Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.

Risk of Second Tumors and T-Cell Lymphoma after CAR T-Cell Therapy.

BACKGROUND: The risk of second tumors after chimeric antigen receptor (CAR) T-cell therapy, especially the risk of T-cell neoplasms related to viral vector integration, is an emerging concern. METHODS: We reviewed our clinical experience with adoptive cellular CAR T-cell therapy at our institution since 2016 and ascertained the occurrence of second tumors. In one case of secondary T-cell lymphoma, a broad array of molecular, genetic, and cellular techniques were used to interrogate the tumor, the CAR T cells, and the normal hematopoietic cells in the patient. RESULTS: A total of 724 patients who had received T-cell therapies at our center were included in the study. A lethal T-cell lymphoma was identified in a patient who had received axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma, and both lymphomas were deeply profiled. Each lymphoma had molecularly distinct immunophenotypes and genomic profiles, but both were positive for Epstein-Barr virus and were associated with DNMT3A and TET2 mutant clonal hematopoiesis. No evidence of oncogenic retroviral integration was found with the use of multiple techniques. CONCLUSIONS: Our results highlight the rarity of second tumors and provide a framework for defining clonal relationships and viral vector monitoring. (Funded by the National Cancer Institute and others.).

Cell-type specific features of circular RNA expression.

Thousands of loci in the human and mouse genomes give rise to circular RNA transcripts; at many of these loci, the predominant RNA isoform is a circle. Using an improved computational approach for circular RNA identification, we found widespread circular RNA expression in Drosophila melanogaster and estimate that in humans, circular RNA may account for 1% as many molecules as poly(A) RNA. Analysis of data from the ENCODE consortium revealed that the repertoire of genes expressing circular RNA, the ratio of circular to linear transcripts for each gene, and even the pattern of splice isoforms of circular RNAs from each gene were cell-type specific. These results suggest that biogenesis of circular RNA is an integral, conserved, and regulated feature of the gene expression program.

A human lymphoma organoid model for evaluating and targeting the follicular lymphoma tumor immune microenvironment.

Heterogeneity in the tumor microenvironment (TME) of follicular lymphomas (FLs) can affect clinical outcomes. Current immunotherapeutic strategies, including antibody- and cell-based therapies, variably overcome pro-tumorigenic mechanisms for sustained disease control. Modeling the intact FL TME, with its native, syngeneic tumor-infiltrating leukocytes, is a major challenge. Here, we describe an organoid culture method for cultivating patient-derived lymphoma organoids (PDLOs), which include cells from the native FL TME. We define the robustness of this method by successfully culturing cryopreserved FL specimens from diverse patients and demonstrate the stability of TME cellular composition, tumor somatic mutations, gene expression profiles, and B/T cell receptor dynamics over 3 weeks. PDLOs treated with CD3:CD19 and CD3:CD20 therapeutic bispecific antibodies showed B cell killing and T cell activation. This stable system offers a robust platform for advancing precision medicine efforts in FL through patient-specific modeling, high-throughput screening, TME signature identification, and treatment response evaluation.

Circulating Tumor DNA Profiling for Detection, Risk Stratification, and Classification of Brain Lymphomas.

PURPOSE: Clinical outcomes of patients with CNS lymphomas (CNSLs) are remarkably heterogeneous, yet identification of patients at high risk for treatment failure is challenging. Furthermore, CNSL diagnosis often remains unconfirmed because of contraindications for invasive stereotactic biopsies. Therefore, improved biomarkers are needed to better stratify patients into risk groups, predict treatment response, and noninvasively identify CNSL. PATIENTS AND METHODS: We explored the value of circulating tumor DNA (ctDNA) for early outcome prediction, measurable residual disease monitoring, and surgery-free CNSL identification by applying ultrasensitive targeted next-generation sequencing to a total of 306 tumor, plasma, and CSF specimens from 136 patients with brain cancers, including 92 patients with CNSL. RESULTS: Before therapy, ctDNA was detectable in 78% of plasma and 100% of CSF samples. Patients with positive ctDNA in pretreatment plasma had significantly shorter progression-free survival (PFS, P < .0001, log-rank test) and overall survival (OS, P = .0001, log-rank test). In multivariate analyses including established clinical and radiographic risk factors, pretreatment plasma ctDNA concentrations were independently prognostic of clinical outcomes (PFS HR, 1.4; 95% CI, 1.0 to 1.9; P = .03; OS HR, 1.6; 95% CI, 1.1 to 2.2; P = .006). Moreover, measurable residual disease detection by plasma ctDNA monitoring during treatment identified patients with particularly poor prognosis following curative-intent immunochemotherapy (PFS, P = .0002; OS, P = .004, log-rank test). Finally, we developed a proof-of-principle machine learning approach for biopsy-free CNSL identification from ctDNA, showing sensitivities of 59% (CSF) and 25% (plasma) with high positive predictive value. CONCLUSION: We demonstrate robust and ultrasensitive detection of ctDNA at various disease milestones in CNSL. Our findings highlight the role of ctDNA as a noninvasive biomarker and its potential value for personalized risk stratification and treatment guidance in patients with CNSL.[Media: see text].

Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

A population-based study of global outcome after moderate to severe traumatic brain injury in children and adolescents.

OBJECTIVE: The primary aim of this study was to evaluate the global outcome longitudinally over 5 years in children and adolescents surviving moderate to severe traumatic brain injury (msTBI) to investigate changes in outcome over time. The secondary aim was to explore how age at the time of injury affected outcome. METHODS: All children and adolescents (aged 0-17 years; subdivided into children aged 0-10 years and adolescents aged 11-17 years) with moderate (Glasgow Coma Scale [GCS] score 9-13) or severe (GCS score ≤ 8) TBI who were admitted to a level I trauma center in Norway over a 10-year period (2004-2014) were prospectively included. In addition, young adults (aged 18-24 years) with msTBI were included for comparison. Outcome was assessed with the Glasgow Outcome Scale-Extended (GOS-E) at 6 months, 12 months, and 5 years after injury. The effect of time since injury and age at injury on the probability of good outcome was estimated by the method of generalized estimating equations. RESULTS: A total of 30 children, 39 adolescents, and 97 young adults were included, among which 24 children, 38 adolescents, and 76 young adults survived and were planned for follow-up. In-hospital mortality from TBI was 7% for children, 3% for adolescents, and 18% for young adults. In surviving patients at the 5-year follow-up, good recovery (GOS-E score 7 or 8) was observed in 87% of children and all adolescents with moderate TBI, as well as in 44% of children and 59% of adolescents with severe TBI. No patient remained in a persistent vegetative state. For all patients, the odds for good recovery increased from 6 to 12 months (OR 1.79, 95% CI 1.15-2.80; p = 0.010), although not from 12 months to 5 years (OR 0.98, 95% CI 0.62-1.55; p = 0.940). Children/adolescents (aged 0-17 years) had higher odds for good recovery than young adults (OR 2.86, 95% CI 1.26-6.48; p = 0.012). CONCLUSIONS: In this population-based study of pediatric msTBI, surprisingly high rates of good recovery over 5 years were found, including good recovery for a large majority of children and all adolescents with moderate TBI. Less than half of the children and more than half of the adolescents with severe TBI had good outcomes. The odds for good recovery increased from 6 to 12 months and were higher in children/adolescents (aged 0-17 years) than in young adults.

Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA.

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors.

Incidence and mortality of moderate and severe traumatic brain injury in children: A ten year population-based cohort study in Norway.

OBJECTIVE: In this study we wanted to estimate population-based rates of incidence and mortality of moderate and severe traumatic brain injury (TBI) in children in one specific region in Norway. METHODS: In the region there are seven acute care hospitals (ACHs) in addition to a Level 1 trauma centre. Of 702 869 inhabitants (2014), 145 395 were children aged 0-16 years. Data were collected during ten years (2004-2014). All children aged 0-16 years with moderate (Glasgow Coma Scale [GCS] score 9-13) or severe (GCS score ≤ 8) TBI admitted to the Level 1 trauma centre were prospectively included. Children treated outside the Level 1 trauma centre were retrospectively included from the ACHs. Children who died from TBI prehospitally were included from the National Cause of Death Registry. Poisson regression was used to estimate incidence rate ratios (with a 95% confidence interval) comparing age, sex, and time periods. RESULTS: A total of 71 children with moderate or severe TBI were identified. Crude incidence rates were 2·4 (95% CI 1·7-3·3) for moderate and 2·5 (95% CI 1·8-3·4) for severe TBI per 100 000 person-years (py). Mortality rate from TBI was 1·2 (95% CI 0·7-1·9) per 100 000 py, and 88% were prehospital deaths. CONCLUSION: The incidence rates and mortality of moderate and severe TBI were low compared to international reports. Most likely explained by successful national prevention of TBI.

Structurally Conserved Primate LncRNAs Are Transiently Expressed during Human Cortical Differentiation and Influence Cell-Type-Specific Genes.

The cerebral cortex has expanded in size and complexity in primates, yet the molecular innovations that enabled primate-specific brain attributes remain obscure. We generated cerebral cortex organoids from human, chimpanzee, orangutan, and rhesus pluripotent stem cells and sequenced their transcriptomes at weekly time points for comparative analysis. We used transcript structure and expression conservation to discover gene regulatory long non-coding RNAs (lncRNAs). Of 2,975 human, multi-exonic lncRNAs, 2,472 were structurally conserved in at least one other species and 920 were conserved in all. Three hundred eighty-six human lncRNAs were transiently expressed (TrEx) and many were also TrEx in great apes (46%) and rhesus (31%). Many TrEx lncRNAs are expressed in specific cell types by single-cell RNA sequencing. Four TrEx lncRNAs selected based on cell-type specificity, gene structure, and expression pattern conservation were ectopically expressed in HEK293 cells by CRISPRa. All induced trans gene expression changes were consistent with neural gene regulatory activity.