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Bladder cancer is the tenth most common cancer worldwide, accounting for over 500,000 new cancer cases and 200,000 cancer-related deaths per year. Depending on the degree of muscle invasiveness and disease spread at the time of diagnosis, 5-year survival rates can range from as high as 96% to as low as 6%. In recent years, use of checkpoint inhibitor treatments for bladder cancer has become more common. Currently, there are five PD-1/PD-L1 checkpoint inhibitors approved by the FDA for treatment of bladder cancer and these treatments have been used across the spectrum of disease, from non-muscle invasive to metastatic disease. However, many patients still do not receive benefit from checkpoint inhibitor treatments. For example, clinical trials have shown response rates of only 20-30% in advanced disease. To improve treatment responses and identify patients who will benefit most from checkpoint inhibitor treatment, a deeper understanding of mechanisms of treatment response is needed. In an effort to explore mechanisms of response to combined PD-1/CTLA-4 checkpoint inhibitor treatment, we generated single cell RNA expression sequencing from a carcinogen-induced mouse model of muscle invasive urothelial carcinoma that included three conditions: untreated tumor, tumor treated with combination treatment, and tumor treated with combination treatment after Cd4+ T-cell depletion. After classifying tumor cells based on per cell detection of somatic variants identified using exome sequencing and assigning non-tumor cell types using SingleR, we performed differential expression, overrepresentation, and gene set enrichment analysis (GSEA) within each cell type, comparing each pairwise set of conditions. This analysis revealed that MSigDB’s hallmark interferon gamma response gene set was overrepresented across multiple cell types. Furthermore, GSEA indicated that with combined checkpoint blockade treatment, endothelial cells showed significant enrichment of upregulated interferon gamma response genes. Functional analysis demonstrated that knocking out Ifngr1 in endothelial cells inhibited the treatment response seen in wild type treated mice. Together, these results indicate that interferon gamma response in endothelial cells is a key mediator of combined PD-1/CTLA-4 checkpoint inhibitor treatment response. Citation Format: Sharon L. Freshour, Bryan Fisk, Timothy Hung-Po Chen, Haolin Shen, Matthew Mosior, Catrina Fronick, Jennifer Bolzenius, Obi L. Griffith, Vivek Arora, Malachi Griffith. Comprehensive single-cell transcriptomic analysis of a mouse model of bladder cancer identifies endothelial cells as a principal target of interferon gamma during response to combined PD-1, CTLA-4 immune checkpoint inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6.

Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X–Y crossing-over unleashed a second dynamic: selfish X–Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of Bos taurus (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X–Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X–Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.

Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Mutations in the DNA methyltransferase 3A ( DNMT3A ) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a −/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a −/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a −/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Significance Current acute myeloid leukemia (AML) risk assessment relies on cytogenetics and gene-sequencing studies but is imperfect, especially for patients with normal karyotypes and intermediate risk. To understand factors associated with excellent responses in these patients, we compared genetic and transcriptional data from patients with first remissions lasting more than 5 y after chemotherapy, to matched controls with first remissions lasting fewer than 2 y. AML cells from patients with early relapse displayed an immunosuppressive phenotype that blocked CD4 T cell activation via the T cell receptor; the long first-remission AML cells did not display this phenotype. Inhibiting LAG3 reversed this immunosuppression in most tested cases. This immunosuppressive phenotype may help to stratify risk of relapse at presentation, and outcomes., Acute myeloid leukemia (AML) patients rarely have long first remissions (LFRs; >5 y) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, standard remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing, and functional immunologic studies, we characterized 28 normal karyotype (NK)-AML patients with >5 y first remissions after chemotherapy (LFRs) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 y (standard first remissions [SFRs]). Our combined analyses indicated that genetic-risk profiling at presentation (as defined by European LeukemiaNet [ELN] 2017 criteria) was not sufficient to explain the outcomes of many SFR cases. Single-cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells or blocking the inhibitory major histocompatibility complex class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell–mediated suppression of CD4+ T cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ Tcell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to ∼1000x median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7; novel mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells, allowing for the analysis for clinically relevant genomic variants in large cohorts of cHL patients.

Getting around the blood–brain barrier The meninges comprise three membranes that surround and protect the central nervous system (CNS). Recent studies have noted the existence of myeloid cells resident there, but little is known about their ontogeny and function, and whether other meningeal immune cell populations have important roles remains unclear (see the Perspective by Nguyen and Kubes). Cugurra et al. found in mice that a large proportion of continuously replenished myeloid cells in the dura mater are not blood derived, but rather transit from cranial bone marrow through specialized channels. In models of CNS injury and neuroinflammation, the authors demonstrated that these myeloid cells have an immunoregulatory phenotype compared with their more inflammatory blood-derived counterparts. Similarly, Brioschi et al. show that the meninges host B cells that are also derived from skull bone marrow, mature locally, and likely acquire a tolerogenic phenotype. They further found that the brains of aging mice are infiltrated by a second population of age-associated B cells, which come from the periphery and may differentiate into autoantibody-secreting plasma cells after encountering CNS antigens. Together, these two studies may inform future treatment of neurological diseases. Science , abf7844, abf9277, this issue p. eabf7844 , p. eabf9277 ; see also abj8183, p. 396

Multiple myeloma (MM) is characterized by the uncontrolled proliferation of plasma cells. Despite recent treatment advances, it is still incurable as disease progression is not fully understood. To investigate MM and its immune environment, we apply single cell RNA and linked-read whole genome sequencing to profile 29 longitudinal samples at different disease stages from 14 patients. Here, we collect 17,267 plasma cells and 57,719 immune cells, discovering patient-specific plasma cell profiles and immune cell expression changes. Patients with the same genetic alterations tend to have both plasma cells and immune cells clustered together. By integrating bulk genomics and single cell mapping, we track plasma cell subpopulations across disease stages and find three patterns: stability (from precancer to diagnosis), and gain or loss (from diagnosis to relapse). In multiple patients, we detect “B cell-featured” plasma cell subpopulations that cluster closely with B cells, implicating their cell of origin. We validate AP-1 complex differential expression (JUN and FOS) in plasma cell subpopulations using CyTOF-based protein assays, and integrated analysis of single-cell RNA and CyTOF data reveals AP-1 downstream targets (IL6 and IL1B) potentially leading to inflammation regulation. Our work represents a longitudinal investigation for tumor and microenvironment during MM progression and paves the way for expanding treatment options., Clonal evolution in multiple myeloma (MM) needs to be understood in both the tumor and its microenvironment. Here the authors perform single-cell multi-omics profiling of samples from MM patients at different stages, finding transitions in the immune cell composition throughout progression.

Virtually all tumors are genetically heterogeneous, containing mutationally-defined subclonal cell populations that often have distinct phenotypes. Single-cell RNA-sequencing has revealed that a variety of tumors are also transcriptionally heterogeneous, but the relationship between expression heterogeneity and subclonal architecture is unclear. Here, we address this question in the context of Acute Myeloid Leukemia (AML) by integrating whole genome sequencing with single-cell RNA-sequencing (using the 10x Genomics Chromium Single Cell 5’ Gene Expression workflow). Applying this approach to five cryopreserved AML samples, we identify hundreds to thousands of cells containing tumor-specific mutations in each case, and use the results to distinguish AML cells (including normal-karyotype AML cells) from normal cells, identify expression signatures associated with subclonal mutations, and find cell surface markers that could be used to purify subclones for further study. This integrative approach for connecting genotype to phenotype is broadly applicable to any sample that is phenotypically and genetically heterogeneous., The advent of single-cell RNA sequencing has revealed significant transcriptional heterogeneity in cancer, but its relationship to genomic heterogeneity remains unclear. Focusing on acute myeloid leukemia samples, the authors describe a general approach for linking mutation-containing cells to their transcriptional phenotypes using single-cell RNA sequencing data.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes categorized on the basis of their core regulatory programs and the expression of signature cytokines. Human ILC3s that produce the cytokine interleukin-22 convert into ILC1-like cells that produce interferon-γ in vitro, but whether this conversion occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets. RNA velocity analysis identified an intermediate ILC3-ILC1 cluster, which had strong directionality toward ILC1s. In humanized mice, the acquisition of ILC1 features by ILC3s showed tissue dependency. Chromatin studies indicated that the transcription factors Aiolos and T-bet cooperated to repress regulatory elements active in ILC3s. A transitional ILC3-ILC1 population was also detected in the human intestine. We conclude that ILC3s undergo conversion into ILC1-like cells in human tissues in vivo, and that tissue factors and Aiolos were required for this process.

BACKGROUND: As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. METHODS: We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. RESULTS: On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes (HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro. CONCLUSIONS: AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.)

Over 94 million domestic cats are susceptible to cancers and other common and rare diseases. Whole exome sequencing (WES) is a proven strategy to study these disease-causing variants. Presented is a 35.7 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. Whole exome sequencing was conducted on 41 cats with known and unknown genetic diseases and traits, of which ten cats had matching whole genome sequence (WGS) data available, used to validate WES performance. At 80 × mean exome depth of coverage, 96.4% of on-target base coverage had a sequencing depth > 20-fold, while over 98% of single nucleotide variants (SNVs) identified by WGS were also identified by WES. Platform-specific SNVs were restricted to sex chromosomes and a small number of olfactory receptor genes. Within the 41 cats, we identified 31 previously known causal variants and discovered new gene candidate variants, including novel missense variance for polycystic kidney disease and atrichia in the Peterbald cat. These results show the utility of WES to identify novel gene candidate alleles for diseases and traits for the first time in a feline model.

To better understand clonal and transcriptional adaptations after relapse in patients with acute myeloid leukemia (AML), we collected presentation and relapse samples from six normal karyotype AML cases. We performed enhanced whole-genome sequencing to characterize clonal evolution, and deep-coverage single-cell RNA sequencing on the same samples, which yielded 142,642 high-quality cells for analysis. Identifying expressed mutations in individual cells enabled us to discriminate between normal and AML cells, to identify coordinated changes in the genome and transcriptome, and to identify subclone-specific cell states. We quantified the coevolution of genetic and transcriptional heterogeneity during AML progression, and found that transcriptional changes were significantly correlated with genetic changes. However, transcriptional adaptation sometimes occurred independently, suggesting that clonal evolution does not represent all relevant biological changes. In three cases, we identified cells at diagnosis that likely seeded the relapse. Finally, these data revealed a conserved relapse-enriched leukemic cell state bearing markers of stemness, quiescence, and adhesion. Significance: These data enabled us to identify a relapse-enriched leukemic cell state with distinct transcriptional properties. Detailed case-by-case analyses elucidated the complex ways in which the AML genome, transcriptome, and immune microenvironment interact to evade chemotherapy. These analyses provide a blueprint for evaluating these factors in larger cohorts. This article is highlighted in the In This Issue feature, p. 1

Here, we report the draft genome sequences of two related Streptomyces sp. strains, JV180 and SP18CM02. Despite their isolation from soils in Connecticut and Missouri (USA), respectively, they are strikingly similar in gene content. Both belong to the Streptomyces griseus clade and harbor several secondary metabolite biosynthetic gene clusters.

Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X–Y crossing-over unleashed a second dynamic: selfish X–Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of Bos taurus (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X–Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X–Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.

The clonal evolution of metastatic colorectal cancer reveals novel insights into cancer biology., Tumor heterogeneity and evolution drive treatment resistance in metastatic colorectal cancer (mCRC). Patient-derived xenografts (PDXs) can model mCRC biology; however, their ability to accurately mimic human tumor heterogeneity is unclear. Current genomic studies in mCRC have limited scope and lack matched PDXs. Therefore, the landscape of tumor heterogeneity and its impact on the evolution of metastasis and PDXs remain undefined. We performed whole-genome, deep exome, and targeted validation sequencing of multiple primary regions, matched distant metastases, and PDXs from 11 patients with mCRC. We observed intricate clonal heterogeneity and evolution affecting metastasis dissemination and PDX clonal selection. Metastasis formation followed both monoclonal and polyclonal seeding models. In four cases, metastasis-seeding clones were not identified in any primary region, consistent with a metastasis-seeding-metastasis model. PDXs underrepresented the subclonal heterogeneity of parental tumors. These suggest that single sample tumor sequencing and current PDX models may be insufficient to guide precision medicine.

Over 94 million domestic cats are considered pets, who, as our companions, are also susceptible to cancers, common and rare diseases. Whole exome sequencing (WES) is a cost-effective strategy to study their putative disease-causing variants. Presented is ~35.8 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. WES was conducted on 41 cats from various breeds with known and unknown diseases and traits, including 10 cats with prior whole genome sequence (WGS) data available, to test WES capture probe performance. A WES and WGS comparison was completed to understand variant discovery capability of each platform. At ~80x exome coverage, the percent of on-target base coverage >20x was 96.4% with an average of 10.4% off-target. For variant discovery, greater than 98% of WGS SNPs were also discovered by WES. Platform specific variants were mainly restricted to a small number of sex chromosome and olfactory receptor genes. Within the 41 cats with ~31 diseases and normal traits, 45 previously known disease or trait causal variants were observed, such as Persian progressive retinal degeneration and hydrocephalus. Novel candidate variants for polycystic kidney disease and atrichia in the Peterbald breed were also identified as well as a new cat patient with a known variant for cystinuria. These results show the discovery potential of deep exome sequencing to validate existing disease gene models and identify novel gene candidate alleles for many common and rare diseases in cats.

Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using three readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Background In solid organ transplantation, donor-derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient from donor cells. Methods Whole-exome sequencing of donor and recipient DNA and single-cell RNA sequencing (scRNA-seq) of five human kidney transplant biopsy cores distinguished immune cell contributions from both participants. DNA-sequence comparisons used single nucleotide variants (SNVs) identified in the exome sequences across all samples. Results Analysis of expressed SNVs in the scRNA-seq data set distinguished recipient versus donor origin for all 81,139 cells examined. The leukocyte donor/recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages displayed inflammatory activation whereas donor macrophages demonstrated antigen presentation and complement signaling. Recipient-origin T cells expressed cytotoxic and proinflammatory genes consistent with an effector cell phenotype, whereas donor-origin T cells appeared quiescent, expressing oxidative phosphorylation genes. Finally, both donor and recipient T cell clones within the rejecting kidney suggested lymphoid aggregation. The results indicate that donor-origin macrophages and T cells have distinct transcriptional profiles compared with their recipient counterparts, and that donor macrophages can persist for years post-transplantation. Conclusions Analysis of single nucleotide variants and their expression in single cells provides a powerful novel approach to accurately define leukocyte chimerism in a complex organ such as a transplanted kidney, coupled with the ability to examine transcriptional profiles at single-cell resolution.

Significance Mutations that reduce the function of the de novo DNA methyltransferase, DNMT3A, are very common in patients with clonal hematopoiesis and hematopoietic malignancies. Restoring the function of DNMT3A in mouse hematopoietic cells deficient for this protein corrects the DNA methylation defect in an ordered fashion, and partially restores abnormalities in gene expression and myeloid population skewing. These data may be relevant for therapeutic strategies designed to restore DNMT3A activity in patients with diseases caused by mutations in this gene., Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a−/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a−/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a−/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Summary Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer’s disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2–/– mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

To detect diverse and novel RNA species comprehensively, we compared deep small RNA and RNA sequencing (RNA-seq) methods applied to a primary acute myeloid leukemia (AML) sample. We were able to discover previously unannotated small RNAs using deep sequencing of a library method using broader insert size selection. We analyzed the long noncoding RNA (lncRNA) landscape in AML by comparing deep sequencing from multiple RNA-seq library construction methods for the sample that we studied and then integrating RNA-seq data from 179 AML cases. This identified lncRNAs that are completely novel, differentially expressed, and associated with specific AML subtypes. Our study revealed the complexity of the noncoding RNA transcriptome through a combined strategy of strand-specific small RNA and total RNA-seq. This dataset will serve as an invaluable resource for future RNA-based analyses.

Background: Previous studies indicate that mutations in signaling (e.g., receptor tyrosine kinases and RAS pathway members) and transcription factor genes are more common in secondary acute myeloid leukemia (sAML) than myelodysplastic syndrome (MDS), suggesting a role in disease progression. However, our understanding of the timing and order of mutation acquisition in these genes remains incomplete without analyzing paired MDS and sAML samples from the same patient. Defining the role of signaling gene mutations during progression should provide biologic insight into clonal evolution and help define prognostic markers for MDS progression. Methods: We banked paired MDS and sAML (and matched skin) samples from 44 patients (median time to progression: 306 days, range 21-3568). We sequenced 44 sAML (+ skin) samples for 285 recurrently mutated genes (RMGs) and 12 samples were selected for enhanced whole genome sequencing (eWGS, genome with deep exome coverage) of MDS and sAML samples (+ skin) to determine clonal hierarchy. Somatic mutations in these 12 samples were validated with high coverage error-corrected sequencing, and clonality was defined in MDS and sAML samples using mutation variant allele frequencies (VAFs). Additionally, error-corrected sequencing for all sAML RMG mutations, plus 40 additional genes, was performed on 43 of the MDS samples. Single cell DNA sequencing (scDNAseq, Mission Bio) was performed on 6 samples. Results: We identified 32 signaling gene mutations in 15 of the 44 sAML samples, with only 11 of 32 mutations (34%) detected in the initial, paired MDS sample (limit of detection; We next asked if low-level ( Finally, we observed that several MDS (n=6) and sAML (n=10) samples had multiple signaling gene mutations, and it was not always clear whether they occurred in the same subclone. We performed scDNAseq of 6 sAML samples with multiple signaling gene mutations (2-4/case). In 5 of 6 cases the signaling gene mutations did not occur in the same subclone. One sample contained 2 subclones with a NRAS and a PTPN11 mutation, with a separate subclone harboring an additional NRAS mutation. In sum, the co-occurrence of two signaling gene mutations in the same subclone is rare, indicating that the presence of multiple signaling gene mutations may be functionally redundant or detrimental to leukemia cells. Conclusions: Rare cells containing signaling gene mutations are present in nearly half of MDS patients who progress to sAML. The high frequency of signaling gene mutations and diverse patterns of clonal evolution (including the loss of one mutation and acquisition of another), suggest that signaling genes are a major driver of progression to sAML. The paucity of subclones with multiple signaling gene mutations suggests a therapeutic vulnerability for mutant cells. Disclosures DiPersio: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Jacoby:AbbVie: Research Funding; Jazz Pharmaceuticals: Research Funding.

Item does not contain fulltext OBJECTIVE: In many rheumatoid arthritis (RA) patients, disease is controlled with anti-tumor necrosis factor (anti-TNF) biologic therapies. However, in a significant number of patients, the disease fails to respond to anti-TNF therapy. We undertook the present study to examine the hypothesis that rare and low-frequency genetic variants might influence response to anti-TNF treatment. METHODS: We sequenced the coding region of 750 genes in 1,094 RA patients of European ancestry who were treated with anti-TNF. After quality control, 690 genes were included in the analysis. We applied single-variant association and gene-based association tests to identify variants associated with anti-TNF treatment response. In addition, given the key mechanistic role of TNF, we performed gene set analyses of 27 TNF pathway genes. RESULTS: We identified 14,420 functional variants, of which 6,934 were predicted as nonsynonymous 2,136 of which were further predicted to be "damaging." Despite the fact that the study was well powered, no single variant or gene showed study-wide significant association with change in the outcome measures disease activity or European League Against Rheumatism response. Intriguingly, we observed 3 genes, of 27 with nominal signals of association (P < 0.05), that were involved in the TNF signaling pathway. However, when we performed a rigorous gene set enrichment analysis based on association P value ranking, we observed no evidence of enrichment of association at genes involved in the TNF pathway (Penrichment = 0.15, based on phenotype permutations). CONCLUSION: Our findings suggest that rare and low-frequency protein-coding variants in TNF signaling pathway genes or other genes do not contribute substantially to anti-TNF treatment response in patients with RA.

To the editor: Quantitative response evaluation in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs) relies on the morphologic quantification of bone marrow (BM) blasts. This process is subject to the operator-dependent quality of BM collection and the interobserver variability

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma, yet it remains only partially characterized at the genomic level. To improve our understanding of the genetic underpinnings of this incurable and clinically heterogeneous disease, whole-exome sequencing was performed on tumor/normal pairs from a discovery cohort of 24 patients with FL. Using these data and mutations identified in other B-cell malignancies, 1716 genes were sequenced in 113 FL tumor samples from 105 primarily treatment-naive individuals. We identified 39 genes that were mutated significantly above background mutation rates. CREBBP mutations were associated with inferior PFS. In contrast, mutations in previously unreported HVCN1, a voltage-gated proton channel-encoding gene and B-cell receptor signaling modulator, were associated with improved PFS. In total, 47 (44.8%) patients harbor mutations in the interconnected B-cell receptor (BCR) and CXCR4 signaling pathways. Histone gene mutations were more frequent than previously reported (identified in 43.8% of patients) and often co-occurred (17.1% of patients). A novel, recurrent hotspot was identified at a posttranslationally modified residue in the histone H2B family. This study expands the number of mutated genes described in several known signaling pathways and complexes involved in lymphoma pathogenesis (BCR, Notch, SWitch/sucrose nonfermentable (SWI/SNF), vacuolar ATPases) and identified novel recurrent mutations (EGR1/2, POU2AF1, BTK, ZNF608, HVCN1) that require further investigation in the context of FL biology, prognosis, and treatment.

BackgroundThe molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear. MethodsWe enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols. ResultsOf the 116 patients, 53 (46%) had bone marrow blast clearance (

The original version of this Article contained errors in the depiction of confidence intervals in the NF1 BCSS data illustrated in Figure 3b. These have now been corrected in both the PDF and HTML versions of the Article. The incorrect version of Figure 3b is presented in the associated Author Correction.

Follicular lymphoma (FL) is the most common indolent non-Hodgkin's lymphoma; however, it remains incurable with conventional therapies and is poorly responsive to checkpoint blockade. Additionally, because FL develops so slowly (and often asymptomatically), a major research focus has been to avoid chemotherapy treatments to limit the potential development of treatment-related side effects and the risk of therapy-related second cancers. The mutational processes that lead to lymphomagenesis and progression also produce tumor-specific mutant antigens (TSMAs) that can be targeted by the immune system to control malignancies. Personalized cancer vaccines designed for these TSMAs represent a promising new strategy for treatment of FL. However, the feasibility of this approach and precisely how to optimize effective vaccine design, informed by next-generation sequencing data, are not fully understood. We hypothesize that (tumor/normal) whole-exome sequencing (WES) and (tumor) RNA sequencing (RNA-Seq) can be used to predict patient HLA typing and neoepitopes to engineer personalized cancer vaccines for FL. DNA and RNA from 58 patients' FL biopsies underwent WES and RNA-Seq. pVACtools and MiXCR predicted potential somatic and B-cell clonotype neoantigens, which were filtered to identify high-quality TSMAs. B-cell oligoclonality was determined by comparison to B-cell receptor (BCR) repertoire profiling of healthy individual lymph nodes. RNA-seq data allowed us to identify expressed TSMAs. Complementary in silico analysis based on mRNA-based peptide reconstruction and custom HLA affinity binding predictions were performed. An average of 52 somatic mutations per patient (range: 2-172) were identified. At least one high-quality TSMA was predicted for 57 of 58 patients. Five or more TSMA candidates were identified for 52 (90%) patients with a mean of 17 predicted peptides per patient (range: 0-45). 81% (813/1,004) of the total predicted TSMA peptides arose from missense mutations, 9% (94/1,004) from indels, and 10% (97/1,004) from BCR. 78% (45/58) of patients have both somatic and BCR vaccine candidates, while 21% (12/58) of patients had only somatic vaccine candidates. No fusion genes were identified within the cohort that could have been a source of neoepitope candidates. Predicted TSMAs were identified in multiple genes recurrently mutated in lymphoma (e.g., BCL2). There was a high prediction concordance with the orthogonal BostonGene Vaccine Module V1 pipeline. These preclinical results led to a first-in-human pilot trial of personalized TSMA vaccine combined with anti-PD-1 mAb for rel/ref FL patients (NCT03121677), with one response observed within 4 patients evaluable for response to date. TSMA peptides suitable for cancer vaccines were identified for most FL patients via next-generation sequencing, MiXCR and pVACtools. This preclinical study suggests that FL patients will be candidates for TSMA vaccine clinical trials, and pilot clinical results provide proof of concept for this approach. Citation Format: Cody A. Ramirez, Felix Frenkel, Olga Plotnikova, Vladislav Belousov, Alexander Bagaev, Elena Ocheredko, Susanna Kiwala, Jasreet Hundal, Zachary L. Skidmore, Marcus Watkins, Michelle Becker-Hapak, Thomas B. Mooney, Jason Walker, Catrina Fronick, Robert Fulton, Robert Schreiber, Nancy L. Bartlett, Brad Kahl, Ravshan Ataullakhanov, Malachi Griffith, Obi Griffith, Todd A. Fehniger. Identification of predicted neoantigen vaccine candidates in follicular lymphoma patients [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-56.