Tisagenlecleucel (tisa-cel), an autologous anti-CD19 CAR T-cell therapy, has significantly improved outcomes in pediatric, adolescents and young adults with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (R/R BCP-ALL). However, 30-50% experience early failure or relapse. We retrospectively analyzed 52 cases of early failures (n = 13) or relapses (n = 39), evaluating post-tisa-cel outcomes and prognostic factors. CD19 antigen loss was the only factor associated with a lower complete remission rate after salvage therapy (OR = 0.16, 95%CI [0.03-0.90], p = 0.04). Median overall survival (OS) was 14.5 months, with a 2-year OS of 37.4% (95%CI [24.4-50.4]), similar between early failure (38.5%) and relapse (37.0%) groups (p = 0.78). Patients with measurable residual disease only at salvage initiation had significantly improved 2-year OS (61.9%, (95%CI [38.1-78.8])) compared to those with overt disease (20.7%, (95%CI [8.4-36.7], p = 0.008)). Factors associated with inferior OS included high pre-infusion tumor burden (HR = 3.57, p < 0.01), and prior inotuzumab ozogamicin exposure (HR = 3.81, p < 0.01). Although salvage therapies and hematopoietic stem cell transplantation benefit some patients, 5 of 18 transplanted patients died from treatment-related toxicity, underscoring the significant associated risks. These findings highlight the poor prognosis of tisa-cel failures and the urgent need for novel strategies.

The clinical significance of PNH Type II white blood cells (WBCs) remains unclear. We assessed the relative percentage (rel%) of Type II neutrophils in 355 patients with a PNH clone ≥ 1% on neutrophils enrolled by 33 flow cytometry laboratories in the 5-year French nation-wide multicenter prospective observational study. We first analyzed 127 of 133 patients with a major PNH clone (≥ 50%) and evaluable Type II neutrophil rel%. Using hemolysis data available for 107 patients, a threshold of 3% Type II neutrophils rel% distinguished two groups: 22 "high Type II" and 105 "low Type II" patients. Hemolysis was less frequent in the "high Type II" group. Thrombosis was more frequent at diagnosis and during cumulative long-term follow-up in this group. High neutrophil rel% ≥ 3% was independently associated with thrombotic events. Among 222 patients with smaller PNH clones (1%-50%), 207 displayed evaluable Type II neutrophil rel%, of which 87 were classified as "high Type II" and 120 as "low Type II". Although no thrombosis was present at diagnosis, two thrombotic events occurred during follow-up in the "high Type II" group, whereas none were observed in the "low Type II" group. Long-term follow-up showed no major changes in Type II neutrophil rel%, irrespective of variations in total clone size. These findings suggest that, regardless of the total PNH clone size, PNH Type II neutrophil rel% is a stable parameter and may represent a reliable predictor of thrombosis, potentially supporting earlier consideration of anticomplement therapy initiation.

Measurable residual disease (MRD) follow-up is recommended for treatment response evaluation in acute myeloid leukemia (AML) clinical trials according to ELN 2025 guidelines. The aim of this study was to implement a standardized follow-up of patients using a harmonized MRD flow approach across 30 French hematology laboratories participating in AML clinical trials. To obtain comparable results, the network established recommendations from wet-lab procedures to clinical reports. We designed a 3-tube panel with mandatory 8-color common markers per tube, according to ELN recommendations, to identify leukemia-associated immunophenotype/different-from-normal (LAIP/DfN) patterns in bulk cells and leukemic stem cell (LSC)-enriched populations in the CD34+CD38- fraction. A backbone of CD34/CD38/CD45/CD117 was used, completed by lineage markers for the first tube, LSC-associated markers for the second tube, and monocytic and differentiation markers for the third tube. This panel can be used in 8-, 10-, and 12-color formats and implemented on multiple conventional flow cytometer platforms. We propose flow cytometer settings adapted to each platform. Harmonization of sensitivity between the four platforms was performed using 8-peak rainbow beads. Immunostaining was performed after bulk lysis. To detect bias between platforms, the staining index was tested using fresh healthy bone marrow samples in parallel on the four platforms. Regular bone marrow quality-control samples were shared among laboratories for wet external quality assessment (EQA) to verify all steps of the protocol. Finally, standardization of the data analysis strategy obtained in the centers was evaluated using dry EQA by sharing MRD FCS data files. The feasibility of this multicenter approach requires harmonization of instrument sensitivity and sample preparation, as well as training and systematic education of analytical operators.

ABSTRACT:The prognostic impact of monocytic differentiation in patients with acute myeloid leukemia (AML) receiving venetoclax (Ven) and azacitidine (Aza) remains unclear. In a prospective cohort of 86 newly diagnosed patients with AML treated with Ven-Aza, we used multiparametric flow cytometry (MFC) to define monoblasts as AML blasts coexpressing ≥2 monocytic markers (CD4, CD36, and CD64) per European LeukemiaNet (ELN) guidelines. Patients with higher monoblasts/CD45+ proportions had lower complete response rates (odds ratio, 0.24; P = .005) and significantly shorter overall survival (OS; 4.0 vs 14.9 months; P = .003). A ≥10% monoblasts/CD45+ threshold, identified via maximally selected rank statistics, stratified patients into monoblasthigh (≥10%) and monoblastlow (<10%) groups. MFC reclassified 20% of French-American-British (FAB) non-M4/5 and 15% of FAB M4/5 cases into monoblasthigh and monoblastlow groups, respectively. Multivariable analysis confirmed monoblasthigh status as an independent adverse prognostic factor for OS (hazard ratio [HR], 1.95; P = .023), with a particularly strong impact in ELN 2024 favorable-risk patients (HR, 2.81; P = .024). Our findings highlight monocytic differentiation, assessed via MFC, as a key predictor of Ven-Aza resistance and poor survival, independent of genetic classification. Given its availability in routine diagnostics, MFC-based monocytic assessment could improve AML risk stratification and treatment decisions in patients eligible for less intensive therapies. This trial was registered at www.clinicaltrials.gov as #NCT05326919.

CD34+CD38- leukemic stem cell (LSC) is a key prognostic marker in AML at diagnosis and follow-up in intensive clinical AML trials (Ngai et al, Haematologica 2025, Plesa et al, Annual ASH Meeting 2024). LSC frequency has been reported as a predictor marker for OS in patients (pts) less-intensively treated with azacitidine (AZA) or decitabine without venetoclax (VEN) (Reuvekamp et al, Leukemia 2025). However, there are no data evaluating the clinical impact of LSC frequency at diagnosis in pts treated with AZA-VEN. In this study, we aim to evaluate the prognosis value of flow LSC at diagnosis in pts enrolled in the prospective real-life observational ALFA-PPP study (NCT04777916). The flow data were obtained from 6 French laboratories using a standardized multicentric approach as previously reported. Methods The quantification of LSC was based on at least one aberrancy in CD34+CD38- fraction using most relevant LSC markers as Mix (CLL1/TIM3/CD97) or CD45RA or CD123 +/- CD90(Thy-1). Multiparametric flow cytometry (MFC) was performed on fresh bone marrow (BM) using a 2-tubes panel with 8 minimal mandatory markers by tube (Canto8C/Navios10C/Lyric12C/DxFlex12C). A backbone of CD34/CD38/CD45/CD117 was completed by HLADR,CD7,CD19,CD56,CD13,CD33,CD36. At least 500,000 to 1 million cells were acquired. The rare events populations (LSC,nHSC,MPP,LMPP”like”) were evaluated using a common gating strategy, independently reviewed by the MRD flow network coordinators. Results Between April 2022 and August 2024, 668/767 pts (87%) registered in 12 centers of the ALFA-PPP were studied by flow LSC at diagnosis:median age,64 years;ECOG-PS 0/1/2/3/unknown,180/328/102/42/16; HCTCI comorbidity index <2 (347) vs ≥2 (321); median WBC, 7.4 G/L; de novo AML, 541 (81%); ELN-2022 risk: 127 (19%) fav, 128 (19.2%) int, 383 (57.3%) adv, 30 (4.5%) unknown. A total of 378 pts were treated with intensive chemotherapy (ICT). At diagnosis, using a 1% of CD45+/ssc BM blast cells cut-off, 140 (37%) were LSC-high and 238 (63%) were LSC-low. LSC-high pts were older than LSC-low pts (median age, 62.5 vs 50 years; p= 0.005). They were 84/160 (52.5%), 32/93 (34.4%) and 19/103 (18.4%) in the adv, int, and fav ELN-2022 risk group respectively (p<0.001). With a median follow-up of 1.9 year, overall survival (OS) was significantly shorter in the LSC-high group (median OS,2.4 years vs not reached; p<0.001). Survival analyses were thus adjusted for age, gender, ECOG-PS, HCTCI, WBC ≥30G/L, de novo vs secondary/therapy-related AML, ELN-2022 risk group, and allogeneic HSCT in CR1 as a time-dependent covariate in multivariate Cox regression models. Interestingly, the negative impact of LSC was observed in the ELN-2022 adverse-risk group (adjusted HR, 1.76 [95% CI, 1.06-2.94]; p= 0.030) while not in the other ELN-2022 risk groups and LSC remained an independent prognostic marker for OS overall (adjusted HR,1.52[1.01-2.28];p= 0.046). Among the remaining 290 pts,170>60years were less-intensively treated with AZA-VEN. Here, using the same 1% of LSC cut-off, 90 (53%) pts were LSC-high and 80 (47%) pts were LSC-low at diagnosis. LSC-high pts were 74/130 (56.9%), 12/24 (50.0%) and 3/12 (25.0%) in the adv, int, and fav ELN-2022 risk group respectively (p= 0.12). Contrary to what observed in ICT-treated pts, no difference in OS was observed here between the LSC-high and LSC-low groups (median OS, 0.86 [0.57-1.14] vs 0.99 [0.43-1.98] years; p= 0.46) even in the adjusted multivariate model (adjusted HR, 0.89 [0.59-1.34]; p= 0.58). These results could be linked to: (i) the specific genomic profile observed in this poor group largely enriched in TP53-mutated AML pts (37/170 ; 24/37 LSC-high ; 13/37 LSC-low);(ii) the biological heterogeneity in the LSC compartment with different stemness signature according to MPP-like vs LMPP-like, and clonal hematopoiesis profile;(iii) the sensitivity of LSC to VEN by disrupting the metabolic machinery (Polyeea et al, Nature 2018). Conclusions In this prospective real-life AML cohort, we confirmed that flow CD34+CD38- LSC at diagnosis allowed better defining the prognosis of intensively-treated AML pts, independently of the ELN-2022 risk classification and especially in the adverse risk group, while this impact was not observed in pts treated with AZA-VEN. Further studies combining LSC flow MRD monitoring, MAC-Score and refined ELN2024 risk stratification should be conducted to clarify the clinical impact and interconnection of these markers in AZA-VEN treated pts.

Hematopoietic stem and progenitor cells (HSPCs) polarize in contact with the bone marrow stromal cells constituting their niche. Given the role of cell polarity in protection against tumorigenesis and the importance of the niche in the progression of acute myeloid leukemias (AMLs), we investigated the polarization capacities of leukemic blasts. Using engineered micro-niches and centrosome position with respect to the contact site with stromal cells as a proxy for cell polarization, we show that AML cell lines and primary cells from AML patient blasts are unable to polarize in contact with healthy stromal cells. Exposure to AML patient-derived stromal cells compromises the polarization of healthy adult HSPCs and AML blasts from patients. When cultured in “bone-marrow-on-a-chip”, stromal cells from a leukemic niche stimulate the migration of healthy HSPCs and AML blast. These results reveal the detrimental influences of both intrinsic transformation and extrinsic contact with transformed stromal cells on the polarization of AML blasts.

Tandem duplications of exon 13 of the UBTF gene ( UBTF- TD) represent a distinct molecular entity of acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS), primarily affecting adolescents and young adults. The typical presentation is characterized by pauciblastic AML or MDS with an excess of blasts (or AML-M6 according to the FAB classification), although AML with hyperleukocytosis forms is also observed, especially in the presence of FLT3-ITD. UBTF-TD is associated with a specific pattern of additional genetic lesions, including WT1 mutations (similar to 50%), FLT3-ITD (similar to 50%), and trisomy 8 (similar to 30%, while similar to 60% of cases have a normal karyotype), and is mutually exclusive with other class-defining lesions (i.e. NPM1 or CEBPA-bZIP mutations and recurrent fusions). Intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation is currently the only evaluated treatment option. The prognosis of this entity remains overall poor, with a high risk of chemotherapy resistance and relapses.

Introduction The distinction between type II and type III cells among the white blood cells (WBC) need to be further investigated since a recent British study reported that the highest proportion of type II red blood cells (RBC) were found in patients with thrombotic PNH (Richards, BJH 2020). Objective We assessed the relative percentage of type II cells among neutrophils in patients displaying a major PNH clone (defined as GPI-deficient cells above 50%) and subsequently in patients with a clone between 1% and 50%. In addition, we aimed to investigate correlation to biological and clinical presentations, notably thrombosis, using data from the 5-year French nation-wide multicenter observational study. Methods All patients, regardless of their age, with a newly or previously diagnosed PNH clone or GPI-deficient cells ≥0.01% on neutrophils, detected in France by flow cytometry (FCM) from 2016 to 2021, were eligible for inclusion in this observatory. For each patient, the baseline assessment was considered as the initial PNH clone detection. Follow-up assessments were conducted in accordance with routine clinical practice. The two principal investigators reviewed all the clinical and biological information, especially FCM raw data files provided by the centers. This study was approved by the national research ethics board. Results Forty-one participating FCM laboratories across France enrolled 352 patients with a PNH clone above 1%. We first focused on the 125 out of 131 patients with a major PNH clone (i-e, ≥ 50%) with evaluable type II neutrophil percentages. Using the 104 patients with available data on hemolysis status, we performed an AUROC test based on the presence of hemolysis, a main feature of patients with major PNH clone. We determined a threshold of 3% of type II neutrophils (the same value was obtained considering raw percentages and relative percentages (r%) among the total deficient cells, that may be useable for patients with a PNH clone between 1 and 50%). This enabled the distinction of two groups: 103 patients with low r% of type II neutrophils (0.00%; IQR [0.00-0.71]) and 22 patients with high r% of type II neutrophils (5.98% [4.75-12.26]; p<0.0001). In addition, type II monocyte r% were higher in the “high type II” group (7.75% [5.13-11.07]) compared to the “low type II” group (0.00% [0.00-0.83]; p<0.0001). However, the total clone size was similar for both groups on WBC (neutrophils: 90.69% [75.44-96.90] vs 92.15% [80.28-98.28]; p=0.66, monocytes: 87.53% [76.10-93.01] vs 91.48% [77.71-96.75]; p=0.69), in the “high type II” and the in the “low type II” group, respectively. Hemolysis was more frequent in “low type II” group: 94.3%(83/88) compared to “high type II” group: 61.1%(11/18) (p=0.0006) for the 106 patients with available data on hemolysis status and a trend to a reduced r% of RBC (0.82% [0.00-31.41] vs 22.19% [0.00-53.14]; p=0.19). Interestingly, thrombosis appears to be more frequent at diagnosis in “high type II” group: 28.6%(6/21) vs 6.3%(6/95) in the “low type II” group (p=0.008) and when analyzing the cumulative events during long-term follow-up (33%(7/21) vs 8.4%(8/95); p<0.005). We then focused on the 205 out of the 221 patients with a lower PNH clone size between 1% and 50% with evaluable type II neutrophil percentages. The relative percentage threshold of 3% allowed the distinction of 118 patients with “low type II neutrophils” and 87 with “high type II”. Hemolysis was more frequent in “low type II' group compared to ”high type II“ group (30% vs 15%; p=0.01). Remarkably, while no thrombosis was present at diagnosis, 2 thrombosis occurred during the follow-up of the ”high type II“ group (respective clone size on neutrophils: 21.77% (r% type II: 5.65%) and 4.74% (r% type II: 60.34%)) and none in the ”low type II“ group. Long-term follow-up up to 14.2 years of 124 patients, of whom 69 were receiving anti-C5, showed that 55 patients (56% treated) displayed an increase of the total clone size (mean: +18.1%), 36 (78% treated) remained stable (+0.0%) and 33 (30% treated) exhibited a decrease (-6.9%). However, we did not observe any major modification of type II neutrophil percentage between the high and low type II groups (-0.3 % vs +0.0%). Conclusion Patients with higher r% of type II neutrophils displayed more thrombosis irrespective of the total PNH clone size. Accurate quantification of type II neutrophils may impact clinical management since a treatment initiation could be considered.

BACKGROUNDDonor cell engraftment is a prerequisite of successful allogeneic hematopoietic stem cell transplantation. Based on peripheral blood analyses, it is characterized by early myeloid recovery and T and B cell lymphopenia. However, cellular networks associated with bone marrow engraftment of allogeneic human cells have been poorly described.METHODSMass cytometry and CITE-Seq analyses were performed on bone marrow cells 3 months after transplantation in patients with acute myelogenous leukemia.RESULTSMass cytometric analyses in 26 patients and 20 healthy controls disclosed profound alterations in myeloid and B cell progenitors, with a shift toward terminal myeloid differentiation and decreased B cell progenitors. Unsupervised analysis separated recipients into 2 groups, one of them being driven by previous graft-versus-host disease (R2 patients). We then used single-cell CITE-Seq to decipher engraftment, which resolved 36 clusters, encompassing all bone marrow cellular components. Hematopoiesis in transplant recipients was sustained by committed myeloid and erythroid progenitors in a setting of monocyte-, NK cell-, and T cell-mediated inflammation. Gene expression revealed major pathways in transplant recipients, namely, TNF-α signaling via NF-κB and the IFN-γ response. The hallmark of allograft rejection was consistently found in clusters from transplant recipients, especially in R2 recipients.CONCLUSIONBone marrow cell engraftment of allogeneic donor cells is characterized by a state of emergency hematopoiesis in the setting of an allogeneic response driving inflammation.FUNDINGThis study was supported by the French National Cancer Institute (Institut National du Cancer; PLBIO19-239) and by an unrestricted research grant by Alexion Pharmaceuticals.

BH3 profiling can assess global mitochondrial priming and dependence of leukemic cells on specific BH3 anti-apoptotic proteins such as BCL-2. In acute myeloid leukemia (AML), proof-of-concept prognostic studies have been performed on archived samples variably accounting for molecular genetics. We undertook a single-center feasibility study of a simplified flow-based assay to determine the absolute mitochondrial priming and BCL-2 dependence in consecutive AML patients. When possible, results on the leukemic fraction were normalized to the cognate lymphocyte population (relative priming and BCL-2 dependence). Samples from 97 (89.8%) of the 108 referred patients were successfully processed. Relative priming and BCL-2 dependence could be determined in 62 (67.4%) and 67 (62.0%) samples, respectively. Absolute mitochondrial priming was lower in patients having previously failed intensive chemotherapy compared to chemotherapy-na & iuml;ve patients (p = 0.01), but its prognostic impact was limited. Conversely, relative BCL-2 independence tended to predict worse EFS (HR = 2.51, p = 0.07) and OS (HR = 2.79, p = 0.10) independently of adverse genetic risk. Our results show that simplified BH3 profiling can be prospectively assessed in AML patients but that its prognostic use may require internal normalization. Future studies should compare its relevance with other functional assays such as ex vivo drug testing or BH3 protein expression.

Introduction MRD monitoring is recommended for treatment response evaluation in clinical AML trials (ELN-2022 guidelines: Heuser et al, PMID 34724563). Besides molecular biology methods, multiparametric flow cytometry represents the most reliable approach. In this study, we evaluated the impact of flow MRD in patients (pts) enrolled in the Backbone Intergroup BIG-1 trial (NCT02416388) from the Acute Leukemia French Intergroup (ALFA-FILO) between 2018 and 2021. For that purpose, a standardized multicentric MRD flow approach based on ELN recommendations and combining LAIP/Dfn and CD34+CD38- Leukemic Stem Cell (LSC) detection as previously reported (Zeijlemaker et al, PMID 30542144; Ngai et al, PMID 36898087) was implemented across 30 French hematology laboratories. Methods Multiparametric flow cytometry was performed on fresh bone marrow (BM) sample using a 2-tubes panel, with minimal mandatory 8-color markers by tube, to characterize the pattern of LAIP (Leukemia Aberrant ImmunoPhenotype)/Dfn (Different from normal) and CD34+CD38- LSCs. Our network has established recommendations from Wet labs procedure to clinical reports using common panels, flow cytometers settings and analysis strategy (Plesa et al, Annual ASH Meeting 2022). A « backbone » using CD34/CD38/CD45/CD117 was used in both tubes, completed by CD7, CD56, CD13, CD33, HLADR, CD19 for the 1st tube and CD90Thy-1, Mix (CD97/CLL1/TIM3), CD45RA, CD123 for the 2nd one. A total of at least 500,000 to one million cells was acquired in each tube. An Assurance Quality Program was implemented in all laboratories during the study. All data were reviewed independently by the MRDflow network coordinator. Results Between 2018 and 2021, 315/1228 pts treated in the BIG-1 trial (median age, 49 years; ELN-2022 risk: 97 favorable, 87 intermediate, 110 adverse, 21 unknown) were studied by flow. No differences were observed between pts with or w/o flow evaluation according to main patient/AML characteristics and outcomes. At diagnosis, using a 1% of CD45+/ssc BM blast cells cut-off, 88/280 pts (31.4%) were LSC-high and 192/280 pts (68.6%) were LSC-low, with a strong correlation between LSC-high incidence and ELN-2022 risk (6.2, 37.3 and 45.6% in the favorable, intermediate and adverse group, respectively; p<0.001). As expected, OS was thus significantly lower in the LSC-high group (3y-OS, 45% [95% confidence interval, 35-56] vs 69% [62-75]; p<0.001). Using a 0.1% of CD45+ BM cells cut-off, the numbers of pts with positive LAIP/Dfn MRD was 90/298 (30.2%) at post-induction (MRD1) and 69/253 (27.3%) at post-consolidation (MRD2) timepoint. For both timepoints, OS was significantly lower in LAIP/Dfn-positive vs LAIP/Dfn-negative pts (3y-OS, 50% [39-60] vs 75% [68-80] for MRD1 and 49% [37-60] vs 78% [71-83] for MRD2; p<0.001 for both). Using a 0.01% of CD45+ BM cells cut-off, the numbers of pts with positive LSC MRD were 57/222 (25.7%) at MRD1 and 38/197 (19.3%) at MRD2. Again, OS was significantly lower in LSC-positive vs LSC-negative pts at both timepoints (3y-OS, 47% [33-59] vs 77% [69-82] for MRD1 and 42% [26-57] vs 80% [73-86] for MRD2; p<0.001 for both). Finally, the “scoring system” was validated when combining both LAIP/Dfn and LSC markers at MRD1 and MRD2. Among 220 pts tested for both markers at MRD1, the LAIP-Dfn/LSC score was -/-, +/-, -/+ and +/+ in 135, 24, 33 and 28 pts, respectively. At 3 years, OS estimates were 78% (70-84), 68% (47-82), 71% (48-85) and 30% (16-46), in these 4 subgroups, respectively (p=0.033). Among 193 pts tested for both markers at MRD2, the LAIP-Dfn/LSC score was -/-, +/-, -/+ and +/+ in 125, 12, 26 and 30 pts, respectively. At 3 years, OS estimates were 82% (74-88), 70% (50-83), 75% (41-91) and 27% (12-44), in these 4 subgroups, respectively (p=0.007). Similar predictive values were observed when censoring patients allografted in first remission at transplant date. Finally, the predictive values of LAIP/Dfn at MRD1 and MRD2, LSC at MRD1 and MRD2, and combined score at MRD2 (but not at MRD1) remained significant after adjustment on the ELN-2022 risk. Conclusions Our first experience demonstrates the feasibility of a multicentric flow MRD approach in AML. We confirm in the prospective French BIG-1 trial that flow MRD monitoring combining LAIP/Dfn and CD34+CD38- LSC allows better defining AML patient prognosis independently of the ELN-2022 risk.

High mitochondrial priming of blasts for apoptosis relative to hematopoietic stem cells, measured by mitochondrial membrane depolarization upon exposure to proapoptotic BH3 peptides, has been suggested to predict longer overall survival (OS) in acute myeloid leukemia (AML).1 Whether its prognostic impact is independent of genetic risk remains unknown. We interrogated the mitochondrial priming of AML cells in patients 60 years or older uniformly treated with intensive chemotherapy in a clinical trial and found that higher blast priming predicted prolonged OS independently of genetic risk. Intensive chemotherapy remains the standard of care of fit adults older than 60 years with newly diagnosed AML. However, results of 7 + 3 based chemotherapy regimens remain disappointing with long-term survival rates below 30%.2 Identifying patients that benefit from intensive chemotherapy thus remains an important area of investigation. Recent efforts have focused on integrating a greater number of genetic lesions to predict OS of intensively treated older AML patients.3,4 However, the predictive power of risk classifiers solely based on (cyto)genetics remains limited.2–4 In recent years, functional precision medicine has emerged as a promising approach to complement oncogenetics in treatment decision making.5 BH3 profiling is one such functional approach, whereby sensitivity to mitochondrial apoptosis triggering agents, including conventional chemotherapy, is estimated by challenging cells with peptides derived from BH3-only proteins and measuring cytochrome C release or loss of mitochondrial transmembrane potential. In a landmark study, high mitochondrial priming of AML blasts relative to their cognate hematopoietic stem cells was found to predict longer OS in univariable analysis.1 A previous study on 62 patients reported that BH3 profiling could predict remission after various cytarabine-based regimens independently of age and cytogenetics.6 Whether BH3 profiling can predict OS in a homogeneously treated AML population independently of up-to-date oncogenetic risk stratification remains unknown. To address this question, we interrogated the mitochondrial priming of primary AML cells in 57 diagnostic samples from patients 60 years or older treated intensively in the ALFA 1200 clinical trial (NCT01966497). The ALFA 1200 trial accrued patients 60 years or older with newly diagnosed AML (de novo, secondary to myelodysplastic syndromes or therapy-related), excluding acute promyelocytic leukemia and Ph-positive AML. Patients received a 7 + 3 induction course with idarubicin as anthracycline. Patients failing to achieve complete remission (CR) or CR with incomplete platelet recovery (CRp) could receive an intermediate-dose cytarabine (IDAC) course. Patients in CR/CRp received 2 such IDAC courses as consolidation and could proceed to allogeneic hematopoietic cell transplantation (allo-HCT). Further details on treatment and centralized molecular genetics and cytogenetics procedures have been previously published.3,4 For this study, cryopreserved mononuclear cells from bone marrow samples collected at inclusion in the ALFA 1200 trial in 65 patients were thawed, washed in with MEMα (Life Technologies) and 10% Fetal Bovine Serum (FBS, BioTec) and a second time in Phosphate Buffer Saline (PBS, Eurobio). 5 × 106 cells were stained with Zombie Aqua Fixable Viability Dye (Bio Legend) according to manufacturer’s instructions, then stained for surface markers with anti-CD34 PE-Cy7 (clone 581), anti-CD38 PerCP-Cy5.5 (clone HB7), anti-CD45 APCH7 (clone 2D1), anti-CD45RA APC (clone HI30), anti-CD3 PC5 (clone SP34-2), anti-CD19 PC5 (clone HIB19), anti-CD20 PC5 (clone 2H7), and anti GPA PC5 (clone GA-R2, all from BD Biosciences), Alexa Fluor anti-CD90 (clone 5E10) and anti-CD123 PE (clone 6H6, both Sony Biotechnology). Cells were then washed and resuspended in DTEB Buffer (135 mM trehalose, 50 mM KCl, 20 µM EDTA, 20 µM EGTA, 5 mM succinate, 0.1% Bovine Serum Albumin, 10 mM HEPES-KOH final pH 7.5, all from Sigma-Aldrich). 6 × 105 cells per condition were incubated in the presence of a BIM peptide (Ac-MRPEIWIAQELRRIGDEFNA-NH2, purity >95%, Proteogenix, France) dissolved in DMSO at the indicated concentrations (1, 0.3, 0.1 µM) for 1 hour in the presence of 0.001% digitonin (Sigma-Aldrich) for cell permeabilization. Cells were then fixated with Formaldehyde 8% in PBS (Sigma-Aldrich). Fixation was terminated by N2 buffer solution (1.7 M Tris, 1.25 M Glycine pH 9.1, both from ThermoFisher). Cells were then labeled with anti-human cytochrome C (CytC) Alexa Fluor 488 (BD Biosciences, clone 6H2.B4) in the presence of adequate amounts of Perm/Wash 10× buffer (BD Biosciences) and incubated overnight at 4°C. All experiments included 2 DMSO 2% sample (without BIM peptide), 1 labeled with anti-Cytochrome C and 1 unlabeled as positive and negative controls for mitochondrial Cytochrome C content, respectively. Flow cytometry acquisition was performed on a BD Fortessa analyser (BD Biosciences) on >200,000 events and analyzed with FlowJo v10 (TreeStar). Mitochondrial priming was defined as the percentage of cytochrome C release based on median fluorescence intensities (MFI) according to the following formula: 1 − ((MFIBIM − MFIFMO)/(MFIDMSO − MFIFMO)) ×100 (%) (Figure 1A). We studied the mitochondrial priming of total leukemic blasts (CD45dim/SSClow, mean 1.06 × 105 cells, interquartile range [IQR] 0.50–1.59) and of Hematopoietic Stem and Progenitor Cells (HSPCs, Lin-/CD34+/CD38−/CD45RA−, mean 2.58 × 104 cells, IQR 0.65–2.82) as internal control (Figure 1B).1 Absolute priming of each cell compartment was measured as the area under the curve (spline interpolation method) of Cyt C release across the 3 BIM concentrations (Figure 1C). All analyses were conducted with R 4.1.0 (cran.r-project.org). Of 65 samples, only 57 could be analyzed, due to low cell viability after thawing in 8 samples. In this study population (31 males, 26 females), median age was 71 years (IQR 67–73) and median white blood cell count was 27.1 × 109/L (IQR 9.0–70.2). AML was de novo, secondary to MDS and therapy-related in 46 (81%), 8 (14%), and 3 (5%) cases, respectively. According to European LeukemiaNet (ELN) 2017 guidelines,2 genetic risk was favorable, intermediate, and adverse in 24 (42%), 9 (16%), and 24 (42%) patients, respectively. Thirty-five (61%) patients achieved CR/CRp. As a continuous variable, relative blast priming (ie, AUC of cytochrome C release in blasts normalized to the AUC in the HSPC compartment) was similar in patients achieving CR/CRp after 1 (n = 34) or 2 (n = 1) course and in those with primary induction failure (n = 19) or early death (n = 3) (Mann-Whitney test P = 0.29). With a median follow-up of 43.1 months, median OS in the study population was 14.6 months. Two-year OS was 60.8% (95% confidence interval [CI], 43.6-84.8), 33.3% (95% CI, 13.2-84.0), and 22.4 (95% CI, 10.4–42.8) in patients with favorable, intermediate, and adverse ELN risk, respectively (log-rank test, P = 0.07). In univariable analysis, higher relative blast priming as a continuous variable was associated with a significantly longer OS (univariable Cox model, hazard ratio (HR) = 0.97, 95% CI, 0.95-0.99, P = 0.045). We empirically assigned patients whose blasts had a global priming >120% of their cognate HSPCs into a “primed” category and all others into an “unprimed” group (Figure 1C). Of the 57 patients, 39 (68%) had “primed” blasts. The genetic profile of primed and unprimed samples was comparable (online Supplement). In univariable analysis, 2-year OS of primed patients was 52.4% (95% CI, 38.5-71.2) compared to 6.8% (95% CI, 1.0-44-8) in unprimed patients (log-rank test P = 0.004, Figure 1D). In a multivariable Cox model accounting for ELN risk (reference: favorable risk), primed status was associated with prolonged OS (HR = 0.39, 95% CI, 0.20-0.78, P = 0.008) independently of intermediate (HR = 1.02, 95% CI, 0.37-2.86, P = 0.96) and adverse (HR = 2.08, 95% CI, 0.98-4.39, P = 0.055) ELN risk. Only 4 patients (3 primed, 1 unprimed) underwent allo-HCT in first remission. Censoring at the time of allo-HCT did not abrogate the independent favorable prognostic value of primed status on OS (HR = 0.44, 95% CI, 0.22-0.88, P = 0.02). Specifically, 17 (70.8%), 5 (55.6%), and 17 (70.8%) patients with favorable, intermediate, and adverse ELN risk had primed blasts, respectively. At 2 years from diagnosis, none of the 11 patients with favorable or intermediate risk AML but unprimed blasts were alive, while 2-year OS was 72.4% (95% CI, 55.9-93.9) in the 22 primed patients with favorable or intermediate risk AML (log-rank test P = 0.002). In contrast, 2-year OS was comparable in primed (25.9%, 95% CI, 11.2-59.7) and unprimed (14.3%, 95% CI, 2.3-87.7) patients with adverse risk AML (log-rank P = 0.4, Figure 1E). Among the 33 patients with favorable or intermediate risk AML achieving CR/CRp, 2-year disease-free survival was 80.0% (95% CI, 62.1-100) in the 15 primed patients, whereas none of the 8 unprimed patients were alive in remission 2 years from remission (log-rank P = 0.007). We previously reported a 3-tier genetic classifier to identify patients older than 60 years benefiting from intensive therapy.4 According to this ALFA classifier, 24, 31, and 2 patients were assigned to the “go-go,” “slow-go,” and “no-go” groups, respectively. Eighteen (75%) and 20 (64.5%) of the “go-go” and “slow-go” patients had primed blasts, respectively. In those patients, relative blast priming also predicted longer OS (HR = 0.46, 95% CI, 0.23-0.94, P = 0.03) independently of the ALFA classifier (HR = 2.11, 95% CI, 0.98-4.52 for slow-go [reference: go-go], P = 0.06). Our study is the first to account for genetic risk to investigate the predictive value of BH3 profiling in uniformly treated AML patients. Our results, albeit on a limited cohort skewed toward proliferative cases, suggest that relative mitochondrial priming could complement genetic risk stratification to identify older patients achieving long-term survival with intensive therapy. Functional assays such as BH3 profiling are currently restricted to specialized labs. Their prospective investigation in larger cohorts is warranted to confirm the emerging role of functional precision oncology in AML.Figure 1.: Relative mitochondrial priming and overall survival in older AML patients treated intensively. (A) Summary of study workflow. (B) Gating strategy for CD45dim/SSClow blasts and live Lin−/CD34+/CD38−/CD45RA− HSPCs. (C) AUC (cubic spline interpolation) of Cytochrome C release in blasts (red) and HSPCs (purple) of 2 exemplary cases, 1 (top) assigned to the “primed” group based on an AUCblasts >120% AUCHSPCs, and 1 (bottom) assigned to the unprimed (AUCblasts ≤120% AUCHSPCs) group. (D) Overall survival probability from study inclusion in patients with primed (n = 39, red curve) or unprimed (n = 18, blue curve) blasts. (E) Overall survival probability from study inclusion in patients with primed (red curve) or unprimed blue curve blasts according to their ELN 2017 risk groups (fav/int favorable or intermediate risk, solid lines, adv adverse risk, dashed lines). All P values from log-rank tests. AML = acute myeloid leukemia; AUC = area under the curve; BMNC = bone marrow mononuclear cell; FSC = forward scatter; HSPC = hematopoietic stem and progenitor cell.ACKNOWLEDGMENTS We are indebted to Sophie Duchez, Christelle Doliger and Niclas Setterblad from the Saint-Louis Research Institute Core Flow Facility, and Céline Decroocq from the Lille University Hospital Tumor Bank. This work was supported by Association Laurette Fugain (ALF2020-01) to R. Itzykson and is part of the THEMA program funded by the Agence Nationale pour la Recherche (ANR IHU-B-2018). AUTHOR CONTRIBUTIONS RI, RDB, and AP designed the study, performed analyses, and drafted the manuscript. RDB, KP, CC, and JP performed experiments. SM, CR, and AP assisted in flow cytometry analyses. LA, TB, CB, ER, DL, TC, HD, and CG accrued patients. CG was the principal investigator of the trial. KCL and HD manage the ALFA cooperative group. All authors reviewed the manuscript and approved its final version. DISCLOSURES The authors have no conflicts of interest to disclose. SOURCES OF FUNDING This study was funded by Association Laurette Fugain (ALF2020-01).

Rationale. The role of bridging therapy, i.e., treatment between apheresis and lymphodepletion (LD) preceding tisagenlecleucel (tisa-cel) infusion for relapsed/refractory B cell acute lymphoblastic leukemia (R/R BCP-ALL) is still unclear. We report a retrospective study on our experience on 81 children, adolescents, and young adults with R/R BCP-ALL who underwent apheresis with an intent to receive tisa-cel infusion, between March 2016 and October 2020. Methods. Bridging therapy intensity was classified into two intensity groups. A high intensity bridging (HIB) regimen, used in 23 patients (pts), was defined as the use of more than 4 intravenous anti-leukemic drugs per week and/or the need of secondary intensification of bridging therapy, the latter being the case in 19 pts (83%). A low-intensity bridging (LIB), in general a combination of vincristine, steroids +/-asparaginase, was used in 58 pts. The aim of the study was to evaluate the impact of HIB versus LIB on tisa-cel efficacy. Factors associated with a failure to infuse tisa-cel were also explored. Results. Patients had a median age of 15 years (range: 1-19). Tisa-cel indication was a primary refractory disease for 5 pts (6%), a first post-allo-HSCT relapse for 47 pts (58%) and a second relapse or more for 29 pts (36%). Patients who received a HIB therapy were more likely to have high-risk genetic features (39% vs 12%, p= .01) and/or a primary refractory disease (17% vs 2%, p=.02). Grade 3+ non-infectious and infectious adverse events during bridging therapy were also more likely in this group, albeit in a non-statistically significant way (35% vs 16 %, p= .07 and 52% vs 34 %, p= .21; respectively). Nine patients (11%) were not infused (disease progression: 3 pts; toxicity: 5 pts (sinusoidal obstruction syndrome: 1 pt, encephalopathy: 2 pts, fungal infection; 1 pt, hemoptysis: 1 pt); and CD19 negativation: 1 pt). A high pre-apheresis tumor burden ( p=.01), the occurrence ( p=.01), and the number of grade ≥ 3 adverse events during bridging period ( p<.01) were associated with a higher risk of tisa-cel non-infusion, while HIB was not ( p=.73). The 72 remaining patients (89%) finally received tisa-cel infusion, 52 (64%) after a LIB therapy and 20 (25%) after a HIB therapy. Despite a comparable BM disease burden at apheresis between the LIB and the HIB groups, patients presenting a high disease burden prior to LD (bone marrow (BM) blasts > 50%) were more frequent in the HIB group ( p< .01). When compared to patients who received a LIB, patients treated with a HIB had a worst, albeit non-statistically different cumulative incidence of relapse (CIR) (SHR 1.94, 95% CI [0.95-3.91], p=.07), an inferior 18-month event-free survival (EFS) (26.9% vs 54.1%; p=.02) and a lower 18-month overall survival (OS) (50.9% vs 88.8%; p<.01). Considering a threshold at 50% BM blasts, patients who presented a high disease burden prior to LD had poorer early response rates (D28 minimal residual disease (MRD) negativity: 85% vs 69%, p< .01), a higher, albeit non-statistically different CIR (SHR 2.27, 95% CI [0.98-5.27], p=.06), an inferior 18-month EFS (26.9% vs 51.3%; p=.02) and a lower 18-month OS (22.6% vs 91.5%; p<.01). We then asked if the bridge intensity, aimed at achieving a low disease burden before LD, could influence patient outcomes. Median percentages of BM blast infiltration were similar after LIB (0%) and HIB therapy (1%, p=.87) in patients with ≤50% BM blast at LD. In these patients, a prior HIB regimen was associated with a higher rate of detectable MRD at D28 (40% vs 8%, p=.02), an inferior 18-month EFS (18.0% vs 57.5%; p=.02) and a higher, albeit non-statistically different CIR (SHR 2.05, 95%CI [0.87-4.88], p=.1). The 18-month OS did not differ between groups (74.0% vs 94.6%; p=.15), suggesting that these patients may respond to further lines of treatment after relapse. The outcome of patients presenting a low disease burden at LD after a HIB regimen was thus similar to those with a high disease burden at LD. Conclusion. This study confirms the prognostic value of a high disease burden pre-LD but also reveals that a low disease burden prior tisa-cel therapy is associated with an impaired outcome when obtained through intensification of the bridging therapy. Additional post-tisa-cel therapies should be considered in this group of patients.

Introduction: MRD follow-up is now mandatory for treatment response evaluation in AML clinical trials (ELN 2017 and 2021 guidelines: Heuser et al,Blood doi:10.1182/blood.2021013626.). Beside to molecular biology methods, multiparameter flow cytometry assay represents the most reliable approach. The aim of this study was to implement an harmonized follow up of the patients using a standardized MRD flow approach across 30 French hematology laboratories participating in AML clinical trials. Methods: To obtain superposable results, the network has established recommendations from Wet labs procedure to clinical reports using common panels, flow cytometers settings and analysis strategy. We designed a 2-tubes panel with minimal mandatory 8c markers by tube, according to ELN recommendations to identify the pattern of LAIP/DfN in bulk cells and the LSC (Leukemia Stem Cells) in CD34+CD38- fraction. A « backbone » CD34/CD38/CD45/CD117 was used, completed by CD7,CD56, CD13, CD33, CD19 for the 1st tube and CD90(Thy-1), Mix (CD97+CLL1+TIM3), CD123, CD45RA for the 2nd one. This panel could be used in 8c,10c,12c (HLADR, CD36, CD10, CD200) implemented on Becton Dickinson (CANTO and LYRIC) or Beckman Coulter (NAVIOS and DxFlex) cytometers (Fig 1A). Harmonization of the sensitivity between platforms (18 BD and 12 BC) was performed using 8 Picks Rainbows calibration beads. Immunostaining was performed after "bulk" lysis technique (NH4Cl). A total of minimal 500 000 cells was acquired in each tube and data were analyzed using DIVA/FACSUITE and KALUZA software. Results: To detect any bias between the platforms, firstly we tested 10 EDTA fresh regenerative marrow samples in parallel at 2 platforms (CANTO and NAVIOS). Similarity of the staining index between positive and negative population were compared between the samples and the 2 platforms showing no differences. Secondly, regular QC (Quality Control Sample), of normal bone marrow (from healthy donor) 1/year (a total of 5 since 2017) was shared among the participants centers(Fig1B).The specific events populations (nHSC, MPP, LMPP"like,etc) were evaluated by centers using a common gating strategy. In a third step, standardization of the data analysis strategy obtained in the centers was subsequently evaluated using a virtual quality control(CQA) by sharing MRD FCS data files. About 90% of the locally analyzed data were included in an interval centralized on the average of the series mean +/- 2 SD. During the follow up of the patients, a series of 18 Web educational meeting was implemented to review and validate cases with difficulties regarding analysis interpretation and confrontation with molecular MRD (transcripts, NPM1, WT1, NGS) and also chimerism in allograft follow-up (mainly cases with clonal hematopoiesis or stressed bone marrow). Finally, the harmonized data generated in our group allowed to perform a Computer aided design (CAD) in assessment of AML MRD flow using proprietary developed R script based upon FlowSom. It performs an automated definition of metacluster with abnormal population (by comparison to a set of reference bone marrow) which are quantified in the CD34+ CD117+ space for all samples (diagnosis, follow up and references marrow). It then extracts the metacluster significantly different from that observed for the reference samples (> mean+6sd. It calculates the MRD in % of WBC CD45+. The limits of sensitivity of the CAD method shows that a MRD remains evaluable by CAD up to 0.04% (50 events in 1 000 000). We compared the results between the conventional and the CAD method on 60 MRD samples. The slope of the correlation line was 0.95 with an R2 of 0.97 (Fig 1C). Using the definition threshold of a positive MRD at 0.1%, the agreement of the results between the two methods was evaluated by the Cohen kappa coefficient of 0.87. Conclusions: This methodological validation protocol is a mandatory step to consider the use of MRD flow in AML clinical trials. Choosing a multicentric approach could be challenging, but our first results are promising and showed the feasibility of this concept when: (i) a straight harmonisation of the instruments sensitivity and samples preparation are established, and (ii) training and systematic education among the analytical operators are regularly performed. Finally, our pilot study shows a very strong correlation between conventional method and unsupervised analyses using data generated by the multicentric network. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background Myelodysplastic syndromes (MDS) are clonal hematopoietic diseases of the elderly characterized by chronic cytopenias, ineffective and dysplastic haematopoiesis, recurrent genetic abnormalities and increased risk of progression to acute myeloid leukemia. A challenge of routine laboratory Complete Blood Counts (CBC) is to correctly identify MDS patients while simultaneously avoiding excess smear reviews. To optimize smear review, the latest generations of hematology analyzers provide new cell population data (CPD) parameters with an increased ability to screen MDS, among which the previously described MDS-CBC Score, based on Absolute Neutrophil Count (ANC), structural neutrophil dispersion (Ne-WX) and mean corpuscular volume (MCV). Ne-WX is increased in the presence of hypogranulated/degranulated neutrophils, a hallmark of dysplasia in the context of MDS or chronic myelomonocytic leukemia. Ne-WX and MCV are CPD derived from leukocytes and red blood cells, therefore the MDS-CBC score does not include any platelet-derived CPD. We asked whether this score could be improved by adding the immature platelet fraction (IPF), a CPD used as a surrogate marker of dysplastic thrombopoiesis. Methods Here, we studied a cohort of more than 500 individuals with cytopenias, including 168 MDS patients. In a first step, we used Breiman’s random forests algorithm, a machine-learning approach, to identify the most relevant parameters for MDS prediction. We then designed Classification And Regression Trees (CART) to evaluate, using resampling, the effect of model tuning parameters on performance and choose the “optimal” model across these parameters. Results Using random forests algorithm, we identified Ne-WX and IPF as the strongest discriminatory predictors, explaining 37 and 33% of diagnoses respectively. To obtain “simplified” trees, which could be easily implemented into laboratory middlewares, we designed CART combining MDS-CBC score and IPF. Optimal results were obtained using a MDS-CBC score threshold equal to 0.23, and an IPF threshold equal to 3%. Conclusions We propose an extended MDS-CBC score, including CPD from the three myeloid lineages, to improve MDS diagnosis on routine laboratory CBCs and optimize smear reviews.