Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.

UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors.

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.

The evolutionary history of 2,658 cancers.

Cancer develops through a process of somatic evolution1,2. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes3. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4, we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection.

Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Genomic ALK alterations in primary and relapsed neuroblastoma.

BACKGROUND: Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse. METHODS: ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively. RESULTS: At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients <18 months. At relapse, ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome. CONCLUSIONS: The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

Telomerase activation by genomic rearrangements in high-risk neuroblastoma.

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

Deep learning for sensitive detection of Helicobacter Pylori in gastric biopsies.

BACKGROUND: Helicobacter pylori, a 2 × 1 µm spiral-shaped bacterium, is the most common risk factor for gastric cancer worldwide. Clinically, patients presenting with symptoms of gastritis, routinely undergo gastric biopsies. The following histo-morphological evaluation dictates therapeutic decisions, where antibiotics are used for H. pylori eradication. There is a strong rational to accelerate the detection process of H. pylori on histological specimens, using novel technologies, such as deep learning. METHODS: We designed a deep-learning-based decision support algorithm that can be applied on regular whole slide images of gastric biopsies. In detail, we can detect H. pylori both on Giemsa- and regular H&E stained whole slide images. RESULTS: With the help of our decision support algorithm, we show an increased sensitivity in a subset of 87 cases that underwent additional PCR- and immunohistochemical testing to define a sensitive ground truth of HP presence. For Giemsa stained sections, the decision support algorithm achieved a sensitivity of 100% compared to 68.4% (microscopic diagnosis), with a tolerable specificity of 66.2% for the decision support algorithm compared to 92.6 (microscopic diagnosis). CONCLUSION: Together, we provide the first evidence of a decision support algorithm proving as a sensitive screening option for H. pylori that can potentially aid pathologists to accurately diagnose H. pylori presence on gastric biopsies.

iRODS metadata management for a cancer genome analysis workflow.

BACKGROUND: The massive amounts of data from next generation sequencing (NGS) methods pose various challenges with respect to data security, storage and metadata management. While there is a broad range of data analysis pipelines, these challenges remain largely unaddressed to date. RESULTS: We describe the integration of the open-source metadata management system iRODS (Integrated Rule-Oriented Data System) with a cancer genome analysis pipeline in a high performance computing environment. The system allows for customized metadata attributes as well as fine-grained protection rules and is augmented by a user-friendly front-end for metadata input. This results in a robust, efficient end-to-end workflow under consideration of data security, central storage and unified metadata information. CONCLUSIONS: Integrating iRODS with an NGS data analysis pipeline is a suitable method for addressing the challenges of data security, storage and metadata management in NGS environments.

Genomic and transcriptomic heterogeneity of colorectal tumours arising in Lynch syndrome.

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Activating ERBB2/HER2 mutations indicate susceptibility to pan-HER inhibitors in Lynch and Lynch-like colorectal cancer.

OBJECTIVE: Microsatellite instability (MSI) is detected in approximately 15% of all colorectal cancers (CRC) and virtually in all cases with Lynch syndrome. The MSI phenotype is caused by dysfunctional mismatch repair (MMR) and leads to accumulation of DNA replication errors. Sporadic MSI CRC often harbours BRAF(V600E); however, no consistent data exist regarding targeted treatment approaches in BRAF(wt) MSI CRC. DESIGN: Mutations and quantitative MSI were analysed by deep sequencing in 196 formalin fixed paraffin embedded (FFPE) specimens comprising Lynch and Lynch-like CRCs from the German Hereditary Nonpolyposis Colorectal Cancer registry. Functional relevance of recurrent ERBB2/HER2 mutations was investigated in CRC cell lines using reversible and irreversible HER-targeting inhibitors, EGFR-directed antibody cetuximab, HER2-directed antibody trastuzumab and siRNA-mediated ERBB2/HER2 knockdown. RESULTS: Quantification of nucleotide loss in non-coding mononucleotide repeats distinguished microsatellite status with very high accuracy (area under curve=0.9998) and demonstrated progressive losses with deeper invasion of MMR-deficient colorectal neoplasms (p=0.008). Characterisation of BRAF(wt) MSI CRC revealed hot-spot mutations in well-known oncogenic drivers, including KRAS (38.7%), PIK3CA (36.5%), and ERBB2 (15.0%). L755S and V842I substitutions in ERBB2 were highly recurrent. Functional analyses in ERBB2-mutated MSI CRC cell lines revealed a differential response to HER-targeting compounds and superiority of irreversible pan-HER inhibitors. CONCLUSIONS: We developed a high-throughput deep sequencing approach for concomitant MSI and mutational analyses in FFPE specimens. We provided novel insights into clinically relevant alterations in MSI CRC and a rationale for targeting ERBB2/HER2 mutations in Lynch and Lynch-like CRC.

Mutational analysis of pulmonary tumours with neuroendocrine features using targeted massive parallel sequencing: a comparison of a neglected tumour group.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows. METHODS: A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes. RESULTS: After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020). CONCLUSIONS: The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.

CGARS: cancer genome analysis by rank sums.

MOTIVATION: Cancer genomes are characterized by the accumulation of point mutations and structural alterations such as copy-number alterations and genomic rearrangements. Among structural changes, systematic analyses of copy-number alterations have provided deeper insight into the architecture of cancer genomes and had led to new potential treatment opportunities. During the course of cancer genome evolution, selection mechanisms are leading to a non-random pattern of mutational events contributing to fitness benefits of the cancer cells. We therefore developed a new method to dissect random from non-random patterns in copy-number data and thereby to assess significantly enriched somatic copy-number aberrations across a set of tumor specimens or cell lines. In contrast to existing approaches, the method is invariant to any strictly monotonous transformation of the input data which results to an insensitivity of differences in tumor purity, array saturation effects and copy-number baseline levels. RESULTS: We applied our approach to recently published datasets of small-cell lung cancer and squamous cell lung cancer and validated its performance by comparing the results to an orthogonal approach. In addition, we found a new deletion peak containing the HLA-A gene in squamous cell lung cancer.

A framework for identification of actionable cancer genome dependencies in small cell lung cancer.

Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3-7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.

Somatic copy number alterations by whole-exome sequencing implicates YWHAZ and PTK2 in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies.

Germline homozygosity and allelic imbalance of HLA-I are common in esophagogastric adenocarcinoma and impair the repertoire of immunogenic peptides.

BACKGROUND: The individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA) and determines the resulting repertoires of potentially immunogenic peptides. METHODS: HLA genotypes and sequences of either (1) 10 relevant tumor-associated antigens (TAAs) or (2) patient-specific mutation-associated neoantigens (MANAs) were used to predict good-affinity binders using an in silico approach for MHC-binding (www.iedb.org). Imbalanced or lost expression of HLA-I-A/B/C alleles was analyzed by transcriptome sequencing. FluoroSpot assays and TCR sequencing were used to determine peptide-specific T-cell responses. RESULTS: We show that germline homozygosity of HLA-I genes is significantly enriched in EGA patients (n=80) compared with an HLA-matched reference cohort (n=7605). Whereas the overall mutational burden is similar, the repertoire of potentially immunogenic peptides derived from TAAs and MANAs was lower in homozygous patients. Promiscuity of peptides binding to different HLA-I molecules was low for most TAAs and MANAs and in silico modeling of the homozygous to a heterozygous HLA genotype revealed normalized peptide repertoires. Transcriptome sequencing showed imbalanced expression of HLA-I alleles in 75% of heterozygous patients. Out of these, 33% showed complete loss of heterozygosity, whereas 66% had altered expression of only one or two HLA-I molecules. In a FluoroSpot assay, we determined that peptide-specific T-cell responses against NY-ESO-1 are derived from multiple peptides, which often exclusively bind only one HLA-I allele. CONCLUSION: The high frequency of germline homozygosity in EGA patients suggests reduced cancer immunosurveillance leading to an increased cancer risk. Therapeutic targeting of allelic imbalance of HLA-I molecules should be considered in EGA.

Systematic screen for tyrosine kinase rearrangements identifies a novel C6orf204-PDGFRB fusion in a patient with recurrent T-ALL and an associated myeloproliferative neoplasm.

Gene fusions involving the catalytic domain of tyrosine kinases (TKs) are found in a variety of hematological and solid tumor malignancies. Clinically, TK fusions have emerged as prime targets for therapy with small molecule kinase inhibitors. Unfortunately, identification of TK fusions has been hampered by experimental limitations. Here, we developed version 2.0 of a genomically based systematic kinase fusion screen and used it to detect a novel imatinib-sensitive C6orf204-PDGFRB fusion in a patient with precursor T lymphoblastic lymphoma (T-ALL) and an associated myeloproliferative neoplasm with eosinophilia. These data validate the ability of this targeted capture-sequencing approach to detect TK fusion events in small amounts of DNA extracted directly from patient samples.

Reconstructing Phylogenetic Relationship in Bladder Cancer: A Methodological Overview.

Bladder cancer (BC) expresses itself as a highly heterogeneous disease both at the histological and molecular level, often occurring as synchronous or metachronous multifocal disease with high risk of recurrence and potential to metastasize. Multiple sequencing studies focusing on both non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) gave insights into the extent of both inter- and intrapatient heterogeneity, but many questions on clonal evolution in BC remain unanswered. In this review article, we provide an overview over the technical and theoretical concepts linked to reconstructing evolutionary trajectories in BC and propose a set of tools and established software for phylogenetic analysis.