T-cell Acute Lymphoblastic Lymphoma Research Articles

Abstract 6246: De novo mutation discovery in a mouse model and a human patient sample of non-Hodgkin's lymphoma

Abstract Cancer encompasses many different diseases, all typically involving an accumulation of genetic alterations. However, despite the genomic complexity of this group of diseases, cancer genomic studies typically rely on sequence alignment to a "reference genome" prior to analysis. Such approaches can provide gene expression quantification of known, annotated genes. However, they miss the detection of fusion genes, novel transcripts, virus insertions, and other genetic insults that may be present in cancer beyond an altered gene expression profile. Here, we develop a workflow for de novo identification and quantification of "outside of reference genome" genetic aberrations. This workflow leverages klue, a method we developed that is based on the compact de Bruijn graph, which can extract sequences uniquely found in a "tumor" sample but not in a "normal" control sample from genomic sequencing data. We first applied this method to an RNA-seq experiment from the EμSRα-tTA/TetO-MYC mouse model, which is a MYC-driven autochthonous transgenic mouse model of T-cell acute lymphoblastic lymphoma (T-ALL). We extracted k-mer contigs present in the mouse tumor samples but are present neither in the spleens of wild-type mice of the same genetic background nor in the standard mouse reference genome assembly. We then mapped RNA-seq reads from the T-ALL mouse tumor samples to these contigs. The contigs with the highest number of mapped reads (i.e. the highest counts) were run through BLAST to determine their identities. The mapped contigs with the highest counts were the tTA transgenic element sequence and the human MYC transgene. This result showcases this workflow's success in isolating and quantifying "reference-absent” tumor-unique sequences. Next, we applied klue to whole genome sequencing (WGS), whole exome sequencing (WES), and RNA-seq data from a patient with B-cell follicular lymphoma (with matched normal specimen) from the Texas Cancer Research Biobank, as this data is unrestricted open access. Tumor-unique contigs in sequencing reads were extracted and analyzed with BLAST and BLAT. In one case, a nonsynonymous point mutation in the SPI1 (also known as PU.1) oncogene was found. The sequencing reads from the tumor sample showed both the "normal variant" of SPI1 as well as the mutant SPI1:H268P, suggesting a heterozygous mutation. Altogether, these results suggest that the idea of first identifying tumor-unique sequences then doing post hoc analysis of those sequences is a viable approach for de novo mutation discovery in cancer genomics. Citation Format: Delaney K. Sullivan, Eissa Albinali, Mayuko Boffelli, Lior Pachter. De novo mutation discovery in a mouse model and a human patient sample of non-Hodgkin's lymphoma [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 6246.

Nelarabine-containing salvage therapy and conditioning regimen in transplants for pediatric T-cell acute lymphoblastic leukemia and lymphoma.

Therapy for relapsed or refractory (r/r) T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) in children is challenging, and new treatment methods are needed. We retrospectively analyzed eight patients with r/r T-ALL (five patients) and T-LBL (three patients) who were treated with nelarabine (NEL) plus etoposide, cyclophosphamide, and intrathecal therapy, administered 3days apart. Five patients achieved a complete response, and the other three achieved a partial response (PR). All patients underwent hematopoietic stem cell transplantation (HSCT) after two cycles of treatment, except for one patient who received one cycle. Three patients who had previously received HSCT were treated with reduced-intensity conditioning regimens, including fludarabine, melphalan, and NEL; one survived for over 5years after the second HSCT. Grade 2 neuropathy occurred in one patient, but other severe toxicities commonly associated with NEL were not observed during NEL administration in combination with chemotherapy. The 2-year overall survival and event-free survival rates were 60.0% and 36.5%, respectively. The addition of NEL to reinduction chemotherapy was useful in achieving remission and did not lead to excessive toxicity. In addition, a conditioning regimen, including NEL, appeared to be effective in patients who had previously undergone HSCT.

Comparative Analysis of Immune Effector Cell-Associated Hematotoxicity (ICAHT) and Infection Incidence Following CD19-Targeted CAR-T Therapy Versus CD7-Targeted CAR-T Therapy

Introduction CD19 CAR-T cell therapy has been revolutionizing the treatment of refractory /relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) over the past decade. While CD7 CAR-T therapy, an innovative approach, has only recently been applied over the last three years in our center to treat R/R T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). While there are studies and guidelines from EBMT addressing hematologic toxicity, specifically Immune Effector Cell-Associated Hematotoxicity (ICAHT), following CD19 CAR-T treatment, the literature is still scarce on ICAHT occurrence post-CD7 CAR-T therapy. Here we conducted a retrospective study aiming at comparing ICAHT and infection incidences following the two distinct CAR-T therapies. Methods Data were extracted from two separate clinical trials. The CD19 CAR is structured with CD19 scFv, CD28-hinge, 4-1BB costimulatory region, and CD3ζ activation domains. T cells were activated by CD3/CD28 microbead 2 days before CD19-CAR lentivirus infection. “Naturally selected” anti-CD7 CAR-T cells (NS7CAR) were transduced an anti-CD7 CAR into bulk peripheral T lymphocytes via a lentivirus vector. NS7CAR is a 2nd generation murine-based CAR-T containing 4-1BB and CD3ζ co-stimulatory domains. All patients received lymphodepletion regimens of fludarabine (30 mg/m 2/d) and cyclophosphamide (300 mg/m 2/d) from Day -5 to Day -3 before CAR-T infusion. A single infusion of either CD19 CAR-T or CD7 CAR-T cells was administrated at a medium dose of 1×10 6/kg. Fitted patients undergo consolidation allogeneic hematopoietic stem cell transplantation (Allo-HSCT) within 1-3 months after CAR-T cell infusion. The hematology toxicity and infection were analyzed during the period from CD19 CAR-T or CD7 CAR-T cell infusion to Allo-HSCT. Hematology toxicity is graded according to “ICAHT: EHA/EBMT Consumption Grading and Best Practice Recommendations” (Table 1). Infection diagnosis includes both etiological diagnosis and imaging diagnosis. Results Between June 1, 2019, and September 22, 2022, 60 patients with R/R B-ALL patients received CD19 CAR-T therapy (https://clinicaltrials.gov NCT04546893, and NCT04792593). Meanwhile, between December 1, 2020, and July 16, 2022, 60 patients with R/R T-ALL/LBL received CD7 CAR-T therapy (NCT04572308, NCT04916860). The CD19 CART group had 19 patients ≤ 14 years old, with a male/female gender ratio of 33:27. The CD7 CART group had 20 patients ≤ 14 years old, with a gender ratio of 49:11. The incidence of severe early ICAHT (Grade III+IV, day 0-30) from CD19 CAR-T cells infusion to allo-HSCT was observed to be 18.3%, which was lower than the incidence of the early ICAHT post CD7 CAR-T therapy at 33.3%, p =0.035. However, the incidence of severe late ICAHT (after day+30) following CD19 CAR-T and the CD7 CAR-T therapy was similar: 27.8% vs. 28.6%, with no statistical difference( p=0.57). There was no statistical difference between CD19 CAR-T and CD7 CAR-T therapy groups in the infection rates with 28.3%vs. 36.7%, p=0.33. In the CD19 CAR-T group, bacterial infections were noted in 10 patients: 6 with Gram-positive bacteria (1 Enterococcus, 1 Streptococcus agalactis, and 4 Staphylococcus), and 4 with Gram-negative bacteria (1 Pseudomonas aeruginosa, 2 Acinetobacter baumannii, and 1 Klebsiella pneumoniae). Viral infections were observed in 7 patients (EBV 2, CMV 2, BKV 3), and 1 had fungal infection. Infection sites included pulmonary infections (7 cases), sepsis (10 cases), and other areas (4 cases). Post-CD7-CAR-T, bacterial infections were observed in 9 cases, 7 with Gram-positive bacteria (6 Staphylococcus, and 1 Streptococcus bradycardia) and 2 with Gram-negative bacteria (1 Pseudomonas aeruginosa, and 1 Klebsiella pneumoniae). Six cases had viral infections (EBV 2, CMV 2, HHV1 type 1, parainfluenza virus 1), and 6 with fungal infections. Infection sites included pulmonary infection (12 cases), sepsis (10 cases), and other areas (5 cases). Conclusions Our study noted a slightly higher incidence of ICAHT within 30 days following CD7 CAR-T infusion compared to CD19 CAR-T therapy. However, there was no statistical difference in the incidence of ICAHT beyond the 30-day. There was no statistical difference for infection rate after CD19 CAR-T and CD7 CAR-T. In both groups, Gram-positive bacteria were primary responsible for bacterial infections.

Gene-Edited Universal CD7 Chimeric Antigen Receptor T Cells for Pediatric T-Cell Acute Lymphoblastic Leukemia or Lymphoblastic Lymphoma

Background: T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are highly aggressive T-lineage malignancies characterized by an infiltration of immature T cells in the bone marrow (BM) and peripheral blood (PB) or extramedullary organ involvements. Effective treatments were elusive, and the long-term survival of patients with refractory/relapsed (R/R) T-ALL/LBL remains poor despite allogeneic hematopoietic stem cell transplantation (allo-HSCT). CAR-T therapies for T-cell malignancies remain underdeveloped due to the shared antigenicity between normal and malignant T cells. CD7 is highly expressed on the surface of T-ALL/LBL T cells and is considered a viable CAR-T therapeutic target. However, CD7-targeting immunotherapies may be compromised by T-cell fratricide and extermination. And one of the biggest challenges with autologous CAR-T cell therapy is to isolate a sufficient number of healthy T cells from malignant T cells. Thus, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR9-associated protein 9 (Cas9)-mediated gene ablation has been used to produce universal CD7-targeted CAR T cells (UCD7-CAR) from suitable healthy donors that no longer express CD7 and TCR. In vitro killing tests, animal tests, and phase 1 clinical trials have preliminarily verified the efficacy and safety of universal CD7 CAR-T cells. Here we investigated the safety and efficacy of the universal CD7 CAR-T therapy in phase 2 clinical trial(NCT05454241) for pediatric R/R T-ALL/LBL patients. Aims: To investigate the efficacy and safety of the universal CD7 CAR-T therapy for pediatric R/R T-ALL/LBL patients. Methods: Patients over three years of age whose disease failed ≥ 2 lines of therapies and had ≥ 80% CD7 expression were eligible, including those who relapsed within 12 months after complete response or relapsed more than 12 months after complete response and failed to achieve remission induced by one or more courses of standard treatment; and patients with recurrence after hematopoietic stem cell transplantation. A lymphodepletion regimen was administered to all patients using fludarabine (30 mg/m2 per day) and cyclophosphamide (500 mg/m2 per day) for three consecutive days (day -5 to day -3) before CAR-T infusion. Patients received one single infusion of UCD7-CAR T cells at a median dose of 2.0×10^6/kg. Consolidative allo-HSCT after remission was allowed after day 28 after UCD7-CART infusion at the discretion of the physician and patient preference. All surviving patients were followed until July 15, 2023; the median follow-up was 109 days (54 to 117 days). Results: Five eligible children, 1 with r/r T-LBL and 4 with r/r T-ALL, were enrolled between December 2022 and April 2023 (Table 1). The median age of the patients was 5 (4-11) years. One girl(Patient 04) had a history of allo-HSCT before enrollment. Patients were heavily pretreated with a median of 3(2-6) lines of therapies. At registration, the median proportion of blasts in 4 T-ALL patients was 47.275% (6.0%-56.0%) in BM by FCM along with CNS(n=2) or testis(n=1) involvement. In contrast, 1 T-LBL patient(patient 02) did not have BM blasts but with bulky mediastinal masses ≥7 cm in diameter. All five patients had no or mild CRS (no CRS, n=1; grade 1, n=3; grade 2, n=1), and most(n=4) did not show symptoms of neurotoxicity. Patient 04, who received a prior allo-HSCT approximately seven months before CAR-T therapy, had a Herpes virus 6 (HHV-6B) encephalitis on day 17 after CAR-T infusion, but no GVHD was observed. On day 28, 4 of 5 (80.0%) patients who had BM involvement achieved MRD-negative complete remission (CR; n=2) or CR with incomplete hematologic recovery (CRi; n=2); but patient 02 with bulky mediastinal masses did not have an obvious response. Until the cutoff date, patient 04 still kept a sustained MRD negative CR; patient 05 relapsed again 49 days after CAR-T infusion, and the other three patients received a subsequent allo-UCBT with a CR state. All the children are still alive. Conclusion:UCD7-CAR T therapy is a safe and highly effective pediatric T-ALL/LBL treatment. More patients and longer follow-ups are needed for validation.

Phase 1 Dose Escalation Study of the Anti-CD7 CAR-T Therapy in Relapsed/Refractory T-Cell Acute Leukemia and Lymphoblastic Lymphoma

Introduction Inspired by the successful experience of CAR-T therapy in B-cell malignancies, its introduction to T-cell tumors is being studied. CD7, highly expressed on T-cell tumors, provides a potential target for CAR-T therapy. However, it is also present on normal T and NK cells, leading to possible fratricide of CAR-T during the production and elimination of T and NK cells after infusion. Moreover, CD7 was suggested to express on parts of hematopoietic stem cells (HSC). Thus, apart from the clinical efficacy and safety, the manufacture and hematopoietic toxicity (HPT) are essential topics related with anti-CD7 CAR-T therapies. Here, we reported the preliminary results of phase I clinical trial of anti-CD7 CAR-T (SENL101, dual CD7-nanobodies with 4-1BB co-stimulation domain and CD7 gene unmodified) to address these issues in CD7 + T-cell acute leukemia and lymphoblastic lymphoma (T-ALL and T-LBL) patients. Methods Informed volunteers were screened according to the inclusion and exclusion criteria. Specially, tumors negative for CD4 and CD8, or the tumor load in peripheral blood less than 1% are required. CD4 and CD8 positive selection was employed for T-cell isolation. Lentivirus encoding CAR vector was transduced in CD7 unedited T-cells. The clinical trial was a single-arm, open-label, dose-escalation and predictive study. One day after a 3-day FC chemotherapy, patients were infused with SENL101 at a dose of 1E6, 2E6 or 4E6/ kg. The efficacy, safety and pharmacokinetics of CAR-T were assessed at specified time points. Results As of July 24 th 2023, 7 patients were treated with the specified dose of SENL101, including 5 T-LBL and 2 T-ALL. The average age was 33 years old, and the median prior therapy lines was 3. 28.6% patients experienced HSC transplantation (HSCT). SENL101 was successfully produced in all patients, with an average cell viability of 87.1% and CAR + rate of 92.3%. Complete response (CR) rate was 100% ( Figure 1A). One patient (E006) received HSCT after CR and continued to survive at the present. One case (E002) relapsed at 3-month follow-up, with CAR copy number16740 /ug DNA and CD7 negative in T and NK cells. Notably, 1 patient (E003) developed B-cell lymphoproliferative disease, with primary disease well controlled. The other 4 patients were in CR or hemogram incomplete-recovered CR (CRi), with the longest remission of 9 months. Unfortunately, 2 patients (E005 and E008) died of sepsis at day 116 and 137. CAR-T expanded robustly in all cases, with a median peak value of 701600 copies/ ug DNA reached at day 14. The median survival of CAR-T in vivo was 133 days. As the removal of CAR-T, the CD7 + T and NK cells recovered. The cytokine release syndrome (CRS) happened in 6 cases, with 5 grade 1-2 CRS and 1 grade 3 CRS. No immune effector cell-associated neurotoxicity syndrome was observed. All patients occurred grade 4 HPT. At day 28, 28.6% grade 4 leukopenia and 57.1% grade 4 thrombocytopenia persisted ( Figure 1B). Infections were observed in 3 out of 7 cases (including 2 CMV, 1 EBV and 2 sepsis). Other AEs, including transaminase elevation (3/7), malaise (2/7), anorexia (2/7), nausea/vomiting/diarrhea (1/7) and flu like symptoms (1/7) were relieved after treatment. Due to the high infectious rate (2 sepsis) and profound reduction of hemocyte in the 2E6/kg group, 1E6/kg was set as dose of an expanded cohort. Conclusions Withoutinterfering CD7,SENL101 can be well manufactured. The treatment was effective for T-ALL/LBL, with tolerable toxicity at a dose of 1E6 cells/kg. SENL101 expanded robustly but persisted shortly, which on the other hand, contributed to the recovery of CD7 + T and NK cells, thereby reducing the risk of infection. An expanded cohort is warranted to verify the long-term benefit of SENL101 for T-cell malignant patients.

Targeting hyperactive platelet-derived growth factor receptor-β signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematologic malignancies. Current treatment consists of intensive chemotherapy leading to 80% overall survival but is associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient, and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient-derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL, and that screening T-ALL/T-LBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

Integration of Genomic Sequencing Drives Therapeutic Targeting of PDGFRA in T-Cell Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma.

Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) or lymphoblastic lymphoma (T-LBL) have limited therapeutic options. Clinical use of genomic profiling provides an opportunity to identify targetable alterations to inform therapy. We describe a cohort of 14 pediatric patients with relapsed or refractory T-ALL enrolled on the Leukemia Precision-based Therapy (LEAP) Consortium trial (NCT02670525) and a patient with T-LBL, discovering alterations in platelet-derived growth factor receptor-α (PDGFRA) in 3 of these patients. We identified a novel mutation in PDGFRA, p.D842N, and used an integrated structural modeling and molecular biology approach to characterize mutations at D842 to guide therapeutic targeting. We conducted a preclinical study of avapritinib in a mouse patient-derived xenograft (PDX) model of FIP1L1-PDGFRA and PDGFRA p.D842N leukemia. Two patients with T-ALL in the LEAP cohort (14%) had targetable genomic alterations affecting PDGFRA, a FIP1-like 1 protein/PDGFRA (FIP1L1-PDGFRA) fusion and a novel mutation in PDGFRA, p.D842N. The D842N mutation resulted in PDGFRA activation and sensitivity to tested PDGFRA inhibitors. In a T-ALL PDX model, avapritinib treatment led to decreased leukemia burden, significantly prolonged survival, and even cured a subset of mice. Avapritinib treatment was well tolerated and yielded clinical benefit in a patient with refractory T-ALL. Refractory T-ALL has not been fully characterized. Alterations in PDGFRA or other targetable kinases may inform therapy for patients with refractory T-ALL who otherwise have limited treatment options. Clinical genomic profiling, in real time, is needed for fully informed therapeutic decision making.

Diagnostic significance of dysregulated miRNAs in T-cell malignancies and their metabolic roles.

T-cell malignancy is a broad term used for a diverse group of disease subtypes representing dysfunctional malignant T cells transformed at various stages of their clonal evolution. Despite having similar clinical manifestations, these disease groups have different disease progressions and diagnostic parameters. The effective diagnosis and prognosis of such a diverse disease group demands testing of molecular entities that capture footprints of the disease physiology in its entirety. MicroRNAs (miRNAs) are a group of noncoding RNA molecules that regulate the expression of genes and, while doing so, leave behind specific miRNA signatures corresponding to cellular expression status in an altered stage of a disease. Using miRNAs as a diagnostic tool is justified, as they can effectively distinguish expressional diversity between various tumors and within subtypes of T-cell malignancies. As global attention for cancer diagnosis shifts toward liquid biopsy, diagnosis using miRNAs is more relevant in blood cancers than in solid tumors. We also lay forward the diagnostic significance of miRNAs that are indicative of subtype, progression, severity, therapy response, and relapse. This review discusses the potential use and the role of miRNAs, miRNA signatures, or classifiers in the diagnosis of major groups of T-cell malignancies like T-cell acute lymphoblastic lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), extranodal NK/T-cell lymphoma (ENKTCL), and cutaneous T-cell lymphoma (CTCL). The review also briefly discusses major diagnostic miRNAs having prominent metabolic roles in these malignancies to highlight their importance among other dysregulated miRNAs.

Nelarabine combination therapy for relapsed or refractory T-cell acute lymphoblastic lymphoma/leukemia.

Nelarabine, an antimetabolite prodrug, is approved as monotherapy for children and adultswith relapsed and refractory T-cell acute lymphoblastic leukemia and lymphoma (R/R T-ALL/LBL), although it is often used in combination regimens. We sought to understand differences in efficacy and toxicity when nelarabine is administered alone or in combination. We retrospectively analyzed 44 consecutive patients with R/R T-ALL/LBL; 29 of whom were treated with combination therapy, most with cyclophosphamide and etoposide (23, 79%) and 15 with monotherapy. The median age was 19 years (range, 2-69), including 18 children (<18 years). After a median of 1 (range, 1-3) cycle of treatment, 24 patients (55%) achieved complete remission, 62% (18/29) with combination therapy and 40% (6/15) with monotherapy (P= .21). Most responders (21, 88%) pursued allogeneic stem cell transplant (alloSCT). Overall survival (OS) was 12.8 months (95% confidence interval, 6.93-not reached) in the entire cohort and was higher in the combination therapy than in the monotherapy group (24-month OS, 53% vs 8%; P= .003). The rate of neurotoxicity was similar between groups (27% vs 17%; P= .46) and grade 3/4 anemia and thrombocytopenia were more frequent in the combination group (76% vs 20%; P< .001% and 66% vs 27%; P=.014, respectively). In a multivariate analysis, nelarabine combination therapy and alloSCT post nelarabine were associated with improved OS (hazard ratio, 0.41; P= .04 and hazard ratio, 0.25; P= .008, respectively). In conclusion, compared with monotherapy, nelarabine combination therapy was well tolerated and associated with improved survival in pediatric and adult patients with R/R T-ALL/LBL.

A phase 1 study to evaluate the safety, pharmacology, and feasibility of continuous infusion nelarabine in patients with relapsed and/or refractory lymphoid malignancies.

Nelarabine is a purine nucleoside analogue prodrug approved for the treatment of relapsed and refractory T-cell acute lymphoblastic leukemia (R/R T-ALL) and lymphoblastic lymphoma (T-LBL). Although effective in R/R T-ALL, significant neurotoxicity is dose-limiting and such neurotoxicity associated with nucleoside analogues can be related to dosing schedule. The authors conducted a phase 1 study to evaluate the pharmacokinetics and toxicity of nelarabine administered as a continuous infusion (CI) for 5days (120hours), rather than the standard, short-infusion approach. Twenty-nine patients with R/R T-ALL/LBL or T-cell prolymphocytic leukemia (T-PLL) were treated, with escalating doses of nelarabine from 100 to 800mg/m2 /day × 5days. The median age of the patients was 39years (range, 14-77years). The overall response rate was 31%, including 27% complete remission (CR) or CR with incomplete platelet recovery (CRp). Peripheral neuropathy was observed in 34% of patients, including four ≥grade 3 events related to nelarabine. Notably, there was no nelarabine-related central neurotoxicity on study. The maximum tolerated dose was not reached. Pharmacokinetic data suggested no relationship between dose of nelarabine and accumulation of active intracellular ara-GTP metabolite. Higher intracellular ara-GTP concentrations were statistically associated with a favorable clinical response. Preliminary evaluation of continuous infusion schedule of nelarabine suggests that the safety profile is acceptable for this patient population, with clinical activity observed even at low doses and could broaden the use of nelarabine both as single agent and in combinations by potentially mitigating the risk of central nervous system toxicities.

A Novel Universal CD7-Targeted CAR-T Cell Therapy for Relapsed or Refractory T-Cell Acute Lymphoblastic Leukemia and T-Cell Lymphoblastic Lymphoma

Background Universal CAR-T (UCAR-T) using gene-editing techniques has attracted significant attention due to its prompt availability for patients. To date, there are only a few clinical case reports of universal CAR-T therapy for T-cell malignancies. Here, we explored the efficacy and safety of CD7-targeted UCAR-T (RD13-01) cells for relapsed or refractory (R/R) T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) in a phase I clinical trial (NCT04620655). Methods RD13-01 is an allogeneic, healthy donor-derived, and CRISPR/Cas9-edited anti-CD7 CAR-T cell product. RD13-01 consists of a CAR with a CD7-binding single-chain variable fragment (scFv), a 4-1BB costimulatory domain, CD3ζ signaling domain, a γc intracellular domain, in addition to an NK inhibitory (NKi) ligand containing an EC1-EC2 extracellular domain of E-cadherin fused to a CD28 co-stimulatory domain. The CAR and the NKi ligand are linked by an F2A self-cleaving peptide. Intravenous fludarabine (25-30 mg/m2/d), cyclophosphamide (300mg/m2/d) and etoposide (100 mg/m2/d) were given to all patients on Day -6 to Day -3 prior to CD7 UCAR-T cell infusion. Results Between December 2020 and May 2022, 10 patients (7 males, 3 females), 7 with T-ALL and 3 with T-LBL were enrolled. Median patient age was 16.5 years (range: 2-27 years). Patients had a median of 4.5 (2-6) prior lines of therapies. Two patients relapsed from a previous allogeneic hematopoietic stem cell transplant (allo-HSCT) within 6 months. In addition, 3 patients who showed no response (NR) to prior autologous CD7 CAR-T therapy were also included. Patient characteristics are shown in Table 1. Four patients harbored high-risk genotypes including STIL-TAL1, TP53, BCR-ABL, and others. By morphology, the median bone marrow (BM) blasts at enrollment were 60% (4%-96.5%). Of the eight patients with peripheral blood (PB) blasts, the median blasts were 27% (9%-85%). At enrollment, 8 patients had extramedullary disease (EMD) including 3 with central nervous system (CNS) involvement (1 CNS-1, 2 CNS-3), 3 with diffuse involvement, and 3 had localized EMD. The median transfection efficiency of the RD13-01 product was 64.6% (range: 59.0%-91.2%). A single dose of CD7 UCAR-T cells were infused to the first 3 enrolled patients at 0.5-1×107 cells/kg. Subsequently, 6 patients received a medium dose of 2×107cells/kg, and 1 received a high dose of 4×107cells/kg. By the cut-off date of July 11, 2021, the median observation time was 153 days (range: 28-315 days). On day 28 post infusion, 8/10 (80%) patients achieved complete remission (CR) in BM/PB, and 7 of the 8 patients were minimal residual disease (MRD) negative. Notably, the 3 patients who had failed prior autologous CD7 CAR-T therapy achieved CR. The first enrolled 2 patients who received the low dose showed NR and withdrew on day 28. Among the 7 patients with EMD, 4 achieved EMD CR at the median day 30, 1 had progression disease (PD) and 2, with NR in BM, withdrew prior to EMD evaluation. With a median time of 47 days (40-67 days) following infusion, 6 patients in CR (including 1 MRD +CR) underwent consolidation allo-HSCT, and 4/6 remained progression-free longest up to 315 days. However, 2/6 relapsed without CD7 lost on day 186 and day 200, respectively, and subsequently died. In addition, 1 CR patient died from bacterial infection on day 35. One PD patient received salvage transplantation and died from relapse on day 150. Post infusion, 9/10 patients experienced grade I cytokine release syndrome (CRS) and 1/10 had grade 3 CRS. Only 1 patient with CNS-3 experienced grade 3 neurotoxicity. By qPCR, CD7 UCAR-T cells reached a median peak level of 1.43×106 copies /μg genomic DNA (1.11×105~4.33×106)copies /μg genomic DNA, which occurred on day 12 (7-24 days). The median peak of circulating CD7 UCAR-T was 47.6% (0.01%~83.9%) occurring on day 7 (6-19 days), as measured by flow cytometry (Figure.1). Conclusions Our phase I trial showed that the allogeneic "off-the-shelf” RD13-01 product was safe and dose-dependently effective in treating patients with heavily pretreated T-ALL/LBL, including those with EMD and prior allo-HSCT, those who already failed autologous CD7 CAR-T therapy, and who could not manufacture autologous CAR-T from the patient's own PB due to high blasts in PB and those with rapid disease progression. Long-term observation and more patients are needed to further evaluate the safety and efficacy of CD7 UCAR-T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of 53 Patients with Relapsed or Refractory (R/R) T-Cell Acute Lymphoblastic Leukemia (T-ALL) and T-Cell Lymphoblastic Lymphoma (T-LBL) Treated with CD7-Targeted CAR-T Cell Therapy

Analysis of 53 Patients with Relapsed or Refractory (R/R) T-Cell Acute Lymphoblastic Leukemia (T-ALL) and T-Cell Lymphoblastic Lymphoma (T-LBL) Treated with CD7-Targeted CAR-T Cell Therapy

Clinical Profile and Induction Outcome of Children with T-cell Acute Lymphoblastic Leukemia and T-cell Lymphoblastic Lymphoma

Objective: To study the clinical profile and induction outcome in children diagnosed with T-cell acute lymphoblastic leukaemia and lymphoblastic lymphoma.&#x0D; Study Design: Cross-sectional study.&#x0D; Place and Duration of Study: Paediatric Oncology Unit, Combined Military Hospital, Rawalpindi Pakistan, from Jan 2012 to Dec 2020.&#x0D; Methodology: One hundered and twenty-one diagnosed patients of T-ALL and LBL were evaluated with bone marrow biopsy and Contrast-enhanced CT scan of the active region after induction chemotherapy with Dexamethasone, Vincristine, Asparaginase and Daunorubicin along with intrathecal Methotrexate. This was carried out on days-8 and 29 to determine the early remission status and end of induction response, respectively.&#x0D; Results: Out of 121 patients, 99(81.8 %) had T–ALL, while 22(18.18%) had LBL. Day-8 assessment showed that 94(77.7%) patients were rapid early responders, 24(19.4%) were slow early responders, while 3(2.4%) patients expired before the day eight assessment. Day-29 bone marrow assessment of 88 T-ALL patients showed complete remission in 77 patients (87.5%), incomplete remission in 5(5.68%) patients and treatment failure in 2(2.27%) patients. 20(93.1%) patients with LBL achieved remission by Day-29, while 2 (0.02%) LBL patients died before the Day-29 assessment.&#x0D; Conclusion: The remission induction rates and mortality rates in our setup are encouraging and comparable to international data. Further improvements can be made by early management of the affected cases and more accessible pediatric oncology health services.

MYC oncogene elicits tumorigenesis associated with embryonic, ribosomal biogenesis, and tissue-lineage dedifferentiation gene expression changes.

MYC is a transcription factor frequently overexpressed in cancer. To determine how MYC drives the neoplastic phenotype, we performed transcriptomic analysis using a panel of MYC-driven autochthonous transgenic mouse models. We found that MYC elicited gene expression changes mostly in a tissue- and lineage-specific manner across B-cell lymphoma, T-cell acute lymphoblastic lymphoma, hepatocellular carcinoma, renal cell carcinoma, and lung adenocarcinoma. However, despite these gene expression changes being mostly tissue-specific, we uncovered a convergence on a common pattern of upregulation of embryonic stem cell gene programs and downregulation of tissue-of-origin gene programs across MYC-driven cancers. These changes are representative of lineage dedifferentiation, that may be facilitated by epigenetic alterations that occur during tumorigenesis. Moreover, while several cellular processes are represented among embryonic stem cell genes, ribosome biogenesis is most specifically associated with MYC expression in human primary cancers. Altogether, MYC's capability to drive tumorigenesis in diverse tissue types appears to be related to its ability to both drive a core signature of embryonic genes that includes ribosomal biogenesis genes as well as promote tissue and lineage specific dedifferentiation.

Nelarabine, etoposide, and cyclophosphamide in relapsed pediatric T-acute lymphoblastic leukemia and T-lymphoblastic lymphoma (study T2008-002 NECTAR).

Children with relapse of T-cell acute lymphoblastic leukemia (T-ALL) or lymphoblastic lymphoma (T-LBL) have a dismal prognosis, largely due to difficulty attaining second remission. We hypothesized that adding etoposide and cyclophosphamide to the nucleoside analog nelarabine could improve response rates over single-agent nelarabine for relapsed T-ALL and T-LBL. This phase I dose-escalation trial's primary objective was to evaluate the dose and safety of nelarabine given in combination with etoposide at 100mg/m2 /day and cyclophosphamide at 330-400mg/m2 /day, each for 5 consecutive days in children with either T-ALL (13 patients) or T-LBL (10 patients). Twenty-three patients were treated at three dose levels; 21 were evaluable for dose-limiting toxicities (DLT) and response. The recommended phase II doses (RP2D) for this regimen, when given daily ×5 every 3weeks, were nelarabine 650mg/m2 /day, etoposide 100mg/m2 /day, and cyclophosphamide 400mg/m2 /day. DLTs included peripheral motor and sensory neuropathies. An expansion cohort to evaluate responses at the RP2D was terminated early due to slow accrual. The overall best response rate was 38% (8/21), with 33% (4/12) responses in the T-ALL cohort and 44% (4/9) responses in the T-LBL cohort. These response rates are comparable to those seen with single-agent nelarabine in this setting. These data suggest that the addition of cyclophosphamide and etoposide to nelarabine does not increase the incidence of neurologic toxicities or the response rate beyond that obtained with single-agent nelarabine in children with first relapse of T-ALL and T-LBL.

Low Expression of CD5 and CD6 Is Associated with Poor Overall Survival for Patients with T-Cell Malignancies.

Background T-cell malignancies (TCMs), including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma (TCL), are highly aggressive and have a poor prognosis. To further understand prognostic stratifications and to design targeted therapies, this study aims to explore novel, potential biomarkers based on alterations in immune costimulatory molecules (CMs) for TCMs. Methods Peripheral blood from 25 de novo T-ALL patients in our clinical center and transcriptome data from 131 to 162 patients with peripheral TCL (PTCL) from the GSE19069 and GSE58445 dataset, respectively, were obtained to assess the expression levels of CMs and their prognostic significance. Results Seven CMs were associated with overall survival (OS). Among these CMs, CD5 and CD6 had the highest pairwise positive correlation (R = 0.69). CD5 and CD6 were significantly down-regulated in TCM patients compared with healthy individuals (HIs), and lower CD5 and CD6 expression was associated with poor OS for both T-ALL and TCL patients, particularly for patients greater than 60 years old. Furthermore, CD5 was positively correlated with CD6 in TCM patients. Compared with patients who were CD5highCD6high, T-ALL and TCL patients who were CD5lowCD6low had poor OS. Importantly, CD5highCD6high was an independent prognostic predictor for OS in T-ALL (HR = 0.39, 95% CI: 0.23–0.65, P < 0.001) and TCL (HR = 0.35, 95% CI: 0.19–0.62, P < 0.001) patients. Conclusions Low expression of CD5 and CD6 was associated with poor OS for TCM patients, and this may be a potential immune biomarker panel for prognostic stratification of TCM patients.

P360: EFFICACY AND SAFETY OF DARATUMUMAB IN PEDIATRIC AND YOUNG ADULT PATIENTS WITH RELAPSED/REFRACTORY T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA OR LYMPHOBLASTIC LYMPHOMA: RESULTS FROM PHASE 2 DELPHINUS STUDY

Background: Approximately 15-20% of pediatric patients with T-cell acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) will be refractory to/relapse after frontline treatment; relapsed disease is associated with poor outcomes. In a phase 2 study, 2 of 7 (28.6%) patients with T-cell ALL in first relapse achieved a complete response (CR) using the vincristine, prednisone, PEG-asparaginase, and doxorubicin (VPLD) reinduction backbone. Daratumumab (DARA), a human IgGκ monoclonal antibody targeting CD38 approved for treating multiple myeloma, has shown preclinical efficacy in ALL models. Aims: Here we report the initial results of DARA plus VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL enrolled in the phase 2, open-label DELPHINUS study. Methods: Eligible patients were aged 1-30 y, had T-cell ALL or LL in first relapse or refractory to 1 prior induction/consolidation regimen, and had a performance status ≥70. DARA (16 mg/kg IV QW) was given with VPLD in Cycle 1 and with methotrexate, cyclophosphamide, cytarabine, and 6-mercaptopurine in Cycle 2. Patients received age/risk-adjusted intrathecal therapy. Response was measured at the end of each cycle by local bone marrow morphology. The primary endpoint for ALL patients was CR rate in pediatric patients at the end of Cycle 1. Patients achieving CR after Cycles 1 or 2 could proceed to allogeneic HSCT off study. Overall response rate (ORR) was defined as CR or CR with incomplete hematological recovery (CRi) at any time before start of subsequent therapy or HSCT. Minimal residual disease (MRD) negativity (<0.01%) at any time before disease progression, start of subsequent therapy, or HSCT was centrally reviewed by flow cytometry. Results: Twenty-four pediatric (age 1-17 y) and 5 young adult (age 18-30 y) ALL patients and 10 LL patients (age 1-30 y) received ≥1 DARA dose. Median (range) age was 10.0 (2-25) y (ALL) and 14.5 (5-22) y (LL); median (range) time from initial diagnosis to first study treatment was 2.0 (0.1-6.1) y (ALL) and 0.8 (0.5-6.0) y (LL). Pediatric ALL patients received a median (range) of 2 (1-3) treatment cycles; young adult ALL patients and LL patients each received 2 (1-2). Among pediatric ALL patients, 10 (41.7%) patients (90% CI, 24.6-60.3) achieved CR at the end of Cycle 1. ORR was 83.3% (CR, 13 [54.2%] patients; CRi, 7 [29.2%] patients) in pediatric and 60.0% (all CR) in young adult ALL patients and 40.0% (all CR) in LL patients. Ten (41.7%) pediatric ALL patients achieved MRD negativity. All pediatric ALL patients had a grade 3/4 TEAE. No pediatric ALL pt discontinued DARA primarily due to AEs and 1 (4.2%) died due to TEAEs (brain edema and hepatic failure) attributed to study treatment but unrelated to DARA. Summary/Conclusion: The addition of DARA to VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL showed initial activity, generating improved response rates compared to those achieved with backbone therapy alone, with a manageable safety profile.

Efficacy and safety of daratumumab (DARA) in pediatric and young adult patients (pts) with relapsed/refractory T-cell acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL): Results from the phase 2 DELPHINUS study.

10001 Background: Approximately 15-20% of pediatric pts with ALL or LL will be refractory to/relapse after frontline treatment; relapsed disease is associated with poor outcomes. In a phase 2 study, 2 of 7 (28.6%) pts with T-cell ALL in first relapse achieved a complete response (CR) using the vincristine, prednisone, PEG-asparaginase, and doxorubicin (VPLD) reinduction backbone. DARA, a human IgGκ monoclonal antibody targeting CD38 approved for treating multiple myeloma, has shown preclinical efficacy in ALL models. We report the initial results of DARA plus VPLD in pediatric and young adult pts with relapsed/refractory T-cell ALL or LL enrolled in the phase 2, open-label DELPHINUS study. Methods: Eligible pts were aged 1-30 y, had T-cell ALL or LL in first relapse or refractory to 1 prior induction/consolidation regimen, and had a performance status ≥70. DARA (16 mg/kg IV QW) was given with VPLD in Cycle 1 and with methotrexate, cyclophosphamide, cytarabine, and 6-mercaptopurine in Cycle 2. Pts received age/risk-adjusted intrathecal therapy. Response was measured at the end of each cycle by local bone marrow morphology. The primary endpoint for ALL pts was CR rate in pediatric pts at the end of Cycle 1. Pts achieving CR after Cycles 1 or 2 could proceed to allogeneic HSCT off study. Overall response rate (ORR) was defined as CR or CR with incomplete hematological recovery (CRi) at any time before start of subsequent therapy or HSCT. Minimal residual disease (MRD) negativity (&lt; 0.01%) at any time before disease progression, start of subsequent therapy, or HSCT was centrally reviewed by flow cytometry. Results: Twenty-four pediatric (age 1-17 y) and 5 young adult (age 18-30 y) ALL pts and 10 LL pts (age 1-30 y) received ≥1 DARA dose. Median (range) age was 10.0 (2-25) y (ALL) and 14.5 (5-22) y (LL); median (range) time from initial diagnosis to first study treatment was 2.0 (0.1-6.1) y (ALL) and 0.8 (0.5-6.0) y (LL). Pediatric ALL pts received a median (range) of 2 (1-3) treatment cycles; young adult ALL pts and LL pts each received 2 (1-2). Among pediatric ALL pts, 10 (41.7%) pts (90% CI, 24.6-60.3) achieved CR at the end of Cycle 1. ORR was 83.3% (CR, 13 [54.2%] pts; CRi, 7 [29.2%] pts) in pediatric and 60.0% (all CR) in young adult ALL pts and 40.0% (all CR) in LL pts. Ten (41.7%) pediatric ALL pts achieved MRD negativity. All pediatric ALL pts had a grade 3/4 TEAE. No pediatric ALL pt discontinued DARA primarily due to AEs and 1 (4.2%) died due to TEAEs (brain edema and hepatic failure) attributed to study treatment but unrelated to DARA. Conclusions: The addition of DARA to VPLD in pediatric and young adult pts with relapsed/refractory T-cell ALL or LL showed initial activity, generating improved response rates compared to those achieved with backbone therapy alone, with a manageable safety profile. Clinical trial information: NCT03384654.

Central nervous system neurotoxicity associated with nelarabine in T-cell acute lymphoblastic leukemia.

Nelarabine, a prodrug of arabinosylguanine has lineage-specific toxicity for T lymphoblasts and is used to treat refractory or relapsed T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma patients. The most commonly observed adverse effects associated with nelarabine are mainly hematological, i.e. neutropenia, anemia, and thrombocytopenia. Additionally, neurological, and gastrointestinal toxicities have been reported. Central nervous system neurotoxicity associated with nelarabine is very rare. A 37-year-old man patient diagnosed with T-cell acute lymphoblastic leukemia had experienced generalized tonic-clonic seizure which lasted for a few seconds and upper extremity weakness after three weeks of the nelarabine infusion. Computed tomography and magnetic resonance imaging have shown periventricular and nucleus caudatus abnormalities. Radiological findings suggested toxic leukoencephalopathy and acute infarct of right nucleus caudatus. After high-dose steroids, intravenous immunoglobulin, and support treatment, his neurologic symptoms disappeared except for mild peroral numbness. However, radiological sequelae persisted despite clinical improvement. Physicians involved in the care of these patients who use nelarabine should be aware of the fact that cerebral toxicity of the nelarabine may occur especially in the presence of predisposing factors. It is crucial to monitor closely those patients receiving nelarabine and also those who have additional predisposing factors for neurotoxicity.

Anticancer effects of disulfiram in T-cell malignancies through NPL4-mediated ubiquitin-proteasome pathway.

T-cell malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma (TCL), are characterized by inferior treatment effects, high heterogeneity, poor prognosis, and a lack of specific therapeutic targets and drugs to improve outcome. Disulfiram (DSF) is a drug used to clinically control alcoholism that has recently been shown to be cytotoxic for multiple cancers. However, the underlying effects and mechanisms of DFS treatment in patients with T-cell malignancies are not well characterized. In this study, we report that DSF promotes apoptosis and inhibits the proliferation of malignant T-cell cell lines and primary T-ALL cells. We provide evidence that DSF exerts anticancer activity in T-cell malignancies by targeting the NPL4-mediated ubiquitin-proteasome pathway. Notably, high expression of NPL4 and 2 ubiquitin-proteasome pathway genes, anaphase-promoting complex subunit 1 (ANAPC1) and proteasome 26S subunit ubiquitin receptor, non-ATPase 2 (PSMD2), was significantly associated with unfavorable overall survival (OS) for patients with TCL and T-ALL (p<0.05). More importantly, the weighted combination of NPL4, ANAPC1, and PSMD2 could visually display the 1-, 3-, and 5-year OS rates for patients with T-cell malignancies in a nomogram model and facilitate risk stratification. Specifically, risk stratification was an independent predictor of OS for patients with T-cell malignancies. In conclusion, DSF might induce apoptosis and inhibit the proliferation of malignant T-cells via the NPL4-mediated ubiquitin-proteasome pathway and offer a potential therapeutic option for T-cell malignancies.