Hemopoiesis and Immune Cell Perturbations during Venetoclax Plus Azacytidine Treatment in Acute Myeloid Leukemia
Authors: Giudice Valentina , Ferrara Idalucia , Gorrese Marisa , D'Alto Francesca , Guariglia Roberto , Luponio Serena , Pezzullo Luca , Mettivier Laura , Bertolini Angela , D'Addona Matteo , De Novellis Danilo , Cuffa Bianca , Serio Bianca , Selleri Carmine 
Published: November 2021

Abstract
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 &lt; 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.