New Blood Based M-Protein Quantification Method 3,000 Times More Sensitive Than Standard SPEP

Abstract Summary of Work The amino acid sequence of the M-protein for multiple myeloma (MM) is unique compared to the polyclonal antibodies in patients' blood. In this study we utilize this uniqueness to develop an ultra-sensitive M-protein detection method with mass spectrometry (MS). The method involves the de novo sequencing of the amino acid sequence of the M-protein from the baseline blood sample collected at the time of diagnosis, and a targeted MS assay to detect and quantify the unique M-protein sequence in the follow-up blood samples. This non-invasive method is purely blood based and is 3,000 and 300 times more sensitive than SPEP and IFE, respectively. De Novo Protein Sequencing The M-protein sequencing is carried out on an MS based platform (REmAb). The effectiveness of de novo protein sequencing has been proven with the sequencing of hundreds of monoclonal antibodies (McDonald et al., Poster 294737, ASMS, 2018). In the current study the M-protein from 4 MM patients' baseline blood samples were successfully sequenced, demonstrating robustness of the method in the presence of the polyclonal background. As little as 50ul of the serum was required for the de novo sequencing. Sensitivity of M-protein Quantification The lower limit of quantification (LLOQ) was studied with a serial dilution experiment. The baseline blood sample of one patient was sequentially diluted with a healthy donor's serum. The peptide sequence unique to the M-protein was monitored with mass spectrometry to detect and quantify the M-protein in the serial dilution. The M-protein could still be detected and quantified when the dilution ratio was 1:10,000 (the amount of M-protein relative to background polyclonal serum IgG). In a separate experiment, synthesized heavy labelled proteotypic peptides were used to estimate the LLOQ in IgG enriched serum at 60 ug/dL, over 3,000 times more sensitive than SPEP (0.2g/dL) (Bergen et al., Clin Chem 62: 1 243-251, 2016) and 300 times more sensitive than IFE (0.02g/dL) (IMWG, British Journal of Haematology, 121, 749-757, 2003). As little as 30ul of serum was required in these experiments for monitoring of M-protein levels. Case Study A targeted MS based assay was developed to monitor the M-protein levels of a serial patient sample set (73-year-old male, treatment: Elotuzumab/Lenalidomide/Dexamethasone, progression free survival 32 months). M-protein levels were quantified in a total of 10 serial samples from partial remission, through complete remission (CR), until relapse over a period of 2 years and 3 months (Figure 1). M-protein could be detected and quantified in all samples collected during the CR period with estimated M-protein levels never below 10mg/dL. Notably, a 2-fold increase in M-protein levels (in comparison to the lowest historic level) could be detected 320 days prior to the timepoint of relapse. The upward trend continued in the next 3 serial CR samples preceding relapse. Except for one sample (see Figure 1, 'CR Before Relapse 12-14-2016'), CR was based on the absence of M-protein in the IFE result in the relevant clinical data. Conclusion The work represents a first step in the application of de novo sequencing and MS based detection for the sensitive monitoring of M-protein levels in serum. It shows a much-improved sensitivity over current standard approaches and has the great potential to provide non-invasive assessment of MRD for multiple myeloma. The preliminary data warrant further development of this MS based non-invasive and highly sensitive M-protein quantification method. Disclosures McDonald: Rapid Novor Inc: Employment, Equity Ownership. Liu:Rapid Novor Inc: Employment, Equity Ownership. Taylor:Rapid Novor Inc: Employment, Equity Ownership. Yang:Rapid Novor Inc: Employment, Equity Ownership. Ma:Rapid Novor Inc: Equity Ownership.

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M-Protein Sequencing and Monitoring in Serum of LC-Only Multiple Myeloma Patients

Blood ◽

10.1182/blood-2021-149785 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4729-4729

Author(s):

Mariya Liyasova ◽

Natalia Migdal ◽

Zac McDonald ◽

Liqiang Yang ◽

Bin Ma ◽

...

Keyword(s):

Multiple Myeloma ◽

Research Funding ◽

Protein A ◽

Disease Status ◽

Protein Sequencing ◽

M Protein ◽

Newly Diagnosed ◽

Protein Levels ◽

Non Invasive ◽

Q Exactive

Abstract Background M-protein, a secreted antibody of malignant plasma cells, is a gold standard biomarker for monitoring the disease status in multiple myeloma (MM) patients (pts). The development of peripheral blood based ultrasensitive (MRD) methods of M protein detection is of high interest and importance. In 20% of MM pts, the M-protein consists of only the light chain (LC) of the immunoglobulin (Ig) molecule. For these LC-only MM pts, disease monitoring is challenging due to low levels of M-protein in serum. Urine protein electrophoresis (UPEP) and immunofixation electrophoresis (IFE), or serum free light chain (FLC) lack the sensitivity and/or specificity to track the M-protein in these pts, while bone marrow-based assays cannot be performed frequently due to their invasive nature. Thus, we evaluated the performance of EasyM, a mass spectrometry(MS)-based, non-invasive, sensitive assay for monitoring M-protein levels in LC-only patients in the MCRN-001 Canadian national and MD Anderson VRD-panobinostat frontline trials. Methods MCRN-001 trial is evaluating enhanced conditioning prior to ASCT for newly diagnosed MM (NDMM). After treatment with bortezomib (BTZ) based induction eligible MM pts received BuMel prior to ASCT. Busulfan was administered via IV at 3.2 mg/kg on days -5 to -3, or days -6 to -4 pre-ASCT (day 0) and melphalan was given at 140 mg/m 2 on day -2 or -3 pre-ASCT. Lenalidomide (LEN) administration began 100 days post-ASCT at 10 mg/d and continued until progressive disease (PD) onset. In the MDACC 2011-0192 frontline study in newly diagnosed MM, transplant-eligible pts received the novel combination of LEN, BTZ, dexamethasone (DEX) and panobinostat (RVD-panobinostat). The IMWG criteria were used to monitor clinical response in both trials. A total of 13 LC-only MM pts were selected for the study. Local IRB approval was obtained prior to the study. To derive the M-protein's full amino acid sequence, FLC was first enriched from the diagnostic serum sample. The FLC enrichment consisted of IgG depletion with protein A/G beads, followed by affinity purification of kappa or lambda LC containing Igs. Non-reducing PAGE was then used to separate FLC monomers, FLC dimers and full-length Igs. Finally, in-gel digestion of FLC monomers and dimers by multiple proteolytic enzymes were analyzed on a Q-Exactive mass spectrometer. Data analysis and sequence assembly were performed with the REmAb protein sequencing platform. To monitor M-protein levels in serum, unique, tryptic peptides from sequenced FLC were selected and quantified in diagnostic and follow-up samples with a PRM assay on a Q-Exactive instrument. Results M-protein sequencing The full FLC sequence was derived for 8 out of 13 (61.5%) LC-only pts. The M-protein was successfully sequenced even when the FLC concentration was as low as 147 mg/L. However, FLC concentration measured by FreeLite assay was not a reliable predictor of our ability to derive the full sequence. The appearance of a sharp FLC monomer and dimer bands on PAGE after FLC enrichment was a better predictor of the M-protein sequencing success. M-protein monitoring The M-protein of one pt did not contain any unique peptides. This pt was excluded for further analysis. In the remaining 7 pts, the M-protein contained at least one unique peptide and could thus be monitored by EasyM. For 6 LC-only pts, the M-protein monitored by EasyM correlated with the disease status measured by serum FLC, UPEP and urine IFE. A separate serial dilution test estimated that the limit of quantification can reach as low as 0.13 mg/L. Figure 1 shows representative data for two LC-only patients. One pt experienced relapse during the study; however, this relapse could not be detected by EasyM. This result could indicate a possible clonal switch at time of disease progression, but further investigation is needed to verify this. Conclusions Due to the rapid turnover and clearance of light chains conventional blood/urine tests have lacked the higher sensitivity needed to monitor disease state in LC only MM patients. To overcome the lower FLC concentration the current study successfully applied LC enrichment strategies to enable the sequencing and high sensitivity monitoring of FLC M-protein by MS in blood. The EasyM LC assay is non-invasive, sensitive, capable of assessing disease status, and has potential to be further investigated as a peripheral blood myeloma response and MRD monitoring biomarker. Figure 1 Figure 1. Disclosures Ma: Rapid Novor Inc.: Current holder of individual stocks in a privately-held company. Reece: Millennium: Research Funding; Amgen: Consultancy, Honoraria; GSK: Honoraria; Karyopharm: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Honoraria; BMS: Honoraria, Research Funding. Manasanch: GSK, Secura Bio,Takeda, Celgene, Sanofi, Janssen and Adaptive Biotechnologies: Consultancy; Sanofi, Quest Diagnostics, Novartis, JW Pharma, Merck: Research Funding. Orlowski: Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, EcoR1 Capital LLC, Genzyme, GSK Biologicals, Janssen Biotech, Karyopharm Therapeutics, Inc., Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, Inc., Sanofi-Aventis, and Takeda P: Consultancy, Honoraria; CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Other: Clinical research funding; Asylia Therapeutics, Inc., BioTheryX, Inc., and Heidelberg Pharma, AG.: Other: Laboratory research funding; Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, Forma Therapeutics, Genzyme, GSK Biologicals, Janssen Biotech, Juno Therapeutics, Karyopharm Therapeutics, Inc., Kite Pharma, Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, I: Membership on an entity's Board of Directors or advisory committees. Trudel: GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Roche: Consultancy; Genentech: Research Funding; Pfizer: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Sanofi: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding.

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Analysis of Patient (Pt) Outcomes By Prior Treatment and Depth of Response in Stratus (MM-010), a Phase 3b Study of Pomalidomide + Low-Dose Dexamethasone (POM + LoDEX) in Patients (Pts) with Relapsed/Refractory Multiple Myeloma (RRMM)

Blood ◽

10.1182/blood.v126.23.1834.1834 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1834-1834

Author(s):

Philippe Moreau ◽

Antonio Palumbo ◽

Paolo Corradini ◽

Michele Cavo ◽

Michel Delforge ◽

...

Keyword(s):

Research Funding ◽

Prior Treatment ◽

M Protein ◽

Employment Equity ◽

Clear Trend ◽

Protein Levels ◽

Treatment History ◽

Depth Of Response ◽

Equity Ownership ◽

Support Research

Abstract Introduction: The introduction of newer agents, such as lenalidomide (LEN) and bortezomib (BORT), has resulted in improved survival outcomes in pts with RRMM (Kumar, Leukemia, 2012), and a greater depth of response has been associated with improved efficacy outcomes in RRMM (Harousseau, Haematologica, 2010). However, once pts become refractory to these newer agents, they experience poorer outcomes, with short overall survival (OS; Kumar, Leukemia, 2012). POM + LoDEX is approved for the treatment of pts with RRMM with ≥ 2 prior treatments, including LEN and BORT. A previous subanalysis of pts treated with POM + LoDEX in the MM-003 trial demonstrated that pts with a deeper response, as measured by a reduction in serum M protein, experienced longer OS and progression-free survival (PFS; San Miguel, ASH 2013). POM + LoDEX was shown to be safe and effective in the phase 3b STRATUS trial (MM-010; Dimopoulos, EHA 2015); here, we present the outcomes in this pt population by prior treatment and depth of response. Patients and Methods: Pts with RRMM (progressive disease [PD] on or within 60 days of last prior treatment) with prior adequate alkylator therapy in whom LEN and BORT treatment failed were eligible. POM 4 mg was administered on days 1 to 21 of a 28-day cycle in combination with LoDEX (40 or 20 mg for pts aged ≤ 75 or > 75 yrs, respectively) on days 1, 8, 15, and 22. Treatment was continued until PD or unacceptable toxicity, and thromboprophylaxis was required for all pts. For this efficacy analysis, pts were grouped according to prior treatment history or reduction in serum M protein level. Results: As of May 4, 2015, 682 pts were enrolled and 676 have received POM + LoDEX. In the intent-to-treat population, the median age was 66 yrs, 56% of pts were male, the median time since diagnosis was 5.3 yrs, and pts had received a median of 5 prior regimens. Approximately half of all pts (54.5%) had received prior thalidomide (THAL), and 186 vs 496 pts had received ≤ 3 vs > 3 prior regimens of therapy, respectively. Most pts were refractory to LEN (96%), BORT (84%), or both LEN and BORT (80%). The overall response rates (ORRs) were generally similar regardless of prior THAL, number of prior regimens, and LEN/BORT refractoriness. Pts who received prior THAL had an ORR of 30.4% compared with 35.2% for pts who did not receive prior THAL (Table). Similarly, for pts with a history of ≤ 3 vs > 3 prior antimyeloma regimens, ORR was 28.5% vs 34.1%. ORR did not appear to be impacted by refractory status to LEN and/or BORT (ORR, 32.1%-32.9%). PFS was comparable and independent of prior THAL exposure, number of prior regimens, and LEN/BORT refractoriness (PFS, 3.9-4.6 mos). Median OS was also similar for pts with or without prior THAL exposure (11.4 mos vs 12.0 mos, respectively), as was OS for pts with ≤ 3 vs > 3 prior regimens (12.8 mos vs 11.9 mos, respectively). Pts with LEN and/or BORT refractoriness had the same median OS of 11.9 mos. Analysis of PFS by serum M protein levels showed that a greater reduction in these levels was associated with a longer median PFS: for pts with a serum M protein reduction of < 25%, ≥ 25% to < 50%, and ≥ 50%, median PFS was 3.0, 4.8, and 7.6 mos, respectively. Conclusions: The efficacy of POM + LoDEX in this heavily pretreated population was independent of treatment history. ORR, OS, and PFS were not significantly impacted by treatment history with THAL, having ≥ 3 prior regimens, or refractoriness to LEN and/or BORT. As shown previously with POM + LoDEX, there was a clear trend toward prolonged PFS in pts with a greater reduction in serum M protein levels. This analysis supports POM + LoDEX as a standard of care for pts with RRMM. Figure 1. Figure 1. Disclosures Moreau: Celgene: Honoraria, Other: Adboard; Takeda: Other: Adboard; Janssen: Other: Adboard; Amgen: Other: Adboard; Novartis: Other: Adboard. Palumbo:Celgene, Millennium Pharmaceuticals, Amgen, Bristol-Myers Squibb, Genmab, Janssen-Cilag, Onyx Pharmaceuticals: Consultancy, Honoraria; Novartis, Sanofi Aventis: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Delforge:Novartis: Honoraria; Celgene Corporation: Honoraria; Janssen: Honoraria; Amgen: Honoraria. Weisel:Amgen: Consultancy, Honoraria, Other: Travel Support; Noxxon: Consultancy; Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Travel Support, Research Funding; Novartis: Other: Travel Support; Onyx: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Other: Travel Support. Knop:Celgene Corporation: Consultancy. de Arriba:MundiPharma: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Speakers Bureau. Simcock:Celgene Corporation: Employment. Miller:Celgene Corporation: Employment, Equity Ownership. Slaughter:Celgene Corporation: Employment, Equity Ownership. Watkins:Celgene Corporation: Employment. Herring:Celgene Corporation: Employment. Biyukov:Celgene: Employment, Equity Ownership. Peluso:Celgene Corporation: Employment, Equity Ownership. Zaki:Celgene Corporation: Employment, Equity Ownership. Dimopoulos:Amgen: Honoraria; Novartis: Honoraria; Genesis: Honoraria; Janssen-Cilag: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Onyx: Honoraria.

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Targeted Mass Spectrometry-Based Serum M-Protein Monitoring for Early Relapse Detection

Blood ◽

10.1182/blood-2019-130251 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4347-4347

Author(s):

Zac McDonald ◽

Mariya Liyasova ◽

Paul Taylor ◽

Xin Xu ◽

Kathleen Gorospe ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Fold Increase ◽

Protein Sequencing ◽

M Protein ◽

Patient Specific ◽

Advisory Committees ◽

Protein Levels ◽

Equity Ownership ◽

Early Relapse Detection

Background For most Multiple Myeloma (MM) patients, M-protein is the gold standard biomarker for disease diagnosis and monitoring. Traditional methods such as serum protein electrophoresis (SPEP) and serum immunofixation electrophoresis (IFE) lack the sensitivity to detect M-protein in patients with minimal residual disease. At ASH 2018, we reported a Mass Spectrometry (MS)-based highly sensitive assay (REmAb) for detecting and quantifying M-protein. This assay exploits the uniqueness of the M-protein sequence and can detect 1 M-protein in 10,000 polyclonal IgG. The current study utilized this established assay to first sequence then monitor M-protein in sera from diagnosis through treatment for patients from the first MCRN-001 Canadian national trial with bortezomib (BTZ)-based pre-induction (PI), augmented high-dose chemotherapy with busulfan + melphalan (BuMel) before ASCT and lenalidomide (Len) maintenance post-ASCT. Methods MCRN-001 trial patients were first induced with BTZ before harvesting stem cells. Eligible MM patients received BuMel prior to ASCT. Busulfan was administered IV at 3.2 mg/kg on days -5 to -3, or days -6 to -4 days pre-ASCT (Day 0) and melphalan was given at 140 mg/m2 on day -2 or -3 pre-ASCT. Len administration began 100 days post-ASCT at 10 mg/d (increased when appropriate to 15 mg/d) and continued until progressive disease (PD) onset. IMWG criteria was used to monitor clinical response and PD. 78 patients enrolled in this trial; only 58 offered consent for M-protein monitoring with MS. Serum samples were acquired at time of pre-induction (PI), before ASCT (screening sample), post-ASCT on day 100, every 3 months for the first year and then every 6 months until PD. The M-protein sequence was determined with the REmAb protein sequencing platform from either PI or screening samples when the PI sample was unavailable. For M-protein quantification, each sample was digested with trypsin prior to analysis by liquid chromatography tandem MS with an Orbitrap Fusion Tribrid or Q-Exactive instrument. Patient-specific, unique tryptic peptides, with at least one peptide from each Heavy and Light chain, were targeted by the MS assay. To monitor changes in M-protein levels per patient post-diagnosis and through treatment to complete remission (CR) and/or PD, this study used MS peak areas of patient-specific unique peptides normalized against peak areas of human serum albumin or spiked-in standard peptides. Results M-protein sequencing In this study, the lowest M-protein serum concentration required for sequencing was 0.2 g/dL. 48 out of 58 (83%) patient-specific M-proteins were sequenced from serum. 5 out of 48 patients sequenced discontinued the study early and were excluded from further analysis. M-protein monitoring 24 out of the 43 achieved CR. The M-proteins of these patients were monitored by MS to study early relapse detection. M-protein levels could be monitored by MS in all these patients from diagnosis through CR to PD onset, even when M-protein was undetectable by SPEP and IFE. A separate serial dilution test estimated that the limit of quantification can reach as low as 0.03 mg/dL. 3 patients who had achieved CR eventually relapsed (PD). In all 3 PD patients, a 2 to 200 fold increase in M-protein in 2 consecutive tests half a year apart was detected by MS 6 months earlier than clinical confirmation of PD by conventional testing. The other 21 had not progressed at time of analysis. In the non-progressor group, the M-protein levels detected by MS either continued to decrease during CR or remained relatively stable. Only 3 out of 21 patients demonstrated a 2-fold increase in M-protein level in any 2 consecutive tests. Figure 1 shows representative data for two patients. Conclusions This study demonstrates that M-protein sequencing and targeted MS assay can be used to monitor serum M-protein levels sensitively even when SPEP and IFE fail to detect M-protein. Based on this assay, a 2-fold increase in serum M-protein levels in the past 6 months can reliably predict disease progression in the next 6 months for patients achieving CR. In the 24 studied patients, the method has 100% sensitivity and 86% specificity at predicting disease progression from CR prior to detection by standard methods. Future work will analyze more patient samples to confirm the findings and investigation into criteria surrounding the 2-fold M-protein increase observed in CR patients and relapse post CR. Disclosures McDonald: Rapid Novor Inc Kitchener: Employment, Equity Ownership, Research Funding. Liyasova:Rapid Novor Inc Kitchener: Employment. Taylor:Rapid Novor Inc Kitchener: Employment. Xu:Rapid Novor Inc Kitchener: Employment. Gorospe:Rapid Novor Inc Kitchener: Employment. Yao:Rapid Novor Inc Kitchener: Employment. Liu:Rapid Novor Inc Kitchener: Employment, Equity Ownership. Yang:Rapid Novor Inc Kitchener: Employment. Ma:Rapid Novor Inc Kitchener: Equity Ownership. Reece:Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; Merck: Research Funding; BMS: Research Funding. Trudel:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Genentech: Research Funding; Sanofi: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Janssen: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: The use of IV busulfan and melphalan as conditioning for myeloma transplants is off-label.

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

Blood ◽

10.1182/blood.v128.22.1650.1650 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1650-1650

Author(s):

Tsila Zuckerman ◽

Stela Gengrinovitch ◽

Ruth Ben-Yakar ◽

Ron Hoffman ◽

Israel Henig ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

De Novo ◽

Intensive Therapy ◽

High Dose ◽

Newly Diagnosed ◽

Employment Equity ◽

Unfit Patients ◽

Equity Ownership ◽

High Doses

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

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Mutations In SETBP1 Occur In 3.1% Of De Novo AML and Show a Distinct Genetic Pattern That Highly Resembles Atypical CML

Blood ◽

10.1182/blood.v122.21.2560.2560 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2560-2560

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Elisabeth Sirch ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Sanger Sequencing ◽

De Novo ◽

Complex Karyotype ◽

Employment Equity ◽

Cytogenetic Abnormalities ◽

Myeloid Malignancies ◽

Genetic Pattern ◽

Equity Ownership ◽

De Novo Aml ◽

Atypical Cml

Abstract Introduction Recently, mutations in SETBP1 (SETBP1mut) have been identified in different myeloid malignancies. We previously determined mutation frequencies in the range of 5-10% in MPN and MDS/MPN overlap, while we found SETBP1 more frequently mutated in atypical CML (32%). SETBP1mut has been shown to associate with CBL and ASXL1 mutations, as well as the cytogenetic abnormalities -7 and i(17)(q10). While SETBP1 mutations have been detected in 3% of s-AML cases, so far no mutations of SETBP1 in de novo AML have been described. Aim To analyze the mutation frequency of SETBP1 mutations in de novo AML with corresponding cytogenetic abnormalities and their respective correlation to clinical data and other gene mutations. Patients and Methods We investigated 422 adult de novo AML patients, diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO classification. SETBP1 was analyzed by Sanger sequencing of the coding region for amino acids 800 to 935. The cohort comprised 229 males and 193 females, the median age was 65.8 years (range: 19.3 – 89.0). Cytogenetics was available in all 422 cases. Based on the previously described association of SETBP1mut with -7 and i(17)(q10) in other myeloid malignancies there was a selection bias to these karyotypes. Cases were grouped according to cytogenetic abnormalities: normal karyotype (n=88) and aberrant karyotype (n=334), i.e. i(17)(q10) (n=15), +14 (n=20), -7 (n=100), other abnormalities (n=129), and complex karyotype (n=114; 44 contained i(17)(q10), +14 or -7). Within the SETBP1mut cases the following molecular markers were analyzed: ASXL1, CBL, CEBPA, FLT3-ITD, FLT3-TKD, IDH1/2, KRAS, NRAS, NPM1, MLL-PTD, RUNX1, SRSF2, TP53 and WT1 by Sanger sequencing, next generation sequencing, gene scan or melting curve analyses. These data were also available in sub-cohorts of SETBP1 negative cases. Results In the total cohort mutations in SETBP1 were detected in 3.1% (13/422) of all cases. SETBP1mut patients were older (median age: 73.5 vs. 65.7 years; p=0.05) and showed a slightly higher white blood cell count (14.5 vs. 13.8x109/L; p<0.001). There was no correlation to gender, hemoglobin level and platelet count. However, analyzing the cytogenetic groups SETBP1mut showed, like in other myeloid malignancies, a strong co-occurrence with -7 and i(17)(q10), since 4/13 SETBP1 positive cases carried a monosomy 7, and 7/13 an i(17)(q10). The two remaining cases showed a trisomy 14 or a complex karyotype that also contained a i(17)(q10). No SETBP1mut was found in any other cytogenetic subgroup. Therefore, SETBP1mut correlated significantly with i(17)(q10) (8/15 i(17)(q10) were SETBP1mut vs. 5/407 in all other karyotypes; p<0.001). Further, we analyzed the association of SETBP1 mutations with other molecular markers. SETBP1mut correlated with ASXL1mut, 9/33 (27%) ASXL1mut patients showed a mutation in SETBP1, while only 2 (1%) showed a SETBP1 mutation in 229 ASXL1 wild type (wt) patients (p<0.001). This was also true for CBLmut, where 4/8 (50%) CBLmut cases were SETBP1mut, while only 8/158 (5%) were SETBP1mut in the group of CBLwt (p=0.001). This was even more prominent in SRSF2mut patients, where all 9 SRSF2mut were also SETBP1mut, while only 4/8 (50%) patients carried a SETBP1 mutation within the SRSF2wt group (p=0.029). In contrast, SETBP1mut were mutually exclusive of mutations in TP53 (0/67 in TP53mut vs. 12/194 in TP53wt; p=0.04), possibly reflecting the exclusiveness of TP53mut in i(17)(q10) patients. There was no correlation to any other analyzed gene mutation. Remarkably, while there was a high coincidence of SETBP1mut, SRSF2mut (9/13) and ASXL1mut (9/11), none of these patients showed mutations in the typical AML markers NPM1, FLT3-ITD, CEBPA, MLL-PTD, or WT1. Comparing the mutational loads of SETBP1, ASXL1 and SRSF2 resulted in SRSF2 having in most cases the highest mutational loads (range: 30-70%) while ASXL1 and SETBP1 showed equal or lower mutational loads (15-50% and 10-50%, respectively), possibly indicating that SRSF2 mutation is a former event followed by ASXL1 and SETBP1 mutation. Conclusions 1) For the first time we describe, that SETBP1 mutations occur in de novo AML. 2) SETBP1 mutations are correlated with a distinct genetic pattern with high association to i(17)(q10), ASXL1 and SRSF2 mutations and are mutually exclusive of TP53mut. 3) Thus, the genetic pattern of SETBP1 mutated AML highly resembles that of atypical CML. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Sirch:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Assessment Of Human Equilibrative Nucleoside Transporter 1 (hENT1) – Expression By Flow Cytometry In Acute Myeloid Leukemia and Myelodysplastic Syndromes and Correlation To Clinical Outcome In Acute Myeloid Leukemia Patients Treated With Intensive Chemotherapy

Blood ◽

10.1182/blood.v122.21.2582.2582 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2582-2582 ◽

Cited By ~ 1

Author(s):

Frauke Bellos ◽

Bruce H. Davis ◽

Naomi B. Culp ◽

Birgitte Booij ◽

Susanne Schnittger ◽

...

Keyword(s):

Flow Cytometry ◽

Myeloid Leukemia ◽

De Novo ◽

Molecular Genetic ◽

Newly Diagnosed ◽

Employment Equity ◽

Equity Ownership ◽

De Novo Aml ◽

Very High ◽

Acute Myeloid

Abstract Background Nucleoside analogs depend on cellular hENT1 expression for entry into cells and cytotoxic activity. Studies suggest low cellular hENT1 levels correlate with poor response to such chemotherapies in solid tumors, data on AML and MDS is scarce. Aim To examine hENT1 expression by multiparameter flow cytometry (MFC) in newly diagnosed AML and MDS and correlate results to morphologic, cytogenetic (CG) and molecular genetic (MG) findings. To examine hENT1 expression with respect to clinical outcome in AML patients (pts) treated with intensive cytarabine-based chemotherapy (CHT). Methods We studied pts with newly diagnosed AML (n=145) and MDS (n=96), 133/108 male/female, median age 67.3 (AML) and 73.3 years (MDS). CG was done in 130 AML and 86 MDS. Pts included 107 de novo AML, 9 t-AML, 29 s-AML; FAB: 9 M0, 27 M1, 50 M2, 9 M3, 21 M4, 8 M4eo, 7 M5, 14 not classified; by CG (MRC): 21 favorable, 75 intermediate, 34 adverse. 91 were de novo MDS, 5 t-MDS; 1 RARS, 17 RCMD-RS, 37 RCMD, 3 5q- syndrome, 3 RAEB-1, 5 RAEB-2, 1 CMML, 24 not classified; 2 IPSS-R very low, 55 IPSS-R low, 8 IPSS-R intermediate, 8 IPSS-R high, 13 IPSS-R very high. hENT1 expression was quantified by a novel four color intracellular staining assay using monoclonal antibodies against hENT1, CD45, CD64 and myeloperoxidase. Median fluorescence intensities (MFI) of hENT1 were determined in myeloid progenitors (MP), granulocytes (G) and monocytic cells (Mo) and correlated to hENT1 MFI in lymphocytes to derive hENT1 index (index). Results No correlation of index to age, gender, hemoglobin level or counts for blasts, WBC or platelets was detected. In AML, we generally saw higher index by trend in the more favorable prognostic subgroups. M3/t(15;17)/PML-RARA+ displayed higher index in MP than non-M3 AML (4.24 vs 2.56, p<0.001). G index was lower in M0 (3.01) vs M1, M2, M4 and M4eo (5.66, 4.34, 5.35, 4.77; p=0.01, 0.028, 0.004, 0.043, respectively) and in M2 compared to M1 and M4 (4.34. vs 5.66 and 5.35, p=0.01 and 0.033, respectively). M2 showed lower MP index than M5 (2.42 vs 2.99, p=0.016). Considering CG, index in MP was higher in favorable vs intermediate and adverse pts (3.05 vs 2.58 and 2.53, p=0.034 and 0.023, respectively), Mo index was higher ín favorable vs adverse pts (3.17 vs 2.71, p=0.044). By MG, higher index in Mo and G was observed in RUNX1-RUNX1T1+ AML (4/83, 4.32 vs 3.04, p=0.01; 8.16 vs 4.60, p=0.002, respectively). Higher index for MP was found in FLT3-ITD mutated (mut) (18/111; 3.19 vs 2.62, p=0.012), CEPBA mut (4/26, 3.15 vs 2.35, p=0.004) and for Mo in NPM1 mut AML (23/104; 3.72 vs 2.84, p=0.02), whereas lower index for MP was found in RUNX1mut pts (13/65; 2.17 vs 2.59, p=0.031). De novo AML displayed higher MP index than s-AML (2.7 vs 2.28, p=0.008). Using lowest quartile of index for MP (2.1185) as cut-off, AML pts in the MRC intermediate group treated with CHT (n=38) had inferior OS if MP index was below vs above this cut-off (OS at 6 months 63% vs. 95%, p=0.017, median follow up 4.6 months). MDS showed lower Mo and MP index than AML (2.68 vs 2.96, p=0.021, 1.84 vs 2.65, p<0.001, respectively). By IPSS-R, significance was reached for higher index in Mo and MP in very low risk compared to low risk pts (3.39 vs 2.54, p=0.013 and 4.07 vs 1.78, p<0.001, respectively), MP in very low compared to intermediate and high risk pts (4.07 vs 1.95, p=0.004; 4.07 vs 1.76, p=0.002), and MP and G in very low vs very high risk pts (4.07 vs 1.71, p=0.005; 5.86 vs 3.85, p=0.001, respectively). IPSS-R intermediate vs poor and very poor showed lower G index (5.47 vs 3.59, p=0.018 and vs 3.85, p=0.034 respectively). Conclusion AML with genetic and molecular genetic good risk profile had higher hENT1 expression in MP, G and Mo, suggesting a causal mechanism for better response to CHT and better outcome. Consequently, AML with poor risk molecular genetics (RUNX1 mut) showed lower levels of hENT1 in MP. The detection of higher levels in FLT3-ITD mut pts is in line with reportedly good response to CHT, overall worse outcome being mostly due to early relapses. Strikingly, we saw differences in outcome in pts treated with CHT according to hENT1 expression with shorter OS in pts with low index for MP. Higher index in de novo AML than s-AML and MDS may be causal for better response to nucleoside-based CHT in de novo AML. Data for MDS may be interpreted accordingly, lower risk cases showing higher index in MP, G and Mo. Further analyses are needed to explore hENT1 expression in AML and MDS more comprehensively. Disclosures: Bellos: MLL Munich Leukemia Laboratory: Employment. Davis:Trillium Diagnostics, LLC: Equity Ownership. Culp:Trillium Diagnostics, LLC: 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. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Myeloid Malignancies with Isolated 7q Deletion Can be Further Characterized By Their Accompanying Molecular Mutations

Blood ◽

10.1182/blood.v126.23.3811.3811 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3811-3811

Author(s):

Claudia Haferlach ◽

Annette Fasan ◽

Manja Meggendorfer ◽

Melanie Zenger ◽

Susanne Schnittger ◽

...

Keyword(s):

Array Cgh ◽

De Novo ◽

Jak2 V617f ◽

Terminal Deletion ◽

Employment Equity ◽

Myeloid Malignancies ◽

Additional Chromosome ◽

Equity Ownership ◽

De Novo Aml ◽

7Q Deletion

Abstract Background: 7q deletions (del(7q)) are recurrent cytogenetic abnormalities. They occur either as the sole abnormality or accompanied by additional chromosome aberrations in AML, MDS, MDS/MPN and MPN. Cases with del(7q) as the sole abnormality are rare and poorly characterized. Aim: In patients with myeloid malignancies and del(7q) as the sole abnormality we determined 1. Type and size of the del(7q) 2. Spectrum of accompanying molecular mutations and their impact on the phenotype. Patients and Methods: 81 cases with myeloid malignancies and del(7q) as the sole abnormality were included in this study. Of these 38 had AML (27 de novo, 7 secondary, 4 therapy-related), 17 MDS (14 de novo, 3 therapy-related), 10 MDS/MPN (9 CMML, 1 MDS/MPN unclassifiable) and 16 MPN. The median age was 72 years (range: 29-89 years). All cases were investigated by array CGH (Agilent, Waldbronn, Germany) and for mutations in ASXL1, CALR, CBL, DNMT3A, ETV6, EZH2, JAK2, KRAS, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, and TP53. Results: Array CGH revealed an interstitial del(7q) in 67 cases, while 14 cases showed terminal del(7q). Further characterization of these deletions using 24 color FISH revealed unbalanced translocations in 10 of the 14 cases with terminal deletion. Partner chromosomes were X, 8, 9, 12, 13, 17 (n=2), 19 (n=2), and 22. The breakpoints on chromosome 7 were diverse ranging from 7q11 to 7q32. In two cases the breakpoint was within the CDK6 gene. In two cases with terminal del(7q) the complete loss of 7q was due to an idic(7)(q11.21). In the remaining two cases the terminal deletion could not be further resolved. In the 67 cases with interstitial del(7q) the size of the del(7q) varied between 1.8 and 158.9 Mb (median: 52.6 Mb). No commonly deleted region could be identified for all cases. However, in 57 cases the deleted region encompassed genomic position 101,912.442 (7q22.1) to 119,608.824 (7q31.31) including 111 genes. The size of the 7q deletion was smaller in cases with interstitial deletion as compared to terminal deletion (57.7 MB vs 70.9 MB, p=0.04) and in MPN as compared to all other entities (48.7 MB vs 62.8 MB, p<0.001). The mutation analyses revealed mutations in TET2 37% (25/67), ASXL1 35% (27/78), RUNX1 26% (18/69), DNMT3A 21% (14/68), SRSF2 18% (13/73), JAK2 V617F 14% (11/79), CBL 9% (7/75), NRAS 9% (7/77), MLL -PTD 5% (4/80), KRAS 5% (3/66), EZH2 4% (3/72), TP53 4% (3/74), SF3B1 4% (3/75), ETV6 3% (2/73), NPM1 3% (2/77), CALR 1% (1/77), MPL 1% (1/76). ASXL1 and TET2 were frequently co-mutated as 56% of ASXL1 mutated cases also harbored a TET2 mutation (p=0.02). 39 cases were analysed for all 16 molecular mutations. The majority of patients (n=27, 69%) had more than one mutation (range: 2-4), 9 patients (23%) had one mutation and in 3 patients (8%) no mutation was detected. The number of mutations per patient was lower in patients <70 years as compared to patients ≥70 years (0, 1,2,3,4 mutations detected in: 23%, 15%, 15%, 46%, and 0% vs 0%, 27%, 27%, 31%, and 15%, p=0.05). CBL mutations were most frequent in CMML (44%) but rare in all other subtypes (5%, p=0.003), while RUNX1 mutations were most frequent in AML (43% vs 9%; p=0.002) and JAK2 V617F mutations most frequent in MPN (50% vs 5%, p<0.001). DNMT3A mutations and MLL -PTD were significantly more frequent in de novo AML than in all other entities (43% vs 11%, p=0.007; 15% vs 0%, p=0.009), while no significant differences in frequency were observed between the different entities for any of the other mutations or the number of mutations per case. In CMML CBL mutations were associated with del(7q) (44%) as CBL mutations were present in only 17% of non del(7q) CMML (n=101, p=0.07). The frequency of RUNX1 mutations was significantly higher in AML with del(7q) as the sole abnormality (43%) as compared to all other AML (n=2273, 21%; p=0.001). Median overall survival (OS) for the total cohort was 25 months and did not differ significantly between AML, MDS, MDS/MPN and MPN (26, 27, not reached, 15 months, respectively). Conclusions: 1. Sizes and localisations of the del(7q) largely overlapped between AML, MDS, MDS/MPN and MPN. 2. 92% of all patients with 7q deletion harbored at least 1 molecular mutation. 3. TET2 and ASXL1 were the most frequently mutated genes and were present at comparable frequencies in all subtypes. 4. AML with del(7q) is closely associated with RUNX1 mutations while CMML with del(7q) frequently harbored CBL mutations suggesting a cooperative leukemogenic potential in these entities. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Fasan:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. 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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The RUNX1 Gene Is Altered in 26% of AML Patients Either By Translocation, Mutation, Gain or Deletion

Blood ◽

10.1182/blood.v124.21.123.123 ◽

2014 ◽

Vol 124 (21) ◽

pp. 123-123

Author(s):

Claudia Haferlach ◽

Niroshan Nadarajah ◽

Wolfgang Kern ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

De Novo ◽

Intermediate Risk ◽

Missense Mutations ◽

Employment Equity ◽

Runx1 Gene ◽

Genetic Abnormalities ◽

Partner Gene ◽

Different Types ◽

Equity Ownership ◽

De Novo Aml

Abstract Background: In AML four types of acquired alterations of the RUNX1 gene have been described: 1. translocations involving RUNX1 leading to fusion genes such as RUNX1-RUNX1T1, 2. molecular mutations, 3. amplifications of RUNX1, 4. Partial or complete deletions of the RUNX1 gene. Aim: To determine the frequency of different RUNX1 alterations and to characterize the spectrum of accompanying genetic abnormalities. Patients and Methods: We screened 726 de novo AML patients (pts) for RUNX1 deletions (del) and translocations using a dual color break-apart probe covering the 5' and 3' part of RUNX1 (MetaSystems, Altlussheim, Germany) and in addition evaluated RUNX1 mutations (mut) by Sanger or next-generation amplicon deep-sequencing. Median age was 67 yrs (range: 18 to 100 yrs). For all patients cytogenetics was available and categorized according to MRC criteria (Grimwade et al. Blood 2010). Partial deletions of RUNX1 as detected by FISH were confirmed by array CGH (Agilent Technologies, Santa Clara, CA). Results: In 89/726 pts (12.3%) abnormalities of the RUNX1 gene were detected by FISH: 10 pts (1.4%) showed a deletion encompassing the whole RUNX1 gene while additional 9 pts (1.2%) showed a partial loss of one RUNX1 copy. A gain of one RUNX1 copy was present in 45/726 (6.2%) pts. In 3 pts a gain of the 5' part of RUNX1 was accompanied by a loss of the 3' part while in 2 pts one copy of the 3' part was gained accompanied by a loss of the 5' part. A translocation affecting the RUNX1 gene was detected in 31 pts (4.3%). The partner gene was RUNX1T1 in 29 pts and located on 16q13 and 18p11 in one pt each. One pt with a RUNX1 translocation also showed a 5' RUNX1 deletion. In 110/726 pts (15.2%) a RUNX1mut was detected. Of these, 16 pts showed two and 5 pts three mutations in RUNX1. Thus, in total 136 mutations were detected in 110 pts: 58 (42.6%) were frameshift, 42 (30.9%) missense, 21 (15.4%) nonsense, 9 (6.6%) splice-site and 6 (4.4%) in-frame insertions/deletions. The RUNX1mut was homozygous in 15 pts, these were predominantly missense mutations (9/15; 60%). Within the subset of pts with RUNX1mut 2 harbored an additional RUNX1del and 9 pts a gain of a RUNX1 copy, while no RUNX1 translocation was present. In AML FAB type M0 both RUNX1mut and RUNX1del showed the highest frequencies (33.3% and 14.8%). 48.4% and 45.2% of cases with RUNX1 translocations were FAB type M1 and M2. While RUNX1mut were most frequent in the cytogenetic intermediate risk group (19.1%; favorable: 2.2%, adverse: 9.7%), RUNX1del were most frequent in pts with adverse risk cytogenetics (9.7%; favorable: 1.1%, intermediate: 0.8%). A comparable distribution was observed for a gain of RUNX1 copies (adverse: 19.4%, favorable: 4.5%, intermediate: 2.6%). With respect to additional molecular mutations all types of RUNX1 alterations were mutually exclusive of NPM1mut. Further, the frequency of DNMT3Amut and CEBPAmut was significantly lower in pts with RUNX1 alterations as compared to those without (14.3% vs. 34.3%; p<0.0001 and 6.4% vs. 13.4%; p=0.012). However, some striking differences between the different types of RUNX1 alterations were detected: ASXL1mut were significantly more frequent in pts with RUNX1mut (36.7%) but rather infrequent in pts with RUNX1del, gain and translocation (12.5%, 6.1%, and 6.7%). A comparable association was noticed for SF3B1mut which were frequent in RUNX1mut pts (23.8%) and rather infrequent in pts with RUNX1del, gain and translocations (0%, 10.5%, and 0%). In contrast, pts with either RUNX1del or RUNX1 gains showed a significantly higher TP53mut frequency (66.7% and 35.3%) as compared to RUNX1mut or RUNX1 translocated pts (7.1% and 4.8%). In the total cohort median overall survival (OS) was 18.7 months and differed significantly between the different types of RUNX1 alterations: for RUNX1 translocations, mutations, gains and deletions it was 35.5, 14.1, 12.4 and 4.3 months. Conclusions: 1. The RUNX1 gene is altered in 26% of AML. 2. All types of RUNX1 alterations predominantly occur in AML M0, M1 and M2 and are rare in the remainder AML. 3. They are mutually exclusive of NPM1 mutations and show a negative association with DNMT3A mutations. 4. While RUNX1 mutations were most frequent in patients with intermediate risk cytogenetics, RUNX1 deletions and gains were most frequent in patients with adverse cytogenetics. 5. Outcome differs significantly and is best in patients with RUNX1 translocations and worst in cases with RUNX1 deletions. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah: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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Results from Ongoing Phase 1/2 Trial of SL-401 in Combination with Pomalidomide and Dexamethasone in Relapsed or Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.5696.5696 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5696-5696 ◽

Cited By ~ 3

Author(s):

Myo Htut ◽

Cristina Gasparetto ◽

Jeffrey Zonder ◽

Thomas G. Martin ◽

Emma C. Scott ◽

...

Keyword(s):

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Phase 1 ◽

Free Light Chain ◽

M Protein ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Stage 1

Abstract Background: The bone marrow microenvironment of many multiple myeloma (MM) patients harbors high quantities of plasmacytoid dendritic cells (pDCs), which are specialized immune cells that express the interleukin-3 receptor (CD123). These pDCs have been shown to augment MM growth and contribute to drug resistance, suggesting that targeting pDCs may offer clinical benefit for MM patients. SL-401, a novel targeted therapy directed to CD123, has previously demonstrated potent preclinical in vitro and in vivo activity against MM cell lines and primary tumor samples via both a direct anti-MM effect and an indirect effect by targeting neighboring pDCs. SL-401 has also demonstrated synergy in these systems when used in combination with traditional MM therapies including pomalidomide (POM). Clinically, SL-401 has demonstrated high levels of anti-tumor activity in patients with an aggressive CD123+ malignancy of pDC origin, namely blastic plasmacytoid dendritic cell neoplasm (BPDCN). SL-401 is currently being evaluated in combination with POM and dexamethasone (DEX) in relapsed or refractory (r/r) MM patients. Preliminary results are reported here. Methods and Results: This multicenter, single arm Phase 1/2 trial of patients with r/r MM includes a lead-in (stage 1) and expansion (stage 2). In stage 1, patients receive SL-401 as a daily IV infusion at 7, 9, or 12 ug/kg/day for days 1-5 of a 28 day cycle as a single agent for the initial run-in cycle (cycle 0) and in combination with standard doses/administration of POM+DEX in cycles 1 and beyond, in a 3x3 design. In stage 2, patients receive SL-401 in combination with POM+DEX at the dose and regimen determined in stage 1. Objectives include characterization of the safety profile of SL-401 in combination with POM+DEX, including determination of the maximum tolerated or tested dose, and detection of efficacy signals including evaluation of tumor response based on International Myeloma Working Group criteria, duration of response, progression-free survival, and translational evaluation of changes in BM microenvironmental pDCs. As of 7-25-16, 2 patients with r/r MM received SL-401 at 7 ug/kg in combination with POM+DEX. The median age was 65 years (range: 63-67 years). The most common treatment-related AEs, all grades, were thrombocytopenia (2/2, both grade 1) and hypoalbuminemia (2/2, both grade 2); there has been no DLT. Rapid onset decrease in a set of myeloma-related laboratory values from pre-SL-401 treatment was observed in both patients after the first combination cycle of SL-401 and POM+DEX. In one patient, serum M-protein decreased from 2.34 to 1.19 g/dL (cycle 1), free light chain kappa decreased from 40.1 to 8.27 mg/dL (cycle 1), and free light chain kappa/lambda ratio decreased from 58.12 to 41.35 (cycle 1). In the other patient, serum M-protein decreased from 1.88 to 0.87 (cycle 1) and then was 0.96 (cycle 3) g/dL, free light chain kappa decreased from 134 to 49.4 (cycle 1) and then was 92.5 (cycle 3) mg/dL, and free light chain kappa/lambda ratio decreased from 638.1 to 76 (cycle 1) and then was 111.45 (cycle 3). Both patients remain on study receiving ongoing SL-401 at 2+ and 4+ months. Dose escalation to 9 ug/kg is planned if a third patient clears the 7 ug/kg cohort. Conclusions:This is the first clinical study to evaluate SL-401 in combination with other agents. SL-401 thus far has been well-tolerated in combination with POM+DEX in r/r MM patients, with no unexpected AEs observed. After the first cycle of SL-401 and POM+DEX combination therapy, 2 of 2 patients experienced a rapid decrease in serum M-protein and remain on SL-401 therapy. Given CD123 expression on microenvironmental immune pDCs and the potential synergy of SL-401 with certain current MM agents including POM, SL-401 may offer a novel therapeutic approach in MM. This Phase 1/2 trial continues to enroll and updated data will be presented. Clinical trial information: NCT02661022. Disclosures Zonder: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Prothena: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pharmacyclics: Other: DSMC membership. Martin:Sanofi: Research Funding; Amgen: Research Funding. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Shemesh:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Chauhan:Stemline Therapeutics: Consultancy. Anderson:Oncopep: Other: Scientific Founder; Acetylon: Other: Scientific Founder; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Hematologic Malignancies Exhibit Selective Vulnerability to Inhibition of De Novo Pyrimidine Biosynthesis By AG-636, a Novel Inhibitor of Dihydroorotate Dehydrogenase in Phase 1 Clinical Trials

Blood ◽

10.1182/blood-2019-123471 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1570-1570

Author(s):

Danielle Ulanet ◽

Victor Chubukov ◽

John Coco ◽

Gabrielle McDonald ◽

Mya Steadman ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Solid Tumor ◽

Research Funding ◽

De Novo ◽

Phase 1 ◽

Cancer Cell Lines ◽

Pyrimidine Biosynthesis ◽

Employment Equity ◽

Equity Ownership

Rapidly proliferating cells reprogram metabolism to support increased biosynthetic demands, a feature that can expose targetable vulnerabilities for therapeutic intervention. A chemical biology screen was performed in an effort to identify metabolic vulnerabilities in particular tumor subtypes, and revealed potent and selective activity of a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic origin. In contrast, cancer cell lines of solid tumor origin exhibited comparatively poor sensitivity. Evaluation of a lymphoma cell line panel demonstrated broad responsiveness to DHODH inhibition, independent of clinical subtype (e.g. ABC, GCB, double-hit). The on-target cellular activity of AG-636 was evaluated by examining the metabolic effects of AG-636 on cells and by evaluating the ability of extracellular uridine to rescue the effects of AG-636 on proliferation and viability. The metabolic changes incurred upon treatment of cells with AG-636 were consistent with a mechanism of action driven by inhibition of DHODH and de novo pyrimidine biosynthesis. Supraphysiologic concentrations of extracellular uridine rescued the effects of AG-636 on growth and viability as well as the effects on metabolism, further confirming on-target activity. The mechanistic basis for differential sensitivity to AG-636 was assessed by comparing the activity of the de novo pyrimidine biosynthesis and uridine salvage pathways in cancer cell lines of hematologic or solid tumor origin with similar proliferative rates. Differential response to AG-636 could not be attributed to varying abilities to utilize the de novo pyrimidine biosynthesis pathway or to salvage extracellular uridine. Real-time imaging of cells treated with AG-636, along with monitoring of extracellular uridine concentrations, demonstrated immediate effects on the viability of lymphoma cell lines in the setting of depleted extracellular uridine. In contrast, solid tumor cell lines were able to maintain growth for an additional period of time, suggestive of adaptive mechanisms to supply pyrimidine pools and/or to cope with nucleotide stress. The high in vitro activity of AG-636 in cancer cells of hematologic origin translated to xenograft models, including an aggressive, patient-derived xenograft model of triple-hit lymphoma and an ibrutinib-resistant model of mantle cell lymphoma in which complete tumor regression occurred. These studies support the development of AG-636 for the treatment of hematologic malignancies. A phase 1 study has been initiated in patients with relapsed/refractory lymphoma (NCT03834584). Disclosures Ulanet: Agios: Employment, Equity Ownership. Chubukov:Agios: Employment, Equity Ownership. Coco:Agios: Employment, Equity Ownership. McDonald:Agios: Employment, Equity Ownership. Steadman:Agios: Employment, Equity Ownership. Narayanaswamy:Agios: Employment, Equity Ownership. Ronseaux:Agios: Employment, Equity Ownership. Choe:Agios: Employment, Equity Ownership. Truskowski:Agios: Employment, Equity Ownership. Nellore:Aurigene Discovery Technologies: Employment. Rao:Firmus Laboratories: Employment, Equity Ownership. Lenz:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Employment, Honoraria, Research Funding, Speakers Bureau. Cooper:Agios: Employment, Equity Ownership. Murtie:Agios: Employment. Marks:Agios: Employment, Equity Ownership.

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