RESUMO
Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.
Assuntos
Neoplasias Renais , Tumor de Wilms , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Anaplasia/genética , Tumor de Wilms/genética , Tumor de Wilms/tratamento farmacológico , Tumor de Wilms/patologia , Mutação , Prognóstico , DNARESUMO
BACKGROUND: Most children with Wilms tumour are successfully treated with multidrug chemotherapy and surgery. These treatments cause severe side effects for the patients, an issue that needs to be addressed by exploring other treatment options with less or no side effects. One option is to complement current therapies with agents that could potentially induce tumour cell differentiation, for example retinoic acid (RA). AIMS: To facilitate quick assessment of an agent's effect on Wilms tumour differentiation by a rapid in vitro model system. METHODS AND RESULTS: Here WiT49 and CCG99-11 Wilms tumour cells were treated with 10 µM RA for 72 h or 9 days. Cultured cells were scraped off from Petri dishes, pelleted and embedded in paraffin in the same way as clinical tumour specimens are preserved. Cell morphology and differentiation were evaluated by analyses of haematoxylin eosin (H&E) and immunohistochemical stainings. Based on H&E, WT1 and CKAE1/3 stainings, RA treatment induced further epithelial differentiation of WiT49 cells, whereas there was no sign of induced maturation in CCG99-11 cells. Ki67 staining showed that RA inhibited cell proliferation in both cell lines. CONCLUSIONS: Our study shows that in vitro culturing of WiT49 and CCG99-11 cells, followed by pelleting and paraffin embedding of cell pellets, could aid in a quick evaluation of potential differentiating agents against Wilms tumour. In addition, our results strengthen previous results that retinoic acid could be a potential complement to regular Wilms tumour treatment.
Assuntos
Antineoplásicos , Neoplasias Renais , Tumor de Wilms , Criança , Humanos , Tretinoína/farmacologia , Tumor de Wilms/tratamento farmacológico , Tumor de Wilms/patologia , Diferenciação Celular , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologiaRESUMO
PURPOSE: While patients with intermediate-risk (IR) Wilms tumors now have an overall survival (OS) rate of almost 90%, those affected by high-stage tumors with diffuse anaplasia have an OS of only around 50%. We here identify key events in the pathogenesis of diffuse anaplasia by mapping cancer cell evolution over anatomic space in Wilms tumors. EXPERIMENTAL DESIGN: We spatially mapped subclonal landscapes in a retrospective cohort of 20 Wilms tumors using high-resolution copy-number profiling and TP53 mutation analysis followed by clonal deconvolution and phylogenetic reconstruction. Tumor whole-mount sections (WMS) were utilized to characterize the distribution of subclones across anatomically distinct tumor compartments. RESULTS: Compared with non-diffuse anaplasia Wilms tumors, tumors with diffuse anaplasia showed a significantly higher number of genetically distinct tumor cell subpopulations and more complex phylogenetic trees, including high levels of phylogenetic species richness, divergence, and irregularity. All regions with classical anaplasia showed TP53 alterations. TP53 mutations were frequently followed by saltatory evolution and parallel loss of the remaining wild-type (WT) allele in different regions. Morphologic features of anaplasia increased with copy-number aberration (CNA) burden and regressive features. Compartments demarcated by fibrous septae or necrosis/regression were frequently (73%) associated with the emergence of new clonal CNAs, although clonal sweeps were rare within these compartments. CONCLUSIONS: Wilms tumors with diffuse anaplasia display significantly more complex phylogenies compared with non-diffuse anaplasia Wilms tumors, including features of saltatory and parallel evolution. The subclonal landscape of individual tumors was constrained by anatomic compartments, which should be considered when sampling tissue for precision diagnostics.
Assuntos
Neoplasias Renais , Tumor de Wilms , Humanos , Neoplasias Renais/patologia , Anaplasia/genética , Estudos Retrospectivos , Filogenia , Tumor de Wilms/genética , Tumor de Wilms/patologiaRESUMO
Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.
Assuntos
Recidiva Local de Neoplasia , Neuroblastoma , Criança , Animais , Humanos , Proteína Proto-Oncogênica N-Myc , Modelos Animais de Doenças , Macrófagos , Microambiente TumoralRESUMO
Cytokines are key regulators of tumor immune surveillance by controlling immune cell activity. Here, we investigated whether interleukin 4 (IL4) has antileukemic activity via immune-mediated mechanisms in an in vivo murine model of acute myeloid leukemia driven by the MLL-AF9 fusion gene. Although IL4 strongly inhibited leukemia development in immunocompetent mice, the effect was diminished in immune-deficient recipient mice, demonstrating that the antileukemic effect of IL4 in vivo is dependent on the host immune system. Using flow cytometric analysis and immunohistochemistry, we revealed that the antileukemic effect of IL4 coincided with an expansion of F4/80+ macrophages in the bone marrow and spleen. To elucidate whether this macrophage expansion was responsible for the antileukemic effect, we depleted macrophages in vivo with clodronate liposomes. Macrophage depletion eliminated the antileukemic effect of IL4, showing that macrophages mediated the IL4-induced killing of leukemia cells. In addition, IL4 enhanced murine macrophage-mediated phagocytosis of leukemia cells in vitro. Global transcriptomic analysis of macrophages revealed an enrichment of signatures associated with alternatively activated macrophages and increased phagocytosis upon IL4 stimulation. Notably, IL4 concurrently induced Stat6-dependent upregulation of CD47 on leukemia cells, which suppressed macrophage activity. Consistent with this finding, combining CD47 blockade with IL4 stimulation enhanced macrophage-mediated phagocytosis of leukemia cells. Thus, IL4 has two counteracting roles in regulating phagocytosis in mice; enhancing macrophage-mediated killing of leukemia cells, but also inducing CD47 expression that protects target cells from excessive phagocytosis. Taken together, our data suggest that combined strategies that activate macrophages and block CD47 have therapeutic potential in acute myeloid leukemia.
Assuntos
Antígeno CD47 , Interleucina-4 , Leucemia Mieloide Aguda , Fagocitose , Animais , Antígeno CD47/metabolismo , Interleucina-4/imunologia , Leucemia Mieloide Aguda/imunologia , Camundongos , Regulação para CimaRESUMO
Malignant rhabdoid tumour (MRT) is a childhood neoplasm of high malignancy characterised by biallelic mutation and/or loss of the epigenetic master regulator SMARCB1, accompanied by no or few other oncogenic drivers. In spite of their generally low mutational burden, an intratumoural T-cell response has been reported in a subset of MRTs, indicating that immune checkpoint inhibition may be considered a viable therapy option for some patients. We assess here the evolution over time and space of predicted neoantigens and indicators of immune checkpoint status in two MRT patients who progressed under treatment. Both patients showed an accumulation of novel clonal and subclonal mutations, including predicted neoantigens, in metastases compared to their inferred ancestral clones in the primary tumours. The first patient had peritoneal metastases from an MRT of the liver. Clonal deconvolution revealed polyclonal seeding from the primary tumour to a single metastatic site, followed by a local subclonal burst of mutations. The second patient had a renal MRT with multiple pulmonary metastases, each of which could be traced back to a single genetically unique founder cell, with formation of novel subclones in two metastases. Both patients showed a regionally heterogeneous landscape of predicted neoantigens and of tumour-infiltrating lymphocytes expressing CD8 and PD1. In both patients, some tumour regions fulfilled established criteria for PD-L1 positivity (> 1% of tumour cells), while others did not. This suggests that even in a tumour type like MRT, with a single driver mutation, there can be heterogeneity in neoantigen repertoire, immune response, and biomarkers for checkpoint blockade among sampled locations. This must be taken into account when assessing progressed MRT patients for checkpoint inhibition therapy. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Neoplasias Renais/genética , Neoplasias Hepáticas/genética , Linfócitos do Interstício Tumoral/patologia , Mutação , Tumor Rabdoide/genética , Proteína SMARCB1/genética , Variações do Número de Cópias de DNA , Humanos , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Linfócitos do Interstício Tumoral/imunologia , Tumor Rabdoide/imunologia , Tumor Rabdoide/patologiaRESUMO
Anaplastic thyroid cancer (ATC) is a rare and extremely malignant tumor with no available cure. The genetic landscape of this malignancy has not yet been fully explored. In this study, we performed whole exome sequencing and the RNA-sequencing of fourteen cases of ATC to delineate copy number changes, fusion gene events, and somatic mutations. A high frequency of genomic amplifications was seen, including 29% of cases having amplification of CCNE1 and 9% of CDK6; these events may be targetable by cyclin dependent kinase (CDK) inhibition. Furthermore, 9% harbored amplification of TWIST1, which is also a potentially targetable lesion. A total of 21 fusion genes in five cases were seen, none of which were recurrent. Frequent mutations included TP53 (55%), the TERT promoter (36%), and ATM (27%). Analyses of mutational signatures showed an involvement of processes that are associated with normal aging, defective DNA mismatch repair, activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity, failure of DNA double-strand break repair, and tobacco exposure. Taken together, our results shed new light on the tumorigenesis of ATC and show that a relatively large proportion (36%) of ATCs harbor genetic events that make them candidates for novel therapeutic approaches. When considering that ATC today has a mortality rate of close to 100%, this is highly relevant from a clinical perspective.
RESUMO
Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock-out Irx3- /Irx5- mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3-/- cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/ß-catenin-signalling. In contrast, IRX5-/- cells displayed activation of Hippo and non-canonical WNT-signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Proteínas de Homeodomínio/metabolismo , Neoplasias Renais/metabolismo , Néfrons/metabolismo , Fatores de Transcrição/metabolismo , Tumor de Wilms/metabolismo , Animais , Carcinogênese , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos Knockout , Morfogênese , Néfrons/crescimento & desenvolvimento , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Tumor de Wilms/genética , Tumor de Wilms/patologia , Via de Sinalização Wnt , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMO
A major challenge to personalized oncology is that driver mutations vary among cancer cells inhabiting the same tumor. Whether this reflects principally disparate patterns of Darwinian evolution in different tumor regions has remained unexplored1-5. We mapped the prevalence of genetically distinct clones over 250 regions in 54 childhood cancers. This showed that primary tumors can simultaneously follow up to four evolutionary trajectories over different anatomic areas. The most common pattern consists of subclones with very few mutations confined to a single tumor region. The second most common is a stable coexistence, over vast areas, of clones characterized by changes in chromosome numbers. This is contrasted by a third, less frequent, pattern where a clone with driver mutations or structural chromosome rearrangements emerges through a clonal sweep to dominate an anatomical region. The fourth and rarest pattern is the local emergence of a myriad of clones with TP53 inactivation. Death from disease was limited to tumors exhibiting the two last, most dynamic patterns.
Assuntos
Mutação/genética , Neoplasias/genética , Criança , Cromossomos/genética , Evolução Molecular , Rearranjo Gênico/genética , Humanos , Proteína Supressora de Tumor p53/genéticaRESUMO
The global methylation profile and the mutational status of 633 specific epigenetic regulators were analyzed in the pediatric tumor clear cell sarcoma of the kidney (CCSK). Methylation array analyses of 30 CCSKs revealed CCSK tumor DNA to be globally hypermethylated compared to Wilms tumor, normal fetal kidney, and adult kidney. The aberrant methylation pattern of CCSKs was associated with activation of genes involved in embryonic processes and with silencing of genes linked to normal kidney function. No epigenetic regulator was recurrently mutated in our cohort, but a mutation in the key epigenetic regulator EZH2 was discovered in one case. EZH2 mRNA was significantly higher in CCSK compared to Wilms tumor and normal kidney, and the EZH2 protein was strongly expressed in more than 90 % of CCSK tumor cells in 9/9 tumors analyzed. This was in striking contrast to the lack of EZH2 protein expression in Wilms tumor stromal elements, indicating that EZH2 could be explored further as a diagnostic marker and a potential drug target for CCSK.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/biossíntese , Neoplasias Renais/genética , Sarcoma de Células Claras/genética , Adulto , Criança , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética , Feminino , Humanos , MasculinoRESUMO
Genetic differences among neoplastic cells within the same tumour have been proposed to drive cancer progression and treatment failure. Whether data on intratumoral diversity can be used to predict clinical outcome remains unclear. We here address this issue by quantifying genetic intratumoral diversity in a set of chemotherapy-treated childhood tumours. By analysis of multiple tumour samples from seven patients we demonstrate intratumoral diversity in all patients analysed after chemotherapy, typically presenting as multiple clones within a single millimetre-sized tumour sample (microdiversity). We show that microdiversity often acts as the foundation for further genome evolution in metastases. In addition, we find that microdiversity predicts poor cancer-specific survival (60%; P=0.009), independent of other risk factors, in a cohort of 44 patients with chemotherapy-treated childhood kidney cancer. Survival was 100% for patients lacking microdiversity. Thus, intratumoral genetic diversity is common in childhood cancers after chemotherapy and may be an important factor behind treatment failure.