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Purpose: To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs).Experimental Design: All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs.Results: Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry.Conclusions: These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582–91. ©2016 AACR.

Supplemental Methods. Table S1. Frequency of hematopoietic subsets in PBMC at all time points for patient samples used for in vitro immunogenicity experiments Table S2: Clinical characteristics of follicular lymphoma cohorts Table S3. Amino acid changes encoded by somatic mutations identified in candidate genes in follicular lymphoma cohort Table S4. Patient HLA class I alleles for in vitro immunogenicity cohort Figure S1. The number of mutant peptides predicted to bind patient-specific HLA class I molecules in the sequencing cohort. Figure S2. Clinical timeline for 13-patient cohort selected for in vitro immunogenicity studies. Figure S3. CD8 status of mutant peptide-reactive T cells.

This table shows the recurrent mutations that were identified as well as the gains/losses at previously reported chromosomal segments in the 39 DAWTs that were selected for in-depth analysis

This table shows all TP53 variant that were identified by Whole Genome or Exome Sequencing in all DAWTs included in this study

This table shows the sequences of the probes used for MLPA

Purpose: A fundamental challenge in the era of next-generation sequencing (NGS) is to design effective treatments tailored to the mutational profiles of tumors. Many newly discovered cancer mutations are difficult to target pharmacologically; however, T-cell–based therapies may provide a valuable alternative owing to the exquisite sensitivity and specificity of antigen recognition. To explore this concept, we assessed the immunogenicity of a panel of genes that are common sites of driver mutations in follicular lymphoma, an immunologically sensitive yet currently incurable disease.Experimental Design: Exon capture and NGS were used to interrogate tumor samples from 53 patients with follicular lymphoma for mutations in 10 frequently mutated genes. For 13 patients, predicted mutant peptides and proteins were evaluated for recognition by autologous peripheral blood T cells after in vitro priming.Results: Mutations were identified in 1–5 genes in 81% (43/53) of tumor samples. Autologous, mutation-specific CD8+ T cells were identified in 23% (3/13) of evaluated cases. T-cell responses were directed toward putative driver mutations in CREBBP and MEF2B. Responding T cells showed exquisite specificity for mutant versus wild-type proteins and recognized lymphoma cells expressing the appropriate mutations. Responding T cells appeared to be from the naïve repertoire, as they were found at low frequencies and only at single time points in each patient.Conclusions: Patients with follicular lymphoma harbor rare yet functionally competent CD8+ T cells specific for recurrent mutations. Our results support the concept of using NGS to design individualized immunotherapies targeting common driver mutations in follicular lymphoma and other malignancies. Clin Cancer Res; 22(9); 2226–36. ©2015 AACR.

This table shows the clinical and pathological characteristics of all 118 DAWTs

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic. Marra and colleagues describe POG570, a pan-cancer, whole-genome, transcriptome and clinical dataset stressing the molecular interactions in advanced and post-therapy cancer patients.

Cervical cancer is the most common cancer affecting sub-Saharan African women and is prevalent among HIV-positive (HIV+) individuals. No comprehensive profiling of cancer genomes, transcriptomes or epigenomes has been performed in this population thus far. We characterized 118 tumors from Ugandan patients, of whom 72 were HIV+, and performed extended mutation analysis on an additional 89 tumors. We detected human papillomavirus (HPV)-clade-specific differences in tumor DNA methylation, promoter- and enhancer-associated histone marks, gene expression and pathway dysregulation. Changes in histone modification at HPV integration events were correlated with upregulation of nearby genes and endogenous retroviruses.

We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A. DNA copy number changes resulted in recurrent 1q gain, MYCN amplification, LIN28B gain, and MIRLET7A loss. Unexpected germline variants involved PALB2 and CHEK2. Integrated analyses support two major classes of genetic changes that preserve the progenitor state and/or interrupt normal development.

Retinitis pigmentosa (RP) is an inherited retinal degeneration (RD) that leads to blindness for which no treatment is available. RP is frequently caused by mutations inRhodopsin; in some animal models, RD is exacerbated by light. Valproic acid (VPA) is a proposed treatment for RP and other neurodegenerative disorders, with a phase II trial for RP under way. However, the therapeutic mechanism is unclear, with minimal research supporting its use in RP. We investigated the effects of VPA onXenopus laevismodels of RP expressing human P23H, T17M, T4K, and Q344ter rhodopsins, which are associated with RP in humans. VPA ameliorated RD associated with P23H rhodopsin and promoted clearing of mutant rhodopsin from photoreceptors. The effect was equal to that of dark rearing, with no additive effect observed. Rescue of visual function was confirmed by electroretinography. In contrast, VPA exacerbated RD caused by T17M rhodopsin in light, but had no effect in darkness. Effects in T4K and Q344ter rhodopsin models were also negative. These effects of VPA were paralleled by treatment with three additional histone deacetylase (HDAC) inhibitors, but not other antipsychotics, chemical chaperones, or VPA structural analogues. In WT retinas, VPA treatment increased histone H3 acetylation. In addition, electron microscopy showed increased autophagosomes in rod inner segments with HDAC inhibitor (HDACi) treatment, potentially linking the therapeutic effects in P23H rhodopsin animals and negative effects in other models with autophagy. Our results suggest that the success or failure of VPA treatment is dependent on genotype and that HDACi treatment is contraindicated for some RP cases.SIGNIFICANCE STATEMENTRetinitis pigmentosa (RP) is an inherited, degenerative retinal disease that leads to blindness for which no therapy is available. We determined that valproic acid (VPA), currently undergoing a phase II trial for RP, has both beneficial and detrimental effects in animal models of RP depending on the underlying disease mechanism and that both effects are due to histone deacetylase (HDAC) inhibition possibly linked to autophagy regulation. Off-label use of VPA and other HDAC inhibitors for the treatment of RP should be limited to the research setting until this effect is understood and can be predicted. Our study suggests that, unless genotype is accounted for, clinical trials for RP treatments may give negative results due to multiple disease mechanisms with differential responses to therapeutic interventions.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic.

Purpose: A fundamental challenge in the era of next-generation sequencing (NGS) is to design effective treatments tailored to the mutational profiles of tumors. Many newly discovered cancer mutations are difficult to target pharmacologically; however, T-cell–based therapies may provide a valuable alternative owing to the exquisite sensitivity and specificity of antigen recognition. To explore this concept, we assessed the immunogenicity of a panel of genes that are common sites of driver mutations in follicular lymphoma, an immunologically sensitive yet currently incurable disease. Experimental Design: Exon capture and NGS were used to interrogate tumor samples from 53 patients with follicular lymphoma for mutations in 10 frequently mutated genes. For 13 patients, predicted mutant peptides and proteins were evaluated for recognition by autologous peripheral blood T cells after in vitro priming. Results: Mutations were identified in 1–5 genes in 81% (43/53) of tumor samples. Autologous, mutation-specific CD8+ T cells were identified in 23% (3/13) of evaluated cases. T-cell responses were directed toward putative driver mutations in CREBBP and MEF2B. Responding T cells showed exquisite specificity for mutant versus wild-type proteins and recognized lymphoma cells expressing the appropriate mutations. Responding T cells appeared to be from the naïve repertoire, as they were found at low frequencies and only at single time points in each patient. Conclusions: Patients with follicular lymphoma harbor rare yet functionally competent CD8+ T cells specific for recurrent mutations. Our results support the concept of using NGS to design individualized immunotherapies targeting common driver mutations in follicular lymphoma and other malignancies. Clin Cancer Res; 22(9); 2226–36. ©2015 AACR.

Oncogenic "driver" mutations are theoretically attractive targets for the immunotherapy of lymphoid cancers, yet the proportion that can be recognized by T cells remains poorly defined. To address this issue without any confounding effects of the patient's immune system, we assessed T cells from 19 healthy donors for recognition of three common driver mutations in lymphoma: MYD88L265P, EZH2Y641F , and EZH2Y641N . Donors collectively expressed the 10 most prevalent HLA class I alleles, including HLA-A*02:01. Peripheral blood T cells were primed with peptide-loaded dendritic cells (DC), and reactive T cells were assessed for recognition of naturally processed mutant versus wild type full-length proteins. After screening three driver mutations across 17-26 HLA class I alleles and 3 × 106-3 × 107 T cells per donor, we identified CD4+ T cells against EFISENCGEII from EZH2Y641N (presented by HLA-DRB1*13:02) and CD8+ T cells against RPIPIKYKA from MYD88L265P (presented by HLA-B*07:02). We failed to detect RPIPIKYKA-specific T cells in seven other HLA-B*07:02-positive donors, including two lymphoma patients. Thus, healthy donors harbor T cells specific for common driver mutations in lymphoma. However, such responses appear to be rare due to the combined limitations of antigen processing, HLA restriction, and T cell repertoire size, highlighting the need for highly individualized approaches for selecting targets.

We report a case of early-onset pancreatic ductal adenocarcinoma in a patient harboring biallelic MUTYH germline mutations, whose tumor featured somatic mutational signatures consistent with defective MUTYH-mediated base excision repair and the associated driver KRAS transversion mutation p.Gly12Cys. Analysis of an additional 730 advanced cancer cases (N = 731) was undertaken to determine whether the mutational signatures were also present in tumors from germline MUTYH heterozygote carriers or if instead the signatures were only seen in those with biallelic loss of function. We identified two patients with breast cancer each carrying a pathogenic germline MUTYH variant with a somatic MUTYH copy loss leading to the germline variant being homozygous in the tumor and demonstrating the same somatic signatures. Our results suggest that monoallelic inactivation of MUTYH is not sufficient for C:G>A:T transversion signatures previously linked to MUTYH deficiency to arise (N = 9), but that biallelic complete loss of MUTYH function can cause such signatures to arise even in tumors not classically seen in MUTYH-associated polyposis (N = 3). Although defective MUTYH is not the only determinant of these signatures, MUTYH germline variants may be present in a subset of patients with tumors demonstrating elevated somatic signatures possibly suggestive of MUTYH deficiency (e.g., COSMIC Signature 18, SigProfiler SBS18/SBS36, SignatureAnalyzer SBS18/SBS36).

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Childhood cancers represent distinct clinical entities, often with unique genomic alterations and therapeutic responses that differ from cancers arising in adults. Pediatric acute myeloid

Summary Type IV pili (T4P) are critical to virulence for Vibrio cholerae and other bacterial pathogens. Among their diverse functions, T4P mediate microcolony formation, which protects the bacteria from host defences and concentrates secreted toxins. The T4P of the two V. cholerae O1 disease biotypes, classical and El Tor, share 81% identity in their TcpA subunits, yet these filaments differ in their interaction patterns as assessed by electron microscopy. To understand the molecular basis for pilus-mediated microcolony formation, we solved a 1.5 A resolution crystal structure of N-terminally truncated El Tor TcpA and compared it with that of classical TcpA. Residues that differ between the two pilins are located on surface-exposed regions of the TcpA subunits. By iteratively changing these non-conserved amino acids in classical TcpA to their respective residues in El Tor TcpA, we identified residues that profoundly affect pilus:pilus interaction patterns and bacterial aggregation. These residues lie on either the protruding d-region of the TcpA subunit or in a cavity between pilin subunits in the pilus filament. Our results support a model whereby pili interact via intercalation of surface protrusions on one filament into depressions between subunits on adjacent filaments as a means to hold V. cholerae cells together in microcolonies.

Meeting abstracts A fundamental challenge in cancer genomics is to design effective, personalized treatments based on the mutational profiles of tumors. Pharmacological targeting of the numerous aberrant pathways found in individual tumors remains exceedingly challenging, but T cell-based therapies

We report the high-quality draft genome sequences of 162 strains of Salmonella enterica subsp. enterica serovar Enteritidis representing diverse phage types and pulsed-field gel electrophoresis (PFGE) profiles. The analysis of these genomes will enable the identification of markers that are useful for differentiating strains of this highly clonal serovar and will provide insights into the evolution, virulence, and epidemiology of the strains.

We report the high-quality draft genome sequences of 162 strains of Salmonella enterica subsp. enterica serovar Enteritidis representing diverse phage types and pulsed-field gel electrophoresis (PFGE) profiles. The analysis of these genomes will enable the identification of markers that are useful for differentiating strains of this highly clonal serovar and will provide insights into the evolution, virulence, and epidemiology of the strains.

Purpose: To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs). Experimental Design: All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs. Results: Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry. Conclusions: These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582–91. ©2016 AACR.

Enterotoxigenic Escherichia coli (ETEC) is a bacterial pathogen that causes diarrhea in children and travelers in developing countries. ETEC adheres to host epithelial cells in the small intestine via a variety of different pili. The CS1 pilus is a prototype for a family of related pili, including the CFA/I pili, present on ETEC and other Gram-negative bacterial pathogens. These pili are assembled by an outer membrane usher protein that catalyzes subunit polymerization via donor strand complementation, in which the N terminus of each incoming pilin subunit fits into a hydrophobic groove in the terminal subunit, completing a β-sheet in the Ig fold. Here we determined a crystal structure of the CS1 major pilin subunit, CooA, to a 1.6-Å resolution. CooA is a globular protein with an Ig fold and is similar in structure to the CFA/I major pilin CfaB. We determined three distinct negative-stain electron microscopic reconstructions of the CS1 pilus and generated pseudoatomic-resolution pilus structures using the CooA crystal structure. CS1 pili adopt multiple structural states with differences in subunit orientations and packing. We propose that the structural perturbations are accommodated by flexibility in the N-terminal donor strand of CooA and by plasticity in interactions between exposed flexible loops on adjacent subunits. Our results suggest that CS1 and other pili of this class are extensible filaments that can be stretched in response to mechanical stress encountered during colonization.

Transgenic models are invaluable tools for researching retinal degenerative disease mechanisms. However, they are time-consuming and expensive to generate and maintain. We have developed an alternative to transgenic rodent models of retinal degeneration using transgenic Xenopus laevis. We have optimized this system to allow rapid analysis of transgene effects in primary transgenic animals, thereby providing an alternative to establishing transgenic lines, and simultaneously allowing rigorous comparisons between the effects of different transgenes.

Transgenic models are invaluable tools for researching retinal degenerative disease mechanisms. However, they are time-consuming and expensive to generate and maintain. We have developed an alternative to transgenic rodent models of retinal degeneration using transgenic Xenopus laevis. We have optimized this system to allow rapid analysis of transgene effects in primary transgenic animals, thereby providing an alternative to establishing transgenic lines, and simultaneously allowing rigorous comparisons between the effects of different transgenes.

Type IV pili (T4P) are critical to virulence for Vibrio cholerae and other bacterial pathogens. Among their diverse functions, T4P mediate microcolony formation, which protects the bacteria from host defences and concentrates secreted toxins. The T4P of the two V. cholerae O1 disease biotypes, classical and El Tor, share 81% identity in their TcpA subunits, yet these filaments differ in their interaction patterns as assessed by electron microscopy. To understand the molecular basis for pilus-mediated microcolony formation, we solved a 1.5 A resolution crystal structure of N-terminally truncated El Tor TcpA and compared it with that of classical TcpA. Residues that differ between the two pilins are located on surface-exposed regions of the TcpA subunits. By iteratively changing these non-conserved amino acids in classical TcpA to their respective residues in El Tor TcpA, we identified residues that profoundly affect pilus:pilus interaction patterns and bacterial aggregation. These residues lie on either the protruding d-region of the TcpA subunit or in a cavity between pilin subunits in the pilus filament. Our results support a model whereby pili interact via intercalation of surface protrusions on one filament into depressions between subunits on adjacent filaments as a means to hold V. cholerae cells together in microcolonies.