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.

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.

Tracing Founder Mutations in Circulating and Tissue-Resident Follicular Lymphoma Precursors.

Follicular lymphomas (FL) are characterized by BCL2 translocations, often detectable in blood years before FL diagnosis, but also observed in aging healthy individuals, suggesting additional lesions are required for lymphomagenesis. We directly characterized early cooperating mutations by ultradeep sequencing of prediagnostic blood and tissue specimens from 48 subjects who ultimately developed FL. Strikingly, CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, and largely distinguished patients developing FL (14/48, 29%) from healthy adults with or without detected BCL2 rearrangements (0/13, P = 0.03 and 0/20, P = 0.007, respectively). CREBBP variants were detectable a median of 5.8 years before FL diagnosis, were clonally selected in FL tumors, and appeared restricted to the committed B-cell lineage. These results suggest that mutations affecting the CREBBP KAT domain are common lesions in FL cancer precursor cells (CPC), with the potential for discriminating subjects at risk of developing FL or monitoring residual disease. SIGNIFICANCE: Our study provides direct evidence for recurrent genetic aberrations preceding FL diagnosis, revealing the combination of BCL2 translocation with CREBBP KAT domain mutations as characteristic committed lesions of FL CPCs. Such prediagnostic mutations are detectable years before clinical diagnosis and may help discriminate individuals at risk for lymphoma development. This article is highlighted in the In This Issue feature, p. 1275.

A randomized trial on the effects of heat stress abatement on environmental conditions and growth, feed efficiency, and concentration of metabolites of pre-weaned female Holstein calves.

Heat abatement strategies for pre-weaned calves are seldom adopted. Our objectives were to determine the effects of adding fans to barns on environmental conditions and growth, feed efficiency, concentration of metabolites and health of pre-weaned female Holstein calves. Calves born from July 15th to 30th of 2019 were eligible for enrollment. At birth (d 0), calves were assigned randomly to: SH (n = 125) - hutch under a barn with no cooling, SHF (n = 101) - hutch under a barn cooled through fans. Body weight (BW) and wither-height were measured at birth and d 68. Calves were evaluated thrice weekly (0700-1000 h) using the Calf Health Scoring Chart (UW-Madison). A sub-sample of hutches (SH = 26, SHF = 25) was evaluated for air velocity and temperature at 1000 and 1600 h thrice weekly and calves housed in these hutches were evaluated for rectal temperature (RT) at 1600 h and respiratory rate (RR) at 1000 and 1600 h. Calves were fed a liquid diet twice a day (d 2-18 = 5.56 L/d; d 19-49 = 7.58 L/d; d 50-56 = 3.84 L/d; d 57-63 = 1.64 L/d) and starter ad libitum starting on d 14. A sub-sample of calves (SH = 56, SHF = 44) had intakes of liquid feed and starter measured daily, BW and wither-height measured weekly from birth to d 68, and blood sampled on d 1, 14, 28, 42, 49, 52, 56, 58, 63 and 65 for the measurement of fatty acids, ß-hydroxybutyrate, and glucose concentrations. The SHF treatment increased air velocity by 0.8 m/sec and reduced air temperature by 0.3 ºC. The SHF treatment reduced RT (38.70 ± 0.03 vs. 38.78 ± 0.02 °C) and the percentage of calves with hyperthermia (RT ≥ 39.2 °C; 20.6 ± 1.9 vs. 30.2 ± 2.0%) at 1000 h. Treatment did not affect feed efficiency (SH = 0.53 ± 0.01, SHF = 0.53 ± 0.01 g of BW gained/g of dry matter intake), nor did it affect BW (SH = 81.6 ± 0.7, SHF = 82.9 ± 0.8 kg) and wither-height (SH = 89.5 ± 0.3, SHF = 90.1 ± 0.3 cm) on d 68. Concentrations of metabolites were not affected by treatment. Cooling the environment through fans reduced RT and the risk of hyperthermia at 1000 h but it did not affect performance of pre-weaned Holstein calves.

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.

Molecular Monitoring of Lymphomas.

Molecular monitoring of tumor-derived alterations has an established role in the surveillance of leukemias, and emerging nucleic acid sequencing technologies are likely to similarly transform the clinical management of lymphomas. Lymphomas are well suited for molecular surveillance due to relatively high cell-free DNA and circulating tumor DNA concentrations, high somatic mutational burden, and the existence of stereotyped variants enabling focused interrogation of recurrently altered regions. Here, we review the clinical scenarios and key technologies applicable for the molecular monitoring of lymphomas, summarizing current evidence in the literature regarding molecular subtyping and classification, evaluation of treatment response, the surveillance of active cellular therapies, and emerging clinical trial strategies.

Heat abatement during the pre-weaning period: effects on lying behavior and disbudding-related responses of male Holstein calves.

Strategies to abate heat stress are seldom adopted for pre-weaned dairy calves and little is known about their effects on behavior and pain sensitivity of youngstock. Our objectives were to determine the effects of heat stress abatement on lying behavior and disbudding-related pain sensitivity, wound healing, and change in intake. Male Holstein calves (n = 60; 0 to 68 d of age) were assigned randomly at birth (d 0) to 1 of 3 treatments: hutch outdoors with 50% of its area covered with plywood (control = 20), hutch in a barn with no cooling (SH = 21), and hutch in a barn with ceiling fans (SHF = 19). Calves were fitted with lying-behavior loggers on the hind leg from d 1 to 68. On d 32 ± 8 (±SD), we disbudded calves using hot iron, 30 min after cornual nerves were blocked with lidocaine. Immediately before (0 h), and at 1, 2, and 3 h after disbudding, we evaluated calves for mechanical nociceptive threshold (MNT) and head (ear flick, head shake, head rubbing) and somatic (tail flicking, foot stamping, restlessness) behaviors. On d 1, 3, 7, and 14 after disbudding, we evaluated the MNT and, on d 7 and 14, we evaluated wound healing (1 = crust, 5 = exudate). We calculated the relative change in milk solids and starter intake from d 0 to 6 relative to disbudding compared with the average of the 72 h preceding the procedure. The lying time was 0.6 h/d greater for the SHF treatment compared with the SH treatment. The control treatment resulted in 3.2 and 4.1 more lying bouts per day than the SH and SHF treatments, respectively; consequently, the control treatment resulted in lying-bout duration 7.7 and 10.9 min/event shorter than the SH and SHF treatments, respectively. We did not detect an effect of treatment on the number of disbudding-related head and somatic behaviors and MNT. The odds of calves having abnormal wound was 3.5 and 3.2 times greater for the control treatment compared with the SH and SHF treatments, respectively. We did not detect an effect of treatment on the relative change in intake of milk solids and starter. Heat abatement improves the welfare of pre-weaned dairy calves and may hasten healing.

Heat abatement during the pre-weaning period: effects on growth, feed efficiency, metabolites, and insulin of male Holstein calves.

Heat stress abatement strategies for pre-weaned dairy calves are seldom evaluated. An experiment was conducted to evaluate the effects of housing calves under a barn and provision of fans to calves housed under a barn on calfhood performance. The experiment was conducted in a dairy in southern Georgia, USA. Male Holstein calves (n = 60; 0 to 68 day of age) were assigned randomly at birth (day 0) to 1 of 3 treatments: hutch outdoors with 50% of its area covered with plywood (control = 20), hutch in a barn with no cooling (SH = 21), and hutch in a barn with ceiling fans (SHF = 19). Body weight (BW) was measured at birth, and total serum protein and wither-height were measured 24 to 48 h after birth. A sub-set of hutches was evaluated for air speed and temperature, and rectal temperature (RT) and respiratory frequency (RF) of calves housed in these hutches were measured at 0900 and 1500 h. Intakes of liquid feed (days 14 to 63) and starter (days 14 to 68) were recorded daily, BW and wither-height were measured weekly, and feed efficiency was calculated weekly. Blood was sampled on days 1, 14, 28, 42, 49, 52, 56, 58, 63, and 65 for the measurement of fatty acids, ß-hydroxybutyrate, glucose, and insulin. The SHF treatment resulted in air velocity 0.56 to 0.83 m/s greater (P < 0.01) than the control and SH treatments, respectively, whereas the control treatment resulted in air temperature 1.2 to 3.2 °C greater (P < 0.01) than the SH and SHF treatments, respectively. The RT of calves in the control treatment was 0.1 to 1.1 °C greater (P ≤ 0.03) than the SH and SHF treatments, respectively, and the control treatment resulted in RF 39.4 to 60.2 mov/min greater (P < 0.01) than the SH and SHF treatments, respectively. Treatment did not (P ≥ 0.27) affect feed efficiency and concentrations of metabolites and insulin, but calves in the control treatment were 2.6 cm shorter (P = 0.03) than calves in the SHF treatments at weaning. Provision of fans to calves housed under a barn reduced RT, RF, but only had a minute impact on wither-height.

Inferring gene expression from cell-free DNA fragmentation profiles.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.

CD20-Targeted Therapy Ablates De Novo Antibody Response to Vaccination but Spares Preestablished Immunity.

To obtain a deeper understanding of poor responses to COVID-19 vaccination in patients with lymphoma, we assessed blocking antibodies, total anti-spike IgG, and spike-specific memory B cells in the peripheral blood of 126 patients with lymphoma and 20 age-matched healthy controls 1 and 4 months after COVID-19 vaccination. Fifty-five percent of patients developed blocking antibodies postvaccination, compared with 100% of controls. When evaluating patients last treated from days to nearly 18 years prior to vaccination, time since last anti-CD20 was a significant independent predictor of vaccine response. None of 31 patients who had received anti-CD20 treatment within 6 months prior to vaccination developed blocking antibodies. In contrast, patients who initiated anti-CD20 treatment shortly after achieving a vaccine-induced antibody response tended to retain that response during treatment, suggesting a policy of immunizing prior to treatment whenever possible. SIGNIFICANCE: In a large cohort of patients with B-cell lymphoma, time since anti-CD20 treatment was an independent predictor of neutralizing antibody response to COVID-19 vaccination. Comparing patients who received anti-CD20 treatment before or after vaccination, we demonstrate that vaccinating first can generate an antibody response that endures through anti-CD20-containing treatment. This article is highlighted in the In This Issue feature, p. 85.

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.

Topical administration of tranexamic acid in primary total hip and total knee arthroplasty.

Major blood loss is a known potential complication in total hip and total knee arthroplasty. We conducted a prospective, stratified, randomized, double-blind, placebo-controlled trial that evaluated 100 patients undergoing total knee or total hip arthroplasty to evaluate the effect on blood loss using the topical application of tranexamic acid. Participants received either 2 g of topical tranexamic acid or the equivalent volume of placebo into the joint prior to surgical closure. Tranexamic acid resulted in a lower mean maximum decline in postoperative hemoglobin levels when compared to placebo (P = 0.013). Patients in the tranexamic acid group demonstrated an improved but non-significant reduction in the units of blood transfused compared to placebo (P = 0.423). There was no clinically significant increase in complications in the tranexamic acid group, including no incidence of venous thromboembolism.