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Bladder Cancer (BLCa) inter-patient heterogeneity is the primary cause of treatment failure, suggesting that patients could benefit from a more personalized treatment approach. Patient-derived organoids (PDOs) have been successfully used as a functional model for predicting drug response in different cancers. In our study, we establish PDO cultures from different BLCa stages and grades. PDOs preserve the histological and molecular heterogeneity of the parental tumors, including their multiclonal genetic landscapes, and consistently share key genetic alterations, mirroring tumor evolution in longitudinal sampling. Our drug screening pipeline is implemented using PDOs, testing standard-of-care and FDA-approved compounds for other tumors. Integrative analysis of drug response profiles with matched PDO genomic analysis is used to determine enrichment thresholds for candidate markers of therapy response and resistance. Finally, by assessing the clinical history of longitudinally sampled cases, we can determine whether the disease clonal evolution matched with drug response., (© 2023. The Author(s).)

Primary prostate cancer (PCa) can show marked molecular heterogeneity. However, systematic analyses comparing primary PCa and matched metastases in individual patients are lacking. We aimed to address the molecular aspects of metastatic progression while accounting for the heterogeneity of primary PCa. In this pilot study, we collected 12 radical prostatectomy (RP) specimens from men who subsequently developed metastatic castration-resistant prostate cancer (mCRPC). We used histomorphology (Gleason grade, focus size, stage) and immunohistochemistry (IHC) (ERG and p53) to identify independent tumors and/or distinct subclones of primary PCa. We then compared molecular profiles of these primary PCa areas to matched metastatic samples using whole-exome sequencing (WES) and amplicon-based DNA and RNA sequencing. Based on combined pathology and molecular analysis, seven (58%) RP specimens harbored monoclonal and topographically continuous disease, albeit with some degree of intratumor heterogeneity; four (33%) specimens showed true multifocal disease; and one displayed monoclonal disease with discontinuous topography. Early (truncal) events in primary PCa included SPOP p.F133V (one patient), BRAF p.K601E (one patient), and TMPRSS2:ETS rearrangements (eight patients). Activating AR alterations were seen in nine (75%) mCRPC patients, but not in matched primary PCa. Hotspot TP53 mutations, found in metastases from three patients, were readily present in matched primary disease. Alterations in genes encoding epigenetic modifiers were observed in several patients (either shared between primary foci and metastases or in metastatic samples only). WES-based phylogenetic reconstruction and/or clonality scores were consistent with the index focus designated by pathology review in six out of nine (67%) cases. The three instances of discordance pertained to monoclonal, topographically continuous tumors, which would have been considered as unique disease in routine practice. Overall, our results emphasize pathologic and molecular heterogeneity of primary PCa, and suggest that comprehensive IHC-assisted pathology review and genomic analysis are highly concordant in nominating the 'index' primary PCa area. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Pan-cancer studies sketched the genomic landscape of the tumor types spectrum. We delineated the purity- and ploidy-adjusted allele-specific profiles of 4,950 patients across 27 tumor types from the Cancer Genome Atlas (TCGA). Leveraging allele-specific data, we reclassified as loss of heterozygosity (LOH) 9% and 7% of apparent copy-number wild-type and gain calls, respectively, and overall observed more than 18 million allelic imbalance somatic events at the gene level. Reclassification of copy-number events revealed associations between driver mutations and LOH, pointing out the timings between the occurrence of point mutations and copy-number events. Integrating allele-specific genomics and matched transcriptomics, we observed that allele-specific gene status is relevant in the regulation of TP53 and its targets. Further, we disclosed the role of copy-neutral LOH in the impairment of tumor suppressor genes and in disease progression. Our results highlight the role of LOH in cancer and contribute to the understanding of tumor progression., Competing Interests: Declaration of interests The authors declare no competing interests. Co-authors D.P. and A.L. are currently at Fondazione Bruno Kessler, Trento, Italy and Biotechnology Research and Innovation Center, University of Copenhagen, Denmark., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutual Exclusivity analysis of genomic aberrations contributes to the exploration of potential synthetic lethal (SL) relationships thus guiding the nomination of specific cancer cells vulnerabilities. When multiple classes of genomic aberrations and large cohorts of patients are interrogated, exhaustive genome-wide analyses are not computationally feasible with commonly used approaches. Here we present Fast Mutual Exclusivity (FaME), an algorithm based on matrix multiplication that employs a logarithm-based implementation of the Fisher's exact test to achieve fast computation of genome-wide mutual exclusivity tests; we show that brute force testing for mutual exclusivity of hundreds of millions of aberrations combinations can be performed in few minutes. We applied FaME to allele-specific data from whole exome experiments of 27 TCGA studies cohorts, detecting both mutual exclusivity of point mutations, as well as allele-specific copy number signals that span sets of contiguous cytobands. We next focused on a case study involving the loss of tumor suppressors and druggable genes while exploiting an integrated analysis of both public cell lines loss of function screens data and patients' transcriptomic profiles. FaME algorithm implementation as well as allele-specific analysis output are publicly available at https://github.com/demichelislab/FaME., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Loss of androgen receptor (AR) signaling dependence occurs in approximately 15%-20% of advanced treatment-resistant prostate cancers, and this may manifest clinically as transformation from a prostate adenocarcinoma histology to a castration-resistant neuroendocrine prostate cancer (CRPC-NE). The diagnosis of CRPC-NE currently relies on a metastatic tumor biopsy, which is invasive for patients and sometimes challenging to diagnose due to morphologic heterogeneity. By studying whole-exome sequencing and whole-genome bisulfite sequencing of cell free DNA (cfDNA) and of matched metastatic tumor biopsies from patients with metastatic prostate adenocarcinoma and CRPC-NE, we identified CRPC-NE features detectable in the circulation. Overall, there was markedly higher concordance between cfDNA and biopsy tissue genomic alterations in patients with CRPC-NE compared with castration-resistant adenocarcinoma, supporting greater intraindividual genomic consistency across metastases. Allele-specific copy number and serial sampling analyses allowed for the detection and tracking of clonal and subclonal tumor cell populations. cfDNA methylation was indicative of circulating tumor content fraction, reflective of methylation patterns observed in biopsy tissues, and was capable of detecting CRPC-NE-associated epigenetic changes (e.g., hypermethylation of ASXL3 and SPDEF; hypomethylation of INSM1 and CDH2). A targeted set combining genomic (TP53, RB1, CYLD, AR) and epigenomic (hypo- and hypermethylation of 20 differential sites) alterations applied to ctDNA was capable of identifying patients with CRPC-NE.

Background: Interrogation of whole exome and targeted sequencing NGS data is rapidly becoming a preferred approach for the exploration of large cohorts in the research setting and importantly in the context of precision medicine. Single-base and genomic region level data retrieval and processing still constitute major bottlenecks in NGS data analysis. Fast and scalable tools are hence needed., Results: PaCBAM is a command line tool written in C and designed for the characterization of genomic regions and single nucleotide positions from whole exome and targeted sequencing data. PaCBAM computes depth of coverage and allele-specific pileup statistics, implements a fast and scalable multi-core computational engine, introduces an innovative and efficient on-the-fly read duplicates filtering strategy and provides comprehensive text output files and visual reports. We demonstrate that PaCBAM exploits parallel computation resources better than existing tools, resulting in important reductions of processing time and memory usage, hence enabling an efficient and fast exploration of large datasets., Conclusions: PaCBAM is a fast and scalable tool designed to process genomic regions from NGS data files and generate coverage and pileup comprehensive statistics for downstream analysis. The tool can be easily integrated in NGS processing pipelines and is available from Bitbucket and Docker/Singularity hubs.

Motivation: Tumor purity (TP) is the proportion of cancer cells in a tumor sample. TP impacts on the accurate assessment of molecular and genomics features as assayed with NGS approaches. State-of-the-art tools mainly rely on somatic copy-number alterations (SCNA) to quantify TP and therefore fail when a tumor genome is nearly euploid, i.e. 'non-aberrant' in terms of identifiable SCNAs., Results: We introduce a computational method, tumor purity estimation from single-nucleotide variants (SNVs), which derives TP from the allelic fraction distribution of SNVs. On more than 7800 whole-exome sequencing data of TCGA tumor samples, it showed high concordance with a range of TP tools (Spearman's correlation between 0.68 and 0.82; >9 SNVs) and rescued TP estimates of 1, 194 samples (15%) pan-cancer., Availability and Implementation: TPES is available as an R package on CRAN and at https://bitbucket.org/l0ka/tpes.git., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Heterogeneity in the genomic landscape of metastatic prostate cancer has become apparent through several comprehensive profiling efforts, but little is known about the impact of this heterogeneity on clinical outcome. Here, we report comprehensive genomic and transcriptomic analysis of 429 patients with metastatic castration-resistant prostate cancer (mCRPC) linked with longitudinal clinical outcomes, integrating findings from whole-exome, transcriptome, and histologic analysis. For 128 patients treated with a first-line next-generation androgen receptor signaling inhibitor (ARSI; abiraterone or enzalutamide), we examined the association of 18 recurrent DNA- and RNA-based genomic alterations, including androgen receptor ( AR ) variant expression, AR transcriptional output, and neuroendocrine expression signatures, with clinical outcomes. Of these, only RB1 alteration was significantly associated with poor survival, whereas alterations in RB1 , AR , and TP53 were associated with shorter time on treatment with an ARSI. This large analysis integrating mCRPC genomics with histology and clinical outcomes identifies RB1 genomic alteration as a potent predictor of poor outcome, and is a community resource for further interrogation of clinical and molecular associations., Competing Interests: Conflict of interest statement: W.A. has consulted for Clovis Oncology, Janssen, ORIC Pharmaceuticals, and MORE Health; has received honorarium from Caret Healthcare; has received travel funds from Clovis Oncology, ORIC Pharmaceuticals, and GlaxoSmithKline; and has received research funding from AstraZeneca, Clovis Oncology, Zenith Epigenetics, and GlaxoSmithKline. G.H., received research funding from Janssen. J.A. is currently employed at AstraZeneca. E.M.V.A. has consulted for Tango Therapeutics, Genome Medical, Invitae, Illumina, Foresite Capital, and Dynamo; has received research support from Novartis and BMS; has equity in Tango Therapeutics, Genome Medical, Syapse, and Microsoft; has received travel reimbursement from Roche/Genentech; and is a coinventor of institutional patents filed on ERCC2 mutations and chemotherapy response, chromatin mutations and immunotherapy response, and methods for clinical interpretation. H.B. has received research funding from Millennium Pharmaceuticals, AbbVie/Stemcentrx, Astellas, Janssen, and Eli Lilly; and is a consultant/advisor for Sanofi Genzyme and Janssen. S.T. is a coinventor for University of Michigan patents on ETS fusion genes, and diagnostic field of use licensed to Hologic/Gen-Probe Inc., which has sublicensed rights to Roche/Ventana Medical Systems; is a consultant for and has received honoraria from Janssen, AbbVie, Sanofi, Almac Diagnostics, and Astellas/Medivation; has performed sponsored research from Astellas/Medivation and GenomeDx; and is an equity holder in and employee of Strata Oncology. J.M. has consulted for AstraZeneca and Janssen; and has received travel support or speaker fees from Astellas, AstraZeneca, Sanofi, and IPSEN. D.B. has received honoraria and travel support from Janssen. H.I.S. has consulted for Astellas, Ferring Pharmaceuticals, Janssen Biotech, Janssen Research and Development, Sanofi Aventis, Clovis Oncology, Merck, and WCG Oncology; is a member of the board of directors for Asterias Biotherapeutics; and has received research support from Illumina Inc., Innocrin, and Janssen. P.W.K. is a board member for Context Therapeutics LLC, has consulted for BIND Biosciences, BN Immunotherapeutics, DRGT, GE Healthcare, Janssen Pharmaceutica, Metamark, New England Research Institutes, OncoCellMDx, Progenity, Sanofi-Aventis, Seer Biosciences Inc., Tarveda Therapeutics, and Thermo Fisher Scientific; serves on data safety monitoring boards for Genentech/Roche and Merck & Co.; and has investment interest in Context Therapeutics, DRGT, Seer Biosciences, Placon, and Tarveda Therapeutics. M.-E.T. has consuled for Janssen. J.S.d. is an advisory board member for AstraZeneca, Astellas, Bayer, Boehringer Ingelheim, Genentech/Roche, Genmab, GSK, Janssen, Merck Serono, Merck Sharp & Dohme, Menarini/Silicon Biosystems, Orion, Pfizer, Sanofi Aventis, and Taiho; and his institution has received funding or other support from AstraZeneca, Astellas, Bayer, Genentech, GSK, Janssen, Merck Serono, MSD, Menarini/Silicon Biosystems, Orion, Sanofi Aventis, and Taiho. P.S.N. is a compensated advisor to Janssen, Astellas, and Roche. M.A.R. is a coinventor on a patent for Gene Fusion Prostate Cancer (Harvard/Michigan) and EZH2 (Michigan) in the area of diagnostics and therapeutics; is a coinventor on a patent filed by Cornell University on AURKA and SPOP mutations in the field of prostate cancer diagnostics; receives royalties on licensing agreements for these inventions; and receives research support from Janssen, Eli Lilly, Millenium, and Sanofi-Aventis. C.L.S. serves on the board of directors of Novartis; is a cofounder of ORIC Pharmaceuticals and coinventor of enzalutamide and apalutamide; is a science advisor to Agios, Beigene, Blueprint, Column Group, Foghorn, Housey Pharma, Nextech, KSQ, Petra, and PMV; and is a cofounder of Seragon, purchased by Genentech/Roche in 2014., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: Phylogenetic studies of bacteria have been based so far either on a single gene (usually the 16S rRNA) or on concatenated housekeeping genes. For what concerns the genus Mycobacterium these approaches support the separation of rapidly and slowly growing species and the clustering of most species in well-defined phylogenetic groups. The advent of high-throughput shotgun sequencing leads us to revise conventional taxonomy of mycobacteria on the light of genomic data. For this purpose we investigated 88 newly sequenced species in addition to 60 retrieved from GenBank and used the Average Nucleotide Identity pairwise scores to reconstruct phylogenetic relationships within this genus., Results: Our analysis confirmed the separation of slow and rapid growers and the intermediate position occupied by the M. terrae complex. Among the rapid growers, the species of the M. chelonae-abscessus complex belonged to the most ancestral cluster. Other major clades of rapid growers included the species related to M. fortuitum and M. smegmatis and a large grouping containing mostly environmental species rarely isolated from humans. The members of the M. terrae complex appeared as the most ancestral slow growers. Among slow growers two deep branches led to the clusters of species related to M. celatum and M. xenopi and to a large group harboring most of the species more frequently responsible of disease in humans, including the major pathogenic mycobacteria (M. tuberculosis, M. leprae, M. ulcerans). The species previously grouped in the M. simiae complex were allocated in a number of sub-clades; of them, only the one including the species M. simiae identified the real members of this complex. The other clades included also species previously not considered related to M. simiae. The ANI analysis, in most cases supported by Genome to Genome Distance and by Genomic Signature-Delta Difference, showed that a number of species with standing in literature were indeed synonymous., Conclusions: Genomic data revealed to be much more informative in comparison with phenotype. We believe that the genomic revolution enabled by high-throughput shotgun sequencing should now be considered in order to revise the conservative approaches still informing taxonomic sciences., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Precision medicine is an approach that takes into account the influence of individuals' genes, environment, and lifestyle exposures to tailor interventions. Here, we describe the development of a robust precision cancer care platform that integrates whole-exome sequencing with a living biobank that enables high-throughput drug screens on patient-derived tumor organoids. To date, 56 tumor-derived organoid cultures and 19 patient-derived xenograft (PDX) models have been established from the 769 patients enrolled in an Institutional Review Board-approved clinical trial. Because genomics alone was insufficient to identify therapeutic options for the majority of patients with advanced disease, we used high-throughput drug screening to discover effective treatment strategies. Analysis of tumor-derived cells from four cases, two uterine malignancies and two colon cancers, identified effective drugs and drug combinations that were subsequently validated using 3-D cultures and PDX models. This platform thereby promotes the discovery of novel therapeutic approaches that can be assessed in clinical trials and provides personalized therapeutic options for individual patients where standard clinical options have been exhausted. Significance: Integration of genomic data with drug screening from personalized in vitro and in vivo cancer models guides precision cancer care and fuels next-generation research. Cancer Discov; 7(5); 462-77. ©2017 AACR. See related commentary by Picco and Garnett, p. 456 This article is highlighted in the In This Issue feature, p. 443 ., (©2017 American Association for Cancer Research.)

Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

Autism spectrum disorders (ASD) are characterized by a high degree of genetic heterogeneity. Genomic studies identified common pathological processes underlying the heterogeneous clinical manifestations of ASD, and transcriptome analyses revealed that gene networks involved in synapse development, neuronal activity, and immune function are deregulated in ASD. Mouse models provide unique tools to investigate the neurobiological basis of ASD; however, a comprehensive approach to identify transcriptional abnormalities in different ASD models has never been performed. Here we used two well-recognized ASD mouse models, BTBR T(+) Itpr3 (tf) /J (BTBR) and Engrailed-2 knockout (En2 (-/-)), to identify conserved ASD-related molecular signatures. En2 (-/-) mice bear a mutation within the EN2 transcription factor homeobox, while BTBR is an inbred strain with unknown genetic defects. Hippocampal RNA samples from BTBR, En2 (-/-) and respective control (C57Bl/6J and En2 (+/+)) adult mice were assessed for differential gene expression using microarrays. A total of 153 genes were similarly deregulated in the BTBR and En2 (-/-) hippocampus. Mouse phenotype and gene ontology enrichment analyses were performed on BTBR and En2 (-/-) hippocampal differentially expressed genes (DEGs). Pathways represented in both BTBR and En2 (-/-) hippocampal DEGs included abnormal behavioral response and chemokine/MAP kinase signaling. Genes involved in abnormal function of the immune system and abnormal synaptic transmission/seizures were significantly represented among BTBR and En2 (-/-) DEGs, respectively. Interestingly, both BTBR and En2 (-/-) hippocampal DEGs showed a significant enrichment of ASD and schizophrenia (SCZ)-associated genes. Specific gene sets were enriched in the two models: microglial genes were significantly enriched among BTBR DEGs, whereas GABAergic/glutamatergic postsynaptic genes, FMRP-interacting genes and epilepsy-related genes were significantly enriched among En2 (-/-) DEGs. Weighted correlation network analysis (WGCNA) performed on BTBR and En2 (-/-) hippocampal transcriptomes together identified six modules significantly enriched in ASD-related genes. Each of these modules showed a specific enrichment profile in neuronal and glial genes, as well as in genes associated to ASD comorbidities such as epilepsy and SCZ. Our data reveal significant transcriptional similarities and differences between the BTBR and En2 (-/-) hippocampus, indicating that transcriptome analysis of ASD mouse models may contribute to identify novel molecular targets for pharmacological studies.

Fourteen mycobacterial strains isolated from pulmonary samples of independent patients in the state of Pará (Brazil), and three strains isolated in Italy, were characterized using a polyphasic approach. Thorough genetic investigation, including whole-genome sequencing, demonstrated that the strains belong to the M. simiae complex, being most closely related to Mycobacterium interjectum. For 14 of the strains, evidence emerged supporting their inclusion in a previously unreported species of the genus Mycobacterium, for which the name Mycobacterium paraense sp. nov. is proposed (type strain, IEC26(T) = DSM 46749(T) = CCUG 66121(T)). The novel species is characterized by slow growth, unpigmented or pale yellow scotochromogenic colonies, and a HPLC mycolic acid profile different from other known mycobacteria. In different genetic regions, high sequence microheterogeneity was detected., (© 2015 IUMS.)