Interleukin-10 receptor signaling promotes the maintenance of a PD-1int TCF-1+ CD8+ T cell population that sustains anti-tumor immunity.

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1hi, functionally impaired CD8+ T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1int subset. Frequencies of PD-1int TCF-1+ CD8+ T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1hi cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8+ T cells and poor survival in CLL and in breast cancer patients. Thus, balance between PD-1hi, exhausted CD8+ T cells and functional PD-1int TCF-1+ CD8+ T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy.

scSNPdemux: a sensitive demultiplexing pipeline using single nucleotide polymorphisms for improved pooled single-cell RNA sequencing analysis.

BACKGROUND: Here we present scSNPdemux, a sample demultiplexing pipeline for single-cell RNA sequencing data using natural genetic variations in humans. The pipeline requires alignment files from Cell Ranger (10× Genomics), a population SNP database and genotyped single nucleotide polymorphisms (SNPs) per sample. The tool works on sparse genotyping data in VCF format for sample identification. RESULTS: The pipeline was tested on both single-cell and single-nuclei based RNA sequencing datasets and showed superior demultiplexing performance over the lipid-based CellPlex and Multi-seq sample multiplexing technique which incurs additional single cell library preparation steps. Specifically, our pipeline demonstrated superior sensitivity and specificity in cell-identity assignment over CellPlex, especially on immune cell types with low RNA content. CONCLUSIONS: We designed a streamlined pipeline for single-cell sample demultiplexing, aiming to overcome common problems in multiplexing samples using single cell libraries which might affect data quality and can be costly.

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. METHODS: Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. RESULTS: Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. CONCLUSIONS: A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. LAY SUMMARY: HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC.

Chinese family with diffuse oesophageal leiomyomatosis: a new COL4A5/COL4A6 deletion and a case of gonosomal mosaicism.

BACKGROUND: Diffuse oesophageal leiomyomatosis (DOL) is a rare disorder characterized by tumorous overgrowth of the muscular wall of the oesophagus. DOL is present in 5 % of Alport syndrome (AS) patients. AS is a rare hereditary disease that involves varying degrees of hearing impairment, ocular changes and progressive glomerulonephritis leading to renal failure. In DOL-AS patients, the genetic defect consists of a deletion involving the COL4A5 and COL4A6 genes on the X chromosome. CASE PRESENTATION: We report a two-generation family (4 individuals; parents and two children, one male and one female) with two members (mother and son) affected with oesophageal leiomyomatosis. Signs of potential renal failure, which characterizes AS, were only apparent in the index patient (son) 2 years and three months after the initial diagnosis of DOL. Blood DNA from the four family members were submitted to exome sequencing and array genotyping to perform a genome wide screening for disease causal single nucleotide (SN) and copy number (CN) variations. Analyses revealed a new 40kb deletion encompassing from intron 2 of COL4A5 to intron 1 of COL4A6 at Xq22.3. The breakpoints were also identified. Possible confounding pathogenic exonic variants in genes known to be involved in other extracellular matrices disorders were also shared by the two affected individuals. Meticulous analysis of the maternal DNA revealed a case of gonosomal mosaicism. CONCLUSIONS: This is the first report of gonadosomal mosaicism associated to DOL-AS.

Association of mutation signature effectuating processes with mutation hotspots in driver genes and non-coding regions.

Cancer driving mutations are difficult to identify especially in the non-coding part of the genome. Here, we present sigDriver, an algorithm dedicated to call driver mutations. Using 3813 whole-genome sequenced tumors from International Cancer Genome Consortium, The Cancer Genome Atlas Program, and a childhood pan-cancer cohort, we employ mutational signatures based on single-base substitution in the context of tri- and penta-nucleotide motifs for hotspot discovery. Knowledge-based annotations on mutational hotspots reveal enrichment in coding regions and regulatory elements for 6 mutational signatures, including APOBEC and somatic hypermutation signatures. APOBEC activity is associated with 32 hotspots of which 11 are known and 11 are putative regulatory drivers. Somatic single nucleotide variants clusters detected at hypermutation-associated hotspots are distinct from translocation or gene amplifications. Patients carrying APOBEC induced PIK3CA driver mutations show lower occurrence of signature SBS39. In summary, sigDriver uncovers mutational processes associated with known and putative tumor drivers and hotspots particularly in the non-coding regions of the genome.

Carbon ion radiotherapy eradicates medulloblastomas with chromothripsis in an orthotopic Li-Fraumeni patient-derived mouse model.

BACKGROUND: Medulloblastomas with chromothripsis developing in children with Li-Fraumeni Syndrome (germline TP53 mutations) are highly aggressive brain tumors with dismal prognosis. Conventional photon radiotherapy and DNA-damaging chemotherapy are not successful for these patients and raise the risk of secondary malignancies. We hypothesized that the pronounced homologous recombination deficiency in these tumors might offer vulnerabilities that can be therapeutically utilized in combination with high linear energy transfer carbon ion radiotherapy. METHODS: We tested high-precision particle therapy with carbon ions and protons as well as topotecan with or without PARP inhibitor in orthotopic primary and matched relapsed patient-derived xenograft models. Tumor and normal tissue underwent longitudinal morphological MRI, cellular (markers of neurogenesis and DNA damage-repair), and molecular characterization (whole-genome sequencing). RESULTS: In the primary medulloblastoma model, carbon ions led to complete response in 79% of animals irrespective of PARP inhibitor within a follow-up period of 300 days postirradiation, as detected by MRI and histology. No sign of neurologic symptoms, impairment of neurogenesis or in-field carcinogenesis was detected in repair-deficient host mice. PARP inhibitors further enhanced the effect of proton irradiation. In the postradiotherapy relapsed tumor model, median survival was significantly increased after carbon ions (96 days) versus control (43 days, P < .0001). No major change in the clonal composition was detected in the relapsed model. CONCLUSION: The high efficacy and favorable toxicity profile of carbon ions warrants further investigation in primary medulloblastomas with chromothripsis. Postradiotherapy relapsed medulloblastomas exhibit relative resistance compared to treatment-naïve tumors, calling for exploration of multimodal strategies.

Chromothripsis in Human Breast Cancer.

Chromothripsis is a form of genome instability by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosomes. Widely assumed to be an early event in tumor development, this phenomenon plays a prominent role in tumor onset. In this study, an analysis of chromothripsis in 252 human breast cancers from two patient cohorts (149 metastatic breast cancers, 63 untreated primary tumors, 29 local relapses, and 11 longitudinal pairs) using whole-genome and whole-exome sequencing reveals that chromothripsis affects a substantial proportion of human breast cancers, with a prevalence over 60% in a cohort of metastatic cases and 25% in a cohort comprising predominantly luminal breast cancers. In the vast majority of cases, multiple chromosomes per tumor were affected, with most chromothriptic events on chromosomes 11 and 17 including, among other significantly altered drivers, CCND1, ERBB2, CDK12, and BRCA1. Importantly, chromothripsis generated recurrent fusions that drove tumor development. Chromothripsis-related rearrangements were linked with univocal mutational signatures, with clusters of point mutations due to kataegis in close proximity to the genomic breakpoints and with the activation of specific signaling pathways. Analyzing the temporal order of events in tumors with and without chromothripsis as well as longitudinal analysis of chromothriptic patterns in tumor pairs offered important insights into the role of chromothriptic chromosomes in tumor evolution. SIGNIFICANCE: These findings identify chromothripsis as a major driving event in human breast cancer.

The landscape of chromothripsis across adult cancer types.

Chromothripsis is a recently identified mutational phenomenon, by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosome(s). Considered as an early event in tumour development, this form of genome instability plays a prominent role in tumour onset. Chromothripsis prevalence might have been underestimated when using low-resolution methods, and pan-cancer studies based on sequencing are rare. Here we analyse chromothripsis in 28 tumour types covering all major adult cancers (634 tumours, 316 whole-genome and 318 whole-exome sequences). We show that chromothripsis affects a substantial proportion of human cancers, with a prevalence of 49% across all cases. Chromothripsis generates entity-specific genomic alterations driving tumour development, including clinically relevant druggable fusions. Chromothripsis is linked with specific telomere patterns and univocal mutational signatures in distinct tumour entities. Longitudinal analysis of chromothriptic patterns in 24 matched tumour pairs reveals insights in the clonal evolution of tumours with chromothripsis.

Cancer gene mutations in congenital pulmonary airway malformation patients.

BACKGROUND: Newborns affected with congenital pulmonary airway malformations (CPAMs) may present with severe respiratory distress or remain asymptomatic. While surgical resection is the definitive treatment for symptomatic CPAMs, prophylactic elective surgery may be recommended for asymptomatic CPAMs owing to the risk of tumour development. However, the implementation of prophylactic surgery is quite controversial on the grounds that more evidence linking CPAMs and cancer is needed. The large gap in knowledge of CPAM pathogenesis results in uncertainties and controversies in disease management. As developmental genes control postnatal cell growth and contribute to cancer development, we hypothesised that CPAMs may be underlain by germline mutations in genes governing airways development. METHODS: Sequencing of the exome of 19 patients and their unaffected parents. RESULTS: A more than expected number of mutations in cancer genes (false discovery rate q-value <5.01×10-5) was observed. The co-occurrence, in the same patient, of damaging variants in genes encoding interacting proteins is intriguing, the most striking being thyroglobulin (TG) and its receptor, megalin (LRP2). Both genes are highly relevant in lung development and cancer. CONCLUSIONS: The overall excess of mutations in cancer genes may account for the reported association of CPAMs with carcinomas and provide some evidence to argue for prophylactic surgery by some surgeons.

Rare variants and de novo variants in mesial temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: We investigated the role of rare genetic variants and of de novo variants in the pathogenesis of mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS). METHODS: Whole-exome sequencing (WES) was performed in patients with MTLE-HS and their unaffected parents (trios). Genes or gene sets that were enriched with predicted damaging rare variants in the patients as compared to population controls were identified. Patients and their parents were compared to identify whether the variants were de novo or inherited. RESULTS: After quality control, WES data from 47 patients (26 female), including 23 complete trios, were available for analysis. Compared with population controls, significant enrichment of rare variants was observed in SEC24B. Integration of gene set data describing neuronal functions and psychiatric disorders showed enrichment signal on fragile X mental retardation protein (FMRP) targets. Twenty-one de novo variants were identified, with many known to cause neuropsychiatric disorders. The FMRP-targeted genes also carried more de novo variants. Inherited compound heterozygous and homozygous variants were identified. CONCLUSIONS: The genetic architecture underlying MTHE-HS is complex. Multiple genes carrying de novo variants and rare variants among FMRP targets were identified, suggesting a pathogenic role. MTLE-HS and other neuropsychiatric disorders may have shared biology.

Evidence for a pre-malignant cell line in a skin biopsy from a patient with Nijmegen breakage syndrome.

BACKGROUND: Nijmegen breakage syndrome is an autosomal recessive disorder characterized by microcephaly, immunodeficiency, hypersensitivity to X-irradiation, and a high predisposition to cancer. Nibrin, the product of the NBN gene, is part of the MRE11/RAD50 (MRN) complex that is involved in the repair of DNA double strand breaks (DSBs), and plays a critical role in the processing of DSBs in immune gene rearrangements, telomere maintenance, and meiotic recombination. NBS skin fibroblasts grow slowly in culture and enter early into senescence. CASE PRESENTATION: Here we present an incidental finding. Skin fibroblasts, derived from a 9 year old NBS patient, showed a mosaic of normal diploid cells (46,XY) and those with a complex, unbalanced translocation. The aberrant karyotype was analysed by G-banding, comparative genomic hybridization, and whole chromosome painting. The exact breakpoints of the derivative chromosome were mapped by whole genome sequencing: 45,XY,der(6)(6pter → 6q11.1::13q11 → 13q21.33::20q11.22 → 20qter),-13. The deleted region of chromosomes 6 harbors almost 1.400 and that of chromosome 13 more than 500 genes, the duplicated region of chromosome 20 contains about 700 genes. Such unbalanced translocations are regularly incompatible with cellular survival, except in malignant cells. The aberrant cells, however, showed a high proliferation potential and could even be clonally expanded. Telomere length was significantly reduced, hTERT was not expressed. The cells underwent about 50 population doublings until they entered into senescence. The chromosomal preparation performed shortly before senescence showed telomere fusions, premature centromere divisions, endoreduplications and tetraploid cells, isochromatid breaks and a variety of marker chromosomes. Inspection of the site of skin biopsy 18 years later, presented no evidence for abnormal growth. CONCLUSIONS: The aberrant cells had a significant selective advantage in vitro. It is therefore tempting to speculate that this highly unbalanced translocation could be a primary driver of cancer cell growth.

Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors.

Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent complex genomic rearrangements (n = 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n = 68) and glioblastomas (n = 32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

Genetic study of congenital bile-duct dilatation identifies de novo and inherited variants in functionally related genes.

BACKGROUND: Congenital dilatation of the bile-duct (CDD) is a rare, mostly sporadic, disorder that results in bile retention with severe associated complications. CDD affects mainly Asians. To our knowledge, no genetic study has ever been conducted. METHODS: We aim to identify genetic risk factors by a "trio-based" exome-sequencing approach, whereby 31 CDD probands and their unaffected parents were exome-sequenced. Seven-hundred controls from the local population were used to detect gene-sets significantly enriched with rare variants in CDD patients. RESULTS: Twenty-one predicted damaging de novo variants (DNVs; 4 protein truncating and 17 missense) were identified in several evolutionarily constrained genes (p < 0.01). Six genes carrying DNVs were associated with human developmental disorders involving epithelial, connective or bone morphologies (PXDN, RTEL1, ANKRD11, MAP2K1, CYLD, ACAN) and four linked with cholangio- and hepatocellular carcinomas (PIK3CA, TLN1 CYLD, MAP2K1). Importantly, CDD patients have an excess of DNVs in cancer-related genes (p < 0.025). Thirteen genes were recurrently mutated at different sites, forming compound heterozygotes or functionally related complexes within patients. CONCLUSIONS: Our data supports a strong genetic basis for CDD and show that CDD is not only genetically heterogeneous but also non-monogenic, requiring mutations in more than one genes for the disease to develop. The data is consistent with the rarity and sporadic presentation of CDD.