Development of a Fast and Reproducible Assay for the Clinical Implementation of Epigenetic Biomarkers to Predict Decitabine Response in Patients with Chronic Myelomonocytic Leukemia

Abstract Chronic myelomonocytic leukemia (CMML) is an aggressive myelodysplastic/myeloproliferative neoplasm represented by abnormal, clonal monocytosis, resistance to conventional chemotherapy, and an elevated risk of transforming into acute leukemia. While allogeneic hematopoietic stem cell transplantation is the only curative treatment option, this is not available to everyone due to either lack of compatible donors or the existence of comorbidities that make the patient ineligible for such procedure. Hypomethylating agents (HMAs) such as Decitabine and Azacitidine have shown efficacy in ∼50% of the patients. However, response to these agents can take 6 months or longer and patients are required to stay on the drug for this period of time, even if only about 50% of them will benefit from it. Therefore, in order to provide the best treatment option at an appropriate timing, robust biomarkers for predicting the response to HMAs are urgently needed. Using genome-wide methylation analysis (ERRBS: enhanced reduced representation bisulfite sequencing), we previously identified 21 epigenetic biomarkers which accurately predicted the response to decitabine in patients with CMML (Meldi K, et al. J Clin Invest. 2015). However, due to the complexity of this assay as well as the genome-wide nature of it, ERRBS is not suitable for the clinical setting. Thus, we sought to develop a novel, targeted assay that can be used in the clinical setting for the implementation of this biomarker as well as to validate the performance of the epigenetic biomarker in an independent cohort of patients. For this purpose, primers targeting the 21 regions included in the epigenetic biomarker were designed with overhang adapter sequences compatible with illumina multi-index system. Genomic DNA was extracted from CD34+ human blood cells and bisulfite conversion was performed using EZ DNA Methylation Kit (ZYMO Research). Multiplex PCR parameters were established by calculating the Gibbs Free Energy (dG) for each primer combination using PrimerSuite program. Four primer pools were determined based on the dG and optimum annealing temperatures at single PCR. Multiplex PCR was performed using FastStart High Fidelity PCR System (Roche) and the combined PCR amplicons were purified by AMPure XP (Beckman Coulter). Purified amplicons were indexed by Nextera XT index kit (illumina) and KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and sequenced on an Illumina MiSeq sequencer (2x150 bp). Sequencing reads were mapped to human reference genome (hg19) using Bowtie2, and cytosine methylation states were determined by Bismark. Twenty out of 21 biomarkers were successfully amplified and sequenced. Using a standard curve generated with in vitro methylated DNA, we demonstrated that the assay accurately captured the different methylation percentages in the standard curve and was highly reproducible, with correlation coefficient r=0.98 between two independent operators (Fig.1). Moreover, analysis of 26 CMML patient samples previously analyzed by ERRBS showed strong correlation between ERRBS and the targeted panel assay (Pearson's r: 0.83-0.98). To test the performance of predicting decitabine response, we analyzed both the ERRBS and the targeted panel assay data of the 26 patients (12 responders, 14 non-responders) using supervised machine learning algorithm, SVM classifier. The two assays showed 100% concordance of predictions with an accuracy of 96%, indicating high reproducibility and predictive performance of the novel targeted panel assay. In summary, we have developed a fast, robust, and highly reproducible MiSeq-based, targeted panel assay for decitabine response prediction in patients with CMML. Figure 1 Figure 1. Disclosures Santini: Astex: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

Phase Ib Study of Oral Panobinostat In Combination with 5-Azacitidine (5-aza) In Patients with Myelodysplastic Syndromes (MDS), Chronic Myelomonocytic Leukemia (CMML), or Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v116.21.4957.4957 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4957-4957 ◽

Cited By ~ 1

Author(s):

Pierre Fenaux ◽

Daniel J DeAngelo ◽

Guillermo Garcia-Manero ◽

Michael Lübbert ◽

Anand P. Jillella ◽

...

Keyword(s):

Febrile Neutropenia ◽

Adverse Events ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Advisory Committees ◽

Dose Cohort ◽

Phase Ib ◽

Myelomonocytic Leukemia

Abstract Abstract 4957 Background: Panobinostat is a potent pan-deacetylase inhibitor (pan-DACi) that causes increased acetylation of target proteins such as HSP90, p53, α-tubulin and HIF-1α which are involved in cell cycle regulation, gene transcription, angiogenesis, and tumor cell survival. Preliminary evidence from phase I trials has demonstrated anti-tumor activity in patients with hematologic malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The advent of hypomethylating agents, such as 5-aza, represent a significant advancement in the treatment of MDS, chronic myelomonocytic leukemia (CMML), and AML. Although an improvement in clinical outcomes has been observed, including increased overall survival in patients with MDS, a substantial number of patients do not benefit from the therapies currently available. Preclinical studies suggest that the combination of a demethylating agent and a pan-DACi represents a rational strategy to reverse silencing of tumor suppressor genes, which contributes to the malignant phenotype, and improve outcomes in patients with MDS and AML. In this study, the combination of the pan-DACi, panobinostat, and the hypomethylating agent, 5-aza, was evaluated in patients with MDS, CMML and AML. Methods: This phase Ib, open-label, multicenter, dose-finding study is comprised of 2 stages: a dose-escalation stage to determine the maximum tolerated dose (MTD) of panobinostat in combination with standard dose 5-aza, and a subsequent expansion stage to evaluate safety, tolerability, and preliminary activity at the MTD dose level. The primary endpoint is incidence of dose-limiting toxicity (DLT) and secondary endpoints include type, duration, frequency, and relationship of adverse events (AEs) to the combination. Exploratory endpoints include clinical response and hematologic improvement according to IWG response criteria, and biomarker analysis of methylation status and expression of disease-associated genes in peripheral blood cells prior to and during therapy. Adult patients with IPSS INT-2 or high-risk MDS, CMML, or AML with multi-lineage dysplasia and ≤ 30% marrow blasts who are candidates for therapy with 5-aza and have not received a prior hypomethylating agent or pan-DACi are eligible for enrollment on the trial. Oral panobinostat was administered on Days (D) 3, 5, 8, 10, 12, and 15, starting at 20 mg, in combination with 5-aza (75 mg/m2 sc D 1–7) during a 28-D cycle. Patients received treatment for ≤ 6 cycles or until progression of disease, incidence of unacceptable toxicity, or withdrawal of consent. Results: To date, 11 patients have been enrolled including 9 patients with MDS, 1 patient with AML and 1 patient with CMML. The median age of patients enrolled on the trial was 69.0 (60-80). Patients have been evaluated at 2 panobinostat dose cohorts; 6 (20 mg) and 5 (30 mg). The AE analysis is based on 9 patients (6 from 20 mg cohort and 3 from 30 mg cohort) and the nature and incidence of AEs observed in the two cohorts were similar. Adverse events regardless of study drug relationship included nausea (4 [44%]), vomiting, fatigue (5 [55%] each) and asthenia (3 [33%]). Grade 3/4 AEs suspected to be treatment related included thrombocytopenia (2 [22%], febrile neutropenia and arthritis (1 [11%] each). Serious adverse events observed included febrile neutropenia, asthenia (2 [22%] each), atrial fibrillation and septic shock (1 [11%] each). One DLT has been observed (grade 4 febrile neutropenia) in the 20 mg panobinostat dose cohort. Conclusions: Panobinostat has been well tolerated up to a dose of 30 mg in combination with 5-aza (75 mg/m2) with dose escalation ongoing. Patients are currently being enrolled at the 40mg dose cohort. The most common AEs observed included febrile neutropenia, thrombocytopenia with one DLT observed (grade 4 febrile neutropenia) in the 20mg panobinostat dose cohort. The current data show that the addition of panobinostat to 5-aza is safe with no unexpected toxicities. Updated data, including safety and preliminary efficacy data will be presented at the meeting. Disclosures: Fenaux: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Off Label Use: Panobinostat is an investigational agent currently being evaluated for the treatment of hematologic and solid malignancies. DeAngelo: Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Winiger: Novartis Pharma AG: Employment. Squier: Novartis: Employment. Li: Novartis: Employment. Ottmann: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Transition of Chronic Myeloid Leukemia to Chronic Myelomonocytic Leukemia As a Tool to Observe Development of Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v126.23.5223.5223 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5223-5223

Author(s):

Jamshid S Khorashad ◽

Srinivas K Tantravahi ◽

Dongqing Yan ◽

Anna M. Eiring ◽

Hannah M. Redwine ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Clonal Evolution ◽

Chronic Myelomonocytic Leukemia ◽

Imatinib Treatment ◽

Advisory Committees ◽

Clonal Architecture ◽

Myelomonocytic Leukemia

Abstract Introduction. Development of abnormal Philadelphia (Ph) negative clones following treatment of chronic myeloid leukemia (CML) patients with imatinib has been observed in 3 to 9% of patients. Here we report on a 77 year old male diagnosed with CML that responded to imatinib treatment and subsequently developed chronic myelomonocytic leukemia (CMML). He achieved major cytogenetic response within 3 months but this response coincided with the emergence of monocytosis diagnosed as CMML. Five months after starting imatinib treatment the patient succumbed to CMML. We analyzed five sequential samples to determine whether a chronological order of mutations defined the emergence of CMML and to characterize the clonal evolution of the CMML population. Materials and Method. Five samples (diagnostic and four follow up samples) were available for analysis. CMML mutations were identified by whole exome sequencing (WES) in CD14+ cells following the onset of CMML, using CD3+ cells as constitutional control. Mutations were validated by Sequenom MassARRAY and Sanger sequencing and quantified by pyrosequencing. Deep WES was performed on the diagnostic sample to determine whether the mutations were present at CML diagnosis. To determine the clonal architecture of the emerging CMML, colony formation assays were performed on the diagnostic and the next two follow-up samples (Samples 1-3). More than 100 colonies per sample were plucked for DNA and RNA isolation. The DNA from these colonies were tested for the presence of the confirmed CMML mutations and the RNA was used for detection of BCR-ABL1 transcript using a Taqman real time assay. Results. Four mutations were identified by Sequenom and WES throughout the patient's time course [KRASG12R, MSLNP462H, NTRK3V443I and EZH2I669M ]. Sequenom did not identify these at diagnosis while deep WES did. Clones derived from colony formation assay revealed three distinct clones present in all samples analysed. Clone 1 had only KRASG12R, clone 2 had KRASG12R, MSLNP462H, and NTRK3V443I, and clone 3 had all four mutations. All clones containing any of these four mutations were BCR/ABL1 negative. Analysis of clonal architecture indicated that KRASG12R was acquired first and EZH2I669M last, while MSLNP462H and NTRK3V443I were acquired in between. These CMML clones increased proportionately as clinical CML metamorphosed into clinical CMML after initiation of imatinib therapy. Consistent with the colony data, pyrosequencing revealed that the ratio between the mutants remained largely stable throughout the follow up period. Conclusion. This case illustrates how targeted therapy impacts clonal competition in a heterogeneous MPN. While the CML clone was dominant in the absence of imatinib, it was quickly outcompeted by the CMML clones upon initiation of imatinib therapy. The clonal architecture analysis, in combination with in vivo kinetics data, suggest that the KRASG12R mutation alone was able to produce a CMML phenotype as clones with just KRASG12R remained at a relatively stable ratio during follow up. Unexpectedly, acquisition of additional mutations, including EZH2I669M as the last mutational event identified in this patient, did not increase clonal competitiveness, at least in the peripheral blood. These data show that clonal evolution may not invariably increase clonal fitness, suggesting that factors other than Darwinian pressures contribute to clonal diversity in myeloproliferative neoplasms. Disclosures Deininger: Gilead: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Overactivation of the NLRP3 Inflammasome in Chronic Myelomonocytic Leukemia KRAS Mutated Patients Can be Detected By the Apoptosis-Associated Speck-like Protein (ASC) and Reverted By IL1β Inhibitors

Blood ◽

10.1182/blood-2021-153686 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3670-3670

Author(s):

Laura Hurtado-Navarro ◽

Ernesto J Cuenca ◽

Eva Soler ◽

Andres Jerez ◽

Helios Martínez-Banaclocha ◽

...

Keyword(s):

Board Of Directors ◽

Nlrp3 Inflammasome ◽

Research Funding ◽

Ex Vivo ◽

Chronic Myelomonocytic Leukemia ◽

Inflammasome Activation ◽

Advisory Committees ◽

Nlrp3 Inflammasome Activation ◽

Myelomonocytic Leukemia

Abstract It has been recently shown that RAS mutations, which occur in 11-38% of Chronic Myelomonocytic Leukemia (CMML), do not only act via RAS/MEK/ERK signaling, but contribute to the disease through NLRP3 inflammasome activation (Hamarsheh, Nat Comm 2020). Despite a therapeutic approach based on NLRP3/IL1β axis blockade, as bring to a stem cell transplantation (SCT) has been proposed, data on the efficacy of IL1β inhibitors in hematopoietic neoplasms is limited. A 55 year old man with previous autoinflammatory episodes (constrictive pericarditis) was diagnosed on September 2020 of CMML-1 KRAS G12D (Inter-2). Due to worsening (orchiepidedymitis, pneumonitis, cellulitis), and the impossibility of performing an SCT at that time, on December 02 2020 he started anakinra (a IL1β receptor antagonist) with good response. Due to new episodes of autoinflammation, anakinra was discontinued (12 April 2021) with severe clinical worsening (heart failure) and no response to diuretic/corticosteroid. After anakinra was restarted (04 May 2021), a progressive improvement was seen, allowing a successful pericardiectomy before an SCT. We obtained blood samples from this patient (at different times) and plasma and whole blood samples from 11 and 5 other CMML KRAS mut patients, respectively. We also included CMML patients without KRAS mutations (KRAS wt) (n=8), with sepsis (n=5) and healthy individuals (n=9). Plasma levels of 15 inflammatory cytokines associated with NLRP3 inflammasome and NFkB pathways were measured using a customized MILLIPLEX ® kit. The inflammasome marker activation assays were conducted as previously published (Martínez García JJ, Nature Comm 2019). Compared to healthy controls, KRAS wt CMML patients did not show differences in any cytokine tested, except IL6, while KRAS mut patients showed significantly higher levels of IL1α, IL1ra, IL18, IL12p40 (associated with NLRP3 inflammasome), IL6, IL8 (associated with NFkB pathway) and M-CSF (Fig. 1A B). Compared to KRAS wt CMML patients, those with KRAS mut showed higher levels of cytokines associated with both the NLRP3 and NFkB pathways, reaching statistical significance for those related with NLRP3 inflammasome. We also observed changes in inflammasome related cytokines before and after anakinra (Table 1). This cytokine profile in the plasma made us analyze the oligomerization of ASC as a marker of inflammasome activation in monocytes of KRAS mut CMML. We found that in all cases of KRAS mut CMML patients around 30 to 80% of monocytes presented oligomers of ASC measured by the time of flight assay, while in healthy donors and KRAS wt CMML patients, ASC oligomerization occurred upon NLRP3 inflammasome activation with lipopolysaccharide (LPS) + ATP or Pyrin inflammasome activation with LPS and Clostridium difficile B toxin (TcdB) (Fig. 2A). Ex vivo activation of PBMCs from KRAS mut CMML patients showed that despite the high percentage of cells with ASC oligomers, very low levels of IL1b released from these cells, even when NLRP3 was activated with LPS+ATP (Fig. 2B), suggesting that this inflammasome is activated in vivo and could not be further activated ex vivo. As control, Pyrin inflammasome activation in PBMCs from KRAS mut CMML was able to induce IL1b release similarly to healthy controls (Fig. 2B). We then found that anakinra treatment of the KRAS mut CMML patient followed in this study, resulted in a decrease of the percentage of monocytes with basal active inflammasomes (Fig. 2C). A little ex vivo activation of the NLRP3 inflammasome was obtained when cells were treated with LPS+ATP, while Pyrin inflammasome was activated at normal levels after LPS+TcdB treatment (Fig. 2D). The inflammasome basal activation increased in the monocytes of the KRAS mut CMML patient after anakinra withdraw and during clinical deterioration and restarting anakinra (second arrow) decreased the basal percentage of monocytes with ASC oligomers (Fig. 2C). Since ASC oligomers are associated to pyroptosis via caspase 1 activation and gasdermin D processing, we then analyzed pyroptotic markers in the plasma of the patient during the time. ASC was increased when monocytes presented elevated percentage of ASC oligomers (Fig. 2E), suggesting that ASC detection could be a promising biomarker. Overall, we show that, in vivo, the NLRP3 inflammasome activation of KRAS mut CMML patients may revert with IL1β blockers. ASC could identify those candidates to receive this therapy. PI18/00316 Figure 1 Figure 1. Disclosures Jerez: Novartis: Consultancy; BMS: Consultancy; GILEAD: Research Funding. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau. Hernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ferrer Marin: Cty: Research Funding; Incyte: Consultancy, Research Funding; Novartis: Speakers Bureau.

Download Full-text

Characteristics and Outcomes Of Patients (pts) With Multiple Myeloma (MM) Who Develop Therapy (t)-Related Myelodysplastic Syndrome (MDS), t-Chronic Myelomonocytic Leukemia (CMML), Or t-Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.1424.1424 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1424-1424

Author(s):

Naveen Pemmaraju ◽

Dhaval Shah ◽

Hagop M Kantarjian ◽

Verena Wagner ◽

Robert Z. Orlowski ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Chemotherapeutic Agents ◽

Chronic Myelomonocytic Leukemia ◽

Advisory Committees ◽

Myelomonocytic Leukemia ◽

Acute Myeloid

Abstract Background There has been a significant improvement in the outcome for patients (pts) with MM over the last decade, mainly due to the availability of immunomodulatory (IMiD) drugs and proteasome inhibitors (PI). The improvement in survival has also increased the risk of second primary malignancies (SPM), such as therapy-related myelodysplastic syndrome (t-MDS), therapy-related chronic myelomonocytic leukemia (t-CMML) or therapy-related acute myeloid leukemia (t-AML). However, little is known about the characteristics and outcomes of pts with t-MDS, t-CMML or t-AML. Methods We aimed to study the characteristics and outcome of pts who developed t-MDS, t-AML and t-CMML as SPM after the treatment of MM. We reviewed our database of pts with MM who were treated at our institution between 1993 and 2011. We identified 49 pts who were diagnosed to have t-MDS, t-CMML, or t-AML. The primary objective of this study was to evaluate the time to develop t-MDS, t-AML and t-CMML, their response to treatment and overall survival. Results Median age of pts at diagnosis of MM was 61 years. Forty-seven (96%) pts had symptomatic MM, while 2 (4%) had asymptomatic myeloma. Forty-seven (95%) pts with symptomatic myeloma received systemic therapy. Eleven (22%) pts were treated with IMiD or PI: lenalidomide 3, thalidomide 6 and bortezomib 2. Thirty-eight (78%) pts were treated with various conventional chemotherapeutic agents including melphalan, cyclophosphamide, doxorubicin, vincristine, etoposide, cisplatin, idarubicin, thiotepa, busulfan, carmustine and cytarabine. Fourteen (28%) pts also received radiation therapy to the affected areas. Twenty (41%) pts underwent high-dose chemotherapy and autologous hematopoietic stem cell transplantation (auto-HCT). Fourteen pts received maintenance therapy after auto-HCT with either thalidomide, lenalidomide, dexamethasone or bortezomib. Median time from the diagnosis of MM to t-MDS, t-CMML or t-AML was 6 years [0 – 24]. Thirty-four (69 %) pts developed t-MDS, 12 (24%) t-AML, and 3 (6%) t-CMML. Median age at diagnosis of t-MDS, t- CMML, or t-AML was 65 years. Twenty-seven (79%) pts with t-MDS and all 12 pts with t-AML had complex/high risk cytogenetics. Most common cytogenetic abnormalities involved chromosome 5 and 7. Thirty four (69%) pts received at least 1 cycle of induction chemotherapy either with conventional chemotherapeutic agents or investigational drugs. Only 9 pts (26%) achieved complete remission (CR). Median duration of CR in these pts was 4 months [1 – 62]. Median overall survival (OS) of pts who received induction therapy was 6.0 months [0-30]. Five (11%) pts received an allogeneic stem cell transplant with three achieving CR. Median OS in this subgroup of pts was 18 months [9 – 23]. Median OS for all 49 pts after diagnosis of t-MDS, t-CMML or t-AML was 6.0 months [0 – 30] Conclusion Development of t-MDS, t-CMML, or t-AML in pts with MM is associated with a poor outcome. These pts in general have complex cytogenetic abnormalities, chemo-resistant disease, a short CR and OS. A better understanding of disease biology and novel therapeutic approaches are warranted. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Qazilbash:Otsuka: Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Download Full-text

Hypomethylating Agent Therapy for Chronic Myelomonocytic Leukemia Does Not Impact Acute Myeloid Leukemia Transformation or Survival

Blood ◽

10.1182/blood-2020-142747 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-7

Author(s):

Sandrine Niyongere ◽

Yamini Kathari ◽

Zeba Singh ◽

Emily J. Vannorsdall ◽

Ashkan Emadi ◽

...

Keyword(s):

Clinical Trial ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Institutional Research ◽

Research Support ◽

Advisory Committees ◽

Clinical Trial Research ◽

Myelomonocytic Leukemia

Background: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). CMML is characterized by persistent blood monocytosis >1 x 109/L, bone marrow dysplasia in one or more hematopoietic cell lines, and increased risk of transformation to acute myeloid leukemia (AML). Our review of SEER Medicare data (Haematologica 2013;98:584) demonstrated that, compared to MDS, CMML has shorter overall survival (OS) and more frequent progression to AML. Hypomethylating agents (HMAs) have become standard therapy for CMML, with reported response rates of 37-69%, but their impact on AML transformation and OS is unclear. Methods: We retrospectively reviewed CMML patients treated at the University of Maryland Greenebaum Comprehensive Cancer Center between January 2000 and December 2019. Clinical characteristics, treatments, AML progression, time to AML progression (TTP), and OS were recorded and analyzed. Descriptive statistics were used for baseline characteristics and Kaplan-Meier analysis was performed for time-to-event data. Statistical analyses were performed using GraphPad Prism 8®. Results: We identified 71 patients with CMML, 82% male and 73% white, with a median age of 69 (range 25 - 96) years; 51% had <10% bone marrow (BM) blasts and 45% had low-risk cytogenetic findings (normal karyotype or -Y). Most patients treated prior to 2005 received hydroxyurea and/or erythropoiesis-stimulating agents or were enrolled on clinical trials, while patients treated since 2005 received HMAs as primary therapy. Median follow-up was 41.1 months. The median OS of the entire cohort was 20 months, with 46% of patients progressing to AML with a median TTP of 11.5 months. By the MD Anderson Prognostic Scoring System at time of diagnosis, CMML was low-risk in 24 patients, intermediate-1 in 16, intermediate-2 in 14, and high-risk in 17. Forty-six patients received HMAs, with an overall response rate (ORR) of 54% (complete response or partial response), while 25 patients did not receive HMAs. Patient and disease characteristics were similar in HMA- and non-HMA-treated patients (Table 1). The estimated OS of HMA-treated patients was 20 months, compared to 14 months for non-HMA-treated patients (p =0.43) (Figure 1). AML transformation occurred in 52% of patients treated with HMAs, with TTP ranging from 3 to 65 months, and in 33% patients not treated with HMAs, with TTP ranging from 5 to 47 months. Most patients receiving HMAs (63%) received ≥ 6 cycles; 46% transformed to AML despite initial response, often in a sudden and unpredictable manner. HMAs were azacitidine in 13 patients, decitabine in 24, azacitidine followed by decitabine in 4, and decitabine followed by azacitidine in 5. Five CMML patients in our cohort underwent allogenic stem cell transplantation. Four of the five relapsed with transformation to AML post transplant, and only one patient remains in remission, 9 months post transplant. Conclusions: Despite a 54% ORR, HMA treatment did not have a significant impact on frequency of AML transformation, or OS in our cohort. Based on our data, favorable response rates previously reported with HMAs and also seen in our patients do not appear to translate into decreased frequency of AML transformation or prolonged OS. Though our study is a retrospective study with inherent selection bias, our results underscore the ongoing need for novel therapies and for clinical trials for CMML patients. Disclosures Niyongere: Kartos Therapeutics: Other: Received clinical trial research support with Kartos Therapeutics ; Forty Seven: Other: Received clinical trial research support with Forty Seven. Emadi:Amgen: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor; NewLink Genetics: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding. Doung:Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: clinical trial research support; Incyte: Other: clinical trial research support; Astex: Other: clinical trial research support; MedPacto: Other: clinical trial research support. Baer:Takeda: Other: Institutional research funding; Oscotec: Other: Institutional research funding; Kite: Other: Institutional research funding; Incyte: Other: Institutional research funding; Forma: Other: Institutional research funding; Astellas: Other: Institutional research funding; AbbVie: Other: Institutional research funding.

Download Full-text

Phenotypic Characterization of Leukemia-Initiating Stem Cells in Chronic Myelomonocytic Leukemia (CMML)

Blood ◽

10.1182/blood-2019-122530 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4223-4223

Author(s):

Gregor Eisenwort ◽

Alexandra Keller ◽

Michael Willmann ◽

Irina Sadovnik ◽

Greiner Georg ◽

...

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Chronic Myelomonocytic Leukemia ◽

Multicolor Flow Cytometry ◽

Advisory Committees ◽

Cd34 Cells ◽

Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived hematopoietic neoplasm characterized by dysplasia, uncontrolled expansion of monocytic (progenitor) cells in the bone marrow (BM) and in the peripheral blood (PB), and an increased risk of progression to secondary acute myeloid leukemia (sAML). Patients with advanced CMML and sAML are often highly resistant to therapy and their prognosis is dismal. It is thought that drug resistance in myeloid malignancies is a quality of leukemia stem cells (LSC), but little is known about CMML-initiating and propagating LSC. We investigated the phenotype and functional behavior of putative CMML-initiating cells in 15 patients with CMML (7 females, 8 males; median age 73 years; range 45-82 years) and 6 with sAML following CMML (1 female, 5 males; median age 67.5 years; range 66-76 years). BM and/or PB samples were examined by multicolor flow cytometry using antibodies against CD34, CD38 and various additional surface markers and target antigens. In a subset of patients, putative stem and progenitor cells (CD34+ cells, CD34+/CD38─ cells and CD34+/CD38+ cells) were FACS-sorted to high purity (>95%) and were employed in xenotransplantation experiments or in drug testing experiments. We found that CMML-initiating and propagating LSC reside within the CD34+/CD38─ fraction of the malignant clone. Whereas highly purified CD34+ cells engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG) 1Eav/MloySzJ (NSGS) mice with full-blown CMML (engraftment rate 44.8±26.0%), no CMML was produced by the bulk of CD34- monocytic cells (engraftment rate 0.8±0.5%; p=0.002). CMML engraftment was also detectable when transplanting unselected mononuclear cells (engraftment rate 19.7±10.9%). By contrast, no leukemic engraftment was produced by CD38+ CMML fractions (engraftment rate 0.1±0.1%; p=0.003), indicating that the NSGS-repopulating CMML LSC reside specifically in a CD34+/CD38- fraction of the clone. In sAML, both the CD34+/CD38- cell fraction (engraftment rate 92.2±6.2%) and the CD34+/CD38+ fraction (engraftment rate 80.5±7.2%) produced engraftment with AML blasts in NSGS mice. In a next step, we established the cell surface phenotype of CD34+/CD38- LSC in CMML and sAML. As assessed by multicolor flow cytometry, CD34+/CD38- CMML cells invariably expressed CD33/Siglec-3, CD117/KIT, CD123/IL3RA, CD133/AC133, CD135/FLT3, and IL-1RAP. In a subset of patients, CMML LSC also expressed CD52 (9/11 patients; 81%), CD114 (3/7 patients; 43%), CD184 (9/12 patients; 75%), CD221 (8/11 patients; 73%) and/or CLL-1 (7/13 patients; 54%). CMML LSC did not express CD25 or CD26. However, in patients with sAML, LSC also displayed CD25 (median fluorescence intensity, MFI: CMML: 0.9 vs. sAML: 23.0; p<0.001). Compared to hematopoietic stem cells in normal BM (NBM), CMML LSC displayed slightly increased levels of CD117/KIT (MFI CMML: 32.5 vs. MFI NBM: 15.0; p=0.019), CD135/FLT3 (MFI CMML: 1.9 vs. MFI NBM: 0.8; p=0.001), CD184/CXCR4 (MFI CMML: 1.6 vs. MFI NBM 0.9; p=0.027), and IL-1RAP (MFI CMML: 1.6 vs. MFI NBM: 0.8; p=0.004). No correlations between surface-marker expression on LSC and the type of CMML (CMML-0/1/2 or dysplastic vs. proliferative CMML) or the clinical course were found. To confirm the clinical relevance of expression of surface target antigens on CMML LSC, we applied the CD33-targeted drug gemtuzumab-ozogamicin (GO). As assessed by combined staining for LSC (CD34+/CD38-) and AnnexinV/DAPI, incubation of CMML LSC with GO (0.001-1 µg/ml) resulted in dose-dependent apoptosis in all donors tested, and the same result was obtained in the monoblastic cell lines THP-1 (GO at 1 µg/ml: 94.2±1.5% vs. control: 12.7±2.2%, p<0.05) and Mono-Mac-1 (GO at 1 µg/ml: 56.4±12.1% vs. control: 10.1±0.6% p<0.05). In conclusion, LSC in CMML and sAML reside within CD34+/CD38─ cell populations that express distinct profiles of surface markers and target antigens. During progression of CMML into sAML, LSC apparently acquire CD25. Characterization of CMML LSC and LSC in sAML should facilitate their enrichment and the development of LSC-eradicating therapies. Disclosures Hoermann: Novartis: Honoraria; Roche: Honoraria. Sperr:Celgene: Consultancy, Honoraria; Novartis: Honoraria. Sill:Astex: Other: Advisory board; Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board. Geissler:Novartis: Honoraria; AOP: Honoraria; Roche: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Ratiopharm: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Pfizer: Honoraria. Deininger:Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Ascentage Pharma: Consultancy, Honoraria; Fusion Pharma: Consultancy; TRM: Consultancy; Sangoma: Consultancy; Adelphi: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy. Valent:Pfizer: Honoraria; Blueprint: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding.

Download Full-text

Phase I Results of a Multicenter Clinical Trial Combining Guadecitabine, a DNA Methyltransferase Inhibitor, with Atezolizumab, an Immune Checkpoint Inhibitor, in Patients with Relapsed or Refractory Myelodysplastic Syndrome or Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2018-99-117658 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1811-1811 ◽

Cited By ~ 2

Author(s):

Casey L. O'Connell ◽

Patricia L. Kropf ◽

Nathan Punwani ◽

Dan Rogers ◽

Richard Sposto ◽

...

Keyword(s):

Clinical Trial ◽

Myelodysplastic Syndrome ◽

Phase I ◽

Board Of Directors ◽

Dose Level ◽

Chronic Myelomonocytic Leukemia ◽

Multicenter Clinical Trial ◽

Advisory Committees ◽

Level 1 ◽

Myelomonocytic Leukemia

Abstract Background: There are no FDA-approved therapies for patients with myelodysplastic syndrome (MDS) in whom hypomethylating agents (HMAs) fail, and mechanisms of resistance are not well-characterized. Preclinical and clinical data suggest that some myeloblasts express PDL-1 and that HMAs can induce expression of PD-1 on T cells, which may result in resistance through immune tolerance. We hypothesized that the addition of the PDL1-inhibitor atezolizumab to guadecitabine, a next generation HMA which has a longer in vivo exposure time, would induce or restore HMA sensitivity in patients with relapsed or refractory (R/R) MDS. Overlapping toxicities were not expected given atezolizumab is not myelosuppressive and guadecitabine does not appear to induce autoimmune or inflammatory conditions. Methods: We are conducting a phase I/II, multicenter clinical trial for adult patients with R/R, intermediate (3+) or high-risk MDS by the revised international scoring system, including chronic myelomonocytic leukemia (CMML). A 3x3 dose escalation design for guadecitabine was used for Phase I, beginning with 30 mg/m2 (Dose level -1) days 1-5 along with a fixed dose of atezolizumab 840mg IV days 8 and 22 of a 28-day cycle. The plan was to escalate to the recommended dose of guadecitabine, 60mg/m2 (Dose Level 1) if no dose limiting toxicities (DLTs) were identified during Dose Level -1. If ≥2/6 DLTs were observed during the Dose Level 1, de-escalation to 45mg/m2 (Dose Level -1.5) would occur. The primary endpoint of phase I was safety and tolerability of the combination. Overall survival (OS) from the on-study date and overall response rates (ORR), based on the 2006 Modified IWG Response Criteria for MDS, were secondary endpoints. Results: Nine patients (5M, 4F, median age 73) with intermediate or higher risk were treated during phase I. Three patients were treated at Dose Level -1 and sustained no DLTs. Similarly, no DLTs were observed among 6 patients treated at Dose Level 1. There were 17 grade 3 or 4 events considered possibly or probably related to the study treatments, the most common of which were: neutropenia (4), thrombocytopenia (4), and leukopenia (4). The median number of treatment cycles was 5 and the treatment duration for each patient is illustrated in Figure 1. Two patients achieved hematologic improvement (HI) and 1 patient achieved CR. Two patients died after coming off of the study (at 4.5 and 9 months respectively) and the median OS has not been reached. Discussion: The combination of guadecitabine at the recommended dose of 60mg/m2 D1-D5 along with atezolizumab 840mg IV d8, 22 was found to be safe with an acceptable toxicity profile in patients with R/R MDS. The ORR was 33% and a phase II study is ongoing. Disclosures Kropf: Celegene: Consultancy; Takeda: Consultancy. Grønbæk:Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen Pharma: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Integrated Transcriptomic and Proteomic Analyses of Inflammasome in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2019-128094 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2991-2991

Author(s):

María Zurdo ◽

Ana M Hurtado López ◽

Tzu Hua Chen-Liang ◽

Helios Martínez-Banaclocha ◽

Laura Palomo ◽

...

Keyword(s):

Board Of Directors ◽

Differentially Expressed Genes ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Differentially Expressed ◽

Advisory Committees ◽

Myelomonocytic Leukemia

Background and aim: Inflammasome and pyroptosis overactivation have recently been associated as fundamental mechanisms in the ineffective hematopoiesis of myelodysplastic syndromes (MDS). Chronic myelomonocytic leukemia (CMML) shares histological and clinical characteristics with MDS but, within clinical differences, It stands out a high association with inflammatory/autoimmune diseases in which a disproportionate activation of inflamasome has been implicated. Our hypothesis is that CMML cases show a higher inflammasome activation with respect to the MDS subset, a relevant difference both in terms of potential therapeutic targets and pathogenic clues. The main objective is to confirm, describe and quantify these differences using high-performance and multi-gene/protein methods. Methods: We performed enhanced RNA-seq in bone marrow mononucleated cells of 27 CMML at diagnosis, 10 MDS and 9 controls (103 million average readings). We selected 116 genes related to the inflammasome and reviewed the differential expression between cases and controls. We evaluated by multiplex immunoassay the profile of 28 cytokines in peripheral blood in 35 CMML patients, 37 MDS and 8 controls. Subsequently, we studied whether these differentially expressed genes / cytokines showed differences in CMML depending on the mutational state of TET2, SRSF2 and ASXL1. Finally, we compared in vitro the degree of activation of inflamasome in the monocytoid component of 8 CMML patients versus 7 controls. Results: In the transcriptomic analysis of the inflamasome genes in patients with CMML, we found 30 of 116 differentially expressed genes compared with healthy controls. Of those 30 genes, 26 showed a pro-inflammatory function and, of them, 18 were up-regulated. Of the 4 differentially expressed genes with an anti-inflammatory function, 3 were significantly under-expressed in CMML patients. We highlight, due to the quantitative difference, the overexpression of two genes coding for monocyte chemotactic proteins, CCL7 and CCL2 (FC = 269.21, p = 0.032; FC = 11.79, p = 0.03) That pro-inflammatory transcriptional profile was not so evident in the cases of MDS: of the 29 differentially expressed genes with pro-inflammatory function, 18 were down-regulated. Subsequently, we designed a customized panel for proteomic analysis including 9 of the 30 differentially expressed genes in CMML. We found that, in a relevant percentage of cases, also proinflammatory cytokines derived from these differentially expressed genes were elevated (62.5%) in peripheral blood of patients, compared to healthy donors; pointing towards the key role of gene transcription in the definition of the pro-inflammatory sense of the proteomic dimension of inflammasome in CMML. Next, we found that those patients with CMML and somatic mutations of TET2 had a higher expression of CCL7 and CCL2 compared to patients with CMML wild type, with a tendency to significance in the first case and significant in the second (FC 11.9, p = 0.15; FC 7.8, p = 0.03). Finally, we conducted in vitro stimulation studies at diagnosis in patients with CMML confirming that the canonical activation of the NLRP3 inflammasome (increased production of IL-1β) is significantly enhanced with respect to control individuals. Conclusion: We describe for the first time, a hyperactivation in CMML, compared with MDS, of the components of the inflammasome. Hyperactivation associated in CMML to a gene transcriptional mechanism and related, in the case of the two most over-expressed genes, CCL2 and CCL7, to the presence of mutations in TET2. Our findings point to new therapeutic targets whose modulation could restore inefficient hemopoiesis and potential diagnostic and prognostic biomarkers in CMML. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Dynamic Epigenetic Landscapes Define Multiple Myeloma Progression and Drug Resistance

Blood ◽

10.1182/blood-2020-142872 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-33

Author(s):

Rafael Renatino-Canevarolo ◽

Mark B. Meads ◽

Maria Silva ◽

Praneeth Reddy Sudalagunta ◽

Christopher Cubitt ◽

...

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Chromatin Accessibility ◽

Refractory Disease ◽

Advisory Committees ◽

Cytogenetic Abnormalities ◽

Genome Wide

Multiple myeloma (MM) is an incurable cancer of bone marrow-resident plasma cells, which evolves from a premalignant state, MGUS, to a form of active disease characterized by an initial response to therapy, followed by cycles of therapeutic successes and failures, culminating in a fatal multi-drug resistant cancer. The molecular mechanisms leading to disease progression and refractory disease in MM remain poorly understood. To address this question, we have generated a new database, consisting of 1,123 MM biopsies from patients treated at the H. Lee Moffitt Cancer Center. These samples ranged from MGUS to late relapsed/refractory (LR) disease, and were comprehensively characterized genetically (844 RNAseq, 870 WES, 7 scRNAseq), epigenetically (10 single-cell chromatin accessibility, scATAC-seq) and phenotypically (537 samples assessed for ex vivo drug resistance). Mutational analysis identified putative driver genes (e.g. NRAS, KRAS) among the highest frequent mutations, as well as a steady increase in mutational load across progression from MGUS to LR samples. However, with the exception of KRAS, these genes did not reach statistical significance according to FISHER's exact test between different disease stages, suggesting that no single mutation is necessary or sufficient to drive MM progression or refractory disease, but rather a common "driver" biology is critical. Pathway analysis of differentially expressed genes identified cell adhesion, inflammatory cytokines and hematopoietic cell identify as under-expressed in active MM vs. MGUS, while cell cycle, metabolism, DNA repair, protein/RNA synthesis and degradation were over-expressed in LR. Using an unsupervised systems biology approach, we reconstructed a gene expression map to identify transcriptomic reprogramming events associated with disease progression and evolution of drug resistance. At an epigenetic regulatory level, these genes were enriched for histone modifications (e.g. H3k27me3 and H3k27ac). Furthermore, scATAC-seq confirmed genome-wide alterations in chromatin accessibility across MM progression, involving shifts in chromatin accessibility of the binding motifs of epigenetic regulator complexes, known to mediate formation of 3D structures (CTCF/YY1) of super enhancers (SE) and cell identity reprograming (POU5F1/SOX2). Additionally, we have identified SE-regulated genes under- (EBF1, RB1, SPI1, KLF6) and over-expressed (PRDM1, IRF4) in MM progression, as well as over-expressed in LR (RFX5, YY1, NBN, CTCF, BCOR). We have found a correlation between cytogenetic abnormalities and mutations with differential gene expression observed in MM progression, suggesting groups of genetic events with equivalent transcriptomic effect: e.g. NRAS, KRAS, DIS3 and del13q are associated with transcriptomic changes observed during MGUS/SMOL=>active MM transition (Figure 1). Taken together, our preliminary data suggests that multiple independent combinations of genetic and epigenetic events (e.g. mutations, cytogenetics, SE dysregulation) alter the balance of master epigenetic regulatory circuitry, leading to genome-wide transcriptional reprogramming, facilitating disease progression and emergence of drug resistance. Figure 1: Topology of transcriptional regulation in MM depicts 16,738 genes whose expression is increased (red) or decreased (green) in presence of genetic abnormality. Differential expression associated with (A) hotspot mutations and (B) cytogenetic abnormalities confirms equivalence of expected pairs (e.g. NRAS and KRAS, BRAF and RAF1), but also proposes novel transcriptomic dysregulation effect of clinically relevant cytogenetic abnormalities, with yet uncharacterized molecular role in MM. Figure 1 Disclosures Kulkarni: M2GEN: Current Employment. Zhang:M2GEN: Current Employment. Hampton:M2GEN: Current Employment. Shain:GlaxoSmithKline: Speakers Bureau; Amgen: Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Siqueira Silva:AbbVie: Research Funding; Karyopharm: Research Funding; NIH/NCI: Research Funding.

Download Full-text

Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemias and Leukemic Stem Cells: Therapeutic Implications

Blood ◽

10.1182/blood.v126.23.2899.2899 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2899-2899

Author(s):

Thierry Guillaume ◽

Virginie Dehame ◽

Patrice Chevallier ◽

Pierre Peterlin ◽

Marc Grégoire ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cell Surface ◽

Board Of Directors ◽

Leukemic Cell ◽

Muc1 Expression ◽

Leukemic Stem Cells ◽

Advisory Committees ◽

Alpha Chain ◽

Myelomonocytic Leukemia

Abstract Monocytic neoplasms comprise a heterogeneous group of hematologic malignancies including chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5), and monocytic sarcoma. Monocytic or granulomonocytic hyperplasia is a finding frequently-if not invariably-shared by these different entities, as is a poor therapeutic outcome in the absence of hematopoietic stem cell transplantation. Cell surface molecules aberrantly expressed or overexpressed by leukemic cells represent potential disease-specific therapeutic targets. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a molecule. MUC1 consists of an extracellular domain containing 20 to 125 tandem repeats of a 20 amino acid-long sequence, followed by a transmembrane domain and a short cytoplasmic tail leading to intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit containing the tandem repeat array bound in a strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. Because the alpha chain binds the cell-bound domains of MUC1 only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on AML cells by flow cytometry. A series of twenty-two AML samples (blood-derived n=12; bone marrow-derived n=10; AML0=2, AML1=2, AML2=10, AML4=1, AML5=5, AML6=2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line stably transfected with human MUC1 DNA served as control. Blasts cells from 5 AML samples highly expressed MUC1, and significantly, all were of monocytic or myelomonocytic lineage (AML4=1, AML5=4). Leukemic stem cells (CD34pos or CD34neg linneg) from the MUC1+ AMLs were examined and likewise found to express MUC1. In addition, AML cell lines MV411, MOLM14, and SHI-1 derived from monocytic leukemic lineage clearly expressed cell surface MUC1, while non- monocytic leukemic cell lines U937, K562, and HL60 had little or no expression. Normal monocytes and monocytes derived from patients with activated monocytosis were also found to express MUC1. Based on these findings we examined MUC1 expression in a series of myelomonocytic leukemia (CMML and JMML). In fifteen CMML samples examined (type 1 n=11, type 2 n=4) (blood n=7, BM n=7) 92%-100% (median 99.7%) of CD14+CD56+ CMML cells bound mAb DMB-5F3 to cell-surface MUC1. CD14+CD16+CD56+ blast cells from 2 pts with JMML were also found to express MUC1 (between 64% and 71 % positive). Based on these findings we conclude that expression of MUC1 is restricted to monocytic and myelomonocytic leukemias and that MUC1 represents an effective target for leukemic immunotherapy. Significantly, anti-MUC1 mAb also targets monocytic leukemic stem cells, reinforcing its therapeutic potential. The fact that the anti-MUC1 antibody DMB-5F3 can enter cells and thereby ferry Ab-bound toxin opens the way for us to demonstrate leukemic cell killing with anti-MUC1 mAb-immunotoxin conjugates. Disclosures Moreau: Bristol-Myers Squibb: 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; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Download Full-text