Positive Acute Lymphoblastic Leukemia Research Articles

A multicentre, randomized trial in adults with de novo Philadelphia-Chromosome positive acute lymphoblastic leukemia to assess the efficacy of ponatinib versus imatinib in combination with low-intensity chemotherapy, to compare end of therapy with indication for SCT versus TKI, blinatumomab and chemotherapy in optimal responders and to evaluate blinatumomab in suboptimal responders (GMALL-EVOLVE).

Introduction Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) is treated as standard of care (SoC) by imatinib based treatment combined with induction and consolidation chemotherapy followed by allogeneic stem cell transplantation (SCT) in first remission. The German Multicenter ALL Study Group for Adult ALL (GMALL) reports about a trial to evaluate the impact of ponatinib based therapy, blinatumomab (blina) treatment for suboptimal responders and the possibility of omission of SoC Allo SCT in optimal responders entitled GMALL-EVOLVE. Methods Herein Imatinib is randomized vs. Ponatinib as frontline treatment combined with chemotherapy, optimal responders also get randomized between SCT and chemo-immunotherapy and suboptimal responders receive immunotherapy before SCT. The trial is registered under the EudraCT number 2022-000760-21. Conclusion This trial will answer several major questions in the treatment of Ph+ALL.

CAR-T Therapy Followed by Hematopoietic Stem Cell Transplantation Can Improve Survival in Children Relapsed/Refractory Philadelphia Chromosome-positive B-cell Acute Lymphoblastic Leukemia.

Philadelphia chromosome (Ph)-positive B-cell acute lymphoblastic leukemia (ALL) has a high complete remission (CR) rate, but relapse and prolonged measurable residual disease remain serious problems. We sought to describe the CR rate measurable residual disease negative rate and address the results and safety of pediatric patients who underwent after receiving chimeric antigen receptor (CAR) specific for CD19 (CAR-19) followed by hematopoietic stem cell transplantation (HSCT) for the treatment of Ph-positive ALL. A descriptive study was conducted at Peking University People's Hospital from September 2013 to January 2021. 13 patients with relapsed/refractory Ph-positive B-ALL who received CAR-T therapy followed by allo-HSCT were included. We concentrated on the overall patient survival and CR rate. The median time between CAR-T therapy and allo-HSCT was 58 days. Among all the patients, the CR rate was 100%, the flow cytometry negativity rate was 84.62%, and the BCR-ABL negativity rate was 53.85% at 1 month after CAR-T infusion. All the patients achieved a major molecular response in 6 months after HSCT. After a median follow-up of 45 months, the 3-year OS rate was 66.7%, and the 3-year DFS rate was 61.5%. The 3-year OS rate of patients with BCR-ABL-positive pre-HSCT was significantly lower than that in the BCR-ABL-negative group (40.0% vs. 85.7%, P=0.042). Also, the same trend was observed for the 3-year DFS rate but did not differ significantly (40.0% vs. 75.0%, P=0.233). CAR-T therapy followed by allo-HSCT can be a safe and effective treatment for Ph-positive B-ALL pediatric patients.

Impact of minimal residual disease response and of status of disease on survival after blinatumomab in B-cell acute lymphoblastic leukemia: results from a real-life study.

Blinatumomab is a bispecific T-cell engager approved for relapsed/refractory and minimal residual disease positive B-cell Acute Lymphoblastic Leukemia. We conducted a retrospective study evaluating the outcome of Blinatumomab. The impact of clinical and treatment-related variables on cumulative incidence of relapse/progression (CIRP), event-free (EFS) and overall survival (OS) was analyzed. From January 2016 to December 2022 50 Ph'- (37) and Ph+ (13) B-ALL patients received Blinatumomab. The median age was 37. Indications to blinatumomab were relapsed/refractory B-ALL in 29 and MRD-positive in 21 patients. Blinatumomab was the 2nd and 3rd line in 40 and in 10 patients, respectively. Twenty patients were treated pre-transplantation, ten were treated for relapse after transplant, twenty were not eligible for transplant. Out of 29 patients treated for relapsed/refractory disease, 16 (55%) achieved complete response and 12 achieved MRD-negativity. Out of 21 patients treated for MRD, 16 (76%) achieved MRD-negativity. At a median follow-up of 46 months the median EFS and OS were 11.5 and 16.2 months. The CIRP was 50%. In univariate analysis age, disease-status (overt vs. minimal disease) at blinatumomab, bridging to transplant after blinatumomab and MRD-response resulted significant for EFS and OS. In multivariate analysis only disease-status and MRD-response retained significance both for EFS and OS. Disease-status and MRD-response resulted significant for EFS and OS also after censoring at HSCT. This retrospective study on B-ALL patients treated with blinatumomab confirms a superior outcome for MRD-responsive over MRD non-responsive patients. Survival depends also on the disease-status prior treatment.

Abstract 5117: CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia

Abstract Survival for pre-B cell Acute lymphoblastic leukemia (B ALL) has improved greatly, however. High risk (HR) subgroups continue to result in significant mortality and morbidity of pediatric oncology patients. Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a lethal disease, often associated with high-risk clinical features. T-ALL therapy has made less improvement than B ALL and continues to represent a significant number of high-risk outcomes. Novel treatment strategies are required to overcome chemotherapy resistance and improve morbidity for HR B ALL. The development of treatments that can overcome chemotherapy resistance is essential for improving survival in T-ALL. Deletions of IKZF1, resulting in decreased IKAROS function, are present in High-risk ALL patients. Casein kinase 2 (CK2), a protein kinase upregulated in ALL, phosphorylates IKAROS preventing DNA binding reducing transcriptional regulation in ALL patients. Past studies have shown that pharmacologic inhibition of CK2 with CX-4945 rescues IKAROS function as a transcriptional regulator. Receptor tyrosine kinase c-KIT is transcriptionally regulated by IKAROS and is often upregulated in cancer. The purpose of this study was to determine whether restoration of IKAROS via CX-4945 would act in a synergistic manner with c-KIT inhibitor imatinib in ALL.Methods/results B-ALL Nalm6 cells were treated with imatinib over 72-hours to establish the half-maximal inhibitory concentration (IC50). The IC50 was used to determine the concentration range of imatinib to be used in combination with CX-4945 in a 72-hour synergy analysis of cytotoxicity.. Cell viability was measured using WST-1 cell proliferation assay. The data were analyzed using Calcusyn and synergy was defined as a combination index below 0.85. The study was repeated to determine a more precise range of synergy. The cytotoxicity of single drug c-KIT inhibitor, Imatinib, in Nalm6 B-ALL yielded an IC50 of 25uM while CK2 inhibitor CX-4945 resulted in 4uM. When a combination was administered with a constant dose of CX-4945 at 4uM and varying concentrations of imatinib, synergy was observed at multiple dose combinations. Conclusion: The data shows that CK2 inhibitor, CX-4945, and c-KIT inhibitor, imatinib, exhibit a synergistic therapeutic effect. Imatinib is indicated for use in Philadelphia positive (Ph+) acute lymphoblastic leukemia so future studies will include testing CX-4945/imatinib synergy in Ph+ BALL where we expect to see more potent synergistic effect. Citation Format: Daniel Gary Bogush, Joseph Schramm, Katarina Dovat, Yali Ding, Chingakham Singh, Sinisa Dovat. CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5117.

Tyrosine kinase inhibitor resistance in de novo BCR::ABL1–positive BCP-ALL beyond kinase domain mutations

AbstractA better understanding of ABL1 kinase domain mutation–independent causes of tyrosine kinase inhibitor (TKI) resistance is needed for BCR::ABL1–positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although TKIs have dramatically improved outcomes, a subset of patients still experiences relapsed or refractory disease. We aimed to identify potential biomarkers of intrinsic TKI resistance at diagnosis in samples from 32 pediatric and 19 adult patients with BCR::ABL1–positive BCP-ALL. Reduced ex vivo imatinib sensitivity was observed in cells derived from newly diagnosed patients who relapsed after combined TKI and chemotherapy treatment compared with cells derived from patients who remained in continuous complete remission. We observed that ex vivo imatinib resistance was inversely correlated with the amount of (phosphorylated) BCR::ABL1/ABL1 protein present in samples that were taken at diagnosis without prior TKI exposure. This suggests an intrinsic cause of TKI resistance that is independent of functional BCR::ABL1 signaling. Simultaneous deletions of IKZF1 and CDKN2A/B and/or PAX5 (IKZF1plus), as well as deletions of PAX5 alone, were related to ex vivo imatinib resistance. In addition, somatic lesions involving ZEB2, SETD2, SH2B3, and CRLF2 were associated with reduced ex vivo imatinib sensitivity. Our data suggest that the poor prognostic value of IKZF1(plus) deletions is linked to intrinsic mechanisms of TKI resistance other than ABL1 kinase domain mutations in newly diagnosed pediatric and adult BCR::ABL1–positive BCP-ALL.

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Avatrombopag Safe and Effective in Treatment of Severe Refractory Thrombocytopenia in a Pediatric Patient with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Following Allogeneic Hematopoietic Stem Cell Transplantation

Avatrombopag Safe and Effective in Treatment of Severe Refractory Thrombocytopenia in a Pediatric Patient with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Following Allogeneic Hematopoietic Stem Cell Transplantation

Dasatinib induced nephrotic range proteinuria in pediatric patient with acute lymphoblastic leukemia with hyper-eosinophilia

Tyrosine kinase inhibitors (TKIs) have improved outcomes for Philadelphia chromosome positive acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). There has been reports of dasatinib induced proteinuria or nephrotic syndrome. We present a case of a 14-year-old male with high-risk B- ALL who developed nephrotic syndrome due to dasatinib. Proteinuria and nephrotic syndrome should be considered as related adverse effects from dasatinib. This is the first reported case of dasatinib induced nephrotic range proteinuria in a patient with high-risk B-ALL with hyper-eosinophilia.

Long-Term Follow up for SWOG 1318: Combination of Dasatinib, Prednisone, and Blinatumomab for Older Patients with Philadelphia-Chromosome (Ph) Positive Acute Lymphoblastic Leukemia (ALL)

Tyrosine kinase inhibitors (TKIs) have improved the outcomes of patients with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Many older patients (pts) are not good candidates for intensive chemotherapy and are treated with TKIs plus corticosteroids or low-intensity chemotherapy. Although the remission rates with this approach have been high, the median disease-free survival (DFS) has been short. Therefore, novel treatment strategies are needed. This trial evaluated the feasibility of combining the TKI dasatinib with prednisone and blinatumomab in older pts with Ph+ ALL. Here we present updated results (median follow-up, 4.3 years). Methods: This trial was activated through the NCTN in January 2015 and closed to accrual in April 2021. Pt eligibility included: age ≥ 65 years; Ph+ or Ph-like ALL (with dasatinib-sensitive fusions or mutations); newly diagnosed or relapsed/ refractory; no evidence of central nervous system (CNS) disease; and adequate organ function. Treatment: For induction, pts received dasatinib 140 mg/d orally (PO) Days 1-56 along with prednisone 60 mg/m 2/d PO Days 1-24. Pts achieving complete remission (CR) or CR with incomplete count recovery (CRi) (Day 28 or Day 56) continued dasatinib until Day 84, followed by 3 cycles of post-remission therapy (PRT) with blinatumomab/ dasatinib. Pts not achieving CR or CRi by Day 56 received re-induction with blinatumomab. The response rate was assessed on Day 35 of blinatumomab. Pts not achieving CR/ CRi could receive a second cycle of blinatumomab. This was followed by 3 cycles of PRT with blinatumomab/ dasatinib. Maintenance therapy consisted of prednisone 60 mg/m 2/d x 5 days every 28 days for a total of 18 cycles, along with dasatinib 140 mg po qd indefinitely. CNS prophylaxis included intrathecal (IT) methotrexate every 4-6 weeks x 8 doses. IT methotrexate was given at least 2 days apart from blinatumomab. Response was assessed at Days 28, 56, and 84, and additional time points were dependent on response. Minimal residual disease (MRD) was assessed centrally by multi-color flow cytometry on Day 28. Statistics: A total of 24 eligible patients were accrued. The primary objective was the feasibility of the combination therapy. The combination was to be deemed feasible if the dose-limiting toxicity (DLT) was < 33%. DLT included: > Grade 3 non-hematologic toxicities (excluding nausea, vomiting, diarrhea) and Grade 4 neutropenia lasting > 42 days. The primary results were reported in 2022 with a median of 2.7 years of follow-up. Herein, we report long-term outcomes for these patients. Results: The median age was 73 years (range 65-87). All pts had newly diagnosed Ph+ ALL; 79% of Ph+ pts had additional cytogenetic abnormalities. During induction, 2 pts experienced treatment-related non-hematologic Grade 4 toxicities. No Grade 4 or higher treatment-related non-hematologic toxicities occurred during post-remission therapy or maintenance. The toxicity of the combination was deemed acceptable. Twenty-two of 24 pts (92%) achieved a CR during dasatinib and prednisone induction therapy. Four did not receive PRT (2 due to adverse events, 1 to receive a transplant, and 1 because of insurance issues). Sixteen pts who achieved CR had MRD data. Six of 16 pts (38%) were MRD undetectable by multi-color flow cytometry on Day 28. Most sites also monitored patients' blood or bone marrow with real-time quantitative PCR to quantify the BCR-ABL1 transcript for molecular response. Of 19 patients analyzed, 17 (89%) were in a major or complete molecular remission at some time point after treatment, with at least 12 of these patients achieving complete molecular remission. Four patients remain on maintenance. The median follow- up for pts who are alive is 4.3 years (range 2.6-6.5 years). The median overall survival (OS) is 6.5 years (95% CI 3-NA) and the median DFS has not been reached as of June 29, 2023. Kaplan-Meier 3-year estimates of OS and DFS are 75% (95% CI 52%-88%) and 72% (95% CI 49%-87%), respectively (Figure). Of the 8 relapses, 4 patients were CD19+ and 3 had T315I mutations. Conclusions: Median DFS and OS with dasatinib and blinatumomab in this older group of Ph+ ALL patients remain excellent despite only 1 patient proceeding to an allogeneic transplant. Further correlative studies (single cell transcriptomics and DNA methylation sequencing) are being planned to evaluate predictors of relapse and will be presented at the meeting.

Chemotherapy-Free Combination of Blinatumomab and Ponatinib in Adults with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Updates from a Phase II Trial

Background Blinatumomab and ponatinib demonstrated efficacy in patients with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). A chemotherapy-free regimen combining both drugs in the frontline setting may mitigate the risk of toxicities and the need for hematopoietic stem cell transplantation (HSCT). Methods In this phase II trial, patients ≥18 years of age with newly diagnosed Ph-positive ALL were eligible. They were required to have a performance status of ≤2, total bilirubin ≤2x the upper limit of normal (ULN), and ALT/AST ≤3x the ULN. Patients with uncontrolled cardiovascular disease or clinically significant central nervous system (CNS) comorbidities (except for CNS leukemia) were excluded. Patients received up to 5 cycles of blinatumomab in combination with ponatinib, followed by ponatinib maintenance for ≥5 years. Ponatinib 30mg daily was given during Cycle 1 and decreased to 15mg daily once a complete molecular response (CMR) was achieved. Patients also received 12 doses of prophylactic intrathecal (IT) chemotherapy with alternating cytarabine and methotrexate. The primary endpoint was the CMR rate. Secondary endpoints included response rates, safety measures, event-free survival (EFS), and overall survival (OS). Results Between June 2018 and May 2023, 62 pts with newly diagnosed Ph-positive ALL were treated. Baseline characteristics are shown in Table 1. Twenty-two patients were in complete remission (CR) at the start of therapy. Among 40 patients evaluable for hematologic response, 39 (98%) achieved CR/CRi; 1 patient had early death. Among 55 patients evaluable for molecular response, 37 (67%) achieved CMR after 1 cycle, and 46 (84%) achieved CMR at any time. Forty-seven patients were evaluated for measurable residual disease (MRD) by next-generation sequencing at a sensitivity level of 10 -6, of whom 43 (91%) were found to have negative MRD. Five of these patients with undetectable MRD by NGS had low-level BCR:ABL1 transcripts detected by PCR at the same time (ranging from 0.01% to 1.23% IS). Four of them had p190 transcript and one had p210. The median follow-up was 17 months (range, 2-61 months). Six patients (10%) relapsed after a median of 21 months of remission (range, 8-24 months): two relapsed in the bone marrow (one with acquired E225V mutation), one had an extramedullary-only relapse (Ph-negative and MYC-rearranged relapse), and three had a CNS-only relapse (after 20, 22, and 23 months). Four patients died (one from intracranial hemorrhage, one from post-procedural hemorrhage, one from brain aneurysm, and one following CNS relapse with intracranial edema and septic shock). One leukemia-related death occurred on study; the estimated 2-year EFS and OS rates were 77% and 89%, respectively (Figure 1). Only one patient underwent HSCT in first CR due to persistently low-level BCR:ABL1 positivity. Among the 52 patients in ongoing remission without HSCT, the median duration of response was 16 months (range, 2-61). Most adverse events were grade 1-2 and were consistent with the known toxicity profile of the two agents. Ponatinib was discontinued in two patients due to possibly related adverse events (cerebrovascular accident and coronary artery stenosis in one patient each). Conclusions The chemotherapy-free combination of blinatumomab and ponatinib is safe and effective in newly diagnosed Ph-positive ALL, with high rates of MRD negativity. Encouraging duration of remission and OS has been observed without the need for HSCT.

Outcomes of Philadelphia Chromosome (Ph) Positive Acute Lymphoblastic Leukemia: An Indian Multi-Institution, Prospective Study from the Haematology Cancer Consortium (HCC)

Introduction One of the commonest genetic abnormalities seen in adult Acute Lymphoblastic Leukemia (ALL) is the reciprocal translocation between chromosomes (Chr.) 9 and 22, leading to the formation of the BCR-ABL fusion gene. This manifests as a shortened Chr 22 (Philadelphia chromosome, Ph+ALL). Despite the widespread use of tyrosine kinase inhibitors (TKI) which target the BCR-ABL fusion protein, Ph+ALL is considered high risk with more relapses and lesser long-term cures. The Haematology Cancer Consortium (HCC) maintains a multi-center database of several hematological malignancies. The ALL database was established in 2018, and the data of the Ph+ALL patients entered into the registry are presented here. The baseline presenting features and induction outcomes were available for all patients, while the survival data is yet to mature. Methods Prospective data of patients (≥ 18 years) with Ph+ ALL (Diagnosed between January 2018 to December 2022) from 10 centers were entered into an independent centralized online data capture system and analyzed for presenting characteristics, treatment, and survival outcomes. Ph+ ALL was defined by the demonstration of t(9,22) [conventional karyotype/ Fluorescent in situ hybridization (FISH)] or the demonstration of BCR-ABL (by polymerase chain reaction) at diagnosis. Treatment (protocol for ALL, TKI used) was per individual-center preference. Intensive protocols (MCP-841, BFM-95, COG), predominantly meant for children, were labeled as “pediatric-type,” and less intense protocols (GMALL, Hyper CVAD, UKALL) were considered “adult-type”. Results In the four years, 1684 patients with ALL registered and 1409 had been tested for Phor BCR-ABL gene, and 292 were labeled as Ph+ALL [Males: 183(63%), Median age: 35 years (18-77); Subtype: B -ALL: 271(93%), T-ALL: 6 (2%), Mixed phenotype: 13 (4%) ; CNS 2 or 3: 60 (18%)]. Of these, 289 underwent treatment. “Pediatric-type” protocols (BFM, MCP841, ICICLE) were used in 241 (84%) patients, followed by the GMALL protocol in 44 patients (15%) and others in 5 patients. TKIs were used in 234 patients (81%) [Imatinib (29%) and Dasatinib (49%)]. Figure 1 shows the baseline characteristics. Induction complications (all grades) were as follows: tumor lysis (18%), pneumonia (11%), neuropathy (15%), pancreatitis (4%), venous thrombosis (5%), and hyperglycemia (44%). After induction, 72% achieved complete remission (CR), 8% were refractory, and 8% died during induction. The rest (12%) were lost from follow-up during induction and were not evaluable. Post-induction minimal residual disease (MRD) status was assessed in 177 patients and was positive in 75 (42%). To date, 21 patients have undergone allogenic transplants. Survival analysis: After a median follow-up of 12 months (0.06-59), 61 patients have relapsed and 57 have died, and 52 (18%) have been lost from follow-up. The estimated 1-year event-free (EFS), relapse-free (RFS), and overall survival (OS) were 85% (S.E:+/-0.027), 87% (+/- 0.033), and 89%(+/- 0.024) respectively ( Figure 2). No factors affecting prognosis were identified in the current analysis. Conclusions In a large prospective database of 12 centers, we found that most adult Ph+ALLs in India are treated with a pediatric-type intensive protocol. Dasatinib is the preferred TKI, followed by imatinib. There was a high degree of treatment abandonment, with an 18% loss from follow-up within six months (including 12% during the induction phase).

Blinatumomab Therapy for Low Level Minimal Residual Disease Detected By Next-Generation Sequencing in Adult B-Cell Acute Lymphoblastic Leukemia

Introduction: Blinatumomab, a bi-specific T-cell engager, has shown clinical benefit in patients with relapsed-refractory and hematologic complete remission with minimal residual disease (MRD)-positive Acute Lymphoblastic Leukemia (ALL). The BLAST trial (Gökbuget N et al., Blood. 2018) demonstrated improved overall survival (OS) and disease-free survival (DFS) in patients with MRD-positive disease (≥0.1%) who achieved MRD response with Blinatumomab. We sought to evaluate the utility of Blinatumomab in low levels of MRD (<0.1%) using clonoSEQ with a sensitivity of (0.0001%). Methods: Adult (18+) B- ALL patients that underwent treatment for B-cell ALL at Norris Comprehensive Cancer Center (NCCC) between 2016 and 2022 who had residual disease tracked using the Next Generation Sequencing (NGS)-based clonoSEQ assay (Adaptive Biotechnologies, Seattle, WA) were included. Patients who received Blinatumomab for MRD positivity were included only if they had a tracked clonoSEQ sequence within one month before starting treatment. Patients who didn't receive Blinatumomab were included if they had an MRD-positive clonoSEQ report. Those who were relapse/refractory when found to be MRD positive were excluded. Cumulative incidence of relapse (CIR) was evaluated using competing risk regression (Fine-Gray method), with death as a competing outcome. At the same time, OS and DFS were analyzed using Cox proportional hazards model. The start time was set to the date of the MRD report, and patients were censored until the last follow-up. Confounder-adjusted KM curves were calculated using direct standardization. Results: 47 patients were included with a median age at diagnosis of 45 years (range: 21-70). Most were Hispanic (N = 33, 70.2%), and the median follow-up time was 12 months. The 3-year OS, DFS, and CIR were 95.4% (95% CI 89.3-100%), 75.1% (95% CI 60.4-93.5%), and 20.3% (95% CI 7.2-38.0%). Twenty-six patients were treated with Blinatumomab (median two cycles, range 1-9), with most (N= 14, 53.8%) having low levels of MRD (<0.1%). Fourteen patients (53.8%) achieved MRD negativity with a median time to MRD negativity of 3.1 months. There were no relapses or death in patients who achieved MRD negativity in the treated group. However, non-responders with Blinatumomab had one death and two relapses. All patients continued Maintenance therapy after receiving Blinatumomab and did not proceed with the transplant. The patients who received Blinatumomab were more likely to be MRD positive on multiparameter flow cytometry (MFC) (60.0% vs. 28.6%, P = 0.042) but had a similar distribution of patient age, treatment type, and receipt of transplant. Additionally, there was a trend toward increased median percent residual cells on clonoSEQ (0.018% vs. 0.003% P =0.244) and flow (0.02% vs 0% P =0.066) in the Blinatumomab treatment group. On univariate analysis, significant predictors of CIR and DFS were flow percent (DFS: HR = 1.54; 95% CI 1.07-2.23; P = 0.019; CIR: HR = 1.83; 95% CI 1.38-2.41; P<0.001) and clonoSEQ residual cell count(DFS: HR = 1.58; 95% CI 1.04-2.41; P = 0.031, CIR: HR = 1.88; 95% CI 1.18-2.97 P=0.008). When controlling for the number of residual cells on close, patients who received Blinatumomab had significantly better CIR(HR = 0.11; 95% CI 0.02-0.69; P = 0.019) and a trend towards improved DFS(HR = 0.21; 95% CI 0.04-1.28; P = 0.092), but no difference in OS. On subgroup analysis of the 31 patients with low levels of MRD (<0.1%), there was no improvement in DFS for patients who were treated with Blinatumomab (HR = 0.41; 95% CI 0.04-3.91; P = 0.44). Of those with low levels of MRD, only two relapsed, and both did not receive Blinatumomab. Conclusions: In B cell ALL, Blinatumomab may offer a CIR benefit to patients with low levels of MRD as detected on clonoSEQ.

Achievement of Undetectable BCR::ABL1 (uBCR::ABL1) Is Predictive of Improved Survival in Philadelphia Chromosome Positive (Ph+ve) Acute Lymphoblastic Leukemia (ALL) Patients Not Receiving Allogeneic Stem Cell Transplantation

Background With the advent of tyrosine kinase inhibitor (TKI)-based therapy the outcome of Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukemia (ALL) has significantly improved. Moreover, patients (pts) who achieved undetectable (u) BCR::ABL1 at 3 months (mo) from the time of initiating therapy, tend to have improved outcomes (Short et al. Blood 2016; 128 [4];504-507). We conducted a retrospective multicenter study to analyze the predictors of achieving uBCR::ABL1 in adult (> 18 years) Ph + ALL pts. Methods A total of 458 adult Ph + ALL pts from 10 US academic institutions, who were diagnosed between May 2003 and April 2023, were evaluated to assess the rates of uBCR::ABL1 at 3 mo after initiating therapy and factors that predict the achievement of molecular response. Continuous variables were summarized as median (range), while categorical variables were reported as frequency (percentage). Unadjusted comparisons of patient/ treatment related characteristics and molecular response were made using a non-parametric test (continuous variables) or Fisher's exact test (categorical variables). Survival curves were estimated using the Kaplan-Meier method and compared between molecular responses via the log-rank test. Results Baseline characteristics The median age of the pts was 53 years (range [R] 19-85), median white blood cell count (WBC) was 17.6 (10 9/L) (R, 1.2-571), 249 (55%) pts had additional cytogenetic (CG) abnormalities, 98 (22%) pts had p210 fusion protein, and 35 (8%) pts had CNS disease at diagnosis. A higher proportion of pts received TKI in combination with intensive chemotherapy (IC) (69%), than TKI + steroids (15%), TKI + low intensity chemotherapy (LC; combination of vincristine, steroids +/- rituximab) (9%), TKI + blinatumomab (3%) and IC without TKI (4%) during induction. During induction, the highest proportion of pts received 2 nd generation TKI combinations (67%; majority dasatinib 98%), compared to imatinib (19%) or ponatinib (10%): 4% of pts did not receive TKI with induction. The complete remission (CR) rate was 94%; measurable residual disease (MRD) negative by flow cytometry 51% (181/352 evaluable pts) and 58.5% (209/354 evaluable pts) of pts had uBCR::ABL1 by PCR at 3 mo from induction. A total of 212 (46%) pts received allogeneic stem cell transplantation (allo-HCT) in CR1, among them 61% of pts received TKI maintenance post allo-HCT. Predictors of uBCR::ABL1 at 3 months Predictors for uBCR::ABL1 at 3 mo are summarized in Table 1. The proportion of pts with WBC > 100 (45% vs 60%, p= 0.04), p210 fusion protein (35% vs 85%, p= <0.001), additional CG abnormality with monosomy 7 (40% vs 61%, p= 0.03), additional somatic myeloid co-mutation (39% vs 61%, p= 0.09; 94 pts evaluable), received induction with steroid plus TKI (41% vs 63%, p= 0.002) or received imatinib based induction (47% vs 61%,p= 0.07) had inferior uBCR::ABL1 rates at 3 mo. Conversely pts who received ponatinib based induction had significantly better uBCR::ABL1 rates (79% vs 57%, p= 0.009). Survival The median relapse free survival (RFS) was 72.3 mo (95% CI; 50.1-94.4), RFS was numerically higher among pts who achieved uBCR::ABL1 at 3 mo but not statistically significant (84.2 mo vs 62.9 mo, p= 0.20). The median overall survival (OS) was 129.2 mo (95% CI; 81.2-177.2), OS was not significantly different among pts who achieved uBCR::ABL1 at 3 mo (129.2) versus no uBCR::ABL1 (not reached; 50% alive at 94 mo), p= 0.47. In sub-set analysis, among non-transplanted pts, RFS was not statistically significant between those who achieved uBCR::ABL1 vs those who did not (38.9 vs 22.0 mo, respectively p= 0.24). However, the median OS was significantly higher in pts who achieved uBCR::ABL1 at 3 mo compared to pts who did not achieve uBCR::ABL1 at 3 mo: 149.3 vs 77.5 mo, p= 0.04 ( Figure 1). Conclusion In this large multicenter study, we observed significantly inferior uBCR::ABL1 rates among pts with WBC > 100, p210 fusion protein, monosomy 7 CG abnormality, pts receiving induction with steroid plus TKI or imatinib based induction. Conversely, uBCR::ABL1 rates were significantly better in pts receiving ponatinib based induction therapy. Overall, we did not see significant impact of uBCR::ABL1 at 3 mo, on RFS or OS. It is likely subsequent therapies and allo-HCT abrogates the negative impact of not achieving uBCR::ABL1 at 3 mo. However, among non-transplanted pts achievement of uBCR::ABL1 at 3 mo showed a significantly improved OS.

Toxicities Associated with Tyrosine Kinase Inhibitor Maintenance Following Allogeneic Hematopoietic Cell Transplantation in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Background Use of tyrosine kinase inhibitor (TKI) maintenance following allogeneic hematopoietic cell transplantation (HCT) in Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia may help to prevent post HCT relapse but is difficult to administer due to toxicity. However, these toxicities and their impact on maintenance is not fully characterized. Herein, we report our experience at a single, large transplant center to describe toxicities encountered with post HCT TKI maintenance in Ph+ ALL. Methods Ph+ ALL patients (pts) who underwent 1st allogeneic HCT at City of Hope between 2003-2021 and received at least one dose of TKI maintenance were included in this retrospective analysis. Baseline characteristics and toxicities were summarized via descriptive analysis, and groups were compared via Fischer test. Survival analyses were calculated from the time of TKI initiation. Results Baseline characteristics are described in Table 1. Of the 186 pts studied, 50 (26.8%) received imatinib and 136 (73.2%) received a newer generation TKI (NG-TKI: 112 dasatinib, 6 nilotinib, 1 bosutinib, 17 ponatinib) at a median time of 78 days (IQR, 41-134) and 91 days (IQR, 58-129), respectively. A total of 96 patients (51.6%) began TKI maintenance within 90 days of HCT. Post HCT TKI was started prophylactically in 149 patients (80.1%) while 37 patients (19.9%) received TKI preemptively for positive MRD. The MRD status at time of HCT differed between the imatinib group and NG-TKI group. In the imatinib group MRD was negative in 33 of 50 patients (66.0%) compared with 78 of 136 patients (57.4%) in the NG-TKI group (P<0.01). There were 12 (24.0%) vs 18 (13.2%) with an unknown MRD-status at HCT in the imatinib vs NG-TKI groups, respectively ( P<0.01). The median follow-up duration for surviving patients in the entire cohort was 5.5 years (range, 1.0-17.5). The median cumulative TKI exposure was 413.5 days (range, 4-2740). The median cumulative imatinib exposure in patients initially receiving imatinib was 328 days (range, 3-2216), while the median cumulative NG-TKI exposure in patients initially receiving NG-TKI was 268.5 days (range, 3-2740), respectively. The median cumulative exposure of dasatinib, nilotinib, bosutinib and ponatinib were 249 days (range, 3-2740), 222 days (range, 114-839), 371 days, and 576 days (range, 72-921), respectively. Twenty-seven (54.0%) vs 75 (55.2%) patients completed >12 months of initial imatinib and NG-TKI maintenance (P=1.0), respectively. Of the treated patients, 78 pts (41.9%) required at least one dose reduction of their initial TKI due to toxicity; 19 of them (10.2%) required ≥2 dose reductions. When imatinib maintenance was used as the initial TKI, a dose reduction due to toxicity was required in 18 patients (36.0%), of whom 7 patients (14.0%) required ≥2 dose reductions. Initial NG-TKI maintenance required a dose reduction due to toxicity in 60 (44.1%), of whom 12 (8.8%) required ≥2 dose reductions. One hundred and thirty-nine patients (74.7%) had their maintenance TKI therapy interrupted, 122 (65.6%) due to toxicity. Sixty-seven patients (36.0%) did not complete their pre-determined maintenance period owing to toxicity; 70 pts (37.6%) discontinued one TKI, and 11 (5.9%) discontinued two TKIs, due to toxicity. The most common reasons for discontinuing the initial TKI are listed in Table 2, including 45.2% of patients due to toxicity, and 12.9% due to progressive disease. Toxicity-related discontinuations occurred within 6 months on that TKI in 63.0% of patients, and 75.3% within the first 12 months. Five-year overall survival and progression-free survival from the start of TKI were similar between the imatinib and NG-TKI group; 83.1% vs 76.9% (P=0.74) and 72.8% vs 70.5% (P=0.63), respectively (Figure 1). Conclusion Toxicities resulting in dose reductions, interruptions, and discontinuation are common in TKI maintenance post HCT. The toxicity profile (mainly GI and blood count related), frequency, and severity of TKIs in the post HCT setting appears to be different than what has been previously reported in the pre-HCT setting, making it a formidable challenge to meet the recommended cumulative TKI maintenance exposure (2 years) per the NCCN guidelines. While the choice of TKI did not appear to impact on long-term outcomes and relapse rates were not excessive in our analysis, further studies are needed to develop tolerable and personalized TKI maintenance strategies.

Blinatumomab in Combination with Immune Checkpoint Inhibitors (ICIs) of PD-1 and CTLA-4 in Adult Patients with Relapsed/Refractory (R/R) CD19 Positive B-Cell Acute Lymphoblastic Leukemia (ALL): Results of a Phase I Study

Background: Blinatumomab (blina) improves outcomes in R/R CD19+ ALL compared to chemotherapy. However, the overall response rate to blina was 44%, and median overall survival (OS) is just 7.7 mos (Kantarjian. NEJM. 2017). Preclinical studies show increased PD-L1 expression on leukemic blasts potentially contributes to relapse after blina. The addition of PD-1 +/- CTLA-4 blockade to blina leads to increased in vitro T cell proliferation and enhanced cytotoxicity (Feucht. Oncotarget 2016). Thus, blockade of co-inhibitory pathways may enhance blina efficacy in vivo. We describe results of a multi-center phase I study combining blina with immune checkpoint inhibitors (ICIs) targeting PD-1 (nivolumab) +/- CTLA-4 (ipilimumab). Methods: In a dose-escalation study, we evaluated the safety, tolerability, and preliminary efficacy of blina with nivolumab (nivo) +/- ipilimumab (ipi) using a 3+3 design. Pts ³16 years-old with R/R CD19+ B-ALL or mixed phenotype acute leukemia (MPAL) were eligible. Pts ³60 years could be untreated. Table 1 presents the dose escalation schema. Expansion cohorts of 6 pts were planned at the maximum tolerated dose (MTD) for blina + nivo AND blina + nivo + ipi. Pts received up to 5 cycles of blina and 1 year of ICIs. Pts removed from the study during the blina lead-in (days 1-10) were replaced. Dose-limiting toxicities (DLTs) were defined as grade 3+ non-hematologic toxicities requiring the permanent discontinuation of treatment in the first 42 days. Primary endpoints were toxicities and MTD. Secondary endpoints included complete remission (CR), MRD-negativity at a sensitivity of 0.01%, duration of response and OS from treatment initiation. Results: Twenty-seven pts were enrolled to 3 dose levels (14 DLA1, 3 DLB1, and 10 DLB-1) from September 2017-December 2022. Six pts were replaced (4 prior to ICI dosing and 2 requiring subsequent therapy in the DLT window). The median age of enrolled pts was 55 (range 24-84), 14 were male (52%), and median baseline BM blasts were 60% (range 0.2-98%). Baseline characteristics are presented in Table 2. A single DLT (G4 infusion reaction and G3 hypotension) occurred among 6 pts at DLA1. Based on this safety and efficacy data, blina + nivo was expanded to include 6 additional pts without additional DLTs. There were 2 DLTs at DLB1 (G5 pneumonitis and G2 GVHD), which led to de-escalation to DLB-1, where there was 1 DLT (G3 delirium). Grade 2+ immune-related adverse events attributable to ICIs included pneumonitis (G2+G5), rash (G2+G3), transaminitis (G2+G3), colitis (G3), and hypothyroidism (G2). Grade 3+ adverse events attributable to blina included neutropenia (5-G4+3-G3), dysphasia (2-G3), weakness (G3), seizure (G4), and transaminitis (1-G4+4-G3). Among 22 pts evaluable for response, the CR rate was 68% (100% MRD-negative). Among 13 pts with >50% BM blasts at baseline, 62% achieved CR. Pts with B-myeloid MPAL (0/2) and a history of extramedullary disease (1/4) were less likely to respond. Nine responders subsequently relapsed including 3 with isolated extramedullary disease and 2 with CD19-negative disease. As shown in Figure 1, relapse-free survival (RFS) at 1 year was 27% (95% CI 10-46), while OS at 1 year was 63% (95% CI 38-79). Twelve pts underwent allogeneic blood or marrow transplant (alloBMT) following study treatment. At 1 year for alloBMT pts, RFS was 51% (95% CI 19-76) and OS was 61% (95% CI 26-83). Ongoing analyses of changes in T cell subpopulations, co-signaling molecule expression, and single cell RNA seq results will be presented. Conclusions: Combination therapy with blina and ICIs in R/R ALL is safe, feasible, and associated with a high MRD-negative response rate. Long-term survival was promising in comparison to prior results with blina monotherapy, especially following consolidation with alloBMT. A randomized trial of blina +/- nivolumab is needed to confirm the benefit of this combination.

Single-Cell Transcriptomic Profiling of T Cells from Blinatumomab-Treated Patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Reveals Circulating CD8 T Cell Subsets Associated with Treatment Response

Background Blinatumomab (Blina), a bispecific CD3xCD19 T-cell engager (BiTE®), is approved for patients with relapsed/refractory (R/R) and measurable residual disease (MRD) positive B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL). However, a significant percentage of patients do not respond to Blina, and the determinants of resistance are currently unknown. Here, we used Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to identify pre-treatment T cell features associated with response. Methods PBMCs of 13 patients with BCP-ALL who received Blina at the LMU Munich University Hospital from 2016 to 2021 were collected before treatment start. CITE-seq(BD Rhapsody Platform) was performed on a total of 34.907 cells isolated via magnetic-activated cell sorting based on the expression of CD3. An AbSeq panel containing 7 surface markers (CD4, CD8, CCR7, CD45RA, CX3CR1, CD127 and PD1) and a customized T-cell targeted RNA sequencing panel (477 genes) were employed. A Monte-Carlo permutation test was used to compare the proportions of cells in different clusters between Responders (R) and Non-Responders (NR). Significant differences were assumed with a fold change threshold of at least 1.5 (log2FC (LFC) ≥ 0.58) and a false discovery rate (FDR) cutoff of < 0.05. Results We used CITE-seq to characterize T cells in peripheral blood (PB) samples obtained from R/R (n=3) and MRD+ (n=10) BCP-ALL patients. Ten patients (9 MRD+, 1 R/R) responded to the Blina treatment. An integrated strategy based on Reciprocal Principal Component Analysis(RPCA) was implemented and bioinformatic analysis of gene expression mapped in two dimensions via uniform manifold approximation and projection (UMAP) identified 12 CD3 pos distinct clusters. Within CD8 pos T cells of R, we found an increased proportion of CCR7 low Granzyme K(GZMK) high Effector Memory-like cells (GZMK high TEM-like, LFC 0.78, 95CI: 0.66 to 0.92; FDR=0.0012) and a reduced percentage of Naïve cells(CCR7 high LEF1 high CD62L high), when compared to NR (LFC -0.59, 95CI: -0.54 to -0.64; FDR =0.0012, Figure1A). Atrend towards an increased proportion of CCR7 lowCX3CR1 highGZMB high terminal effector-like cells (GZMB high TE-like, LFC 0.57, 95CI: 0.45-0.70; FDR=0.0013) was also noted in CD8 pos T cells of R vs NR. Peripheral CD4 pos T cells of R exhibited a tendency towards an increased proportion of GZMK high cytotoxic T cells (LFC 0.43, 95CI: 0.26 to 0.58; FDR=0.0019) and a reduced percentage of FOXP3 high T regulatory cells (LFC -0.19, 95CI: -0.03 to -0.34; FDR=0.025), compared to NR. Furthermore, we investigated gene expression differences between R and NR cells in each cell cluster. Naïve T cells (CD8 and CD4) of NR showed a significantly increased (LFC>1, FDR<0.05, Figure1B) PRDM1 (Blimp-1) expression, a key promoter of T-cell terminal differentiation that is known to be involved in CD19 CAR-T dysfunction. CD8GZMB high TE-likecells of NR showed upregulated expression of genes involved in chemokine production and cytokine signaling (CCL3, IFNGR1) and downregulated LAIR2, a decoy receptor protecting CD8 T cells from collagen-induced exhaustion. Additionally, TOX, a regulator of T cell exhaustion, was moderately upregulated in CD8GZMB high TE-like cells of NR (LFC 0.41, FDR=0.007). Conclusions Taken together, we were able to identify CD8 T cell subsets that are associated with a response to Blina treatment, as well as gene sets involved in BiTE-mediated T-cell function. Interestingly, the PRDM1 overexpression we found in both CD4 and CD8 naive T cells of Non-Responders has already been linked to T-cell dysfunction in the context of CD19 CAR-T cell therapy. We plan to confirm the results of our analysis in an independent patient cohort. Concomitantly, we will enrich the dataset with results from multi-parameter flow cytometry and in vitro T-cell functional assays conducted using longitudinally collected PB samples from Blina-treated patients. Figure1 A)Log Fold Change(log FC) of the proportion of Responders vs Non Responders CD8 pos cells in each cluster. The dots represent the observed logFC, the horizontal line the bootstrapped 95%Confidence Intervals; PTGDR2= prostaglandin D2 receptor 2; ns= not significant B) Differentially expressed genes in Non Responders vs Responders CD8 pos naïve T cells. FDR=False Discovery Rate; CCL5= Chemokine (C-C motif) ligand 5 ; PRDM1= PR/SET Domain 1;SGK1= serum/glucocorticoid regulated kinase 1.

Beyond Mitigating Immune-Related Adverse Events: Glucocorticoids Ameliorate Bispecific Antibody-Mediated T-Cell Exhaustion I n Vitro

Following the approval of blinatumomab in 2015 T-cell recruiting bispecific antibodies (BsAbs) became the established standard-of-care for relapsed or refractory (r/r) and minimal residual disease positive B-cell precursor acute lymphoblastic leukemia. More recently, there has been a notable increase in the approval of additional BsAb constructs for malignancies such as r/r follicular lymphoma, r/r large B-cell lymphoma, and advanced multiple myeloma (MM), with further approvals anticipated. Despite the clinical success, administration of BsAbs is commonly linked to dose-limiting immune-related adverse events (IrAEs), such as cytokine release syndrome, possibly compromising treatment efficacy. Hence, current BsAb as well as CAR T approvals integrate prophylactic and acute glucocorticoid (GC) application upon occurrence of IrAEs. Surprisingly, despite their immunosuppressive nature, GC administration had no negative impact on response or survival of MM patients receiving CAR T-cell therapy (Wang 2022). Based on these data, we hypothesize that the GC-mediated downregulation of BsAb-induced T-cell function has a dual effect: mitigating IrAEs and ameliorating T-cell exhaustion. Here we tested the effect of simultaneous administration of the GC dexamethasone (dexa) and a CD3xCD19 targeting BsAb on T-cell function and exhaustion. We first evaluated the short-term effect of dexa exposure on BsAb-mediated cytotoxicity by co-culturing healthy donor (HD) T cells with CD19 + cell lines and stimulating with a CD3xCD19 BsAb alone or in combination with 300nM dexa for 3-5 days. In fact, the addition of dexa decreased BsAb-mediated tumor cell lysis against OCI-Ly1 cells after 3 days, but not after 5 days. Surprisingly, however, T-cells showed a significantly increased expansion upon dexa exposure after 3 and 5 days (day 3: Ki-67 MFI ratio BsAb=337.2, +dexa=676.0, p=0.008; day 5: CD2 +-fold change to day 0 BsAb only=7.1; +dexa=11.6; p=0.01). In cytotoxicity assays against murine Ba/F3 cells transduced with human CD19 with or without the co-stimulatory molecule CD86, T cells showed significantly reduced BsAb-mediated cytotoxicity and proliferation upon dexa exposure in the absence of CD86. Intriguingly, however, T-cell function was rescued upon CD86 co-expression. To evaluate long-term effects of GCs on T-cell function and exhaustion, we next used our established long-term in vitro model system to mimic continuous stimulation of T cells with a BsAb ± GCs. HD T cells were co-cultured with CD19 + OCI-Ly1 cells for 28 days and continuously stimulated with a CD3xCD19 BsAb alone or in combination with dexa. T-cell fitness was assessed longitudinally. Surprisingly, but in line with our short-term data, we found increased CD8 + T-cell expansion upon BsAb +dexa co-exposure in long-term cultures (abs. CD8 + cell count day 21 BsAb=1.613, +dexa=34.583). Furthermore, immunophenotyping revealed similar upregulation of the checkpoint inhibitors PD-1, Tim-3, Lag-3 after 7 days. On day 21, however, Lag-3 expression on dexa exposed CD4 + and CD8 + T cells was decreased (MFI ratio BsAb=10.5, +dexa=1.7, p=0.02). Inflammatory cytokine (IL-2, IFN-γ, TNF-α) levels were decreased in the supernatant of dexa-exposed vs BsAb only co-cultures. However, low levels of IFN-γ were maintained in the dexa co-exposed condition over 24 days (mean level IFN-γ day 24 BsAb=53pg/ml, +dexa=1756 pg/ml). Finally, T cells were isolated from cultures every 7 days and tested in 72h cytotoxicity assays, without further exposure to dexa. Intriguingly, T cells previously exposed to dexa maintained a significantly higher cytotoxicity over 28 days (mean % specific lysis day 21 BsAb=34.2, +dexa=90.3, p=0.004). Taken together, our data demonstrate a positive impact of GCs on BsAb-mediated T-cell expansion in short- and long-term setting. Characterization of T cells upon long term BsAb exposure demonstrated an ameliorating effect of GCs on BsAb-induced T-cell exhaustion. Our data suggest that dampening of T-cell activity by GC administration in BsAb-treated patients could serve two purposes: managing IrAEs while simultaneously ameliorating T-cell exhaustion and improving T-cell persistence. In future studies we will further dissect the effect of dexa on the transcriptional landscape of BsAb-stimulated T cells by performing RNA sequencing and also explore the effect of GCs on CAR T-cell function.

A Phase II Study of Low-Intensity Chemotherapy (Mini-Hyper-CVD) and Ponatinib Followed By Blinatumomab and Ponatinib in Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Background Patients with Philadelphia chromosome (Ph)-positive B-cell acute lymphoblastic leukemia (ALL) treated with Hyper-CVAD and ponatinib achieve deep responses and long-term overall survival (OS). We aim to investigate if a reduced intensity regimen of mini-Hyper-CVD (mini-HCVD) with sequential blinatumomab (blina) in combination with ponatinib may further improve outcomes while mitigating the toxicity of intensive chemotherapy and the need for hematopoietic stem cell transplantation (HSCT). Methods Patients age ≥18 years with newly diagnosed (ND) or relapsed/refractory (R/R) Ph-positive ALL, or chronic myeloid leukemia in lymphoid blast phase (CML-LBP) were eligible. Other inclusion criteria included adequate liver, renal and cardiac function, and ECOG performance status ≤2. Patients with baseline central nervous system (CNS) involvement were not excluded. All patients had BCR::ABL1 transcripts and fluorescence in situ hybridization (FISH) for t(9;22) done at baseline. Morphological analysis of FISH patterns was performed in ND patients to identify if the BCR::ABL1 signal was observed in myeloid cells (e.g., segmented neutrophils). Four cycles (C) of mini-HCVD alternating with methotrexate (MTX) + cytarabine were followed by blina 28µg/d given for four weeks every six weeks during C5-8. Patients received ponatinib 30mg daily initially, with a dose reduction to 15mg daily once in complete molecular remission (CMR). CMR was defined as undetectable BCR::ABL1 with an assay sensitivity of 0.01%. Rituximab was given for CD20-positive disease. Maintenance ponatinib, vincristine and prednisone for 15 cycles with blina + ponatinib every three cycles was given, followed by ponatinib for at least five years. All patients without CNS disease received 12 intrathecal injections of MTX or cytarabine. Results 20 patients (12 ND, 4 R/R, 4 CML-LBP) were treated between November 2019 and May 2023. Baseline characteristics are shown in Table 1. One patient with CML-LBP had received prior dasatinib while in chronic phase. The predominant BCR::ABL1 transcript was p190 in 7 (58%) ND patients. All patients achieved a complete remission (CR). Among patients in the ND, R/R, and CML-LBP cohorts, CMR was achieved in 10/12 (83%), 3/4 (75%), and 4/4 (100%) patients, respectively. In the ND cohort, six (50%) patients achieved CMR after C1 and eight (67%) after C3. Five (42%) ND patients had documented BCR::ABL1 signal in myeloid cells by FISH. Four of them achieved CMR, after C1, C3, C4 and C3 maintenance respectively. With a median follow-up of 25 months (range, 2-42), the 2-year continuous remission duration (CRD) and OS rates were 93% and 83% for the entire cohort (Figure 1), and 90% and 82% in the ND cohort, respectively. In the ND cohort, one (8%) patient had isolated CNS relapse (during C4 of maintenance with blina and ponatinib), three (25%) patients died (two in CR due to COVID-19 and one of HSCT complications), and eight (67%) patients are in remission without HSCT. No patients relapsed in the R/R cohort; one patient underwent HSCT, one patient died in CR from MTX-associated disseminated necrotizing leukoencephalopathy, and two patients are in remission without HSCT. None of the CML-LBP relapsed; one patient underwent HSCT in CR. Ponatinib dose was reduced in two patients prior to obtaining CMR (one had pancreatitis, one had cardiomyopathy). One patient switched from ponatinib to dasatinib due to a pulmonary embolism in C2. No patients required dose modification of blina. The 60-day mortality rate was 0%. Conclusion In patients with Ph-positive ALL, the combination of mini-HCVD and ponatinib followed by sequential blina and ponatinib yielded excellent outcomes with durable remissions. The toxicity profile was favorable and HSCT was avoided in most patients with ND disease. Longer follow-up is needed to confirm the durability of responses.

A Study on Heterogeneity and Early Response to Chemotherapy in Pediatric ETV6-RUNX1 Positive Acute Lymphoblastic Leukemia By RNA-Seq Expression Profile

Objective ETV6-RUNX1 positive ALL is the most common type in childhood acute lymphoblastic leukemia(ALL), and it is also a type with good prognosis. However, there are still recurrent or refractory cases in this type, and there is still heterogeneity among individuals.In this research, we investigate the heterogeneity of ETV6-RUNX1 positive ALL in children by RNA-seq expression profile, and to explore the early response to chemotherapy and microenvironment characteristics of different clusters, so as to provide evidences for the exploration of genetic high-risk factors and clinical personalized diagnosis and treatment of such children. Method From September 2019 to June 2021, 72 children with newly diagnosed ETV6-RUNX1 positive ALL in standard risk group were enrolled and treated with CCLG-ALL-2018 chemotherapy protocol, which were all detected by RNA-seq technology. The sequencing results were analyzed by cluster analysis and bioinformatics analysis. We explored the early response to chemotherapy and microenvironment characteristics of the different clusters. Result ETV6-RUNX1 positive ALL were divided into three groups according to the RNA-seq expression profile including 16 cases (22.2%) in ER1 group, 35 cases (48.6%) in ER2 group and 21 cases (29.2%) in ER3 group. The number of patients with co-negative minimal residual disease (MRD) by flow cytometry (MRD-FCM) and next-generation sequencing (MRD-Gene) on the 15th day of chemotherapy in ER1 and ER3 groups was significantly different (3/16 vs. 13/21, P=0.0178), while there were 17 cases (17/35) with co-negative MRD in ER2 group after fifteen-day chemotherapy. It showed that the proportion of NK cells ( P=0.056) and CD8 + T cells ( P=0.047) in peripheral blood in ER1 group were both higher than those in ER3 group. Compared with ER3 group, ER1 group showed low expression of RPL41 and high expression of MRPS28 and SERPIND1, which were mainly involved in stress, proliferation and drug resistance of cells and the differentiation of hematopoietic stem cell. In addition, ER1 group showed lower expression of ribosome-related genes and higher activation of mTOR pathway than ER3 group. Conclusion The heterogeneity in gene expression profile were presented in children with ETV6-RUNX1 positive ALL. Compared with ER3 group, patients in ER1 group may have a poorer early response to chemotherapy with a relatively higher risk due to the stronger proliferation and drug resistance of leukemic cells probably. For patients in ER1 group, even if MRD is negative on the 15th day of themotherapy, it may be necessary to strengthen the observation of early response and consider the treatment for escalated risk in time.

A Phase I/II Study to Investigate the Addition of Venetoclax to Dasatinib and Steroids in Patients with Newly Diagnosed or Relapsed Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL): The Venda Trial, in Progress

Background: Acute Lymphoblastic Leukemia (ALL) is a hematologic malignancy of immature lymphocytes of either B cell or T cell origin. One particularly high -risk subset is Ph+ ALL, characterized by a translocation of chromosome 9 and 22, which generates the constitutively active BCR-ABL1 fusion tyrosine kinase capable of driving proliferation and survival of leukemic cells. Currently treatment options include multi-agent chemotherapy along with an ABL-specific tyrosine kinase inhibitor (TKI), though these regimens can lead to significant toxicity, particularly in older adults. Venetoclax is a potent and selective small molecule inhibitor of the B-cell lymphoma 2 (BCL-2) anti-apoptotic protein. Strong mechanistic and preclinical data have demonstrated strong synergistic activity between dasatinib and venetoclax I xenograft models and ex vivo patient samples, and provide an impetus to further explore the combination of BCR-ABL and BCL-2 inhibition in vivo. Based on these data, we designed a clinical trial to investigate the safety and preliminary efficacy of venetoclax in combination with the second generation TKI dasatinib in Ph+ ALL. Study Design and Methods This is an open label, Phase I/II investigator initiated trial evaluating the addition of venetoclax to dasatinib and steroids (with Rituximab in CD20+ patients). Phase I is the safety and dose-finding portion of the trial combining the BCL2 inhibitor venetoclax with dasatinib and steroids during the first 28 days of induction followed by dasatinib and venetoclax for the final 48 days of induction in newly diagnosed and relapsed Ph+ ALL and mixed phenotype acute leukemia (MPAL) patients, including patients with molecular only relapse. Primary inclusion criteria include a histologically confirmed diagnosis of Ph+ ALL or MPAL. Newly diagnosed Ph+ ALL or MPAL patients may not have received leukemia-directed therapy other than for steroids or hydrea, and relapsed patients must have not had prior dasatinib exposure. Key exclusion criteria include subjects with CML in myeloid blast crisis or Ph+ AML, ECOG < 2 and patients with a history of pleural effusions. n order to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D), a Bayesian toxicity probability interval design applied to a pre-specified targeted dose limiting toxicity rate of 30%. Three dose cohorts investigating increasing doses of venetoclax will be enrolled. Once the RP2D dose has been identified, a Phase II expansion will examine preliminary efficacy of the combination. The primary endpoint is the rate of measureable residual disease (MRD) negative remission rate as assessed by both BCR-ABL RT-PCR and next generation sequencing (NGS) after induction. Secondary endpoints include relapse-free survival (RFS) and overall survival (OS). Correlative studies include BH3 profiling (measure of apoptosis) and ex vivo screening to dasatinib and venetoclax alone and in combination. Consolidation phase with blinatumomab plus dasatinib and venetoclax is planned for the cohort expansion group. Enrollment began in September 2022 and is expected to be complete by September 2025. This trial is registered at clinicaltrials.gov (NCT04872790).