells was assessed in cytotoxicity assays against autologous myeloma cells. Results: ADC-136 demonstrated the most potent cytotoxicity against the MM cells with an IC50 of 6pM at day 6 following a single dose treatment. ADC-129 showed cell killing with an IC50 of 30pM, while DARA did not exhibit appreciable cytotoxicity. Regarding the cell therapy approach, patients' T and NK cells were effectively transduced, showing a CD38A2-CAR expression ranging between 11-68%. In functional assays, CAR-T and CAR-NK cells were assayed against autologous myeloma cells, where they exhibited an increase in target cell cytotoxicity, compared to the untransduced cells. Summary/Conclusion: Altogether, our preliminary findings demonstrate that CD38 targeting using CD38A2-based immunotherapies could be a viable therapeutic approach in R/R MM patients previously exposed to DARA. Currently, an anti-CD38 CAR-T therapy based on CD38A2 is being evaluated in Phase 1 studies in R/R MM patients by Sorrento Therapeutics, Inc. Disclosures Zhou: Sorrento Therapeutics Inc: Employment, Equity Ownership. Ma:Sorrento Therapeutics Inc: Employment, Equity Ownership. Zhu:Sorrento Therapeutics Inc: Employment, Equity Ownership. Zhang:Sorrento Therapeutics Inc: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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Impact of Amotosalen/UVA Pathogen Inactivated Platelet Components on Outcomes after Allogeneic Hematopoetic Stem Cell Transplantation: A Single Center Analysis

Blood ◽

10.1182/blood-2019-122330 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1167-1167

Author(s):

Andreas S. Buser ◽

Laura Infanti ◽

Andreas Holbro ◽

Joerg Halter ◽

Sabine Gerull ◽

...

Keyword(s):

Stem Cell ◽

Cox Regression ◽

Univariate Analysis ◽

Conditioning Regimen ◽

Disease Status ◽

Risk Scores ◽

Employment Equity ◽

Stem Cell Source ◽

Equity Ownership ◽

The Impact

Background: Platelet component (PC) transfusion is required for allogeneic hematopoietic stem cell transplantation (HCT) recipients. Contamination with infectious pathogens (bacteria, viruses, or protozoa) and T-cells is a risk factor for transfusion-transmitted infection (TTI) and transfusion associated graft-versus-host disease (TA-GVHD). Pathogen inactivation (PI) treatment of PC with amotosalen-UVA (PI-PC, INTERCEPT Blood System, Cerus Corp) in platelet additive solution (PAS) without bacterial screening, gamma irradiation, CMV serology, and with 7-day storage has been the standard of care in Switzerland since 2011 to manage risk of TTI and TA-GVHD. PI-PC have replaced conventional PC (C-PC) prepared in PAS with gamma irradiation and 5 day storage. We previously reported platelet usage in two consecutive five year periods at the University Hospital of Basel. Mean PI-PC dose was higher (3.0 vs. 2.8 x 1011, p=0.001) and mean storage duration longer (4.2 vs. 3.4 days: p=0.001) than with C-PC. PC expiration wastage was reduced with 7-day PI-PC storage vs. 5-day storage (1.5% vs. 8.7%). For HCT recipients, days of PC support; PC use per patient; and RBC use per patient were similar, despite 24.3% lower corrected count increments (CCI) with PI-PC. Now, we report the impact of these observations on treatment related mortality (TRM) and overall survival (OS) 100 days after HCT. Patients and Methods: A two-period retrospective cohort study was conducted to evaluate PI-PC impact on outcomes of consecutive first allogeneic HCT recipients from January 2006 to December 2010 (Period 1, P1), when gamma-irradiated apheresis C-PC were used, and Period 2 (P2) from January 2011 to December 2017, when apheresis and whole blood-derived PI-PC were used. The review utilized 100-day OS and 100-day TRM to determine the impact of PI-PC on HCT outcomes. Descriptive statistics were used for continuous variables and log-rank analysis for survival outcomes. Univariate analysis was performed using Pearson χ2 statistics. Multivariate Cox regression modelling analyses included: PC period (P1, P2), donor match (HLA identical/twin, matched related, matched unrelated), disease state (early, intermediate, late), and conditioning regimen (reduced intensity, myeloablative) with TRM as the outcome. This was an IRB approved single-center analysis. Results: In P1 and P2, 256 and 557 consecutive first-time allogeneic HCT recipients were included, respectively. By univariate analysis, the distribution of European Group for Bone Marrow Transplantation (EBMT) risk scores (grouped 0-2, 3-4, 5-7) and mean patient age were higher during P2 (p = 0.001 and p <0.001, respectively). Primary disease status (p = 0.039); stem cell source (p <0.001); GVHD prophylaxis with ATG (p <0.001); total body irradiation (p <0.001); and conditioning regimen (p <0.001) were different between P1 and P2. Donor match (p=0.084) and disease status (p = 0.628) were similar in P1 and P2. TRM at day 100 post HCT was significantly less (31/557, 5.5%) for PI-PC recipients in P2 vs. C-PC recipients in P1 (37/256, 14.5%, p<0.001). Overall proportion of survivors at day 100 post HCT was significantly greater for PI-PC recipients (507/557, 91.0 %) compared to C-PC recipients (209/256, 81.6%, p <0.001). By multivariate Cox regression analysis, P2 with PI-PC component support was associated with improved TRM (p = 0.001; adjusted hazard ratio 0.433; 95% confidence interval: 0.262, 0.716). Donor match (p = 0.019), disease state (p = 0.022), and myeloablative conditioning (p = 0.034) were associated with significantly poorer TRM (Table). Stem cell source was not significant (p=0.157) in the model. Hemorrhage was reported as cause of death in 1/50 (2.0%) patients during P2 with PI-PC and 4/47 (8.5%) patients during P1 with C-PCs. Conclusions: Universal implementation of PI-PC in routine with extended storage to 7 days in P2 was associated with reduced TRM and better overall survival 100 days post HCT, despite transplantation of older patients with higher EBMT risk scores. Multivariate analysis revealed an adjusted hazard ratio of 0.433 (95% C.I. 0.262, 0.716) for TRM by 100 days, suggesting better outcomes in P2. This retrospective analysis at a single site indicated that PI-PC treated with amotosalen /UVA stored up to 7 days did not have a negative impact on TRM and OS in HCT recipients, and was an integral part of improving clinical outcomes at our institution. . Table. Disclosures Heim: Novartis: Research Funding. Irsch:Cerus Corporation: Employment, Equity Ownership. Lin:Cerus Corporation: Employment, Equity Ownership. Benjamin:Cerus Corporation: Employment, Equity Ownership. Corash:Cerus Corporation: Employment, Equity Ownership.

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Astarabine, a Novel Leukemia-Targeted Cytarabine Composition Allows, for the First Time, the Delivery of High Cytarabine Doses for Older or Unfit Patients with Acute Leukemia. Results of an Ongoing Phase I/IIa Study

Blood ◽

10.1182/blood.v128.22.1650.1650 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1650-1650

Author(s):

Tsila Zuckerman ◽

Stela Gengrinovitch ◽

Ruth Ben-Yakar ◽

Ron Hoffman ◽

Israel Henig ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

De Novo ◽

Intensive Therapy ◽

High Dose ◽

Newly Diagnosed ◽

Employment Equity ◽

Unfit Patients ◽

Equity Ownership ◽

High Doses

Abstract Introduction: Therapy of acute myeloid leukemia (AML) has not changed significantly during several decades. High-dose cytarabine, although used as the first-line treatment for AML since 1970s and as a second-line treatment for acute lymphoblastic leukemia (ALL), is associated with severe side effects, such as cerebellar toxicity and bone marrow suppression. Hence, while the incidence of AML increases with age, doses of cytarabine are significantly attenuated or the drug is entirely excluded from the regimen used in older adults due to its potential toxicities, particularly in individuals with hepatic or renal dysfunction. Astarabine is a new composition of cytarabine covalently bound to asparagine. It is designed to target cytarabine to leukemic blasts, thus avoiding extramedullary toxicity. Leukemic cells, which are dependent on an external source of amino acids in general and asparagine in particular, due to their high metabolic rate, have a relatively increased uptake of Astarabine. Inside the blasts, Astarabine is cleaved to cytarabine, enabling targeted killing and relative sparing of normal hematopoiesis. As such, Astarabine may serve as an ideal therapy for leukemia, particularly for delivering high doses of cytarabine to medically unfit or older adults who otherwise can be given supportive therapy only. The aim of this study was to evaluate the safety and optimal dose of Astarabine in refractory/relapsed or medically unfit patients with acute leukemia. Methods: This Phase I/IIa prospective open label study enrolled patients aged ≥18 years with relapsed/refractory or newly-diagnosed acute leukemia unfit for intensive therapy, as judged by the treating physician. The study was approved by the Rambam IRB (approval #0384-11). Patients were enrolled into 6 Astarabine escalating-dose cohorts, each composed of 3-6 patients. Treatment was administered as a 1-hour single daily infusion for 6 days. For cohorts 1-4, Astarabine doses for each infusion were 0.5g/m2, 1.5g/m2, 3g/m2 and 4.5g/m2. The doses were reduced by 50% for patients >50 years. Since dose limiting toxicity (DLT) was not reached in cohorts 1-4, the study was extended to include cohorts 5 and 6 with daily Astarabine doses of 4.5g/m2 and 6g/m2, respectively, with no dose reduction for patients >50 years old. Results: The outcome of 15 patients is reported herein. Six patients with a median age of 64 years (range 27-81) had refractory/relapsed AML, 9 patients with a median age of 80 years (range 70-90) were newly diagnosed (secondary AML - 6, de-novo AML - 2, de-novo ALL - 1) and unfit for intensive therapy. Astarabine treatment was well-tolerated. Two patients died (one from pneumonia and one from sudden death 2 weeks from end of treatment) before completing 30 days post-treatment and hence were excluded from the outcome analysis. Response to the treatment was observed in the bone marrow of 6 of the 7 newly-diagnosed patients for whom bone marrow analysis was available, 3 of whom had a continuous complete remission (CR) for 4 (ongoing), 8, and 10 months post-treatment, and 3 had a continuous partial remission (PR) for 3,7, and 7 (ongoing) months. The median overall survival (OS) of the patients with CR/PR is 7 months to date (table 1). No significant response was observed in the relapsed/refractory patients, with a median OS of 2.5 months. Twelve patients died from disease progression. Conclusions: Astarabine, a new composition of leukemia-targeted cytarabine, is safe and very well tolerated, even in patients over 80 years of age, resulting in response in 6 of 7 newly diagnosed patients with acute leukemia. To the best of our knowledge, this is the first report permitting high-dose of cytarabine, considered a cornerstone of leukemia therapy, to be given to a population of patients that heretofore did not have this option. Further dose escalation studies are currently ongoing at a cytarabine-equivalent dose of 4.5 and 6 g/m2/day. A phase II study is planned to confirm these encouraging results and define the use of Astarabine for patients otherwise unable to receive high doses of cytarabine. Disclosures Zuckerman: BioSight Ltd: Consultancy, Research Funding. Gengrinovitch:BioSight Ltd: Employment, Equity Ownership, Patents & Royalties: Inventor all of the patents. Ben-Yakar:BioSight Ltd: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor of all patents.

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Sensitivity of Childhood Acute Lymphoblastic Leukemia (ALL) to Idarubicin Is Significantly Higher Than to Daunorubicin Treatment Based on Ex Vivo Apoptosis Induction.

Blood ◽

10.1182/blood.v104.11.4495.4495 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4495-4495

Author(s):

Aram Prokop ◽

Banu Bagci ◽

Guenaelle Lingfeld ◽

Lucia Badiali ◽

Karin Garbrecht ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Response Rate ◽

De Novo ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Cell Populations ◽

Apoptosis Induction ◽

Good Tolerability ◽

Childhood All

Abstract Anthracyclines, especially daunorubicin, play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and the relapsed ALL in childhood. In the present study, primary lymphoblasts isolated from 65 children with de novo ALL (median: 5.8 years; range: 1.9 – 16.9 years) and relapsed ALL (median: 12.7 years; range: 1.3 – 17.9 years) were treated with daunorubicin (10 mmol/l) or idarubicin (2 mmol/l) in vitro. We could show that both anthracylines induce apoptosis, as evidenced by measurement of genomic DNA fragmentation. Interestingly, daunorubicin only induced modest apoptosis, whereas idarubicin displayed a significantly stronger apoptosis inducing effect. Furthermore the treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in good response in most of the resistant cell populations. Out of the 65 patients analysed in this study 23 were female (13 de novo ALL, 10 relapsed ALL) and 42 were male (29 de novo ALL, 13 relapsed ALL). Primary lymphoblasts were obtained by bone marrow aspiration and separated by centrifugation over Ficoll. Within these cell populations following immunologic subgroups were found: 35 c-ALL, 10 pre-B-ALL, 7 pro-B-ALL, 10 T-ALL and 3 pre-T-ALL. Daunorubicin induced apoptosis in 33 out of 65 lymphoblast populations (response rate 50.8 %). Nevertheless, a far higher response rate was observed for idarubicin with 59/65 (90,8 %) (p < 0.008), if response is defined as apoptosis induction higher than 1 %. Daunorubicin-resistance was found in 32/65 (49,2 %), resistance to both was observed in 6/65 (9,2 %). Treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in significant apoptosis induction in 26 out of 32 cell populations (81,3 %). We clearly demonstrated here that the in vitro treatment of lymphoblasts from children with de novo or relapsed ALL with idarubicin induces significantly higher response rates than daunorubicin treatment. The ex vivo sensitivity of daunorubicin-resistant lymphoblasts of childhood ALL to idarubicin treatment reflects the better potency of idarubicin to induce apoptosis and to overcome daunorubicin resistance. These data prompted us to study the clinical relevance of idarubicin in ongoing clinical trials to improve existing therapeutic regiments. First clinical data point to a good tolerability of idarubicin in the treatment of relapsed ALL in childhood.

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TT30, a Novel Human Protein Therapeutic, Selectively Modulates the Complement Alternative Pathway by Targeted Supplementation of Local Factor H Activity.

Blood ◽

10.1182/blood.v114.22.3021.3021 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3021-3021 ◽

Cited By ~ 2

Author(s):

V. Michael Holers ◽

Istvan Mazsaroff ◽

Hillary Akana ◽

Christopher G. Smith ◽

J. Woodruff Emlen ◽

...

Keyword(s):

Cell Surface ◽

Potent Inhibitor ◽

Ex Vivo ◽

Alternative Pathway ◽

Factor H ◽

Employment Equity ◽

Complement Alternative Pathway ◽

Equity Ownership ◽

The Complement System

Abstract Abstract 3021 Poster Board II-997 The complement system is activated through three pathways: classical, lectin/mannose and alternative. Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases including atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration (AMD). The complement system is also centrally involved in many hemolytic disorders, including paroxysmal nocturnal hemoglobinuria (PNH) where the CAP initiates complement activation resulting in intravascular hemolysis (IVH) after engagement of C5 and formation of the membrane attack complex (MAC). Systemic neutralization of C5 with the anti-C5 monoclonal antibody, eculizumab, abrogates IVH when plasma concentrations are maintained above the minimal efficacious concentration (Cmin = 35 μg/mL). However, because eculizumab does not inhibit CAP activity prior to C5, C3 fragments (C3frag) continue to covalently bind to and accumulate on PNH red blood cells (RBCs). Clearance by the reticuloendothelial system of PNH RBCs that are C3frag-coated is a putative cause of extravascular hemolysis (EVH) in eculizumab-treated patients. In order to selectively modulate CAP activity, we developed TT30, a novel therapeutic 65kD fusion protein linking the first four short consensus repeat (SCR) domains of human complement receptor type 2 (CR2/CD21) with the first five SCR of human factor H (fH). CR2 SCR1-4 encompasses the antigen-fixed C3frag (iC3b, C3dg and C3d) binding domain. Factor H is the primary soluble phase, negative regulator of CAP activity functioning via the SCR1-5 domains. The unique mechanism of TT30 utilizes CR2 SCR1-4 to recognize and bind to C3frag on cells in which complement activation is occurring, thus delivering cell surface-targeted inhibition of CAP activity via fH SCR 1-5. TT30 both prevents CAP-dependent hemolysis of rabbit RBCs in human serum and blocks accumulation of C3frag on the RBC surface. By design, TT30 should also be a potent inhibitor of the CAP, but with minimal inhibition of the complement classical (CCP) and mannose (lectin; CMP) pathways. To test this hypothesis, we utilized sensitive pharmacodynamic assays that allow in vitro or ex vivo assessment in an ELISA format of individual complement pathway activity present in human serum. In this format, TT30 is a potent and selective inhibitor of CAP activity in normal human complement-preserved serum, with EC50 and EC100 values of ∼0.1 and 1 μg/mL serum. As predicted by the use of fH in its construction, TT30 is a much less potent inhibitor of the CCP and CMP, with EC100 values of ∼65 μg/mL. By contrast, in these assays a monoclonal and polyclonal anti-C5 antibody each demonstrate non-selective inhibition of CAP and CCP activity at all effective concentrations. TT30 activity is dependent upon CR2 binding to C3frag, as an anti-CR2 monoclonal antibody reverses the surface inhibition of CAP activity. This surface-targeting approach to delivering fH SCR1-5 results in a molecule with a 10-fold potency gain in CAP inhibition relative to added purified fH and an ∼30-fold potency gain relative to the total fH present in the serum used in the assay. TT30 administered as a single IV injection at 20 mg/kg to rats, rabbits and monkeys results in Cmax values of ∼400, 500 and 300 μg/mL and concentration-dependent inhibition of CAP activity. At serum concentrations of TT30 that induced maximal (100%) inhibition of systemic CAP activity for up to 12 hours, CCP activity is modestly (∼35-60%) inhibited for only 2 hours. CAP activity returns to baseline levels in a predictable fashion. Pharmacokinetic analysis indicates no gender-related differences and the expected scaling of parameters across species. TT30 is pharmacologically active in monkeys, rabbits and mice. TT30 administered as a single subcutaneous injection at 20 mg/kg to monkeys results in Cmax values of ∼25 μg/mL, and EC100 values identical to those observed with IV administration, but with a 3-fold prolongation of the maximal pharmacodynamic effect. The novel therapeutic TT30 has been shown in vitro and ex vivo to deliver cell surface-targeted control of CAP activation with minimal CCP and CMP inhibition and effective blockade of C3frag accumulation and MAC formation. As a result, TT30 has potential utility for the treatment of complement-mediated diseases such as PNH, AMD and aHUS, in which cell surface-targeted control of CAP activation may be clinically beneficial. Disclosures Holers: Taligen Therapeutics: Employment, Equity Ownership, Patents & Royalties, Research Funding. Mazsaroff:Taligen Therapeutics: Employment. Akana:Taligen Therapeutics: Employment. Smith:Taligen Therapeutics: Employment. Emlen:Taligen Therapeutics: Employment, Equity Ownership. Marians:Taligen Therapeutics: Employment. Horvath:Taligen Therapeutics: Employment.

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Use of Genome-Wide Expression Analysis to Optimize An Ex Vivo clinical Protocol for 16,16-Dimethyl Prostaglandin E2 Enhancement of Umbilical Cord Blood In Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v116.21.1451.1451 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1451-1451

Author(s):

Caroline Desponts ◽

David Robbins ◽

Thuy Le ◽

Annie Chi ◽

Scott Thies ◽

...

Keyword(s):

Ex Vivo ◽

Treatment Protocol ◽

Employment Equity ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Biologic Activity ◽

Genome Wide ◽

Cd34 Cells ◽

Equity Ownership ◽

Genome Wide Expression

Abstract Abstract 1451 A systematic investigation was performed to optimize the treatment protocol for ex vivo incubation of human hematopoietic stem cells (HSCs) with 16,16-dimethyl prostaglandin E2 (FT1050) prior to transplantation. This protocol is part of an ongoing Phase Ib clinical trial of FT1050-enhanced double cord blood (CB) transplantation after reduced intensity conditioning. FT1050 has been previously shown in vertebrate models to improve the engraftment potential of HSCs from bone marrow (BM) and CB after a brief ex vivo treatment. In these models, treatment of BM or CB with FT1050 was performed for 1 to 2 hours at 4 °C, followed by a wash and subsequent cell infusion into the recipient (North et al. Nature 2007, Hoggatt et al. Blood 2009). Several groups have demonstrated that under these conditions, FT1050-treated cells have an engraftment advantage over vehicle treated cells. The objective of the current investigation was to identify a set of conditions that maximizes the biologic activity of FT1050. Genome-wide expression analysis and cAMP assays were used to optimize the ex vivo FT1050 treatment protocol with respect to concentration, time and temperature. Using this approach, hundreds of up- and down-regulated genes were identified in FT1050-treated CD34+ cells. These signature genes include upregulation of CXCR4, a known mediator of HSC homing via SDF-1a, and CREB, a key gene involved in cAMP signaling. Results from these experiments demonstrated that FT1050 concentrations above 10 μM did not result in increased levels of biologic activity. In terms of duration of incubation, cAMP activity reached maximal levels within 30 minutes of exposure while a 2 hour treatment period was necessary to maximize the changes in gene expression. Finally, the biologic activity of FT1050 was highly sensitive to temperature, with treatment of cells at 37 °C yielding larger changes in cAMP production and gene expression as compared to incubation of cells at 25 °C and 4 °C. The biological effects of FT1050 on subsets of CD34+ cells isolated from CB were also determined. Interestingly, the stem/progenitor subsets of CD34+ cells (Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA- and Lin-CD34+) had a greater response to FT1050 relative to the lineage positive cells. The different conditions were also evaluated using CFU-C and 7-AAD assays. No evidence of adverse effects were observed. Based upon these findings, the ongoing clinical trial incorporates the optimized FT1050 ex vivo treatment protocol (10 μM for 120 minutes at 37 °C). Disclosures: Desponts: Fate Therapeutics, Inc.: Employment, Equity Ownership. Robbins:Fate Therapeutics, Inc.: Employment, Equity Ownership. Le:Fate Therapeutics, Inc.: Employment, Equity Ownership. Thies:Fate Therapeutics, Inc.: Employment, Equity Ownership. Mendlein:Fate Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Grayson:Fate Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Multani:Fate Therapeutics, Inc.: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

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Report of a Phase II Trial of Single-Agent BiTE® Antibody Blinatumomab in Patients with Minimal Residual Disease (MRD) Positive B-Precursor Acute Lymphoblastic Leukemia (ALL).

Blood ◽

10.1182/blood.v114.22.840.840 ◽

2009 ◽

Vol 114 (22) ◽

pp. 840-840 ◽

Cited By ~ 5

Author(s):

Max S Topp ◽

Gerhard Zugmaier ◽

Nicola Goekbuget ◽

Peter Kufer ◽

Mariele Goebeler ◽

...

Keyword(s):

T Cells ◽

Phase Ii ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Target Cells ◽

Molecular Response ◽

Intensive Chemotherapy ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 840 Introduction: In patients with B-precursor ALL, presence of MRD after induction therapy or at any time point later predicts a hematological relapse, despite continued intensive chemotherapy or/and an allogeneic hematological stem cell transplantation (HSCT). Blinatumomab (MT103) targets the CD19 antigen, and is a member of a novel class of bispecific BiTE® antibodies that redirect T cells for lysis of target cells. A phase II study was conducted in collaboration with the German Multicenter Study Group on Adult Lymphoblastic Leukemia (GMALL) in patients with MRD-positive B precursor ALL. Methods: B–precursor ALL patients in complete hematological remission with either persistent or reappeared MRD at any time after consolidation I of front-line therapy were included. One treatment cycle of blinatumomab is a 4-week continuous i.v. infusion, which can be followed by allogeneic HSCT or in case of response by repeated consolidation cycles of blinatumomab with 2-week treatment-free intervals. The dose level at enrollment is 15 μg/m2/day. In patients, who do not respond within four cycles of treatment, the dose can be increased to 30 μg/m2/day. Molecular response is assessed by quantitative PCR of either individual rearrangements of immunoglobulin/TCR-genes or specific genetic aberrations such as bcr/abl or MLL-AF4. Results: Nineteen patients have been treated to date and 16 patients are already evaluable for response. Thirteen of 16 evaluable patients went into molecular complete remission (CR) already after one cycle of blinatumomab. Three patients had a stable MRD level. Of note, 10 of the responding 13 patients had never achieved a molecular CR before blinatumomab treatment despite multiple treatment cycles including tyrosine kinase inhibitors in case of Ph-positive ALL. Two patients in molecular CR had an extra-medullary relapse one in testis and one in cerebro-spinal fluid, both representing immunological niches with limited accessibility for T cells. One patient with stable MRD level had a medullary relapse. All other patients are still relapse-free. None of the patients with molecular CR has shown a medullary relapse to date. The maximum follow-up of molecular CR has been 12 months. Most common adverse events (AEs) included lymphopenia, pyrexia, leucopenia and hypoimmunoglobulinemia. Only one patient had to be discontinued because of a fully reversible epileptical seizure. All other AEs resolved during treatment. Overall, treatment with blinatumomab was well tolerated. Response data of all 21 patients will be presented at ASH. Conclusions: Treatment with blinatumomab converted MRD-positive B–precursor ALL into molecular CR in 13 of 16 evaluable patients with refractory disease as indicated by persistent MRD after intensive chemotherapy. This amounts to a response rate of 81% providing thus the rationale for introducing blinatumomab as a novel agent in the treatment of B–precursor ALL. Disclosures: Zugmaier: Micromet: Employment, Equity Ownership. Goekbuget:Micromet: Consultancy, Research Funding. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Klinger:Micromet: Employment, Equity Ownership. Degenhard:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Nagorsen:Micromet: Employment, Equity Ownership. Riethmueller:Micromet: Consultancy, Equity Ownership. Bargou:Micromet: Consultancy, Equity Ownership, Patents & Royalties.

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