The meninges are important for brain development and pathology. Using single-cell RNA sequencing, we have generated the first comprehensive transcriptional atlas of neonatal mouse meningeal leukocytes under normal conditions and after perinatal brain injury. We identified almost all known leukocyte subtypes and found differences between neonatal and adult border-associated macrophages, thus highlighting that neonatal border-associated macrophages are functionally immature with regards to immune responses compared with their adult counterparts. We also identified novel meningeal microglia-like cell populations that may participate in white matter development. Early after the hypoxic–ischemic insult, neutrophil numbers increased and they exhibited increased granulopoiesis, suggesting that the meninges are an important site of immune cell expansion with implications for the initiation of inflammatory cascades after neonatal brain injury. Our study provides a single-cell resolution view of the importance of meningeal leukocytes at the early stage of development in health and disease.
Abstract Cure rates of classical Hodgkin Lymphoma (cHL) in children and young adult patients currently exceed 90%. Nonetheless, survivors are confronted with chronic therapy-related health conditions such as infertility, cardiovascular disease, and high rates of novel second cancers. This calls for development of new targeted and less toxic treatments. cHL is characterized by a low frequency (~0.1-5%) of malignant Hodgkin Reed-Sternberg (HRS) cells, while most of the tissue is composed of nonmalignant immune cells. It is thought that the HRS cells depend on interactions with the tumor microenvironment (TME) for their survival. Indeed, a vast number of interactions between different immune and HRS cells have been reported; however, most of these reports are based on immunohistochemistry or in vitro studies. Here, we systematically characterized the in vivo interactions by applying single-cell RNA sequencing (scRNAseq) to nine primary pediatric and adolescent cHL biopsies and three noncancerous control biopsies of reactive lymph nodes. With scRNAseq, we first sorted live cells to get an unbiased overview of the TME. Then we used a previously published flow cytometry antibody panel to enrich for HRS cells, allowing us to directly assess interactions on a per-tumor basis. Tumor cell identity was confirmed by marker expression as identified by pathology, single-cell copy-number status and the ratio of immunoglobulin kappa/lambda expression. Immune cell identity was determined by canonical marker expression. Using the scRNAseq data, we found that the TME expression profiles in cHL and control biopsies mostly overlap but harbor some differences. First, we identified genes that are consistently overexpressed in HRS cells. These included transcription factors, neural markers, multiple interleukins and other signaling molecules. Second, the extensively described immunosuppressive interactions between HRS, T and NK cells (expressing CTLA-4, TIM-3, and LAG-3) were the strongest and most common interactions that we could identify in HL but not in noncancerous reactive lymph nodes. Third, while the inflammation in the reactive lymph nodes was driven by IFN-g signaling, this pathway was inactive in HL tumors. Other interactions like recruitment of CXCR3+ and CCR4+ T cells, CD47 signaling and interleukin signaling were less pronounced in cHL compared to the controls or were less consistent between tumors. These findings were validated in bulk RNA sequencing of 45 HL tumors. A model arises in which the presence of HRS cells induces inflammation that in most ways resembles lymph node infections. This inflammation and HRS survival are controled by patient-specific interactions between HRS cells and the TME, and by T cell exhaustion, which is universal and the most essential interaction in cHL. Citation Format: Jurrian K. de Kanter, Thanasis Margaritis, Auke Beishuizen, Marijn Scheijde-Vermeulen, Liset Westera, Arianne M. Brandsma, Ruben van Boxtel, Friederike Meyer-Wentrup. Single-cell RNA sequencing reveals that childhood classical Hodgkin Lymphoma resembles normal inflammation except for T cell exhaustion [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A37.
<div>Abstract<p>Childhood cancer survivors are confronted with various chronic health conditions like therapy-related malignancies. However, it is unclear how exposure to chemotherapy contributes to the mutation burden and clonal composition of healthy tissues early in life. Here, we studied mutation accumulation in hematopoietic stem and progenitor cells (HSPC) before and after cancer treatment of 24 children. Of these children, 19 developed therapy-related myeloid neoplasms (t-MN). Posttreatment HSPCs had an average mutation burden increase comparable to what treatment-naïve cells accumulate during 16 years of life, with excesses up to 80 years. In most children, these additional mutations were induced by clock-like processes, which are also active during healthy aging. Other patients harbored mutations that could be directly attributed to treatments like platinum-based drugs and thiopurines. Using phylogenetic inference, we demonstrate that most t-MN in children originate after the start of treatment and that leukemic clones become dominant during or directly after chemotherapy exposure.</p>Significance:<p>Our study shows that chemotherapy increases the mutation burden of normal blood cells in cancer survivors. Only few drugs damage the DNA directly, whereas in most patients, chemotherapy-induced mutations are caused by processes similar to those present during normal aging.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-12-8-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 1825</a></i></p></div>
Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.
Abstract Therapy-related myeloid neoplasms (t-MN) arise as a complication of chemo- and/or radiotherapy. Although t-MN can occur both in adult and childhood cancer survivors, the mechanisms driving therapy-related leukemogenesis likely vary across different ages. Chemotherapy is thought to induce driver mutations in children, whereas in adults pre-existing mutant clones are selected by the exposure. However, selective pressures induced by chemotherapy early in life are less well studied. Here, we use single-cell whole genome sequencing and phylogenetic inference to show that the founding cell of t-MN in children starts expanding after cessation of platinum exposure. In patients with Li-Fraumeni syndrome, characterized by a germline TP53 mutation, we find that the t-MN already expands during treatment, suggesting that platinum-induced growth inhibition is TP53- dependent. Our results demonstrate that germline aberrations can interact with treatment exposures in inducing t-MN, which is important for the development of more targeted, patient-specific treatment regimens and follow-up.
Genetic instability is a major concern for successful application of stem cells in regenerative medicine. However, the mutational consequences of the most applied stem cell therapy in humans, hematopoietic stem cell transplantation (HSCT), remain unknown. Here we characterized the mutation burden of hematopoietic stem and progenitor cells (HSPCs) of human HSCT recipients and their donors using whole-genome sequencing. We demonstrate that the majority of transplanted HSPCs did not display altered mutation accumulation. However, in some HSCT recipients, we identified multiple HSPCs with an increased mutation burden after transplantation. This increase could be attributed to a unique mutational signature caused by the antiviral drug ganciclovir. Using a machine learning approach, we detected this signature in cancer genomes of individuals who received HSCT or solid organ transplantation earlier in life. Antiviral treatment with nucleoside analogs can cause enhanced mutagenicity in transplant recipients, which may ultimately contribute to therapy-related carcinogenesis.
Abstract Childhood cancer survivors are confronted with various chronic health conditions like therapy-related malignancies. However, it is unclear how exposure to chemotherapy contributes to the mutation burden and clonal composition of healthy tissues early in life. Here, we studied mutation accumulation in hematopoietic stem and progenitor cells (HSPC) before and after cancer treatment of 24 children. Of these children, 19 developed therapy-related myeloid neoplasms (t-MN). Posttreatment HSPCs had an average mutation burden increase comparable to what treatment-naïve cells accumulate during 16 years of life, with excesses up to 80 years. In most children, these additional mutations were induced by clock-like processes, which are also active during healthy aging. Other patients harbored mutations that could be directly attributed to treatments like platinum-based drugs and thiopurines. Using phylogenetic inference, we demonstrate that most t-MN in children originate after the start of treatment and that leukemic clones become dominant during or directly after chemotherapy exposure. Significance: Our study shows that chemotherapy increases the mutation burden of normal blood cells in cancer survivors. Only few drugs damage the DNA directly, whereas in most patients, chemotherapy-induced mutations are caused by processes similar to those present during normal aging.
