Hemopoiesis and Immune Cell Perturbations during Venetoclax Plus Azacytidine Treatment in Acute Myeloid Leukemia

Treatment of acute myeloid leukemia (AML) in elderly is still challenging. Indeed, high-dose chemotherapy followed by hematopoietic stem cell transplantation with myeloablative regimens is not always feasible because patients are often unfit and have several comorbidities; however, they frequently show multiple negative prognostic factors and have a worse overall survival compared to younger adults. Venetoclax, the first-in-class Bcl-2 antagonist and first approved for treatment of chronic lymphocytic leukemia, inhibits the anti-apoptotic functions of Bcl-2 inducing apoptosis and tumor growth arrest. Venetoclax is also used in combination with azacytidine, or decitabine, or low-dose cytarabine for treatment of elderly newly diagnosed AML. However, several mechanisms of resistance have already been described, such as increased expression of other anti-apoptotic proteins by the leukemic clone. In this case series, we investigated hematopoiesis and immune cell perturbations during venetoclax plus azacytidine treatment in elderly AML patients. A total of six AML patients (M/F, 2/4; median age, 71 years old; range, 63-79 years) were retrospectively evaluated at the Hematology and Transplant Center, University Hospital "San Giovanni di Dio e Ruggi d'Aragona", Salerno, Italy. Patients received a diagnosis of AML based on the 2016 World Health Organization (WHO) criteria and chemotherapy with azacytidine 75 mg/m 2 daily for 7 days per cycle and venetoclax 70 mg/daily. Two patients were NPM1 mutated (one of them also had mutated IDH1, VAF 27.2%), while all subjects had FLT3 wild type. Based on the 2017 European LeukemiaNet risk classification, two patients had favorable risk and four intermediate. Median follow-up was 10.1 months (range, 4.9-16.6 months), and all patients were in partial or complete remission at the time of writing. Flow cytometry immunophenotype, complete blood counts (CBCs), and WT1 expression levels were performed at diagnosis and after every cycle of therapy as per our institutional guidelines. In our case series, leukemic cells were already decreased after the first cycle of therapy (blasts by flow cytometry + SD, 54.7+39.9% vs 4.2+5.4%, diagnosis vs post I cycle; P = 0.0671; paired t-test performed), while normal granulocytes detected by flow cytometry recovered only after the third cycle of therapy (20.7+23.7% vs 53+6.6%, diagnosis vs post III cycle; P = 0.1396; uncorrected Fisher's mixed model performed). Treated patients also displayed a contextual decreased in WT1 expression levels (normalized WT1 copy number + SD, 1810+2723 copies vs 201+132.9 copies, diagnosis vs post I cycle; P = 0.2660; paired t-test performed). Platelet count tended to increase after the first cycle (P = 0.0680); however, at the end of the second cycle, half of patients were again thrombocytopenic (platelets < 100 x 10 3/µL). Interestingly, percentage of lymphocytes detected by flow cytometry were significantly increased after the second cycle of azacytidine plus venetoclax compared to baseline and after the first cycle of therapy (mean+SD, 13.5+13.3% vs 48+8.7%, diagnosis vs post II cycle; P = 0.0167; and vs 28+11.3%, vs post III cycle; P = 0.0480), likely because an increase in Natural Killer (NK) cell frequency peaking after the second cycle (mean+SD, 19.4+4.4% vs 32.5+15.1%, diagnosis vs post II cycle; P = 0.1383). Moreover, five out of six patients displayed expansion of plasma cells detected by flow cytometry in the bone marrow after the first cycle: in particular, one case patient had expansion of aberrant CD45-/dimCD38++CD138++CD56+CD19- plasma cells, while one subject showed only a transient appearance of clonal plasma cells after the second cycle. No differences in bone marrow monocyte frequencies were described during treatment. Our preliminary results added evidence to efficacy and safety of the combination of venetoclax and azacytidine in treatment of elderly AML in a real-world setting. These drugs might synergistically function on hematopoiesis by inducing apoptosis of neoplastic cells while favoring differentiation of other lineages, as suggested by the expansion of plasma cells, or triggering NK-mediated immunosurveillance. However, prognostic and clinical significance of plasma cell and NK cell expansion in the setting of AML treatment needs to be further explored in larger prospective cohorts. Disclosures No relevant conflicts of interest to declare.

Venetoclax and Azacitidine Therapy in Elder Acute Myeloid Leukemia: A Retrospective Evaluation of Real-World Experience

Background：To investigate the efficacy of venetoclax combined with azacytidine in the treatment of elderly patients with relapsed and refractory (R/R) acute myeloid leukemia（AML). Methods： The clinical data of 9 elderly AML patients over 65 years old, including 5 with R/R AML, using venetoclax and azacytidine were retrospectively analyzed. Results： Six males and 3 females with a median age of 71 years were included in this study, of which four patients had at least one relapse, and one patient did not get go into remission after 4 cycles of azacytidine monotherapy, deeming it refractory. Four patients had AML with myelodysplasia-related changes (AML-MRC). After 1 to 13 cycles of treatment using venetoclax and azacytidine, one of the 9 patients died early due to long duration of neutropenia and severe pulmonary infection caused by drugs. and six of the remaining 8 patients obtained complete response or complete response with incomplete hematologic recovery (CR/ CRi) , including five R/R patients. One patient did not respond to treatment after two cycles. For the side effects of the treatment, granulocytopenia occurred in all patients, and neutropenia occurred in 8 patients, lasting for an average of 10.5 (6-15) days and was most obvious in the second to third week of treatment. Three patients with TP53 gene mutation positive had following different outcomes. One relapsed patient achieved progression free remission (PFS) for 16 months up to date, and a second patient achieved complete remission but relapsed two months thereafter. Another patient had complete remission in myelology for 4months, but the variant allele fraction value (VAF) gradually increased, indicative that the disease was about to progress. Conclusion：Venetoclax combined with azacytidine regimen in elderly patients is an effective and well tolerated rescue scheme for R/R AML.The patients with TP53 mutation with lower VAF may be benifit from Venetoclax and azacytidine. Severe infection caused by neutropenia is an adverse reaction worthy of attention in the treatment process of the regimen.

PD-1 and TIGIT Are Highly Co-Expressed on CD8+ T Cells in AML Patient Bone Marrow

Despite the great success of immune-checkpoint inhibitor (ICI) treatment for multiple cancers, evidence for the clinical use of ICIs in acute myeloid leukemia (AML) remains inadequate. Further exploration of the causes of immune evasion in the bone marrow (BM) environment, the primary leukemia site, and peripheral blood (PB) and understanding how T cells are affected by AML induction chemotherapy or the influence of age may help to select patients who may benefit from ICI treatment. In this study, we comprehensively compared the distribution of PD-1 and TIGIT, two of the most well-studied IC proteins, in PB and BM T cells from AML patients at the stages of initial diagnosis, complete remission (CR), and relapse-refractory (R/R) disease after chemotherapy. Our results show that PD-1 was generally expressed higher in PB and BM T cells from de novo (DN) and R/R patients, while it was partially recovered in CR patients. The expression of TIGIT was increased in the BM of CD8+ T cells from DN and R/R patients, but it did not recover with CR. In addition, according to age correlation analysis, we found that elderly AML patients possess an even higher percentage of PD-1 and TIGIT single-positive CD8+ T cells in PB and BM, which indicate greater impairment of T cell function in elderly patients. In addition, we found that both DN and R/R patients accumulate a higher frequency of PD-1+ and TIGIT+ CD8+ T cells in BM than in corresponding PB, indicating that a more immunosuppressive microenvironment in leukemia BM may promote disease progression. Collectively, our study may help guide the combined use of anti-PD-1 and anti-TIGIT antibodies for treating elderly AML patients and pave the way for the exploration of strategies for reviving the immunosuppressive BM microenvironment to improve the survival of AML patients.

The Impact of DNMT3A Status on NPM1 MRD Predictive Value and Survival in Elderly AML Patients Treated Intensively

Minimal residual disease (MRD) is now a powerful surrogate marker to assess the response to chemotherapy in acute myeloid leukemia (AML). DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. DNMT3A status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. Only 20.4% of FLT3-ITD patients reached ≥ 4log MRD1 reduction compared to 47.5% in FLT3wt cases. A 4log reduction of NPM1 MRD was associated with a better outcome, even in FLT3-ITD mutated patients, independent of the allelic ratio. DNMT3A negative patients who reached a 4log reduction had a superior outcome to those who did not (HR = 0.23; p < 0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identifies a subgroup of patients at high risk of relapse.

Inferior Outcome of Addition of the Aminopeptidase Inhibitor Tosedostat to Standard Intensive Treatment for Elderly Patients with AML and High Risk MDS

Treatment results of AML in elderly patients are unsatisfactory. We hypothesized that addition of tosedostat, an aminopeptidase inhibitor, to intensive chemotherapy may improve outcome in this population. After establishing a safe dose in a run-in phase of the study in 22 patients, 231 eligible patients with AML above 65 years of age (median 70, range 66–81) were randomly assigned in this open label randomized Phase II study to receive standard chemotherapy (3+7) with or without tosedostat at the selected daily dose of 120 mg (n = 116), days 1–21. In the second cycle, patients received cytarabine 1000 mg/m2 twice daily on days 1-6 with or without tosedostat. CR/CRi rates in the 2 arms were not significantly different (69% (95% C.I. 60–77%) vs 64% (55–73%), respectively). At 24 months, event-free survival (EFS) was 20% for the standard arm versus 12% for the tosedostat arm (Cox-p = 0.01) and overall survival (OS) 33% vs 18% respectively (p = 0.006). Infectious complications accounted for an increased early death rate in the tosedostat arm. Atrial fibrillation was more common in the tosedostat arm as well. The results of the present study show that the addition of tosedostat to standard chemotherapy does negatively affect the therapeutic outcome of elderly AML patients.

Measurable residual disease in elderly acute myeloid leukemia: results from the PETHEMA-FLUGAZA phase 3 clinical trial

The value of measurable residual disease (MRD) in elderly patients with acute myeloid leukemia (AML) is inconsistent between those treated with intensive vs hypomethylating drugs, and unknown after semi-intensive therapy. We investigated the role of MRD in refining complete remission (CR) and treatment duration in the phase 3 FLUGAZA clinical trial, which randomized 283 elderly AML patients to induction and consolidation with fludarabine plus cytarabine (FLUGA) vs 5-azacitidine. After consolidation, patients continued treatment if MRD was ≥0.01% or stopped if MRD was &lt;0.01%, as assessed by multidimensional flow cytometry (MFC). On multivariate analysis including genetic risk and treatment arm, MRD status in patients achieving CR (N = 72) was the only independent prognostic factor for relapse-free survival (RFS) (HR, 3.45; P = .002). Achieving undetectable MRD significantly improved RFS of patients with adverse genetics (HR, 0.32; P = .013). Longer overall survival was observed in patients with undetectable MRD after induction though not after consolidation. Although leukemic cells from most patients displayed phenotypic aberrancies vs their normal counterpart (N = 259 of 265), CD34 progenitors from cases with undetectable MRD by MFC carried extensive genetic abnormalities identified by whole-exome sequencing. Interestingly, the number of genetic alterations significantly increased from diagnosis to MRD stages in patients treated with FLUGA vs 5-azacitidine (2.2-fold vs 1.1-fold; P = .001). This study supports MRD assessment to refine CR after semi-intensive therapy or hypomethylating agents, but unveils that improved sensitivity is warranted to individualize treatment and prolong survival of elderly AML patients achieving undetectable MRD.

Impact of DNMT3a Status on Post Induction NPM1 MRD Predictive Value on Survival in Elderly AML Patients Treated Intensively

Minimal residual disease is now a powerfull surrogate marker to assess response to chemotherapy in acute myeloid leukemia (AML). In younger adults, NPM1 MRD has recently demonstrated to be a favorable predictive marker for EFS and OS independently of fms-like tyrosine kinase-3 internal tandem duplications (FLT3-ITD) status. However, there is very few datas regarding predictive value of NPM1 MRD in elderly patients treated with intensive chemotherapy. Moreover, numerous studies have suggested the negative impact of DNMT3a mutation in NPM1 AML patients, especially in those with concurrent FLT3-ITD mutation. In this study, we aimed to investigate the impact of DNMT3a status on post induction NPM1 MRD1 predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1 mutated AML in two French institutions (Lyon, Lille) were analyzed retrospectively. Median age of the entire cohort was 66.1 years old (range 60-78.2). An FLT3-ITD mutation was evidenced in 52 of 138 patients (37.6%) with a median FLT3-ITD AR of 0.53 (range, 0.05-3). With a median follow-up of 19.61 months (0.07-128.4), the overall CR rate was 89.9% with no influence of DNMT3a or FLT3 mutational status on the probability of CR. In this elderly cohort of NPM1mut patients, a 4log reduction of NPM1 bone marrow (BM) MRD1 was associated with better outcome (median OS: NR vs 13.4 months, HR=0.35, p&lt;0.01)(Figure A). Overall, DNMT3 status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. However, only 9/44 (20.4%) FLT3-ITD patients reached ≥ 4log MRD1 reduction whereas 38/80 FLT3wt (47.5%) were good molecular responders (p&lt;0.001). FLT3-ITD mutated patients who achieved a 4log reduction had a superior outcome compared to those who did not (HR=0.34; 95% CI, 0.16 to 0.70; P &lt;0.001). Similarly, NPM1mut FLT3wt patients with a 4log reduction in NPM1 BM-MRD1 had a longer OS (3-year OS, 68.1%; 95% CI, 48.8 to 82.9) than those without good molecular response (3-year OS, 46.5%; 95% CI, 30.2 to 61.7)(Figure B). DNMT3a negative patients who achieved a 4log reduction had a superior outcome to those who did not reached at least a 4log reduction (HR=0.23; 95% CI, 0.07 to 0.72; P &lt;0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and leukemia free survival (LFS) in DNMT3amut patients. DNMT3amut patients has a very poor LFS which was even worst in poor NPM1 MRD1 responders compared to those who reached at least 4log reduction (median LFS: 8.3 months vs 17.4 months, HR = 0.48, 95% CI, 0.25-0.91, p=0.023)(Figure C). In multivariate analysis, only DNMT3a mutational status and a 4-log reduction in NPM1 BM-MRD were significantly associated with survival. Based on these results, we identified among NPM1 positive patients 3 groups with distinct prognosis, based on FLT3-ITD, DNMT3a status and NPM1 BM-MRD post induction response (NPM1 scoring system)(Figure D). When compared to ELN 2017 intermediate risk group (AUC=0.695), NPM1 scoring system (NPM1 SS) was more accurate for OS prediction in patients within intermediate (AUC=0.833) and unfavorable (AUC=0.863) NPM1 SS risk group. However, there was no significant difference in AUC between NPM1 SS favorable and ELN 2017 favorable risk group. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3a also identify a subgroup of patients at very high risk of relapase, despite good molecular responses. As hematopoietic stem cell transplantation (HSCT) might improve OS in elderly patients, DNMT3a positive AML elderly patients should be considered for HSCT or post induction maintenance strategies, even within the favorable ELN risk group. Figure Disclosures Sujobert: Gilead/Kyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding.