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Introduction: Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. Adverse events are common, and neurotoxicity is one of the most important. However, the physiopathology is unknown and neuropathologic information is scarce. Materials and methods: Post-mortem examination of 6 brains from patients that underwent CAR T-cell therapy from 2017 to 2022. In all cases, polymerase chain reaction (PCR) in paraffin blocks for the detection of CAR T cells was performed. Results: Two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. Two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. The neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly CD8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. Microbiological studies were negative for neurotropic viruses, and PCR failed to detect CAR T -cells. Another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. The remaining 4 cases only showed a mild patchy gliosis and microglial activation, and CAR T cells were detected by PCR only in one of them. Conclusions: In this series of patients that died after CAR T-cell therapy, we predominantly found non-specific or minimal neuropathological changes. CAR T-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings., Free Neuropathology, Bd. 3 (2022)

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. Adverse events are common, and neurotoxicity is one of the most important. However, the physiopathology is unknown and neuropathologic information is scarce. Materials and methods: Post-mortem examination of 6 brains from patients that underwent CAR T-cell therapy from 2017 to 2022. In all cases, polymerase chain reaction (PCR) in paraffin blocks for the detection of CAR T cells was performed. Results: Two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. Two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. The neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly CD8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. Microbiological studies were negative for neurotropic viruses, and PCR failed to detect CAR T -cells. Another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. The remaining 4 cases only showed a mild patchy gliosis and microglial activation, and CAR T cells were detected by PCR only in one of them. Conclusions: In this series of patients that died after CAR T-cell therapy, we predominantly found non-specific or minimal neuropathological changes. CAR T-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings.

We describe 36 patients with splenic marginal zone lymphoma (SMZL) with transformation (SMZL-T), including 15 from a series of 84 patients with SMZL diagnosed at the Hospital Clinic of Barcelona (HCB) and 21 diagnosed with SMZL-T in other centres. In the HCB cohort, the cumulative incidence of transformation at 5 years was 15%. Predictors for transformation were cytopenias, hypoalbuminaemia, complex karyotype (CK) and both the Intergruppo Italiano Linfomi (ILL) and simplified Haemoglobin, Platelet count, lactate dehydrogenase (LDH) and extrahilar Lymphadenopathy (HPLL)/ABC scores (P, This work was supported by grants from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (PI17/01061 to Sílvia Beà, PI19/00887 to Armando López-Guillermo), Fundacion AECC/CIBERONC (PROYE18020BEA to Sílvia Beà), and CIBERONC (CB16/12/00334 to Dolors Colomer, and CB16/12/00225 to Elías Campo), Generalitat de Catalunya Suport Grups de Recerca AGAUR, 2017-SGR-709 (to Sílvia Beà) 2017-SGR-1009 (to Dolors Colomer), 2017-SGR-1142 (to Elías Campo), Ministerio de Ciencia e Innovación (MCI) [Grant No. RTI2018-094274-B-I00 (to Elías Campo)] and the European Regional Development Fund ‘Una manera de fer Europa’. Elías Campo is an Academia Researcher of the ‘Institució Catalana de Recerca i Estudis Avançats’ (ICREA) of the Generalitat de Catalunya.

Introduction Outcomes for follicular lymphoma (FL) patients are generally good. Clinical and biological variables have been studied to identify patients at higher risk of early relapse, which markedly impacts survival. However, the genetic landscape of FL according to its clinical behavior (need of treatment and timing of relapse) is not well characterized, which was the aim of the present study. Patients and Methods We included 67 samples from 55 grade 1-3A FL patients from a single institution [55 samples at diagnosis (D), and 12 at relapse (R)]. Five groups were defined according to their clinical behavior: never required treatment after >5 years (y) of follow-up [never treated (NT), n=7]; treated and did not relapse after >9 y of follow-up, [non-relapsed (NR), n=19]; treated and relapsed beyond 24 months of frontline therapy [late relapse (LR), n=14]; treated and relapsed within 24 months of frontline treatment [early relapse (ER), n=11]; and primary refractory (PR, n=4). Of the 48 treated patients, 96% received R-CHOP. Patients developing histologic transformation were not included. DNA was extracted from FFPE tissue biopsies. Copy number alterations (CNA) were assessed in 50 D and 11 R samples [OncoScan CNV Assay (Thermofisher)], and single nucleotide variants (SNV) and insertions/deletions (indels) in 51 D and 10 R samples using a B-cell malignancy NGS panel examining 121 genes (SureSelectXT, Agilent Technologies). Genes and genomic regions were considered altered if they harbored SNV/indels and/or CNA. For comparisons and plotting, only the 52 D samples with both NGS and CNA data were considered. Non-parametric statistical tests were used. Results Median age was 56 y (range, 26−79), and 30 patients (55%) were female. Forty-two patients (76%) had stage III-IV disease, without significant differences among groups. Thirteen patients (25%) had a high-risk FLIPI score, a percentage that was higher in the LR and PR groups (50 and 67%, respectively, P=0.02). With a median follow-up of 12.9 y, 10-y overall survival estimates were 71, 100, 74, 82, and 0% for NT, NR, LR, ER, and PR patients, respectively. We detected CNA in all samples, with a median number of 7 (range, 1−27) for D samples, and of 6 (2−19) for R samples. We also identified SNV/indels in all samples, with a median number of 10 (1−23) for D samples, and of 9 (3−18) for R samples. The most commonly altered genes at diagnosis were KMT2D (82%), CREBBP (73%), SPEN (38%), TNFRSF14 (38%), ARID1A (33%), and BCL2 (33%) (Figure). There were no significant differences in the number of altered genes/regions among the five groups. Genes or regions with significantly different alteration frequency among groups were: CARD11 (57, 12, 0, 9, and 0% for NT, NR, LR, ER, and PR, respectively, P=0.014), CD70 (12% for NR, 50% for PR, and 0 for the remaining groups P=0.026), HIST1H1B (29% for NT, and 0% for the remaining groups, P=0.020), HVCN1 (43, 6, 0, 18, and 0% for NT, NR, LR, ER, and PR, respectively, P=0.048), KLHL6 (12% for NR, 75% for PR, and 0 for the remaining groups, P=0.002), PRKCB (0, 6, 0, 18, 50%, P=0.037), and 13q14.2-q14.3 loss (DLEU1/DLEU2) (24% for NR, 50% for PR, and 0 for the remaining groups, P=0.015). Two genes had a significantly lower alteration rate comparing the cases with a favorable (NT, NR, LR) and more aggressive (ER, PR) clinical behavior: CIITA (22 vs. 53%, P=0.043), and PRKCB (3 vs. 27%, P=0.044). Of the 51 patients for which the m7-FLIPI was calculated, six (12%) had a high-risk score. Of note, none of the high-risk patients belonged to the NT or NR groups. No significant differences were found in the number of altered genes/regions between D and R, or in the alteration frequency of each gene/region. We identified the presence of an ancestral common precursor cell with a divergent evolution in all paired cases. The median percentage of shared alterations between D and R was 50% (range 28−63%). Conclusions In this comprehensive genetic analysis of 55 FL patients, categorized into five groups according to their clinical behavior, alterations in the chromatin modifying genes KMT2D and CREBBP were the most frequent, and CIITA and PRKCB were more frequently altered in cases with a shorter duration of response. These data warrant further study of the mechanisms underlying these mutations, mutual exclusivity/co-occurrence, and relationships with B-cell biology and the tumor microenvironment. Disclosures Nadeu: Janssen: Honoraria. Giné:Gilead: Research Funding; Janssen: Research Funding; Roche: Research Funding. Armando:Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding.

Introduction: Previous studies have highlighted the potential of cell-free DNA (cfDNA) to assess the mutational profile and copy number alterations (CNA) in diffuse large B-cell lymphoma (DLBCL), usually performed in tissue biopsies, both at diagnosis and relapse. In addition, the quantitative levels of cfDNA might be a surrogate of the lymphoma tumor burden and could therefore predict response to therapy, progression-free survival (PFS) and overall survival (OS). The aim of this study was to analyze the mutational profile and CNA at diagnosis using cfDNA, to compare these results with those obtained from formalin-fixed paraffin-embedded (FFPE) biopsies, and to estimate the correlation of pre-treatment levels of cfDNA with FDG-PET/CT Total Metabolic Tumor Volume (TMTV) and their impact in the outcome of DLBCL patients. Methods: We included 79 patients (41M/38F , median age 63 years) diagnosed with DLBCL according to the WHO criteria in a single institution between 2016 and 2018. All patients received chemoimmunotherapy. After frontline treatment, 56 patients achieved a complete (CR) response, 4 partial response, 16 were refractory, including 7 early deaths, and in 3 cases the response was not yet evaluable. Samples were obtained at diagnosis before starting treatment. cfDNA extraction was performed from 2-4 mL of plasma collected in PAXgene Blood ccfDNA tubes (Qiagen) and size distribution of DNA fragments was analyzed using the Agilent 2100 Bioanalyzer. Tumor genomic DNA (gDNA) was isolated from FFPE diagnostic tissue biopsies. A panel of 115 genes was enriched using a hybridization capture-based protocol from 10-30 ng of cfDNA and 150 ng of gDNA (ThruPlex Tag-seq kit and SureSelectXT enrichment reagents, Agilent Technologies) and sequenced in a MiSeq instrument (Illumina). CNA were examined using CNVkit software toolkit. cfDNA levels were reported as haploid genome equivalents per mL of plasma and expressed as a base 10 logarithm (log hGE/mL). Quantitative analysis of TMTV was performed using the semiautomatic MIM software, with a fixed SUV>2.5 thresholding method for segmentation. Results: A median of 15.6 ng/mL (range: 4-754 ng/mL) of cfDNA was obtained. At least one mutation could be detected in 69/79 cases (87.3%). The median number of mutations per sample was 6 (range: 0-41 mutations). The genes most frequently mutated at diagnosis in plasma samples were KMT2D, TP53, TNFRSF14, MYD88, BCL2, SOCS1, CREBBP, MYC and EP300. In 45 cases, paired FFPE samples were available. Sensitivity of cfDNA to detect mutations in baseline FFPE samples was 69% (95%CI: 64.1-73.9). In 28 of the 45 cases, >70% of the mutations were observed both in the cfDNA and FFPE samples. In the remaining 17 cases, the number of mutations identified in cfDNA was lower than the one observed in the FFPE sample (Figure 1a). Of note, most of the cases in which mutations were not detected in the cfDNA corresponded to localized stages (11/17, 65%) and/or primary extranodal DLBCL (12/17, 71%). The CNA profile in cfDNA was similar to that reported in tissue, with recurrent deletions of TNSFRS14 (11%), TNFAIP3 (14%), CDKN2A (7%), or TP53 (7%), as well as gains of REL (7%) or BCL2 (5%), among others (Figure 1b). Median pre-treatment TMTV (N=48) was 292 cm3 (0-4,171 cm3). Higher TMTV predicted for a poorer PFS (HR 4.85; p=0.005). cfDNA concentration significantly correlated with TMTV (R=0.546, p3 log hGE/mL) had a significantly lower CR rate, PFS, and OS than those with low levels (CR rate, 47% vs. 92%, p Conclusions: cfDNA represents a valuable tool to easily assess the genomic landscape and tumor burden in DLBCL, as well as to predict response to therapy and outcome in these patients. Figure 1 Disclosures Gine: Janssen: Other: Travel expenses, Research Funding; Roche: Other: Travel expenses, Research Funding; Gilead: Other: Travel expenses, Research Funding. Lopez-Guillermo:Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Janssen: Research Funding.