Lineage tracing of normal human development and childhood cancers

From fertilisation onwards, the cells of the human body continuously experience damage to their genome, either from intrinsic causes or from exposure to mutagens. While the vast majority of DNA damage is repaired and the genome is replicated with extremely high fidelity, cells steadily acquire single nucleotide variants throughout life. Since cells pass these genetic changes on to their descendants, mutations shared between any two cells therefore imply a shared developmental path. In essence, these somatic mutations connect all cells together into one large phylogenetic tree of human development with the zygote at the root. Reconstructing phylogenies of human development requires readouts of somatic mutations present in single cells. Recently, low-input whole-genome sequencing following laser-capture microdissection has allowed us to reliably call somatic mutations in distinct single-cell derived physiological units, such as colonic crypts and endometrial glands, while retaining spatial information on a microscopic level. In this way, I reconstructed large-scale phylogenies of cells from many different organs of three individuals. These phylogenetic trees recapitulate the early stages of embryonic development and asymmetric cell allocation in the blastocyst, as well as later clonal expansions such as benign prostatic hyperplasia and neoplastic polyp formation. In a similar way, I also used somatic mutations to investigate the emergence of paediatric cancer, which is thought to be closely linked to aberrations in development. In the context of phylogenetic analyses of tumours, mutations shared between childhood cancers and different normal tissues can shed light on the embryonic lineage of tumours and may reveal the precise juncture at which tumours began to form. Accordingly, I studied the origin of Wilms tumour, the most common childhood cancer of the kidney. I discovered that these tumours often arise from large tissue-resident precursor clones residing in the normal kidney. These embryonal precursors represent an early clonal expansion driven by H19 hypermethylation. Lastly, using somatic mutations I discovered that the human placenta is made up of large clonal patches of closely related trophoblast cells. Comparing early embryonic mutations between placental lineages and umbilical cord DNA, which is derived from the inner cell mass, revealed that in approximately half of the cases, a trophectodermal lineage shares no somatic mutations with the umbilical cord. Furthermore, in a quarter of cases, the umbilical cord is entirely derived from a progenitor later than the zygote. This indicates a natural early segregation between these lineages and a pathway to generate confined placental mosaicism. This dissertation as a whole provides a new framework to study normal and aberrant human development from whole-genome sequencing. The ability to reconstruct developmental lineages retrospectively can answer fundamental questions about human development and carcinogenesis

Институт правовой охраны земель Российской Федерации, как предмет сравнительно-правового анализа земельно-правового регулирования в странах СНГ

У статті автором проведено порівняльно-правовий аналіз інституту правової охорони земель у країнах СНД на прикладі Росії. Ключові слова: oхорона земель, охорона земель Російської Федерації, землекористування, охорона навколишнього середовища, деградація, консервація землі.В данной статье отображена попытка автора провести сравнительно-правовой анализ института правовой охраны земель в странах СНГ на примере Российской Федерации и Украины. Ключевые слова: oхрана земель, охрана земель Российской Федерации, землепользование, охрана окружающей среды, деградация, консервация земли.The attempt of author to conduct the comparative-legal analysis of institute of legal safeguard of earths in the countries of the CIS on the example of Russian Federation and Ukraine is represented in this article. Key words: land’s preservation, Russian Federation’s land preservation, land tenure, preservation of the environment, land’s degeneration, land’s conservation

Lineage-Independent Tumors in Bilateral Neuroblastoma

Childhood tumors that occur synchronously in different anatomical sites usually represent metastatic disease. However, such tumors can be independent neoplasms. We investigated whether cases of bilateral neuroblastoma represented independent tumors in two children with pathogenic germline mutations by genotyping somatic mutations shared between tumors and blood. Our results suggested that in both children, the lineages that had given rise to the tumors had segregated within the first cell divisions of the zygote, without being preceded by a common premalignant clone. In one patient, the tumors had parallel evolution, including distinct second hits in SMARCA4, a putative predisposition gene for neuroblastoma. These findings portray cases of bilateral neuroblastoma as having independent lesions mediated by a germline predispositio

An in vitro stem cell model of human epiblast and yolk sac interaction.

Human embryogenesis entails complex signalling interactions between embryonic and extra-embryonic cells. However, how extra-embryonic cells direct morphogenesis within the human embryo remains largely unknown due to a lack of relevant stem cell models. Here, we have established conditions to differentiate human pluripotent stem cells (hPSCs) into yolk sac-like cells (YSLCs) that resemble the post-implantation human hypoblast molecularly and functionally. YSLCs induce the expression of pluripotency and anterior ectoderm markers in human embryonic stem cells (hESCs) at the expense of mesoderm and endoderm markers. This activity is mediated by the release of BMP and WNT signalling pathway inhibitors, and, therefore, resembles the functioning of the anterior visceral endoderm signalling centre of the mouse embryo, which establishes the anterior-posterior axis. Our results implicate the yolk sac in epiblast cell fate specification in the human embryo and propose YSLCs as a tool for studying post-implantation human embryo development in vitro.</i

Clonal hematopoiesis and therapy-related myeloid neoplasms following neuroblastoma treatment.

Therapy-related myeloid neoplasms (TMN) constitute one of the most challengingcomplications of cancer treatment.1 Whilst understanding of TMN pathogenesis remains fragmentary, genomic studies in adults have thus far refuted the notion that TMN simply result from cytotoxin-induced DNA damage.2–4 Analysis of the preclinical evolution of a limited number of adult TMN have retraced the majority of cases to clonal haematopoiesis (CH) that predates cytotoxic treatment and lacks the mutational footprint of genotoxic therapies.2–6 Balanced translocations, generally attributed to treatment with topoisomerase II inhibitors, are implicated in a minority of TMN.1 TMN is a leading cause of premature death in childhood cancer survivors, and affects 7-11% of children treated for high-risk neuroblastoma and sarcoma.7,8 However, the origin of pediatric TMN remains unclear. Targeted sequencing of known cancer genes detects CH in ~4% of children following cytotoxic treatment,6,9 whereas CH is vanishingly rare in young individuals in the general population.10,11 Moreover, to our knowledge, no cases of childhood TMN have been retraced to pretreatment CH. In light of these observations, we asked whether a broader driver landscape had eluded targeted CH screens in pediatric cancer patients and/or whether therapy-induced mutagenesis may be an under-recognised catalyst of CH and TMN in this patient group

MYC amplifications are common events in childhood osteosarcoma.

Funder: Bone Cancer Research Trust; Id: http://dx.doi.org/10.13039/100011719Funder: The Tom Prince Cancer TrustFunder: Wellcome Trust; Id: http://dx.doi.org/10.13039/100010269Funder: Jean Shanks Foundation – Pathological Society Clinical FellowshipFunder: UCL Experimental Cancer CentreFunder: UCLH Biomedical Research Centre; Id: http://dx.doi.org/10.13039/501100012317Funder: National Institute for Health Research; Id: http://dx.doi.org/10.13039/501100000272Osteosarcoma, the most common primary malignant tumour of bone, affects both children and adults. No fundamental biological differences between paediatric and adult osteosarcoma are known. Here, we apply multi-region whole-genome sequencing to an index case of a 4-year-old child whose aggressive tumour harboured high-level, focal amplifications of MYC and CCNE1 connected by translocations. We reanalysed copy number readouts of 258 cases of high-grade osteosarcoma from three different cohorts and identified a significant enrichment of focal MYC, but not CCNE1, amplifications in children. Furthermore, we identified four additional cases of MYC and CCNE1 coamplification, highlighting a rare driver event which warrants further investigation. Our findings indicate that amplification of the MYC oncogene is a major driver of childhood osteosarcoma, while CCNE1 appears recurrently amplified independent of age

Somatic Evolution in Non-neoplastic IBD-Affected Colon.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis

A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre.

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development