Periodic Reporting for period 1 - BEAT B-cell Lymphoma (BEAT B-cell Lymphoma: Biomarkers of Effective CART19 Assessed in the Tissue Microenvironment)
Periodo di rendicontazione: 2024-05-01 al 2026-04-30
Sintesi del contesto e degli obiettivi generali del progetto
Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are among the most common lymphoid malignancies worldwide and remain associated with substantial morbidity and mortality despite major therapeutic advances. In particular, patients with relapsed or refractory disease often face limited treatment options and poor prognosis. The introduction of CD19-directed chimeric antigen receptor T-cell therapy (CART19) has represented a major breakthrough in the treatment landscape of aggressive B-cell lymphomas, offering the possibility of long-term remission in patients who previously had very limited therapeutic prospects. Nevertheless, a substantial proportion of patients either fail to respond to CART19 therapy or eventually relapse after an initial response. The biological mechanisms underlying treatment resistance, durability of response, and toxicity remain incompletely understood.
Current clinical biomarkers for CART19 response are largely derived from bulk tumor characteristics, peripheral blood measurements, or generalized clinical risk factors. However, these approaches insufficiently capture the complexity of the tumor tissue microenvironment (TME), which plays a central role in shaping anti-tumor immune responses. Increasing evidence suggests that interactions between malignant B-cells, endogenous immune cells, stromal elements, and infused CART19 cells critically determine therapeutic efficacy. Yet these interactions remain poorly characterized directly within patient tissues at high spatial and molecular resolution.
The BEAT B-cell Lymphoma project addresses this unmet need by investigating the tissue-level determinants of CART19 response in B-cell lymphomas using advanced spatial and computational profiling technologies. The project aims to identify predictive and mechanistic biomarkers associated with effective CART19 activity within the native lymphoma microenvironment. By integrating histopathology, multiplex tissue imaging, molecular profiling, and computational bioinformatics, the project seeks to generate a detailed systems-level understanding of how CART19 therapy remodels lymphoma tissues and how pre-existing microenvironmental states influence therapeutic outcomes.
The overall objectives of the project are:
To characterize the spatial organization and cellular composition of the lymphoma tissue microenvironment before and after CART19 therapy.
To identify tissue-based biomarkers associated with treatment response, resistance, and relapse.
To develop computational and bioinformatic frameworks capable of integrating high-dimensional spatial and molecular datasets derived from lymphoma tissues.
To improve biological understanding of CART19-tumor interactions in human patient samples and generate hypotheses for future therapeutic optimization strategies.
To strengthen translational bridges between pathology, immunology, and computational oncology, thereby supporting the development of more personalized immunotherapy approaches.
Overall, BEAT B-cell Lymphoma aims to generate biologically meaningful and clinically actionable insights into the tissue ecology of CART19 therapy. By improving understanding of the determinants of treatment response within lymphoma tissues, the project seeks to contribute to more effective, personalized, and durable immunotherapeutic strategies for patients with B-cell lymphomas.
Current clinical biomarkers for CART19 response are largely derived from bulk tumor characteristics, peripheral blood measurements, or generalized clinical risk factors. However, these approaches insufficiently capture the complexity of the tumor tissue microenvironment (TME), which plays a central role in shaping anti-tumor immune responses. Increasing evidence suggests that interactions between malignant B-cells, endogenous immune cells, stromal elements, and infused CART19 cells critically determine therapeutic efficacy. Yet these interactions remain poorly characterized directly within patient tissues at high spatial and molecular resolution.
The BEAT B-cell Lymphoma project addresses this unmet need by investigating the tissue-level determinants of CART19 response in B-cell lymphomas using advanced spatial and computational profiling technologies. The project aims to identify predictive and mechanistic biomarkers associated with effective CART19 activity within the native lymphoma microenvironment. By integrating histopathology, multiplex tissue imaging, molecular profiling, and computational bioinformatics, the project seeks to generate a detailed systems-level understanding of how CART19 therapy remodels lymphoma tissues and how pre-existing microenvironmental states influence therapeutic outcomes.
The overall objectives of the project are:
To characterize the spatial organization and cellular composition of the lymphoma tissue microenvironment before and after CART19 therapy.
To identify tissue-based biomarkers associated with treatment response, resistance, and relapse.
To develop computational and bioinformatic frameworks capable of integrating high-dimensional spatial and molecular datasets derived from lymphoma tissues.
To improve biological understanding of CART19-tumor interactions in human patient samples and generate hypotheses for future therapeutic optimization strategies.
To strengthen translational bridges between pathology, immunology, and computational oncology, thereby supporting the development of more personalized immunotherapy approaches.
Overall, BEAT B-cell Lymphoma aims to generate biologically meaningful and clinically actionable insights into the tissue ecology of CART19 therapy. By improving understanding of the determinants of treatment response within lymphoma tissues, the project seeks to contribute to more effective, personalized, and durable immunotherapeutic strategies for patients with B-cell lymphomas.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
During the reporting period, substantial progress was achieved toward the scientific and technical objectives of the BEAT B-cell Lymphoma project. The work performed combined translational clinical investigation, molecular pathology, spatial profiling, and computational analysis focused on understanding the biological determinants and consequences of CART19 therapy in B-cell lymphomas.
A major scientific achievement of the fellowship was the completion and publication of a high-impact translational study addressing the safety and biological characterization of secondary malignancies arising after CAR T-cell therapy. This work was published in the New England Journal of Medicine (DOI: 10.1056/NEJMoa2401361) with the fellow serving as first author. The follow-up paper that dives deeper in specific CAR T-cell related toxicities is under review, a preprint is deposited (https://doi.org/10.21203/rs.3.rs-7746241/v1(si apre in una nuova finestra)).
In this published study, a comprehensive institutional analysis was performed on 724 patients treated with adoptive CAR T-cell therapies since 2016. The work addressed emerging international concerns regarding the potential development of secondary T-cell neoplasms associated with viral vector integration following CAR T-cell therapy. A lethal secondary T-cell lymphoma occurring after axicabtagene ciloleucel treatment for diffuse large B-cell lymphoma was deeply investigated using an integrated molecular and cellular profiling strategy. Multiple orthogonal genomic, immunophenotypic, and molecular techniques were applied to interrogate the tumor tissue, CAR T-cell product, and normal hematopoietic compartment. The analyses demonstrated that the original B-cell lymphoma and the secondary T-cell lymphoma represented biologically distinct malignancies, while also identifying shared associations with Epstein–Barr virus positivity and clonal hematopoiesis involving DNMT3A and TET2 mutations. Importantly, no evidence of oncogenic retroviral vector integration was identified despite extensive testing using complementary methodologies.
This work represents a significant contribution to the international CART19 field by helping clarify the biological basis and rarity of secondary tumors after CAR T-cell therapy. Furthermore, the study established a translational framework for future investigation of clonal relationships, viral vector safety monitoring, and long-term surveillance strategies in cellular immunotherapy. The project therefore directly contributed to improving understanding of real-world CART19-associated risks and informed ongoing international regulatory and clinical discussions surrounding CAR T-cell safety.
In parallel with these clinically oriented investigations, major technical and methodological progress was achieved in the spatial biology component of the fellowship. Pilot studies were successfully initiated and performed using multiple advanced spatial profiling platforms across distinct lymphoma entities and tissue contexts.
These activities included:
Spatial transcriptomic profiling using 10x Genomics Visium and VisiumHD in follicular lymphoma samples (8 cases). A computational pipeline converting visiumHD data to single cell transcriptional fingerprints was created and shared licence free with the community at https://github.com/noordenbos/visiumhd_singlecell(si apre in una nuova finestra).
Spatial proteomic profiling in diffuse large B-cell lymphoma using multiplexed tissue imaging (70 cases). A manuscript in preparation describing ingrated analysis of this spatial proteomic profiling with genomic and transcriptomic profiling. For this project the computational pipeline 'Unhuddle' was created to resolve signal in densely packed tissue regions, as is common in lymphoma, — or "cell huddles" — in multiplex spatial proteomics, where traditional absolute segmentation introduces 'neighbor noise' and blur the phenotypic signal. The code base and detailed user instructions are shared licence free with the community at https://github.com/noordenbos/Unhuddle(si apre in una nuova finestra).
To support the central objectives of the project, a highly customized and disease-focused spatial transcriptomic panel was designed for use with the 10x Genomics Xenium platform. The resulting approximately 2,000-gene panel was specifically optimized for investigating CART19 biology and lymphoma microenvironment interactions. Importantly, the panel incorporates several innovative features that extend beyond conventional lymphoma profiling panels, including probes targeting:
CAR vector-associated transcripts,
Epstein–Barr virus (EBV)-related transcripts,
B-cell receptor (BCR) transcripts,
T-cell receptor (TCR) transcripts,
immune activation and exhaustion programs,
lymphoma-specific oncogenic pathways,
stromal and microenvironmental components.
This panel design constitutes a major technical achievement of the project because it enables simultaneous interrogation of malignant B-cells, endogenous immune populations, infused CAR T-cell signatures, viral biology, and adaptive immune receptor dynamics directly within intact tissue architecture.
In addition, extensive biobanking and cohort-building activities were completed during the reporting period. Tissue blocks were collected and curated from patients with relapsed/refractory diffuse large B-cell lymphoma and follicular lymphoma treated with CART19 therapy. These collections include:
pre-treatment tissue samples, a subset with paired post-relapse tissue samples for both FL and DLBCL cohorts.
samples originating from an innovative CART19 dosing trial in relapsed/refractory DLBCL.
The establishment of these clinically annotated tissue cohorts provides an important translational resource for ongoing and future spatial profiling analyses within the project.
Overall, the work completed during the reporting period substantially advanced the project objectives. The fellowship has already generated impactful translational findings in the CART19 field, established novel spatial biology resources and methodologies for lymphoma research, and created unique clinically annotated datasets that will support subsequent mechanistic and biomarker-focused investigations.
A major scientific achievement of the fellowship was the completion and publication of a high-impact translational study addressing the safety and biological characterization of secondary malignancies arising after CAR T-cell therapy. This work was published in the New England Journal of Medicine (DOI: 10.1056/NEJMoa2401361) with the fellow serving as first author. The follow-up paper that dives deeper in specific CAR T-cell related toxicities is under review, a preprint is deposited (https://doi.org/10.21203/rs.3.rs-7746241/v1(si apre in una nuova finestra)).
In this published study, a comprehensive institutional analysis was performed on 724 patients treated with adoptive CAR T-cell therapies since 2016. The work addressed emerging international concerns regarding the potential development of secondary T-cell neoplasms associated with viral vector integration following CAR T-cell therapy. A lethal secondary T-cell lymphoma occurring after axicabtagene ciloleucel treatment for diffuse large B-cell lymphoma was deeply investigated using an integrated molecular and cellular profiling strategy. Multiple orthogonal genomic, immunophenotypic, and molecular techniques were applied to interrogate the tumor tissue, CAR T-cell product, and normal hematopoietic compartment. The analyses demonstrated that the original B-cell lymphoma and the secondary T-cell lymphoma represented biologically distinct malignancies, while also identifying shared associations with Epstein–Barr virus positivity and clonal hematopoiesis involving DNMT3A and TET2 mutations. Importantly, no evidence of oncogenic retroviral vector integration was identified despite extensive testing using complementary methodologies.
This work represents a significant contribution to the international CART19 field by helping clarify the biological basis and rarity of secondary tumors after CAR T-cell therapy. Furthermore, the study established a translational framework for future investigation of clonal relationships, viral vector safety monitoring, and long-term surveillance strategies in cellular immunotherapy. The project therefore directly contributed to improving understanding of real-world CART19-associated risks and informed ongoing international regulatory and clinical discussions surrounding CAR T-cell safety.
In parallel with these clinically oriented investigations, major technical and methodological progress was achieved in the spatial biology component of the fellowship. Pilot studies were successfully initiated and performed using multiple advanced spatial profiling platforms across distinct lymphoma entities and tissue contexts.
These activities included:
Spatial transcriptomic profiling using 10x Genomics Visium and VisiumHD in follicular lymphoma samples (8 cases). A computational pipeline converting visiumHD data to single cell transcriptional fingerprints was created and shared licence free with the community at https://github.com/noordenbos/visiumhd_singlecell(si apre in una nuova finestra).
Spatial proteomic profiling in diffuse large B-cell lymphoma using multiplexed tissue imaging (70 cases). A manuscript in preparation describing ingrated analysis of this spatial proteomic profiling with genomic and transcriptomic profiling. For this project the computational pipeline 'Unhuddle' was created to resolve signal in densely packed tissue regions, as is common in lymphoma, — or "cell huddles" — in multiplex spatial proteomics, where traditional absolute segmentation introduces 'neighbor noise' and blur the phenotypic signal. The code base and detailed user instructions are shared licence free with the community at https://github.com/noordenbos/Unhuddle(si apre in una nuova finestra).
To support the central objectives of the project, a highly customized and disease-focused spatial transcriptomic panel was designed for use with the 10x Genomics Xenium platform. The resulting approximately 2,000-gene panel was specifically optimized for investigating CART19 biology and lymphoma microenvironment interactions. Importantly, the panel incorporates several innovative features that extend beyond conventional lymphoma profiling panels, including probes targeting:
CAR vector-associated transcripts,
Epstein–Barr virus (EBV)-related transcripts,
B-cell receptor (BCR) transcripts,
T-cell receptor (TCR) transcripts,
immune activation and exhaustion programs,
lymphoma-specific oncogenic pathways,
stromal and microenvironmental components.
This panel design constitutes a major technical achievement of the project because it enables simultaneous interrogation of malignant B-cells, endogenous immune populations, infused CAR T-cell signatures, viral biology, and adaptive immune receptor dynamics directly within intact tissue architecture.
In addition, extensive biobanking and cohort-building activities were completed during the reporting period. Tissue blocks were collected and curated from patients with relapsed/refractory diffuse large B-cell lymphoma and follicular lymphoma treated with CART19 therapy. These collections include:
pre-treatment tissue samples, a subset with paired post-relapse tissue samples for both FL and DLBCL cohorts.
samples originating from an innovative CART19 dosing trial in relapsed/refractory DLBCL.
The establishment of these clinically annotated tissue cohorts provides an important translational resource for ongoing and future spatial profiling analyses within the project.
Overall, the work completed during the reporting period substantially advanced the project objectives. The fellowship has already generated impactful translational findings in the CART19 field, established novel spatial biology resources and methodologies for lymphoma research, and created unique clinically annotated datasets that will support subsequent mechanistic and biomarker-focused investigations.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
The BEAT B-cell Lymphoma project generated several advances beyond the current state of the art in lymphoma immunotherapy research, spatial biology, and computational pathology.
A major contribution of the project was the publication of a first-author study in the New England Journal of Medicine (DOI: 10.1056/NEJMoa2401361) addressing secondary malignancies after CAR T-cell therapy. Through comprehensive molecular characterization of a lethal secondary T-cell lymphoma arising after CART19 treatment, the study demonstrated the absence of detectable oncogenic viral vector integration and established a framework for future safety monitoring and investigation of clonal relationships in cellular immunotherapy. These findings contributed to ongoing international discussions regarding long-term CART19 safety.
The project further advanced the field through implementation of high-dimensional spatial profiling approaches in lymphoma tissue, including 10x Genomics Visium, Visium HD, multiplex spatial proteomics, and development of a customized approximately 2,000-gene 10x Genomics Xenium panel optimized for CART19 biology. The panel uniquely integrates detection of CAR-associated transcripts, Epstein–Barr virus transcripts, B-cell receptor and T-cell receptor biology, immune activation and exhaustion programs, and lymphoma-associated oncogenic pathways within intact tissue architecture.
To support analysis of these complex datasets, novel computational tools were developed and openly shared with the scientific community. These include the Unhuddle GitHub repository; A pipeline for resolving signal contamination in densely packed lymphoma tissues during multiplex spatial proteomic analysis, and the visiumhd_singlecell GitHub repository; A framework for conversion of Visium HD data into single-cell transcriptional fingerprints. Further uptake and translational implementation of the project results will require validation in larger multicenter cohorts, integration of spatial biomarkers into prospective CART19 clinical studies, and continued development of standardized computational workflows for spatial omics analysis. Open sharing of computational tools and international collaboration will support broader adoption of the methodologies developed during the fellowship. In the longer term, supportive clinical and regulatory frameworks will be important for translation of spatial biomarker strategies into precision immunotherapy settings.
A major contribution of the project was the publication of a first-author study in the New England Journal of Medicine (DOI: 10.1056/NEJMoa2401361) addressing secondary malignancies after CAR T-cell therapy. Through comprehensive molecular characterization of a lethal secondary T-cell lymphoma arising after CART19 treatment, the study demonstrated the absence of detectable oncogenic viral vector integration and established a framework for future safety monitoring and investigation of clonal relationships in cellular immunotherapy. These findings contributed to ongoing international discussions regarding long-term CART19 safety.
The project further advanced the field through implementation of high-dimensional spatial profiling approaches in lymphoma tissue, including 10x Genomics Visium, Visium HD, multiplex spatial proteomics, and development of a customized approximately 2,000-gene 10x Genomics Xenium panel optimized for CART19 biology. The panel uniquely integrates detection of CAR-associated transcripts, Epstein–Barr virus transcripts, B-cell receptor and T-cell receptor biology, immune activation and exhaustion programs, and lymphoma-associated oncogenic pathways within intact tissue architecture.
To support analysis of these complex datasets, novel computational tools were developed and openly shared with the scientific community. These include the Unhuddle GitHub repository; A pipeline for resolving signal contamination in densely packed lymphoma tissues during multiplex spatial proteomic analysis, and the visiumhd_singlecell GitHub repository; A framework for conversion of Visium HD data into single-cell transcriptional fingerprints. Further uptake and translational implementation of the project results will require validation in larger multicenter cohorts, integration of spatial biomarkers into prospective CART19 clinical studies, and continued development of standardized computational workflows for spatial omics analysis. Open sharing of computational tools and international collaboration will support broader adoption of the methodologies developed during the fellowship. In the longer term, supportive clinical and regulatory frameworks will be important for translation of spatial biomarker strategies into precision immunotherapy settings.