CAR-T Cells in Acute Myeloid Leukemia: Where Do We Stand?

Despite recent advances, the prognosis of acute myeloid leukemia (AML) remains unsatisfactory due to disease recurrence and the development of resistance to both conventional and novel therapies. Engineered T cells expressing chimeric antigen receptors (CARs) on their cellular surface represent one of the most promising anticancer agents. CAR-T cells are increasingly used in patients with B cell malignancies, with remarkable clinical results despite some immune-related toxicities. However, at present, the role of CAR-T cells in myeloid neoplasms, including AML, is extremely limited, as specific molecular targets for immune cells are generally lacking on AML blasts. Besides the paucity of dispensable targets, as myeloid antigens are often co-expressed on normal hematopoietic stem and progenitor cells with potentially intolerable myeloablation, the AML microenvironment is hostile to T cell proliferation due to inhibitory soluble factors. In addition, the rapidly progressive nature of the disease further complicates the use of CAR-T in AML. This review discusses the current state of CAR-T cell therapy in AML, including the still scanty clinical evidence and the potential approaches to overcome its limitations, including genetic modifications and combinatorial strategies, to make CAR-T cell therapy an effective option for AML patients.

ATP-Binding Cassette Subfamily G Member 2 in Acute Myeloid Leukemia: A New Molecular Target?

Despite the progress in the knowledge of disease pathogenesis and the identification of many molecular markers as potential targets of new therapies, the cure of acute myeloid leukemia remains challenging. Disease recurrence after an initial response and the development of resistance to old and new therapies account for the poor survival rate and still make allogeneic stem cell transplantation the only curative option. Multidrug resistance (MDR) is a multifactorial phenomenon resulting from host-related characteristics and leukemia factors. Among these, the overexpression of membrane drug transporter proteins belonging to the ABC (ATP-Binding Cassette)-protein superfamily, which diverts drugs from their cellular targets, plays an important role. Moreover, a better understanding of leukemia biology has highlighted that, at least in cancer, ABC protein's role goes beyond simple drug transport and affects many other cell functions. In this paper, we summarized the current knowledge of ABCG2 (formerly Breast Cancer Resistance Protein, BCRP) in acute myeloid leukemia and discuss the potential ways to overcome its efflux function and to revert its ability to confer stemness to leukemia cells, favoring the persistence of leukemia progenitors in the bone marrow niche and justifying relapse also after therapy intensification with allogeneic stem cell transplantation.

Correlation between IPSET-t risk at diagnosis and subsequent hemorrhage in patients with essential thrombocythemia; a single institution experience.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by an increased risk of thrombotic and hemorrhagic events, that represent the leading causes of mortality and morbidity. Currently, while thrombotic risk is assessed through the IPSET-t and r-IPSET scores, there is no specific prognostic tool used to predict hemorrhagic risk in ET. The aim of the study was to define incidence and risk factors connected to hemorrhagic events by retrospectively analyzing 308 ET patients diagnosed between 1996 and 2022 at the Division of Hematology of Udine and treated according to the current international guidelines. According to molecular status, 193 patients (62.7%) were JAK2 mutated, 66 (21.4%) had a CALR mutation, 14 (4.5%) had a MPL mutation, 21 patients (6.8%) were "triple negative," and 14 patients (4.5%) were not evaluable. According to IPSET-t score, 49.7% patients were at high, 24.3% at intermediate, and 26.0% at low-risk, respectively. Twelve (3.9%) patients experienced bleeding at ET diagnosis, while 24 (7.8%) had at least one hemorrhagic event during follow-up at a median time of 103 months (range: 1-309). Forty hemorrhagic events were totally recorded and defined as minor in 22 cases, moderate in 11 cases, and severe in 7 cases. Cumulative incidence (CI) of hemorrhage at 10 and 20 years was 6.0% and 12.0%, respectively. A statistically significant correlation between hemorrhagic risk and IPSET-t score emerged: 10 years hemorrhage CI was 3.2% for low-risk, 2.9% for intermediate-risk, and 9.8% for high-risk patients, respectively (p=0.002). We found no correlation between hemorrhagic risk and gender or mutational status. Results of our study highlight the validity of IPSET-t score in predicting individual hemorrhagic risk among ET patients, suggesting a possible role of IPSET-t scoring system as a global evaluator for vascular events in ET patients.

B-cell prolymphocytic leukemia with P53 abnormalities successfully treated with bendamustine and rituximab: a report of three cases.

Background: B-cell prolymphocytic leukemia (B-PLL) is a rare mature B-cell tumor with an aggressive clinical course and poor prognosis. It is characterized by prominent splenomegaly and prolymphocytes exceeding 55% of the lymphoid cells in the blood. Purine analog-based chemo-immunotherapy is the first-line therapy for B-PLL. Owing to its rarity, there are few reports on the efficacy of bendamustine and rituximab (BR) regimen. Our study presents three cases of BR being effective in the treatment of B-PLL and provides experience for clinical treatment. Case Description: This report describes the cases of three male patients (median age: 66 years old) who initially presented with abdominal discomfort. Physical examinations and imaging revealed splenomegaly, while a peripheral blood (PB) smear revealed a prolymphocyte count exceeding 70% of the lymphoid cells. Therefore, the three patients were diagnosed with B-PLL. Further molecular detection showed that they harbored P53 abnormalities (17p deletion/TP53 mutation) associated with resistance to conventional chemotherapies. In addition, one of the patients had a highly complex karyotype and multiple gene mutations. All patients underwent four cycles of BR, and two of them received two further cycles of rituximab monotherapy. Ultimately, the patients achieved a complete response (CR) that lasted for 25, 33, and 34 months, respectively, with a median follow-up time of 34 months. The adverse events of the BR mainly included a grade 3 haematological toxicities. Also, the treatment was well-tolerated. Conclusions: This case series suggests that BR regimen is promising for bringing deep remission to patients with B-PLL. Prospective trials are still required for further elucidation.

Checkpoint Inhibitors in Acute Myeloid Leukemia.

The prognosis of acute myeloid leukemia (AML) remains unsatisfactory. Among the reasons for the poor response to therapy and high incidence of relapse, there is tumor cell immune escape, as AML blasts can negatively influence various components of the immune system, mostly weakening T-cells. Since leukemic cells can dysregulate immune checkpoints (ICs), receptor-based signal transductors that lead to the negative regulation of T-cells and, eventually, to immune surveillance escape, the inhibition of ICs is a promising therapeutic strategy and has led to the development of so-called immune checkpoint inhibitors (ICIs). ICIs, in combination with conventional chemotherapy, hypomethylating agents or targeted therapies, are being increasingly tested in cases of AML, but the results reported are often conflicting. Here, we review the main issues concerning the immune system in AML, the main pathways leading to immune escape and the results obtained from clinical trials of ICIs, alone or in combination, in newly diagnosed or relapsed/refractory AML.

ABCG2 in Acute Myeloid Leukemia: Old and New Perspectives.

Despite recent advances, prognosis of acute myeloid leukemia (AML) remains unsatisfactory due to poor response to therapy or relapse. Among causes of resistance, over-expression of multidrug resistance (MDR) proteins represents a pivotal mechanism. ABCG2 is an efflux transporter responsible for inducing MDR in leukemic cells; through its ability to extrude many antineoplastic drugs, it leads to AML resistance and/or relapse, even if conflicting data have been reported to date. Moreover, ABCG2 may be co-expressed with other MDR-related proteins and is finely regulated by epigenetic mechanisms. Here, we review the main issues regarding ABCG2 activity and regulation in the AML clinical scenario, focusing on its expression and the role of polymorphisms, as well as on the potential ways to inhibit its function to counteract drug resistance to, eventually, improve outcomes in AML patients.

BCR::ABL1 levels at first month after TKI discontinuation predict subsequent maintenance of treatment-free remission: A study from the "GRUPPO TRIVENETO LMC".

We analyzed BCR::ABL1 expression at stop and in the first month after discontinuation in 168 chronic myeloid leukemia patients who stopped imatinib or 2nd generation tyrosine kinase inhibitors (2G-TKIs) while in sustained deep molecular response. Patients were divided among those who maintained response (group 1, n = 123) and those who lost major molecular response (group 2, n = 45). Mean BCR::ABL1 RNA levels 1 month after discontinuation were higher in group 2 than in group 1 (p = 0.0005) and the difference was more evident 2 months after stop (p < 0.0001). The same trend was found both for imatinib and 2G-TKIs. A receiver operating characteristic (ROC) analysis to determine a threshold value of BCR::ABL1 at 1 month after discontinuation identified a cut-off value of 0.0051%, with 92.2% specificity, 31.7% sensitivity and a likelihood ratio of 4.087.

Analysis of m6A-related signatures associated with the tumor immune microenvironment and predict survival in acute myeloid leukemia.

Background: Most previous studies have focused on the intrinsic carcinogenic pathways of tumors; however, little is known about the potential role of N6-methyladenosine (m6A) methylation in the tumor immune microenvironment (TIME). To better diagnose and treat acute myeloid leukemia (AML), we sought to examine the correlation between m6A regulatory factors and immune infiltration in cases of AML. At the same time, a prognostic model was constructed to predict the survival of AML. Methods: We extracted data from The Cancer Genome Atlas (TCGA) database, including ribonucleic acid sequencing (RNA-seq) transcriptome data and data on the corresponding clinical characteristics of AML patients. We identified two m6A modification patterns with distinct clinical outcomes and found a significant relationship between them. Simultaneous discovery of distinct m6A clusters associated with the tumor immune microenvironment [immune cell types and Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm] are closely related. Next, we implemented Lasso (Least Absolute Shrinkage and Selection Operator) Cox regression to build a predictive model in the 2-m6A regulator TCGA dataset to further explore m6A prognostic features in AML, and perform correlation validation. Results: We identified 2 molecular subtypes (Clusters 1 and 2) by the consistent clustering of significant m6A regulators in AML. Cluster 2 was associated with a higher immune score and obvious immune cell infiltration, and thus patients in Cluster 2 had a poorer prognosis than those in Cluster 1 (P<0.05). Additionally, the 2 m6A-related signatures representing the independent prognostic factors in AML were screened to construct a prognostic risk-score model. We found that patients with low-risk scores had higher immune scores than those with high-risk scores (P<0.05). Conclusions: Our research confirmed that m6A methylation plays an important role in AML. Further provide new directions for the prognosis and treatment of AML.

Present and Future Role of Immune Targets in Acute Myeloid Leukemia.

It is now well known that the bone marrow (BM) cell niche contributes to leukemogenesis, but emerging data support the role of the complex crosstalk between AML cells and the BM microenvironment to induce a permissive immune setting that protects leukemic stem cells (LSCs) from therapy-induced death, thus favoring disease persistence and eventual relapse. The identification of potential immune targets on AML cells and the modulation of the BM environment could lead to enhanced anti-leukemic effects of drugs, immune system reactivation, and the restoration of AML surveillance. Potential targets and effectors of this immune-based therapy could be monoclonal antibodies directed against LSC antigens such as CD33, CD123, and CLL-1 (either as direct targets or via several bispecific T-cell engagers), immune checkpoint inhibitors acting on different co-inhibitory axes (alone or in combination with conventional AML drugs), and novel cellular therapies such as chimeric antigen receptor (CAR) T-cells designed against AML-specific antigens. Though dozens of clinical trials, mostly in phases I and II, are ongoing worldwide, results have still been negatively affected by difficulties in the identification of the optimal targets on LSCs.

BCL-2 Expression in AML Patients over 65 Years: Impact on Outcomes across Different Therapeutic Strategies.

BCL-2 overexpression has been associated with resistance to chemotherapy and reduced survival in acute myeloid leukemia (AML), but few data are available in elderly patients, a subset accounting for majority of AML cases and with dismal prognosis. We retrospectively analyzed 113 AML patients aged ≥65 years treated with 3 + 7 chemotherapy (n = 51) or hypomethylating agents (HMAs) (n = 62), evaluating the role of BCL-2 expression on complete remission (CR) and overall survival (OS). BCL-2 was expressed in 81 patients (72%), more frequently in those with unfavorable cytogenetic-molecular risk. CR was achieved in 34.5% cases, without differences according to BCL-2 expression or induction therapy. In the whole population 1-year OS was 39%, similar in BCL-2+ and BCL-2- cases. In BCL-2 positive patients OS was superior with HMAs (56% vs. 25% with 3 + 7; p = 0.02), while no advantage for HMA was found in BCL-2 negative cases (36% vs. 27% for 3 + 7). Therapy with HMAs was the only factor associated with longer OS in BCL-2+ AML by multivariable analysis. Use of HMAs, possibly in combination with BCL-2 inhibitors, appears to be particularly appealing in BCL2+ AML, where it is associated with superior survival.

The serological prevalence of SARS-CoV-2 infection in patients with chronic myeloid leukemia is similar to that in the general population.

BACKGROUND: Patients with hematological malignancies are at an increased risk of SARS-CoV-2 disease (COVID-19) and adverse outcome. However, a low mortality rate has been reported in patients with chronic myeloid leukemia (CML). Preclinical evidence suggests that tyrosine kinase inhibitors (TKIs) may have a protective role against severe COVID-19. METHODS: We conducted a cross-sectional study of 564 consecutive patients with CML who were tested for anti-SARS-CoV-2 IgG/IgM antibodies at their first outpatient visit between May and early November 2020 in five hematologic centers representative of three Italian regions. RESULTS: The estimated serological prevalence of SARS-CoV-2 infection in patients with CML after the first pandemic wave was similar to that in the general population (about 2%), both at national and regional levels. CML patients with positive anti-SARS-CoV-2 serology were more frequently male (p = 0.027) and active workers (p = 0.012), while there was no significant association with TKI treatment type. Only 3 out of 11 IgG-positive patients had previously received a molecular diagnosis of COVID-19, while the remainders were asymptomatic or with mild symptoms. CONCLUSIONS: Our data confirm that the course of SARS-CoV-2 infection in patients with CML is generally mild and reassure about the safety of continuing TKIs during the COVID-19 pandemic. Furthermore, we suggest that patients with CML succeed to mount an antibody response after exposure to SARS-CoV-2, similar to the general population.

Impact of Concomitant Aberrant CD200 and BCL2 Overexpression on Outcome of Acute Myeloid Leukemia: A Cohort Study from a Single Center

Objective: CD200 and BCL2 overexpression is independently associated with inferior survival in acute myeloid leukemia (AML), and these two factors are frequently co-expressed; however, no data are available on the role of concomitant aberrant CD200 and BCL2 expression on outcome of AML patients. We aimed to elucidate the prognostic role of CD200/BCL2 co-expression and its association with specific leukemia subsets. Materials and Methods: We analyzed 242 adult AML patients uniformly treated with intensive chemotherapy, evaluating the impact of CD200 and BCL2 expression on complete remission (CR), disease-free survival, and overall survival (OS). Results: CD200 and BCL2 were expressed in 139 (57.4%) and 137 (56.6%) cases, respectively, with 92 patients (38%) displaying double positivity (DP), 58 (24%) displaying double negativity (DN), and 92 patients expressing only either CD200 (n=47) or BCL2 (n=45). CR was achieved in 71% of cases, being less frequent in DP patients (60%) compared to other groups (76%-81%, p<0.001). In the whole population 3-year OS was 44%, being lower in DP patients (28%) than in patients with single CD200 or BCL2 expression (47%) or DN cases (60%; p=0.004). Other factors associated with worse OS were advanced age, CD34 positivity, secondary AML, and high white blood cell count at diagnosis; combining these 4 factors with CD200/BCL2 DP, we identified 6 groups with significantly different rates of survival (3-year OS ranging from 90% to 0%). Conclusion: Our data support a synergistic effect of CD200 and BCL2 in AML cells, conferring an enhanced survival capacity in a permissive microenvironment and resulting in worse prognosis.

Primary analysis of JUMP, a phase 3b, expanded-access study evaluating the safety and efficacy of ruxolitinib in patients with myelofibrosis, including those with low platelet counts.

Ruxolitinib is a potent Janus kinase (JAK) 1/JAK2 inhibitor approved for the treatment of myelofibrosis (MF). Ruxolitinib was assessed in JUMP, a large (N = 2233), phase 3b, expanded-access study in MF in countries without access to ruxolitinib outside a clinical trial, which included patients with low platelet counts (<100 × 109 /l) and patients without splenomegaly - populations that have not been extensively studied. The most common adverse events (AEs) were anaemia and thrombocytopenia, but they rarely led to discontinuation (overall, 5·4%; low-platelet cohort, 12·3%). As expected, rates of worsening thrombocytopenia were higher in the low-platelet cohort (all grades, 73·2% vs. 53·5% overall); rates of anaemia were similar (all grades, 52·9% vs. 59·5%). Non-haematologic AEs, including infections, were mainly grade 1/2. Overall, ruxolitinib led to meaningful reductions in spleen length and symptoms, including in patients with low platelet counts, and symptom improvements in patients without splenomegaly. In this trial, the largest study of ruxolitinib in patients with MF to date, the safety profile was consistent with previous reports, with no new safety concerns identified. This study confirms findings from the COMFORT studies and supports the use of ruxolitinib in patients with platelet counts of 50-100 × 109 /l. (ClinicalTrials.gov identifier NCT01493414).

ABCG2 and CD200 define patients at high risk of relapse in ELN favorable subgroup of AML.

OBJECTIVE: Overexpression of ABCG2 and CD200 has been independently associated with poor outcome in acute myeloid leukemia (AML). However, no data are available on the role of these two factors in patients with core-binding factor (CBF)-positive or FLT3-negative/NPM1-mutated cytogenetically normal (CN) AML. METHODS: We analyzed 65 adult AML patients with CBF+ (n=16) or FLT3-/NPM1+ CN (n=49), evaluating clinical and biological factors associated with complete remission attainment, leukemia-free survival (LFS) and overall survival (OS). RESULTS: ABCG2 was expressed in 36 (55%) cases, and CD200 was positive in 33 (51%) cases, six at high levels. Both ABCG2 and CD200 positivity have a negative impact on relapse risk: 3-year LFS was 51% vs 82% in ABCG2+ cases (RR 3.3), 49% vs 82% in CD200+ patients (RR=4.4), and 25% in CD200- high cases (RR=17.1). ABCG2 and CD200 affected also OS with 3-year OS of 39% in ABCG2+ (compared to 71% in ABCG2-; RR=2.6) and CD200+ (compared to 68% in CD200-; RR=2.5) patients. CONCLUSIONS: Our data confirm a negative impact of ABCG2 and CD200 overexpression also in AML patients considered at favorable risk according to ELN cytogenetic/molecular classification.

High CD200 expression is associated with poor prognosis in cytogenetically normal acute myeloid leukemia, even in FlT3-ITD-/NPM1+ patients.

Overexpression of CD200, a trans-membrane protein belonging to the immunoglobulin superfamily, has been associated with poor prognosis in patients with acute myeloid leukemia (AML). As few data are available in the subset of cytogenetically-normal (CN) AML, we retrospectively evaluated the correlations between CD200 expression and response to therapy in a series of 139 adults with CN-AML. CD200 was expressed in 67/139 (48%) cases; 18 of them (28%) expressed CD200 at high intensity. No differences in CD200 expression rate were observed according to age, WBC count, type of leukemia, FLT3 or NMP1 mutation, and CD56 expression. A higher incidence of CD200 expression was observed in CD34+ cases (P<0.0001) and in BCL2+ patients (P=0.04). Complete remission (CR) was evaluable achieved in 98 patients (70%): 56/71 (79%) in CD200- and 47/67 (63%) in CD200+ patients (P=0.03), with a lower CR rate in patients with high CD200 intensity (9/18, 50%). CD200 expression had a negative impact on long-term outcome. CD200 expression, per se, did not impact on disease-free survival (DFS), but cases with high CD200 expression had a lower 3-year DFS compared to CD200-negative and low-expressing ones (0% vs 65% vs 68%, P=0.019). Three-year overall survival (OS) was 51% in CD200- and 27% in CD200+ patients (P=0.01), with a significant difference among cases with low or high CD200 expression (35% vs 0%, P=0.001). CD200 high expression defined a group with very poor DFS and OS also among the 37 FLT3-/NPM1+: 3-year DFS and OS were 88% and 60% in CD200-, 50% and 32% in CD200 low and 0% and 0% in CD200 high patients, respectively (P=0.01 for DFS and P=0.05 for OS). Our data suggest a negative impact of CD200 expression in CN-AML, with a further worsening in high-expressing cases, also in the subset of FLT3-/NPM1+ patients.