67th ASH Annual Meeting

Background:
Epstein-Barr virus (EBV)-positive lymphoproliferative disorders can exhibit overlapping Hodgkin-like morphological features but show a wide spectrum of clinical behaviours, and include indoent diseases such as EBV-positive mucocutaneous ulcer (EBVMCU), and malignant systemic diseases such as EBV+ classical Hodgkin lymphoma (EBVCHL) and EBV+ diffuse large B-cell lymphoma (EBVDLBCL). Diagnostic distinction of these lesions is imperative to ensure appropriate treatment; however, there is a dearth of diagnostic biomarkers to differentiate these lesions. Furthermore, the underlying biological basis of this divergent behaviour is unclear. This study aimed to identify the immunobiological and genetic basis of this divergent clinical behaviour using multimodal spatial profiling of the immune microenvironment and tumour cells, and single-cell genomic sequencing.

Methods:
Multiplex immunofluorescence (mIF) and Nanostring GeoMX spatial profiling (GeoMX-DSP) were performed on formalin-fixed paraffin-embedded (FFPE) sections, comparing indolent EBVMCU to malignant EBVCHL and EBVDLBCL. Single HRS and HRS-like cells were isolated from EBVMCU, EBVCHL and EBVDLBCL using the DepArray platform, and low-pass whole genome sequencing was performed. Chromosome 9 ploidy and PDL1/PDL2 amplification status were assessed by DNA fluorescent in situ hybridisation (FISH) combined with CD30 immunofluorescence. PDL1 protein expression was assessed by PAX5/PDL1 dual chromogenic immunohistochemistry.

Results:
MIF showed an increase in CD4+ T-regulatory and PDL1+IDO1+M1 macrophages in EBVMCU (p<0.05), whilst EBVDLBCL showed an increase in immunosuppressive IDO1+M2 macrophages (p<0.05). Gene set enrichment analysis performed on GeoMX-DSP data showed that EBVMCU was characterised by marked antiviral responses, with upregulation of pathways involved in T-regulatory chemotaxis in HRS cells (odds ratio (OR) 120; p <0.0001), interferon-alpha; (OR 50; p <0.0005) and interferon-beta (OR 40; 0 < 0.0004) responses in macrophages. Furthermore, EBVMCU HRS-like cells, in comparison to EBVDLBCL HRS-like cells, showed upregulation of type 1 interferon responses (OR 20; p< 0.00025). Differential gene expression analysis shows increased expression of tumour suppressor CRIP1 (FDR>0.001), chemotactic CXCL10 and pro-inflammasome IL1RN (FDR>0.001) in EBVMCU, and upregulation of anti-inflammasome IL1R2 (FDR>0.001) in EBVCHL and EBVDLBCL. Low-pass whole-genome sequencing of single HRS/HRS-like cells showed few copy number variations in EBVMCU, whilst EBVCHL and EBVDLBCL showed greater genomic complexity. Moreover, EBVCHL showed recurrent chromosome 9 amplifications. On chromosome9/PDL1/PDL2 FISH, EBVMCU showed no chromosome 9 aneuploidy above 3n compared to 50% in EBVDLBCL and 33% EBVCHL. Amplification of PDL1/PDL2 (>2n+1) was not present in EBVMCU or EBVDLBCL, but was present in 66% of cHL. Assessment of proteomic expression of PDL1 by immunohistochemistry showed ubiquitous, strong expression of PDL1 in HRS cells in EBVCHL, whilst HRS-like cells in EBVMCU and EBVDLBCL showed patchy weak expression.

Conclusions: EBVMCU showed evidence of active inflammation, T-regulatory cell chemotaxis and antiviral response, whereas EBV+cHL and EBVDLBCL showed evidence of immune permissive, and immune suppressive microenvironments, with loss of inflammasome related signalling and decreased tumoursuppressor mechanisms. EBVMCU showed little genomic complexity whilst EBVDLBCL and EBVCHL showed greater genomic complexity. Chromosome 9 amplification was a recurrent aberration in EBVMCU. FISH for PDL1/L2 and PDL1 immunohistochemistry are potential diagnostic discriminatory biomarkers.

Angioimmunoblastic T-cell lymphoma (AITL) is a rare subtype of mature T-cell lymphoma (TCL) that arises from T follicular helper (TFH) cells and is characterized by distinct clinicopathologic features, poor prognosis, and resistance to current treatments. A hallmark of AITL is its complex lymphoma microenvironment (LME), in which neoplastic TFH cells typically comprise only 10–30% of the infiltrate, complicating diagnosis. The heterogeneous cellular composition of the LME, along with subtle morphological features and frequent overlap with other reactive lymphoid processes or classic Hodgkin lymphoma, often results in delayed diagnosis or misdiagnosis. This persistent diagnostic challenge, together with poor therapeutic responses, underscores the need to further define the transcriptional and spatial programs of neoplastic TFH cells and their environment. To address this, we performed single-cell RNA sequencing on five AITL lymph node specimens. After quality filtering, cells expressing 200–10,000 genes with fewer than 10% mitochondrial transcripts were retained. Seurat-based integration was used to mitigate batch effects and cluster cells by transcriptional similarity. Malignant TFH cells were identified by co-expression of ten canonical TFH genes (CD3E, CD4, BCL6, ICOS, PDCD1, CXCR5, CXCL13, IL21, CTLA4 and CD40LG) alongside monoclonal T-cell receptor expansions via scRepertoire. Gene set enrichment revealed consistent upregulation of oxidative phosphorylation signatures, implicating altered mitochondrial metabolism, and expression of central memory-associated transcripts. Notably, a subset of malignant TFH cells expressed FOXP3, a hallmark of regulatory T cells, suggesting phenotypic plasticity and context-dependent adoption of regulatory programs. Immunofluorescence validation confirmed FOXP3 expression in neoplastic cells. To study the LME, we performed sequential forty‑plex immunofluorescence (COMET Lunaphore) in 14 AITL and 3 reactive lymph nodes. In AITL we identified two distinct microenvironmental archetypes. Both niches exhibited marked CD40 upregulation relative to controls, but with distinct cellular localizations: in the M1‑skewed archetype CD40 was confined primarily to macrophages, whereas in the M2‑skewed niche it was predominantly B‑cells. CD40 single immunostaining was conducted in 200 additional cases, including other T-cell lymphomas and autoimmune disorders, and confirmed that while CD40-driven immune activation is a hallmark of AITL, elevated CD40 expression was also observed in a subset of other T-cell lymphomas. In our AITL PDX models using NSG mice, CD40 expression persisted in tumorinfiltrating B cells (patient derived), reinforcing the potential role of the CD40-CD40L axis in tumormicroenvironment crosstalk (PMID: 35921527). Altogether, our data indicates that neoplastic TFH cells in AITL possess phenotypic plasticity, adopting both Treg‑like and central memory expression programs. These cells reside within two distinguishable microenvironmental archetypes which may underlie aspects of diagnostic complexity and variable clinical behavior. The consistent overexpression of CD40 across these niches suggests it could serve as a biomarker for stratifying microenvironmental contexts and monitoring therapeutic responses. While these findings provide preliminary insight into the cellular and spatial heterogeneity of AITL, further studies in larger patient cohorts are necessary to confirm these observations and to explore the feasibility of targeting the CD40-CD40L axis or related pathways as part of future therapeutic strategies.

Background
Immunohistochemistry is essential to the pathological diagnosis of formalin-fixed paraffinembedded (FFPE) tissue biopsies for suspected lymphomas. At present, multiple pathologist-selected single-plex chromogenic stains are performed on separate tissue sections, potentially leading to tissue exhaustion, incomplete diagnosis, and re-biopsy in a proportion of cases. Comprehensive multiplex immunohistochemistry (mIHC) on a single tissue section, such as sequential immunofluorescence (seqIF™, PMID 37813886) performed on the COMET™ platform, could potentially overcome these shortcomings, but the diagnostic utility of mIHC in pathological tissue assessment has not yet been evaluated.

Methods
A 38-marker seqIF™panel comprising most markers required for lymphoma diagnosis was optimised on the automated staining and imaging COMETTM platform (Lunaphore, a Bio-Techne brand), including 37 antibodies against immune lineage proteins and 1 RNA probe against EBER. Each stain was evaluated compared to standard clinical chromogenic stains by semi-quantitative clinical assessment scoring (CAS). 15 retrospective lymphoma cases, comprising 3 Hodgkin lymphomas, 6 low-grade lymphomas, 2 high-grade lymphomas, 2 T-cell lymphomas, and 2 reactive lymphadenopathies were processed on the COMET™. Each case was evaluated by 5 hematopathologists to assess diagnostic utility and usability of the seqIF™ panel alongside a haematoxylin and eosin stain of the tissue. Diagnoses rendered on seqIF™ were correlated with the diagnoses previously rendered on single-plex-chromogenic stains.

Results
All 38 markers in the seqIF™ panel showed equal, or better, CAS scores compared to standard-ofcare single-plex chromogenic markers. Diagnostic assessment of the 15 cases (75 interactions) showed 100% correlation with the diagnosis rendered using chromogenic single-plex stains. In 5/75 (6%) of diagnostic interactions, the comprehensive seqIF™ panel contained a marker crucial to diagnosis, which was not selected in the initial panel by the assessing pathologist. Usability of the seqIF™ panel was rated equivalent to chromogenic staining in 37/75 (49.3%) interactions, and better than chromogenic staining in 38/75 (50.8%) interactions. Assessment of co-expression was rated equal to chromogenic staining 71/74 (91%) of interactions.

Conclusions
This is the first investigation into the diagnostic utility of a comprehensive mIHC panel, such as seqIF™, for lymphoma, demonstrating that the technique is safe and effective for lymphoma diagnosis. Widespread adoption of comprehensive seqIF™ for diagnosis could improve turnaround times, facilitate interpretation, reduce diagnostic error and obviate the requirement for repeat biopsies due to tissue exhaustion.

Introduction
The tumor microenvironment (TME) plays a central role in the efficacy of T cell-engaging immunotherapies, including CD20×CD3 bispecific antibodies (BsAbs) in lymphoma. In follicular lymphoma (FL), multiplex immunofluorescence (mxIF) and single-cell RNAseq show disruption of nodal architecture and T cell subtype content (Radtke et al., Cancer Cell 2024). Spatially resolved transcriptomics (ST) offers a powerful approach to characterize the TME and cell interactions in situ. We applied ST and mxIF to analyze the TME in FL and nodal marginal zone lymphoma (MZL) tumors from patients receiving first-line CD20×CD3 BsAb mosunetuzumab (Mosun) in our investigator-sponsored phase 2 trial (NCT04792502).

Methods
Trial subjects (n=52) had FL or MZL requiring first-line therapy due to symptoms or tumor burden. They received Mosun for 4 cycles, followed by Mosun alone in case of interim complete response (CR) or Mosun + lenalidomide otherwise. We performed 10x Visium ST on formalin-fixed biopsies collected pre-treatment (n=12) or after 4-6 cycles of Mosun (paired n=2 biopsies), as well as single-cell resolved mxIF (Lunaphore COMET; n=4 pre-treatment biopsies). Spatial domains were defined by unsupervised clustering of integrated ST data, with further differential gene expression (via DESeq2), pathway enrichment (MSigDB and curated immune gene sets from Tumuluru et al. Blood 2025), and cellcell interaction analyses (via CellChat). TME composition was inferred by spatial deconvolution (CARD) and CytoSPACE using scRNA-seq references. mxIF images were processed by QuPath, with InstanSeg segmentation and cell-type assignment supported by a random-forest classifier. Spatial domains were manually annotated and cellular neighborhoods defined based on each cell’s 100 nearest neighbors.

Results
Across all baseline FL (n=10; 8 with CR, 2 refractory) and MZL (n=2, with CR) samples, four consistent spatial domains were identified: neoplastic follicle/germinal center (GC), border zone (BZ), interfollicular (IF), and stromal. The BZ showed the highest density of CD8⁺ T cells and LAMP3⁺ dendritic cells (DC; P<0.001), and intense CCR7–CCL19/21 signaling. LAMP3+ DC were the major source of CCL19CCR7 interactions targeting naïve/central memory CD8+ T cells, and Galectin 9–TIM-3 interactions targeting exhausted CD8+ T cells. Enriched BZ gene sets were related to T cell activation, differentiation, migration, and cytotoxicity, including upregulation of IL7R, GZMB, CD69, CCR7, SELL, ITGAX, CXCL9, and CCL19/21. Stromal signatures showed activated and CXCL10⁺ high endothelial venules in the BZ, suggesting active T cell recruitment. Notably, cytotoxic CD8⁺ T cells were excluded from the GC (P<0.001), and many exhibited an exhausted transcriptional program. On mxIF images (n=4 FL tumors encompassing 500K cells), BZ was significantly enriched with CD8+ T cells (compared with GC), as well as DCs and Tregs (compared with either GC or IF; P<.001). CD4+ and CD8+ T cells overexpressed PD-1 in GC and BZ compared with other domains (P<.001). In neighborhood analysis, BZ showed the highest entropy (P<0.001 vs. other zones), indicating greater cellular diversity. A specimen from a FL refractory to Mosun showed disruption of GC/BZ morphology with expansion of BZ cellular neighborhood rich in Tregs, macrophages, and LAG3+CD8+ T cells. Paired FL specimens pre- and on-treatment (n=2 pairs) showed absence of B-cells in one sample ontreatment, but residual GC-like areas in another. Expanded BZ areas were depleted of B cells, showing abundance of CD8+ T cells and macrophages, with overexpression of T cell activation and exhaustion signatures. Upregulated T cell-related genes included LCK, ZAP70, CCR7, TCF7, LEF1, as well as the inhibitory receptors LAG3 and VSIR. There were increased interactions involving CD47 and Galectin 9. Residual GC areas remained devoid of T cells but showed increased macrophage and cDC1 content and increased frequency of interactions involving CTLA4-CD80 and CD70-CD27 compared with pre-treatment.

Conclusions
This spatial analysis of TME identifies the perifollicular BZ as a hub for T cell recruitment and activity in FL, expanding during mosunetuzumab therapy. Increased macrophage content and T cell regulatory mechanisms occurring on therapy support exploration of rational BsAb-based combinations with agents that modulate T cell function, macrophage activity, or immune checkpoints to enhance therapeutic efficacy.

B-cell lymphomas encompass a wide range of malignancies, with clinical features ranging from indolent to aggressive. The diagnosis of these malignancies can routinely entail the evaluation of up to 20 immunohistochemistry (IHC) markers for complete characterization. An additional challenge is that analysis of these lymphomas requires inference of co-expression patterns from single-plex IHC on serial tissue sections, leading to significant subjectivity and inaccuracy. Increasingly, molecular and cytogenetic tests must also be performed placing additional demands on tissue material. At the same time, core needle biopsies, rather than full excisional biopsies, have become standard for lymphoma diagnosis, further limiting the material available for analysis (Seviar et al. 2020). Here, we present the development, validation, and evaluation of a 22-marker sequential immunofluorescence (seqIF, Rivest et al. 2023) panel for the diagnosis and prognostication of B-cell lymphomas that can be performed on a single single FFPE section on the Lunaphore COMET automated staining and imaging platform. Each marker was robustly validated through blinded hematopathologist review and compared to clinical IHC results from the same case. Validation was performed by converting each single marker expression from fluorescence to an RGB image with nuclei represented in a color similar to hematoxylin and the marker of interest in a color similar to DAB, which is routinely used in clinical practice. These sets of single-plex pseudo-colored images were then evaluated by a hematopathologist for diagnosis and classification, as well as comparison to the original IHC stains for that case. We further evaluated the effect of tissue section and tissue block age on performance of this panel and showed excellent results on tissue blocks more than10 years since collection. The COMET system can process four tissue slides in parallel in under 16 hours (overnight run) for this panel, which is compatible with expected clinical IHC turnaround times. We next evaluated the utility of this seqIF panel in the classification and diagnosis of indolent and aggressive B-cell lymphoma types including small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, large B-cell lymphoma, and other subtypes. As controls, we included benign lymph nodes, as well as biopsies exhibiting progressive transformation of germinal centers, follicular hyperplasia, and interfollicular hyperplasia, all of which can mimic malignancy and present diagnostic challenges. We constructed a tissue microarray (TMA) from these cases (n=85) to recapitulate the limited diagnostic material received from core biopsies. Again, blinded hematopathology review of pseudo single-plex images was performed, leading to a 100% concordance (marker positive/negative) between the COMET generated data and diagnostic IHC at the single marker level. Furthermore, there was no discordance between classification of a tissue as malignant vs benign, or indolent vs aggressive between the seqIF images and the clinical pathology report. Expression patterns of markers such as BCL2 and CMYC in malignant B-cells in large B-cell lymphoma have prognostic significance; however, other cell types within the tumor microenvironment can express these proteins, which makes the tumor specific quantification challenging. Our seqIF panel allows for accurate quantification of malignant B-cell co-expression of multiple prognostic markers, including BCL2, BCL6, CMYC, TP53, and KI67, as well as classification markers CD10, BCL6, and MUM1. With theimplementation of cell segmentation, this process of co-expression analysis can be fully automated, producing precise quantifications from tens of thousands of cells. Overall, we demonstrate the utility of a seqIF panel in the classification and prognostication of B-cell lymphomas. This panel has equal performance characteristics to single-plex clinical IHC in both qualitative marker assessment, tissue classification and B-cell lymphoma classification. Additionally, as the system performs the analysis on a single FFPE tissue section that can be reused for downstream applications, it preserves precious material for cytogenetic and molecular prognostic studies. Automated image analysis with the help of machine learning classifiers to assess for potential clinical diagnostic use and independent validation on a second set of diagnostic samples is ongoing.

Introduction
Infection and immune-driven inflammation of the gut often result in severe intestinal dysfunction or even end-organ disease. Intestinal dysfunction negatively affects outcome of allogeneic hematopoietic stem cell (HSCT) recipients. Cellular senescence (CS) represents a stress response mechanism. It is considered as a program of innate immunity and exhibits immunomodulatory characteristics. The study of DNA-damage and CS markers in nucleated cells of the intestinal stroma including innate and adaptive immunity cellular effectors infiltrating the intestinal stroma was performed. Correlations between DNA-damage and immune responses and the effect of common pre- and peritransplant risk factors responses were analyzed.

Methods
Using the multiplex immunofluorescence (Lunaphore CometTM) we analyzed a tissue microarray marked with 34 antibodies targeting immune cells, CS, apoptosis and inflammation from 27 colonic biopsies of 24 patients who developed lower gastro-intestinal (GI) symptoms after allogeneic HSCT. CD3 and CD68 markers were used to identify Tlymphocytes and macrophages infiltrating intestinal stroma, respectively. Promyelocytic leukemia protein (PML) and p53-binding protein 1 (53BP1) nuclear bodies as DNA-damage markers and CS marker p16INK4a were analyzed in all nucleated cells and in CD3 and CD68 cellular subsets of the intestinal stroma. Nuclear markers were quantified per nuclei count and per nuclear area. Immune cells were quantified per cell count and per stromal area. Mean fluorescence intensity was measured for every marker.

Results
Median time of GI symptoms onset was 64 days (12-903). GI symptoms stage III-IV were present in 71 % of recipients. The median time of onset of GI symptoms stage III-IV was 45 days (range 12-134), whereas the time of onset of GI symptoms stage I-II was 126 days (range 64-903) (p<0.01). Acute graft versus host disease (GVHD) was diagnosed in 71 % of patients. Overlap syndrome and chronic GVHD developed in 21 and 30 % of patients, respectively. Nuclear PML foci count positively correlated with the count of nuclear 53BP1 foci (R=0.53, p<0.01). Importantly, positive correlation was found between the count of 53BP1 foci and p16INK4a positivity (R=0.50, p<0.01). Increased PML nuclear foci count correlated with augmented infiltration with CD68+ macrophages and CD3+lymphocytes (R=0.46, p=0.02, R=0.45, p=0.02), respectively. Also, increased 53BP1 nuclear foci correlated with enriched infiltration with CD68+ macrophages and CD3+lymphocytes (R=0.61, p<0.01, R=0.39, p=0.04), respectively. Increased counts of 53BP1 foci were observed in patients who developed GI symptoms earlier than the median time onset (p=0.07). Also, patients presenting with severe GI symptoms (stage III-IV) had increased counts of 53BP1 nuclear foci (p=0.02). There was a positive correlation found between the time of lower GI symptoms onset and the number of CD3+ T-cells positive for p16INK4a (p=0.03). Patients classified as late onset acute GVHD/overlap syndrome had increased expression of p16INK4a in the intestinal stromal cells (p=0.08). Combined immunosuppression was associated with augmented counts of PML (p=0.03) and 53BP1 (p=0.06) nuclear foci in CD3+ lymphocytes, respectively. Moreover, in CD3+ lymphocytes, 53BP1 nuclear foci were increased in HLAmismatched recipients (p<0.01).

Conclusion
Organ-intrinsic immune reconstitution favors successful HSCT outcome. DNA-damage response and CS elicit immunomodulatory effects implying innate immune responses that may provide transitional substitute for restoring adaptive immunity but may become deleterious in long time perspective. Deciphering their roles in the context of HSCT-related complications may unravel novel treatment venues.