A Comprehensive Cytogenetic and Molecular Genetic Characterization of Patients with T-PLL Revealed Two Distinct Genetic Subgroups and JAK3 Mutations As an Important Prognostic Marker

Abstract Background: T-cell prolymphocytic leukemia (T-PLL) is a rare, mature T-cell neoplasm with poor prognosis. Only few T-PLL cases have been analyzed with regard to cytogenetic and molecular genetic aberrations so far. Therefore, we performed a comprehensive characterization of patients with T-PLL, including the identification of potential correlations between the respective markers and their impact on prognosis. Methods: The cohort comprised 47 T-PLL cases (32 male, 15 female). Median age was 69.8 years (range: 32.7-86.6 years). Diagnosis of T-PLL was assigned by immunophenotyping and cytomorphology. All 47 patients were further investigated using (i) chromosome banding analysis (CBA), (ii) interphase FISH to determine the copy number state for TP53 and ATM and chromosomal rearrangements of TCRA/D and TCL1 and (iii) array CGH. Next-generation amplicon deep-sequencing was performed to analyze mutations in ATM,BCOR, TP53 (n=47, respectively); JAK1 (n=44) and JAK3 (n=45) were analyzed by Sanger sequencing. Clinical follow-up data was available for 43 patients. Results: In all 47 cases, chromosomal abnormalities and/or molecular mutations were detected. Combining CBA and FISH data, an inv(14)(q11q32)/t(14;14)(q11;q32) was observed in 37/47 (78.7%) cases, a t(X;14)(q27;q11) in 3 cases (6.4%) and an i(8)(q10) in 17/47 (36.2%) cases. ATM deletions were detected in 27/47 (57.5%), TP53 deletions in 11/47 (23.4%) patients. Array CGH analyses revealed additional gains and losses of specific chromosomal regions, mainly affecting 7q (deletions in region 7q34-7q36; n=16), 12p (deletions in 12p12-12p13; n=11) and 22q (deletions in 22q11-q12 with a concomitant gain of 22q12-q13; n=8). Regarding molecular analyses, the most frequently mutated gene was ATM (34/47; 72.3%). Mutations in TP53 were found in 7/47 (14.9%) and in BCOR in 4/47 (8.5%) patients. Mutations of JAK1 were found in 3/44 (6.8%), and of JAK3 in 8/45 (17.8%) cases. ATM and TP53 frequently carried a mutation of one allele and a deletion of the other: 23/34 (67.6%) cases with ATM mutation also showed an ATM deletion and in 5/7 (71.4%) cases with TP53 mutation also a TP53 deletion was detected. Regarding chromosomal aberrations, all cases with i(8)(q10) harbored a TCRA/D rearrangement and an ATM mutation, whereas TP53 mutations were not present in any case with i(8)(q10). ATM mutations were found to be correlated to TCRA/D rearrangements (33/40 TCRA/D+ cases, 82.5%; 1/7 TCRA/D- cases, 14.3%; p<0.001). In contrast, TP53 mutations were predominantly observed in patients without TCRA/D rearrangement (4/7 TCRA/D- cases, 57.1%; 3/40 TCRA/D+ cases, 7.5%; p=0.008). Additionally, all three JAK1 mutations were detected in cases with a TCRA/D rearrangement. When splitting the cohort into patients ≤60 years (n=13) and >60 years (n=34), JAK1 mutations (0/12 vs. 3/32) and mutations/deletions in the TP53 gene were detected exclusively in patients >60 years (TP53mut: 0/13 vs. 7/34; TP53del: 0/13 vs. 11/34). JAK3 mutations were also found predominantly in older patients (1/12; 8.3% vs. 7/33; 21.2%). Median overall survival (OS) was 27.4 months. No influence on OS was found for mutations and/or deletions of ATM, TP53, BCOR orJAK1 or aberrations of chromosomes 8 or 14. The age of patients was found to impact OS (median OS, ≤60 years: 29.0 months vs. >60 years: 15.9 months), although this was not significant (p=0.077). However, OS was found to be significantly shorter in patients with JAK3 mutation compared to patients without JAK3 mutation (median OS, 5.1 months vs. 29.1 months; p=0.009). Conclusions: Genetic abnormalities were revealed in all 47 cases with T-PLL. Two distinct genetic subgroups of T-PLL were identified: A large subset, comprising 81% of patients, showed abnormalities involving the TCRA/D locus activating the proto-oncogenes TCL1 (14q32) or MTCP1 (Xq28). This subgroup had higher frequencies of i(8)(q10) and of ATM mutations, while the second group was characterized by a higher frequency of TP53 mutations (figure). Further, JAK3 mutations were identified as an important prognostic marker, showing a significant negative impact on OS. Figure 1: Genetic abnormalities in T-PLL Figure 1:. Genetic abnormalities in T-PLL mut=mutation, del=deletion, TCRA/D=rearrangements involving TCRA/D, TCL1=rearrangements involving TCL1, MTCP1= rearrangements involving MTCP1 Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger: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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Comparison of TP53 Alterations in Hematological Malignancies

Blood ◽

10.1182/blood.v126.23.4819.4819 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4819-4819

Author(s):

Anna Stengel ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

Susanne Schnittger ◽

Melanie Zenger ◽

...

Keyword(s):

Burkitt Lymphoma ◽

Tp53 Mutation ◽

Hematological Malignancies ◽

Complex Karyotype ◽

Mutation Load ◽

Employment Equity ◽

Copy Number State ◽

Tp53 Mutations ◽

Cytogenetic Aberrations ◽

Equity Ownership

Abstract Background: TP53 is altered in ~50% of human cancers. Alterations include mutations and deletions. Both frequently occur together, supporting the classical "two-hit" hypothesis for tumor-suppressor genes. Aim: Comparison of TP53 mutation/deletion patterns in different hematological malignancies, including AML, MDS, ALL, Burkitt lymphoma, CLL and T-PLL. We analyzed (i) the frequencies of TP53 mutations and deletions, (ii) the types of mutation, (iii) the mutation load, (iv) the correlations to cytogenetic aberrations, (v) the age dependency, and (vi) impact on survival. Patient cohort and methods: A total of 3383 cases (AML: n=858, MDS: n=943, ALL: n=358, Burkitt lymphoma: n=25, CLL: n=1148 and T-PLL: n=51) were analyzed for TP53 deletions by interphase FISH determining the copy number state and for TP53 mutations by next-generation amplicon deep sequencing. Karyotype data was available for all cases. Results: Overall, alterations in TP53 were detected in 361/3383 cases (11%; 186 cases with mutation only (mut only), 51 cases with deletion only (del only), 124 cases with mutation and deletion (mut+del)). Regarding the respective entities, the highest frequency of TP53 alterations was observed in patients with Burkitt lymphoma (total alteration frequency: 56%, mut+del: 12%, mut only: 44%, no case del only). Alterations in TP53 also occured with a high incidence in patients with T-PLL (total: 30%; mut+del: 10%; mut only: 4%; del only: 16%) followed by cases with ALL (total: 19%; mut+del: 6%; mut only: 8%; del only: 5%) and AML (total: 13%; mut+del: 5%; mut only: 7%; del only: 1%). By contrast, TP53 alterations occurred less frequently in patients with CLL (total: 8%; mut+del: 4%; mut only: 3%; del only: 1%) and MDS (total: 7%; mut+del: 1%; mut only: 5%; del only: 1%). Missense mutations were found to be the most abundant mutation type in all entities analyzed with a frequency ranging from 71% - 88%. In all entities mainly one mutation per case was detected; however, MDS cases were found to harbour a statistically increased proportion of cases with two mutations compared to the other entities (p = 0.003). High TP53 mutation loads were detected in T-PLL (median: 88%) and AML (47%), whereas the lower ones were found in ALL (28%), Burkitt lymphoma (39%), MDS (39%), and CLL (36%). A strong correlation of TP53 alterations with a complex karyotype was observed in AML (of patients with TP53 alteration: 5% with normal karyotype, 67% with complex karyotype, 28% with other aberrations), ALL (16% normal, 45% complex, 39% other), MDS (14% normal, 53% complex, 33% other), and T-PLL (20% normal, 47% complex, 33% other). By contrast, in CLL and Burkitt lymphoma, TP53 alterations were mainly correlated with other aberrations (CLL: 10% normal, 30% complex, 60% other; Burkitt: 29% normal, 0% complex, 71% other). TP53 mut and TP53 mut+del were significantly more frequent in patients ≥ 60 vs < 60 years in AML (9% vs. 2% for mut only, p < 0.001; 7% vs. 2% for mut+del, p = 0.001) and ALL (12% vs. 6% for mut only, p < 0.001; 13% vs. 3% for mut+del, p = 0.001). By contrast, no such differences were observed for patients with CLL, MDS, T-PLL and Burkitt lymphoma. Moreover, TP53 alterations (especially of TP53 mut+del) had a significant negative impact on OS in all entities except for T-PLL and Burkitt lymphoma, most probably due to their overall short OS or due the lower number of cases. Conclusion: The frequency of TP53 mutations and/or deletions as well as the mutation load clearly varied between different hematological malignancies with the highest incidence of TP53 mut in patients with Burkitt lymphoma (56%) and a rather low frequency in CLL (7%) and MDS (6%). TP53 del were frequent in patients with T-PLL (26%) and Burkitt lymphoma (12%) and are hardly found in MDS cases (2%). TP53 alterations are correlated to higher age in AML and ALL. Moreover, alterations in TP53 are correlated to a short OS and to a complex karyotype, with the exception of Burkitt lymphoma and CLL, were they were found to be associated to other cytogenetic aberrations. Thus, TP53 mutations and deletions need further investigation in the future, especially regarding their clinical impact in different hematologic entities. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comprehensive Investigation of 225 Patients with Myeloid Malignancies and Erythroid Hyperplasia (≥50%) Demonstrates That Acute Erythroid Leukemia (AEL, according WHO Classification 2008) Differs Significantly From MDS but Overlaps with Other AML Subtypes and Pure AEL Regarding Clinical and Genetic Features.

Blood ◽

10.1182/blood.v116.21.1672.1672 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1672-1672

Author(s):

Ulrike Bacher ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

...

Keyword(s):

Who Classification ◽

Molecular Genetic ◽

Erythroid Cells ◽

Employment Equity ◽

Not Otherwise Specified ◽

Erythroid Hyperplasia ◽

Genetic Abnormalities ◽

Genetic Features ◽

Equity Ownership ◽

Who 2008

Abstract Abstract 1672 Introduction: According to the WHO 2008, acute erythroid leukemia (AEL) is defined by erythroid hyperplasia ≥50% and ≥20% of myeloblasts of non-erythroid cells but <20% of all nucleated cells. “Pure AEL” is defined by ≥80% of erythropoiesis without relevant myeloblasts. Cases with erythroid hyperplasia ≥50% and ≥20% of myeloblasts of all nucleated cells are classified as acute myeloid leukemia (AML-MRC/NOS), while presence of <20% of myeloblasts of non-erythroid cells assigns cases to myelodysplastic syndrome (MDS). As the separation of MDS/AML with erythroid hyperplasia in different categories is still under debate, we studied 225 patients with MDS/AML and ≥50% erythroid cells in bone marrow (BM) for cytomorphology, cyto-/molecular genetics, and prognosis. Patients/Methods: The cohort consisted of 225 pts (f/78; m/147; median age, 68.8 yrs; 18.5–88.4 yrs) with BM erythroid hyperplasia (≥50%) and different myeloid subtypes strictly defined according to WHO 2008: MDS: n=107; AML-MRC/NOS: n=32; AEL: n=79, pure AEL: n=7 (the WHO cohort “AML with recurrent genetic abnormalities” was excluded). All pts were investigated by MGG staining of BM and chromosome banding/FISH. In addition, we performed analysis for the NPM1 mutations (n=126 investigated), FLT3-ITD (n=135), MLL-PTD (n=136), and NRAS mutations (n=90). Results: MDS subtypes were as follows: RA: n=18; RARS: n=18; RCMD: n=21; RCMD-RS (WHO 2001): n=26; RAEB-1: n=22; RAEB-2: n=2. Most AML pts were categorized as “AML with myelodysplasia related changes; AML-MRC” (27/30 cases; 90%); 3 pts were classified as “AML, not otherwise specified; AML-NOS”, 2 pts were not evaluable for this aspect. We first compared the MDS cohort (n=107) with the AML cohort (all 118 pts with AML-MRC/NOS, AEL, and pure AEL): Overall survival (OS) was better in MDS than in the AML cohort (median: not reached vs. 13.9 months; p<0.001). In contrast, OS showed no significant differences across the AML-NOS/MRC, AEL, and pure AEL subgroups (9.3 vs. 13.9 vs. 6.1 months; n.s.). In the total cohort, aberrant karyotypes (KTs) were detected in 105/225 pts (46.7%) and were associated with inferior median OS when compared to normal KTs (aberrant KTs: 12.5 months vs. normal KTs: not reached; p<0.001). Aberrant KTs were more frequent in the AML categories when compared to MDS (69/118; 58.5%; vs. 36/107; 33.6%; p<0.001), but showed no significant differences across the different AML subgroups: AML-MRC/NOS: 20/32; 62.5%; AEL: 44/79; 55.7%; pure AEL: 5/7; 71.4%; n.s.). Performing cytogenetic risk categorization according to revised MRC criteria (Grimwade, 2010) for the whole cohort, unfavorable KTs showed an inferior prognosis compared to intermediate KTs (unfav. KTs: 65/225; 28.9%; median OS: 7.6 months; vs. intermed. KTs: 160/225; 71.1%; not reached; p<0.001). The pts from the AML cohort more frequently had unfav. KTs than those with MDS (AML cohort: 50/118; 42.4% vs. MDS: 15/107; 14.0%; p<0.001). Unfav. KTs were similarly distributed in the AML cohort (AML-MRC/NOS: 14/32; 43.8%; AEL: 32/79; 40.5%; pure AEL: 4/7; 57.1). Regarding the molecular markers, we detected the NPM1mut in 25/126 investigated (19.8%; MDS: 0/43; AML cohort: 22/91; 24.2%), FLT3-ITD in 5/135 (3.7%; MDS: 0/43; AML cohort: 5/92; 5.4%), MLL-PTD in 12/136 (8.8%; MDS: 2/44; 4.5%; AML: 10/92; 10.9%), and NRASmut in 4/90 (4.4%; MDS: 1/42; 2.4%; AML: 3/48; 6.2%). Mutation frequencies did not differ significantly in the MDS vs. AML categories or across the AML-MRC/NOS, AEL, and pure AEL subgroups. Conclusions: MDS with erythroid hyperplasia (≥50%) was clearly separated from the AML cohort (consisting of AML-MRC/NOS, AEL, and pure AEL, all with ≥50% of erythropoiesis) by less adverse cytogenetics and by improved survival. In contrast, no significant differences were observed across the different acute leukemia subentities regarding prognosis and cyto-/molecular genetic features. These data support the separation of MDS and AML with ≥50% of erythroid precursors according to the WHO classification. However, with respect to different AML subgroups, the separation to AEL, pure AEL, and AML-MRC/-NOS having ≥50% erythropoiesis seems arbitrary: these AML subtypes show no significant differences regarding prognosis or genetic risk profiles. This argues in favor of a combined group of AML with erythroid hyperplasia aiming to facilitate the definition for clinical studies and the development of therapeutic strategies. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comprehensive Cytogenetic and Molecular Genetic Characterization of T-PLL Identifies for the First Time BCOR Mutations in a Lymphatic Disease.

Blood ◽

10.1182/blood.v120.21.2539.2539 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2539-2539

Author(s):

Vera Grossmann ◽

Alexander Kohlmann ◽

María Abáigar ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

...

Keyword(s):

Mutation Frequency ◽

Molecular Genetic ◽

Normal Karyotype ◽

Wild Type ◽

Chromosome 14 ◽

Genetic Aberrations ◽

Tp53 Mutations ◽

Equity Ownership ◽

First Time

Abstract Abstract 2539 Introduction: T-cell prolymphocytic leukemia (T-PLL) is a rare mature post-thymic T-cell neoplasm with aggressive clinical course and a median overall survival of less than one year. Due to the rareness of this disease (∼2% of cases of mature lymphocytic leukemias in adults) only few cases with cytogenetic and molecular genetic aberrations have been reported so far. However, almost 75% of T-PLL cases are reported to harbor chromosome 14 abnormalities involving the TCRA/D locus resulting in the aberrant activation of the proto-oncogenes TCL1A or MTCP1. Aim: Perform a comprehensive cytogenetic and molecular characterization of T-PLL. Patient and Methods: The cohort comprised 36 T-PLL cases diagnosed between 10/2005 and 07/2012 (23 male, 13 female patients). Median age was 71.0 yrs (range: 26.8–82.8 yrs). According to the WHO classification all T-PLL cases were characterized using immunophenotyping and cytomorphology. Patients were further investigated using chromosome banding analysis (CBA) (n=30), FISH for deletions of ATM (n=30), TP53 (n=29), and 13q (n=26), and CGH arrays (n=3, Human CGH 6×630K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation analyses for BCOR and TP53 were performed using amplicon sequencing (Roche 454, Branford, CT). Results: Aberrant karyotypes were observed by CBA in 25/30 cases (83.3%). However, the 5 remaining cases with normal karyotype were due to insufficient proliferation of the T-PLL clone as gains, losses and rearrangements were detected in these 5 cases using FISH analyses. In more detail, combined CBA and FISH data revealed in 20/30 (66.7%) cases an inv(14)(q11q32)/t(14;14)(q11;q32)/TCRA/D-TCL1A (n=18) or t(X;14)(q27;q11)/TCRA/D-MTCP1 (n=2). Further, a gain of chromosome 8q and concomitant loss of 8p was observed in 13/30 (43.3%) cases. In addition, in 10/25 (40.0%) cases a 6q deletion and in 7/25 (28.0%) an 12p deletion were observed. Based on FISH data, deletions were detected of ATM in 19/30 (63.3%) cases, TP53 in 7/29 (24.1%), and 13q in 9/26 (34.6%) cases. In addition, 3 cases were studied using array CGH. Hereby, an intragenic deletion in the BCOR gene was observed in one patient. BCOR is a BCL6 corepressor and located on chromosome Xp11.4. BCOR mutations were recently described in cases with AML. BCOR mutation frequency was determined at 3.8% in AML with normal karyotype and mutations were associated with shorter overall and event-free survival. The deletion in BCOR identified in one of our cases and the TP53 deletions in 7 T-PLL cases prompted us to screen 35 cases for molecular mutations in these two genes. Overall, BCOR mutations were detected in 5/35 (14.3%) patients and TP53 mutations in 4/35 (11.4%) cases. In total, 7 missense, one frame-shift and one nonsense mutations were found. Median mutation load was 90.0% for BCOR (range: 38–100%) and 80.0% (59–87%) for TP53. Next, we performed correlation analyses between mutations in BCOR and TP53, rearrangements involving chromosome 14, deletions of ATM, TP53, 6q, 12p, and 13q and gain of 8q. Here, BCOR was not associated with any of these parameters. In contrast, TP53 mutations were accompanied in all 4 cases by TP53 deletions, while only 3/24 TP53 wild-type cases harbored a TP53 deletion (P=0.002). In addition, only one of 4 TP53 mutated cases harbored a chromosome 14 rearrangement while 18/25 (72%) TP53 wild-type cases did (P=0.105). In line with this result, TP53 deletions were also negatively associated with chromosome 14 rearrangements (2/7 vs 17/22, P=0.030). Further, all cases with a gain of 8q harbored a 14q rearrangement (13/13 vs 8/18 without gain of 8q, P=0.001). Conclusions: 1. CBA, FISH and mutation analysis of TP53 and BCOR revealed genetic abnormalities in all 36 analyzed cases. 2. The most frequent abnormality involved the TCRA/D locus (14q11) (20/30; 66.7%) activating the proto-oncogenes TCL1A on chromosome 14q32 (90.0%) or MTCP1 on chromosome Xq28 (10.0%). 3. Deletions were detected for ATM (63.3%), TP53 (24.1%), 6q (40.0%), 13q (34.6%), 12p (28.0%), and a gain was detected for the long arm of chromosome 8 (43.3%). 3. In addition to the detection of TP53 mutations in 11.4%, BCOR mutations were observed for the first time in a lymphatic malignancy with a mutation frequency of 14.3%. 4. The prognostic relevance of such cytogenetic and molecular genetic aberrations has to be determined in T-PLL, given that in myeloid malignancies both BCOR and TP53 are associated with shorter OS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-129025 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3921-3921 ◽

Cited By ~ 1

Author(s):

Cesar Sommer ◽

Hsin-Yuan Cheng ◽

Yik Andy Yeung ◽

Duy Nguyen ◽

Janette Sutton ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Target Cells ◽

Employment Equity ◽

T Cell Therapy ◽

Cell Therapies ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

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Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Blood ◽

10.1182/blood-2019-122787 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5603-5603 ◽

Cited By ~ 1

Author(s):

Cherie Tracy Ng ◽

Jeanette Ampudia ◽

Robert J. Soiffer ◽

Jerome Ritz ◽

Stephen Connelly

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Gvhd ◽

Vehicle Control ◽

Control Group ◽

Employment Equity ◽

Equity Ownership

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

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Effective Targeting of PRAME-Positive Tumors with Bispecific T Cell-Engaging Receptor (TCER®) Molecules

Blood ◽

10.1182/blood-2019-129958 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3368-3368

Author(s):

Sebastian Bunk ◽

Martin Hofmann ◽

Felix Unverdorben ◽

Meike Hutt ◽

Gabriele Pszolla ◽

...

Keyword(s):

T Cell ◽

Tumor Cells ◽

Half Life ◽

Normal Tissue ◽

Malignant Diseases ◽

Proof Of Concept ◽

Employment Equity ◽

Target Peptide ◽

Equity Ownership ◽

Nog Mice

T cell receptors (TCRs) naturally recognize human leukocyte antigen (HLA)-bound peptides derived from foreign and endogenous proteins regardless of their extracellular or intracellular location. Preferentially expressed antigen in melanoma (PRAME) has been shown to be expressed at high levels in a variety of cancer cells while being absent or present only at very low levels in normal adult tissues except testis. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia and myeloma cells. Immunotherapy with bispecific T cell engagers has emerged as a novel and promising treatment modality for malignant diseases, however, antibody-based approaches (ie. blinatumomab) are restricted to few surface antigens such as CD19 or BCMA. Immatics has developed bispecific T cell-engaging receptors (TCER®) that are fusion proteins consisting of an affinity-maturated TCR and a humanized T cell-recruiting antibody with an effector function-silenced IgG1 Fc part. TCER® molecules confer extended half-life together with antibody-like stability and manufacturability characteristics. The molecular design allows for effective redirection of T cells towards target peptide-HLA selectively expressed in tumor tissues. Here we present proof-of-concept data from a TCER® program targeting a PRAME-derived peptide bound to HLA-A*02:01. We confirmed the abundant presence of the target peptide-HLA in several cancer indications and its absence in relevant human normal tissues by using the XPRESIDENT® target discovery engine, which combines quantitative mass spectrometry, transcriptomics and bioinformatics. Yeast surface display technology was used to maturate the stability and affinity of a parental human TCR recognizing PRAME with high functional avidity and specificity. During maturation we applied XPRESIDENT®-guided off-target toxicity screening, incorporating the world's largest normal tissue immunopeptidome database, to deselect cross-reactive candidate TCRs. The maturated TCRs were engineered into the TCER® scaffold and production in Chinese hamster ovary (CHO) cells generated highly stable molecules with low tendency for aggregation as confirmed during stress studies. Following TCR maturation, the TCER® molecules exhibited an up to 10,000-fold increased binding affinity towards PRAME when compared to the parental TCR. The high affinity correlated with potent in vitro anti-tumor activity requiring only low picomolar concentrations of TCER® molecules to induce half-maximal lysis of tumor cells expressing the target at physiological levels. Furthermore, using a tumor xenograft model in immunodeficient NOG mice, we could demonstrate significant growth inhibition of established tumors upon intravenous injection of TCER® molecules. Pharmacokinetic profiling in NOG mice determined a terminal half-life of more than 4 days, compatible with a once weekly dosing regimen in patients. For the safety assessment, we measured killing of more than 20 different human normal tissue cell types derived from high risk organs. Notably, we could confirm a favorable safety window for selected TCER® molecules, which induced killing of most normal tissue cells only at significantly higher concentrations than required for killing of tumor cells. To further support safety of TCER® molecules, we also performed a comprehensive characterization of potential off-target peptides selected from the XPRESIDENT® normal tissue database based on its high similarity to the sequence of the target peptide or based on data from alternative screening approaches. In summary, the efficacy, safety and manufacturability data to be presented provide preclinical proof-of-concept for a novel bispecific T cell-engaging receptor (TCER®) molecule targeting PRAME for treatment of various malignant diseases. Disclosures Bunk: Immatics: Employment. Hofmann:Immatics: Employment. Unverdorben:Immatics: Employment. Hutt:Immatics: Employment. Pszolla:Immatics: Employment. Schwöbel:Immatics: Employment. Wagner:Immatics: Employment. Yousef:Immatics: Employment. Schuster:Immatics: Employment. Missel:Immatics: Employment. Schoor:Immatics: Employment. Weinschenk:Immatics: Employment, Equity Ownership. Singh-Jasuja:Immatics: Employment, Equity Ownership. Maurer:Immatics: Employment. Reinhardt:Immatics: Employment, Equity Ownership.

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Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽

10.1182/blood.v114.22.2744.2744 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2744-2744

Author(s):

Xiaochuan Chen ◽

Rhona Stein ◽

Chien-Hsing Chang ◽

David M. Goldenberg

Keyword(s):

T Cell ◽

B Cells ◽

Cell Lines ◽

Hairy Cell ◽

Employment Equity ◽

Cross Presentation ◽

Equity Ownership ◽

Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Occurrence of T-Lymphocytes with Aberrant Immunophenotypes Suggestive of Mature T-Cell Neoplasms In Patients with Hairy Cell Leukemia

Blood ◽

10.1182/blood.v116.21.4165.4165 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4165-4165

Author(s):

Wolfgang Kern ◽

Susanne Schnittger ◽

Claudia Haferlach ◽

Torsten Haferlach

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Hairy Cell Leukemia ◽

Initial Diagnosis ◽

Hairy Cell ◽

Cell Leukemia ◽

Employment Equity ◽

Cell Counts ◽

Equity Ownership

Abstract Abstract 4165 Mature T-Cell Neoplasms (MTN) comprise a heterogeneous group of diseases with largely varying clinical courses ranging from indolent cases which are asymptomatic for years to aggressive cases requiring immediate therapy. The incidence of MTN is increasing with age, however, particularly due to the large group of cases with very indolent clinical course MTN are considered underdiagnosed to a significant portion. Published data indicates that T-lymphocytes with aberrant immunophenotype, i.e. double-positive cell expressing both CD4 and CD8, are present in healthy subjects, however, their frequency in general amounts to clearly less than one percent of total leukocytes. With increasing age, these T-lymphocytes with aberrant immunophenotype are detected in more subjects and at higher frequencies suggesting a higher incidence of mature T-cell neoplasms or at least of pre-malignant conditions in these cases. The diagnosis of hairy cell leukemia has not yet been linked to a higher frequency of these T-lymphocytes with aberrant immunophenotype or of MTN. Following the identification of various cases with both a diagnosis of hairy cell leukemia and the presence of T-lymphocytes with aberrant immunophenotype in our laboratory we hypothesized that both of these conditions co-occur at a higher rate than would be expected by chance. We therefore retrospectively evaluated multiparameter immunophenotyping results of 338 patients diagnosed with hairy cell leukemia (newly diagnosed or during follow-up) between August 2005 and July 2010 for the presence of an increased percentage (more than 1%) of T-lymphocytes with an aberrant immunophenotype. We identified 31 such patients, i.e. 9.2% of all patients with hairy cell leukemia. 17 were identified at initial diagnosis and 14 during follow-up after therapy for hairy cell leukemia. The patients` ages ranged from 43.4 to 89.3 years (median, 62.1 years), 21 were male. The aberrant immunophenotype comprised the coexpression of CD3, CD4, and CD8 in all cases and in addition of CD56 in 25/31 (80.6%) cases. The median values and ranges for blood cell counts amounted to: WBC, 2.6 ×10e9/l, 1.0–24.6 ×10e9/l; hemoglobin, 12.7 g/dl, 7.3–16.5 g/dl; thrombocytes, 116 ×10e9/l, 24–258 ×10e9/l. The percentage of T-lymphocytes with an aberrant immunophenotype (compared to all leukocytes) ranged from 1% to 22% (median, 4%); the respective concentrations ranged from 0.013 ×10e9/l to 0.984 ×10e9/l (median, 0.122 ×10e9/l). The concentrations of T-lymphocytes with an aberrant immunophenotype tended to be higher in cases at follow-up as compared to those at initial diagnosis, although this difference was not significant (mean±SD, 0.266±0.275 ×10e9/l vs. 0.144±0.119 ×10e9/l). In four of the 31 patients with T-lymphocytes with an aberrant immunophenotype molecular genetic analysis of T-cell receptor (TCR) rearrangement was performed. In 3/4 patients both TCR beta and gamma were found rearranged and in one of these also TCR delta was rearranged, however, in 1/4 patient no TCR rearrangement was present. This data indicates that T-lymphocytes with aberrant immunophenotypes are present in patients with hairy cell leukemia much more often and at higher concentrations than in the general population. This data therefore suggests that the incidence of mature T-cell neoplasms may be higher in hairy cell leukemia patients. Clinical symptoms and findings like cytopenia and splenomegaly therefore may not be attributable to hairy cell leukemia alone which may have significant therapeutic implications. It is suggested to monitor for T-lymphocytes with aberrant immunophenotypes in patients with hairy cell leukemia and to perform analysis for TCR rearrangements in positive cases. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger: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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Impact of Expression of BAALC, CDKN1B, ERG, EVI1, and MN1 on Prognosis and Their Association with Karyotype, FLT3-ITD, NPM1 and MLL-PTD Status In Adult AML: A Comprehensive Study on 286 Cases

Blood ◽

10.1182/blood.v116.21.953.953 ◽

2010 ◽

Vol 116 (21) ◽

pp. 953-953

Author(s):

Claudia Haferlach ◽

Alexander Kohlmann ◽

Sonja Schindela ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

...

Keyword(s):

Molecular Genetic ◽

Normal Karyotype ◽

Genetic Alterations ◽

Complex Karyotype ◽

Employment Equity ◽

Aberrant Expression ◽

Cox Regression Analysis ◽

Total Cohort ◽

Equity Ownership ◽

Low Expression

Abstract Abstract 953 Introduction: The WHO classification in 2008 listed for the first time aberrant expression of genes as molecular genetic alterations affecting outcome in AML. High expression of BAALC, ERG and MN1 were shown thus far to be associated with unfavorable outcome in normal karyotype AML (AML-NK). In addition high EVI1 expression was suggested to predict poor outcome. Recently, our group identified low expression of CDKN1B as a favorable prognostic marker. The aim of this study was to evaluate the expression of BAALC, CDKN1B, ERG, EVI1 and MN1 in AML comprising all cytogenetic risk groups with respect to their association with distinct cytogenetic and known molecular genetic subgroups and their impact on prognosis. Patients/Methods:: Expression levels of BAALC, CDKN1B, ERG, EVI1 and MN1 were determined by oligonucleotide microarrays (HG-U133 Plus 2.0, Affymetrix) in 286 AML (t(15;17) n=15; t(8;21) n=16; inv(16) n=7; normal karyotype n=99; 11q23/MLL-rearrangements n=10; complex karyotype n=51; other abnormalities n=88). Patients were further analyzed for mutations in NPM1, FLT3-ITD, CEPBA and MLL-PTD. Results: Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varied significantly between genetic subgroups: While t(15;17), t(8;21) and 11q23/MLL-rearrangements were associated with low CDKN1B expression, AML-NK and NPM+ cases showed a higher CDKN1B expression. Lower BAALC expression was observed in AML with t(15;17), 11q23/MLL-rearrangement and AML-NK as well as in FLT3-ITD+ AML and in NPM1+ AML, while in AML with other abnormalities a higher BAALC expression was observed. ERG expression was lower in AML with 11q23/MLL-rearrangement and normal karyotype, while it was higher in AML with complex karyotype. Low EVI1 expression was observed in AML with t(15;17), t(8;21), inv(16) and AML-NK, while it was higher in AML with 11q23/MLL-rearrangements. Low MN1 expression was associated with t(15;17), t(8;21) and AML-NK, while it was increased in cases with inv(16) or other abnormalities. Next, Cox regression analysis was performed with respect to overall survival (OS) and event free survival (EFS). In the total cohort high BAALC and ERG expression as continuous variables were associated with shorter OS and EFS while CDKN1B, EVI1 and MN1 had no impact. Furthermore the cohort was subdivided into quartiles of expression for each gene. After inspection of the survival curves the cut-off for high vs low expression was set as follows: BAALC: 75th percentile, CDKN1B: 25th percentile, ERG and MN1: 50th percentile. For EVI1 expression pts were separated into expressers (n=44) and non-expressers (n=242). Low CDKN1B expression was associated with longer OS and EFS in the total cohort (p=0.005, not reached (n.r.) vs 14.9 months (mo); p=0.013, 31 vs 9.7 mo). High BAALC expression had no impact on OS, but was associated with shorter EFS in the total cohort as well as in AML with intermediate cytogenetics and AML with other abnormalities (p=0.032, 6.2 vs 13.0 mo; p=0.027, 5.1 vs 11.3 mo; p=0.006, 2.3 vs 14.8 mo). High ERG expression was significantly associated with shorter OS and EFS in the total cohort (p=0.002, 12.5 mo vs n.r.; p=0.001, 8.1 vs 15.7 mo) as well as in AML-NK (p=0.001, 11.3 mo vs n.r.; p=0.010, 7.2 vs 22.1 mo). OS was also shorter in AML with unfavorable karyotype (p=0.048, median OS 9.3 mo vs n. r.). With respect to MN1 high expressers had a significantly shorter OS and EFS in the total cohort (p=0.004, 12.3 mo vs. n.r.; p=0.001, 8.1 vs 16.7 mo) as well as in AML-NK (p=0.001, 9.7 mo vs n.r.; p=0.001, 5.1 vs 22.1 mo). In a multivariate analysis including CDKN1B, ERG and MN1 all parameters retained their impact on OS as well as on EFS, while BAALC lost its impact on EFS. Adding MLL-PTD, NPM1+/FLT3-ITD-, favorable and unfavorable karyotype into the model demonstrated an independent significant adverse impact on OS for MLL-PTD (p=0.027, relative risk (RR): 2.38) and ERG expression (p=0.044, RR: 1.59) only. In the respective analysis for EFS only favorable karyotype showed an independent association (p=0.002, RR: 0.261). Conclusion: 1) Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varies significantly between cytogenetic subgroups. 2) BAALC as a continuous variable and CDKN1B, ERG and MN1 as dichotomized variables are independently predictive for OS and EFS in AML. 3) ERG expression even retains its independent prediction of shorter OS if cytogenetic and other molecular genetic markers are taken into account. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela: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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Single Cell Network Profiling (SCNP) Functionally Characterizes FLT3 Pathway Deregulation in Non-M3 Acute Myeloid Leukemia (AML) and Provides Prognostic Value Independent From Mutational Status

Blood ◽

10.1182/blood.v118.21.2512.2512 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2512-2512

Author(s):

Alessandra Cesano ◽

Santosh Putta ◽

Kavita Mathi ◽

David B. Rosen ◽

Urte Gayko ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Induction Therapy ◽

Mutational Load ◽

Receptor Signaling ◽

Employment Equity ◽

Cell Network ◽

Mutational Status ◽

Equity Ownership

Abstract Abstract 2512 Background: FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations (FLT3 ITD+) result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients (pts) with AML; however, substantial heterogeneity in clinical outcomes still exists within both the FLT3 ITD+ and FLT3 ITD- AML subgroups, suggesting alternative mechanisms of disease relapse not accounted for by FLT3 mutational status. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Previously, we reported the use of this assay to functionally characterize FLT3 receptor signaling in healthy bone marrow and AML samples (Rosen et al. PLoS One 2010). By applying it to a separate cohort of samples collected from elderly non-M3 AML pts at diagnosis, a subclassification of AML samples beyond their “static” molecular FLT3 ITD status was generated (Rosen et al. ASH 2010 Abstr 2739). Specifically, FLT3 ITD- AML samples displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD+ AML samples. Conversely, FLT3 ITD+ AML samples displayed more homogeneous induced signaling, with the exception of those with low mutational load, which had profiles more analogous to FLT3 ITD- AML samples. Due to the small numbers of pts in that exploratory study (n=44 [38 FLT3 ITD- and 6 FLT3 ITD+ pts]), an independent study was undertaken to confirm the observations, as well as to evaluate their clinical relevance (i.e., association with disease free survival (DFS) following anthracycline/cytarabine-based induction therapy). Methods: SCNP was performed as previously described on cryopreserved bone marrow or peripheral blood samples collected prior to anthracycline/cytarabine-based induction therapy from 104 elderly (>60y) non-M3 AML pts enrolled on ECOG trial 3999 or 3993 for whom ITD mutational status (including % mutational load), response and DFS data were available. Samples included 85 FLT3 ITD- and 19 FLT3 ITD+ AML, 30 and 8 of which, respectively, were collected from patients who achieved complete remission (CR). Objectives: The primary study objective was to confirm that levels of FLT3 ligand (FLT3L)-induced signaling (as measured by changes in intracellular phospho-S6 level) are more homogeneous in FLT3 ITD+ than in FLT3 ITD- myeloblasts. Four FLT3 ITD+ groups were pre-defined based on % mutation load (>0, 30%, 40%, or 50%). In addition, FLT3 ITD mutational status and signaling data from the SCNP assay (FLT3L and stem cell factor-induced phospho-S6 signaling and cytarabine/daunorubicin-induced apoptosis [cleaved PARP]) were combined to mathematically model their association with DFS among pts who achieved CR. DFS was defined as time from date of confirmed CR to date of relapse or death. Results: As shown in Figure 1a, our previous observations that variance in FLT3L-induced signaling is higher in FLT3 ITD- AML samples than in FLT3 ITD+ ones and that variance is decreased with increasing mutational load were verified in this study (Levene Test for FLT3 ITD- vs FLT3 ITD+ 50 p value=0.023). Further, when the association of DFS with FLT3 ITD mutational status and signaling data from the SCNP assay was measured using a Cox Proportional-Hazards model, the SCNP data were shown to provide independent information from FLT3 ITD mutational status (p =0.0115 for FLT3L-induced phospho-S6 signaling, Figure 1b). Conclusions: These data add to the growing body of evidence that, even within currently accepted risk stratification groups, AML is a heterogeneous disease. Functional characterization of FLT3 receptor signaling deregulation using SCNP provides prognostic information independent from FLT3 ITD mutational status and allows for more accurate pt stratification by functionally defining DFS risk sub-groups. Characterization of FLT3 signaling deregulation by SCNP could ultimately aid in the improved clinical management of AML pts and help identify candidates for FLT3 receptor inhibitor studies. Disclosures: Cesano: Nodality: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Mathi:Nodality: Employment. Rosen:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership.

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