Large-scale Genome Sequencing Research Articles

Challenges in returning results in a genomic medicine implementation study: the Return of Actionable Variants Empirical (RAVE) study

To inform the process of returning results in genome sequencing studies, we conducted a quantitative and qualitative assessment of challenges encountered during the Return of Actionable Variants Empiric (RAVE) study conducted at Mayo Clinic. Participants (n = 2535, mean age 63 ± 7, 57% female) were sequenced for 68 clinically actionable genes and 14 single nucleotide variants. Of 122 actionable results detected, 118 were returnable; results were returned by a genetic counselor—86 in-person and 12 by phone. Challenges in returning actionable results were encountered in a significant proportion (38%) of the cohort and were related to sequencing and participant contact. Sequencing related challenges (n = 14), affecting 13 participants, included reports revised based on clinical presentation (n = 3); reports requiring corrections (n = 2); mosaicism requiring alternative DNA samples for confirmation (n = 3); and variant re-interpretation due to updated informatics pipelines (n = 6). Participant contact related challenges (n = 44), affecting 38 participants, included nonresponders (n = 20), decedents (n = 1), and previously known results (n = 23). These results should be helpful to investigators preparing for return of results in large-scale genomic sequencing projects.

Driver mutations in acute myeloid leukemia.

The mutational landscape of acute myeloid leukemia (AML) has revised diagnostic, prognostic, and therapeutic schemata over the past decade. Recurrently mutated AML genes have functional consequences beyond typical oncogene-driven growth and loss of tumor suppresser function. Large-scale genomic sequencing efforts have mapped the complexity of AML and trials of mutation-based targeted therapy has led to several FDA-approved drugs for mutant-specific AML. However, many recurrent mutations have been identified across a spectrum from clonal hematopoiesis to myelodysplasia to overt AML, such as effectors of DNA methylation, chromatin modifiers, and spliceosomal machinery. The functional effects of these mutations are the basis for substantial discovery. Understanding the molecular and pathophysiologic functions of key genes that exert leukemogenic potential is essential towards translating these findings into better treatment for AML.

Glutamate receptors from diverse animal species exhibit unexpected structural and functional diversity.

The identification of AMPA, kainate and NMDA glutamate receptor subtypes by Watkins and colleagues underlies much of our understanding of excitatory synaptic transmission in the central nervous system of animals. Ongoing large scale genome sequencing projects in species for which physiological analysis of receptor function is challenging are resulting in identification of numerous eukaryotic glutamate receptor ion channels in the animal kingdom of life. On the basis of sequence similarity, these are frequently classified into the three vertebrate subtypes, initially identified using subtype selective ligands. Recent work reveals unexpected ligand binding profiles for these newly identified glutamate receptors, for example, kainate receptors on which NMDA acts as a competitive antagonist, and high affinity homomeric glycine activated glutamate receptors. Structural studies reveal that only subtle changes in the ligand binding domain, often identified only in retrospect, underlie different patterns of ligand binding, and that the biology of glutamate receptors is more complex than first anticipated.

Darwinian Shift: A General Approach for Establishing Evidence and Mechanism of Natural Selection

Increasingly sensitive and large scale genome sequencing has revealed a massive breadth of heterogeneity between clones in competitive environments. The study of large sets of genome data from the epithelial tissues of healthy patients have shown how mutationally diverse aged tissues become over time. In the interpretation of such datasets, a fundamental question is whether the mutations are selected for in competitive processes, or they arise through neutral drift. The ratio of synonymous to non-synonymous mutations (dn/ds) is commonly used to establish evidence of selection. Other techniques attempt to score codons or residues as “pathogenic” through training complex models against of diverse datasets, and using this score to weight subsequent analysis of mutations. In both cases, whilst evidence of selection may be found, there exist known limitations to the techniques and interpretation of the functional impact requires further work. Here we present “Darwinian Shift”, a workflow for using user selected metrics of protein or gene activity to establish evidence and mechanisms of selection. Testing against published healthy patient datasets, we show that through uses of conservation scores and mutations that induce residue swaps, the approach can reproduce the findings of existing techniques in the literature to establish evidence of selection. We go on to show how we can use folding energies, calculated with FoldX and Rosetta alongside more abstract measures of disorder, can identify an overlapping, alternative set of genes that undergo misfolding due to selected-for mutations. We also use high throughput molecular dynamics simulations of the transmembrane helix of NOTCH1 to explore how mutations change the orientation of the helix.

Recent advances in functional genomics for parasitic nematodes of mammals.

Human-parasitic nematodes infect over a quarter of the world's population and are a major cause of morbidity in low-resource settings. Currently available treatments have not been sufficient to eliminate infections in endemic areas, and drug resistance is an increasing concern, making new treatment options a priority. The development of new treatments requires an improved understanding of the basic biology of these nematodes. Specifically, a better understanding of parasitic nematode development, reproduction and behavior may yield novel drug targets or new opportunities for intervention such as repellents or traps. Until recently, our ability to study parasitic nematode biology was limited because few tools were available for their genetic manipulation. This is now changing as a result of recent advances in the large-scale sequencing of nematode genomes and the development of new techniques for their genetic manipulation. Notably, skin-penetrating gastrointestinal nematodes in the genus Strongyloides are now amenable to transgenesis, RNAi and CRISPR/Cas9-mediated targeted mutagenesis, positioning the Strongyloides species as model parasitic nematode systems. A number of other mammalian-parasitic nematodes, including the giant roundworm Ascaris suum and the tissue-dwelling filarial nematode Brugia malayi, are also now amenable to transgenesis and/or RNAi in some contexts. Using these tools, recent studies of Strongyloides species have already provided insight into the molecular pathways that control the developmental decision to form infective larvae and that drive the host-seeking behaviors of infective larvae. Ultimately, a mechanistic understanding of these processes could lead to the development of new avenues for nematode control.

The genetics of intellectual disability: advancing technology and gene editing.

Intellectual disability (ID) is a neurodevelopmental condition affecting 1–3% of the world’s population. Genetic factors play a key role causing the congenital limitations in intellectual functioning and adaptive behavior. The heterogeneity of ID makes it more challenging for genetic and clinical diagnosis, but the advent of large-scale genome sequencing projects in a trio approach has proven very effective. However, many variants are still difficult to interpret. A combined approach of next-generation sequencing and functional, electrophysiological, and bioinformatics analysis has identified new ways to understand the causes of ID and help to interpret novel ID-causing genes. This approach offers new targets for ID therapy and increases the efficiency of ID diagnosis. The most recent functional advancements and new gene editing techniques involving the use of CRISPR–Cas9 allow for targeted editing of DNA in in vitro and more effective mammalian and human tissue-derived disease models. The expansion of genomic analysis of ID patients in diverse and ancient populations can reveal rare novel disease-causing genes.

Analysis of RNA sequences of 3636 SARS-CoV-2 collected from 55 countries reveals selective sweep of one virus type.

Background & objectives:SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) is evolving with the progression of the pandemic. This study was aimed to investigate the diversity and evolution of the coronavirus SARS-CoV-2 with progression of the pandemic over time and to identify similarities and differences of viral diversity and evolution across geographical regions (countries).Methods:Publicly available data on type definitions based on whole-genome sequences of the SARS-CoV-2 sampled during December and March 2020 from 3636 infected patients spread over 55 countries were collected. Phylodynamic analyses were performed and the temporal and spatial evolution of the virus was examined.Results:It was found that (i) temporal variation in frequencies of types of the coronavirus was significant; ancestral viruses of type O were replaced by evolved viruses belonging to type A2a; (ii) spatial variation was not significant; with the spread of SARS-CoV-2, the dominant virus was the A2a type virus in every geographical region; (iii) within a geographical region, there was significant micro-level variation in the frequencies of the different viral types, and (iv) the evolved coronavirus of type A2a swept rapidly across all continents.Interpretation & conclusions:SARS-CoV-2 belonging to the A2a type possesses a non-synomymous variant (D614G) that possibly eases the entry of the virus into the lung cells of the host. This may be the reason why the A2a type has an advantage to infect and survive and as a result has rapidly swept all geographical regions. Therefore, large-scale sequencing of coronavirus genomes and, as required, of host genomes should be undertaken in India to identify regional and ethnic variation in viral composition and its interaction with host genomes. Further, careful collection of clinical and immunological data of the host can provide deep learning in relation to infection and transmission of the types of coronavirus genomes.

Abstract ED01-02: Successes and failures of targeted drug development in pediatric tumors

Abstract Eighty percent of children diagnosed with cancer are cured with multimodality therapy including cytotoxic chemotherapy. However, up to 40% of survivors of childhood cancer have at least one serious chronic medical condition as a consequence of curative therapy. Thus, patients, families, clinicians, researchers, and regulatory agencies enthusiastically endorse the development of targeted therapy to improve survival, decrease life threatening acute toxicity, and late effects of therapy for childhood cancer. Genomic characterization demonstrates that the tumor mutational burden in childhood cancers is low. For children with cancer a small number of drugs, such as NTRK inhibitors have had major unequivocal successes including initial regulatory approvals that include specific dosing in children. Other molecularly targeted drugs have achieved moderate success, however, many have failed despite strong pre-clinical rationale. As of June 2019, 25 drugs have been FDA approved for use in children with cancer including five tyrosine kinase inhibitors (3 BCR-ABL inhibitors and 2 NTRK inhibitors), and one mTOR inhibitor. A single drug, dinutuximab, was approved for a pediatric specific indication in high-risk neuroblastoma. This parallels recent European Medicines Agency authorizations that include indications for treating childhood cancer in 10 of 177 anti-cancer drug approvals. Similar to adults, the era of targeted therapies in children with cancer began with imatinib. Drugs targeting BCR-ABL in chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) in children include imatinib, dasatinib, and nilotinib. Point mutations in the kinase domain of the BCR-ABL fusion protein result in resistance to imatinib. Dasatinib, nilotinib, and ponatinib were developed to overcome resistance. Each has a specific role in caring for children with CML or Ph+ ALL. Ph-like ALL, a high-risk acute leukemia in children and young adults, is characterized by genomic alterations and activation of signaling pathways including JAK/STAT, amenable to inhibition tyrosine kinase inhibitors. The landmark age and histology agnostic global approval of larotrectinib and entrectinib was based on durable objective responses in patients with tumors harboring NTRK fusions enrolled on early phase clinical trials. Larotrectinib has produced dramatic objective responses in children and adults with cancer harboring NTRK fusions including a very rare cancer, infantile fibrosarcoma which is NTRK fusion positive in at least 95% cases. In addition to potent inhibition NTRK fusions, initial clinical trials of larotrectinib included adults and children as well as a pediatric appropriate formulation. Children with brain tumors harboring NTRK1,2,3 or ROS1 fusions have had rapid, durable objective responses to entrectinib. MAP kinase pathway aberrations occur in childhood cancer including rhabdomyosarcoma and brain tumors. Everolimus is approved for patients with subependymal giant cell astrocytoma. Dabrafenib and vemurafenib have produced durable objective responses in patients with pediatric low-grade glioma harboring BRAF V600E mutations. Selumetinib has been FDA approved for the treatment of plexiform neurofibromas in children with neurofibromatosis type 1 and may have activity in children and adolescents with solid tumors harboring MAP kinase aberrations. Overall, the success of MAP kinase inhibition in children with cancer has been modest, leading to consideration for combinatorial approaches. Crizotinib is approved for the treatment of ALK-positive metastatic NSCLC and has activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and inflammatory myofibroblastic tumors, both associated with ALK fusions. Trials are ongoing to determine the activity of crizotinib and other ALK inhibitors in children with neuroblastoma with known translocations, mutations or amplifications in the ALK gene. Clinical trials of multi-targeted kinase inhibitors including cabozantinib, regorafenib, sorafenib and vandetanib have demonstrated activity in a variety of solid tumors. Clinical trials of these agents have been based on mechanistic rationale and pre-clinical evaluation rather than biomarker selection. Through large scale genomic sequencing efforts in the US and Europe actionable molecular alterations including mutations, amplifications and fusions have been identified in a variety of childhood cancers. Compared to cancers in adults, these molecular aberrations occur less frequently in most cancers in children. However, due to dramatic objective responses and the potential for improved response with decreased toxicity, efforts to evaluate and incorporate targeted agents into multimodality therapy for children with cancer remains a major focus. To date, multi-targeted kinase inhibitors and drugs that target fusion oncoproteins that are histology-agnostic oncogenic drivers in cancers that occur in both child and adults have been most successful. Understanding the characteristics of successful target- drug pairs is critically important to the prioritization of new drugs and clinical trials in children as well as the development of new therapies for pediatric specific targets. Citation Format: Elizabeth Fox. Successes and failures of targeted drug development in pediatric tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr ED01-02. doi:10.1158/1535-7163.TARG-19-ED01-02

Alu insertion polymorphisms shared by Papio baboons and Theropithecus gelada reveal an intertwined common ancestry

BackgroundBaboons (genus Papio) and geladas (Theropithecus gelada) are now generally recognized as close phylogenetic relatives, though morphologically quite distinct and generally classified in separate genera. Primate specific Alu retrotransposons are well-established genomic markers for the study of phylogenetic and population genetic relationships. We previously reported a computational reconstruction of Papio phylogeny using large-scale whole genome sequence (WGS) analysis of Alu insertion polymorphisms. Recently, high coverage WGS was generated for Theropithecus gelada. The objective of this study was to apply the high-throughput “poly-Detect” method to computationally determine the number of Alu insertion polymorphisms shared by T. gelada and Papio, and vice versa, by each individual Papio species and T. gelada. Secondly, we performed locus-specific polymerase chain reaction (PCR) assays on a diverse DNA panel to complement the computational data.ResultsWe identified 27,700 Alu insertions from T. gelada WGS that were also present among six Papio species, with nearly half (12,956) remaining unfixed among 12 Papio individuals. Similarly, each of the six Papio species had species-indicative Alu insertions that were also present in T. gelada. In general, P. kindae shared more insertion polymorphisms with T. gelada than did any of the other five Papio species. PCR-based genotype data provided additional support for the computational findings.ConclusionsOur discovery that several thousand Alu insertion polymorphisms are shared by T. gelada and Papio baboons suggests a much more permeable reproductive barrier between the two genera then previously suspected. Their intertwined evolution likely involves a long history of admixture, gene flow and incomplete lineage sorting.

Insular Microbiogeography: Three Pathogens as Exemplars.

Traditional taxonomy in biology assumes that life is organized in a simple tree. Attempts to classify microorganisms in this way in the genomics era led microbiologists to look for finite sets of 'core' genes that uniquely group taxa as clades in the tree. However, the diversity revealed by large-scale whole genome sequencing is calling into question the long-held model of a hierarchical tree of life, which leads to questioning of the definition of a species. Large-scale studies of microbial genome diversity reveal that the cumulative number of new genes discovered increases with the number of genomes studied as a power law and subsequently leads to the lack of evidence for a unique core genome within closely related organisms. Sampling 'enough' new genomes leads to the discovery of a replacement or alternative to any gene. This power law behaviour points to an underlying self-organizing critical process that may be guided by mutation and niche selection. Microbes in any particular niche exist within a local web of organism interdependence known as the microbiome. The same mechanism that underpins the macro-ecological scaling first observed by MacArthur and Wilson also applies to microbial communities. Recent metagenomic studies of a food microbiome demonstrate the diverse distribution of community members, but also genotypes for a single species within a more complex community. Collectively, these results suggest that traditional taxonomic classification of bacteria could be replaced with a quasispecies model. This model is commonly accepted in virology and better describes the diversity and dynamic exchange of genes that also hold true for bacteria. This model will enable microbiologists to conduct population-scale studies to describe microbial behaviour, as opposed to a single isolate as a representative.

Genome survey analysis and SSR loci mining of Bupleurum falcatum

Buplewrum falcatum is a traditional Chinese medicine,which is mainly used for the treatment of cold and liver protection. B. falcatum is dominantly cultivated in Japan as well as planted in China,Korea and other countries and regions. In order to determine the appropriate sequencing strategy,the genome survey before large-scale genome sequencing is needed. This survey can provide information about the size and complexity of the whole genome of the target species. In the present study,the next generation sequencing technology( Illumina Hiseq 2000) was used to analyze the genome size and complexity of B. falcatum. In addition,SSR loci were analyzed from the sequenced data. Primer 3 was used to design specific primers and 33 pairs of primers were randomly selected for PCR with template DNA of B. falcatum,and the PCR system and optimal annealing temperature were screened. A total of 288. 64 G genome sequence data was obtained,and the estimated genome size of B. falcatum was 2 119. 58 Mb. The measured genome data depth was138×; the rate of heterozygosity was 1. 84%; and the ratio of repeat sequence was 83. 89%. It is speculated that the genome of B. falcatum is complex. The preliminary assembly was performed with K-mer = 41,and the contig N50 was 224 bp,the total length 896. 97 Mb,the scaffold N50 313 bp,and the total length was 922. 67 Mb. A total of 91 377 SSR sequences were detected in the sequenced genome data which were distributed in 70 809 unigenes.The main type is dinucleotide repeats,with 49 680 sequences,accounting for70. 16%. Among the 33 pairs of primers randomly synthesized according to the obtained SSR sequences,21 pairs were successfully amplifying the target sequences. The results will be helpful for later large scale genome sequencing and SSR molecular markers development for germplasm identification and trait mapping.

Clinical utility of genomic sequencing.

Identifying pathogenic variation underlying pediatric developmental disease is critical for medical management, therapeutic development, and family planning. This review summarizes current genetic testing options along with their potential benefits and limitations. We also describe results from large-scale genomic sequencing projects in pediatric and neonatal populations with a focus on clinical utility. Recent advances in DNA sequencing technology have made genomic sequencing a feasible and effective testing option in a variety of clinical settings. These cutting-edge tests offer much promise to both medical providers and patients as it has been demonstrated to detect causal genetic variation in ∼25% or more of previously unresolved cases. Efforts aimed at promoting data sharing across clinical genetics laboratories and systematic reanalysis of existing genomic sequencing data have further improved diagnostic rates and reduced the number of unsolved cases. Genomic sequencing is a powerful and increasingly cost-effective alternative to current genetic tests and will continue to grow in clinical utility as more of the genome is understood and as analytical methods are improved. The evolution of genomic sequencing is changing the landscape of clinical testing and requires medical professionals who are adept at understanding and returning genomic results to patients.

Archaeogenetics and human evolution: the ontogeny of a biological discipline

ABSTRACT Genomics and ancient DNA methods have revolutionized many areas of biology, including human evolution. Recently we have seen significant advances in archaeogenetics including the use of large-scale genomic datasets to track human movements globally. In addition, advances in ancient and modern genomics have enabled researchers to resolve a range of issues of importance to indigenous people. Most notable among these is the repatriation of ancient remains to their kin i.e. to what Aboriginal Australians refer to as their ‘Place and Country’. From an historical perspective, new fields of science can be characterized as moving through three stages beginning with description, followed by a focus on mechanisms/functions and finally the formulation of experiments and hypothesis testing. The new science of archaeogenetics is currently in the descriptive stage. It involves large-scale genome sequencing and the use of explanatory narratives. Our analysis suggests that a mature formulation lies in the future.

Variant calling and quality control of large-scale human genome sequencing data.

Next-generation sequencing has allowed genetic studies to collect genome sequencing data from a large number of individuals. However, raw sequencing data are not usually interpretable due to fragmentation of the genome and technical biases; therefore, analysis of these data requires many computational approaches. First, for each sequenced individual, sequencing data are aligned and further processed to account for technical biases. Then, variant calling is performed to obtain information on the positions of genetic variants and their corresponding genotypes. Quality control (QC) is applied to identify individuals and genetic variants with sequencing errors. These procedures are necessary to generate accurate variant calls from sequencing data, and many computational approaches have been developed for these tasks. This review will focus on current widely used approaches for variant calling and QC.

Large-scale ruminant genome sequencing provides insights into their evolution and distinct traits.

The ruminants are one of the most successful mammalian lineages, exhibiting morphological and habitat diversity and containing several key livestock species. To better understand their evolution, we generated and analyzed de novo assembled genomes of 44 ruminant species, representing all six Ruminantia families. We used these genomes to create a time-calibrated phylogeny to resolve topological controversies, overcoming the challenges of incomplete lineage sorting. Population dynamic analyses show that population declines commenced between 100,000 and 50,000 years ago, which is concomitant with expansion in human populations. We also reveal genes and regulatory elements that possibly contribute to the evolution of the digestive system, cranial appendages, immune system, metabolism, body size, cursorial locomotion, and dentition of the ruminants.

Noncoding deletions reveal a gene that is critical for intestinal function

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

Genome sequencing analysis identifies Epstein-Barr virus subtypes associated with high risk of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) infection is ubiquitous worldwide and is associated with multiple cancers, including nasopharyngeal carcinoma (NPC). The importance of EBV viral genomic variation in NPC development and its striking epidemic in southern China has been poorly explored. Through large-scale genome sequencing of 270 EBV isolates and two-stage association study of EBV isolates from China, we identified two non-synonymous EBV variants within BALF2 strongly associated with the risk of NPC (odds ratio (OR) = 8.69, P=9.69×10−25 for SNP 162476_C; OR = 6.14, P=2.40×10−32 for SNP 163364_T). The cumulative effects of these variants contributed to 83% of the overall risk of NPC in southern China. Phylogenetic analysis of the risk variants revealed a unique origin in Asia, followed by clonal expansion in NPC-endemic regions. Our results provide novel insights into NPC endemic in southern China and also enable the identification of high-risk individuals for NPC prevention.

PI3Kα inhibitors sensitize esophageal squamous cell carcinoma to radiation by abrogating survival signals in tumor cells and tumor microenvironment.

Radiotherapy is one of the standard therapies for esophageal squamous cell carcinoma (ESCC), but the efficacy is far from desirable. Large scale genome sequencing reveals PI3Kα is frequently hyper-activated in ESCC. We found that ESCC cells harboring alterations in PI3K pathway were more resistant to radiation and combination of a clinical PI3Kα-selective inhibitor CYH33 and radiation synergistically inhibited cell proliferation in 14 ESCC cell lines. Radiation induced phosphorylation of FOXO1 and Akt, which sensitized ESCC cells to PI3Kα inhibitors. Both S1PR3 and DNA-PK contributed to radiation-induced Akt phosphorylation, which were revealed to be collectively dependent on PI3Kα. By contrast, constitutively active Akt abrogated the synergism between PI3Kα inhibitors and radiation. PI3Kα inhibition enhanced radiation-induced DNA damage, G2/M arrest and apoptosis. Combination of CYH33 and radiation significantly inhibited the growth of xenografts derived from ESCC patients, which was accompanied with abrogation of radiation-induced phosphorylation of Akt and filtration of M2-like macrophages. Taken together, combination of CYH33 and radiation possesses synergism in ESCC, which provides promising rationale to test this combinatorial regimen in ESCC patients.

The Status and Impact of Clinical Tumor Genome Sequencing.

Since the discovery that DNA alterations initiate tumorigenesis, scientists and clinicians have been exploring ways to counter these changes with targeted therapeutics. The sequencing of tumor DNA was initially limited to highly actionable hot spots-areas of the genome that are frequently altered and have an approved matched therapy in a specific tumor type. Large-scale genome sequencing programs quickly developed technological improvements that enabled the deployment of whole-exome and whole-genome sequencing technologies at scale for pristine sample materials in research environments. However, the turning point for precision medicine in oncology was the innovations in clinical laboratories that improved turnaround time, depth of coverage, and the ability to reliably sequence archived, clinically available samples. Today, tumor genome sequencing no longer suffers from significant technical or financial hurdles, and the next opportunity for improvement lies in the optimal utilization of the technologies and data for many different tumor types.

The changing scenario of non-Down syndrome acute megakaryoblastic leukemia in children.

Pediatric non-Down-syndrome acute megakaryoblastic leukemia (non-DS-AMKL) is a heterogeneous subtype of leukemia that has historically been associated with poor prognosis. Until the advent of large-scale genomic sequencing, the management of patients with non-DS-AMKL was very difficult due to the absence of reliable biological prognostic markers. The sequencing of large cohort of pediatric non-DS-AMKL samples led to the discovery of novel genetic aberrations, including high-frequency fusions, such as CBFA2T3-GLIS2 and NUP98-KDM5 A, as well as less frequent aberrations, such as HOX rearrangements. These new insights into the genetic landscape of pediatric non-DS-AMKL has allowed refining the risk-group stratification, leading to important changes in the prognostic scenario of these patients. This review summarizes the most important molecular pathogenic mechanisms of pediatric non-DS-AMKL. A critical discussion on how novel genetic abnormalities have refined the risk profile assessment and changed the management of these patients in clinical practice is also provided.