Outcomes of Allogeneic Stem Cell Transplantation for AML and MDS Based on Pre-Transplant MRD Status By Next-Generation Sequencing

Abstract Background After induction therapy for acute myelogenous leukemia (AML), the presence of minimal residual disease (MRD) by targeted next-generation sequencing (NGS) during complete remission (CR) predicts relapse and survival, particularly after exclusion of pre-leukemic mutations. MRD assessment is not routinely performed for AML prior to transplant, partly because consensus regarding assay methodology, appropriate timing, interpretation of results, and therapeutic value prior to SCT is lacking. We therefore sought to describe the rates of mutational clearance and correlate these with relapse rates post-transplant. Methods We conducted a retrospective review of sequential AML or myelodysplastic syndrome (MDS) patients undergoing allogeneic hematopoietic cell transplant (alloHCT) at our institution between 2014 and 2017. There were 119 patients with AML/MDS who were treated with either myeloablative or reduced intensity conditioning regimens. Of the 119 patients transplanted, 60 had both pre- and post-treatment NGS results and were included in the analysis. 56 patients had somatic mutations on initial NGS and were therefore eligible for mutational clearance analysis. Twelve patients were in active disease and excluded from further analyses. The remaining patients (n=44) represent the core dataset. Blood and/or marrow specimens were analyzed via a clinical NGS panel targeting 68 leukemia-associated genes. Median coverage (across 88 samples) was 2817 reads. Mutations were considered persistent if present at variant allele frequencies (VAF) ≥ 1% for single nucleotide variants (SNV) or ≥ 2 copies for insertions and deletions (indels). Validated laboratory reporting practice at our institution reports VAF > 4% for SNVs and ≥ 1% for indels with a minimum of 250 total reads. We therefore defined three levels of mutational clearance on the basis of the VAF of residual mutations: VAF for SNV <1% (and/or indels ≤1 copy), between 1-4% (and/or indels <1% and ≥ 2 copies), and >4% (and/or indels > 1%). Patients with ≥ 1 mutation meeting these thresholds were designated NGS(-), NGS-low and NGS(+), respectively. The median follow-up was 332 days. Results On review of NGS data, 120 mutations were present in initial sequencing, with 64 mutations persistent in pre-transplant samples from 26 patients. The most commonly mutated genes from initial samples were FLT3 (18), ASXL1 (11), TET2 (10), NPM1 (9), RUNX1 (8), SRSF2 (8), and DNMT3A (7) (Figure 1A). Mutational clearance varied widely, with the putative pre-leukemic genes DNMT3A, TET2, and ASXL1 (DTA) demonstrating low rates of mutational clearance (Figure 1A). Mutations persisting below the validated reporting threshold were present in 20 patients, including 10 patients otherwise negative by NGS. There were 16 patients categorized as NGS(+), 10 NGS-low, and 18 NGS(-), with relapse rates of 31%, 22%, and 30%, respectively. No difference in relapse risk was observed between NGS(-) and NGS-low subgroups (p = 0.72), and no RFS benefit was observed for patients without persistent mutations > 4% relative to the NGS(+) subgroup (p = 0.56, Figure 1B). Recent work has shown a survival benefit in AML patients in CR without persistent mutations that is enhanced when DTA genes were excluded from the analysis (Jongen-Lavrencic, NEJM 2018). In our cohort, after exclusion of DTA mutations, 6 patients were reclassified by mutational clearance status, and 2 were excluded from the analysis as they had only DTA mutations in pre-treatment samples. Similar to the more comprehensive cohort, no RFS benefit based on NGS status was observed in the post-transplant period (p = 0.42, Figure 1C). Conclusions There were similar outcomes regardless of molecular MRD findings by NGS for patients with advanced myeloid malignancies who were in morphologic CR prior to alloHCT. These results contrast with those in the published literature that address a more uniform patient population of clinical trial participants, not all of whom went on to transplant. Further detailed analyses from larger more homogeneous populations will be useful to determine the prognostic significance of MRD by NGS prior to allogeneic HCT. Figure 1 Figure 1. Disclosures Frey: Servier Consultancy: Consultancy; Novartis: Consultancy. Perl:Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Daiichi Sankyo: Consultancy. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding; Amgen: Consultancy; Janssen: Consultancy. Porter:Genentech: Other: Spouse employment; Kite Pharma: Other: Advisory board; Novartis: Other: Advisory board, Patents & Royalties, Research Funding. Gill:Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Equity Ownership; Novartis: Research Funding.

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Long-Term Follow-up of Follicular Lymphoma (FL) Patients (pts) Demonstrating Undetectable Minimal Residual Disease (MRD) Using a Next-Generation Based DNA Assay: Support for FL As a Curable Disease

Blood ◽

10.1182/blood-2019-127262 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2813-2813

Author(s):

Maryam Sarraf Yazdy ◽

Umair Jarral ◽

Chao Yin ◽

Frank Kuhr ◽

Allison P. Jacob ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Comprehensive Cancer Center ◽

Cancer Center ◽

Allogeneic Bone ◽

Next Generation ◽

Advisory Committees ◽

Generation Sequencing

BACKGROUND FL is the most common indolent non-Hodgkin lymphoma (NHL). While very responsive to therapy, it has been considered incurable. Nonetheless, pts remaining in remission >24 months appear to have a survival comparable to an age-matched population without NHL (Casulo et al, J Clin Oncol, 33:2516, 2015), and some are free of disease for many years and die from unrelated events. METHODS Adult pts were accrued from the Lombardi Comprehensive Cancer Center Lymphoma clinic. Pts were required to have histologically confirmed FL or transformed FL that was previously treated resulting in a complete remission, and were required to be free of progression at any time > 24 months following completion of treatment without intervening therapy. Original diagnostic samples were retrieved and subjected to clonality assessment using Adaptive's next generation sequencing (NGS) MRD assay , a research version of clonoSEQ®; (Adaptive Biotechnologies, Seattle, WA) that leverages multiplex PCR followed by NGS to identify and track rearrangements of IgH, V-J, D-J and IgK/L loci as well as translocations in Bcl1/2 IgH. Lymph node biopsy from time of original diagnosis was assessed to identify trackable clonotypes, which were found in 37/43 patients. Peripheral blood was assayed upon entry onto the study and every 6 months thereafter by the NGS-MRD assay to monitor MRD. Samples are being collected every 6 months during follow-up, and the results are being correlated with clinical outcome. RESULTS Of the 60 eligible pts who signed consent 41% were females, with a median age at diagnosis of 56 yrs (21-75) and median age at treatment of 56 years (21-75). Twenty six had received one prior line of treatment (LOT), 4 had 2, 6 had 3, and 1 had 5. The most common immediately prior line of therapy included bendamustine and rituximab (BR, n=16); rituximab, cyclophosphamide, adriamycin, vincristine, prednisone (RCHOP, n=6); double-monoclonal antibody containing regimens(rituximab-galiximab; rituximab-epratuzumab (n=3)), radioimmunotherapy (n=3), and allogeneic bone marrow transplant (n=2). The media follow-up since the start and completion of most recent therapy was 62 months (range 25-183 and 32-193, respectively). Of the 60 pts for whom original biopsy slides were obtainable, the quality was inadequate to amplify the DNA in 18. In another 5 pts, the sample was polyclonal and a dominant rearrangement could not be identified. In 32 of the 37 pts (86.5%), samples were negative at enrollment to this study at a level of detection of 10-5. By prior LOT, samples were negative in 25 of 26 following 1st line; 1 of 4 following 2nd line; 5 of 6 following 3rd line; and in the one pt after 5th line. In all but 1 pt, the assay has remained negative on subsequent determinations as shown in the spider plot (Fig 1). The 5 positive patients had been followed for a median of 85 (56-118) months. Additional follow-up is underway to determine if positive pts will eventually relapse. CONCLUSIONS These data are the first to demonstrate that a high proportion of FL pts in a prolonged clinical remission have undetectable DNA by sensitive next generation sequencing, without evidence of clinical progression, and are potentially cured of their disease. Figure 1 Disclosures Yazdy: Bayer: Honoraria, Speakers Bureau; Genentech: Research Funding; Abbvie: Consultancy; Octapharma: Consultancy. Kuhr:Adaptive Biotechnologies: Employment, Other: shareholder. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Cheson:Epizyme: Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding; Portola: Research Funding; Kite: Research Funding; Gilead: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Symbios: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trillium: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy, Research Funding.

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Sensitivity, Reproducibility and Clinical Utility Of Next-Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain Mutation Screening: Results From The CML Work Package Of The Iron-II (Interlaboratory RObustness Of Next-Generation Sequencing) International Study

Blood ◽

10.1182/blood.v122.21.3824.3824 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3824-3824 ◽

Cited By ~ 2

Author(s):

Simona Soverini ◽

Thomas Ernst ◽

Alexander Kohlmann ◽

Caterina De Benedittis ◽

Mary Alikian ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Mutation Detection ◽

Mutation Screening ◽

Kinase Domain ◽

Clinical Samples ◽

Next Generation ◽

Advisory Committees ◽

Generation Sequencing

Abstract Background and Aims In chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL1 mutation status is an essential component of the therapeutic decision algorithm. Capillary Sanger sequencing (SS) is currently the gold standard for mutation screening of the BCR-ABL1 kinase domain (KD), despite key technical limitations including limited sensitivity and no discrimination between compound and polyclonal mutations. Benchtop next-generation sequencers have recently been introduced as potential diagnostic platforms and there is growing interest in their clinical application. In the framework of the IRON-II (Interlaboratory RObustness of Next-generation sequencing) international consortium, 10 laboratories from 7 countries (Italy, Germany, United Kingdom, Spain, Austria, Turkey, Czech Republic) have engaged in the set-up, standardization and validation of a laboratory-developed screening assay for BCR-ABL1KD mutations based on the Roche 454 amplicon deep-sequencing technology. Methods Fusion primers were designed to generate four partially overlapping amplicons by nested reverse transcription (RT)-polymerase chain reaction (PCR), the first amplification step needed to select for the translocated ABL1 allele. Fusion primers were barcoded with multiplex identifiers (MIDs) consisting of 10-base pair tags allowing multiplexing of twelve clinical samples (forty-eight amplicons) in a single NGS run. The assay was designed in a ready-to-use 96-well plate format containing lyophilized oligonucleotide primers. Results Different primer designs and primer-MID combinations were evaluated for their performances. Sequencing runs generated an average of 97,432 reads (range, 59,459-151,335). For the primer design selected for further evaluation, the coverage per amplicon ranged between 1,449 and 5,997 sequencing reads. To explore the sensitivity and accuracy of the assay, serial dilutions of BaF3 cell lines harboring four different known mutations (Y253F, E255K, T315I, M351T) into an unmutated BaF3 cell line (50%:50%; 25%:75%; 10%:90%; 5%:95%; 2%:98%; 1%:99%) were sequenced in parallel in two distinct laboratories (Bologna and Jena). In both centers, results showed a high linearity of mutation calling and accuracy of mutation detection and quantitation over the entire range of dilutions, down to 1% mutation abundance. Intra-run reproducibility and inter-run reproducibility were confirmed by a series of experiments in which a set of samples was resequenced in the same and in independent runs, respectively, with and without repetition of the RT and PCR steps. Importantly, we demonstrated that reproducibility could be maintained over a wide dynamic range of amplicon coverage (from 100 to 5,000 independent sequencing reads). A total of 554 clinical samples (2,216 amplicons) were analyzed by the 10 laboratories - including 517 clinical samples analyzed in parallel by NGS and SS and 30 clinical samples analyzed in parallel by NGS, SS and conventional pyrosequencing. Three hundred and ninety-four of 398 (99%) variants detected by SS were also detected by NGS. In addition, comparison between NGS, SS and conventional pyrosequencing results showed very good concordance with respect to the estimation of variant abundance. NGS allowed to detect additional, low level mutations (>1% but<10-15%, i.e. undetectable by SS) in 294/554 (53%) samples. In a subset of twenty randomly selected samples, low level mutations were confirmed by independent methods (restriction fragment length polymorphism or allele-specific oligonucleotide-PCR). Compound mutations as against polyclonality could be resolved in all the clinical samples harboring multiple mutations mapping 450 bp apart or closer. Longitudinal retrospective analysis of CML and Ph+ ALL clinical samples showed that NGS could have identified TKI-resistant mutations earlier than SS, thus allowing more timely therapeutic intervention. Conclusions Our results indicate the technical feasibility, accuracy and robustness of NGS for BCR-ABL1 KD mutation screening and represent an important step forward towards its routine application in a clinical setting. An international ring trial to test inter-laboratory reproducibility of BCR-ABL1 mutation detection by NGS is now about to start. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Machova Polakova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Lion:Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hochhaus:ROCHE: Research Funding. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

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NPM1 mutations Using Deep Amplicon Sequencing and Broad Next Generation Sequencing at the Time of Complete Remission Is Informative to Predicting Risk of Relapse Following Intensive Chemotherapy

Blood ◽

10.1182/blood-2019-130856 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1329-1329

Author(s):

Bhavana Bhatnagar ◽

Shelley Orwick ◽

Nyla A. Heerema ◽

Alison R. Walker ◽

Alice S. Mims ◽

...

Keyword(s):

Next Generation Sequencing ◽

Complete Remission ◽

Board Of Directors ◽

Research Funding ◽

Amplicon Sequencing ◽

Next Generation ◽

Research Support ◽

Advisory Committees ◽

Npm1 Mutation ◽

Generation Sequencing

Introduction: NPM1 gene mutations are a common molecular aberration in acute myeloid leukemia (AML). In the absence of concurrent high FLT3-ITD ratio mutations (>0.5), NPM1 mutations typically associate with higher complete remission (CR) rates following intensive induction chemotherapy. NPM1 mutations have been shown to be stable markers of persistent disease or impending relapse during CR or complete remission with incomplete count recovery (CRi). Given the clinical implications that persistent NPM1 mutations can have during CR/CRi, we used Deep Amplicon sequencing on CR/CRi bone marrow (BM) samples collected from adult de novoNPM1-mutated AML patients to determine the ability of NPM1 mutations at both a high and lower sensitivity next generation sequencing methods and also the presence of additional clonal abnormalities on relapse risk. Methods: We performed targeted next generation sequencing (NGS) analysis in addition to NPM1 Deep Amplicon sequencing on paired BM or blood samples collected from 38 newly diagnosed NPM1-mutated AML patients during CR/CRi after successful induction (1-2 courses of 7 + 3) and, if available, at relapse. NPM1 mutated NGS libraries were prepared using a KAPA HyperPlus Kit (Roche, Pleasanton, CA) and xGen Lockdown Probes (IDT, Coralville, IA). Libraries were sequenced using the Illumina HiSeq 4000 (Illumina, San Diego, CA). GATK's MuTect2 was used to perform variant calling. Variant allele frequency (VAF) cut-off for the NGS panel was 0.05 (5%) with the exception of hotspot variants in IDH1 (R132) and IDH2 (R140) where variants detected to a level of 0.01 (1%) were included. The VAF cut off used for NPM1 Deep Amplicon sequencing was 0.00012 (0.012%). Results: Targeted NGS analysis and NPM1 Deep Amplicon sequencing had exceptional concordance at the level of detection of VAF= 0.05 (Figure 1). Of 38 patients, 23 patients had undetectable NPM1 mutations as analyzed through NPM1 Deep Amplicon sequencing of whom 9 (38.1%) relapsed. In contrast, 15 patients were positive by NPM1 Deep Amplicon sequencing and 9 (60%) relapsed. Only 4 patients had detectable persistent NPM1 mutations after induction according to both detection techniques and two of these relapsed. We next examined the potential impact of clearing both NPM1 mutation and co-occurring mutations together on relapses (Figure 2). A total of 15 patients cleared all of their clonal abnormalities and 5 (27%) relapsed. In contrast, of the 23 patients who did not clear the NPM1 mutation and/or another co-occurring mutation at remission, 14 (61%) have relapsed. Eleven of the relapsed patients had relapse samples available of whom all had persistent NPM1 mutation at this time. Paired CR/CRi and relapsed samples showed acquisition or recurrence of several other mutations, most notably FLT3-ITD, IDH1, and IDH2 which are all targetable with small molecule therapeutics. Conclusions: The use of Deep Amplicon sequencing to identify NPM1 mutations at a lower detection threshold compared to standard NGS techniques was more sensitive, but did not appear to fully inform relapse rates in NPM1-mutated AML patients after receipt of induction therapy. The appearance of other AML-associated mutations, identified together with NPM1 at time of remission, was more frequent among patients relapsing. These pilot data provide support for concurrent assessment of Deep Amplicon sequencing together with a broad standard NGS AML mutational assay to further enhance risk stratification of NPM1-mutated patients. Additionally, while NPM1 clones are present in all patients examined at the time of relapse, persistence or development of targetable clones justifies repeat broad NGS sequencing at this time. Figure Disclosures Bhatnagar: Novartis and Astellas: Consultancy, Honoraria; Cell Therapeutics, Inc.: Other: Research support; Karyopharm Therapeutics: Other: Research support. Mims:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees. Behbehani:Fluidigm corporation: Other: Travel funding. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S.

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Utilization of Next Generation Sequencing Identifies Potentially Actionable Mutations with Prognostic Significance in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.2929.2929 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2929-2929

Author(s):

Jie Wang ◽

Jennifer J.D. Morrissette ◽

E. Paul Wileyto ◽

Stephen J. Schuster ◽

Alexandra Vandegrift ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Genetic Alterations ◽

Telomere Maintenance ◽

Next Generation ◽

Advisory Committees ◽

Mek Inhibitors ◽

Signal Transduction Inhibitors ◽

Generation Sequencing

Abstract Introduction: CLL is a clinically and biologically heterogeneous disease; cytogenetic evaluation with fluorescence in situ hybridization (FISH) is routinely used to guide therapy. For example, del17p is associated with chemoimmunotherapy (CIT) refractoriness and decreased survival. The use of kinase inhibitors (KI) has improved clinical outcomes; however, some pts progress on KI and require subsequent therapies. Next generation sequencing (NGS) can further define genetic alterations that may act in concert to drive malignancy, and identify pathways that can be targeted with novel approaches. Here we describe the mutational landscape of a cohort of 57 CLL pts treated at the University of Pennsylvania and identify potentially targetable pathways for intervention. Methods: We identified 57 pts who underwent analysis of tumor DNA using NGS (2013-2015) and analyzed clinical characteristics, genetic mutations, and progression free survival (PFS). NGS was performed on an Illumina MiSeq using a 33 gene amplicon-based panel developed at our center with detection limit of 5% allele frequency with a minimum depth of coverage of 250x. We used custom bioinformatic pipelines combining open source tools and custom algorithms for analysis. Pathogenic mutations were defined as those that have been reported in studies with functional data. Results: Of the57 pts who underwent NGS the median age at NGS was 65.5 yr (range 17.7-90.7), 65% were male, 21% patients received CIT alone, 21% patients received KI alone and 39% pts received both CIT and KI, and 32% received KI in relapse. 74% (42/57) of pts had at least one genetic mutation identified by NGS. The median number of mutations per pt was 1 (range 0-8). 25% of pts had ≥ unique 3 mutations). Mutations in 24 unique genes (n=94) were identified and were categorized as likely pathogenic (69%), variants of uncertain significance (27%), or likely benign (4%). The most frequently mutated genes were ATM (20%), SF3B1 (12%), NOTCH1 (10%), DNMT3A (7%), and TP53 (7%). We identified 19 low frequency gene mutations, which in aggregate affect 24.5% of the pt cohort (Table). The median PFS for CIT pts was 31.4 mo (median f/u 15 mo) and 8 mo for KI pts (median f/u 4 mo). Using Cox regression, the presence of ≥ 1 mutation was associated with an inferior PFS (Figure) following CIT when controlled for del11q status (HR 3.1, p=.05) or complex karyotype (HR 3.4, p=.03) and a PFS trend when controlled for del17p (HR 2.7, p=.08). Pts with ≥ 4 mutations had a shorter PFS on ibrutinib (Ibr) compared to those with fewer mutations (p=0.0002). Conclusion: NGS identifies several mutations that may be targetable using agents which have not been tested in CLL. The presence of a mutation identified by NGS predicts for inferior PFS on CIT, and the presence of ≥ 4 mutations predict early treatment failure on Ibr. These genetic alterations demonstrate the diversity of pathways that are involved in CLL biology. These results support a rationale for clinical trial design using a precision medicine approach selecting therapies which are already available in practice based on individual pt genetic profiles. Table 1. Mutation Events Summary Putative Pathway Frequency (%) Potential Therapy DNA Damage and Cell Cycle Control 32 (34) ATMTP53XPO1STAG2 19 (20.2)7 (7.4)5 (5.3)1 (1.1) PARP inhibitorsSelective inhibitors of Nuclear Export RNA Processing 20 (21.3) SF3B1XPO1TBL1XR1PRPF40BZRSR2 11 (11.7)5 (5.3)2 (2.1)1 (1.1)1 (1.1) Epigenetic modification 11 (11.7) DNMT3ATET2 7 (7.4)4 (4.3) DNA methyltransferase inhibitors RAS-RAF-MEK-MAPK 10 (10.6) BRAFKRASNRASNF1 5 (5.3)2 (2.1)2 (2.1)1 (1.1) BRAF inhibitorsRAS/MEK inhibitors RAF/MEK inhibitors Transcriptional regulation activity 10 (10.6) BCORPHF6TBL1XR1ASXL1 4 (4.3)2 (2.1)2 (2.1)2 (2.1) Notch Signaling 9 (9.6) Notch1 9 (9.6) Notch inhibitors Inflammatory Pathways 3 (3.2) MYD88BIRC3 1 (1.1)2 (2.1) B cell receptor signal transduction inhibitors Cellular metabolism 2 (2.2) IDH1IDH2 1 (1.1)1 (1.1) IDH inhibitors Telomere maintenance 2 (2.1) POT1 2 (2.1) Chromatin modification 2 (2.1) ZMYM3 2 (2.1) Figure 1. Figure 1. Disclosures Schuster: Phamacyclics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Research Funding; Hoffman-LaRoche: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Gilead: Research Funding; Genentech: Consultancy. Rago:Gilead Sciences: Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Porter:Genentech: Other: Spouse employment; Novartis: Other: IP interest, Research Funding. Dwivedy Nasta:Millenium: Research Funding; BMS: Research Funding. Svoboda:Seattle Genetics: Research Funding; Celgene: Research Funding; Celldex: Research Funding; Immunomedics: Research Funding. Loren:Merck: Research Funding. Mato:Pronai Pharmaceuticals: Research Funding; Celgene Corporation: Consultancy, Research Funding; Genentech: Consultancy; Pharmacyclics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy; TG Therapeutics: Research Funding; Gilead: Consultancy, Research Funding.

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Elucidation of Additional Mutations By Next-Generation Sequencing Is of Clinical Significance in Patients with Rare MPNs and MDS/MPN Overlap Syndromes

Blood ◽

10.1182/blood.v128.22.4260.4260 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4260-4260

Author(s):

Martin MJ Kirschner ◽

Mirle Schemionek ◽

Matthias Begemann ◽

Susanne Isfort ◽

Kristina Feldberg ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Hot Spot ◽

Clinical Impact ◽

Next Generation ◽

Advisory Committees ◽

Overlap Syndromes ◽

The Absolute ◽

Generation Sequencing

Abstract Introduction: Recently, next-generation sequencing (NGS) has revolutionized the molecular characterization and understanding of several hematologic entities, including myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS)/MPN overlap syndromes. Nevertheless, the frequency and clinical impact of the mutations detected by NGS, remain largely unclear, especially in rare MPN which were analyzed in this study. Methods: Thus, we established a novel amplicon-based NGS panel, comprising genes that are known to be recurrently mutated in MPN and/or MDS/MPN. Hot spot regions or all exons of the following 32 genes were chosen: ABL, ASXL1, BARD, CALR, CBL, CEBPA, CHEK2, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NFE2, NRAS, PDGFRA, PTPN11, RUNX1, SETBP1, SF3A1, SF3B1, SH3B2 (LNK), SRSF2, TCF12, TET2, TP53, U2AF1. After establishing this panel, peripheral blood samples of 19 patients, which were diagnosed with CMML(10), aCML(2), MPNu(1), MDS/MPNu or other MPN(6), were analyzed on a MiSeq® illumina sequencer. Variants were only analyzed if the absolute coverage at each SNV site was >50 reads, and the absolute coverage of the mutant allele was 10 or more reads and its relative coverage exceeded 10%. Results: Mean coverage of the run was 1516 reads with good Phred-score quality parameters (>84% of called bases with Q-score >= 30). In 300 bidirectional cycles, a yield of nearly seven gigabases of sequencing data was reached. One out of 19 analyzed patients was excluded from analysis due to insufficient DNA quality. In 89% of the samples(16/18), mutations were detected which had not previously been known to be present in these patients. TET2 (50%, 9/18) and SETBP1 mutations were the most common (44%, 8/18). As expected, TET2 mutations were spread over the entire gene and SETBP1 mutations were restricted to the known hot spot region (exon 4, c.2602-c.2620). Additionally, CSF3R mutations were detected in 22% (4/18) of patients. Epigenetic regulator genes were also affected as EZH2 mutations were detected in 17% (3/18), ASXL1 mutations in 39% (7/18) and IDH1/2 mutations were found in 6% (1/18) of all samples, whereas DNMT3A mutations were not present. Further mutations were found in the following genes: CBL (11%), ETV6 (6%), JAK2V617F (6%), KRAS (11%), NRAS (11%), PTPN11 (6%), SH2B3 (6%) and SRSF2 (11%). Besides previously known mutations, several novel variants could be detected. All but one patient harbored more than one of these mutations. Furthermore, clinical correlates and morphologic and cytogenetic subtypes of each patient were available to associate with the NGS data of individual patients. For example, the one patient with a solitary NRAS c.35G>A (amino acid: p.G12D) mutation showed the most aggressive clinical course in our cohort with transformation to AML only 7 months after first diagnosis of CMML. Moreover, CSF3R mutations have been shown to confer sensitivity to ruxolitinib and may thus open up new avenues of treatment for our patients. Conclusion: In a cohort of unclassified MPN and rare MDS/MPN subtypes, NGS is a powerful tool to characterize samples more extensively. Our data suggests that a more comprehensive understanding of the mutational spectrum may have important clinical impact in individual patients, including diagnosis, prognosis, and more specific treatment. Disclosures Isfort: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; BMS: Honoraria; Mundipharma: Other: Travel; Amgen: Other: Travel; Hexal: Other: Travel. Brümmendorf:Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Dissecting Genomic Aberrations In CML and Bcr-Abl Negative Myeloproliferative Neoplasms By The Use Of Multiplex-PCR and Parallel Resequencing

Blood ◽

10.1182/blood.v122.21.1612.1612 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1612-1612

Author(s):

Martin M. Kirschner ◽

Mirle Schemionek ◽

Claudia Schubert ◽

Nicolas Chatain ◽

Stephanie Sontag ◽

...

Keyword(s):

Next Generation Sequencing ◽

Multiplex Pcr ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Tp53 Mutation ◽

Jak2 V617f ◽

Next Generation ◽

Advisory Committees ◽

Generation Sequencing

Abstract Introduction Next-Generation Sequencing holds the promise of comprehensive analysis of molecular aberrations in human malignancies and therapeutic approaches individually tailored to each patient. Methods We investigated the use of a multiplex-PCR (TruseqAmplicon Cancer Panel, Illumina) of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes to identify mutations in a cohort of patients with myeloproliferativeneoplasms (MPN). After signed informed consent, samples from 59 patients with MPN (19 MF [8 PMF, 11 post-PV/ET-MF], 14 PV, 10 ET, 10 CML, 4 HES, and 2 SM), two patients with reactive erythrocytosis, and two anonymized healthy controls as well as six myeloid cell lines (K562, HEL, HMC1, SUPB15, HL60, U937) were analyzed on a Miseq sequencer (Illumina), using 250 ng of genomic DNA from peripheral blood -derived cells. Results Altogether, the quality of the sequencing runs was very good, with Q30 values above 90%. 151 bidirectional cycles were performed, yielding between 2 and 6 Gigabases of sequencing data.Healthy donor and reactive erythrocytosis samples showed several SNPs but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of a TP53 frameshift mutation (c.405_406insC; in 98% of transcripts) in K562, JAK2 V617F (100%) and TP53 M133K (99%) mutations in HEL, two heterozygous KIT mutations (V560G in 51% and D816V in 52%) and a TP53 C277F (16%) mutation in HMC1, while SUP-B15, HL60, and U937 showed no abnormality in the tested gene set.JAK2 V617F was present in all PV, 4 of 10 ET, and 14 of 19 MF patients.The JAK2 V617F allele burden was significantly higher in MF than ET (p=0.026) but not PV (71+/-27% vs. 33+/-22% vs. 55+/-29%, respectively). Further analysis detected a previously described G12V NRAS mutation (13% of transcripts) in a patient with JAK2 V617F negative PMF and an additional IDH1 R132H mutation (24%) in a JAK2 V617F positive (46%) MF patient with 20% basophils and hyperhistaminemia. Another JAK2 V617F positive (31%) MF sample showed an E255G ABL mutation (10%). One patient with JAK2 V617F negative ET showed an ERBB2 A847D sequence variant (50%). Moreover, an S935N CSF1R mutation (17%) and a V125G IDH1 mutation (9%) were each detected in one case of PV, but the biological relevance remains unclear so far. Four patients with CML-CP (n=3) or –AP (n=1) showed subclones with sequence variants in the HNF1A gene, with two S304P changes (9 and 10% of transcripts) and two 872delC mutations (6 and 5%), the latter of which have already been implicated in colon cancer. Two patients with CML-CP showed KIT mutations (a V532I mutation and a known oncogenic mutation V530I). This latter patient also harbored the known E255K ABL mutation – leading to imatinib resistance. Interestingly, this patient showed a good response to dasatinib (which is also active against KIT) but not to bosutinib (which has no activity against KIT). These data suggest that HNF1A and KIT may play a role in CML pathogenesis. One patient with lymphoid BC/Ph+ ALL who had a T315I ABL mutation and was treated with ponatinib, was found to harbor a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib had led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation may have caused ponatinib resistance in this patient. Additionally, other not yet defined aberrations may have been responsible for the observed resistance. Finally, while both SM patients were negative for KIT D816V, one of them harbored a KRAS 436G>A(146A>T) mutation (34%) which is a known oncogene in colorectal cancer and may thus also play a role in SM pathogenesis. We are currently generating induced pluripotent stem cells from patients harboring selected mutations described above in order to better be able to study the functional properties of genetically unstable malignant stem cell populations. Conclusion Amplicon-based next-generation sequencing may uncover additional oncogenic mutations in patients with MPN, potentially explaining therapy resistance and opening new therapeutic options for individual patients. Disclosures: Off Label Use: Two individual patients mentioned that were treated with ponatinib or bosutinib within compassionate use trials before these drugs were approved for the indication. Bruemmendorf:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria. Koschmieder:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

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Prompt CR Plus Consolidation Therapy Yields Improve Survival after Allogeneic Transplantation for AML Patients Receiving Myeloablative, but Not Reduced-Intensity Conditioning: A CIBMTR Analysis

Blood ◽

10.1182/blood-2021-149962 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 414-414

Author(s):

Michael M. Boyiadzis ◽

Marcos J.G. de Lima ◽

Mei-Jie Zhang ◽

Karen Chen ◽

Christopher S. Hourigan ◽

...

Keyword(s):

Multivariate Analysis ◽

Board Of Directors ◽

Research Funding ◽

Disease Status ◽

Advisory Board ◽

Consolidation Therapy ◽

Advisory Committees ◽

Induction Cycle ◽

Relapse Rates ◽

Reduced Intensity

Abstract Leukemia relapse and treatment related mortality (TRM) remain major obstacles for successful allogeneic hematopoietic cell transplantation (allo-HCT). The number of induction cycles using intensive chemotherapy at AML diagnosis to achieve complete remission (CR) and the number of consolidation cycles and disease status at the time of allo-HCT for patients with acute myeloid leukemia (AML) may each affect TRM and relapse rates. We investigated the impact of the number of induction/consolidation cycles and disease status on the success of allo-HCT in 3113 AML patients reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) (2008-2019). They received allo-HCT in first CR or with persistent leukemia (primary induction failure-PIF) receiving myeloablative (MA) or reduced-intensity (RIC) conditioning. 1473 AML patients (median age, 47 years) in CR received MAC; 862 (58%) achieved CR after 1 cycle of intensive induction chemotherapy and 74% of these had no evidence of measurable residual disease (MRD). 454 (31%) patients required 2 cycles to CR (72 % MRD negative) and 157 (11%) patients (69% MRD negative) after ≥ 3 cycles. The overall survival (OS), relapse and TRM by induction cycle number is shown in Table 1. Multivariate analysis demonstrated that CR after 1 cycle led to higher OS vs. 2 cycles (HR 1.32 95%CI 1.11-1.56, p< 0.01) or ≥ 3 cycles (HR 1.47 95%CI 1.16-1.87, p< 0.01), while OS after 2 cycles or ≥ 3 cycles were similar (HR 1.2 95%CI 0.87-1.4, p=0.38). Higher TRM was observed in patients receiving 2 or ≥ 3 cycles vs. only 1 induction cycles (HR 1.34 95%CI 1.05-1.72, p< 0.02). Relapse risk was greater in those needing ≥ 3 cycles to achieve CR. Consolidation therapy after CR was associated with improved OS vs. no consolidation therapy (HR 1.57 95%CI 1.24-1.99, p< 0.01). The need for ≥2 induction cycles plus consolidation therapy was associated with higher TRM (HR 1.34 95%CI 1.05-1.72, p< 0.02). 1162 AML patients (median age, 63 years) in CR received allo-HCT after RIC; 714 (61%) achieved CR after 1 cycle of induction chemotherapy (72% MRD negative); 310 (27%) patients after 2 cycles (67% MRD negative) and 138 (12%) patients (58% MRD negative) after ≥ 3 cycles (Table 1). Multivariate analysis demonstrated that the number of induction cycles did not affect the OS or TRM. Relapse risk was greater in patients requiring ≥2 cycles to achieve CR. The use of consolidation therapy did not affect OS or TRM. MRD status at the time of allo-HCT did not have a significant impact on OS, TRM and relapse rates after either MA or RIC conditioning. 478 AML patients received allo-HCT after PIF (328 patients with MAC [median age, 51 years], 150 patients RIC [median age, 61 years], Table 1). After MAC, OS and relapse were significantly worse in PIF patients compared to any CR patients (p<0.01). After RIC, relapse was significantly more frequent in PIF patients vs. CR patients after 1 or more induction cycles (p<0.01). TRM was similar for PIF vs CR patients after MAC or RIC allo-HCT. These data demonstrate that among patients eligible for allo-HCT, the need for only one induction cycle to achieve CR, particularly when combined with consolidation therapy is associated with better outcomes after MA conditioning. Achieving CR prior to allo-HCT needing one or more induction cycles is associated with lower relapse rates and improved OS compared to patients with PIF that receive allo-HCT. Figure 1 Figure 1. Disclosures de Lima: BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hourigan: Govt. COI: Other. Litzow: Omeros: Other: Advisory Board; Pluristem: Research Funding; Jazz: Other: Advisory Board; AbbVie: Research Funding; Amgen: Research Funding; Actinium: Research Funding; Astellas: Research Funding; Biosight: Other: Data monitoring committee. Saber: Govt. COI: Other. Weisdorf: Incyte: Research Funding; Fate Therapeutics: Research Funding.

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Myeloablative Fractionated Busulfan Conditioning Regimen with Sorafenib in Patients with AML: Results of Phase I Clinical Trial

Blood ◽

10.1182/blood-2021-152450 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 556-556

Author(s):

Uday R. Popat ◽

Roland Bassett ◽

Peter F. Thall ◽

Amin M. Alousi ◽

Gheath Alatrash ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Conditioning Regimen ◽

Advisory Board ◽

License Agreement ◽

Advisory Committees ◽

Post Transplant ◽

Scientific Advisory Board ◽

Scientific Advisory

Abstract Background: Myeloablative conditioning can be given safely to older patients by administering busulfan over a longer period (fractionated busulfan regimen) than the standard four-day regimen. (Popat, et al Lancet Haematology 2018). This longer conditioning regimen duration allows the addition of oral targeted agents like sorafenib, which may be synergistic with conditioning chemotherapy and thus further improve disease control. Therefore, we added sorafenib to fludarabine and fractionated busulfan regimen (f-bu) in a phase 1 dose-finding trial studying 4 different doses of sorafenib with f-bu (NCT03247088). Here we report the results of this trial. Methods: Between 3/2018 and 6/2021, 24 patients with AML aged 18 to 70 years with adequate organ function and 8/8-HLA matched related or unrelated donors were enrolled prospectively. The dose of sorafenib was varied among the four values 200, 400, 600, and 800 mg administered from day -24 to -5. Dose-limiting toxicity (DLT) was defined as grade 3 or higher regimen-related non-hematologic, non-infectious, non-GVHD toxicity occurring between day -24 and day 3. The Bayesian Model Averaging Continual Reassessment Method (BMA-CRM) with target DLT probability 0.30 was used to choose doses for successive cohorts of 3 patients. The first cohort was treated at the lowest sorafenib dose 200, with all successive cohorts' doses chosen adaptively by the BMA-CRM. The doses and schedules of busulfan and fludarabine were fixed, with f-Bu dose targeting an area under the concentration vs time curve (AUC) of 20,000 ± 12% μmol.min given over 3 weeks. The first two doses of busulfan (80 mg/m2 IV each) were administered on days -20 and -13 on an outpatient basis. The last four Bu doses were calculated to give a total course AUC of 20,000 ± 12% μmol.min and were given as inpatient following each dose of Flu 40 mg/m2 on days -6 through -3. GVHD prophylaxis was post-transplant cyclophosphamide (PTCy) 50mg/kg on days 3 and 4 and tacrolimus. Recipients of unrelated donor grafts also received MMF. All patients were eligible to receive post-transplant maintenance sorafenib after engraftment. Results: The median age was 52 years (range, 30-70). Disease status was CR in 16 (66.6%) patients, CRi in 5 (20.8%), and advanced in 3 (12.5%). Adverse risk karyotype was present in 10 (41.7%) patients. MRD was present in 13 (54.2%). 9 (38%) had mutated flt3. The donor was unrelated in 14 (58%), and peripheral blood stem cells were the graft source in 21(87.5%). Due to the absence of DLTs, the BMA-CRM assigned 200mg, 400mg, 600mg, and 800mg of sorafenib, respectively, to the first 4 cohorts, and the next 4 cohorts were given 800mg. Only 2 dose-limiting skin toxicities were seen, one in cohort 3 with 600mg of sorafenib and the second in cohort 6 with 800mg of sorafenib. 800mg was the final recommended phase 2 dose. The median follow-up in 20 surviving patients was 7.6 months and 1-year progression free survival was 89% (95% CI 75-100%). Other outcomes are summarized in Table 1. Conclusion: Sorafenib can be safely added to the fractionated busulfan regimen. Early data on efficacy appear promising, with an 89% PFS at 1 year of follow up. Figure 1 Figure 1. Disclosures Popat: Bayer: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding. Hosing: Nkarta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Rezvani: Bayer: Other: Scientific Advisory Board ; AvengeBio: Other: Scientific Advisory Board ; Navan Technologies: Other: Scientific Advisory Board; GSK: Other: Scientific Advisory Board ; Virogin: Other: Scientific Advisory Board ; Affimed: Other: License agreement and research agreement; education grant, Patents & Royalties, Research Funding; Pharmacyclics: Other: Educational grant, Research Funding; Caribou: Other: Scientific Advisory Board; GemoAb: Other: Scientific Advisory Board ; Takeda: Other: License agreement and research agreement, Patents & Royalties. Qazilbash: Bristol-Myers Squibb: Other: Advisory Board; Biolline: Research Funding; Amgen: Research Funding; Oncopeptides: Other: Advisory Board; NexImmune: Research Funding; Angiocrine: Research Funding; Janssen: Research Funding. Daver: Daiichi Sankyo: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Abbvie: Consultancy, Research Funding; Hanmi: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Sevier: Consultancy, Research Funding; Novimmune: Research Funding; Trovagene: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Ravandi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Honoraria; Novartis: Honoraria; Xencor: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Astex: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Prelude: Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Shpall: Magenta: Consultancy; Bayer HealthCare Pharmaceuticals: Honoraria; Magenta: Honoraria; Adaptimmune: Consultancy; Novartis: Consultancy; Navan: Consultancy; Novartis: Honoraria; Takeda: Patents & Royalties; Affimed: Patents & Royalties; Axio: Consultancy. Mehta: CSLBehring: Research Funding; Kadmon: Research Funding; Syndax: Research Funding; Incyte: Research Funding.

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Allogeneic Transplantation for Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia with Post-Transplantation Cyclophosphamide: Assessing the Importance of Conditioning Regimen, Donor Choice, and Tyrosine Kinase Inhibitor Use

Blood ◽

10.1182/blood-2020-141294 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 44-45

Author(s):

Jonathan Webster ◽

Leo Luznik ◽

Hua-Ling Tsai ◽

Philip H. Imus ◽

Amy E. DeZern ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Conditioning Regimen ◽

Advisory Board ◽

Categorical Variables ◽

Advisory Committees ◽

Post Transplant ◽

Scientific Advisory Board ◽

Scientific Advisory ◽

Donor Type

Background: Contemporary trials in adult Ph+ ALL patients with TKIs continue to show improved outcomes with allogeneic blood or marrow transplantation (alloBMT) in first remission (CR1) (Chalandon. Blood. 2015 AND Ravandi. Blood Adv. 2016). These studies have relied on myeloablative conditioning (MAC) and largely required an HLA-matched donor. Post-transplant survival in Ph+ ALL has been shown to be similar between patients transplanted with reduced-intensity conditioning (RIC) and MAC, but the incidence of relapse after RIC is higher (Bachanova. Leukemia. 2014). Post-transplant TKI maintenance reduces the incidence of relapse (Brissot. Haematologica. 2015), but this strategy has not specifically been investigated after RIC. Additionally, HLA-haploidentical donor transplants using post-transplant cyclophosphamide (PTCy) as a component of graft-versus-host disease (GVHD) prophylaxis have comparable outcomes to HLA-matched transplants (McCurdy. Haematologica. 2017). We analyzed outcomes among patients who universally received PTCy and attempted post-transplant TKI prophylaxis to determine the importance of remission status (CR1 vs. later), conditioning regimen, donor type, and TKI choice. Methods: The bone marrow transplant database at Johns Hopkins was queried for adult patients with de novo Ph+ ALL who received alloBMT using PTCy between January 2008 and August 2018. Characteristics of patients were summarized and compared using the student's T test for continuous variables and Fisher's exact test for categorical variables. Estimators of OS and RFS were reported using the Kaplan-Meier method. Differences in time-to-event outcomes were estimated using Cox proportional hazards model. Results: A total of 81 transplants for Ph+ ALL were performed: 69 (85%) in CR1 and 12 (15%) in second or greater remission (CR2+). The demographics are presented in Table 1 and separated by conditioning regimen [MAC vs. nonmyeloablative (NMAC)] for transplants in CR1. The cumulative incidences of grade 2-4 and grade 3-4 aGVHD at 1 year were 33% (95% CI, 23% to 44%) and 9% (95% CI, 3% to 15%), respectively. The incidence of moderate or severe cGVHD at 2 years was 8% (95% CI, 2% to 13%). Nearly all patients (91.4%) initiated a post-transplant TKI at a median of 56 days. Overall, 44.4% of patients were able to take a TKI on ≥85% of nonrelapse days from day 31-395 post-transplant. AlloBMT in CR1 (compared to CR2+) improved RFS (HR=0.25, p=0.0002) and pre-transplant minimal residual disease (MRD) by flow cytometry (MFC) was associated with decreased RFS (HR=2.57, p=0.039). The presence of pre-transplant MRD by PCR did not confer an increased risk of relapse (HR 1.12, p=0.84). Among the 69 patients transplanted in CR1, the 5-year OS was 77.6% (95% CI, 64.8% to 86.2%) and RFS was 67% (95% CI, 52.4-76.5%). As shown in Figure 1, the use of NMAC versus MAC (HR 0.37, p=0.02) and dasatinib versus imatinib at diagnosis (HR 0.21, p=0.007) led to improved relapse-free survival (RFS) in univariate analyses. Neither donor type (with the majority being haploidentical) nor recipient age ≥60 affected RFS. Post-transplant TKI prophylaxis was discontinued prior to relapse in 20 patients among whom 12 remain in an MRD-negative remission, 4 died of non-relapse causes, 3 relapsed, and 1 developed recurrent MRD controlled by a TKI. The median duration of post-transplant TKI prophylaxis prior to discontinuation was 46.5 months in those who remain in treatment-free remission versus 15.6 months in those who relapsed (p=0.01). Eighteen relapses occurred on maintenance therapy, and 90% of tested cases were positive for a kinase domain mutation conferring resistance to the TKI in use at relapse. No significant difference in the median time to TKI initiation post-transplant was noted between those who relapsed on maintenance and those who did not (70 days vs. 55 days, p=0.6). All patients in ongoing remission were MRD-negative by PCR at their most recent evaluation. Conclusions: AlloBMT with PTCy in Ph+ ALL was most effective when performed in CR1 with negative MFC for MRD. The initiation of post-transplant TKI prophylaxis was nearly universal. Among patients transplanted in CR1, the best results were achieved in patients treated with dasatinib at diagnosis (5-year RFS 83%) and NMAC (5-year RFS 73.1%). Thus post-transplant TKI prophylaxis appeared to overcome any relapse control advantage for MAC, yielding better outcomes with NMAC. Disclosures Webster: Amgen: Consultancy; Pfizer: Consultancy. Luznik:WindMil Therapeutics: Patents & Royalties: Patent holder; Genentech: Research Funding; Merck: Research Funding, Speakers Bureau; AbbVie: Consultancy. DeZern:Abbvie: Consultancy; Astex: Research Funding; MEI: Consultancy; Celgene: Consultancy, Honoraria. Pratz:Jazz Pharmaceutical: Consultancy; Millennium: Research Funding; Daiichi Sankyo: Research Funding; Agios: Other: Scientific Advisory Board, Research Funding; Celgene: Other: Scientific Advisory Board; Boston BioMedical: Consultancy; Astellas: Other: Scientific Advisory Board, Research Funding; AbbVie: Other: Scientific Advisory Board, Research Funding. Levis:Astellas: Honoraria, Research Funding; Menarini: Honoraria; Amgen: Honoraria; FujiFilm: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria. Gojo:Amgen: Research Funding; Merck: Research Funding; Genentech: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Amphivena: Research Funding. Bolanos-Meade:Incyte: Other: DSMB Fees. Dalton:Eli Lilly: Research Funding; AbbVie: Research Funding. Jain:Takeda: Consultancy, Honoraria; Bristol Myer Squibb: Other: for advisory board participation; CareDx: Other: Advisory Board. Ali:Celgene: Membership on an entity's Board of Directors or advisory committees. Borrello:Celgene: Research Funding; Aduro: Patents & Royalties; WindMIL Therapeutics: Other: Founder , Research Funding. Wagner-Johnston:ADC Therapeutics, Regeneron, CALIB-R, Verastem: Membership on an entity's Board of Directors or advisory committees. Smith:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Clinical Utility of a Multi-Gene Next-Generation Sequencing Myeloid Panel in an Academic Hematology Practice

Blood ◽

10.1182/blood-2019-124445 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1408-1408

Author(s):

Pedro G Vianna ◽

Richard D. Press ◽

Henning Stehr ◽

Fei Yang ◽

Linda Gojenola ◽

...

Keyword(s):

Clinical Trial ◽

Clinical Practice ◽

Targeted Therapy ◽

Next Generation Sequencing ◽

Board Of Directors ◽

Research Funding ◽

Pathogenic Variant ◽

Advisory Committees ◽

Management Guidance ◽

Generation Sequencing

Background Next-generation sequencing (NGS) panels have created an unprecedented opportunity to interrogate a broad array of variants in a multiplex fashion. Few data have demonstrated how NGS testing impacts diagnosis and treatment decisions. In this retrospective analysis, we evaluated the clinical application of NGS myeloid panels (MP) in patients (pts) evaluated by physicians in the Stanford Division of Hematology. Methods The study was approved by the Stanford University IRB. The cohort consisted of 1,015 pts ≥18 years of age (median 66; range 18-96 years; 51% female). A total of 1,213 MPs obtained from peripheral blood (n=568) or bone marrow (n=645) from March 2017 to June 2018 were analyzed at Stanford (n=761) using the 54-gene TruSight® Myeloid Sequencing Panel (Illumina, San Diego, CA) or Oregon Health & Science University (n=452) using the GeneTrails® Hematologic Malignancies 76-Gene Panel. Electronic medical records were reviewed from t-3 to t+9 months from when MPs were obtained to assess physician reasoning for MP acquisition, documentation of results, and how results were clinically applied. We defined three categories of MP acquisition: 1) diagnostic clarification, 2) prognostication and/or management guidance, or 3) minimal residual disease (MRD) monitoring. We analyzed changes in clinical management, including addition of targeted or non-targeted therapeutics, clinical trial eligibility, or other practice recommendations. Results Of the 1,213 MPs, 882 (73%) demonstrated at least one pathogenic/ likely pathogenic variant (median 2; range 1-8). Median turn-around-time was 18 days (range 7-31) and average cost was $2,600. Of all MPs, 462 (38%) were obtained for diagnostic clarification, 732 (60%) for prognostication / management guidance in pts with known myeloid (n=701) or lymphoid (n=31) neoplasms, and 19 (2%) for MRD monitoring, although the assay was not designed for this indication. MPs were ordered to clarify a diagnosis for the following reasons: unexplained cytopenia(s) (n=199), molecular profiling for a suspected hematolymphoid neoplasm (n=156), unexplained -cytosis (n=86), and testing for other lab abnormalities (e.g. elevated serum tryptase, paraproteinemia) (n=18), or signs (e.g. splenomegaly, splenic vein thrombosis)(n=3). A pathogenic/likely pathogenic variant was found in 294 (64%) pts, confirming or establishing the presence of a myeloid (n=266) or lymphoid neoplasm (n=7) or resulting in a diagnosis of CHIP (n=9) or CCUS (n=12) in pts who did not meet 2016 World Health Organization diagnostic criteria for a hematolymphoid neoplasm (Fig. 1). Of the 732 MPs ordered for prognostication/ management guidance, 272 MPs (37%) were obtained in the initial workup of non-APL AML pts. The frequency of favorable (21%), intermediate (55%), and adverse risk (24%) genetics according to the European LeukemiaNet stratification in non-APL AML is shown in Figure 2a, which also denotes the frequency of favorable (9%) or adverse risk (70%) variants in MDS, and adverse risk variants in MF (52%), MDS/MPN (68%), and advanced systemic mastocytosis (44%). Among MPs obtained for prognostication/ management guidance, 163 (22%) led to a modification in clinical practice, divided between 132 (18%) which led the physician to change therapy (e.g. FDA-approved targeted therapy, clinical trial, or FDA-approved therapy, such as hypomethylating agents in MDS) and 31 (4%) resulted in a non-therapeutic change (e.g. expedited HSCT referral or more frequent follow-up)(Fig. 2b). 87 pts with a myeloid neoplasm had 184 repeat MPs for relapsed/refractory disease (n=52), transformation to higher-risk MDS or AML (n=45) or progressive cytopenias (n=87). Among these MPs, 38 (21%) identified a new pathogenic/likely pathogenic variant of which 29% (n=11) led to either a) initiation of targeted therapy with enasidenib in relapsed IDH2+ AML (n=3) or midostaurin for secondary FLT3+ AML from MDS (n=3), or b) consideration for a clinical trial with a splicing modulator for MDS characterized by a splicing variant (n=5). Conclusion In our academic hematology practice, two-thirds of MPs ordered for diagnostic clarification identified a pathogenic/likely pathogenetic variant that helped to confirm or establish a new diagnosis of a hematolymphoid neoplasm, CHIP, or CCUS. In addition, approximately 20% of MPs ordered for prognostication/ management guidance led to a change in clinical practice. Disclosures Gotlib: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Promedior: Research Funding; Pharmacyclics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Allakos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Deceiphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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