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In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 1534-1534

Abstract: Background: Recently, the oral multitargeted small molecule FLT3 inhibitor midostaurin (M) was approved for treatment of FLT3-mutated AML in combination with standard chemotherapy. In the international RATIFY (NCT00651261) trial, addition of M led to superior overall and event-free survival compared to placebo, thus defining a new standard of care in this AML subset (Stone RM et al. NEJM 2017). Although not powered for subgroup analyses, M showed consistent effects across all FLT3 mutation strata [tyrosine kinase domain (TKD); internal tandem duplication (ITD) with low (0.05-0.7; ITDlow) or high ( 〉 0.7; ITDhigh) allelic ratio] suggesting significant off-target activity beyond FLT3 inhibition. Aim: We aimed to comprehensively profile the mutational landscape of FLT3 mutated (FLT3mut) AML in a large, well characterized cohort of patients (pts) treated within the RATIFY trial using a high-throughput targeted sequencing (HTS) approach. Methods: HTS was performed on the entire coding region of 262 genes involved in hematologic malignancies including 20 genes that encode kinases targeted by M (M kinome, MK). Pretreatment peripheral blood (PB; 14%) or bone marrow (BM; 86%) specimens were available from 475 (66%) of 717 FLT3mut AML RATIFY pts. Libraries were prepared using SureSelectXT custom solutions (Agilent). Paired-end sequencing was carried out on a HiSeq platform (Illumina). FLT3 mutation (mut) status was available for all pts [TKD: 24%; ITD: 76% (ITDlow: 45%; ITDhigh:31%)], and cytogenetic data for 454 pts (96%). Results: An average sequencing depth of 978x was obtained for all pts. In sum, 1815 mut (missense: 49%; indels: 40%; nonsense: 7%; other: 3%) were identified with a mean of 3.8 mut per pt (FLT3 strata; TKD: 4; ITDlow: 4; ITDhigh: 3.6).Overall, recurrent mut ( 〉 5% of all pts) were found in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), RUNX1 (11%), NRAS (11%), PTPN11 (9%), ASXL1 (8%), IDH1 (8%), IDH2 (7%; R140 only), and SMC1A (6%). In contrast, TP53 (1%) and biallelic CEPBA (1%) mut were rare events. This was also true for aberrations of the MK (7% in total) with KIT (2%), MAP3K11 (1%), and NTRK3 (1%) being most frequently mutated. When stratified according to FLT3mut type, mut in NRAS (24% vs 7%, p 〈 .0001), SMC1A (10% vs 4%, p=.02), and KIT (4% vs 1%, p=.02) occurred significantly more often in TKD than ITD groups, respectively, whereas WT1 (13% vs 24%, p=.018) was more frequently co-mutated in the ITD group. In general, pts in the TKD group had significantly more mut in genes encoding for cohesin (TKD: 29% vs ITD: 16%, p=.004) and signaling (TKD: 40% vs ITD: 24%, p=.001) proteins compared to ITD pts, who had significantly more mut in transcription genes (TKD: 37% vs ITD: 48%, p=.03). Based on the mut and cytogenetic data, we next sought to assign all FLT3mut pts to the 11 recently defined molecular AML classes (Papaemmanuil E et al. NEJM 2016). The majority fell into two classes, namely the NPM1 (N; 62%) and the chromatin-spliceosome (CS; 15%) classes, underscoring the significance of FLT3mut as the driver in these particular genomic classes. Other class-defining lesions were rare or absent in this cohort [inv(16): 2%; t(8;21): 2%; t(11q23;x): 2%; t(6;9): 1%, TP53-aneuploidy: 1%; CEBPAbiallelic: 1%; IDH2R172: 0%]. In 14% of all pts categorization was not possible (no or 〉 1 class-defining lesion), emphasizing the need for further refinement of this classification. When focusing on these two groups, N and CS had comparable FLT3mut patterns (TKD: 24% vs 21%; ITDlow: 44% vs 45%; ITDhigh: 32% vs 33%), whereas N more frequently correlated with a normal karyotype (N: 91% vs CS: 63%). With respect to clinical characteristics, no differences between N and CS in terms of age, white blood cells, platelets, PB and BM blasts, as well as history of MDS were observed. Conclusion: In this comprehensive sequencing approach, we could further delineate the molecular pattern of FLT3mut AML. Here, FLT3-ITD and -TKD groups exhibited remarkable differences in cooperating pathways, highlighting distinct biologic features in the leukemogenesis of FLT3mut AML. Overall, mut of MK genes were rare events, not fully explaining the complexity of M off-target effects. Understanding the underlying disease mechanism will potentially provide useful information on prognosis and prediction of response to M. Further analyses including correlation with clinical outcome are ongoing. Support: U10CA180821, U10CA180861, U10CA180882, U24CA196171 Disclosures Bullinger: Janssen: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Bayer Oncology: Research Funding. Gathmann:Novartis: Employment. Larson:Ariad/Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding. Medeiros:Genentech: Employment; Celgene: Consultancy, Research Funding. Tallman:ADC Therapeutics: Research Funding; AROG: Research Funding; BioSight: Other: Advisory board; Orsenix: Other: Advisory board; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; Cellerant: Research Funding. Tiecke:Novartis: Employment. Pallaud:Novartis: Employment. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Stone:Otsuka: Consultancy; Jazz: Consultancy; Cornerstone: Consultancy; Fujifilm: Consultancy; Arog: Consultancy, Research Funding; Pfizer: Consultancy; Sumitomo: Consultancy; Novartis: Consultancy, Research Funding; Ono: Consultancy; Orsenix: Consultancy; Merck: Consultancy; Argenx: Other: Data and Safety Monitoring Board; AbbVie: Consultancy; Agios: Consultancy, Research Funding; Amgen: Consultancy; Astellas: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Döhner:AROG Pharmaceuticals: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2018-99-115842

Language: English

Publisher: American Society of Hematology

Publication Date: 2018

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 435-435

Abstract: Introduction: Internal tandem duplication of the FLT3 gene (FLT3-ITD), resulting in duplication of 3 to more than hundreds of nucleotides, are present in approximately 25% of adults with newly diagnosed AML. Several studies have shown that ITD mutations are associated with poor prognosis due to a high relapse rate, in particular in case of a high mutant to wild-type allele ratio and/or insertion site in the beta1-sheet of the tyrosine kinase domain-1 (beta1-sheet). Aims: To investigate the relationship between ITD insertion site and patient outcome, Roche 454 next generation sequencing (NGS) was performed in 452/555 (81.4%) FLT3-ITD positive patients (pts) enrolled into the RATIFY trial (NCT00651261). Results: NGS identified 908 ITDs with up to 9 ITDs per case (1 ITD: n=210, 46.5%; 2 ITDs: n=131, 29.0%; 3 ITDs: n=58, 12.8%; 4 ITDs: n=24, 5.3%; 5 ITDs: n=18, 4.0%; 6 ITDs: n=3, 0.7%; 7 ITDs: n=7, 1.5%; 9 ITDs: n=1, 0.2%). Median ITD-size was 45 nucleotides (range, 6-246); all ITDs were in-frame with direct head-to-tail orientation. According to the 4 functional groups, 488 ITDs (53.7%) were located within the juxtamembrane domain (JMD), 155 ITDs (17.1%) within the hinge region, 211 ITDs (23.2%) within the beta1-sheet, and 54 ITDs (5.9%) 3´of beta1-sheet. ITD size strongly correlated with insertion site, in that the more C-terminal the insertion site, the longer the size of the inserted fragment (P 〈 .001). In 242 pts (53.5%) featuring multiple ITD clones, 698 concurrent integration sites were delineated, with coexistent integration sites within JMD being the most frequent interaction (37%) followed by JMD and beta1-sheet (13.5%), and within beta1-sheet (7.8%). NPM1 mutations (NPM1mut) were present in 203/358 pts (56.7%). Correlation of ITD insertion site with NPM1mut revealed a significantly lower incidence of NPM1mut in pts with insertion located within the hinge region (50/106, 47.2% vs 153/252, 60.7%; P=.02) and 3´of beta1-sheet (14/41, 34.1% vs 189/317, 59.6%; P=.002), whereas NPM1mut were significantly more frequent in pts with insertions affecting JMD (143/235, 60.9% vs 60/123, 48.8%; P=.03). Clinical characteristics differing among the 4 functional ITD groups were gender and WBC. Pts with insertions 3´of beta1-sheet were predominantly male (28/46, 60.9% vs 178/406, 43.8%; P=.03); pts with JMD insertions exhibited lower WBC (median 36.5 vs 52.7 x109/L; P=.03). Complete remission (CR) was achieved within 60 days in 248/452 pts (54.9%). To evaluate the impact of ITD insertion site on response to induction, a logistic regression model was used. ITD insertion sites were categorized in (i) only in beta1-sheet, (ii) in beta1-sheet and other sites, and (iii) outside the beta1-sheet. Other variables were ITD mutant to wild-type allelic ratio (fragment analysis, cutoff at 0.5), number of ITDs per patient, log2 of WBC counts, age, NPM1mut, and midostaurin treatment. In this model, only number of ITDs predicted lower CR rate (OR, 0.72; 95% CI, 0.57-0.90), while NPM1mut was a favorable marker for CR (OR, 2.69; 95% CI, 1.70-4.28). Median follow-up for survival was 60.6 months (mo); median event free survival (EFS) and overall survival (OS) were 3.9 mo and 24.4 mo, respectively. The 4-year EFS and OS rates were 21.0% (95% CI, 17.2%-24.8%) and 42.6% (95% CI, 37.9%-47.4%), respectively. Survival analysis according to categorized insertion site groups showed that pts exhibiting insertion exclusively in the beta1-sheet had significantly inferior OS (P=.014) compared to the other two groups. Multivariate models for OS and EFS including hematopoietic stem-cell transplantation (HSCT) as a time-dependent covariate revealed WBC counts as unfavorable and NPM1mut as favorable for both endpoints; further unfavorable factors were older age and exclusive insertion in the beta1-sheet (HR, 1.49; 95% CI, 1.01-2.20) for OS and number of ITDs (HR, 1.15; 95% CI, 1.04-1.28) for EFS; HSCT was a favorable factor only for EFS (HR, 0.66; 95% CI, 0.44-0.99). Midostaurin treatment was associated with in trend improved EFS (HR, 0.81; 95% CI, 0.63-1.03) and OS (HR, 0.77; 95% CI, 0.57-1.02). Conclusions: In this large cohort of 452 FLT3-ITD mutated AML treated within the RATIFY trial the negative prognostic impact of beta1-sheet insertion site of FLT3-ITD could be confirmed. Further analyses to investigate potential predictive effects of midostaurin treatment are ongoing. Support: U10CA180821, U10CA180882, U24CA196171, (CCSRI) #704970. Disclosures Du: Novartis: Employment. Gathmann:Novartis: Employment. Larson:Ariad/Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding. Medeiros:Celgene: Consultancy, Research Funding; Genentech: Employment. Tallman:Orsenix: Other: Advisory board; Cellerant: Research Funding; AROG: Research Funding; AbbVie: Research Funding; BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Tiecke:Novartis: Employment. Pallaud:Novartis: Employment. de Witte:Amgen: Consultancy, Research Funding; Novartis: Research Funding; Celgene: Honoraria, Research Funding. Niederwieser:Novartis: Research Funding; Miltenyi: Speakers Bureau. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Bullinger:Bristol-Myers Squibb: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau. Döhner:Pfizer: Research Funding; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2018-99-116149

Language: English

Publisher: American Society of Hematology

Publication Date: 2018

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 135, No. 5 ( 2020-01-30), p. 371-380

Abstract: Patients with acute myeloid leukemia (AML) harboring FLT3 internal tandem duplications (ITDs) have poor outcomes, in particular AML with a high (≥0.5) mutant/wild-type allelic ratio (AR). The 2017 European LeukemiaNet (ELN) recommendations defined 4 distinct FLT3-ITD genotypes based on the ITD AR and the NPM1 mutational status. In this retrospective exploratory study, we investigated the prognostic and predictive impact of the NPM1/FLT3-ITD genotypes categorized according to the 2017 ELN risk groups in patients randomized within the RATIFY trial, which evaluated the addition of midostaurin to standard chemotherapy. The 4 NPM1/FLT3-ITD genotypes differed significantly with regard to clinical and concurrent genetic features. Complete ELN risk categorization could be done in 318 of 549 trial patients with FLT3-ITD AML. Significant factors for response after 1 or 2 induction cycles were ELN risk group and white blood cell (WBC) counts; treatment with midostaurin had no influence. Overall survival (OS) differed significantly among ELN risk groups, with estimated 5-year OS probabilities of 0.63, 0.43, and 0.33 for favorable-, intermediate-, and adverse-risk groups, respectively (P & lt; .001). A multivariate Cox model for OS using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable revealed treatment with midostaurin, allogeneic HCT, ELN favorable-risk group, and lower WBC counts as significant favorable factors. In this model, there was a consistent beneficial effect of midostaurin across ELN risk groups.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.2019002697

Language: English

Publisher: American Society of Hematology

Publication Date: 2020

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7