Level Of Residual Disease Research Articles

Deuterated water labeling in ibrutinib-treated patients with CLL: leukemia cell kinetics correlate with IGHV, ZAP-70, and residual disease

AbstractDeuterated (“heavy”) water labeling in CLL patients demonstrates that IGHV unmutated and ZAP-70+ patients have higher blood and tissue CLL death rates on ibrutinib therapy, resulting in lower measurable residual disease levels with long-term ibrutinib treatment. This trial was registered at www.clinicaltrials.gov as #NCT01752426.

Infections Deaths in Acute Lymphoblastic Leukemia More Frequent in High Risk Leukemia

Abstract Background Acute lymphoblastic leukemia (ALL) in children currently has more than 90% survival in higher resource countries. However, in less resource countries, the survival rates are lower and infections are frequent causes. Risks for infections and poor outcomes vary during the chemotherapy phases. There is a paucity of data concerning the frequencies of the chemotherapy phase where the event is more probable. Our primary objective is to describe infections frequencies and outcomes of infection in children and also to look for association with risk factors. Methods Patients (Pts) were treated according to ALLIC-BFM-2010 protocol, a simpler branch of the BFM-AEIOP protocol and accrued prospectively. The main question is comparing outcomes of two randomizations using higher dose chemotherapy based on the leukemia risk. The risk severity was based on levels of measurable residual disease (<0.1, 0.1-10, >10 %,) and categorized in high (HR), intermediate (IR), and low (LR) risks. In SR Pts with B-cell-precursor ALL two doses of daunorubicin were prescribed in induction compared with four doses in all others. In consolidation, high-dose methotrexate was administered at 5g/m2 for SR/IR T-cell ALL and at 2g/m2 for SR/IR B-precursor-ALL. Consolidation for HR pts consisted of three intensive polychemotherapy blocks. Only T-ALL and HR pts age > 1 year received prophylactic cranial radiotherapy (12 Gy). Therapeutic cranial radiotherapy was reserved for pts with initial CNS. We performed a descriptive analysis, comparison of frequencies and proportions using Fisher test/Ji2. For time to event proportion we used Cox Analysis. Results 2232 de novo ALL pts were analyzed. Median age 5y (IQR 3-10), 56.3% were males. 2.1% had Down Syndrome; 24% categorized as HR, 56% as IR, and 20% as LR. The Immunophenotype analysis mostly B lineage (88.8%) and less T lineage (11.2%). The majority (96.7%) of pts achieved complete remission. Estimated mean time of survival was 8.8 years (ES 0.099, CI 95% 8.38-8.77) and the overall survival rate was 74 % (0.01). Trial wide, 518 (23.2%) pts died, 59 (2.6%) pts had criteria for clinical sepsis and 459 (20%) for not related sepsis causes. According to phase of the protocol, in less than one month from diagnosis, 21 events (35.6%) were observed; from 2-3 months 9 events (15.3%), from 4-6 months 6 events, (10.2%) and afterwards 23 events (39%) (p-value 0.000). No difference was found on T vs B immunophenotype, sex, age or Down syndrome. (P-value NS). However significant differences were found on risk groups 26/538 pts (4.8%) died on HR, 24/1248 (1.9%) in IR and 9/433 (2.1%) on standard risk (p-value 0.009). There was an association between the rates of sepsis and death on pts randomized to I and II protocols (p-value 0.007 and P-value 0.007). Conclusion In this large cohort of pts, we could demonstrate association between sepsis and mortality in those high-risk groups and in those pts randomized to higher chemotherapy doses. These results will allow to establish strategies to prevent infections or maximize medical interventions preventing poor infectious outcomes in high-risk groups.

Association Between Measurable Residual Levels and Infection Mortality in Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) outcomes have improved over the years. Better AML disease risk classification, achieved by measuring minimal residual disease (MRD) levels, has contributed to improved treatment and survival. Even though the role of MRD in survival is still preliminary, results are promising. However, infections are still a problem to circumvent. In our previous protocol LMA-GATLA 2007 we found a 30% incidence of sepsis related death (SRD), and they were more frequent during or after first induction. The overall survival of that study was 51.4 % (0.38). In this study, we aimed to analyze the mortality rates due to infection; and determine if there is an association of the MRD levels and infection mortality rates; also, we will analyze overall outcomes of AML. Methods Patients were accrued form the National Registry GATLA LMA 2021 protocol. Disease risks were classified based on cytogenetics and molecular abnormalities as standard risk (SR), high risk (HR), and very high risk (VHR). MRD determinations were performed using flow cytometry after 1st induction with mitoxantrone, anthraciclines and etoposide (MAE) protocol, at day 22 and 29 and after 2nd induction with high dose citarabine and mitoxantrone (HAM). We used descriptive statistics (analysis by frequency), compared categorical variables using chi-square or χ2 test and Fisher test, and time to event calculation techniques. Results 95 patients have been accrued, 51.6 % were males. The median age was 9 years of age (IQR 3-13), and 31% were SR, 22% HR, and 47% VHR. MRD levels < 0.1% were seen in 55.5% post first induction, 92% post intensification and 85% post consolidation. Estimated probability of overall survival was 65% (ES 0.6) at 45.7 (2.7) months, (CI 95% 40.3-51,1). 23 pts (24%) died; 11 (48 %) for progression, 7 (30%) for sepsis, for non-sepsis toxicity 3 (13%), other causes 2 pts (9%). Hazard ratio of death with MRD > 0.1% was 2.35 (p=0.06, 95% CI 0.043-1.756). The incidence of SRD was 7.4%, 4 patients had sepsis with MRD < 0.1% (57 %), after HAM /consolidation phase; and 3 patients with > 0.1% (43%) when in relapse (p= 0.028). The SRD episodes were independent from MRD levels, however patients on remission showed SRD as late events, because no patient developed SRD during or after first induction. Conclusion Overall survival results have improved with current protocol. MRD seems a valuable tool to estimate response, levels >0.1 % are associated with increased risk of death. However, better infection prevention and care are required to improve further the outcomes, especially those associated with higher MRD levels.

Delineation of features, responses, outcomes, and prognostic factors in pediatric PDGFRB-positive acute lymphoblastic leukemia patients: A retrospective multicenter study.

PDGFRB fusions in acute lymphoblastic leukemia (ALL) is rare. The authors identified 28 pediatric PDGFRB-positive ALL. They analyzed the features, outcomes, and prognostic factors of this disease. This multicenter, retrospective study included 6457 pediatric patients with newly diagnosed PDGFRB fusion ALL according to the CCCG-ALL-2015 and CCCG-ALL-2020 protocols from April 2015 to April 2022 in 20 hospitals in China. Of these patients, 3451 were screened for PDGFRB fusions. Pediatric PDGFRB-positive ALL accounted for only 0.8% of the 3451 cases tested for PDGFRB. These patients included 21 males and seven females and 24 B-ALL and 4 T-ALL; the median age was 10 years; and the median leukocyte count was 29.8 × 109/L at baseline. Only one patient had eosinophilia. Three patients had an IKZF1 deletion, three had chromosome 5q31-33 abnormalities, and one suffered from a complex karyotype. The 3-year event-free survival (EFS), overall survival (OS), and cumulative incidence of relapse (CIR) were 33.1%, 65.5%, and 32.1%, respectively, with a median follow-up of 25.5 months. Twenty patients were treated with chemotherapy plus tyrosine-kinase inhibitors (TKIs) and eight were treated without TKI. Complete remission (CR) rates of them were 90.0% and 63.6%, respectively, but no differences in EFS, OS, or CIR. Univariate analyses showed patients with IKZF1 deletion or measurable residual disease (MRD) ≥0.01% after induction had inferior outcomes (p<.05). Pediatric PDGFRB-positive ALL has a poor outcome associated with high-risk features. Chemotherapy plus TKIs can improve the CR rate, providing an opportunity for lower MRD levels and transplantation. MRD ≥0.01% was a powerful adverse prognostic factor, and stratified treatment based on MRD may improve survival for these patients. Pediatric acute lymphoblastic leukemia patients with PDGFRB fusions are associated with high-risk clinical features such as older age, high white blood cell count at diagnosis, high measurable residual disease after induction therapy, and increased risk of leukemia relapse. Chemotherapy plus tyrosine-kinase inhibitors can improve the complete remission rate and provide an opportunity for lower measurable residual disease (MRD) levels and transplantation for pediatric PDGFRB-positive acute lymphoblastic leukemia (ALL) patients. The MRD level was also a powerful prognostic factor for pediatric PDGFRB-positive ALL patients.

Risk factors for positive post-transplantation measurable residual disease in patients with acute lymphoblastic leukemia.

The level of measurable residual disease (MRD) before and after transplantation is related to inferior transplant outcomes, and post-hematopoietic stem cell transplantation measurable residual disease (post-HSCT MRD) has higher prognostic value in determining risk than pre-hematopoietic stem cell transplantation measurable residual disease (pre-HSCT MRD). However, only a few work has been devoted to the risk factors for positive post-HSCT MRD in patients with acute lymphoblastic leukemia (ALL). This study evaluated the risk factors for post-HSCT MRD positivity in patients with ALL who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 1683 ALL patients from Peking University People's Hospital between January 2009 and December 2019 were enrolled to evaluate the cumulative incidence of post-HSCT MRD. Cox proportional hazard regression models were built for time-to-event outcomes. Multivariate analysis was performed to determine independent influencing factors from the univariate analysis. Both in total patients and in T-cell ALL or B-cell ALL, pediatric or adult, human leukocyte antigen-matched sibling donor transplantation or haploidentical SCT subgroups, positive pre-HSCT MRD was a risk factor for post-HSCT MRD positivity (P<0.001 for all). Disease status (complete remission 1 [CR1] vs. ≥CR2) was also a risk factor for post-HSCT MRD positivity in all patients and in the B cell-ALL, pediatric, or haploidentical SCT subgroups (P=0.027; P=0.003; P=0.035; P=0.003, respectively). A risk score for post-HSCT MRD positivity was developed using the variables pre-HSCT MRD and disease status. The cumulative incidence of post-HSCT MRD positivity was 12.3%, 25.1%, and 38.8% for subjects with scores of 0, 1, and 2-3, respectively (P<0.001). Multivariate analysis confirmed the association of the risk score with the cumulative incidence of post-HSCT MRD positivity and relapse as well as leukemia-free survival and overall survival. Our results indicated that positive pre-MRD and disease status were two independent risk factors for post-HSCT MRD positivity in patients with ALL who underwent allo-HSCT.

How I treat newly diagnosed acute lymphoblastic leukemia.

Treatment algorithms differ for adult patients with Philadelphia-negative (Ph-) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). For Ph- ALL intensive induction-consolidation chemotherapy using "pediatric-inspired" protocols is a standard of care. Allogeneic hematopoietic cell transplantation (allo-HCT) from either an HLA-matched sibling, unrelated or haploidentical donor should be considered for patients with high estimated risk of relapse. Inadequate response at the level of measurable residual disease (MRD) is the strongest adverse prognostic factor. Patients with B-ALL and detectable MRD should be treated with blinatumomab. In the future, the use of blinatumomab and/or inotuzumab ozogamycin in addition to first-line chemotherapy may become a new standard of care reducing the role of allo-HCT. For patients with Ph+ ALL, tyrosine kinase inhibitors (TKI) are the most important components of treatment protocols, while the intensity of chemotherapy may be reduced. Allo-HCT is recommended for all patients treated with imatinib along with low-intensity chemotherapy. Results of phase-II studies using front-line dasatinib or ponatinib in sequence or in combination with blinatumomab are very promising. Such a strategy may allow the avoidance of systemic chemotherapy. The future role of allo-HCT in this context appears uncertain.

Abstract PO4-13-11: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results in early TNBC, many patients do not respond, and chemotherapy remains the main treatment. High recurrence rates in TNBC are in part due to its inherent molecular heterogeneity and sub-clonal diversity, whereby cells present in minority sub-populations escape therapeutic pressure. Recurrent genomic alterations are not selected for upon therapy resistance in neoadjuvant chemotherapy (NAC) resistant TNBC, however sub-clonal transcriptionally defined cells are “primed” to drive resistance and are pre-existing, suggesting phenotypic heterogeneity is governed by inheritable activation of epigenetically defined regulatory regions. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Driver DNA mutations and copy number alterations (CNA’s) were identified using a clinically validated targeted capture panel. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine meta-clusters across the dataset. A distinct immune-like MC was contributed by epithelial G1 cells, suggesting a cross-talk of epithelial cells with spatially proximal immune cells. Two MCs were dominated by cells from ChemoR patients and were defined by pathways related to hypoxia, EMT, and extra-cellular matrix signalling. Using scATAC profiles, we identified that the sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of distal enhancer chromatin modification in regulating MC transcription. None of the meta-clusters were dominated by tumours with specific genetic mutations or CNA’s. Spatial ATAC and RNA profiling of a subset of tumours identified spatial epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and associated with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommati, Shefali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-11.

Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia.

Minimal residual disease (MRD) based risk stratification criteria for specific genetic subtypes remained unclear in childhood acute lymphoblastic leukemia (ALL). Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group CCLG-2008 protocol, MRD was assessed at time point 1 (TP1, at the end of induction) and TP2 (before consolidation treatment) and the MRD levels significantly differed in patients with different fusion genes or immunophenotypes (P all < 0.001). Moreover, the prognostic impact of MRD varied by distinct molecular subtypes. We stratified patients in each molecular subtype into two MRD groups based on the results. For patients carrying BCR::ABL1 or KMT2A rearrangements, we classified patients with MRD < 10-2 at both TP1 and TP2 as the low MRD group and the others as the high MRD group. ETV6::RUNX1+ patients with TP1 MRD < 10-3 and TP2 MRD-negative were classified as the low MRD group and the others as the high MRD group. For T-ALL, We defined children with TP1 MRD ≥ 10-3 as the high MRD group and the others as the low MRD group. The 10-year relapse-free survival of low MRD group was significantly better than that of high MRD group. We verified the prognostic impact of the subtype-specific MRD-based stratification in patients treated with the BCH-ALL2003 protocol. In conclusion, the subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL.

The impact of varying levels of residual disease following cytoreductive surgery on survival outcomes in patients with ovarian cancer: a meta-analysis

BackgroundResidual disease following cytoreductive surgery in patients with ovarian cancer has been associated with poorer survival outcomes compared with no residual disease. We performed a meta-analysis to assess the impact of varying levels of residual disease status on survival outcomes in patients with ovarian cancer who have undergone primary cytoreductive surgery or interval cytoreductive surgery in the setting of new therapies for this disease.MethodsMedline, Embase, and Cochrane databases (January 2011 – July 2020) and grey literature, bibliographic and key conference proceedings, were searched for eligible studies. Fixed and random-effects meta-analyses compared progression and survival by residual disease level across studies. Heterogeneity between comparisons was explored via type of surgery, disease stage, and type of adjuvant chemotherapy.ResultsOf 2832 database and 16 supplementary search articles screened, 50 studies were selected; most were observational studies. The meta-analysis showed that median progression-free survival and overall survival decreased progressively with increasing residual disease (residual disease categories of 0 cm, > 0–1 cm and > 1 cm). Compared with no residual disease, hazard ratios (HR) for disease progression increased with increasing residual disease category (1.75 [95% confidence interval: 1.42, 2.16] for residual disease > 0–1 cm and 2.14 [1.34, 3.39] for residual disease > 1 cm), and also for reduced survival (HR versus no residual disease, 1.75 [ 1.62, 1.90] for residual disease > 0–1 cm and 2.32 [1.97, 2.72] for residual disease > 1 cm). All comparisons were significant (p < 0.05). Subgroup analyses showed an association between residual disease and disease progression/reduced survival irrespective of type of surgery, disease stage, or type of adjuvant chemotherapy.ConclusionsThis meta-analysis provided an update on the impact of residual disease following primary or interval cytoreductive surgery, and demonstrated that residual disease was still highly predictive of progression-free survival and overall survival in adults with ovarian cancer despite changes in ovarian cancer therapy over the last decade. Higher numerical categories of residual disease were associated with reduced survival than lower categories.

Minor population of NK lymphocytes with CD19 coexpression

Single reports were published concerning a minor subpopulation of NK cells with weak coexpression of the B cell antigen CD19 in the patients’ blood and bone marrow. The frequency and relative number of CD56+CD19+dim cells is virtually not assessed, and there is no data on their phenotypic characteristics, as well as the connection of this subpopulation with any disease state. The purpose of the present study was to assess the frequency, relative quantity and phenotypic characteristics of CD56+CD19+dim lymphocytes in blood of patients referred for assessment of the lymphocyte subpopulation profile. Peripheral blood of immunocompromised individuals was taken, and subpopulation composition of lymphocytes was determined using eight-color flow cytometry (markers: CD3, CD4, CD8, CD19, CD25, CD45, CD56, HLA-DR). To estimate incidence of the CD56+CD19+dim subpopulation, we have carried out a retrospective analysis of LMD files on 1210 studies for 935 patients (average age, 39.8±14.7 years old) including 84 children under 18 years old. The study was performed repeatedly for some patients. Phenotyping of CD56+CD19+dim cells was performed using a panel of antibodies to B cell, T/NK cell antigens. The occurrence of blood samples containing CD56+CD19+dim was 1.2%, with a relative content of 2.1±1.9% among total lymphocyte population (0.8±0.6% of leukocytes). Long-term persistence of the subpopulation was noted in the patients throughout the entire observation period. The comparison of specific marker expression by NK CD56+CD19+dim, and CD56+CD19- cells revealed high expression of CD2, CD57, reduced expression density of CD7, CD16, CD38. The phenotype of the studied NK cell subpopulation was as follows: CD56+dimCD19+dimCD2+brightCD7+dimCD11c+CD16+dimCD38+dimCD45RA+CD57+CD94+dimNKG2D+CD3-CD4-CD5-CD20-CD21-CD25-CD45R0-CD62L-CD79b-CD117-, with variable expression of CD8 and HLA-DR. The phenotype is consistent with activated terminally differentiated adaptive NK associated with cytomegalovirus infection. The individuals with CD56+CD19+dim had a history of CMV-infection and reactivation of chronic EBV-infection at the time of the study. A probable cause of CD19 coexpression may be trogocytosis of B cell membrane fragments by natural killer cells during active EBV-infection. CD56+CD19+dim lymphocytes can reach noticeable values thus altering the results of studies performed by flow cytometry. The errors are most likely to occur upon assessing the minimal residual disease levels in acute B cell leukemias. The minor CD56+CD19+dimNK subpopulation may be detected in routine immunological analysis. Its functional features and association with certain disorders require further studies.

Abstract B059: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results, many patients do not respond, and chemotherapy remains the main treatment. High TNBC recurrence rates are in part due to its molecular heterogeneity. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods: Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results: Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine MC’s across the dataset, including a distinct immune-like MC. Two MCs were dominated by cells from ChemoR patients and were defined by hypoxia, EMT, and extra-cellular matrix signalling. Sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of enhancer regulation of MC transcription. Spatial ATAC and RNA profiling of a subset of tumours identified spatially epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions: Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommatis, Shafali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B059.

Pre-Transplant Levels of Multiparametric Flow-Cytometry Measurable Residual Disease (MFC-MRD) Predict Outcomes in Children with Acute Myeloid Leukemia Given Allogeneic Hematopoietic Stem Cell Transplantation

Background: Measurable residual disease assessed by multiparametric flow-cytometry (MFC-MRD) is largely used in trials for childhood de novo acute myeloid leukemia (AML) in the early phase of treatment, to stratify patients into different risk groups and to tailor treatment. Despite this, unlike acute lymphoblastic leukemia, the impact of MFC-MRD before allogeneic hematopoietic stem cell transplantation (HSCT) on patients' outcomes has yet to be established. The aim of this study was to evaluate the prognostic role of MFC-MRD before HSCT in a cohort of children with AML treated in centers belonging to Associazione Italiana Ematologia/Oncologia Pediatrica (AIEOP) cooperative group. Patients and Methods: We analyzed MFC-MRD in 208 pediatric patients affected by de novo AML who underwent a first allo-HSCT after a myeloablative conditioning regimen in an AIEOP Centre between 12/2011 and 12/2021. Assessment of MFC-MRD was performed on BM sample within 60 days from the transplantation date. Analysis of 5-10 color-panel MFC-MRD was centrally performed at the Laboratory of Diagnosis and Research, University of Padova, according to standardized operating procedures previously described (Buldini et al., BJH 2017). We defined MFC-MRD positivity as the presence of at least 50 leukemic events of 500,000 acquired nucleated cells. Five-year overall (OS) and event-free survival (EFS) were estimated using Kaplan and Meier method and differences between groups were tested using the log-tank test; the cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated using the Fine&amp;Gray method to take into account the respective competitive risks. Results: Median age at HSCT of these 208 patients was 7.8 years (range 0.4-23.3); 168 patients (80.8%) were transplanted in CR1, 30 (14.4%) in CR2 and 10 (4.8%) in CR3 or more advanced disease. Forty-eight patients (23.1%) were transplanted from a matched family donor, 85 (40.9%) from an unrelated donor (59 fully-matched and 26 mismatched), while 75 (36%) from a partially matched family donor. The source of stem cells was bone marrow in 117 patients (56.2%), peripheral blood in 81 (39%) and cord blood in 10 (4.8%). Median time from MRD evaluation and HSCT was 22 days (range, 6-57). Overall,168 out of 208 (78.9%) patients were MFC-MRD negative in the pre-HSCT BM, while 44/208 (21.1%) showed a positive MFC-MRD. Among those, MFC-MRD was &amp;lt; 0.1% in 12/44 (27%) patients, 0.1-&amp;lt;1% in 16 patients (36.5%) and ≥ 1% in 16 patients (36.5%). The 5-year OS and EFS of the whole cohort were 79.2% and 65.7%, respectively, while CIR and NRM were 24.7% and 8.6%. Notably, patients with negative MFC-MRD had a significantly better 5-yr OS and EFS (Fig.A) as compared to those with any MFC-MRD positivity (OS 86.3% vs 50.2%; EFS 71.6% vs 42.4%, p &amp;lt;0.0001 in both analysis). CIR significantly differed between patients with MFC-MRD negative and positive (21.1% vs 43.9%, p=0.001) (Fig.B). We also observed a higher NRM in patients with MFC-MRD positivity (13.6%) as compared to those with negative MFC-MRD (7.2%), although this was not statistically significant (p=0.1). Disease status (CR1 vs CR2 vs other CR) did also influence EFS (69.9% vs 55.6% vs 23.3%, p&amp;lt;0.001), OS (83.2% vs 66.6% vs 40.6%, p=0.02) and CIR (21.1% vs 37.7% vs 66.6%, p&amp;lt;0.001), while other variables (including year of HSCT, type of donor and stem cell source employed) did not. In a multivariable model for OS, EFS and CIR, including MFC-MRD, disease status and year of HSCT, MFC-MRD (pos vs neg) remained statistically significant (HR for OS 3.5, p=0.0004; HR for EFS 2.38, p=0.003; HR for CIR 2.14, p=0.02). In addition, advanced disease phase was an independent risk factor for CIR (HR 1.81, p=0.008). Conclusion: These data document the independent prognostic value of MFC-MRD before HSCT in a large cohort of pediatric AML patients, using a centralized evaluation by 5-10 color MFC. Patients with negative MFC-MRD before HSCT have a significantly lower risk of treatment failure. In light of these results, we recommend monitoring MFC-MRD before HSCT in pediatric AML patients as a critical tool for therapy-intervention decision before or after HSCT (e.g., additional treatment for improving leukemia control or less intensive/shorter GvHD prophylaxis) with the aim to improve patients' outcomes.

Single Cell Transcriptomic Analysis of B-Cell Acute Lymphoblastic Leukemia (B-ALL) Reveals Correlation between Leukemia Cell State, Genetic Alterations and Clinical Outcome

I.I., A.G.X.Z, Q.G., L.G.P.: co-first authors; J.E.D., C.G.M.: co-senior authors Introduction: Genomic analyses of bulk ALL samples have improved our understanding of the genetic basis and risk stratification of B-ALL, but do not directly examine intratumor heterogeneity or enable inference of leukemia developmental state and cell of origin. Methods: We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) (10X Genomics 5'v2) and compared them to a scRNA-seq reference map of normal human B-cell development. Results: Analysis of heterogeneity of inferred DNA copy number alterations at single cell level showed that aneuploid ALL with near haploidy and hyperdiploid harbor chromosomal losses or gains in all blasts, consistent with early, synchronous origin rather than sequential accumulation. A subset of TCF3::PBX1 samples harbored clones with progressive overexpression of genes surrounding PBX1 which scWGS confirmed caused by accumulation of DNA gains. Extensive intra-sample heterogeneity was observed by non-negative matrix factorization and driven by five consensus gene expression signatures: cell cycle (S; G2/M), metabolism, differentiation and inflammation. Signature composition refined leukemic subtyping; for example, within DUX4-r B-ALL two groups were discerned: one with higher inflammation score, expression of stem cell genes and worse outcome and one with lower inflammation score and expression of Pro-B related genes. To understand variation in developmental states among B-ALL blasts, we first developed an atlas of human B cell development. Marker genes elucidated from bulk RNA-seq on purified human HSC, MPP, LMPP, MLP, CLP, Pre-Pro-B, Pro-B, Pre-B, and B populations guided the development of a new scRNA-seq atlas of human B cell development comprising 130,085 cells spanning 90 fetal, pediatric, and adult samples from 8 studies (Fig 1A). Unexpectedly, we found that human CLPs retain transcriptional programs governed by CEBPA; in vitro and in vivo functional assays validated the capacity of CLPs for extensive myeloid differentiation prior to lineage restriction at the Pro-B stage. We next mapped our 89 B-ALL sc samples to precise cellular states along B cell development and clustered samples by leukemia cell composition. As expected, most samples exhibited similarity of GEP to that of pro-B cells, and this was most pronounced among hyperdiploid samples. High pre-B abundance was observed in MEF2D-r and TCF3::PBX1, while an HSC/MPP/LMPP enriched cluster encompassed ZNF384-r and a subset of DUX4-r B-ALL. Notably, one ZNF384-r B-ALL patient with LMPP involvement at diagnosis retained these LMPP-like cells at relapse despite a lineage switch to AML. Finally, we identified a group of patients with high early lymphoid (MLP, CLP, Pre-Pro-B) abundance representing a subset of Ph+ and KMT2A-r samples (Fig. 1B). We next utilized marker genes from each B-ALL cell state to estimate their relative abundance within a bulk RNA-seq cohort of 2046 B-ALL patients. In addition to validating these associations between genomic class and leukemia cell state, we identified age-dependent patterns of B-ALL state involvement wherein early lymphoid involvement is highest in infancy and adulthood while Pro-B involvement is highest in childhood. In childhood, high-risk disease is associated with higher early lymphoid ( P=4.9e-12) and lower Pro-B ( P=0.0085) abundance. Further, patients with residual disease (RD) levels above 1% are enriched for higher upfront early lymphoid abundance ( P=1e-7) and lower Pro-B ( P=4.1e-5) and Pre-B ( P=2.2e-11) abundance compared to patients with RD &amp;lt; 0.01%. Finally, B-ALL cell state improved resolution of previously reported transcriptional heterogeneity within defined genomic classes. Early lymphoid abundance discerns between two subtypes within Ph+ (Kim Nat Gen 2023) with AUC=0.953 ( P=2.4e-9), as well as two subtypes within KMT2A-r (Brady Nat Gen 2022) with AUC=0.996 ( P=1.1e-6). Notably we find that KMT2A::AFF1 fusions produce early lymphoid-enriched disease compared to KMT2A::MLLT3 ( P=0.0024) and KMT2A::MLLT10 ( P=0.0037), wherein the latter two fusions exhibit Pre-B enrichment ( P=0.025 and P=0.0037, respectively). Conclusions: Understanding variation in transcriptional programs and developmental states of B-ALL blasts by sc transcriptome refines existing clinical and genomic classifications and provides novel prognostic markers.

Impact of Sequence Uniqueness on MRD Monitoring in NGS Immunoglobulin Sequencing: An Analysis of Ig Loci Among >1200 Diffuse Large B-Cell Lymphoma Patients Tested By Clonoseq

Introduction Diffuse Large B-Cell Lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma that most commonly affects older adults. While ~80% of patients achieve complete response to frontline therapy, roughly half are cured while the other half relapse and receive salvage treatment such as transplant or CAR-T therapy, either of which may be curative. Significant efficacy in later lines has been demonstrated with the recently approved bispecific therapies. Due to the aggressiveness of DLBCL and currently available therapies that offer a potential cure, there is growing interest in a monitoring tool that provides additional information to standard disease monitoring where clinical imaging may fall short. clonoSEQ Ò is a next-generation sequencing CLIA LDT which monitors measurable residual disease (MRD) levels from ctDNA of DLBCL patients Methods The clonoSEQ assay sequences the V(D)J regions (specifically, the CDR3) of Ig loci. A baseline (calibration) sample containing high tumor burden is sequenced to determine the clonotypic/dominant sequence(s). The clonoSEQ algorithm assigns a uniqueness score to each dominant sequence based on analyses of Ig locus V, D, and J segments and non-templated nucleotides. This score reflects the probability a that sequence could be independently recreated in a non-malignant cell, including in another person, and is incorporated into the limit of detection (LOD). Consequently, sequences with a lower uniqueness must be observed at a higher rate to be above the LOD and reflect confidence they are tumor related in subsequent MRD samples. Previous reports have explored the uniqueness distribution of Ig loci, but did not consider the impact of LOD on report interpretation. Using data from our database, we assessed ID sample calibration rates and Ig loci uniqueness among patients with orders from the clinical offering and from clinical trials. Our database contains 3657 dominant sequences from 1268 patients. 969 of these patients are from 27 clinical studies, and 302 are from use of the commercially available CLIA LDT assay. Results: Overall, 1535 ID samples have been received and included several different sample tissues and preparation methods. 1285 (83.7%) of these calibrated. Calibration was most successful in FFPE and gDNA preparations from lymphatic tissue or extranodal masses with 806/883 (91.3%). Least successful sample types included whole blood (48/106 [45.3%] calibrated) and skin (9/28 [32.1%] calibrated) which is likely a reflection of disease burden not being present in these tissues among a large proportion of the patients tested. For samples run on the clinical offering where the sample requirements are more stringent, the most common and most successfully calibrated sample type is FFPE lymph node tissue with a calibration rate of 95.2%. Across the population of 1268 DLBCL patients and 3657 dominant sequences, there was a range of 1-9 dominant sequences (avg 2.89; med 3). We assessed the distribution of uniqueness across the Ig loci in these patients. Fig 1a reflects a higher median uniqueness of IgH (VJ), IgH (DJ), and IgL clonotypes relative to IgK. Fig 1b indicates the uniqueness score for each patient with only one dominant sequence, with just 31 (2.4%) of all subjects with expected ‘backgrounds’ of &amp;gt; 1/100,000 of all Ig sequences. Conclusions Our analysis of &amp;gt;1200 DLBCL patients demonstrates that identification of dominant Ig sequences is more successful using lymph tissue and that the majority of these patients rely on multiple dominant sequences for disease tracking. In a small subset of patients (2.4%), disease is only trackable using one lower uniqueness sequence. In these cases, the LOD is particularily informative in providing guidance to contextualize the probability the sequence may not be associated with the disease being monitoried.

Role of Allogeneic Stem Cell Transplantation in Preventing Relapse in Adult BCR::ABL1-like Acute Lymphoblastic Leukemia

Background Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by the activation of several kinase pathways that promote treatment resistance leading to poor survival of patients at all ages. Despite the use of tyrosine kinase inhibitors in the Ph-like ABL-class subtype (15% Ph-like ALL), there is not a consensus approach for treatment of most patients (JAK-class especially), and the role of allogeneic stem cell transplantation (alloSCT) has scarcely been explored so far (i.e., mandatory vs. MRD-oriented). Objective To assess the outcome of Ph-like patients enrolled in the ongoing ALL19 trial from the Programa Español de Tratamientos en Hematología (PETHEMA) for adults (18-60y) with Ph-negative ALL, and to evaluate the effectiveness of alloSCT in first complete remission (CR). Methods In the ALL19 trial, patients are allocated to alloSCT according both to the end of induction (EOI) measurable residual disease (MRD) level and to high-risk genetic markers such as low-hypodiploidy, KMT2A rearrangement, TP53 biallelic alteration and concomitant deletion of IKZF1 and CDKN2A/B. Therefore, BCR::ABL1-like subtype was not a criteria for early transplantation unless meeting either poor MRD clearance at EOI or concomitant deletion of IKZF1+CDKN2A/B. Patients with MRD≥0.01% on day+35 (EOI) and/or high-risk genetics were assigned to early alloSCT (preceded by 1 cycle of consolidation chemotherapy) while those with good MRD clearance and standard risk genetics received early and delayed consolidation (3 cycles each) and maintenance therapy. Bone marrow or peripheral blood samples were analyzed by G-banding+FISH, SNP array (750K Affymetrix, Thermo Fisher) and next generation sequencing (NGS, with a custom DNA gene panel, Illumina) at diagnosis in 4 reference laboratories. MRD was centrally assessed by next generation flow cytometry with a sensitivity up to 2x10 -6. Results Twenty-four Ph-like out of 248 adult BCP ALL (10%) were detected. Baseline characteristics were similar between Ph-like and the remaining BCP-ALL. Most Ph-like ALL patients showed the JAK-class profile (20/24 [83%]: 17/24 CRLF2 rearrangement[r], 1/24 JAK2r, 1 BLNK::DNTT and 1 JAK2 R683 mutation), 3/24 belonged to the ABL-class subtype (2 ABL2r, 1 NUP214::ABL1) and 1 patient was identified by RNAseq (gene expression profiling similar to BCR::ABL1 ALL) without identifying any rearrangement. Regarding secondary genetic alterations, 17/24 (71%) patients showed the IKZF1 plus profile, 10/23 (43%) harbored JAK2 R683 mutation, 4/23 N/KRAS mutations, 2/23 CRLF2 mutation and 1/23 showed mutations in several genes such as IKZF1, NR3C1 or FLT3, among others. There were no significant differences in the probability of achieving CR between Ph-like and the remaining BCP ALL (15/20 [75%] vs. 137/162 [85%], p=0.334). However, EOI MRD level of Ph-like individuals was significantly poorer (MRD&amp;lt;0.01% Ph-like 4/15 [27%] vs. other BCP-ALL 85/136 [63%], p=0.007). By intention to treat, most Ph-like patients (17/19, 89%) were allocated to alloSCT (vs. 91/153 [59%] in the other BCP-ALL, p=0.011) due to either EOI MRD≥0.01% (9/18), IKZF1+CDKN2A/B concomitant deletions (3/18) or both (6/18). The 2-y overall survival (OS) in both groups was not significantly different (57% [95% CI, 27%-78%] vs. 67% [55%-77%, p=0.304]) (Figure 1). Interestingly, most Ph-like patients died due to treatment-related toxicity (2 transplant-related mortality, 1 CART-related, 1 during induction, 1 in CR and 1 by COVID-19 infection). The 2-y cumulative incidence of relapse (CIR) was also similar (Ph-like 27% [8%-50%] vs. other BCP-ALL 35% [25%-45%], p=0.946) (Figure 1). Conclusions Despite the short follow up of this series, our results show that Ph-like ALL patients have poorer EOI MRD clearance and higher rate of alloSCT realization than the remaining BCP ALL patients, without significant differences in outcome. This suggests that early alloSCT in first CR might overcome the poor prognosis of Ph-like ALL patients. Funding Funded in part by: PI10/01417 y FIS PI19/01183 Ministerio de Salud Carlos III, JCyL_SA118P20, FMM21/002_AP176752021, “La Caixa” Foundation, Pfizer Spain, Fundación PETHEMA. This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No IMI001-07. The JU receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA.

Frontline Consolidation with Nelarabine for Adults with High-Risk T-Cell Acute Lymphoblastic Leukemia. Results of the Graall-2014/T Atriall Phase 2 Study

Rationale. Acute lymphoblastic leukemia (ALL) with unfavorable biology and/or high minimal residual disease (MRD) levels are at high-risk of disease recurrence. Based on the results of the GRAALL-2005/T study (Trinquand A et al, JCO 2013; Beldjord K et al, Blood 2014), T-ALL patients enrolled in the GRAALL-2014/T trial were classified at high-risk (HR) if presenting an unfavorable 4-gene classifier (absence of NOTCH1/FBXW7 mutations or presence of RAS/PTEN alterations) and/or a post-induction IG/TR MRD1 ≥ 10 -4 (including the need of a salvage course to reach a complete remission [CR]). In the phase 2 ATRIALL sub-study, HR patients were offered to receive nelarabine (NELA) during consolidation and maintenance or as bridge to allogeneic hematopoietic stem cell transplantation (HSCT). The aim of the present report is to compare their outcome to that of similar patients treated in the same GRAALL-2014/T trial but without frontline NELA. Method. Between November 2015 and May 2022, the GRAALL-2014/T included 325 patients with T-ALL. Among these patients, 199 presented with HR features. The ATRIALL sub-study was activated in April 2017. ATRIALL eligibility criteria were defined as HR features, no CNS involvement at baseline, and continuous CR before to start consolidation 2 (first NELA course) at week 12. Based on the results of the COG phase 3 study, the protocol was amended in January 2021 to include patients with CNS involvement at baseline. NELA was given at 1,500 mg/sqm/day at day 1,3 and 5 in combination with etoposide and cyclophosphamide for a maximum of 5 courses during consolidation (2 cycles) and maintenance (3 cycles). Patients with MRD1 ≥ 10 -3 and/or with post-consolidation 1 MRD2 ≥ 10 -4 were eligible for HSCT after one NELA course. A total of 121 patients were included. One patient was excluded due to overt relapse before to start NELA. Eight patients had CNS involvement at baseline. A total of 33 patients included in the GRAALL-2014/T met initial ATRIALL criteria but were not included in the sub-study either before ATRIALL activation (N=21), or during accrual (N=10), or after accrual completion (N=2) and received standard of care (SOC) without NELA. The ATRIALL/CNS- cohort (N=112) was compared to the control cohort (N=33) for MRD response, cumulative incidence of relapse (CIR), disease-free survival (DFS) and overall survival (OS). Results. The median follow-up in the ATRIALL study (n=120) was 2.9 years (95%CI, 2.5-3.5). At 3 years, the CIR, DFS, and OS were 28% (95%CI, 20-37), 67% (95%CI, 55-76), and 71% (95%CI, 61-79), respectively. Patients from the ATRIALL/CNS- (N=112) and control (N=33) cohorts exhibited similar characteristics, except for a longer median follow-up in the control cohort (Figure A). Patients included in the ATRIALL study reached lower post-consolidation MRD3 levels (p=0.05). Among patients with MRD1 ≥10 -4, undetectable MRD3 was achieved in 36/55 (65%) after NELA versus 5/16 (31%) after SOC (p=0.02). When censoring at HSCT time, NELA was associated with a significantly reduced CIR (SHR=0.48; 95%CI, 0.23-0.99; p=0.045) and a trend for a prolonged DFS (HR=0.52; 95%CI, 0.26-1.07; p=0.075) (Figure B,C). This difference did not reach significance when patients were not censored at HSCT time. Indeed, high-risk patients not eligible for HSCT benefited from NELA in terms of CIR (SHR=0.43; 95%CI, 0.20-0.98; p=0.045) and DFS (HR=0.46; 95%CI, 0.21-1.03; p=0.06). Patients with non-ETP ALL had a significantly improved CIR (SHR=0.03; 95%CI, 0.17-0.89; p=0.025) and DFS (HR=0.47; 95%CI, 0.22-0.99; p=0.048), whereas ETP-ALL patients did not benefit from NELA over SOC. There was no benefit of prior NELA in patients who received alloHSCT per protocol. Conclusion. While NELA did not yield an overall improved outcome in the study population, benefits were observed in favorable MRD responders and non-ETP patients. Additional prospective studies are needed to further delineate the specific patient subgroups that might benefit from NELA.

CRLF2 Expression in Mexicans with Acute Lymphoblastic Leukemia across the Age Spectrum

Introduction Acute lymphoblastic leukemia (ALL) is a hematological malignancy that primarily affects children also occurs in adults. Certain subtypes of ALL, such as Philadelphia chromosome-like ALL (Ph-like ALL), present unique challenges and are associated with poor outcomes. Alterations in the CRLF2 (Cytokine Receptor-like Factor 2) gene have emerged as critical contributors to the pathogenesis of Ph-like ALL. CRLF2 gene rearrangements result in the overexpression of CRLF2 protein, leading to dysregulated signaling pathways and enhanced leukemogenesis. The prevalence and clinical implications of CRLF2 overexpression in Mexicans with ALL remain underexplored. Hence, this study aimed to investigate the prevalence of CRLF2 overexpression in Mexican patients with ALL across different age groups. Patients and Methods: We conducted a prospective study on a cohort of ALL patients from a single institution in Mexico that cares for patients across the age spectrum. Consecutive patients with a diagnosis of ALL were enrolled in the study and were assessed for CRLF2 overexpression using flow cytometry and fluorescent in situ hybridization (FISH). Patients were treated with a local BFM-based chemotherapy regimen according to age and risk stratification. In adolescents and young adults ≥16, a pediatric-inspired regimen was used and in patients with positive measurable residual disease (MRD) allogeneic hematopoietic cell transplantation was sought. To determine the impact of CRLF2 overexpression on clinical characteristics and treatment response, we analyzed various parameters, including response rates, MRD, and survival. Statistical analyses were performed using SPSS 25, encompassing descriptive analysis, chi-squared tests, and survival analysis with the Kaplan-Meier method. Results: A total of 79 patients were enrolled with a median age of 17 years (2-70 years), 34 were female and 45 males with a median white blood cell count of 10.1x103/L (.63-649.57). Our findings revealed that 21 out of 79 patients (26.6%) exhibited CRLF2 overexpression. The proportion of CRLF positive ALL increased with age; it was 20.5% in patients aged 0-16 years, 34.6% in 17-39 and 40% in &amp;gt;60 years. There were no statistically significant differences observed in white blood cell counts, central nervous system involvement, response to treatment, or minimal residual disease levels in CRLF2 positive or negative patients (Table 1). In the Kaplan-Meier survival analysis, it was observed that positive CRLF2 expression was significantly associated with lower overall survival (OS) with a median of 24.9 months (0.2-65.4 months); with 85.8% OS at 2 years in CRLF2 negative vs 41.2% in CRLF2 positive patients (Figure 1). Event-free survival rate at 2 years was also lower with 70.8% vs 35.3% at 2 years in CLRF2 positive patients. Moreover, this association was found to be consistent across all age subgroups, including patients aged 0-16, 17-39, and over 40 years (P &amp;lt; 0.05). Conclusion: Our study highlights the significance of CRLF2 overexpression as a cost-effective biomarker indicative of an unfavorable prognosis in ALL across the age spectrum. Early detection of this alteration through flow cytometry plays a vital role in risk stratification and should prompt the implementation of tailored therapeutic interventions to improve clinical outcomes. Additionally, our study underscores the importance of conducting population-specific research to gain comprehensive insights into the genetic landscape and disease characteristics of ALL in different ethnic groups.

Using Peripheral Blood (PB) Measurable Residual Disease (MRD) Levels to Predict

CONCLUSIONS Time-limited treatments for CLL are under investigation but the optimal target for treatment cessation is not fully clear. Bone marrow (BM) measurable residual disease (MRD) is usually the most sensitive measure but would be too invasive to guide treatment in a routine setting. The iwCLL threshold of BM uMRD4 (&amp;lt;0.01%) has been demonstrated to be a powerful indicator of improved outcomes across multiple different trials and the aim of this analysis is to determine the feasibility of using PB MRD analysis to identify patients who have attained BM uMRD4. FLAIR (ISRCTN01844152) is an open-label, randomised, controlled, phase 3 trial comparing ibrutinib plus rituximab (IR) with fludarabine, cyclophosphamide and rituximab (FCR), subsequently amended to compare ibrutinib plus venetoclax (I+V) and ibrutinib alone (I) with FCR. Paired PB &amp; BM samples were scheduled at: (1) response assessment 9 months after randomization in all arms (n=1086); (2) end of treatment after 72 months in the ibrutinib-containing arms (n=137, most I/I+V participants have not reached this timepoint yet); and (3) confirmation of BM uMRD4 for initiation of planned stopping rules in participants with sustained PB uMRD4 defined as &amp;lt;0.01% PB MRD at 3 timepoints over 6 months (n=188 in the I+V arm). MRD analysis was performed using an ERIC-compliant 8-colour flow cytometry assay targeting acquisition of 2.2 million events (detection limit MRD5/0.001%). As the target could not always be met due to sample/laboratory limitations, stringent uMRD4 assessing 500 thousand leucocytes (detection limit 0.005%) was also evaluated. Table 1 shows the BM vs. PB MRD levels for different treatment arms and timepoints. The greatest discrepancy was observed in participants receiving FCR with a median 0.8log higher disease in the BM vs. PB. Participants receiving ibrutinib monotherapy had similar PB and BM MRD levels while the addition of either rituximab or venetoclax was associated with slightly higher (median &amp;lt;0.1 log) BM vs. PB MRD levels for both IR and I+V arms. The difference did not persist in the IR arm but remained during I+V treatment in some cases, with median &amp;gt;0.59 log higher BM vs. PB MRD levels in 29/182 participants achieving PB uMRD4 but with detectable BM MRD. Table 1 also shows the proportion of cases that attain BM uMRD4 according to different PB MRD thresholds. For participants with rituximab exposure in the past 3 months, BM uMRD4 was attained in 75-88% of participants with PB uMRD5, and only 43-76% of participants with stringent uMRD4. For participants with PB dMRD5 (0.001-0.01% MRD), only 14-52% achieved BM uMRD4. However, participants on I+V with either PB uMRD5 or stringent uMRD4 attained BM uMRD4 in 94-96% of cases. Planned cessation of treatment in the FLAIR trial was guided by sustained PB uMRD4, defined as 3 successive results with &amp;lt;0.01% PB disease over six months with confirmation of BM uMRD4 at the final time point. This strategy in the I+V arm is highly effective in achieving improved progression-free and overall survival compared to FCR [see related abstract]. Sustained PB uMRD4 led to BM assessments in 188 participants of which 173/188 (92%) attained BM uMRD4. High sensitivity PB MRD gave concordant results to BM MRD4 analysis in 91% of participants (172/188), while 9/188 (5%) had PB dMRD5 / BM uMRD4 (7/9 had detectable BM MRD in the range 0.002-0.007% and 2/9 had BM uMRD5/&amp;lt;0.001%) and 7/188 (4%) had PB uMRD5/stringent uMRD4 but with persistent BM dMRD4 (median 0.02% BM MRD, range 0.01-0.055%). PB MRD level correlates closely with BM MRD level but there is lower concordance at the 0.01%/MRD4 threshold after recent cessation of therapeutic antibody. PB uMRD4 is not a suitable target for treatment cessation as the majority of patients with 0.001%-0.01% PB MRD have &amp;gt;0.01% BM disease. For the identification of people attaining BM uMRD4 (&amp;lt;0.01%) on I+V treatment, sustained PB uMRD4 was &amp;gt;90% effective. For translation into MRD-guided treatment in future routine clinical practice, BM assessment may be replaced with PB MRD monitoring if the 0.001%/MRD5 threshold is applied.

Prognostic Role of Molecular MRD Variations during Treatment of Pediatric AML: A Retrospective AIEOP AML2013/01 Study

Background. Measurable residual disease (MRD) is a key parameter to evaluate response to cytotoxic treatment in leukemia patients and, thus, to guide therapeutic strategies. In fact, the identification of persisting leukemia cells after chemotherapy is associated with an increased risk of disease recurrence, and many patients with acute myeloid leukemia (AML) experiencing relapse have a fatal outcome. Many somatic genetic aberrations are under investigation for assessing molecular quantitative MRD (qMRD). However, while flow cytometry is already recognized as useful in measuring residual disease levels, the impact of qMRD monitoring is still debated. Methods and Results. We set up a qPCR assay to quantify mRNA fusion or mutated transcripts for 23 AML rearrangements identified at diagnosis in 247 patients (67% of the whole population) enrolled in our recently concluded AIEOP-AML2013/01 trial, with a sensitivity reaching 10 -5/-6. We measured qMRD after the induction therapy courses (n=202) and identified 66 cases with qMRD&amp;gt;1x10 -3 (cut-off determined by ROC curve method, qMRD with respect to disease levels found at disease onset) and 136 with qMRD&amp;lt;1x10 -3 after the II induction course. Patients with qMRD&amp;gt;1x10 -3 had a significantly worse OS and EFS with respect to patients with qMRD&amp;lt;1x10 -3 (77% vs 89%, p=0.016 and 61% vs 78%, p=0.016, respectively). qMRD assessment within each AML risk group, namely Standard (SR, n=64), Intermediate (IR, n=38) or High (HR, n=101), showed that the persistence of qMRD&amp;gt;1x10 -3 after the II induction course in SR patients did not correlated with worse EFS with respect to patients who reduced qMRD levels below the cut-off, mainly due to NPM1 mutated patients all relapsing (7/7) with qMRD&amp;lt;1x10 -3 after the second induction course. By contrast, qMRD&amp;gt;1x10 -3 was a predictive variable able to discriminate, among the patients assigned to the IR (EFS 46% vs 81% for IR, p=0.009) those children with poorer outcome. For the HR patients, qMRD 1x10 -3 predicted a worse, although not statistically significant, outcome (61% vs 80% for HR, p=0.08). When we interrogated qMRD role within the different genetic subtypes, we showed that qMRD significantly impacted on EFS of patients harboring t(8;21) RUNX1-RUNX1T1 (47% for qMRD&amp;gt;1x10 -3vs 88% for qMRD&amp;lt;1x10 -3, p=0.004), this finding corroborating results previously published by our group. A similar trend, although not significant, was also obtained for patients harboring isolated FLT3-ITD mutation, who had a dismal EFS when qMRD levels after the II induction course were above 1x10 -3 (40% (n=5) vs 71% (n=7) for qMRD&amp;lt;1x10 -3, p=ns). qMRD reduction for KMT2A-rearranged AML cases and for other genetic markers having a lower incidence in AML, needs to be evaluated in larger cohorts to obtain robust results. Additionally, we interrogated if qMRD maintains the same clinical relevance when measured after the III or IV chemotherapy blocks. We calculated proper cut-off for this analysis by ROC curve method, finding again 1x10 -3 for the third chemotherapy cycle; we observed that AML cases with qMRD&amp;gt;1x10 -3 had a worse EFS with respect to patients with lower levels of qMRD (39% n=30 vs 80% n=82, p&amp;lt;0.0001). As concerns the IV chemotherapy block, we calculated an appropriate cut-off corresponding to 1x10 -4, and once again we confirmed that patients with qMRD above the cut-off displayed a lower probability of experiencing adverse events (EFS 49% n=46 vs 85% n=65, p=0.0003). In conclusion, this is the first study that considers a serial and continuous qMRD monitoring all along an entire trial demonstrating a significant prognostic role of qMRD after II, III and IV therapy courses. These findings demonstrate that qMRD represents a useful tool in supporting therapeutic decisions and suggest that harmonization of currently used molecular MRD detection methods is the best solution to face MRD challenge in pediatric AML. Further comparisons between molecular and cytofluorimetric methods will increase the chances to identify those patients at high risk of relapse, despite negative MRD measured by flow cytometry.

Characteristics of Molecular Genetic Mutations and Their Correlation with Prognosis in Adolescent and Adult Patients with Acute Lymphoblastic Leukemia

Introduction: The prognosis of acute lymphoblastic leukemia (ALL) in adolescents and adults is poor, and recurrence is an important cause of their death. Changes of genetic information play a vital role in the pathogenesis and recurrence of ALL; however, the impact of molecular genetic mutations on disease diagnosis and prognosis remains unexplored. This study aimed to explore the frequency spectrum of gene mutations and their prognostic significance, along with the minimal residual disease (MRD) level and hematopoietic stem cell transplantation (HSCT), in adolescent and adult patients aged ≥15 years with ALL. Methods: The basic characteristics, cytogenetics, molecular genetics, MRD level, treatment regimen, and survival outcome of patients with untreated ALL (≥15 years) were collected, and the correlation and survival analysis were performed using the SPSS 25.0 and R software. Results: This study included 404 patients, of which 147 were selected for next-generation sequencing (NGS). NGS results revealed that 91.2% of the patients had at least one mutation, and 67.35% had multiple (≥2) mutations. NOTCH1, PHF6, RUNX1, PTEN, JAK3, TET2, and JAK1 were the most common mutations in T-ALL, whereas FAT1, TET2, NARS, KMT2D, FLT3, and RELN were the most common mutations in B-ALL. Correlation analysis revealed the mutation patterns, which were significantly different between T-ALL and B-ALL. In the prognostic analysis of 107 patients with B-ALL, multivariate analysis showed that the number of mutations ≥5 was an independent risk factor for overall survival and the RELN mutation was an independent poor prognostic factor for event-free survival. Discussion: The distribution of gene mutations and the co-occurrence and repulsion of mutant genes in patients with ALL were closely related to the immunophenotype of the patients. The number of mutations ≥5 and the RELN mutation were significantly associated with poor prognosis in adolescent and adult patients with ALL.