T cell immune deficiency rather than chromosome instability predisposes patients with short telomere syndromes to squamous cancers.

Patients with short telomere syndromes (STS) are predisposed to developing cancer, believed to stem from chromosome instability in neoplastic cells. We tested this hypothesis in a large cohort assembled over the last 20 years. We found that the only solid cancers to which patients with STS are predisposed are squamous cell carcinomas of the head and neck, anus, or skin, a spectrum reminiscent of cancers seen in patients with immunodeficiency. Whole-genome sequencing showed no increase in chromosome instability, such as translocations or chromothripsis. Moreover, STS-associated cancers acquired telomere maintenance mechanisms, including telomerase reverse transcriptase (TERT) promoter mutations. A detailed study of the immune status of patients with STS revealed a striking T cell immunodeficiency at the time of cancer diagnosis. A similar immunodeficiency that impaired tumor surveillance was documented in mice with short telomeres. We conclude that STS patients' predisposition to solid cancers is due to T cell exhaustion rather than autonomous defects in the neoplastic cells themselves.

The genomic landscape of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Exploration of Coding and Non-coding Variants in Cancer Using GenomePaint.

GenomePaint (https://genomepaint.stjude.cloud/) is an interactive visualization platform for whole-genome, whole-exome, transcriptome, and epigenomic data of tumor samples. Its design captures the inter-relatedness between DNA variations and RNA expression, supporting in-depth exploration of both individual cancer genomes and full cohorts. Regulatory non-coding variants can be inspected and analyzed along with coding variants, and their functional impact further explored by examining 3D genome data from cancer cell lines. Further, GenomePaint correlates mutation and expression patterns with patient outcomes, and supports custom data upload. We used GenomePaint to unveil aberrant splicing that disrupts the RING domain of CREBBP, discover cis activation of the MYC oncogene by duplication of the NOTCH1-MYC enhancer in B-lineage acute lymphoblastic leukemia, and explore the inter- and intra-tumor heterogeneity at EGFR in adult glioblastomas. These examples demonstrate that deep multi-omics exploration of individual cancer genomes enabled by GenomePaint can lead to biological insights for follow-up validation.

Therapy-induced mutations drive the genomic landscape of relapsed acute lymphoblastic leukemia.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

Resolving medulloblastoma cellular architecture by single-cell genomics.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Analysis of error profiles in deep next-generation sequencing data.

BACKGROUND: Sequencing errors are key confounding factors for detecting low-frequency genetic variants that are important for cancer molecular diagnosis, treatment, and surveillance using deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, PCR enrichment, and sequencing. In this study, we use current NGS technology to systematically investigate these questions. RESULTS: By evaluating read-specific error distributions, we discover that the substitution error rate can be computationally suppressed to 10-5 to 10-4, which is 10- to 100-fold lower than generally considered achievable (10-3) in the current literature. We then quantify substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets. We find that error rates differ by nucleotide substitution types, ranging from 10-5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10-4 for A>G/T>C changes. Furthermore, C>T/G>A errors exhibit strong sequence context dependency, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. We also find that more than 70% of hotspot variants can be detected at 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression. CONCLUSIONS: We present the first comprehensive analysis of sequencing error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS analysis accuracy both experimentally and computationally, ultimately enhancing the precision of deep sequencing.

Insights into glycogen metabolism in chemolithoautotrophic bacteria from distinctive kinetic and regulatory properties of ADP-glucose pyrophosphorylase from Nitrosomonas europaea.

Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO(2) via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.