Azacitidine Therapy Research Articles

Single-cell analysis of bone marrow CD8+ T cells in Myeloid Neoplasms reveals pathways associated with disease progression and response to treatment with Azacitidine.

CD8+ T cells are crucial for antitumor immunity. In higher-risk myelodysplastic neoplasms (HR-MDS) and acute myeloid leukemia (AML), CD8+ T cells exhibit altered functionality. To address their role in the course of the disease, we performed in-depth immunophenotypic analysis of 104 pre-treatment bone marrow (BM) samples using mass and flow cytometry and observed an increased frequency of the CD57+CXCR3+ subset of CD8+ T cells in patients who failed azacitidine (AZA) therapy. Furthermore, an increased baseline frequency (>29%) of the CD57+CXCR3+CD8+ T cell subset was correlated with poor overall survival. We performed scRNA-seq to assess the transcriptional profile of BM CD8+ T cells from treatment-naive patients. The response to AZA was positively associated with the enrichment of IFN-mediated pathways, whereas an enhanced TGF-β signaling signature was observed in non-responders. Our results suggest that targeting CD8+ T cells with inhibitors of TGF-β signaling in combination with AZA is a potential therapeutic strategy for HR-MDS and AML.

Overexposure to venetoclax is associated with prolonged-duration of neutropenia during venetoclax and azacitidine therapy in Japanese patients with acute myeloid leukemia.

An observational study was conducted to evaluate the pharmacokinetics of venetoclax and its impact on the efficacy and safety for Japanese patients with acute myeloid leukemia (AML) treated with venetoclax and azacitidine therapy. The association between the plasma concentration, after the first cycle of azacitidine and venetoclax therapy, and the efficacy and safety was evaluated in 33 patients with untreated or relapsed/refractory AML. Full dose of venetoclax was administered to all patients. Venetoclax treatment was 28day long in 82% of patients; the relative dose intensity of azacitidine was 82%. Trough concentration was significantly higher among patients with complete remission (CR) and CR with incomplete hematologic recovery (CRi) than those with the morphologic leukemia-free state and partial remission, and no response groups (P = 0.01). Median duration of grade 3 neutropenia was 28 days (range 8-46 days). Area under the concentration-time curve (AUC0-24) was significantly higher among patients with protracted grade 3 neutropenia (≥ 28 days) than those with a shorter duration (< 28 days) (P = 0.03); multivariate analysis revealed that a higher AUC0-24 was a significant predictor of a longer duration of neutropenia (odds ratio 54.3, P = 0.007). Plasma concentrations of venetoclax were variable in Japanese patients with AML. Higher plasma concentrations were associated with CR/CRi and protracted grade 3 neutropenia. Therefore, it is essential to adjust the duration of venetoclax administration based on individual pharmacokinetic data to limit total drug exposure, reduce severe neutropenia, and achieve higher efficacy.

A Phase 1A/B Combination Study of Pegargiminase (ADI-PEG 20), Venetoclax and Azacitidine in Patients with Acute Myeloid Leukemia

Background and Significance: Outcomes for adverse-risk acute myeloid leukemia (AML), especially in older and/or unfit patients and those with relapsed/refractory disease, remain poor despite several new drug approvals over the last decade. A promising area for drug development in AML resides in targeting tumor metabolism. The metabolism therapy of pegargiminase (ADI-PEG 20), an agent consisting of the arginine-degrading enzyme arginine deiminase combined with polyethylene glycol, has demonstrated preclinical and clinical efficacy as monotherapy and in combination in AML. ADI-PEG 20 has been shown to be most effective when tumors are deficient in argininosuccinate synthetase (ASS1), the rate limiting step in the urea cycle to form arginine. AML is one such cancer, which appears to be addicted to arginine, with about 90% of AML patient samples being deficient in ASS1 expression. The focus of this phase 1A/B study is to explore combination treatment with ADI-PEG 20 added to standard front-line venetoclax and azacitidine therapy in AML. Study Design and Methods: This study (NCT05001828) is an open label, single arm, multicenter phase 1 trial for patients with newly diagnosed adverse-risk AML unfit for intensive chemotherapy or relapsed/refractory AML. Eligible patients must be ≥18 years old and are ineligible if determined favorable-risk by European LeukemiaNet (ELN) criteria. The primary objective is to determine the recommended phase 2 dose (RP2D) of ADI-PEG 20 administered by intramuscular injection weekly in combination with venetoclax and azacitidine. Secondary objectives include evaluating preliminary evidence of antitumor activity and determining the pharmacodynamics and immunogenicity of ADI-PEG 20 in combination with venetoclax and azacitidine. Patients receive ADI-PEG 20 via intramuscular injection on day -3 and then weekly along with the combination regimen of azacitidine 75 mg/m 2 for 7 days and oral venetoclax on days 1-28 of the first cycle and days 1-21 for subsequent cycles. Subjects may remain on study treatment for a maximum of 24 cycles (96 weeks). The study is designed with a lead-in group exploring 2 dose levels of ADI-PEG 20 and an RP2D expansion group with 18 subjects each in 2 cohorts: Cohort 1 including previously treated (relapsed/recurrent) or refractory AML and Cohort 2 including untreated AML with high-risk features and ineligible for intensive chemotherapy. In total, 48 to 60 subjects are planned across up to 5 centers in the United States. In the RP2D cohorts, the target response rates for the relapsed/refractory and untreated AML with high-risk features are 25% and 55%, respectively. Safety is assessed by comparing adverse events, laboratory results, and physical examination results. A greater than 20% clinically significant drug-related grade 3 or higher toxicity rate occurring in the first cycle is considered unacceptable. To assess efficacy, the number and percent of subjects' objective response rate, duration of response, and overall survival (OS) will be summarized. Pharmacodynamics using peripheral blood arginine and citrulline levels will be summarized for all subjects by time point for the observed value as well as for the change from baseline value and by duration of arginine depletion and citrulline increase. Immunogenicity, determined by antibodies to ADI-PEG 20, will be summarized for all subjects by time point for the observed value as well as for the change from baseline value. Semiquantitative assessment of ASS1 expression by immunoblots and/or immunohistochemistry or other appropriate means will be summarized descriptively. Demographics, clinical characteristics (including duration of response), and safety data of the patients will be summarized using descriptive statistics. For the efficacy analysis, we will estimate the response rate along with the 95% credible interval. The association between response and patient's clinical characteristics will be examined by Wilcoxon's rank sum test or Fisher's exact test, as appropriate. The distribution of time-to-event endpoints, such as OS and others, will be estimated using the method of Kaplan and Meier. Comparisons of time-to-event endpoints by important subgroups will be made using the log-rank tests. The trial is currently enrolling and presents a novel and promising therapeutic strategy for patients with high-risk newly diagnosed and relapsed/refractory AML.

A Systematic Review of Venetoclax and Azacitidine: Effectiveness and Safety for Newly Diagnosed and Relapsed Acute Myeloid Leukemia Patients

Introduction: Acute Myeloid Leukemia (AML) remains the most prevalent acute leukemia in adults, often associated with a bleak prognosis. Current treatment recommendations for elderly AML patients incorporate a combination of venetoclax (Ven) and azacitidine (AZA) following trials that underscored Ven's efficacy. This review seeks to analyze and summarize the literature concerning the effectiveness and safety of the Ven + AZA regimen for adult patients with newly diagnosed (ND) and relapsed/refractory (R/R) AML, and those unsuitable for intensive chemotherapy. Materials and Methods: Following PRISMA guidelines, a comprehensive electronic literature search was conducted across PubMed, Embase, Clinicaltrials.gov, Cochrane Library, and Web of Science from inception to 2023. MeSH terms and relevant keywords for (Leukemia, Myeloid, Acute) AND (Venetoclax) AND (Azacitidine) were used. From 327 initially identified articles, 12 studies were selected for inclusion, and citation searching further added 2 studies, summing up to 14. We analyzed data from a combined patient pool of 1256. Results: The review encompassed 13 non-randomized and 1 randomized clinical trials. The median patient age was 75.2 years. Primary outcomes comprised complete remission (CR) and complete remission with incomplete count recovery (CRi), yielding a pooled CR/CRi of 57.8%. Secondary outcomes included an overall response rate (ORR) of 61.1%, and a median overall survival (mOS) of 8.94 months with a median follow-up duration of 13.25 months (Table 1). Notably, high-grade adverse effects were predominantly hematological (anemia, neutropenia, thrombocytopenia, febrile neutropenia) while low-grade effects primarily involved gastrointestinal symptoms (nausea, vomiting, diarrhea, constipation, decreased appetite, etc). Conclusion: The current data suggest that the Ven + AZA regimen is both efficacious and well-tolerated for treating newly diagnosed and relapsed/refractory AML. Cytopenias across all three cell lines emerged as the most reported adverse effects of Ven + AZA therapy. Ongoing Phase III clinical trials are expected to enhance our understanding of this regimen's efficacy and safety profile, solidifying its role in standard AML therapy.

Lenzilumab in Addition to Azacitidine Improves Complete Response Rates in Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a rare cancer orchestrated by granulocyte-macrophage colony-stimulating factor (GM-CSF), a pro-inflammatory cytokine that drives leukemic monocyte proliferation. Standard of care (SOC) for CMML treatment includes azacitidine (AZA), with a complete response (CR) rate of 16-21% 1,2. The PREcision Approach to CHronic Myelomonocytic Leukemia (PREACH-M; ACTRN12621000223831) trial investigates novel CMML therapies directed by molecular profiling. Lenzilumab (LENZ; Humanigen, Inc., Short Hills, NJ) a proprietary Humaneered® first-in-class monoclonal antibody with best-in-class off-rate and affinity that neutralizes GM-CSF. PREACH-M interim results show that LENZ/AZA improves hematologic parameters, decreases spleen size, and dampens pro-inflammatory responses in CMML with RAS-pathway mutations. This report details the objective clinical responses from an interim analysis of the first 11 subjects who completed at least three months LENZ/AZA treatment. Methods: PREACH-M is a Phase 2/3 non-randomized, uncontrolled, open-label trial in 72 adults aged at least 18 years, newly diagnosed with WHO 2016 criteria for CMML. Key exclusion criteria include prior treatment with investigational agents; radiotherapy within 28 days before treatment; treatment with G-CSF within 7 days of screening; GM-CSF within 28 days of screening; and uncontrolled medical conditions. Subjects exhibiting RAS-pathway mutations ( NRAS, KRAS, CBL) receive 24 cycles (every 28 days) of AZA (SC; 75 mg/m2 for 7 days) and LENZ (IV; 552 mg; d1 &amp; d15 of cycle 1 and d1 only for all subsequent cycles); while those with only TET2 mutations receive the same AZA regimen and sodium ascorbate (IV; 30 g for 7 days [15 g for 1st dose only, 30 g thereafter if no evidence of tumor lysis syndrome]; PO; 1.1g on all other days). Subjects who complete 24 cycles of treatment are followed every 6 months for an additional 24 months. The primary endpoint is the frequency of complete response (CR) or partial response (PR) during the first 12 cycles according to Savona Criteria. Secondary endpoints include responses according to modified 2006 International Working Group criteria, 2 year overall survival, and symptom improvement. Results: As of July 2023, 15 subjects were enrolled in the LENZ/AZA arm (8 females, 7 males with mean age 69; mean white cell count 21x10 9/L, mean Hb 121 g/L; mean platelet count, 74x10 9/L, mean blast count, 10.1%). Mutations included; CBL (47% of subjects), NRAS (27%), KRAS (47%), NRAS and KRAS (13%), and TET2 (93%). Subjects exhibited CPSS-MOL scores, of intermediate risk 1 (n=1), intermediate risk 2-3 (n=8), and high risk 4-6 (n=6). All the 11 evaluable subjects at 3 months responded to LENZ/AZA. CR was achieved within 3 cycles in 55% of subjects. Six of the subjects demonstrated CR including 2 with a high risk CPSS-MOL profile and 8 achieved either CR or complete marrow response (blasts&amp;lt;5%) within 12 months. One subject had a platelet response, 1 subject each had PR and stable disease with blasts &amp;lt;5%. CMML progression was absent and 1 subject became eligible for allogeneic transplant. These findings exceed historical CR rates for hypomethylating agents (16%; 95%CI, 12-21% 1 and 21%;13-29% 2). Self-reported symptom scores from the standardized MPN-SAFFS improved from baseline (mean of 22 vs 12, P=0.06). Fifteen grade 3 and 9 grade 4 adverse events were reported of which 2 were “probably” ascribed to both LENZ/AZA and 7 were “possibly” ascribed to LENZ. No unexpected adverse events were observed. Conclusion: Interim analysis of the PREACH-M trial demonstrated that GM-CSF neutralization with LENZ/AZA, for the treatment of CMML with RAS-pathway mutations resulted in 55% CR, achieved early in treatment, durability up to 18 months, thus far, and no unexpected serious adverse events. These data suggest CMML is driven by a non-redundant cytokine that responds to immunotherapy. 1. Xu Y, Guo R, Miao M, Zhang G, Lan J, Jin J. Real-world data on efficacy and safety of azacitidine therapy in chronic myelomonocytic leukemia in China: results from a multicenter, retrospective study. Invest New Drugs 2022;40(5):1117-1124. DOI: 10.1007/s10637-022-01283-x. 2. Zheng X, Lv L, Li X, Jiang E. Efficacy and Safety of Hypomethylating Agents in Chronic Myelomonocytic Leukemia: A Single-Arm Meta-analysis. Glob Med Genet 2022;9(2):141-151. DOI: 10.1055/s-0042-1744157.

Olutasidenib Alone or in Combination with Azacitidine Induces Durable Complete Remissions in Patients with mIDH1 Myelodysplastic Syndromes/Neoplasms (MDS)

Introduction Olutasidenib is a potent, selective, oral, small-molecule inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1). Olutasidenib is approved in the US for the treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML) based on the pivotal cohort (n=153) of a registrational Phase 1/2 trial (NCT02719574), which demonstrated a rate of complete remission (CR) or CR with partial hematologic recovery (CRh) of 35%, with a duration of response of 25.9 months. The Phase 1/2 study also enrolled patients with mIDH1 myelodysplastic syndromes/neoplasms (MDS), and here we report results from this subset of 22 patients. Methods The Phase 1/2 open-label study enrolled 332 adult patients with mIDH1 AML or intermediate-, high-, or very high-risk MDSfrom 57 centers in 9 countries. Inclusion criteria included an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 and adequate liver and renal function. Patients were R/R to prior therapy or, if treatment-naïve (TN), had to be eligible for azacitidine therapy (to be enrolled in a combination therapy cohort). Olutasidenib was administered at 150 mg BID (n=3) or 100-150 QD (n=3) as monotherapy or in combination with azacitidine (n=16), given at 75 mg/mm 2 daily for 7 consecutive days, in 28-day cycles. The primary efficacy endpoint for MDS was the CR rate, based on response criteria of the International Working Group in MDS. Secondary efficacy endpoints included overall response (CR/PR/marrow CR), time to response, duration of response and safety. Results Twenty-two patients with MDS were enrolled, including 6 who received monotherapy (4 R/R and 2 TN) and 16 who received combination therapy (11 R/R and 5 TN). Baseline demographics and disease characteristics are shown in Table 1. The age ranged from 59-87 years, and 59% of the study population were male. Seven patients were treatment-naïve and 15 were R/R with 1-4 prior treatment regimens. For the pooled Phase 1 and 2 data (n=22), 6 (27%) patients achieved CR and 7 (32%) patients achieved marrow CR with no PRs, generating a 59% overall response rate. The median time to response was 2.0 months. The median duration of response was not reached (NR) at 30.1+ months ( Table 2). The overall response rate was 2/6 (33%) for monotherapy and 11/16 (69%) for combination therapy. Three of 6 patients received monotherapy at a lower dose (100-150 QD) than the approved dose level, and none responded. Of the 3 patients who received monotherapy at the approved dose level (150mg BID), 2 (66%) responded. In the 7 TN patients, the overall response rate was 86%. In the 15 R/R patients, the overall response rate was 47%. All patients with MDS experienced at least 1 treatment-emergent adverse event (TEAE). The most frequent TEAEs in the study were nausea, constipation, vomiting, thrombocytopenia, neutropenia, diarrhea, and fatigue. Grade 3 TEAEs occurred in 19/22 (86%) patients, and Grade 4 TEAEs in 9/22 (41%). The most frequent Grade 3/4 TEAEs reported were cytopenias. Grade 3 transaminitis occurred in 4 patients. Grade 3 differentiation syndrome was reported in 1 patient, who discontinued treatment after 23 days due to pneumonia, which lead to respiratory failure and death. TEAEs resulting in death occurred in 5 patients and included disease progression (3), pneumonia (1, mentioned above) and chest fungal infection (1). The overall safety profile in patients with MDS was consistent with what has been reported in patients with AML in this study (Watts, et al., Lancet Hematol, 2022; de Botton et al., Blood Adv, 2023). Conclusions Olutasidenib, both as monotherapy and in combination with azacitidine, induced durable remissions in patients with intermediate-, high-, or very high-risk MDS. Patients had varying treatment backgrounds, including treatment-naïve and up to four prior regimens. This treatment had a tolerable and manageable safety profile. These encouraging results, which warrant further investigation with a larger number of patients, show that olutasidenib has clinically meaningful activity in patients with mIDH1 MDS.

Cytokine and Mutation Profiling Reveal Patterns of Complete Remission Rates with Lenzilumab Combination Therapy in Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is characterized by accumulation of classical CD14+CD16- inflammatory monocytes driven in part by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a pro-inflammatory cytokine. Standard of care in CMML includes hypomethylating agents such as azacytidine (AZA), with complete response (CR) rates of 16-21% 1,2 and no reliable biomarkers that predict response. The complete pro-inflammatory profile of CMML is unknown and no treatment addresses the hematologic aberrations of CMML. Lenzilumab (LENZ; Humanigen, Inc., Short Hills, NJ) is a proprietary Humaneered ® first-in-class monoclonal antibody with best-in-class off-rate and affinity that neutralizes GM-CSF. The interim analysis of the PREcision Approach to CHronic Myelomonocytic Leukemia (PREACH-M; ACTRN12621000223831) trial showed LENZ/AZA treatment in 11 subjects with RAS-pathway ( NRAS/ KRAS/ CBL) mutations resulted in 8 subjects with complete response or optimal marrow response and improvements in hematological parameters. This report describes the cytokine profiles and systemic C-reactive protein levels of these subjects. Methods: PREACH-M is a Phase 2/3 non-randomized, open-label trial in 72 subjects, aged at least 18 years, with newly diagnosed CMML based on the WHO 2016 criteria; and RAS-pathway mutations at a variant allele frequency ≥3%. Subjects received 24 cycles (every 28 days) of AZA (SC; 75 mg/m 2 for 7 days) and LENZ (IV; 552 mg; d1 &amp; d15 of cycle 1 and d1 only for all subsequent cycles). Subjects without RAS-pathway mutations received the same AZA regimen and sodium ascorbate (IV; 30g for 7 days (15g for 1st dose only; 30g thereafter if no evidence of tumor lysis syndrome); PO; 1.1g on all other days). Cytokine profiling from bone marrow plasma was performed after 4,7,12 and 24 months using Milliplex Human Cytokine/Chemokine Magnetic bead panel and compared with 24 age-matched healthy subjects. Unsupervised hierarchical clustering with Ward's method sought distinct CMML patterns based on cytokine expression. C-reactive protein (CRP) was determined from blood samples using a routine assay. Variant allele frequencies were determined from bone marrow mononuclear cells using a 41-myeloid panel using Illumina Hi-Seq with a depth of 1000x. Results: As of July 2023, 15 subjects were enrolled in the LENZ/AZA arm (8 females, 7 males with mean age 69; mean white cell count, 21x10 9/L; mean Hb, 121 g/L, mean platelet count; 74x10 9/L, mean blast count, 10.1%). CRP decreased from a median of 5.6 at baseline to 2.1 mg/L after 6 months of LENZ/AZA (P=0.02). GM-CSF and other pro-inflammatory cytokines were increased in CMML bone marrow plasma compared with healthy subjects. Hierarchical clustering of all subjects revealed 2 patterns of CMML, distinct from healthy subjects. Cluster “INNATE-1” was comprised of medium increases in inflammatory cytokines associated with innate immune response and M1 macrophage activation (IFN-g, IL-1b, TNFa, IL-12p70, IL-12p40, IL-17, Fractalkine, MCP-3). Cluster “INNATE-2” was associated with extreme increases of these cytokines. The mean pro-inflammatory innate immune score (sum of z-scores) was 20-fold greater in INNATE-2 compared with INNATE-1 ( P=0.055). Somatic mutations SRSF2, WT1, PHF6 were enriched in INNATE-2. All patients (5/5) in INNATE-1 showed 100% response (CR or optimal marrow response resulting in &amp;lt;5% blasts) to AZA/LENZ. Two subjects (2/6) in INNATE-2 demonstrated partial responses or haematological improvement thus far. Conclusion: CMML is a disorder of profound innate immune activation, driven by GM-CSF and other pro-inflammatory cytokines. Early treatment with LENZ/AZA, a precision immunotherapeutic approach, leads to a) efficacy in INNATE-1 that exceeds historical CR rates for hypomethylating agents 1,2; and b) evolving efficacy in INNATE-2, in which pro-inflammatory activity is more robust. 1. Xu Y, Guo R, Miao M, Zhang G, Lan J, Jin J. Real-world data on efficacy and safety of azacitidine therapy in chronic myelomonocytic leukemia in China: results from a multicenter, retrospective study. Invest New Drugs 2022;40(5):1117-1124. DOI: 10.1007/s10637-022-01283-x. 2. Zheng X, Lv L, Li X, Jiang E. Efficacy and Safety of Hypomethylating Agents in Chronic Myelomonocytic Leukemia: A Single-Arm Meta-analysis. Glob Med Genet 2022;9(2):141-151. DOI: 10.1055/s-0042-1744157.

Selinexor in Combination with Azacitidine (AZA) in Patients with Myelodysplastic Syndrome (MDS): A Real-World Retrospective Study

Background: Patients with higher-risk MDS face limited treatment options and poor outcomes. While HMAs are approved as frontline therapy for higher-risk MDS, primary failure occurs in 50% of HMA-treated patients, and most responders progress within 2 years. There is an unmet need to improve the efficacy of AZA frontline therapy while maintaining a tolerable safety profile. Preclinical studies have demonstrated that the combination of selinexor and azacitidine synergistically inhibits MDS cell proliferation and arrests the cell cycle at the G2/M phase (Yixuan Guo, 2022). The combination of selinexor and azacitidine may be a promising approach for treating MDS. Here, we present this single-center, real-world evidence supporting the use of selinexor in combination with AZA in MDS patients. Methods: Medical records of 19 patients with MDS receiving selinexor in combination with AZA at the First Affiliated Hospital of Soochow University between Jan 2022 and March 2023 were reviewed. Inclusion criteria were a pathologically confirmed diagnosis of MDS, and receipt of ≥1 cycle of azacitidine in combination with selinexor. HMA-naïve patients and those previously treated with HMAs were included. Patients with a diagnosis other than MDS or progression to AML before treatment with selinexor + AZA were excluded. Patients received selinexor 40-60 mg QW orally and AZA 75 mg/m 2 intravenously or subcutaneously on days 1-5 or 7 of each 28-day cycle. Responses were evaluated per the International Working Group criteria. Results: A total of 19 pts (median age, 54 y [range, 13-68 y]) were treated (Table 1). IPSS-R risk was high or very high in 15.8% and 68.4% of pts, respectively. IPSS-M risk was high or very high in 10.5% and 78.9% of pts, respectively. 57.9% of pts were previously treated with HMA. 63.2% had myelofibrosis, 42.1% had hepatosplenomegaly, 31.6% had a TP53 mutation, and 63.2% had poor or very poor-risk cytogenetics. As of Jun 10, 2023 at a median follow-up of 5.9 months (range 1.5-17.4 months), eight pts died (42.1%) due to disease progression. For the total cohort, the overall response rate (ORR) was 73%. In pts with worse survival prognosis, such as relapse and refractory, high and very high in IPSS-R, high and very high in IPSS-M, myelofibrosis and TP53 mut, the ORR was 64%, 81%, 76%, 75% and 83%, respectively (Figure 1). In seven pts with splenomegaly, six pts had reduced spleen volume, and one pt had no significant change by abdominal palpation or imaging examination. The 6-month overall survival (OS) was 74.7% in total pts, and 70.0% in pts previously exposed to HMA (Figure 2a, 2b). Selinexor + AZA therapy improved survival of patients with poor prognosis, such as TP53 mut (0.099), MF (+) (0.613), ≥high in IPSS-R (0.938) (Figure 2c, 2d, 2e). The survival of six pts who received transplantation after combination therapy was significantly prolonged, with 6-month OS was 83.3% (Figure 2f). Conclusion: Selinexor + AZA combination therapy was well tolerated with promising efficacy in patients with untreated or relapse/refractory MDS, including those with TP53 mutation, myelofibrosis, and higher risk in IPSS-R.

Comparative analysis of hypomethylating agents as maintenance therapy after allogeneic hematopoietic stem cell transplantation for high-risk acute leukemia

We retrospectively analyzed the outcomes of 136 consecutive patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) at our center. Among them, 76 cases used hypomethylating agents (decitabine, n = 40; azacitidine, n = 36) as post-transplant maintenance therapy, whereas 60 contemporaneous patients did not adopt maintenance therapy. The 3-year incidences of relapse in two groups were 16.6% and 39.2% (p = .001). The 3-year OS and DFS in maintenance group were 84.0% and 78.6%, which were remarkably improved than in control group (60.0% and 58.0%) (p = .004, p = .011). Moreover, the 3-year relapse rates for patients receiving decitabine and azacitidine therapy were 8.5% and 25.0%, respectively (p = .019). Patients utilizing decitabine had more common possibility of grade 3–4 neutropenia than azacitidine (20.0% vs. 2.8%, p = .031). These results indicate that maintenance therapies using hypomethylating agents could reduce the risk of post-transplant recurrence, resulting into remarkable superior survival. Decitabine might lower relapse after allo-HSCT with somewhat more severe myelosuppression when being compared to azacitidine.

Next-generation sequencing of baseline genetic mutations and outcomes of eltrombopag and azacitidine therapy in patients with myelodysplastic syndromes and thrombocytopenia: Data from the SUPPORT clinical trial.

Eltrombopag has been previously shown to be effective in reversing azacitidine-mediated thrombocytopenia. This was further investigated in the SUPPORT trial, a phase III study assessing the efficacy/safety of eltrombopag plus azacitidine in patients with intermediate- to high-risk myelodysplastic syndromes and thrombocytopenia. The results did not support a clinical benefit for the addition of eltrombopag to azacitidine. We investigated if the somatic mutational profiles in the patient cohort were associated with treatment outcomes. Based on the available data, we observed no imbalance in the mutational profiles between treatment arms or a clear association between identified somatic mutations and clinical outcomes.

Solute Carrier Family 29A1 Mediates In Vitro Resistance to Azacitidine in Acute Myeloid Leukemia Cell Lines.

Azacitidine (AZA) is commonly used hypomethylating agent for higher risk myelodysplastic syndromes and acute myeloid leukemia (AML). Although some patients achieve remission, eventually most patients fail AZA therapy. Comprehensive analysis of intracellular uptake and retention (IUR) of carbon-labeled AZA (14C-AZA), gene expression, transporter pump activity with or without inhibitors, and cytotoxicity in naïve and resistant cell lines provided insight into the mechanism of AZA resistance. AML cell lines were exposed to increasing concentrations of AZA to create resistant clones. 14C-AZA IUR was significantly lower in MOLM-13- (1.65 ± 0.08 ng vs. 5.79 ± 0.18 ng; p < 0.0001) and SKM-1- (1.10 ± 0.08 vs. 5.08 ± 0.26 ng; p < 0.0001) resistant cells compared to respective parental cells. Importantly, 14C-AZA IUR progressively reduced with downregulation of SLC29A1 expression in MOLM-13- and SKM-1-resistant cells. Furthermore, nitrobenzyl mercaptopurine riboside, an SLC29A inhibitor, reduced 14C-AZA IUR in MOLM-13 (5.79 ± 0.18 vs. 2.07 ± 0.23, p < 0.0001) and SKM-1-naive cells (5.08 ± 2.59 vs. 1.39 ± 0.19, p = 0.0002) and reduced efficacy of AZA. As the expression of cellular efflux pumps such as ABCB1 and ABCG2 did not change in AZA-resistant cells, they are unlikely contribute to AZA resistance. Therefore, the current study provides a causal link between in vitro AZA resistance and downregulation of cellular influx transporter SLC29A1.

High rate of invasive fungal infections during early cycles of azacitidine for patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a form of cancer that is characterized by infiltration of the bone marrow, blood, and other tissues by proliferative, clonal, abnormally differentiated, and occasionally poorly differentiated cells of the hematopoietic system. Patients with acute myeloid leukemia (AML) receiving azacitidine (AZA) alone or in combination with venetoclax (VEN-AZA) are at increased risk for invasive fungal infections (IFIs). We compared the incidence and risk of IFI during these treatment regimens in a single Taiwan hospital. A total of 61 patients with AML received at least one course of AZA in the hematology ward of China Medical University Hospital (Taichung, Taiwan) between September 2012 and June 2020. Thirty-eight patients (62.3%) received AZA monotherapy; 23 (37.7%) received VEN-AZA. Incidence rates of probable and proven IFI were 18% and 1.6%, respectively, during AZA treatment. One proven case of Fusarium spp. infection was isolated by skin and soft tissue culture. Most (75%) IFI cases occurred during the first cycle of AZA therapy. Half of all IFI cases occurred in patients with prolonged neutropenia. The risk of IFI was significantly higher for the European LeukemiaNet (ELN) nonfavorable-risk group (intermediate- and adverse-risk group) versus the ELN favorable-risk group and for patients with prolonged neutropenia versus those without (P<0.05 for both comparisons). In this study, median OS did not differ significantly between patients with and without IFIs during AZA-containing regimens (14.6 months vs 13.7 months; P=0.59). The incidence of IFI was high in this AML cohort treated with AZA-containing regiments in Taiwan. The majority of IFI cases occurred during the early cycles of AZA (cycles 1-2). Prospective studies are needed to determine the optimal choice of antifungal prophylaxis agent during VEN-AZA therapy for AML.

A retrospective assessment of real-world experience with venetoclax and azacitidine therapy in elderly acute myeloid leukemia.

This study aimed to examine the effect of venetoclax coupled with azacytidine in treating older adults with relapsed and refractory (R/R) acute myeloid leukemia (AML). The clinical data of 10 senior patients with AML over 65 years old who were treated with venetoclax and azacytidine, including six patients with R/R AML, were retrospectively evaluated. This study comprised seven males and three females with a median age of 71 years. Five patients had at least one relapse, and one patient did not achieve remission after four cycles of azacytidine monotherapy, considering it resistant. AML with myelodysplasia-related changes was found in four cases. One of the 10 patients died early after 1-13 cycles of venetoclax plus azacytidine treatment due to a protracted period of neutropenia and severe lung infection induced by medications. Six of the remaining nine patients, including six R/R patients, achieved a complete remission (CR) or a CR with incomplete hematologic recovery (CRi). After two cycles of therapy, one patient did not react. Neutropenia lasted an average of 10.5 (6-15) days in all patients, with the most severe cases occurring in the second and third weeks of therapy. Three patients who tested positive for the TP53 gene mutation had the following outcomes: One relapsed patient has been in progression-free remission (PFS) for the past 24 months, whereas another has been in full remission but relapsed 2 months later. Another patient experienced complete remission in myelology for 4 months, but the variable allele fraction (VAF) value steadily rose, suggesting that the illness was on the verge of progressing. IDH2 gene alterations were found in three of four patients who obtained maintained CR for more than 18 months following recurrence. Venetoclax in combination with azacytidine is a successful and well-tolerated therapy for R/R AML in the elderly. Venetoclax and azacytidine may help patients with TP53 mutations and reduce VAF. The IDH2 mutation might be a good predictor of veneclax sensitivity. A notable adverse response in the treatment phase of the regimen is severe infection induced by neutropenia.

XPO1 Overexpression Is a Mechanism of Resistance to Eprenetapopt and 5-Azacitidine Therapy That Can be Therapeutically Exploited for the Treatment of TP53 Mutated Myeloid Malignancies

XPO1 Overexpression Is a Mechanism of Resistance to Eprenetapopt and 5-Azacitidine Therapy That Can be Therapeutically Exploited for the Treatment of TP53 Mutated Myeloid Malignancies

Combination Azacitidine and Lenalidomide: A Novel Relapse Prophylaxis Option in Patients after Allogeneic Stem-Cell Transplantation for Acute Myeloid Leukemia

Introduction: Some patients with acute myeloid leukemia (AML) will still relapse after receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Once relapse, the salvage options are limited and difficult. Therefore, novel drug regimens to prevent relapse after transplantation are required. Both azacitidine (AZA) and lenalidomide (LEN) possess significant antitumor activity effect in myeloid neoplasms. LEN as maintenance or pre-emptive therapy in the first 3 months post-transplantation was reported to induce severe graft-versus-host disease (GVHD), but AZA has been shown to ameliorate GVHD in transplantation. We therefore examined the efficacy and safety of combined AZA/LEN prophylactic regimen after transplantation. Patients and Methods: AML patients who had received allo-SCT were treated with AZA (75 mg/m2 for 7 days) followed by low dose of LEN on days 10 to 28. The above is one cycle, a total of 8-10 cycles. The course of treatment can be appropriately changed according to the patient's tolerance and condition. The primary endpoint is cumulative incidence of relapse (CIR), the secondary endpoints are Overall survival (OS), Disease-free survival (DFS), adverse events (AEs). Results: A total of 28 patients received azacytidine and low-dose lenalidomide prophylaxis after allo-SCT. According to risk stratification, 71% (20/28) patients were at high risk and 29% (8/28) were at intermediate risk. Median chemotherapy numbers before transplantation were 4 (3-8) cycles. The transplantation model includes Haplo-identical (46%), HLA-identical sibling (14%), and Unrelated donor (40%). With a median follow-up of 387 (94-1073) days, 9 patients relapsed (including molecular relapse and cytological relapse), among them, 4 patients had molecular relapse and MRD was changed into negative again after treatment with this AZA/LEN regimen, and other 5 patients developed cytologically relapsed, so this program was terminated, and they received new treatment, but all still alive now. The remaining 23 have survived so far, with disease free. The 1-year CIR is 26 %, and 2-year CIR is 41 %. The 1-year DFS is 74%, and the 2-year DFS is 59%. OS is 100%. Sequential AZA and LEN prophylaxis therapy was well tolerated. About adverse events: a total of 8 patients had grade 3-4 neutropenia, none had agranulocytosis with fever; 3 patients had grade 3-4 thrombocytopenia; Grade 3 transaminase elevations occurred in 2 patients. Four patients developed chronic GVHD after AZA/LEN regimen with a score of 1-2, and no patient developed acute GVHD.

Venetoclax Rapidly and Strongly Enhances the Phospholipase C Response to Azacitidine Therapy in Myelodysplastic Syndromes

Venetoclax Rapidly and Strongly Enhances the Phospholipase C Response to Azacitidine Therapy in Myelodysplastic Syndromes

Treatment with Oral Azacitidine in Elderly Patients with Higher Risk Myelodysplastic Syndromes after Response to Subcutaneous Azacitidine

Background Hypomethylating agents (HMAs) are the standard of care for (elderly) IPSS-R higher risk MDS (HR-MDS) patients, but HMA treatment for those patients who are not eligible for hematopoietic stem cell transplant has to be continued for very long time to maintain efficacy. This prolonged continuous treatment renders patients dependent on hospital and on caregivers, and as a result, decreases QoL and too often provokes premature interruption of therapy. Oral azacitidine is approved for maintenance therapy of acute myeloid leukemia patients in complete remission after intensive chemotherapy, and it has shown activity in lower risk MDS. Oral decitabine has been recently approved by FDA for treatment of MDS. There are no data regarding maintenance of response achieved by sc azacitidine in HR MDS by switching to the oral formulation. Aim The primary objective of the study is to explore the feasibility of replacing subcutaneous (sc) azacitidine by the oral formulation, in patients with HR-MDS and responding to sc azacitidine as well as the maintenance or improvement of the achieved hematological response (time to relapse/progression).Methods A monocentric, pilot phase 2 study (ClinicalTrials.gov: NCT04806906) was planned to enroll 10 subjects with confirmed diagnosis of MDS or dysplastic non proliferative CMML, IPSS-R higher risks, aged ≥ 65 years, in CR/CRi, PR or SD with HI stable for additional 2 cycles after response at 4-6 cycles of azacitidine therapy, hence a total of at minimum 6 to 8 cycles of sc azacitidine. At screening, morphological MDS diagnosis, cytogenetic analysis, IWG response assessment, bone marrow and peripheral blood sampling for flow cytometric analysis, NGS evaluation of somatic mutations, DNA methylation analysis and HRQoL are performed. During treatment, subjects are assessed for safety, tolerability and efficacy of oral azacitidine. Assessments during treatment include monitoring for AEs, monitoring for loss of response and SPMs, physical examination, vital signs and weight measurement, ECOG performance status, hematology and serum chemistry, serum ferritin level, concomitant medications, therapies and procedures, review of bone marrow aspirate or biopsy. Patients receive 300 mg oral azacitidine for the first 14 days of each 28-day treatment cycle until no longer receiving the benefit or until progression. Dose of oral azacitidine can be de-escalated based on toxicity. Besides feasibility of treatment with oral azacitidine, we evaluate safety/tolerability, time to treatment discontinuation, and patient-reported outcomes, utilizing the EQ-5D, and measures of healthcare resource utilization. DNA methylation pattern of bone marrow CD34 positive cells at diagnosis, after response to sc azacitidine, and after 2 and 4 cycles of oral azacitidine is also determined by ERRBS for all patients. Results As per August 1, 2022, with a follow up ranging from 1-10 months, 5 HR MDS patients have been enrolled in the study. Median age is 81 yrs (79-86). Male/female ratio: 4/1. At start of therapy with sc azacitidine IPSS-R risk was high 3/5, very high 2/5. Median number of cycles of sc azacitidine is 25 (7-56), median number of cycles of oral azacitidine is 4 (1-9). The dose of 300mg/day was maintained for all patients. Pattern of DNA methylation determined by ERRBS is available for all treated patients and confirms the activity of oral azacitidine. Two patients evolved to AML after 9 and 4 cycles of oral azacitidine, with progression free survival (PFS) from onset of therapy of 17 and 12 months respectively. None of those 2 patients had adverse events during therapy. Treatment was interrupted after 2 cycles in 1 patient following a GI event grade 3 and following patient decision; 2 patients are still ongoing without report of hematological or non-hematological toxicity. At present 1/5 patients died with AML. Conclusions. The patients included in this study were HR MDS, particularly frail, not eligible to investigational studies because of poor compliance and very old median age. The administration of oral azacitidine in patients who responded to sc azacitidine is feasible, the treatment is very well tolerated with no AEs different from those observed in sc azacitidine treatment. Two of five treated patients experienced progressive disease after >12 months of HMA therapy from diagnosis. Oral azacitidine may allow prolonged and continuous treatment in frail HR MDS, optimizing outcome.

Molecular, Epigenetic, and Immune Landscape of TP53-mutated (TP53-M) Acute Myeloid Leukemia (AML) and Higher Risk Myelodysplastic Syndromes (HR-MDS)

Introduction: Mutations in TP53 confer an adverse prognosis in patients (pts) with AML and HR-MDS. Comprehensive analyses of the genetic, epigenetic, and immunologic landscape of TP53-M AML and HR-MDS are lacking, especially associations of allelic status of TP53 with immune and epigenetic disease phenotype. Methods: Using a large, randomized study with extensive and prospective clinical, histopathological, cytogenetic, molecular, epigenetic, and immunologic annotations, we compared data from 61 TP53-M (37 AML, 24 HR-MDS) and 144 TP53 wild-type (TP53-WT; 89 AML, 55 HR-MDS) pts. Pts were randomized to azacitidine (AZA) monotherapy or AZA + anti-PDL1 antibody durvalumab, which did not improve clinical outcomes (Zeidan et al. Blood Advances 2022). A 38 gene-targeted myeloid mutation analysis from screening bone marrow (BM) was performed at Munich Leukemia Laboratory. Only pathogenic TP53 variants with variant allele frequency (VAF) ≥2% were included. Pts with a common SNP or VUS in the TP53 gene were classified as TP53-WT. Multi-hit TP53 mutations were defined as previously published by Grob et al. (Blood 2022) which confirmed that TP53-M AML and HR-MDS can be grouped as one entity. BM aspirates from screening and cycle 3 day 22 (C3D22) were used to study gene expression profiles by RNA-sequencing and immune phenotyping by flow cytometry. Gene set enrichment analysis (GSEA) using the MSigDB and H (Hallmark) gene sets as provided by the msigdbr R package were performed. Flow cytometry data were analyzed into a collection of 170 reportable subpopulations in the immune cell and immune checkpoint panel and 68 reportables in the tumor/blast panel. DNA methylation of peripheral blood samples was assessed using Illumina's Infinium Human Methylation EPIC methylation array pre-treatment and at C2D1. Global DNA methylation scores (GDMS) and focal DNA demethylation in PD-1, PD-L1 and PD-L2 regulatory regions were analyzed. Results: Of the 61 TP53-M and 144 TP53-WT pts, 85.4% and 10.4% had complex karyotype, respectively. TP53-M pts had fewer co-occurring mutations compared with TP53-WT (mean number of mutations: 1.8 [SD: 0.77] vs 2.9 [SD: 1.4]; P <0.001). Distinct mutational patterns by TP53 mutational status were noted (Figure 1). The median VAF for TP53 mutations was 40%, and 90% had a VAF ≥10%. 22 (11.0%) and 41 (20.5%) pts were mono-hit and multi-hit, respectively. Global DNA methylation was independent of TP53 mutation status at baseline when comparing mono-hit, multi-hit and wild-type. AZA therapy led to statistically significant decrease in GDMS independent of TP53 mutation status (Figure 2). Various loci of PD-1, PD-L1 and PD-L2 genes showed differences in methylation status by TP53 mutation status, which were reversed on treatment. However, methylation of 2 PD-L2 gene loci (cg07211259 and cg11299543) remained significantly lower for multi-hit TP53-M compared to TP53-W at C2D1. Patterns of differentially expressed genes were similar for multi-hit TP53-M vs wild-type and for all TP53-M vs wild-type. The number of differentially expressed genes was higher in the AML cohort compared to MDS pts both at baseline (1887 vs 78) and after C3 (116 vs 0) with TP53-M samples having more differentially expressed genes. ZNF560 was the gene with the strongest differential expression by TP53 mutation status. TP53-M pts had higher expression of T-cell genes (e.g., IL7R), markers of proliferation (MKI67), and PD-L1 (CD274) expression compared to TP53-W at baseline. Genes in hallmark clusters of heme metabolism, inflammatory response, cell cycle regulation, and TGF-β signaling pathways were enriched in TP53-M pts in GSEA. TP53-M pts had higher median levels of PD-L1-positive progenitors (5.6% vs 2.4%; p=0.006) and less myeloid progenitor cells (58% vs 75%; p=0.005) than TP53-WT at baseline. However, there was no shift towards a more exhausted T-cell phenotype among TP53-M pts (CD3+CD4+ PD1+ TIM3+ T-cells and CD3+CD8+PD1+TIM3+ T-cells as % of CD3+CD4+ T-cells and CD3+CD8+ T-cells, respectively). Conclusion: Although an increased population of cytotoxic T-cells suggest an activated immune system, we also found the upregulation of inhibitory immune checkpoints such as PD-L1 in TP53-mut pts suggesting an immunosuppressive microenvironment as a potential contributor to the poor prognosis of TP53-M AML and MDS with similar findings across disease type (HR-MDS vs AML) and mono- vs multi-hit TP53 mutation status. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Combination of Decitabine Plus Venetoclax Is an Active Salvage Therapy for AML Post HR-MDS Progressed during Treatment with Azacitidine Based Regimens

Background: Higher risk IPSS-R myelodysplastic syndromes (HR-MDS) are characterized by high propensity to transform into acute myeloid leukemia (AML). Azacitidine (AZA) and decitabine (DAC) are standard of care for HR-MDS and overall response rates (ORR) range from 25 to 40% with a median overall survival (OS) of 13-17 months. Responses to hypomethylating agents (HMA) are transient and disease progression almost inevitable. These observations prompted to evaluate the ongoing novel combination therapies with HMAs as backbone. At present, acute myeloid leukemia (AML) post HR MDS treated with HMAs have very scarce therapeutic salvage options. The oral inhibitor of BCL-2 Venetoclax (VEN) has been approved for treatment of de novo AML in combination with either HMAs. Scarce data are available describing possible salvage treatment with DAC+VEN after AZA failure in HR MDS progressed to AML. Aims and Methods: We evaluated at MDS UNIT, AOU Careggi-University of Florence, 6 consecutive patients with AML post HR MDS. Patients were treated as outpatients with decitabine plus venetoclax after progression during AZA treatment. IPSS-R calculated before starting AZA was high (4 pts) and very high (2 pts). All patients had previously received standard regimen of azacitidine 75 mg/mq s.c. for 5+2 days every 28 days; median number of cycles was 19 (3-49). 3 pts were primary refractory to AZA, 1 obtained CR and 2 stable disease with hematological improvement. All patients received DAC 20 mg/mq i.v. for 5 days and VEN 400 mg/day for 14 days every 28 days. Results: Median age was 72, all patient were male. At progression, median bone marrow blasts were 25% (range 20-90). Five patients had a normal karyotype and 1 had trisomy of chromosome 8. All patients had at least 1 somatic mutation, with a median of 4 mutated genes (1-8), RUNX1 (2 pts), DNMT3A (3 pts), TET2 (3 pts), ASXL1 (2 pts). None presented IDH1/2 mutation, nor FLT3 ITD or TP53. None of them was eligible for intensive chemotherapy nor hematopoietic stem cell transplant. Median number of cycles of DAC + VEN was 5.5 (2-14), overall response rate was 50% with 2 complete responses and 1 partial response. Median duration of response was 8 months. Response to DAC+VEN was independent from achievement and duration of response to previous AZA therapy and from mutational status. The most frequent side effect was febrile neutropenia G4 with infection: 3 bacterial pneumonia (2 G3 and 1 G2), 2 esophageal candidiasis (1 G2 e 1 G3) and 1 sepsi (G3) with 4/6 who required hospitalization for severe infections. Median OS from starting DAC+VEN was 21 months: 3 patients died during treatment (1 pt died after 2 cycles, 1 after 3 cycles and 1 after 14 cycles): 1 for bleeding and 3 for infection; 1 pt died 5 months after interruption of treatment for relapse of AML. 2 patients are still in treatment. Conclusions: Combination of Decitabine plus Venetoclax seems to have a role as salvage therapy in AML post AZA-treated HR MDS. In consideration of the absence of effective treatments, our extremely preliminary data could indicate a feasible option for a subset of patients without actionable mutations permissive of rescue with target therapy.

Replicated Adsorption Technique used to Resolve ALX148 Interference in the Immunohematology Reference Laboratory

Abstract Introduction/Objective Increased expression of CD47 by cancer cells inhibits macrophages' phagocytic activity through CD47-SIRPα interactions, allowing evasion of the innate immune system. Several drugs on the market have been developed to target this interaction, including monoclonal therapeutics and fusion proteins. ALX148 is a genetically engineered SIRPα fusion protein with an inactivated Fc portion. CD47 is expressed highly on red blood cells (RBC), resulting in strong interference in pre-transfusion compatibility testing of patients receiving ALX148. Consequently, trial patients are at increased risk of transfusion-related adverse events or delay of needed transfusion. Few resolutions of ALX148 interference to assess underlying alloantibody development have been reported. One such examiniation demonstrated x6 linear papain-treated RBC adsorptions removed ALX148 interference. This study aimed to replicate these findings. Methods/Case Report Three patients receiving combination ALX148 (dose 6.57-20mg/mL) and Azacitidine therapy with broad-reactive RBC antibody reactivity were selected. High volume alloadsorptions (8:1 RBC:plasma) were performed with papain-treated rr (ccdee) RBCs. Adsorptions were incubated at 37C for 10 minutes. Polyethylene glycol (PEG) was utilized in test tube indirect antiglobulin testing (IAT). Results (if a Case Study enter NA) Initial reactivity strength was 3-4+ at PEG-IAT and saline-IAT with a three-cell screening RBC reagent. Alloadsorbed plasma was non-reactive at IAT with a three-cell screening RBC reagent enhanced by PEG. Drug interference was removed following three times high volume papain-treated alloadsorption. Conclusion Cancer immunotherapies have transformed the standard of care in oncology. Despite the evident clinical success, the medical laboratory has been challenged with adapting to cancer drug therapies capable of causing interference in routine laboratory testing. Clinical trials of ALX148 are associated with interference in pre-transfusion compatibility testing. The use of extended phenotype matching for RBC transfusion can be utilized but incurs additional time and resources. These results suggest high volume linear papain RBC alloadsorptions may be incorporated into antibody resolution for patients receiving ALX148.