French-American-British Research Articles

PTBP1 is a potential indicator of disease progression in acute myeloid leukemia.

This study aimed to verify a novel potential indicator of disease progression in acute myeloid leukemia (AML) patients. Bone marrow samples were collected from 27 AML patients and 27 controls without hematological malignancies. Polypyrimidine tract-binding protein 1 (PTBP1) expression in bone marrow samples was measured, and the association of PTBP1 with the French-American-British (FAB) classification, cytogenetics, risk stratification, and complete remission (CR) rate was analyzed. The correlation between PTBP1 and Ki-67/p53 expression in AML patients was ultimately evaluated. The results showed that PTBP1 mRNA and protein levels were greater in AML patients than in controls. PTBP1 expression was able to distinguish between AML patients and controls (area under the curve, 0.8601; 95% confidence interval, 0.7632-0.9570). Furthermore, PTBP1 expression was associated with an increased frequency of internal tandem duplication mutations within FMS-like tyrosine kinase-3 (FLT3) and a complex karyotype, while PTBP1 expression was not correlated with FAB classification, monosomal karyotype, isolated biallelic CCAAT/enhancer-binding protein α (CEBPA) mutation, or nucleophosmin 1 (NPM1) mutation in patients with AML. Moreover, PTBP1 expression was associated with a poorer prognosis according to risk stratification and a lower CR rate in AML patients. In addition, PTBP1 expression was positively correlated with the expression of the proliferation marker Ki-67 and negatively correlated with the expression of the apoptosis marker p53 in AML patients. Overall, PTBP1 is a viable biomarker that contributes to the risk prediction and the determination of potential drug targets for AML.

Expression and Prognostic Value of a Novel B7-H3 (CD276) Antibody in Acute Myeloid Leukemia.

Despite recent advances in immunophenotyping, the prognosis of acute myeloid leukemia (AML) is still mainly estimated using age and genetic markers. As the genetic heterogeneity of AML patients is high, flow cytometry-based classification with appropriate biomarkers can efficiently complement risk stratification and treatment selection. An increased expression of B7-H3 (CD276), an immune checkpoint protein, has been reported and associated with poor prognosis. However, the available data are limited and heterogeneous. Here, we used a novel, proprietary murine anti-B7-H3 8H8 antibody for the flow cytometric analysis of B7-H3 expression in AML blasts from 77 patients. Our antibody reliably detected substantial B7-H3 expression in 62.3% of AML patients. B7-H3 expression was higher in the monocytic French-American-British (FAB) M5 group and in intermediate and poor risk patients according to the European Leukemia Network. Using receiver operating characteristics (ROCs), we identified a specific fluorescence intensity cut-off of 4.45 to discriminate between B7-H3high and B7-H3low expression. High B7-H3 expression was associated with shorter overall survival (OS) and progression-free survival (PFS). In conclusion, we have developed a novel B7-H3 antibody that serves as a new tool for the detection of B7-H3 expression in AML and may help to facilitate risk stratification and treatment selection in AML patients.

Correlation between P-glycoprotein and CD34 Expressions in Leukemic Cells from De novo Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a clonal cancer that develops from myeloid progenitors. P-glycoprotein (Pgp) is the membrane transporter encoded by the multidrug resistance (MDR1) gene, which is responsible for the efflux of several chemotherapeutic drugs for the treatment of AML. Pgp’s physiological role in normal cells is unknown. This contrasts with the lack of clarity on Pgp’s activity as an efflux pump in neoplastic or cancer cells. The association between Pgp and CD34 expression in adult patients with de novo AML was investigated in this study. Methods: Thirty participants were enrolled between November 2022 and April 2023. Immunophenotyping, Pgp percentage, and CD34 expression examinations using bone marrow aspirate blood were performed using immunoflowcytometry. The analysis of the correlation test was performed by Spearman correlation. Results: Patients with de novo AML had a median age of 42.5 years. The bone marrow aspiration results of adult patients with AML reveal the following distribution according to the French–American–British classification: 30% of patients have AML M1, 30% have AML M2, 13.33% have AML M3, 3.33% have AML M4, 16.66% have AML M5, and 6.66% have AML M7. There was no statistical correlation between Pgp and CD34 (P = 0.063), neither Pgp and leukocyte count (P = 0.442). Conclusions: Although no correlation between Pgp with CD34 or leukocyte count, it is necessary to consider the role of other members of the ABC transporter superfamily to further examine its relationship with CD34.

RNA expression-based risk stratification of de novo acute myeloid leukemia.

e18503 Background: Prognostication of acute myeloid leukemia (AML) at initial diagnosis relies on the identification of certain underlying genetic abnormalities as proposed by the European LeukemiaNet (ELN) risk classification. However, AML is highly heterogenous at the molecular level and standard guidelines are often not straightforwardly applicable. In this study, we explored gene expression-based risk stratification strategies and found that FLT3 and NPM1 transcript level profiles can predict overall survival in de novo AML. Methods: The Cancer Genome Atlas AML (TCGA-LAML) project’s normalized RNA-Seq transcriptome profile (n=158) published in 2013 was analyzed with MATLAB software. "High” or “low” level of each gene was determined by the median RPKM of the cohort used as cutoff. P-values were obtained from Cox proportional hazards regression model. Results: The low-FLT3 level group was associated with superior median OS (mOS) compared to high-FLT3 group (24 vs. 16 months; p = 0.02), whereas high-NPM1 level group was associated with superior mOS compared to low-NPM1 group (26 vs. 12 months; p = 0.03). The mOS for high-FLT3/low-NPM1 and low-FLT3/high-NPM1 groups were 9 months and 27 months, respectively (p = 0.001). In high-FLT3/low-NPM1 and low-FLT3/high-NPM1 groups, composition of Cancer and Leukemia Group B (CALGB) cytogenetic risk category [favorable, intermediate, adverse] was [9%, 73%, 18%] and [32%, 49%, 19%], respectively; composition of French-American-British (FAB) classification [M0, M1, M2, M3, M4, M5, M6, M7] was [15%, 27%, 3%, 6%, 24%, 24%, 0%, 0%] and [3%, 14%, 41%, 19%, 22%, 0%, 0%, 3%], respectively; composition of FLT3/NPM1c mutations [ wt/wt, mut/wt, wt/mut, mut/mut, NOS] was [45%, 18%, 18%, 18%, 0%] and [61%, 14%, 7%, 14%, 2%], respectively. Conclusions: We demonstrate a de novo AML risk categorization method solely based on FLT3 and NPM1 RNA-Seq transcript levels. The mOS of high-FLT3/low-NPM1 group resembled that of ELN "adverse" risk group, whereas the mOS of low-FLT3/high-NPM1 group resembled that of ELN "favorable" risk group. High-FLT3/low-NPM1 group was composed of fewer "favorable” but more “intermediate” CALGB risk categories compared to low-FLT3/high-NPM1 group. Notably, considerably more FAB M0, M1 and M5 but fewer M2 and M3 classes were enriched in high-FLT3/low-NPM1 group compared to low-FLT3/high-NPM1 group. We also note approximately half of the cases analyzed were FLT3 and NPM1 wild-types and speculate FLT3 and NPM1 transcript profiles may represent a global oncogenic state regardless of the underlying FLT3 or NPM1 mutations. This is also supported by the previously reported FLT3 mutation-like transcriptomic profiles observed in FLT3 wild-type AML cases1). Reference: 1) Mosquera Orgueira A, et al. Gene expression profiling identifies FLT3 mutation-like cases in wild-type FLT3 acute myeloid leukemia. PLoS One. 2021 Feb 16;16(2):e0247093

Immunotherapy-relevance of a candidate prognostic score for Acute Myeloid Leukemia

BackgroundAcute Myeloid Leukemia (AML) exhibits a wide array of phenotypic manifestations, progression patterns, and heterogeneous responses to immunotherapies, suggesting involvement of complex immunobiological mechanisms. This investigation aimed to develop an integrated prognostic model for AML by incorporating cancer driver genes, along with clinical and phenotypic characteristics of the disease, and to assess its implications for immunotherapy responsiveness. MethodsCritical oncogenic driver genes linked to survival were identified by screening primary effector and corresponding gene pairs using data from The Cancer Genome Atlas (TCGA), through univariate Cox proportional hazard regression analysis. This was independently verified using dataset GSE37642. Primary effector genes were further refined using LASSO regression. Transcriptomic profiling was quantified using multivariate Cox regression, and the derived prognostic score was subsequently validated. Finally, a multivariate Cox regression model was developed, incorporating the transcriptomic score along with clinical parameters such as age, gender, and French-American-British (FAB) classification subtype. The ‘Accurate Prediction Model of AML Overall Survival Score’ (APMAO) was developed and subsequently validated. Investigations were conducted into functional pathway enrichment, alterations in the gene mutational landscape, and the extent of immune cell infiltration associated with varying APMAO scores. To further investigate the potential of APMAO scores as a predictive biomarker for responsiveness to cancer immunotherapy, we conducted a series of analyses. These included examining the expression profiles of genes related to immune checkpoints, the interferon-gamma signaling pathway, and m6A regulation. Additionally, we explored the relationship between these gene expression patterns and the Tumor Immune Dysfunction and Exclusion (TIDE) dysfunction scores. ResultsThrough the screening of 95 cancer genes associated with survival and 313 interacting gene pairs, seven genes (ACSL6, MAP3K1, CHIC2, HIP1, PTPN6, TFEB, and DAXX) were identified, leading to the derivation of a transcriptional score. Age and the transcriptional score were significant predictors in Cox regression analysis and were integral to the development of the final APMAO model, which exhibited an AUC greater than 0.75 and was successfully validated. Notable differences were observed in the distribution of the transcriptional score, age, cytogenetic risk categories, and French-American-British (FAB) classification between high and low APMAO groups. Samples with high APMAO scores demonstrated significantly higher mutation rates and pathway enrichments in NFKB, TNF, JAK-STAT, and NOTCH signaling. Additionally, variations in immune cell infiltration and immune checkpoint expression, activation of the interferon-γ pathway, and expression of m6A regulators were noted, including a negative correlation between CD160, m6A expression, and APMAO scores. ConclusionThe combined APMAO score integrating transcriptional and clinical parameters demonstrated robust prognostic performance in predicting AML survival outcomes. It was linked to unique phenotypic characteristics, distinctive immune and mutational profiles, and patterns of expression for markers related to immunotherapy sensitivity. These observations suggest the potential for facilitating precision immunotherapy and advocate for its exploration in upcoming clinical trials.

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells.

Abstract 3400: Single-cell proteogenomics analysis of AML samples treated with decitabine and venetoclax reveals divergent treatment escape mechanisms

Abstract The combination therapy of hypomethylating agents and venetoclax (HMA+Ven) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist including relapse and treatment resistance, to which our understanding remains incomplete. Previous studies have revealed genetic (RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms. In this context, we hypothesized that integrative assessment of both genetic and phenotypic factors could further unveil the contributors of HMA+Ven resistance. To determine the predictors of treatment response, we first analyzed the baseline characteristics of 208 newly diagnosed, secondary, and relapse/refractory AML patients treated with Decitabine and Venetoclax (Dec+Ven). While complex cytogenetics, KRAS and TP53 mutations were significantly associated with treatment resistance, phenotypic factors such as bone marrow cell compositions or French-American-British (FAB) classifications were not. To identify the factors of resistance at the cellular level, we performed a single-cell targeted DNA-antibody sequencing (DAb-seq) on a subset of 33 patients. In total, 60 multi-timepoint bone marrow samples were analyzed, which included five patients with paired baseline-relapse timepoints. From all five patients, we identified that stem-like cells were largely eradicated and replaced by more differentiated cells such as monocyte-like or erythroid-like cells. At relapse, we identified three patterns of phenotypic shifts: monocytic shift, erythroid shift, and mixed monocytic/erythroid shift. The phenotypic shifts were not always associated with the expansion of new genetic mutations, except for in two cases: one patient had monocytic shift accompanied by the expansion of KRAS mutation, and another patient had erythroid shift accompanied by the expansion of NRAS mutation. To examine the mechanisms behind the phenotypic shifts, we performed a single-cell RNA sequencing on three of the relapsed patients. As a result, we confirmed the upregulation of BCL2L1 in the patient with erythroid shift (N=1), and MCL1 in the patients with monocytic shifts (N=2), but not BCL2 in both. These findings are consistent with known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. To summarize, we conclude both genetic and phenotypic factors play a role in Dec+Ven resistance. Most importantly, we report that divergent phenotypic transitions, from stem-like toward more differentiated phenotypes (monocytic or erythroid), were frequently observed in AML relapse following Dec+Ven therapy. Close monitoring of phenotypic alterations during HMA+Ven treatment may facilitate the precise identification of AML patients predisposed to relapse. Citation Format: Yi June Kim, Zongrui Li, Abhishek Maiti, Tomoyuki Tanaka, Hidetaka Uryu, Ken Furudate, Kala Hayes, Naval Daver, Naveen Pemmaraju, Tapan M. Kadia, Farhad Ravandi, Nicholas J. Short, Maro Ohanian, Gautam Borthakur, Nitin Jain, Ghayas C. Issa, Lucia Masarova, Steven M. Kornblau, William G. Wierda, Sanam Loghavi, Sa A. Wang, Guillermo Garcia-Manero, Hagop Kantarjian, Courtney D. DiNardo, Marina Y. Konopleva, Koichi Takahashi. Single-cell proteogenomics analysis of AML samples treated with decitabine and venetoclax reveals divergent treatment escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3400.

Outcomes of intracranial hemorrhage in critically ill patients with acute leukemia: Results of a retrospective cohort study

BackgroundAdmission to the intensive care unit (ICU) is frequently required for patients with acute leukemia (AL) because of life-threatening complications such as intracranial hemorrhage (IH). In this study, we evaluated the impact of IH on survival and neurological outcomes in this population. MethodsThis was a single-center retrospective cohort study including adult patients with AL requiring ICU admission and experiencing IH between 2002 and 2019 at Saint Louis Hospital. Leukemia type was determined according to the French–American–British classification. Brain imaging (either computed tomography or magnetic resonance imaging) was available for all the patients. The primary endpoint of the study was to describe the clinical and biological characteristics and evaluate the mortality and neurological outcome of patients hospitalized in the ICU with newly diagnosed AL and IH. The secondary endpoint was to identify predictive factors of IH in these patients. ResultsThirty-five patients with AL were included, median age of the patients was 59.00 (interquartile range [IQR]: 36.00–66.00) years. Twenty-nine patients (82.9%) had acute myeloid leukemia, including 12 patients with acute promyelocytic leukemia. Thrombocytopenia was constant, and 48.5% of patients had disseminated intravascular coagulation (DIC). At ICU admission, the median Sequential Organ Failure Assessment score was 5 (IQR: 3–9). The median time between AL onset and IH was 2.0 (IQR: 0.0–9.5) days. The ICU and hospital mortality rates were 60.0% (n =21) and 65.7% (n=23), respectively. In univariate analysis, mechanical ventilation and stupor were associated with mortality, but DIC and acute promyelocytic leukemia were not. Upon multivariate analysis, stupor or coma was the only factor significantly associated with a poor outcome (odds ratio = 8.56, 95 % confidence interval: 2.40 to 30.46). ConclusionIH is associated with a high mortality rate in AL patients, with stupor or coma at the onset of intracranial bleeding being independently associated with poor outcomes.

Myelomonocytic and monocytic acute myeloid leukemia demonstrate comparable poor outcomes with venetoclax-based treatment: a monocentric real-world study.

Venetoclax (VEN), a BCL-2 inhibitor, has transformed treatment strategies for elderly and unfit acute myeloid leukemia (AML) patients by significantly improving response rates and survival. However, the predictive factors for VEN efficacy differ from traditional chemotherapy. The clinical relevance of the FAB (French-American-British) monocytic subtype, including M4 and M5, has been debated as a marker for VEN resistance. This real-world study examined 162 newly diagnosed (ND) and 85 relapsed/refractory (R/R) AML patients who received VEN-based therapy at West China Hospital, Sichuan University, from January 2019 to January 2023. We retrospectively collected clinical and treatment data from electronic medical records. The median age of the cohort was 55.5years (range: 16.5-83.5). The composite complete remission (cCR) rate in the entire cohort was 60.7%. Specifically, among newly diagnosed (ND) patients, FAB monocytic subtypes exhibited lower cCR compared to non-monocytic subtypes (55.1% vs. 76.3%, P = 0.007). Additionally, there were no significant differences observed between M4 and M5 subtypes, both in the ND group (61.7% vs. 40.9%, p = 0.17) and the R/R group (38.2% vs. 40%, p > 0.9). Furthermore, the median follow-up was 238 (range: 7-1120) days. ND patients with monocytic subtypes had shorter overall survival compared to non-monocytic subtypes (295days vs. not reached, p = 0.0017). Conversely, R/R patients showed no such difference (204 vs. 266days, p = 0.72). In summary, our study suggests that the FAB monocytic subtype can predict VEN resistance and shorter survival in ND AML patients. Moreover, there is no significant distinction between M4 and M5 subtypes.

High Mitochondrial Protein Expression as a Potential Predictor of Relapse Risk in Acute Myeloid Leukemia Patients with the Monocytic FAB Subtypes M4 and M5.

AML is a highly aggressive and heterogeneous form of hematological cancer. Proteomics-based stratification of patients into more refined subgroups may contribute to a more precise characterization of the patient-derived AML cells. Here, we reanalyzed liquid chromatography-tandem mass spectrometry (LC-MS/MS) generated proteomic and phosphoproteomic data from 26 FAB-M4/M5 patients. The patients achieved complete hematological remission after induction therapy. Twelve of them later developed chemoresistant relapse (RELAPSE), and 14 patients were relapse-free (REL_FREE) long-term survivors. We considered not only the RELAPSE and REL_FREE characteristics but also integrated the French-American-British (FAB) classification, along with considering the presence of nucleophosmin 1 (NPM1) mutation and cytogenetically normal AML. We found a significant number of differentially enriched proteins (911) and phosphoproteins (257) between the various FAB subtypes in RELAPSE patients. Patients with the myeloblastic M1/M2 subtype showed higher levels of RNA processing-related routes and lower levels of signaling related to terms like translation and degranulation when compared with the M4/M5 subtype. Moreover, we found that a high abundance of proteins associated with mitochondrial translation and oxidative phosphorylation, particularly observed in the RELAPSE M4/M5 NPM1 mutated subgroup, distinguishes relapsing from non-relapsing AML patient cells with the FAB subtype M4/M5. Thus, the discovery of subtype-specific biomarkers through proteomic profiling may complement the existing classification system for AML and potentially aid in selecting personalized treatment strategies for individual patients.

C-Kit and Flt3 mutation status in acute myeloid leukaemia with cytogenetic correlation and prognosis: A series of 75 cases

Recent WHO classification (5 edition) has included Acute Myeloid Leukaemia (AML) with mutations in NPM1, CEBPA as separate entities along with AML with cytogenetic abnormalities in AML with recurrent genetic abnormalities. Even though C-kit and FLT3 mutations in AML have no diagnostic importance, prognostic significance in different subtypes of AML for the presence of these mutations are not the same.To assess the correlation between French American British (FAB) AML classification, specific cytogenetic abnormalities with C-kit, and FLT3 mutation in AML and to evaluate the prognosis and survival of AML patients with respect to cytogenetic abnormalities and C-kit and FLT3 mutation status. Retrospectively all AML cases in which C-kit and FLT3 mutation status was assessed were retrieved; C-kit D816V mutation status had been assessed by Real time PCR; For FLT3 mutation, both Internal tandem duplication (ITD) and D835V mutation status had been assessed using PCR and gel electrophoresis; The data regarding morphological with immunophenotypic diagnosis, conventional karyotyping, FISH for translocation 8;21 (t (8;21)) and inversion16 / translocation16;16 (t (16;16) were also retrieved in all these cases along with follow-up from hospital records. Total 75 cases were included; Male/ Female ratio was 1.21:1 (41/34); Median age was 31 (Range: 2 - 64); 18 cases had translocation 8;21 (t (8;21)); 3 cases showed inversion16 / translocation16;16 (t (16;16); Out of the 18 cases which showed t (8;21), 10 cases had associated loss of sex chromosome. Eight cases had C-kit D816V mutation; three of which had t (8;21) while two had inversion 16. 12 cases had FLT3 mutations among which nine were ITD while three had D835V mutation. On karyotyping, one of these cases showed hyperdiploidy while the majority had normal karyotype. A single case had both C-kit D816V mutation and FLT3 D835V mutation with inversion 16 on karyotyping; Most common type of AML in both cases with FLT3 mutation and C-kit mutation was AML-M2 (FAB); Commonest karyotyping abnormality for cases with C-kit mutation was t(8;21); while for FLT3 mutation, the majority had normal karyotype; The single case which had both C-kit D816V mutation and FLT3 D835V mutation was alive event-free at three-year follow-up. Both FLT3 ITD and TKD mutations had a worse prognosis in our study. However, AML cases with C-kit mutation had a similar prognosis comparable to C kit negative cases. A large-scale study is required to elucidate the significance of this.

Measurable Residual Disease As Assessed By Flow Cytometry As Predictor of Survival in Pediatric AML Patients

Introduction Pediatric acute myeloid leukemia (pedAML) is a heterogeneous group of hematological malignancies classified according to morphology, immunophenotyping, and genomics. The optimal adjustment of therapy based on level of response is not well defined. Aim: Herein, we present the results of a retrospective study aiming to analyze the prognostic value of measurable residual disease (MRD) as determined by flow cytometry (FC-MRD) in pediatric AML. Patients and Methods All pediatric and adolescent patients (aged 0-18 years old) with AML treated in our unit between 1/2013-6/2023 were included in this study. French American British (FAB) morphological classification, immune-phenotype analysis, and molecular characterization were performed on diagnosis, and all patients were treated according to AML-BFM protocols. Final risk classification was based on evaluation of response and on genetic abnormalities at diagnosis. MRD was assessed by FC-MRD after Induction 1 and 2, and, either after course 5 or before hematopoietic stem cell transplantation (HSCT). Complete Remission (CR) was defined as regenerative or normocellular bone marrow with <5% blasts; in peripheral blood ≥1.0x10 9/L neutrophil granulocytes and ≥80x10 9/L thrombocytes or Remission with partial regeneration ( CRp: regenerative or normocellular bone marrow with <5% blasts; in the blood: ≥1.0x10 9/L leukocytes, ≥0.5x10 9/L, neutrophil granulocytes and ≥50x10 9/L, self-sustained platelets), lasting at least 4weeks; no extramedullary disease. Overall survival (OS) was defined as the time interval from the treatment initiation date to the date of death due to any cause. Progressive free survival (PFS) was defined as the time interval from the response date to the date of disease relapse or death due to any cause. The probabilities of OS and PFS were estimated with the Kaplan-Meier method. Results Overall, there were 52 patients (median age at diagnosis:7 years, 37males/15 females) diagnosed with AML, of whom: 40 patients with de novo AML, 5 patients with therapy- or myelodysplasia-related AML and 7 patients with mixed-lineage leukemia. Cytogenetics were considered favorable for 16 (31.0%), intermediate for 4 (7.6%), and adverse for 32 (61.4%) patients. Standard-risk patients (n=16) received only chemotherapy, whereas all high-risk patients, and intermediate-risk patients with available matched donors, underwent HSCT should they have reached remission. Forty-four patients (84.6%) achieved complete response (CR). Among these patients, 43 patients (97.7%) achieved CR after 1 course of induction, and 1 patient (2.3%) required at least 2 courses. Twenty-four patients (46.1%) underwent HSCT during either their first (20/24) or second (4/24) CR. Overall, 12 patients (23.07%) died, 4 patients due to resistant disease, 3 patients due to relapse, 2 patients due to serious complications upon diagnosis, 1 patient due to infection and 2 patients due to transplant-related complications. With a median follow-up time of 3.7 years (range 0.1-11.1years), the five-year OS and PFS of the entire cohort was 74.0% and 70.0%, respectively. The level of FC-MRD was a significant prognostic factor for OS and PFS. More specifically, achievement of FC-MRD < 0.1% at all time points was associated with a significant improvement in PFS and OS. (figure 1). FC-MRD ≥ 0.1% after course 5 or before HSCT was the only independent adverse prognostic factor that was statistically significant for both progression-free (P=0.001) and overall survival (P<0.001) (figure1). Conclusions We investigated the association between FC-MRD and survival in Greek pediatric patients with AML. In our cohort, achieving an MRD status <0.1% according to FC is associated with significant improvement in the outcome of pediatric patients with AML.

Molecular Classification of Chronic Myelomonocytic Leukemia: Results of the Analysis of an International Cohort of 2,471 Patients

Background. Chronic myelomonocytic leukemia (CMML) is a rare myeloid neoplasm that shares both dysplastic and proliferative features and includes patients with highly heterogeneous clinical manifestations and prognosis. Despite known co-mutation patterns that are suggesting of a CMML diagnosis such as TET2 and SRSF2 mutations, the current diagnosis and classification schemes rely solely on morphological criteria that fail to capture the genomic heterogeneity of the disease. Here we perform an unsupervised analysis using only genomic features to generate a novel classification schema of CMML patients with unique clinical features. Methods. A cohort of 2,471 CMML patients defined according to WHO 2016 criteria from European and US centers was analyzed. Bayesian network analysis and hierarchical Dirichlet processes were used to identify genomic associations and subgroups as a basis to define a molecular classification of CMML. Bayesian networks allow to infer the structure of conditional dependencies among mutations. Dirichlet processes were applied to define clusters capturing broad dependencies among all gene mutations and cytogenetic abnormalities. Multivariate logistic regression analysis was applied to compare clinical and hematological characteristics among different groups. ( JCO 2021 39:11, 1223-1233) Results. Baseline clinical and molecular characteristics are summarized in Table 1. We identified 11 clusters defined according to specific genomic features (Table 2). Two clusters were primarily defined by the presence of mutations in splicing genes SRSF2 and ZRSR2, frequently associated with TET2 mutations. Patients assigned to these clusters showed myelodysplastic features according to French American British (FAB) criteria, had a low rate of peripheral blood cytopenias, and low blast counts. Additionally, two clusters were characterized by the same mutations in splicing genes (i.e., SRSF2 or ZRSR2 with associated TET2), together with co-mutations in genes that correlate with a more aggressive phenotype ( RUNX1, ASXL1, EZH2). These groups showed overlapping myelodysplastic and myeloproliferative features by FAB, intermediate-risk per prognostic scores, and decreased survival as compared with the first 2 clusters. A distinct cluster was characterized by SF3B1 gene mutations (frequently associated with TET2 or DNMT3A). The SF3B1 cluster was marked by a dysplastic phenotype and more severe anemia (with a high rate of transfusion-dependence) with respect to the other genomic groups. Four clusters (27.8% of the overall population) were characterized by the presence of mutations in signal transduction and tyrosine kinase pathways genes ( NRAS/KRAS, CBL, JAK2 and SETBP1 often associated with ASXL1/TET2, respectively). Patients within these groups showed a mostly proliferative phenotype and intermediate survival. The presence of JAK2 mutations appeared to confer a better prognosis compared with other proliferative clusters. A minority of patients (4.6%) were assigned to two high-risk clusters, defined by TP53 mutations (often biallelic) with complex karyotype, and AML-like mutations ( NPM1, IDH1/2, FLT3), respectively. Patients in these clusters were predominantly dysplastic by FAB, had a high prevalence of severe cytopenias, higher blast counts, and a dismal outcome. Finally, we identified a cluster of patients defined by the absence of gene mutations and chromosomal abnormalities (4.5%). The majority of patients assigned to this cluster were characterized by a dysplastic phenotype, had low absolute monocyte counts, and favorable outcome. A subset of patients (18.9%) could not be assigned to a specified cluster in this preliminary analysis. An independent validation of this molecular classification is currently being conducted on a US prospective cohort (n=650). Conclusion. Our results provide proof of concept for a molecular classification of CMML based on the identification of homogeneous genomic signatures and associated clinical features. 15% of patients showed clear overlapping features with other myeloid neoplasms, thus providing the rational to refine the boundaries among different clinical entities. A molecular classification of CMML may provide a basis to define a next-generation prognostic tool and improve clinical decision-making.

Myelomonocytic/Monocytic Subtypes Are More Resistant to Venetoclax-Based Therapy in Acute Myeloid Leukemia Patients: A Monocentric, Real-Life Retrospective Study

Background： Venetoclax(VEN)-based regimens have seen broad adoption in newly diagnosed patients with acute myeloid leukemia (AML) who are not candidates for intensive induction chemotherapy. Nonetheless, a sizable proportion of AML patients do not respond. Finding biomarkers that can predict treatment results is crucial. FAB (French, American, British) classification system provides a well-described and clinically associated means to segregate AML patients by virtue of myeloid differentiation status. Whether FAB classification can be used as a predictor marker for VE-based therapy is controversial. In the Chinese population of patients with AML, we conducted this single center retrospective study to examine the predictive value of monocytic and myelomonocytic differentiation on the response to VEN-based treatment. Methods: The clinical features, treatment information, and clinical outcomes of AML patients who received at least one dose of VEN treatment at West China Hospital for more than seven days between January 1, 2019, and September 8, 2022 were retrospectively examined in this study. We classified the patients into two groups based on the FAB classification system: the primitive group (includes M0/M1/M2, abbreviated as PRI) and the myelomonocytic/monocytic group (includes M4/M5, abbreviated as M/M). AML response to VEN therapy was determined by the 2017 European Leukemia Net response criteria after one cycle of VEN treatment. The overall response rate (ORR) was defined as the combination of CR, CRi, PR. From the first day of VEN use until the patient dies or the end of follow-up, the overall survival (OS) time is calculated. Logistic regression and COX regression were used to examine the variables that influence ORR and survival. All statistical tests were two-sided, and p-values of 0.05 or lower were considered significant. All statistical analyses were performed using RStudio 4.2.2. Results: There were 267 patients in all, with 162 newly diagnosed (M/M n=69; PRI n=93) and 85 relapsed/refractory (M/M n=44; PRI n=41). The ORR for M/M is 63.77%, while that for PRI is 84.95% among newly diagnosed patients (p=0.018). The ORR for M/M is 40.91% and PRI is 68.29% among relapsed/refractory patients (p=0.0114, Figure1). In addition, we identified (i) FAB classification M/M subtypes, (ii) previously diagnosed MDS/MPN, (iii) IDH1/IDH2 mutations, (iv) relapsed/refractory event, (v) transfusion requirement, and (vi) TP53 mutation as significant factors predicting the response of all patients using univariate logistic regression analysis. M/M subtypes and MDS/MPN also trended toward independent predictors in the multivariate analysis. We identified (i) FAB Classification M/M subtype, (ii) adverse risk AMLs according to the 2022 ELN classification, (iii) CEBPA mutation, and (iv) TP53 mutation as significant factors predicting the risk of all patients using univariate COX regression analysis. M/M subtype and TP53 also trended toward independent predictors in the multivariate analysis (Table1). OS was significantly longer in the PRI than in the M/M(median OS: 621 days vs 266 days, hazard ratio 0.56, 95% CI: 0.39-0.80, p=0.0011). We compared the overall survival of various illness conditions; OS still was significantly longer in the PRI group than in the M/M group among newly diagnosed patients (median OS: newly diagnosed: 1124 days vs 295 days, HR: 0.47, 95% CI: 0.24-0.83, p=0.0017; refractory/relapsed: 204 days vs 266 days, HR 0.68: 95% CI: 0.45-1.97, p=0.72). Conclusion： Our research demonstrates that the response to VEN may differ depending on how advanced the AML is, with PRI patients being more sensitive and M/M patients being more resistant, particularly in newly diagnosed individuals. Previous research has discovered that AML cells with Monocyte/erythroid/Megakaryocyte differentiation are less sensitive to BCL-2 inhibition in vitro, indicating that the differentiation stage of AML may affect its sensitivity to BCL-2 family inhibitors. This is likely due to an increase in MCL1 family expression and a decrease in BCL2 family expression during cell differentiation. In conclusion, M/M subtype may be able to predict VENs resistance and the worse prognosis for patients treated with VEN-based regimens. Legends to figures Figure 1: Differences in ORR and NR (no response) between M/M and PRI in newly diagnosed and relapsed/refractory patients Table1: Regression analysis results

Epigenetic Classification of Acute Myeloid Leukemia Revealed By Genome-Wide Chromatin Profiling

Background Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease and is predominantly defined by genetic abnormalities and morphology. However, many lines of evidence suggest the role of epigenetic alterations in the pathogenesis and the classification of AML, whereas no large-scale comprehensive analyses of both genetic and epigenetic alterations in AML have been reported. Methods We enrolled a total of 1,564 primary AML cases (524 from Japanese and 1,040 from Swedish cohorts), whose diagnostic samples were subjected to ATAC sequencing together with targeted capture sequencing using an in-house gene panel including 331 known AML driver genes and an additional 1,158-1,317 probes for copy number detection. We also performed RNA (N=1,398) and H3K27ac-targeted ChIP sequencing (N=80). Normal bone marrow samples from 25 donors as well as 15 remission samples were analyzed by ATAC-seq as a control. Results ATAC-seq revealed approximately 300,000 reproducible peaks in primary AML. Most peaks were found in gene-distal regions, and explained a large variance across samples. Among all peaks, 72% were not detected in control samples and thus, specific to AML. Analysis of the correlations between ATAC-seq accessibility and gene expression revealed a subset of regulatory elements that regulate nearby gene expression; each peak was linked to a median of 1 gene (0-31), while each gene was associated with a median of 11 peaks (0-110). We next estimated the cellular composition of the bulk AML samples by deconvolution analysis using ATAC-seq data. The predominance of hematopoietic stem cell (HSC), monocyte, and erythroid signatures was associated with French-American-British (FAB) subtypes of minimal differentiation/without maturation subtypes (M0/M1), myelomonocytic/monoblastic/monocytic subtypes (M4/M5), and erythroid subtype (M6), respectively, suggesting bulk ATAC-seq of AML samples reflected the differentiation potential of leukemic cells. The ATAC-based clustering identified 11 distinct epigenetic subgroups, including three well-known genetic classes defined by gene fusions, such as PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11. By contrast, some genetic classes defined by a single mutation, such as NPM1 or CEBPA mutation, were further classified into multiple distinct epigenetic subgroups. For example, the NPM1-mutated subtype was separated into four subgroups based on the varying degrees of ATAC signatures associated with HSC and monocytes. Each subgroup of NPM1-mutated AML showed different clinical characteristics such as white blood cell counts, age, and prognosis, and co-mutation pattern in FLT3, DNMT3A, and IDH1/ IDH2 mutations. Gene ontology analysis revealed that the subgroup of NPM1-mutated AML with high HSC signature showed upregulated TGF-β signaling, while one with monocytic signature showed upregulated TNF-α signaling and interferon-γ response. Next, we evaluated transcription factor (TF) binding sites in a genome-wide manner, using TF motifs and foot printing in upregulated ATAC-peaks in each subgroup and revealed distinct profiles of activated TFs in each epigenetic subgroup of NPM1-mutated AML. In the subgroup characterized by high HSC signature, HSC-related TFs that belong to MECOM, HOX, and RUNX families, were more active. By contrast, other TFs, such as IRF, CEBP, and JUN/FOS families, were more enriched in the subgroup showing high monocyte signature. By developing a prediction model for these subgroups of NPM1-mutated AML based on the gene expression profile, we reproduced these subgroups and validated clinical and molecular features in an external Beat AML cohort. Utilizing ex vivo drug sensitivity dataset in the Beat AML, we identified candidate drugs showing subgroup-specific effectiveness such as panobinostat for NPM1-mutated AML with monocytic differentiation. Finally, ChIP-seq analysis identified a number of super-enhancers in the vicinity of leukemia-related TFs and oncogenes. Conclusion Through comprehensive profiling of chromatin accessibility and gene mutations/expression in a large cohort of AML patients, we demonstrate the epigenetic heterogeneity and identify novel AML subgroups that had unique epigenetic and clinical features. Our findings highlight the role of epigenetic alterations in AML pathogenesis and impact on molecular classification of AML.

Combination of Liposomal Mitoxantrone, Venetoclax, Homoharringtonine, and Olverembatinib (HQP1351) (MVHO) in Pediatric Patients with Refractory or Relapsed Acute Myeloid Leukemia (AML): Case Series

Introduction: AML accounts for up to 25% of all pediatric acute leukemias. Although overall survival (OS) in pediatric AML has increased to 70% in recent years because of enhanced risk stratified intensive chemotherapy and hematopoietic stem cell transplantation. Unfortunately, more than 30% of patients experience relapse. Further increase intensity of chemotherapy was proven unfeasible for the toxicity and treatment related mortality. Hence, specific targeted therapy has been being explored to treat pediatric AML in recent decade. Because of the heterogeneity of AML and multiple pathways involved in leukemogenesis, single agent targeting one pathway was effective in limited cases. In this study, we designed a regimen to block multiple pathways with combination of Liposomal Mitoxantrone, Venetoclax, Homoharringtonine, and Olverembatinib (MVHO) to investigate its efficacy and safety in treatment of refractory or relapsed AML. Methods: We enrolled patients with relapsed or newly diagnosed AML with poor prognosis with adverse molecular abnormalities, such as NUP98 rearrangements, FUS::ERG, CBFA2T3::GLIS2, and del7/7q, who did not achieve complete remission (CR) after first-line chemotherapy. Patients were administered the MVHO regimen, which included 1 dose of liposome mitoxantrone 8mg/m 2; venetoclax, 300 to 350mg/m 2 once daily, from day1 to day 7, (dose escalation for up to 3days from 50mg/m 2 if high tumor burden occur); homoharringtonine 2mg/m 2 once daily, from day1 to day 7; and Olverembatinib (HQP1351) 20-30mg/m 2 every other day, for 4 doses from day 1 to day 7. Prophylactic oral levofloxacin and posaconazole were administered from day 8 through whole myelosuppression period of every cycles. One cycle (28-35 days, depending on hematopoietic recovery) response, total response, cumulative incidence of relapse (CIR), event-free survival (EFS), hematologic toxicity, and infection during this study were evaluated. Results: A total of 18 patients were enrolled (9 boys and 9 girls), with a median (range) age of 7.3 (4 months-13 years) years, and underwent a total of 27 cycles of MVHO therapy. Half of the patients underwent 1 cycle and the other half, 2 cycles. Per French-American-British (FAB) classification criteria: patients had myelodysplastic syndrome (n = 2); mixed-phenotype acute leukemia (n = 2); and AML subtypes M7 (n = 4), M2 (n = 4), M5 (n = 4), M4 (n = 1), and M0 (n = 1). The median (range) follow-up time was 131 (26-256) days. After cycle one, the objective response rate (ORR, CR plus CR with incomplete hematological recovery [Cri] and partial response [PR]) was 94.4% (17/18), and the remission rate (CR + Cri) was 72.2% (13/18). The total ORR and remission rate before transplantation were 83.3% (15/18) and 66.7% (12/18), respectively. 8 patients among them reached minimum residual disease (MRD) negative remission. For 6 patients with relapsed AML, the ORR and one cycle remission rate was 100% and 66.7%, respectively. A total of 3 patients discontinued because of no response or disease progression, and 12 patients underwent hematopoietic stem cell transplantation (HSCT), after which 1 patient experienced relapse. Remaining 2 patients are being underwent further MVHO cycles. The CIR was 6.7% (1/15). The 8-month mean (± SD) EFS was 60.1% (±19%) and the OS, 100%. Among 26 cycles of MVHO treatment, there were no associated fatal infections or bleeding events. There were 21 episodes of breakthrough infection occurred in 26 cycles of MVHO. There was 1 case of septic shock, and the incidence of ≥grade 3 infection was 80.7%, which included pneumonia, bloodstream infection. Common grade 4 treatment-related adverse events were neutropenia, which was experienced by 100% of patients, and grade 4 thrombocytopenia, which was experienced by 46.1%. Most notably, platelets were stably normal in nearly 20% (5/26) MVHO cycles. Conclusions: MVHO therapy was effective and reasonably well tolerated in pediatric patients with refractory or relapsed AML, suggesting that it may comprise a suitable first-line treatment option for pediatric AML patients.

Clinical Characteristics And Treatment Outcome Of Paediatric Non-Hodgkin's Lymphoma At A Tertiary Care Hospital In Pakistan.

To highlight the clinical characteristics of paediatric patients presenting with non-Hodgkin's lymphoma, treatment toxicities, and outcome. The retrospective study was conducted at the Aga Khan University Hospital, Karachi, and comprised data of all paediatric patients aged 0-18 years diagnosed with non-Hodgkin's lymphoma from 2010 to 2020. Demographic characteristics, presentation, treatment provided, complications, if any, and treatment outcome were recorded. Data was analysed using SPSS 21. Of the 92 patients, 69(75.0%) were males. The overall mean age was 14.35±5.80 years. The most common presenting complaint was pyrexia 42(45.7%), the most common diagnosis was Burkitt lymphoma 40(43.5%), the most common complication related to gastrointestinal issues 8(15.7%), and most toxicities were reported with the use of FAB-LMB96 (French American-British Mature B-Cell Lymphoma 96) for B-cell non-Hodgkin's lymphoma 23 (45.1%). Mortality was the outcome in 17(18.5%) cases, while 19(20.7%) patients were lost to follow-up. PFS and OS was 60.4%, and OS 81.3% respectively at 10 years follow-up, median PFS was 17.5 months ([IQR]: 4.5-43.5 months) (p=0.011) and median OS was 33.5 months (IQR: 19.5-84 months) (p=0.007). Early recognition of symptoms, specialist care, and proper planning can decrease treatment-related complications that result in abandonment.

Outcomes and challenges treating pediatric acute myeloid leukemia: a retrospective analysis of patients treated at the Fundación Valle del Lili between 2011 and 2020

IntroductionAcute myeloid leukemia is a challenging disease, due to a poor prognosis in developing countries. Herein, we aim to describe the clinical characteristics and outcomes after chemotherapy and transplantation. MethodsA retrospective analytic observational study was performed with patients under 18 years of age with newly diagnosed acute myeloid leukemia treated at a referral center in Colombia. Two groups were compared: induction therapy (IT) and induction therapy plus consolidation (IT + C). The survival analysis was performed using the Kaplan–Meier method. ResultsWe analyzed 34 patients diagnosed with acute myeloid leukemia; 20 received hematopoietic stem cell transplantation. Most were French-American-British (FAB) classification types M1, M5 and M0. The transplantation was haploidentical in 65%, conditioning was myeloablative in 67% and graft-versus-host disease prophylaxis was performed with post-transplant cyclophosphamide in 70%. Overall, the 5-year survival was 52% and the overall 5-year survival in the transplanted group was 80%. There were 16 deaths; in the IT group, n = 12, and in the IT + C group, n = 4. In the former, the main cause of death was septic shock and in the latter, it was relapse. ConclusionTransplantation is a safe option. Receiving treatment and supportive measures in hematopoietic stem cell transplantation units is necessary to avoid infections, especially during induction cycles.

Assessing the expression of differentiation antagonizing non-protein coding RNA (DANCR) in newly diagnosed Egyptian acute myeloid leukemia patients

BackgroundRole of Long non-coding RNAs in cancer research in the recent years have been highlighted with evidence to their involvement in cancer disease pathogenesis and progression. One of these emerging long non-coding RNAs is differentiation antagonizing non-protein coding RNA (DANCR). DANCR distinct expression in different cancers and implication in tumor signaling pathways made it a promising therapeutic target for cancer.The purpose of this study was to evaluate DANCR expression in de novo acute myeloid leukemia (AML) patients and to assess DANCR expression in relation to cytogenetics and French American British (FAB) AML classification as well as correlate DANCR expression with patients’ response to treatment.The present study included 60 newly diagnosed AML patients and 30 healthy subjects as controls. Relative DANCR expression was done using real time qPCR method.ResultsDANCR was significantly downregulated in AML patients compared to controls (p = 0.038). In addition, DANCR showed significantly lower expression in M4 and M5 compared to M0, M1, and M2 groups (p < 0.001). Furthermore, DANCR expression was significantly downregulated in cytogenetically normal AML patients compared to the controls (p = 0.011).ConclusionSignificant downregulation of DANCR in AML suggests a potential tumor suppressor role and variable expression of DANCR among AML subtypes suggests that DANCR action may be different among AML subtypes. Also, M1 subtype patients with higher DANCR expression were less refractory to treatment and therefore less resistant to cytarabine.

The Role of CXCR1, CXCR2, CXCR3, CXCR5, and CXCR6 Ligands in Molecular Cancer Processes and Clinical Aspects of Acute Myeloid Leukemia (AML).

Acute myeloid leukemia (AML) is a type of leukemia known for its unfavorable prognoses, prompting research efforts to discover new therapeutic targets. One area of investigation involves examining extracellular factors, particularly CXC chemokines. While CXCL12 (SDF-1) and its receptor CXCR4 have been extensively studied, research on other CXC chemokine axes in AML is less developed. This study aims to bridge that gap by providing an overview of the significance of CXC chemokines other than CXCL12 (CXCR1, CXCR2, CXCR3, CXCR5, and CXCR6 ligands and CXCL14 and CXCL17) in AML's oncogenic processes. We explore the roles of all CXC chemokines other than CXCL12, in particular CXCL1 (Gro-α), CXCL8 (IL-8), CXCL10 (IP-10), and CXCL11 (I-TAC) in AML tumor processes, including their impact on AML cell proliferation, bone marrow angiogenesis, interaction with non-leukemic cells like MSCs and osteoblasts, and their clinical relevance. We delve into how they influence prognosis, association with extramedullary AML, induction of chemoresistance, effects on bone marrow microvessel density, and their connection to French-American-British (FAB) classification and FLT3 gene mutations.