B-cell Precursor Acute Lymphoblastic Leukemia Research Articles

Optimized Method for Cell Surface Protein Identification on Primary B-Cell Precursor Acute Lymphoblastic Leukemia Cells

Optimized Method for Cell Surface Protein Identification on Primary B-Cell Precursor Acute Lymphoblastic Leukemia Cells

The Role of Mitochondrial RNA from B-Precursor Acute Lymphoblastic Leukaemia Cells in Generating a Chemoprotective Bone Marrow Niche

Treatment resistant cells in precursor B-cell Acute Lymphoblastic Leukaemia (B-ALL) likely reside in a protective bone marrow niche with nestin-expressing mesenchymal stromal cells (MSC) (Duan et al Cancer Cell 2014). Previous work from our laboratory demonstrated that an 'activated MSC' niche, in which MSC have acquired features of cancer associated fibroblasts (CAF), develops in response to reactive oxygen species (ROS)-inducing chemotherapy. Mitochondria are actively transferred along tunneling nanotubes from activated MSC to B-ALL cells and can 'rescue' the cells from chemotherapy (Burt, et al Blood 2019). However, work from other groups suggests that chemoresistant ALL sub-clones exist prior to chemotherapy exposure, carrying a gene expression signature enriched for stemness, mitochondrial metabolism, stress responses and chromatin remodelling (Dobson et al Cancer Discovery 2020). We hypothesised that B-ALL cells may themselves directly activate bone marrow stromal cells and induce niche-formation prior to treatment, leading to ab-inito chemoresistance. To test this hypothesis, we first used our previously-described, in vitro niche model wherein either the HS27a MSC cell line or healthy donor MSCs were co-cultured with either B-ALL cell lines or primary patient B-ALL cells. Three of the five B-ALL cell lines and three of the four primary B-ALL samples that were tested induced activation of the MSCs, as characterised by cytomorphological changes identified by confocal microscopy, up-regulation of CAF-relevant gene expression and increased IL6, IL8 and CCL2 protein secretion. The ability of the B-ALL cell lines to activate MSC correlated directly with the intrinsic ROS level of the cell as determined by CellROX assay. Mitochondrial transfer from the cell-activated MSC to B-ALL cells, as measured by mitotracker dye transfer, occurred exactly as we had seen when we had used chemotherapy to activate the stroma. In vivo, the MSC-activating ALL cell lines also activated murine MSCs when established as xenografts in NOD-SCID gamma (NSG) mice. Immunohistochemistry of NSG murine femora revealed a very significant expansion in nestin+ cells after exposure to niche-induing ALL cells compared to non-niche-inducing controls. We aimed to determine the mechanism behind the B-ALL induced MSC activation. We determined that it was contact-independent, arising from exposure of MSC to ALL cells in transwell but also from exposure to B-ALL cell conditioned media alone. RNA sequencing of MSC after treatment with conditioned medium from activating B-ALL cells compared with non-activating controls unexpectedly showed that most highly upregulated pathways in the MSC-activating scenario were those involved in RNA sensing. Thus, we sought evidence for the role of mitochondrial dsRNA, as it had been recently shown to activate the anti-viral signalling pathway (Dhir et al, Nature 2018). Using the J2 anti-double stranded (ds) RNA monoclonal antibody in both confocal and flow cytometric assays, we observed very significantly higher levels of dsRNA in MSC which had been exposed to stromal-activating B-ALL cell conditioned medium than in non-activating B-ALL or MSC alone controls (see panel for image). We used the small molecule VBIT4, a voltage-dependent anion channel oligomerization inhibitor that decreases mitochondrial nucleic acid release to block this phenomenon in B-ALL cells. We quantified expression of a selection of stromal cell activation marker genes as well as genes in the RNA sensing pathway after treatment with VBIT4-exposed B-ALL cell CM or non-treated control. We observed a 2 to 4-fold reduction in expression of IL6 and CCL2 after VBIT4 exposure, compared to control. In the stromal cell RNA sensing pathway, there was a 15-fold reduction in expression of RIG-I and a 2 to 4-fold reduction in the interferon pathway genes MX2, IFIH1, MAVS and OAS1. Experiments to determine whether knock down of components of the antiviral sensing pathway also abrogates stromal activation and further murine experiments are ongoing. We propose a model in which ROS-induced mitochondrial dysfunction leads to release of dsRNA which in turn activates bone marrow stromal cells leading to protective niche formation and chemoprotection of acute lymphoblastic leukaemia cells despite lack of prior exposure to cytotoxic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Data Confirm the Superiority of Total Body Irradiation-Containing Conditioning Regimen in Comparison to a Chemotherapy-Based Preparation in Children with Acute Lymphoblastic Leukemia Above the Age of 4 Years Given an Unmanipulated Allograft. Results of the Forum Randomized Clinical Trial

Background Conditioning regimen plays a crucial role in determining the outcomes of patients with hematological malignancy treated with allogeneic hematopoietic stem cell transplantation (HSCT). In a multicenter, international, randomized, phase 3 clinical Trial (FORUM study), we documented (Peters C, et al. J Clin Oncol 2021) that children with acute lymphoblastic leukemia (ALL) given total body irradiation (TBI) in combination with etoposide had a superior probability of 2-year overall (OS) and event-free survival (EFS) in comparison to patients given either one of two myeloablative chemo-conditioning regimens. In this report, we assessed whether the benefit deriving from the use of TBI was sustained over time. Patients and methods Patients <18 years at diagnosis, 4-21 years at HSCT, in complete morphological remission (CR) pre-HSCT, and with an HLA-compatible related or unrelated donor (UD) were randomly assigned 1:1 to a myeloablative conditioning regimen with fractionated TBI and etoposide or fludarabine, thiotepa, and either busulfan (BU) or treosulfan (TREO). Children transplanted from a matched sibling donor (MSD) received cyclosporine A only as graft-versus-host disease (GvHD) prophylaxis, whereas recipients of UD HSCT also received short-term methotrexate and anti-thymocyte globulin (ATG). Further details on transplant procedure have been described elsewhere (Peters C, et al. J Clin Oncol 2021). Results Between 04/2013 and 12/2018, 413 patients were randomly assigned to receive either TBI (212) or a chemotherapy-based conditioning (201). Compliance with random assignment was 92%. Sixty-five percent of patients were male, 72% had B-cell precursor ALL, 73% were transplanted from a UD, and in 82% of patients bone marrow was the stem cell source employed. Fifty-four percent of patients were in CR1 at time of allograft, while 40% and 4% were transplanted in CR2 and CR3, respectively. Patient's outcomes were updated on February 1st, 2022. With a median follow-up of 3.7 years (range, 0.3-7.9), the 3-year probability of OS of patients allocated to TBI/etoposide or to the chemotherapy-based conditioning regimens was 90±2% and 71±3% (p<0.001), respectively, while that of 3-year EFS was 81±3% and 59±4% (p<0.001). OS and EFS remain comparable in patients receiving BU or TREO in combination with fludarabine and thiotepa. In detail, OS and EFS of the 102 patients given BU, fludarabine and thiotepa were 73±5% and 69±5% respectively, while those of the 99 patients treated with TREO, fludarabine and thiotepa were 62±5% and 61±5%. Multivariate analysis confirmed that the use of chemotherapy-based regimens correlated with worse OS [Hazard Ratio, HR, 2.63, 95% confidence interval, CI, (1.61- 4.31), p<0.001] and EFS [HR 2.30, 95%CI (1.58- 3.35) p<0.001]. Transplantation in >CR1 predicted a worse EFS in multivariate analysis, as well [HR 1.67, 95% CI (1.14- 2.45) p=0.009]. Patients given TBI benefited from a lower cumulative incidence of relapse (Figure 1A), as well as from a lower 3-year cumulative incidence of non-relapse mortality (2+1% vs. 10+2% in the chemo arm, p=0.007). The 3-year cumulative incidence of chronic GvHD was comparable between the 2 arms (15+3% vs. 12+2% in the TBI and chemo arm, respectively, p=n.s.). We also analyzed the probability of GvHD/relapse-free survival (GRFS) considering both relapse and chronic GvHD as events; children prepared with TBI had a better 3-year GRFS probability (Figure 1B) as compared to chemo-prepared patients. Five patients (all transplanted from an UD) developed secondary malignancies: 4 of them had received TBI. Conclusions With a significantly longer follow-up, we confirm that the use of TBI during the conditioning regimen is associated with better outcomes in children and adolescents with ALL above the age of 4 years transplanted in CR, mainly due to a lower risk of leukemia recurrence. The number of cases of secondary malignancies, particularly in patients offered radiotherapy, deserves particular attention and underlines the importance of continuous safety monitoring over time to comprehensively evaluate the long-term sequels observed in this population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Histone Deacetylase Inhibition Restores Therapy Response in TP53mutated Acute Lymphoblastic Leukemia to Chemotherapy

Background: Although TP53 is the most frequently mutated gene in cancer, in Acute Lymphoblastic leukemia (ALL), mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse TP53 aberrations are found in about 12% of the patients and predict a very poor outcome. As a consequence, relapsed TP53 deleted ALL is now classified as 'very high risk, together with t(1;19) and t(17;19) ALL. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance not only to classical chemotherapeutics, but also newly introduced immunotherapies such as bi-specific T cell engagers such as Blinatumomab or CAR-T cell therapies. Therefore, there is an urgent clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods: Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wildtype and knockout models in the TP53WT B-cell precursor ALL (BCP-ALL) cell lines Nalm6 and RCH-ACV. After confirming the presence of TP53 deletions, we treated these isogenic models with a panel of treatment protocol drugs, demonstrating that TP53 loss negatively affects response to therapy to most contemporary drugs used in the treatment of relapsed ALL. To identify drug combinations that may reverse TP53 mediated therapy resistance, we assessed the viability of these isogenic models in a drug screen containing 198 (pre)clinical drugs. The most interesting targets were subsequently combined with the previously tested treatment protocol drugs to determine potential synergies. Effects on cell viability were determined by amine exposure and MTT assay and confirmed by measuring PARP cleavage. Additionally, positive drug interactions were validated in vivo using immunocompromised mice engrafted with RCH-ACV p53KO cells expressing a luciferase reporter gene. Results: We observed that loss of p53 protects BCP-ALL cell lines Nalm6 and RCHACV from drug-induced apoptosis, leading to increased resistance to all drugs used currently in the treatment of relapsed ALL, except for Asparaginase. In a subsequent drug screen, we found that the histone deacetylase (HDAC) 1/2 inhibitor romidepsin shows strong single agent activity at single nanomolar concentrations. Moreover, romidepsin was found to strongly synergize with contemporary ALL treatment drugs in TP53KO and TP53WT cells (Figure 1A). This effect was strongest in TP53KO cells, effectively reversing therapy resistance induced by TP53 loss. Gene expression profiling suggest that romidepsin may lead to reactivation of TP53 target genes in response to cellular stress, even in the absence of functional p53. Finally, preliminary mouse experiments using TP53 deleted RCH-ACV cells showed that romidepsin improves the response to cytarabine in TP53 aberrant leukemia in vivo (Figure 1B) without signs of added toxicity. A mouse trial testing a potential synergy between romidepsin and cytarabine using TP53 deleted patient derived xenografts (PDX) is currently underway. Conclusions: Loss of p53 protects BCP-ALL cells from currently used (chemo)therapy strategies. Romidepsin can restore response to therapy in TP53 deleted ALL, presumably by reactivation of p53 target genes in response to cellular stress. Therefore, romidepsin may be used to improve therapy response in relapsed TP53 deleted ALL. Since romidepsin already has FDA approval for use in other hematological malignancies, our findings may be rapidly translated into clinical practice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Prognostic Effect of IKZF1 Deletions in ETV6::RUNX1 and High Hyperdiploid Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and recent cure rates approach 90% on first line therapy. IKZF1 deletions have been reported as an unfavorable prognostic factor and are rare in ETV6::RUNX1 and high hyperdiploid (HeH) ALL, the two cytogenetic subgroups with the most favorable prognosis. Due to its rarity, the prognostic effect of IKZF1deletions within this subset of patients remains unclear. Therefore, we assessed the prognostic impact of IKZF1deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients. We performed this retrospective analysis on data of children and adolescents of 1-18 years with B-cell precursor ALL diagnosed in 1991-2016 and treated on one of 16 trials from 9 study groups, of which 6 were minimal residual disease (MRD) guided. Cytogenetic, fluorescence in-situ hybridization, and RT-PCR analyses of pre-treatment bone marrow samples to determine ploidy and fusion gene status were performed locally. Trials using MRD for risk stratification applied either PCR or flow cytometry analyses. The IKZF1 status was determined by multiplex ligation-dependent probe amplification by each individual study group. 3% of ETV6::RUNX1 (n=26) cases had an IKZF1 deletion; this affected survival adversely overall (5-year event free survival [EFS]: 79% vs. 92%, p = 0.02) (Table 1). However, in minimal residual disease (MRD) guided protocols, IKZF1 did not affect outcome in ETV6::RUNX1 cases ( 5-year EFS: 100% vs. 93%, p = 0.34) (Table 1). 9% of HeH cases had an IKZF1 deletion (n=85); this adversely affected survival overall (5-year EFS: 76% vs. 89%, p = 0.006), and in MRD-guided protocols (73% vs. 88%, p = 0.004) (Table 1). Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (Hazard ratio of EFS [95% CI]: 2.00 [1.20-3.30]) (Table 1). As the prognostic effect of IKZF1 deletions has been shown to vary by MRD risk group, we examined the prognostic effect of IKZF1deletions in HeH cases with detectable MRD <5% at end of induction in more detail. Of these cases, 10% carried an IKZF1deletion (n=34) which negatively affected outcome (5-year EFS: 78% vs. 92%, p = 0.01) Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in this intermediate MRD HeH ALL (Hazard ratio of EFS [95% CI]: 2.90 [1.20-6.80]). However, cases with an IKZF1 deletion had significantly higher MRD values than their wild-type counterparts (p = 0.03, Figure 1). In conclusion, our analysis of a large composite cohort consisting of 16 trials shows that IKZF1deletions do not affect survival in ETV6::RUNX1 ALL when treated on MRD-guided protocols. In contrast, our data shows that in HeH ALL IKZF1deletions are associated with lower survival rates, higher relapse rates, and higher MRD values. Future results of current trials such as the ALLTogether will likely reveal whether risk stratification predominantly reliant on MRD is adequate for HeH patients or whether stratification by copy number alteration profile, including IKZF1 status, or by other methods would be more suitable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD19-CAR-T Cells Are Effective and Safe Treatment of Post-Transplant Relapse in Pediatric and Young Adult Patients with B-Lineage ALL: Real-World Data from Germany

Introduction: Patients with precursor-B acute lymphoblastic leukemia (pre-B ALL) which have relapsed subsequent to allogeneic hematopoietic stem cell transplantation (HSCT), have relapsed twice, or were resistant to upfront therapy have a dismal prognosis. CD19-directed chimeric antigen receptor (CAR) T cells have evolved as potent immune therapy for such patients. Tisagenlecleucel (Tisa-cel) is a commercial CD19-directed autologous CAR-T cell product. The CAR contains the co-stimulatory element 4-1BB to promote in vivo persistence and proliferation. Thus this therapy is in principle competent to serve as a stand-alone therapy. Based on the results of the ELIANA trial (Maude et al. NEJM 2017), Tisa-cel was approved by FDA and EMA for pediatric/young adult B-ALL in 2017 and 2018. In clinical trials, stringent inclusion criteria narrow down the risk profiles of trial subjects. Treatment in clinical trials provides close guidance to physicians and patients during the trial. However, post approval reality leads to treatment of patients with higher and more variable risk profiles treated in many centers. With approval by the local ethics committees, we performed a retrospective study asking all CAR T cell centers in Germany approved for treatment with Tisa-cel (Kymriah®) to provide treatment data reflecting post approval reality, in order to identify patients who might most benefit from this novel, complex and expensive treatment. Patients: Twenty-one centers included all pediatric and young adult patients with pre-B ALL (n=81) who had received Tisa-cel between 8/2018-1/2022, allowing for a minimal follow-up of 5 months of surviving patients. Median age was 11.5 years (range: 1.0-25.0 years), 53 (65.4%) were male and 28 (34.6%) female, median body weight was 41.6 kg (range: 8.0 kg-135 kg). 65 patients (80.2%) had relapsed after HSCT, 15 (18.5%) had suffered a 2nd relapse without HSCT and 1 had primary refractory disease. For bridging therapy, 3 patients (3.7%) received Blinatumomab, 4 (4.9%) inotuzumab, 72 (88.9%) received low-dose chemotherapy. One patient was bridged with a high-dose chemotherapy; 1 patient with extra-medullary disease did not receive any bridging therapy. Results: Cytokine release syndrome (CRS) was observed in 55 (67.9%) patients (grade I-II: n=50, Grade III-IV: n=4 and grade V: n=1); immune cell-associated neurological syndrome (ICANS) was seen in 6 (7.4%) patients (grade I: n=2, grade III-IV: n=4). At day 28, 72 patients (88.9%) were in CR, 8 (9.9%) in NR. One patient (1.2%) succumbed to CRS. At last follow-up, 42 patients (60%) remained in remission, 34 patients had relapsed, 1 died of TRM, 3 of NRM, 1 had developed secondary MDS. This translated into an event-free survival probability (pEFS) of 43.9% (±6.5), an of overall survival (pOS) of 52.9% (±7.0), and a cumulative incidence of relapse (CIR) of 48.8% (±6.4) at 2 years post CAR T cell transfusion. CIR CD19 neg. was 33.8% (±6.1%) and CIR CD19 pos. was 15% (±4.2%). Interestingly, there was no difference between children and adult patients with regard to pEFS and pOS. A trend towards a higher pEFS (n=16, 55.0±12.7%) compared to patients receiving CAR-T for relapse after alloHSCT (n=65, 41.8±7.2%) was observed (p=0.630). Time to relapse after HSCT was a strong predictor for outcome. Patients relapsing within 6 months of HSCT had a pEFS (pOS) of 17.2±9.6% (15.6±9.6%), pEFS (pOS) for those relapsing later was 53.8±9.0% (74.5±8.0%) p=0.001), Figure 1. Risk factors: For the subgroup with post HSCT relapse, multivariable Cox-regression analysis considering age, time from HSCT to relapse, extra-medullary relapse, remission status at start of lymphodepleting chemotherapy (LDC) and LDC dosing revealed that time from HSCT to relapse >6 months’ post HSCT as a single factor reduced the risk to 0.381 and 0.221, respectively (p=0.001 and p=0.003), Table 1. Conclusion: In our analysis providing the first real-world experience in a patient cohort highly enriched for patients with post HSCT relapse, 40% of ALL patients with poor prognosis were rescued with a single dose of Tisa-cel. Our novel finding that patients post HSCT had a far better EFS if relapse occurred beyond 6 months might be attributable to more competent T-cell system with more native T-cells with higher plasticity for proliferation, expansion and persistence. This is currently a matter of investigation and may in future allow to implement strategies to optimize CAR T cell therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Olverembatinib Based Regimen in Patients with Philadelphia Chromosome-Positive Advanced Leukemias: A Real-World, Single-Center Experience

Background Philadelphia chromosome-positive (Ph+) advanced leukemias including chronic myeloid leukemia blast phase and acute lymphoblastic leukemia have poor outcomes. Olverembatinib, a novel BCR-ABL tyrosine kinase inhibitor, has anti-tumor activity in Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia cells(CML) and tumor models, is currently approved for treating resistant CML patients with T315I mutation by the National Medical Products Administration in China, and in phase Ib clinical trials in the US for ponatinib resistant and/or intolerant CML patients. Olverembatinib also inhibited p-KIT, p-AKT, p-ERK1/2, and p-STAT3 to a higher extent than ponatinib, and suppresses pre-B ALL cells through inhibition of the SRC kinase and PI3K/AKT pathways and may be a potential therapeutic agent for the management of pre-B ALL. However, the efficacy of Olverembatinib based regimen in Philadelphia chromosome- positive advanced leukemias is still unknown. Methods We conducted a retrospective study on patients with Philadelphia Chromosome- Positive Advanced Leukemias including Ph+ CML (n=11) and Ph+ ALL (n=5) who received a Olverembatinib-based regimens in our center. Results The trial included 11 patients with Ph+ CML and 5 patients with Ph+ ALL who had relapsed after transplantation. Among the 11 CML patients, there were 2 CML-AP, 6 chronic myeloid leukemia-myeloid blast phase (MBP), 2 chronic myeloid leukemia-lymphoblastic bast phase(LBP), 1 chronic myeloid leukemia- myeloid and lymphoblastic mixed blast phase. Median age was 34 years and the median follow-up time was 4 months. Also, two (18.2%) CML-AP patients were treated witholverembatinib alone. Six (54.5%) CML-BP patients were treated with olverembatinib in combination with demethylating agents. And two patients with LBP received VDP and stem cell transplantation (SCT)-based regimens combined with olverembatinib. Treatment for patients with chronic myeloid leukemia- myeloid and lymphoblastic mixed blast phase is consistent with patients with MBP. The overall response rate of 8 evaluable patients was 100%. CR/CRi rate was 100%, and CCyR/PCyR rates 62.5% vs 37.5%, respectively. In addition, 6 patients achieved major molecular responses (MMR). the remaining three patients were not yet due for evaluation. Of 5 patients with ph+ ALL that relapsed after transplantation, 1patient was treated with blinatumomab and olverembatinib, 2 patients were treated with intensive chemotherapy and olverembatinib followed by donor lymphocyte infusion(DLI), The treatment for the remaining 2 patients was Anti-CD19 chimeric antigen receptor T cell (CAR-T) technology followed by olverembatinib, and this 5 Ph+ ALL patients achieved complete molecular remission(CMR). In addition, treatment-related adverse reactions included cytopenias, skin pigmentation, proteinuria and high triglycerides, all grades less than 2. No deaths have occurred. Conclusion Olverembatinib-based regimens show encouraging activity and safety in very heavily pre-treated, advanced Ph+ leukemias.

Identifying Patterns of Neurocognitive Dysfunction through Direct Comparison of Patients with Leukemia, CNS Tumors, and Sickle Cell Disease

Background. Innovations in pediatric oncology have led to advances that take the place of older, more toxic therapies, making it possible for more children to survive. As this population grows, it is essential to monitor and carefully study late effects. Perhaps most insidious and far reaching in its effect on the daily lives of survivors is neurocognitive dysfunction that impacts school performance, occupational attainment, and quality of life. The populations at greatest risk for neurocognitive dysfunction in the pediatric hematology oncology population include children with central nervous system (CNS) tumors, leukemia (ALL), and sickle cell disease (SCD). What is known about neurocognitive dysfunction in these populations has come from studying these groups separately and with heterogeneous tools, putting each group on a different "measuring stick." Methods. The current study seeks to quantify and compare the magnitude and type of neurocognitive dysfunction between groups of children with CNS tumors, pre-B ALL, and SCD by employing a common methodology to directly compare these groups to each other. Neurocognitive function was assessed using the NIH Toolbox Cognition Battery. Children between the ages of 7-12 were included without pre-existing neurological disorders. Children with CNS tumors or pre-B ALL were within 18 months of completing therapy. Children with SCD possessed the SS or Sβ0 genotype, regularly took hydroxyurea, had no history of overt stroke, and did not receive chronic transfusions. NIH Toolbox Cognition Battery scores are presented as age-adjusted standard scores with a mean of 100, standard deviation of 15. Effect sizes were calculated using Cohen's d statistic. We hypothesized that these groups would perform significantly differently from normative data and differently from each other, with children with CNS tumors showing the greatest deficits, followed by children with SCD, with children with ALL showing the fewest deficits. Results. Fifty-three patients were included in analysis (n = 11 CNS tumor, n = 27 ALL, n = 15 SCD). Children with ALL demonstrated a large, negative effect in processing speed (median=86, IQR 65-95; Cohen's d = -0.93) and medium, negative effects in working memory (median=91, IQR 84.5-105.5, d = -0.6) and attentional control (median= 92, IQR 80-102, d = -0.6; Figure 1). Children with CNS tumors experienced medium deficits in cognitive flexibility (median=90, IQR 84-97, d = -0.67) and large, negative effects in processing speed (median=77, IQR 69-95, d = -1.53) and attentional control (median=88, IQR 76.5-93, d = -0.8). Children with SCD demonstrated medium, negative effects in episodic memory (median=92.5, IQR 85-102.8, d = -0.5). For the overall cognitive fluid composite score, children with SCD performed in the average range (median=99.5, IQR 83.2-105), while children diagnosed with malignancies demonstrated poorer performance with large effect sizes (ALL median=87, IQR 82-95, d = -0.87; CNS tumor median=84, IQR 74.5-95.5, d = -1.07). Conclusions. Preliminary analyses suggest that at-risk groups of children with ALL, CNS tumors, and SCD experience neurocognitive deficits differently from one another in multiple domains. Future studies should employ findings like these to develop and implement disease- and domain-specific interventions to reverse or alleviate neurocognitive deficits. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Highly Parallel Discovery of Synthetic Knockin Sequences for Enhanced Cancer Immunotherapies

Introduction T cells engineered to express transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged as powerful treatment options for some malignancies. However, while CAR T cells have induced impressive initial response rates in patients with B-precursor acute lymphoblastic leukemia (ALL) and lymphoma, they fail to mediate long-term relapse-free survival in 40-60% of patients and have not been successful in most solid tumors. For both TCR- and CAR-based approaches, T cell persistence and long-term functionality can be diminished by chronic antigen stimulation or tonic signaling. One approach to enhance the efficacy of cell therapies that has been explored by several groups is the overexpression of specific transcription factors (e.g. AP-1/ATF transcription factors such as c-JUN or BATF); however, more systematic gain-of-function screens in human CAR T cells have not yet been done. Methods and Results Here, we introduce CRISPR-based modular pooled knockin (ModPoKI) screens to evaluate hundreds to thousands of different constructs in human T cells and their potential to overcome TCR or CAR T cell dysfunction. We generated two barcoded ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs) to identify those that confer a fitness advantage to TCR or CAR T cells. The libraries were combined with either an NY-ESO-1-specific TCR, an anti-CD19 CAR or a high affinity anti-GD2 CAR that is known to induce tonic signaling-based dysfunction. The libraries and the TCR/CAR were engineered as polycistronic sequences that were non-virally integrated into the TRAC (T cell receptor alpha chain constant) locus of primary human T cells. We performed >20 unique ModPoKI screens including bead-based single stimulation, repetitive stimulation with target cells and tonic stimulation (GD2 CAR model). While constructs containing known AP-1/ATF transcription factors BATF and BATF3 showed increased abundance across multiple screens, transcription factor AP4 (TFAP4) knockin (KI) constructs were more clearly enriched after repetitive stimulation and tonic signaling, suggesting potentially undescribed benefits in exhaustion-prone environments. We next validated single knockins of TFAP4 or a control construct (truncated nerve growth factor receptor, tNGFR) in combination with the GD2 or the CD19 CAR. TFAP4 KI led to increased levels of cytokine release, proliferation and target cell killing compared to conventional CAR T cells. Moreover, TFAP4 KI mediated increased tumor control and survival in an in vivo model. Interestingly, we observed elevated levels of CD25 (IL2RA) expression on TFAP4 KI T cells and enrichment of genes related to IL2/STAT5 pathways which could hint at a potential benefit in situations of IL-2 competition/presence of regulatory T cells. Lastly, we aimed to discover specific combinations of TFs that work in concert to enhance T cell fitness in the setting of tonic CAR signaling. We created a ~10,000-member library (102 x 102 - pairwise combinations of 100 transcription factors plus two controls) cloned into constructs with the GD2 CAR and performed a tonic signaling ModPoKI screen. Analysis of the constructs that increased the most in relative abundance highlighted that several of the top performing constructs included either TFAP4, BATF, BATF3 or a combination of TFAP4 and BATF(3), suggesting that TFAP4 and BATF(3) are key transcription factors that can coordinately drive increased T cell fitness upon repetitive stimulation. Validation analyses confirmed that the combinatorial KI of a BATF-TFAP4 construct confers highest cytotoxic capacity in vitro and in vivo compared to single knockin (BATF-RFP or RFP-TFAP4) or control knockin (RFP-tNGFR) GD2 CAR constructs. Conclusions In conclusion, ModPoKI screens allow for customizable parallel evaluation and functional characterization of hundreds to thousands of different T cell constructs. Using clinically relevant screening modalities, we nominated candidate genes that can synthetically re-write the T cell response to repetitive antigen exposure or tonic signaling and thus have the potential to program more effective TCR or CAR T cell therapies for cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inotuzumab Ozogamicin Induction Followed By Standard Chemotherapy Yields High Remission Rates and Promising Survival in Older (>55 Years) Patients with De Novo B-Lymphoblastic Leukemia (GMALL-Initial1 Trial)

Introduction: Acute lymphoblastic leukemia (ALL) represents approx. 5% of all newly diagnosed leukemias in patients (pts) between 55 and 70 years of age. Incorporation of novel antibody-based therapies in the first line induction therapy bears the potential of a significantly increased response rates and survival together with a reduction of treatment toxicity. Methods: This open label phase II study (ClinicalTrials.gov, identifier: NCT03460522) of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia was conducted at 13 centers. Pts aged >55 years with newly diagnosed Ph/BCR-ABL negative B-precursor ALL (at least CD20 positivity of leukemic blasts) were eligible. The 1st induction cycle consisted of InO 0.8mg/m2 on day1 and 0.5mg/m2 on d8 and d15 together with dexamethasone 10 mg/m2 (day7-8, day14-17) and one intrathecal injection (i.th.) of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dex). The 2nd and 3rd induction cycle consisted of InO 0.5mg/m2 on day 1, 8 and 15 plus i.th. MTX/AraC/Dex. Pts achieving a complete remission (CR) were offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/ AraC/ cyclophosphamide / Dex.) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-MP/MTX. The primary endpoint was event free survival (EFS) at 12-months follow-up. An event was defined as any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up was considered to qualify the experimental treatment as very promising for additional testing. Results: 43 out of the 45 pts included were analyzed regarding the primary end point. Two pts were excluded per protocol from further analysis, as they received only one cycle of induction or withdraw consent. At diagnosis, median age was 64 years (range 56-80 years, 20 pts ≥65 years and 12 pts ≥70 years), median bone marrow blast count was 80% and median CD22 expression on leukemic blasts was 69% (range 21-99). 38 pts were diagnosed with a common- and 5 with a pro-B ALL. 42/43 pts completed all 3 cycles of induction therapy. All 43 pts achieved a complete remission (CR/CRi), mainly after the 1st induction. Median time between start of 1st induction to start 2nd induction, from 2nd to 3rd induction and from 3rd induction to 1st consolidation therapy was 21 days, 28 days and 30 days, respectively. Results of minimal residual disease (MRD), measured by real-time quantitative PCR, are available for all 43 pts, with 23/43 (53%) and 31/42 (74%) pts being MRD negative after 2nd and 3rd induction therapy, respectively. With a median follow-up of 697 days, the probability of OS at 1 and 2 years is 91% (95% C.I. 82-99%) and 81% (95% C.I. 69-94%), respectively. No patient died within the first 6 months after start of induction therapy. 5 events occurred within the first year after study inclusion, 3 deaths in CR and 2 relapsed ALL, resulting in event rate of 12% and a probability of EFS at 1 year (primary endpoint of the study) of 88% (95% CI 79-98). EFS at 2 years was 73% (95% CI 59-88). So far, 7 pts with persisting/reappearance of MRD (2 pts) or relapse (5 pts) were treated with blinatumomab. 9 pts received an aSCT (4 pts in CR1 and 5 pts >CR1), 6 of these pts are alive in CR. Regarding adverse events (AEs) during induction cycles 1, 2 and 3, most common AEs ≥CTC grade 3 reported were leukocytopenia (in 74%, 19% and 2% of all cases, respectively), anemia (37%, 5%, 0%), thrombocytopenia (49%, 7%, 2%) and elevation of liver enzymes (14%, 5%, 0%). One patient was reported with suspected veno occlusive disease (after 2nd induction therapy). Classical factors which may be associated with inferior survival outcomes like age, diagnosis of pro-B ALL, disease burden at diagnosis, co-morbidity or no MRD negative CR after induction had no significant impact on OS or EFS. Conclusion: Three cycles of InO as induction therapy for the treatment of older pts with de novo acute B-lymphoblastic leukemia resulted in a very high remission rate. Given that our study met the primary end point (predefined event rate at 1 year ≤40%, observed event rate 12%), these promising study results could be a rationale for integrating InO induction therapy in future treatment recommendations. Ideally, this novel induction therapy should be evaluated in prospective randomized trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Outcomes of Childhood Picalm:MLLT10+ Acute Leukemias: An International Retrospective Study

Introduction: Acute leukemia with translocation t(10;11)(p12-13;q14-21) results in generation of the PICALM:MLLT10 fusion gene, and is distinct from t(10;11)(p11-13;q23) which gives rise to KMT2A (formerly MLL) gene rearrangement. Defective PICALM function leads to aberrant hematopoiesis, while defective MLLT10 function impairs chromatin-mediated gene regulation. The PICALM:MLLT10 gene has been reported in patients with Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) predominantly T-cell, Acute Undifferentiated Leukemia (AUL) and Mixed Phenotype Acute Leukemia (MPAL). MLLT10 interacts directly with DOT1L in a complex that regulates the methylation of H3K79. MLLT10 fusions are thought to misdirect DOT1L to the promoters of HOXA genes, leading to hypermethylation of H3K79. This hypermethylation causes constitutive activation of HOXA activity and prevents cell maturation and differentiation. The clinico-biological characteristics, response to conventional treatments, and survival outcomes of childhood PICALM:MLLT10+ leukemias are poorly understood. Methods: This was an international, multicenter, retrospective cohort study. Data were submitted by ten International-BFM (I-BFM) study Group-AML and ALL committees and by the Children`s Oncology Group (COG)-AML Study committee. For study inclusions, patients were required to be ≤ 21 years of age at diagnosis, diagnosed between January 1995 and December 2019 with de novo ALL, AML, AUL or MPAL and PICALM:MLLT10 fusion-positive. Patients with KMT2A-rearranged leukemia or acute promyelocytic leukemia were excluded. Kaplan Meier curves were employed for survival analysis with Cox proportional hazard modelling to compare effect of AML vs ALL therapy, and receipt of hematopoietic stem cell transplant (HSCT) vs no HSCT, on survival. Pearson's Chi Squared was employed as a non-parametric measure to determine the effect of end of induction-1 (EIO-1) MRD positive vs negative status on incidence of relapse. P value cut-off for data to reach statistical significance was <0.05. Results: Ninety-eight children with acute leukemia were included, 60 were males. Median age at diagnosis was 12 years (IQR:9-14.8 years) and median initial white cell count was 40.5 x 109/L (IQR:13.8-168.9). Fifty-five (55.6%) patients were diagnosed with T-ALL, 39 (39.4%) with AML, while precursor-B ALL, AUL and MPAL were each diagnosed in single patients; 1 subtype diagnosis was unknown. Ten (10.1%) patients presented with CNS disease and 13 (13.1%) presented with non-CNS extramedullary manifestations. Of 66 with cytogenetic/molecular data available, 33 (50%) had additional cytogenetic-molecular aberrations; NOTCH1, WT1 and RAS pathway mutations predominated. Most patients received an initial chemotherapy regimen guided by their disease immunophenotype: 58/98 (58.6%) received an ALL-like chemotherapy regimen upfront, 38 (38.4%) received an AML-like regimen, 1 (1.0%) a hybrid regimen, 1 initial chemotherapy regimen was unknown. Five-year event-free survival (EFS) was 50.4% (CI 39.1-64.9). For AML patients, 5-year EFS was 22.5% (CI 9.75-52.0) and for ALL 67.7% (CI 54.2-84.7), p=<0.001. Five-year overall survival (OS) was 56.3% (CI 46.3-68.5). For AML patients, 5-year OS was 26.7% (CI 13.3-53.6) and for ALL 75.7% (CI 64.6-88.8), p=<0.001. Thirty-eight patients died, 22 (57.9%) deaths were disease-related, 4 (10.5%) treatment-related. Patients who received HSCT in CR1, demonstrated a trend towards an inferior 5-year EFS (23.7% vs 56.5%) and OS (42.6% vs 64.5%) than those who did not; however, analysis was limited by small sample size. Four (22.2%) HSCT recipients had ALL but most, 14 (77.8%), had AML, which may account for the unfavourable outcomes associated with HSCT. There was no statistically significant difference in rate of relapse in the EOI-1 MRD-positive compared with MRD-negative patients (p=0.49). Of those who relapsed, 25 (75.8%) had an isolated bone marrow relapse, 3 (9.1%) isolated CNS, 3 (9.1%) other sites and, for 2 patients, site of relapse was unknown. Conclusions: Children with PICALM:MLLT10+ AML have inferior 5-year EFS and OS outcomes compared to their T-ALL counterparts. MRD status at EOI-1 was not predictive of relapse. Targeted therapies with DOT1L inhibitors or DNA demethylating agents should be explored, particularly for patients with AML.

Dermatologic Adverse Events in Acute Lymphocytic Leukemia Patients Treated with Bispecific T-Cell Engager Blinatumomab

Blinatumomab is a novel FDA-approved bi-specific T-cell engager (BiTE) that significantly improves survival (vs standard chemotherapy) in adults with relapsed or refractory Philadelphia chromosome positive or negative B-cell precursor ALL (B-ALL) and is associated with high rates of clearing persistent measurable residual disease. Though the best known adverse effects associated with blinatumomab include cytokine release syndrome and neurologic toxicity, dermatologic adverse events (DAE) have also been reported in 16% of patients in clinical trials (FDA, 2018). In this study, we aimed to assess the incidence of DAEs in B-ALL patients treated with blinatumomab and characterize their associated clinical and histopathologic features. We conducted a retrospective analysis of B-ALL patients treated with blinatumomab monotherapy at Memorial Sloan Kettering Cancer Center from 1/1/2012 - 10/1/2021 (N=45). Seven patients experienced a total of nine DAEs (15.6%). Median number of blinatumomab cycles at time of DAE onset was one (range cycles 1-3). Median time to DAE onset following blinatumomab induction was 40 days (range 5-115 days). The most common DAEs were rashes (N=4); types included eczema, folliculitis, papular dermatitis, and panniculitis. Other DAEs (N=5) included new onset psoriasis, seborrheic dermatitis, acne, facial erythema, and nail dystrophy. All DAEs were grade 1, many DAEs did not require treatment, and no DAEs warranted interruption or discontinuation of blinatumomab. Four of the 7 patients with DAEs developed CRS at any point during blinatumomab therapy (ASTCT consensus grade 1, N=3; grade 2, N=1). Six of 7 patients with DAE achieved undetectable minimal residual disease (MRD) in the course of blinatumomab therapy or maintained MRD negativity. This is the most comprehensive study to report incidence and clinico-morphologic patterns of DAEs associated with blinatumomab monotherapy. Blinatumomab DAEs may be more common in patients who experience CRS; this finding may support that DAEs are the result of increased circulating cytokines secondary to effective immune activation by blinatumomab. DAEs are mild and we recommend that patients with severe rash during blinatumomab infusion are carefully evaluated for other etiologies of rash.

Bone Marrow Plasma Proteome Analysis By Differential Ultracentrifugation and Tandem Mass Spectrometry Allow for Characterization of Leukemia-Derived Extracellular Vesicles in Patients with Acute Lymphoblastic Leukemia

In recent years, the interplay of leukemic cells with the bone marrow niche has emerged as a potential contributing factor for the survival, therapy resistance and relapse-initiating capacity of leukemic cell subsets in ALL. This is known as early, environment-mediated drug resistance (EMDR). The role for leukemia-derived extracellular vesicles (LEVs) in this interplay remains largely unexplored. In vitro studies and PDX models have demonstrated that LEVs are internalized by bone marrow stromal cells (BMSC), leading to BMSC reprogramming and metabolic switch to glycolysis, with beneficial effects on leukemic cell survival as a result. This suggests that LEVs could also play a role in leukemic cell survival in ALL during chemotherapy, however, studies examining LEVs in patients are warranted. We performed fractionation by differential ultracentrifugation and subsequent mass spectrometry analysis of bone marrow plasma samples (n=12) collected at time of diagnosis (n=6, one sample was processed at 30, 60 and 120 min after sampling to assess the potential effect of contamination with platelet-derived EVs), during (n=2) and after induction therapy (n=4) in patients with B-cell precursor ALL (n=4). We isolated four fractions: A (2,000g pellet): Enriched in platelets/apoptotic bodies, M (20,000g pellet, enriched in microvesicles: large, membrane-derived EVs), E (150,000g pellet, enriched in exosomes), and S (150,000g supernatant, mostly soluble proteins), as well as R (raw untreated plasma), and we subjected these to mass spectrometry-based quantitative proteomics in 3 technical replicates. We examined protein composition and enriched pathways in the fractions and investigated associations of EV protein levels with the size of the leukemic cell fraction in the bone marrow. Flowcytometric data was used to confirm cell distributions/surface marker expression. Examining the protein composition of the five fractions (A, M, E, S, and R) showed that although the highest number of proteins was found in the A fraction (2803 proteins), the M fraction (2477 proteins) had the highest proteome diversity (Figure 1). Focusing on the M fraction, we compared diagnostic with follow up samples (blast percentage day 0: 92, 95, and 72, day 15: 0.4 and 1.3, day 29: 0.05, 0.2, 0, and 0.001, one day 0 sample excluded due to poor readout quality). We found significant upregulation of B-cell associated proteins at diagnosis, incl. HLA-DR, CD19, and CD22, along with the stem cell marker CD34, while T-cell and platelet-associated proteins did not show this pattern. Flow cytometry demonstrated only very few healthy B-cells in the samples at time of diagnosis (5, 0.8, and 0.6% vs. 25, 3, 32 and 21% on day 29) and showed all the leukemic clones to be CD34positive, confirming the BCP-ALL leukemic origin of the EVs. Examining the proteins most closely correlated to the percentage of blasts across all the samples, we found several proteins associated with poor survival, cell migration/adhesion and chemoresistance in acute leukemia, including CD97 (r=0.85, p=0.006), PLXNB2 (r=0.72, P=0.037), and ITGA4 (r=0.6, p=0.097). Gene set enrichment analysis of the proteins showing the most prominent differences between day 0 and day 15/29 using Reactome identified a variety of metabolic processes, but also oncogenic MAPK signaling (NES 1.94, p-adj=0.005), integrin signaling (NES 1.94, p-adj=0.003), and regulation of expression of SLITs and ROBOs (NES 2.1, p-adj=3x10-7) as enriched among the day 0 samples. Interestingly, we recently found genes within the latter two selectively upregulated in leukemias with poor induction therapy response (Modvig et al, Molecular Oncology, 2022). Taken together, this suggests that LEVs indeed could play a role in leukemic cell adherence and EMDR in the bone marrow niche. In conclusion, LEV content can be reliably characterized in bone marrow plasma of patients with ALL through differential ultracentrifugation and mass spectrometry-based proteomics. LEV protein composition reflects processes such as leukemic adhesion/migration, survival and drug resistance, carefully suggesting a role for LEVs in EMDR, but larger studies in patients are warranted. The identification of LEVs and key pathways in the leukemic-stromal cell interplay/EMDR can lead to development of new drugs blocking this cross-talk, potentially contributing to an improved relapse-free survival in patients with ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fractionated Inotuzumab Ozogamicin Combined with Low-Intensity Chemotherapy in Older Patients with Newly Diagnosed CD22+ Philadelphia Chromosome (Ph)-Negative B-Cell Precursor (BCP) Acute Lymphoblastic Leukemia (ALL): Results of the EWALL-INO Study

Introduction. We report here the results of the EWALL-INO Phase 2 study (ClinicalTrials.gov: NCT03249870) evaluating the activity and safety of frontline inotuzumab ozogamicin (INO), an anti-CD22 antibody conjugated to calicheamicin, combined to low-intensity chemotherapy in 131 older patients with CD22+ Ph-negative BCP-ALL. Methods. This single arm prospective study has been conducted in several European centers from the European Working group on adult ALL (EWALL). Eligibility criteria were patients aged ≥55 years, with an ECOG performance status ≤2, newly diagnosed with CD22+ (20% or more of positive blast cells) Ph-negative BCP-ALL without central nervous system (CNS) involvement. After a prephase including 5 days (D) of dexamethasone (DEX) 10mg per D and intrathecal (IT) methotrexate (MTX), the induction regimen was delivered in 2 parts. Induction part I (Ind1) consisted of one triple IT, vincristine (VCR) 2 mg (1 mg over 70 years of age) D1 D8 D15 D22 and DEX 20 mg D1D2 D8D9 D15D16 D22D23 combined with 3 injections of INO (0.8 mg/m² D1, 0.5 mg/m² D8 and D15). Induction part II (Ind2) was offered to patients in CR or CRp (CR with platelets <100 x 109/L) after Ind1 or as salvage therapy. Ind2 consisted of DEX 20mg D1D8, cyclophosphamide (CY) 300 mg/m² D1 to D3, one triple IT D2 and 2 injections of INO (0.5 mg/m² D1 and D8). Patients in CR/CRp were programmed to receive 6 blocks of consolidation (Ara-C 1.5g/m²/12h adapted to renal clearance D1D2 and DEX 10mg/12h D1D2, cycles 1 and 4; MTX 1.5 g/m² over 24h D1, VCR 1 or 2 mg D1, one triple IT D2 and 6-mercaptopurin (6-MP) D1 to D7, cycles 2 and 5; CY 500 mg/m² D1D2, VP16 75 mg/m² D1D2, one triple IT D2 and MTX 25 mg/m² D1, cycles 3 and 6) followed by a POMP maintenance (VCR, 6-MP, MTX, DEX) during 18 months. Allogeneic hematopoietic stem cell transplant (HSCT) was allowed after at least 3 blocks of consolidation at the discretion of the investigator. The primary endpoint of the study was overall survival (OS) with an expected 1-year OS rate of 70%. The evaluable population consisted of patients who received at least 1 dose of INO. Minimal residual disease (MRD) was assessed on bone marrow aspirates after Ind1 and Ind2 using quantification of clonal molecular rearrangements. The study was planned to recruit up to 130 patients. All patients provided informed consent. Results. Between 12/2017 and 03/2022, 6 screen failures occurred (including one Ph+ ALL) and 131 patients were enrolled and received at least one dose of INO. These 131 patients were considered for the intent-to-treat analysis. However, among them, 5 patients had CD22 expression <20% and 3 had CNS involvement. Patient characteristics are shown on Table 1. After Ind1, the CR/CRp rate was 88.5% (116/131, 9 CRp) and 4/131 (3%) patients had died. MRD <10-4 was achieved in 56 of 97 (56.6%) CR/CRp tested patients. A total of 120 patients received Ind2 (115 CR/CRp patients, as 1 death in CR occurred between Ind1 and Ind2 and 5 patients received Ind2 after failure of Ind1) and 118 patients eventually achieved CR/CRp (112 CR, including 3 late CR after Ind2; 6 CRp) resulting in a CR/CRp rate of 90% (118/131) after Ind1 + Ind2. No patient died during Ind2. MRD <10-4 was achieved in 80 of 99 CR/CRp tested patients (80.8%) after Ind2. Overall, the full planned INO dosing was administered to 108 patients (82.5%). With a median follow-up of 15 months (range 3-42), 29 patients relapsed and 50 patients died, including 29 deaths after relapse. Only 11/118 CR/CRp patients received an HSCT (9.4%). With a 1-year OS of 72.5% (95% CI, 64-81), the primary endpoint was reached. At 2 years, OS was 53.6% (95% CI, 44-64). Leukemia- free survival was 64.9% (95% CI, 56-74) at 1 year and 49.8% (95% CI, 40-61) at 2 years. (Figure 1) A grade 3-4 liver toxicity was observed in 21 patients (16%), including 3 patients (2.2%) with transient sinusoidal obstruction syndrome (SOS), 2 during Ind1 and 1 after HSCT. Conclusion. These results confirm that fractionated INO combined to low-intensity chemotherapy is a very active and well tolerated frontline therapy for older patients with CD22+ Ph-neg BCP-ALL, as previously reported in the MDACC INO/mini-HCVD study (Jabbour et al. Cancer 2019). An updated analysis will be provided at ASH meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blinatumomab during Consolidation in High-Risk Philadelphia Chromosome (Ph)-Negative B-Cell Precursor (BCP) Acute Lymphoblastic Leukemia (ALL) Adult Patients: A Two-Cohort Comparison within the Graall-2014/B Study

Blinatumomab during Consolidation in High-Risk Philadelphia Chromosome (Ph)-Negative B-Cell Precursor (BCP) Acute Lymphoblastic Leukemia (ALL) Adult Patients: A Two-Cohort Comparison within the Graall-2014/B Study

Reduced Intensity Reinduction Followed By Blinatumomab Achieves Excellent MRD Clearance with Reduced Toxicity and Facilitates Timely Transplant in Children and Young People with Relapsed B-Precursor Acute Lymphoblastic Leukemia

Introduction Despite the emergence of effective novel immunotherapies, the outcome of relapsed pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains poor due to treatment-related mortality (TRM) and resistant disease. In response to accumulating evidence on the efficacy and low toxicity of blinatumomab in relapsed B-ALL and the availability of highly active CD19-targeted chimeric antigen receptor T-cell therapy (CART), we redesigned the United Kingdom (UK) relapse treatment pathway, based on two key guiding principles: 1) to reduce chemotherapy burden and associated toxicity through a move away from the highly intensive ALL R3 backbone towards increased reliance on blinatumomab to clear disease, and 2) to identify treatment failures early and direct them to CART, given the poor responses reported with hematopoietic stem cell transplant (HSCT) in this context. Methods Patients were treated with UKALL induction chemotherapy (<16yr: 3-drugs; >16yr: 4-drugs) until adequate blast clearance. Patients with blasts <5% at Day 15 progressed directly to blinatumomab; remaining patients continued to Day 28. Following reinduction, patients received up to 2 cycles of blinatumomab as a single agent. HR patients (early/very early relapses and/or HR cytogenetics) proceeded to HSCT once minimal residual disease (MRD) <0.01%. Those with high-risk (HR) isolated central nervous system (CNS) disease had the option to proceed directly to HSCT following CSF blast clearance without blinatumomab. All Standard risk (SR) patients continued on chemotherapy alone. Patients who were refractory after re-induction (>25% disease), blinatumomab (MRD >0.01%), or for whom there was no available donor were considered for CART. All patients were required to be discussed at a national tumour board, and data were subsequently collected every 3 months for analysis. Events were defined as death, relapse, and secondary cancer. Results Between September 2018 and July 2022, 111 patients were treated on the pathway; 63 (56.8%) were HR and 48 (43.2%) were SR. There were 26 patients >15yr, 3 Infant ALL, 2 Ph+ ALL and 4 with Down Syndrome. Induction therapy achieved disease reduction to <5% and CNS clearance in 98 patients (88%). Of the 90 patients with BM involvement, 52.2% required only 15 days of induction therapy. Induction failure (>25% blasts) occurred in 6.3%, comparable to the 7.1% using the R3 induction in the COG AALL1331 trial (p=1.00); 6/7 patients with induction failure successfully underwent CART infusion. The rate of end of induction MRD <0.01% in the HR group was also similar to COG AALL1331 (30% vs. 27.4%, p=0.669). Importantly, there were no cases of TRM with the reduced intensity induction, compared to 2.4-4% reported with the ALL R3 induction (p=0.06). Ninety patients received blinatumomab, of whom 87% achieved a complete MRD response, with grade 3 or 4 treatment-related toxicity in 11 patients (12.2%). Amongst HR patients, 86.8% underwent HSCT, significantly more than in the blinatumomab-treated IR/HR arm of COG AALL1331 (66%, p=0.02). With a median follow-up of 12 months (range 2-49), 19% of HR patients and 8.3% of SR patients have relapsed, comparable to results achieved in the blinatumomab-treated arm of the COG AALL1331 trial. There were no deaths in the SR group, and 12 deaths in the HR group (6 due to relapse/refractory disease, 6 due to post-HSCT TRM). Conclusions Our findings demonstrate that a reduced intensity reinduction can achieve equivalent disease control to the more intensive ALL R3 induction, with a substantial reduction in TRM. Although follow-up is short, consolidative blinatumomab achieved very high rates of MRD clearance, allowing a greater number of HR patients to reach potentially curative therapy with HSCT, the key predictor of outcome in the COG AALL1331 trial. We propose that our strategy represents a new standard of care for reinduction therapy in relapsed childhood B-ALL. DO and AV are joint senior authors Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutational Mechanisms in Pediatric Acute Lymphoblastic Leukemia with Multiple Relapses

Pediatric acute lymphoblastic leukemia (ALL) is generally marked by a low mutational load at initial diagnosis, which increases in subsequent relapses, partly because of hypermutation in a subset of cases (Waanders et al., 2020, Blood Canc. Disc., 1:96). This increased mutational load can be driven by intrinsic mutational processes or could be therapy related. In this study, we have investigated these mutational processes in pediatric ALL patients who suffered multiple relapses, to determine which processes are active in (relapsed) ALL and how they behave during disease progression before and after therapy. We identified 63 historical cases of ALL with multiple relapses, and selected patients by availability of material and blast percentage above 70% in multiple leukemic samples. If viable material with less than 70% blasts was available, mononuclear cells were enriched by sorting for CD10 positivity. We collected and whole genome sequenced 97 tumor samples from 28 ALL patients (27 precursor B-ALL and 1 T-ALL) with 2 (n=17) or more (n=11) relapses, and 44 remission samples. Somatic single nucleotide variants (SNVs) were called by Mutect2. SNVs were clustered per sample based on their dynamics in time and all clusters in all patients were included for de novo signature extraction using the R package MutationalPatterns. A mutational process was deemed active in a sample if >150 SNVs and >10% of SNVs in a cluster were attributed to a mutational process by de novo signature extraction. The majority of ALL patients with multiple relapses had a mutational load above average, compared to an unselected diagnosis cohort, which often increased during disease progression. Six cases (21%) passed the hypermutation threshold of 1.3 mutations/Mb as previously defined (Waanders et al., 2020). However, mutational signature analysis revealed that mutational processes were active in a much larger proportion of cases. We extracted 7 single base substitution (SBS) signatures, of which 4 were known COSMIC signatures (cosine similarity >0.95) and 2 were similar to known COSMIC signatures (cosine similarity >0.8). At time of diagnosis 4 distinct mutational processes were found to be active alone or in combination in 10 cases (36%), which is much higher than an unselected diagnosis cohort (13%). These processes included APOBEC-associated mutagenesis (SBS2 & SBS13), a mutational pattern resembling UV-like damage (SBS7a), a process that may be caused by reactive oxygen species (SBS18-like) and an unknown process (Fig. 1). By clustering SNVs per patient in time we followed the activity of mutational processes and identified rising and falling (sub)clones. APOBEC mutagenesis was found to be an ongoing process, sometimes appearing for the first time at relapse (Fig. 1B), although when xenografted in mice (n=2) no continued mutagenesis was observed. SBS7a or SBS18-like mutagenesis always occurred already at diagnosis. One patient, who relapsed 5 times, acquired at each presentation between 500-1500 new SNVs with a specific pattern that did not match any known cosmic mutational signatures, and may represent a novel intrinsic mutational mechanism. At time of relapse, additional (mostly treatment-related) mutational processes became active, eventually affecting 17/28 (61%) patients (Fig. 1A), much higher than observed in cases with single relapses (~40%; Li et al., 2020, Blood, 135:41). We observed the thiopurine-associated SBS87 in 9 (32%) patients, and an unexplained SBS86-like mutational signature in 2 patients (Fig. 1B). Furthermore, we noticed SBS17-like mutagenesis in one patient. The etiology of SBS17 is as yet not established, but it has been proposed to be caused by reactive oxygen species. To conclude, ALL patients with multiple relapses showed an increased prevalence and large variety of interplaying mutational processes at diagnosis (36%) and final relapse (61%), with intrinsic or therapy induced origins. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blinatumomab Prior to CAR-T Cell Therapy-A Treatment Option Worth Consideration for High Disease Burden.

The optimal bridging therapy before CAR-T cell infusion in pediatric relapsed or refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL) still remains an open question. The administration of blinatumomab prior to CAR-T therapy is controversial since a potential loss of CD19+ target cells may negatively impact the activation, persistence, and, as a consequence, the efficacy of subsequently used CAR-T cells. Here, we report a single-center experience in seven children with chemorefractory BCP-ALL treated with blinatumomab before CAR-T cell therapy either to reduce disease burden before apheresis (six patients) or as a bridging therapy (two patients). All patients responded to blinatumomab except one. At the time of CAR-T cell infusion, all patients were in cytological complete remission (CR). Four patients had low positive PCR-MRD, and the remaining three were MRD-negative. All patients remained in CR at day +28 after CAR-T infusion, and six out of seven patients were MRD-negative. With a median follow-up of 497 days, four patients remain in CR and MRD-negative. Three children relapsed with CD19 negative disease: two of them died, and one, who previously did not respond to blinatumomab, was successfully rescued by stem cell transplant. To conclude, blinatumomab can effectively lower disease burden with fewer side effects than standard chemotherapeutics. Therefore, it may be a valid option for patients with high-disease burden prior to CAR-T cell therapy without clear evidence of compromising efficacy; however, further investigations are necessary.

Trends of Vancomycin-Resistant Enterococcus Infections in Cancer Patients.

Objective Vancomycin-resistant Enterococcus (VRE) is an important cause of infection in immunocompromised populations. In Pakistan, very limited data are available regarding Enterococcus infection and its outcomes. We conducted this study to evaluate the trends including risk factors, treatment options, and outcomes of infections due to vancomycin-resistant enterococci in cancer patients in Pakistan. Methods We conducted a retrospective observational study. We extracted data from medical records of our center over a period of seven years. All admitted cancer patients with any vancomycin-resistant Enterococcus positive culture were included. The following parameters were evaluated: age, gender, type of cancer, febrile neutropenia, prior antibiotics, admission, comorbidities, system-wise infections (including bacteremia, catheter-related infection, pneumonia, urinary tract infections, intra-abdominal infection, bone and joint infections, skin and skin structure infections), intensive care unit admission, and 30-day all-cause mortality. Frequencies of infections, mortality, and drug susceptibility were evaluated over the course of seven years. Results Risk factors for enterococcal infection included prior exposure of piperacillin/tazobactam (n=209, 86.7%), meropenem (n=132, 54.8%), vancomycin (n=126, 52.3%), metronidazole (n=67, 27.8%), prior admission for more than 48 hours (n=198, 82.2%), and comorbidities (n=76, 31.5%), with acute kidney injury being most common (n=72, 95%) followed by diabetes mellitus (n=70, 92.1%). Precursor B cell acute lymphoblastic leukemia (pre-B ALL) was the most common malignancy in which infection occurred (n=54, 38.3%). Among patients who developed infection, 46% (n=111) had febrile neutropenia. Enterococcus species caused infection in 61% (n=147) and Enterococcus faecium in 39% (n=94). Bacteremia occurred in 45.2% (n=109) patients followed by urinary tract and intra-abdominal infection; 45.6% (n=110) patients were admitted to ICU, and 30-day all-cause mortality was 44.8% (n=108). Linezolid sensitivity was 100%. The total number of enterococci infections decreased over seven years. Frequency of E. species infection, bacteremia, intra-abdominal, skin-related infections, and recurrent infection also decreased, but the frequency of E. facium infections, ICU admission, and 30-day all-cause mortality was increased. Conclusion VRE infections have become less frequent but more severe in recent years with increase in mortality. Prior use of antibiotics (including piperacillin/tazobactam, vancomycin, carbapenems, and metronidazole), diagnosis of hematological malignancy, febrile neutropenia, diabetes mellitus, and renal failure are the risk factors for VRE infection. Bacteremia was the most common infection with high mortality rate. All strains remain sensitive to linezolid. Patients with these risk factors should be worked up for VRE and can be treated with linezolid empirically.

Primary Chronic Myelogenous Leukemia Blast Crisis with Precursor B Lymphoblastic Leukemia, a Case Report

Abstract Introduction/Objective Primary chronic myelogenous leukemia (CML) blast crisis at the initial disease presentation is rare. Most CML blast crisis cases present with increased myeloblasts with a minority of patients showing lymphoblastic leukemia. Differentiating primary CML lymphoblast crisis from de novo acute lymphoblastic leukemia may represent a diagnostic challenge to both pathologists and treating clinicians. This distinction is important as it has significant implications on patient management. Methods/Case Report A 15-year-old male patient was admitted to our University Hospital for hyperleukocytosis. Patient reportedly had weight loss with occasional sweats and cervical lymphadenopathy. She was found to have massive splenomegaly. Peripheral blood showed hyperleukocytosis with predominance of granulocytes at all maturation stages ranging from blast to segmented neutrophils with increased blasts. Subsequent bone marrow findings were consistent with extensive involvement by B-lymphoblastic leukemia. Ten-color Flow cytometry showed approximately 30% blasts with rare lymphocytes and monocytes. The blasts revealed precursor B-lymphoblastic immunophenotypic expression of CD45, CD10, CD19, CD20, CD22, CD34, CD38, CD200, HLA-DR and TdT expression. The results were similar to that of peripheral blood. Granulocytes showed abnormal maturation pattern with increased immature precursors and partial expression of CD4 and CD56 with no abnormalities detected in lymphocytes. In this case, while the bone marrow findings are consistent with B-lymphoblastic leukemia, the peripheral blood findings are consistent with blast phase of chronic myeloid leukemia. Further evaluation by cytogenetic and molecular studies confirmed the presence of Philadelphia chromosome, p210 transcripts, and rearrangement of BCR-ABL1, which supported the impression of precursor B-lymphoblastic leukemia in primary blast phase of CML. The patient was treated with tyrosine kinase inhibitor combined chemotherapy and went into remission. She has been followed up without significant complications for a year. Results (if a Case Study enter NA) NA. Conclusion The diagnosis of CML in primary lymphoblastic crisis is rare and needs to be systemically excluded before giving the diagnosis of de novo BCR-ABL1-positive acute lymphoblastic leukemia. If the patient does not have splenomegaly or previous leukocytosis, it needs cytological examination and extensive molecular analyses.