The 29th Annual Meeting of the European Hematology Association (EHA) was grandly held from June 13-16, 2024, in Madrid, Spain. As the largest international conference in the field of hematology in Europe, it attracts experts and scholars from around the world each year to share and discuss innovative concepts and the latest scientific and clinical research results in hematology. At this year's conference, Professor Xiaohui Zhang's team from Peking University People's Hospital performed outstandingly, with four studies selected for oral presentation, and one study awarded the YoungEHA Best Abstract Award, making it one of the four global award-winning studies of the year and the only one from China to receive this honor. This achievement not only highlights the team's exceptional strength but also showcases the potential and capabilities of Chinese researchers. "Oncology Frontier - Hematology Frontier" specially invited Professor Xiaohui Zhang and his team members for a roundtable discussion to share their research findings, covering non-invasive diagnostic techniques, mechanisms of CAR-T cell therapy toxicity in relapsed/refractory T-cell leukemia, prognosis models for mixed phenotype acute leukemia (MPAL), and innovative clinical research in immune thrombocytopenia (ITP).From left to right: Mankaran Singh (Newcastle University, UK), An Zhuoyu (Peking University People’s Hospital, China), Nili Furer (Weizmann Institute of Science, Israel), Fieke Hoff (University of Texas Southwestern Medical Center, USA)
Invited Oral Presentation 1
S336 – Novel Non-Invasive Diagnostic Technology for T-ALL/LBL Based on Ultra-Sensitive Biosensors
Dr. Ting Wang presented the oral lecture.
Background: T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a malignant tumor caused by the malignant proliferation of immature T cells, severely affecting white blood cells and lymphocytes, characterized by high invasiveness and rapid progression. Currently, the diagnosis of T-ALL/LBL relies on immunotyping, an invasive, expensive, and technically demanding method. Invasive tissue biopsy is the gold standard for obtaining molecular data and classifying lymphoma patients into genetic subtypes. However, for critically ill patients, those with inoperable tumors, or those with poor compliance, surgical intervention is not feasible, preventing tissue biopsy. Thus, there is an urgent need for a convenient, sensitive, and specific method to detect gene rearrangements, high-frequency mutations associated with drug resistance, or EBV-related infections. Existing methods such as first-generation sequencing (FGS) and next-generation sequencing (NGS) have limitations, including low sensitivity, long processing time, and high cost. To overcome these drawbacks, developing a low-cost, ultra-sensitive biosensor for non-invasive diagnosis holds great potential.
Objective: To develop a CRISPR-based high-sensitivity, high-specificity multifunctional biosensor for the non-invasive diagnosis of T-ALL/LBL.
Methods: In this study, researchers developed an accurate and sensitive diagnostic method for T-ALL/LBL using a CRISPR/Cas9-Cas12a biosensor. This method uses an sgRNA
complex with single-strand nicking activity, a strand displacement DNA polymerase, and two primers with Cas9n cleavage site sequences to promote DNA cycling amplification through primer, extension, nicking, and displacement reactions. The amplified fragments are then detected by a crRNA-triggered Cas12a reaction, and the results can be visualized under a blue light or measured using a fluorescence reader. Additionally, this method is suitable for low-cost point-of-care (POC) testing based on lateral flow biosensors. The researchers validated this method using gene models and samples from 120 T-ALL/LBL patients.
Results: The CRISPR/Cas biosensor demonstrated high accuracy, specificity, and sensitivity in diagnosing T-ALL/LBL, showing great potential in nucleic acid programmable detection. Based on this, researchers designed a Cas9n-Cas12a nucleic acid detection biosensor for detecting gene rearrangements and mutations in T-ALL/LBL. Detecting high-frequency gene mutations and fusions is crucial for accurate diagnosis and treatment of T-ALL/LBL. The researchers selected the following mutation sites: DNMT3A, ETV6, IDH2, JAK1, JAK3, and SETD2 in ALL and FLT3; NRAS/KRAS, DNMT3A, IDH1, and IDH2 in LBL. This method could detect significant gene mutations and fusions in complex biological samples at levels as low as 0.1%. The CRISPR detection results of 120 T-ALL/LBL patients were compared with qPCR and NGS as reference controls, showing 100% specificity. EBV is a ubiquitous carcinogenic virus associated with lymphoma, especially in immunosuppressed patients such as those with T-ALL/LBL or transplant recipients. This study developed a simple and sensitive method for EBV detection using Cas9-Cas12a and lateral flow biosensors. The method also identifies EBV-encoded RNA (EBER) and microRNA-191. In a 20 μL reaction system, the detection limit (LOD) achieved was as low as 1×10-8 ng (0.99 pM), equivalent to 1.2 copies per reaction, comparable to PCR and superior to existing CRISPR-based detection methods.
Conclusion: The method features single-copy detection limits and single-base discrimination capability, with sensitivity up to 0.1%, completing the diagnostic process within 40 minutes. This method is significant for early screening, point-of-care applications, and treatment guidance in T-ALL/LBL.
Dr. Ting Wang: My research focuses on developing and applying biosensor analysis technology, primarily in non-invasive diagnostics of hematological diseases. The technology aims to provide rapid and sensitive diagnostic methods and real-time monitoring and tracking of treatment efficacy. Specifically, ultra-sensitive biosensors play a vital role in non-invasive diagnostics of hematological diseases, offering key molecular diagnostic information for monitoring treatment efficacy and prognosis evaluation. This method holds great promise for non-invasive diagnostics and liquid biopsy applications.
Invited Oral Presentation 2
S335 – Antigen Epitope Editing of Hematopoietic Stem Cells to Mitigate Off-Target Toxicity in Dual-Targeted Immunotherapy for T-Cell Leukemia
Dr. Guoling Wang presented the oral lecture.
Background: The treatment of relapsed/refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) poses significant challenges. Although CD7 CAR-T therapy shows a complete remission rate of 85%-90% in the short term, long-term follow-up reveals that the 24-month progression-free survival rate and overall survival rate are 36.8% and 42.3%, respectively. Among patients with relapsed disease, 66.7% (4/6) experienced CD7 antigen loss-induced negative relapse, while 80% (4/5) of non-relapsed deaths were due to severe infections. These findings indicate that the long-term efficacy of CD7 CAR-T therapy is severely affected by CD7 antigen loss and infection-related deaths. To overcome these limitations, this study proposes an antigen epitope editing hematopoietic stem cell transplantation combined with a novel dual-targeted CAR-T therapy strategy. Dual targeting of CD7 and CD5 can reduce treatment failure caused by single-target loss, while antigen epitope editing of HSCT renders blood cells “invisible” to CAR-T, aiming to improve treatment success rates and reduce toxicity to the immune system.
Methods: Researchers constructed tan 5/7-4 1BB-CD3ζ CAR vectors by linking humanized heavy-chain antibodies targeting CD5/CD7 in series, then packed lentivirus and infected T cells to create nano dual CAR-T cells. Using antigen-antibody docking and alanine scanning mutagenesis techniques, researchers identified binding epitopes of dual CARs to their respective antigens and screened single amino acid mutants that could not bind to each other through Western blot and flow cytometry. Based on single-base mutants, sgRNAs were designed to guide single-base editors for antigen epitope base editing of hematopoietic stem cells. Finally, NSG mice were used to evaluate the immune reconstitution ability and resistance to dual-target CAR-T recognition and killing after epitope editing of HSCs.
Results: In preliminary clinical trials, genome sequencing analysis of CD7-negative relapsed patients revealed frame-shift mutations causing antigen loss in tumor cells. To reduce treatment failure caused by single-target loss, researchers designed and constructed nano tan 5/7 CAR or tan7 CAR-T cells. In vitro co-culture experiments showed that these cells effectively killed CD5/CD7 single-positive or multi-positive tumor cells, with higher killing ratios for T-ALL cell lines such as CCRF-CEM/JURKAT compared to single-positive CAR-T cells and control T cells.
In previous trials, researchers observed that CD7+ T cells were rapidly cleared within 15 days after infusion, with only CD7- T cells remaining in patients who received CD7 CAR-T infusion. Analysis indicated that CD7- T cells primarily consisted of effector/memory phenotype cells, lacked naive cell populations, had reduced thymic output levels, and decreased TCR diversity. scRNA-seq analysis of CD7- T cells revealed that CD7- T cells mainly comprised CD8+GZMK+ cells. Pseudotime analysis showed CD7- CD8+ T cells distributed at both ends of the trajectory, with a small portion at the starting end and the majority of CD7- CD8+ GZMK+ T cells at the late stage of T cell differentiation. To reduce off-target toxicity to the immune system, researchers performed alanine scanning mutagenesis on both antigens, screening single-base mutants that did not bind to the antibody through flow cytometry. Based on these mutants, sgRNAs were designed to guide ABE single-base editors for efficient, non-double-strand break precise editing of hematopoietic stem cells. Epitope-edited HSC cells retained normal immune reconstitution function, and the differentiated normal blood cells were not recognized or killed by dual CARs.
Conclusion: This study proposes an antigen epitope editing hematopoietic stem cell transplantation combined with a novel dual-targeted CAR-T therapy strategy. Dual targeting of CD7 and CD5 can reduce treatment failure caused by single-target loss, while antigen epitope editing of HSCT renders blood cells “invisible” to CAR-T, aiming to improve the success rate of r/r T-ALL patient treatment and reduce immune system toxicity.
Dr. Guoling Wang: This report focuses on cell therapy toxicity related to relapsed/refractory T-cell leukemia. Currently, the clinical prognosis for relapsed/refractory T-cell leukemia is very poor. However, recent clinical trials have shown that CD7 CAR-T therapy achieves remarkable results in treating this disease. Despite this, the therapy faces challenges such as negative relapse and infections. To address these challenges, we plan to conduct antigen site mutation editing at the hematopoietic stem cell level. Minimal editing aims to reduce toxicity to the hematopoietic system and promote the development of dual-target CAR-T. This strategy is expected to solve current issues like infection risks and negative relapse.
