Purpose: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. Experimental Design: GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. Results: GD2 was expressed in 82.68% of MB tumors. The SHH and G3–G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood–brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. Conclusions: Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

NPM1 is the most frequently mutated gene in adults with acute myeloid leukemia (AML). The interaction between mutant NPM1 (NPM1c) and exportin-1 (XPO1) causes aberrant cytoplasmic dislocation of NPM1c and promotes the high expression of homeobox (HOX) genes, which is critical for maintaining the leukemic state of NPM1-mutated cells. Although there is a rationale for using XPO1 inhibitors in NPM1-mutated AML, selinexor administered once or twice per week did not translate into clinical benefit in patients with NPM1 mutations. Here, we show that this dosing strategy results in only a temporary disruption of the XPO1-NPM1c interaction, limiting the efficacy of selinexor. Because the second-generation XPO1 inhibitor eltanexor can be administered more frequently, we tested the antileukemic activity of prolonged XPO1 inhibition in NPM1-mutated AML models. Eltanexor caused irreversible HOX downregulation, induced terminal AML differentiation, and prolonged the survival of leukemic mice. This study provides essential information for the appropriate design of clinical trials with XPO1 inhibitors in NPM1-mutated AML.

Background/Objectives: Minimal residual disease (MRD) negativity is associated with improved outcomes in acute myeloid leukemia (AML) patients. In this retrospective observational real-life case series study, we investigated the efficacy and safety of venetoclax dose adjustment in unfit AML patients and the role of WT1 expression levels as a surrogate marker of MRD monitoring. Methods: A total of 24 consecutive unfit AML patients treated with azacytidine and venetoclax were enrolled in this study, and MRD monitoring was performed by flow cytometry as per international guidelines and by WT1 expression levels assessed by RT-qPCR. Dose adjustment of venetoclax was decided based on MRD status and the onset of grade > 2 neutropenia. Results: The overall response rate was 87.5%, and 16 patients achieved a response already at the first re-evaluation (66.7%). No statistically significant differences were observed between patients who received the standard dose and those with venetoclax dose adjustment in terms of overall survival (19.6 months vs. 30.1 months, respectively; p = 0.9428) and progression-free survival (not reached vs. 22.1 months, respectively; p = 0.3865), although a numerical trend toward lower relapse rates was observed in subjects with late (33.3%) or early and late dose reduction (37.5%) compared to those who had dose adjustment only at the first re-evaluation (75%) (p = 0.3014). The toxicity rate was 33.3% in patients who had early and late dose adjustments, which was lower than that observed with early adjustment (58.3%) and than that reported in the VIALE-A study (84%). Conclusions: Reduced-dose venetoclax regimens (from 28 to 21 days per cycle) in unfit AML patients do not affect response rates or survival and are associated with comparable rates of neutropenia and infectious events, supporting flexible dosing strategies based on patient response and side effects. In addition, WT1 expression could serve as a reliable marker for MRD monitoring.

Background/Objectives: Clonal evolution is mainly defined based on the appearance or expansion of clones harboring specific somatic mutations and/or cytogenetic abnormalities, whereas few studies have investigated immunophenotypic heterogeneity assessed by flow cytometry and its relationship with disease progression. In this study, flow cytometry immunophenotyping of acute myeloid leukemia (AML) was carried out to identify phenotypic subclones based on antigen expression and to investigate clonal sweep. Methods: A total of 24 patients diagnosed with AML followed at the Hematology and Transplant Center of Salerno were included. Bone marrow or peripheral blood specimens were subjected to flow cytometry immunophenotyping and leukemic cell characterization. Phenotypic profiles were also compared to molecular alterations detected by next-generation sequencing. Results: We found that flow cytometry-defined clonal heterogeneity was more complex than molecular heterogeneity at diagnosis and disease relapse. Flow cytometry enabled the identification of small phenotypic subclones that were not detected by molecular profiling and that, in several cases, expanded over time, consistent with a phenotypic clonal sweep. The presence of small clones was associated with shorter progression-free survival and overall survival. Conclusions: Flow cytometric clonal heterogeneity, especially the presence of small clones (defined by antigen expression from 2 to 30%), may serve as an additional prognostic factor in AML. Immunophenotyping integrated with molecular data may improve risk stratification, enhance measurable residual disease assessment, and contribute to a more personalized disease monitoring strategy.

Supplemental Figure 14. Orthotopic PDX mouse model of human MED-411 FH mCherry/Luc cell line to evaluate anti-tumor activity of CAR.GD2 T-cells.

Supplemental Figure 16. Evaluation of the efficacy of AP1903 CAR.GD2 T-cells infiltrating tumor in in vivo MB xenograft mouse model.