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Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC.Significance:The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis.This article is highlighted in the In This Issue feature, p. 2945

Maintenance of immunological homeostasis between tolerance and autoimmunity is essential for the prevention of human diseases ranging from autoimmune disease to cancer. Accumulating evidence suggests that p53 can mitigate phagocytosis-induced adjuvanticity thereby promoting immunological tolerance following programmed cell death. Here we identify Inhibitor of Apoptosis Stimulating p53 Protein (iASPP), a negative regulator of p53 transcriptional activity, as a regulator of immunological tolerance. iASPP-deficiency promoted lung adenocarcinoma and pancreatic cancer tumorigenesis, while iASPP-deficient mice were less susceptible to autoimmune disease. Immune responses to iASPP-deficient tumors exhibited hallmarks of immunosuppression, including activated regulatory T cells and exhausted CD8+ T cells. Interestingly, iASPP-deficient tumor cells and tumor-infiltrating myeloid cells, CD4+, and γδ T cells expressed elevated levels of PD-1H, a recently identified transcriptional target of p53 that promotes tolerogenic phagocytosis. Identification of an iASPP/p53 axis of immune homeostasis provides a therapeutic opportunity for both autoimmune disease and cancer.

Immune checkpoint inhibitors (ICIs) lead to durable responses in a subset of patients with cancer, but most patients do not respond to ICI, prompting interest in combining immunotherapy with other therapeutic regimens. Preclinical evidence supports the potential for therapeutic synergy between immunotherapy and radiation therapy through modulation of the tumor microenvironment and antitumor immune responses. Local therapy also has the potential to overcome localized sites of relative immune suppression and resistance. Prospective clinical trials have been initiated to test these hypotheses in the clinic as well as to investigate the toxicities and adverse events associated with combination immunotherapy and radiation therapy. In this review, we discuss the emerging results from prospective clinical trials of combination immunotherapy and radiation therapy, the safety and efficacy of their combination, concordance with preclinical and retrospective data, and some of the remaining open questions to be addressed by future clinical trials.

In response to the coronavirus disease-2019 (COVID-19) pandemic, we developed and launched a student-led telemedicine program in Chelsea. From April to November 2020, over 200 student volunteers contacted over 1000 patients to assess COVID-19 symptoms, provide counseling, and triage patients. Through a retrospective cohort study, we determined that student triage decision was associated with patient outcomes, including hospitalization status, COVID-19 test administration, and COVID-19 test result. These results quantify the outcomes of a student-led telemedicine clinic to combat the ongoing pandemic and may serve as a model for implementation of similar clinics to alleviate mounting health care system burden.

Though it is widely believed that chronic immunosuppressive medications increase the severity of coronavirus disease 2019 (COVID‐19) illness, there is little data to support this. We performed a retrospective study of COVID‐19 positive patients diagnosed at a single academic medical center between March 10, 2020 and October 13, 2020. A total of 835 patients diagnosed with COVID‐19 by polymerase chain reaction were included (median age 64 years; 52% female). Of these, 46 (5.5%) had a prescription for an immunosuppressive therapy before diagnosis, most commonly oral steroids (20, 43%), mycophenolate (12, 26%), or tacrolimus (11, 24%). Patients on immunosuppressive therapy with COVID‐19 had increased mortality (30% vs. 17%, p = 0.036; odds ratio 2.1, 95% confidence interval 1.11–4.04), which remained significant (p = 0.040) after performing multivariate logistic regression controlling for gender, age, race, and comorbidity status. Laboratory markers of inflammation were uniformly elevated in both patients on or not on immunosuppressive therapies who died, but lymphocytes and neutrophils were decreased in both COVID‐19 patients on immunosuppressive therapies who died and who remained alive. These findings demonstrate that COVID‐19 disease is more severe in patients taking prior immunosuppressive medications. This finding emphasizes the need for aggressive monitoring and supportive care for immunosuppressed patients who are diagnosed with COVID‐19., Highlights COVID‐19 patients on immunosuppression at the time of diagnosis have increased mortality.Laboratory markers of inflammation are elevated in severe COVID‐19 in both patients on and non on prior immunosuppression.Age is not associated with longer hospitalization in COVID‐19 patients on prior immunosuppression.

Affinity matured self-reactive antibodies are found in autoimmune diseases like systemic lupus erythematous. Here we used fate-mapping reporter mice and single cell transcriptomics coupled to antibody repertoire analysis to characterize the post-germinal center (GC) B cell compartment in a new mouse model of autoimmunity. Antibody secreting cells (ASCs) and memory B cells (MemBs) from spontaneous GCs grouped into multiple subclusters. ASCs matured into two terminal clusters, with distinct secretion, antibody repertoire and metabolic profiles. MemBs contained FCRL5+ and CD23+ subsets, with different in vivo localization in the spleen. GC-derived FCRL5+ MemBs share transcriptomic and repertoire properties with atypical B cells found in aging and infection and localize to the marginal zone, suggesting a similar contribution to recall responses. While transcriptomically diverse, ASC and MemB subsets maintained an underlying clonal redundancy. Therefore, self-reactive clones could escape subset-targeting therapy by perpetuation of self-reactivity in distinct subsets.

The germinal center serves as a site of B cell selection and affinity maturation, critical processes for productive adaptive immunity. In autoimmune disease tolerance is broken in the germinal center reaction, leading to production of autoreactive B cells that may propagate disease. Follicular T cells are crucial regulators of this process, providing signals necessary for B cell survival in the germinal center. Here, we review the emerging roles of follicular T cells in the autoreactive germinal center. Recent advances in immunological techniques have allowed study of the gene expression profiles and repertoire of follicular T cells at unprecedented resolution. These studies provide insight into the potential role follicular T cells play in preventing or facilitating germinal center loss of tolerance. Improved understanding of the mechanisms of T cell help in autoreactive germinal centers provides novel therapeutic targets for diseases of germinal center dysfunction.

Pathogenic autoantibodies contribute to tissue damage and clinical decline in autoimmune disease. Follicular T cells are central regulators of germinal centers, although their contribution to autoantibody-mediated disease remains unclear. Here we perform single cell RNA and T cell receptor (TCR) sequencing of follicular T cells in a mouse model of autoantibody-mediated disease, allowing for analyses of paired transcriptomes and unbiased TCRαβ repertoires at single cell resolution. A minority of clonotypes are preferentially shared amongst autoimmune follicular T cells and clonotypic expansion is associated with differential gene signatures in autoimmune disease. Antigen prediction using algorithmic and machine learning approaches indicates convergence towards shared specificities between non-autoimmune and autoimmune follicular T cells. However, differential autoimmune transcriptional signatures are preserved even amongst follicular T cells with shared predicted specificities. These results demonstrate that follicular T cells are phenotypically distinct in B cell-driven autoimmune disease, providing potential therapeutic targets to modulate autoantibody development., Follicular T cells regulate germinal centre reactions, but their phenotypes in autoimmune disease are unclear. Using a mouse model of autoantibody disease, the authors show that autoimmune follicular T cells differ by TCR clonotype and transcriptional profile from non-autoimmune follicular T cells.

Significance SCLC is a deadly disease for which treatment outcomes have not improved significantly for over 30 y due to the lack of effective new therapies. Large-scale sequencing studies have identified many recurrently mutated genes in human SCLC tumors, whose functions remain poorly understood. We have adapted the CRISPR-Cas9 system to rapidly model mutations in target genes in a mouse model of SCLC. Using this system, we show that the gene p107 functions as a tumor suppressor gene in SCLC. Furthermore, loss of p107 confers a distinct tumor phenotype from loss of its close relative p130. Our results demonstrate the utility of our system for better understanding the genetic factors that contribute to SCLC progression., Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130. Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.

There is currently limited clinical ability to identify COVID-19 patients at risk for severe outcomes. To unbiasedly identify metrics associated with severe outcomes in COVID-19 patients, we conducted a retrospective study of 835 COVID-19 positive patients at a single academic medical center between March 10, 2020 and October 13, 2020. As of December 1, 2020, 656 (79%) patients required hospitalization and 149 (18%) died. Unbiased comparisons of all clinical characteristics and mortality revealed that abnormal pH (OR 8.54, 95% CI 5.34–13.6), abnormal creatinine (OR 6.94, 95% CI 4.22–11.4), and abnormal PTT (OR 4.78, 95% CI 3.11–7.33) were most significantly associated with mortality. Correlation with ordinal severity scores confirmed these associations, in addition to associations between respiratory rate (Spearman’s rho = −0.56), absolute neutrophil count (Spearman’s rho = −0.5), and C-reactive protein (Spearman’s rho = 0.59) with disease severity. Unsupervised principal component analysis and machine learning model classification of patient demographics, laboratory results, medications, comorbidities, signs and symptoms, and vitals are capable of separating patients on the basis of COVID-19 mortality (AUC 0.82). This retrospective analysis identifies laboratory and clinical metrics most relevant to predict COVID-19 severity. Supplementary Information The online version contains supplementary material available at 10.1007/s10238-021-00730-y.

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. Significance: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945

Mouse models with inducible neoantigens have been historically difficult to generate because of leaky expression of antigens in the thymus, which causes central tolerance in developing CD8 and CD4 T cells. Attempts to resolve this problem using existing genetic tools have been unsuccessful. We developed the iNversion INducible Joined neoAntigen (NINJA) mouse model that uses RNA splicing, DNA recombination, and three levels of regulation to prevent neoantigen leakiness and allow tight control over the induction of neoantigen expression. We describe the development of these genetic tools and their use for obtaining tumor cell lines with inducible neoantigen expression. Moreover, we show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cells responses upon neoantigen induction in peripheral tissues. Thus, NINJA fills a long-standing gap in the field and will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, autoimmune diseases, and developing tumors.

Pathogenic autoantibodies contribute to tissue damage and clinical decline in autoimmune disease. Follicular T cells are central regulators of germinal centers, although their role in epitope spreading towards autoantigens remains unclear. We performed single cell RNA and T cell receptor (TCR) sequencing of follicular T cells in autoantibody-mediated disease, allowing for analyses of paired transcriptomes and unbiased TCRab repertoires at single cell resolution. A minority of clonotypes were preferentially shared amongst autoimmune follicular T cells, and clonotypic expansion was associated with differential gene signatures in autoimmune disease. Antigen prediction using algorithmic and machine learning approaches revealed convergence towards shared specificities between non-autoimmune and autoimmune follicular T cells. However, differential autoimmune transcriptional signatures were preserved even amongst follicular T cells with shared predicted specificities. These results demonstrate that follicular T cells are phenotypically distinct in B cell-driven autoimmune disease, providing potential therapeutic targets to modulate autoreactive epitope spreading. Supported by grants from NIH (R01AI130307, R01AR074105, T32GM007753).

PURPOSE/OBJECTIVE(S) Radiation (RT)-associated lymphopenia may adversely affect treatment outcomes, particularly in the era of immunotherapy. We sought to determine dosimetric factors (DF) correlated with lymphopenia following palliative RT in a cohort of patients with advanced cancer treated with anti-PD-1 immune checkpoint inhibitors (ICIs). MATERIALS/METHODS We included patients with metastatic lung cancer, melanoma, or renal cell carcinoma who were treated with either pembrolizumab or nivolumab and received palliative RT to an extracranial site. Baseline and nadir absolute lymphocyte count (ALC) within 6 weeks of RT were recorded. Heart, lungs, liver, kidneys, spleen, bone, and large blood vessels (LBV) were contoured for each RT course, and various DF were extracted from the corresponding dose-volume histograms (DVHs). To model RT dose to circulating lymphocytes, we also input these DVHs into a whole-body blood flow model that simulates the spatiotemporal distribution of blood particles in organs based on blood volumes and flow rates from ICRP-89. Associations between DF with ALC nadir and ALC change were assessed with Spearman correlation coefficients. DF with the strongest correlations were dichotomized at the median and evaluated for association with grade 3+ lymphopenia (ALC < 500cells/mL) post-RT by univariable logistic regression. RESULTS We analyzed 55 patients who underwent 80 total courses of palliative RT; most (94%) were treated with 3D conformal RT. Doses to whole body, bone, and LBV were negatively correlated with ALC nadir, with the strongest correlations seen at V15 (rs = -0.38, -0.43, and -0.36, P = 0.0004, 0.0001, and 0.001, respectively). Doses to other organs were not significantly correlated with ALC nadir. The modeled dose to circulating lymphocytes was also negatively correlated with ALC nadir and ALC change (for D2%, rs = -0.31 and -0.38, P = 0.005 and 0.0007, respectively). Associations between these DF and grade 3+ lymphopenia are shown in the Table. CONCLUSION RT dose to whole body, bone, and large blood vessels were correlated with lymphopenia in patients treated with palliative RT and anti-PD-1 ICIs. The modeled dose to circulating lymphocytes in our dynamic model correlated with lymphopenia as well, validating our model and enabling future investigation into dose rate and fractionation effects. These dose parameters may help guide RT planning to minimize lymphopenia risk, though additional studies are required to elicit the relative importance of each factor.

In an autoimmune environment, B cells spontaneously participate and are selected in germinal centers (GC). In previous work, we investigated epitope spreading in a mixed bone marrow chimera model that combines B cells from lupus-like mice with those from wild type (WT) B6 mice (Degn et al Cell 2017). In the current study, we identify novel biomarkers of autoimmune B cells at the population and single cell level. Cohorts of autoimmune and WT immunized chimeric mice were prepared. To track WT-derived B cells, we used the tamoxifen inducible AID-CreERT2-EYFP mice. WT chimeras were immunized and boosted. Splenic B cells were sorted based EYFP+ signal and processed for droplet-based gene expression and BCR repertoire single cell RNAseq. To detect specific traits of autoreactive B cells relative to immunized, unsupervised cluster analysis, differential gene expression and pseudotime analysis were used. Results show higher antibody repertoire diversity in the autoimmune relative to immune B cells, but striking similarity for transcriptomic usage. However, distinct gene expression was identified for the autoreactive B cells such as CXCL10 usage by GC B cells and SLPI expression by autoreactive plasma cells. Importantly, we were able to identify DN2- and DN4-like memory B cells in both conditions. DN2 cells, presumed to derive directly from naïve B cells, have been recently pointed as a source of pathogenic extrafollicular antibody secreting cells in humans. Overall, results reveal the variety of characteristics that B cells, in their many states, adopt in the response to self and should open the way to new approaches to control their pathogenicity in autoimmune disease.

Infiltration of regulatory T (Treg) cells into many tumor types correlates with poor patient prognoses. However, mechanisms of intratumoral Treg cell function remain to be elucidated. We investigated Treg cell function in a genetically-engineered mouse lung adenocarcinoma model and found Treg cells suppress anti-tumor responses in tumor-associated tertiary lymphoid structures (TA-TLS). TA-TLS have been described in human lung cancers, but their function remains to be determined. TLS in this model were spatially associated with >90% of tumors and facilitated interactions between T cells and tumor-antigen presenting dendritic cells (DCs). Costimulatory ligand expression by DCs and T cell proliferation rates increased in TA-TLS upon Treg cell depletion, leading to tumor destruction. Thus, we propose Treg cells in TA-TLS can inhibit endogenous immune responses against tumors, and targeting these cells may provide therapeutic benefit for cancer patients., National Cancer Institute (U.S.) (Cancer Center Support Grant P30-CA14051), Howard Hughes Medical Institute, National Institutes of Health (U.S.) (Grants 1 U54 CA126515-01, R01-CA185020-01 and T32 GM007753), Damon Runyon Cancer Research Foundation, John D. Proctor Foundation (Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award), Lung Cancer Research Foundation, Howard Hughes Medical Institute (Investigator), Daniel K. Ludwig Scholar

SUMMARY Objectives: Anterior temporal lobectomy is curative for many patients with disabling medically refractory temporal lobe epilepsy, but carries an inherent risk of disabling verbal memory loss. Although accurate prediction of iatrogenic memory loss is becoming increasingly possible, it remains unclear how much weight such predictions should have in surgical decision making. Here we aim to create a framework that facilitates a systematic and integrated assessment of the relative risks and benefits of surgery versus medical management for patients with left temporal lobe epilepsy. Methods: We constructed a Markov decision model to evaluate the probabilistic outcomes and associated health utilities associated with choosing to undergo a left anterior temporal lobectomy versus continuing with medical management for patients with medically refractory left temporal lobe epilepsy. Three base-cases were considered, representing a spectrum of surgical candidates encountered in practice, with varying degrees of epilepsy-related disability and potential for decreased quality of life in response to post-surgical verbal memory deficits. Results: For patients with moderately severe seizures and moderate risk of verbal memory loss, medical management was the preferred decision, with increased quality-adjusted life expectancy. However, the preferred choice was sensitive to clinically meaningful changes in several parameters, including quality of life impact of verbal memory decline, quality of life with seizures, mortality rate with medical management, probability of remission following surgery, and probability of remission with medical management. Significance: Our decision model suggests that for patients with left temporal lobe epilepsy, quantitative assessment of risk and benefit should guide recommendation of therapy. In particular, risk for and potential impact of verbal memory decline should be carefully weighed against the degree of disability conferred by continued seizures on a patient-by-patient basis.

Traditional cloning methods have limitations on the number of DNA fragments that can be simultaneously manipulated, which dramatically slows the pace of molecular assembly. Here we describe GMAP, a Gibson assembly-based modular assembly platform consisting of a collection of promoters and genes, which allows for one-step production of DNA constructs. GMAP facilitates rapid assembly of expression and viral constructs using modular genetic components, as well as increasingly complicated genetic tools using contextually relevant genomic elements. Our data demonstrate the applicability of GMAP toward the validation of synthetic promoters, identification of potent RNAi constructs, establishment of inducible lentiviral systems, tumor initiation in genetically engineered mouse models, and gene-targeting for the generation of knock-in mice. GMAP represents a recombinant DNA technology designed for widespread circulation and easy adaptation for other uses, such as synthetic biology, genetic screens, and CRISPR-Cas9., Howard Hughes Medical Institute, Amgen Inc. (Massachusetts Institute of Technology. Undergraduate Research Opportunities Program), Massachusetts Institute of Technology (Peter J. Eloranta Fellowship)

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.

Although chromosomal deletions and inversions are important in cancer, conventional methods for detecting DNA rearrangements require laborious indirect assays. Here we develop fluorescent reporters to rapidly quantify CRISPR/Cas9-mediated deletions and inversions. We find that inversion depends on the non-homologous end-joining enzyme LIG4. We also engineer deletions and inversions for a 50 kb Pten genomic region in mouse liver. We discover diverse yet sequence-specific indels at the rearrangement fusion sites. Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks. These reporters allow mechanisms of chromosomal rearrangements to be investigated. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0680-7) contains supplementary material, which is available to authorized users.

Importance Conclusive intraoperative pathologic confirmation of diffuse infiltrative glioma guides the decision to pursue definitive neurosurgical resection. Establishing the intraoperative diagnosis by histologic analysis can be difficult in low-cellularity infiltrative gliomas. Therefore, we developed a rapid and sensitive genotyping assay to detect somatic single-nucleotide variants in the telomerase reverse transcriptase ( TERT ) promoter and isocitrate dehydrogenase 1 ( IDH1 ). Observations This assay was applied to tissue samples from 190 patients with diffuse gliomas, including archived fixed and frozen specimens and tissue obtained intraoperatively. Results demonstrated 96% sensitivity (95% CI, 90%-99%) and 100% specificity (95% CI, 95%-100%) for World Health Organization grades II and III gliomas. In a series of live cases, glioma-defining mutations could be identified within 60 minutes, which could facilitate the diagnosis in an intraoperative timeframe. Conclusions and Relevance The genotyping method described herein can establish the diagnosis of low-cellularity tumors like glioma and could be adapted to the point-of-care diagnosis of other lesions that are similarly defined by highly recurrent somatic mutations.