Canine Oncopanel: A capture-based, NGS platform for evaluating the mutational landscape and detecting putative driver mutations in canine cancers.

Canine cancer, a significant cause of mortality in domestic dogs, is a powerful comparative model for human cancers. Revealing genetic alterations driving the oncogenesis of canine cancers holds great potential to deepen our understanding of the cancer biology, guide therapeutic development, and improve cancer management in both dogs and people. Next generation sequencing (NGS) based-diagnostic panels have been routinely used in human oncology for the identification of clinically-actionable mutations, enabling tailored treatments based on the individual's unique mutation profiles. Here, we report the development of a comprehensive canine cancer gene panel, the Canine Oncopanel, using a hybridization capture-based targeted NGS method. The Canine Oncopanel allows deep sequencing of 283 cancer genes and the detection of somatic mutations within these genes. Vigorous optimization was performed to achieve robust, high-standard performance using metrics of similar cancer panels in human oncology as benchmarks. Validation of the Canine Oncopanel on reference tumour samples with known mutations demonstrated that it can detect variants previously identified by alternative methods, with high accuracy and sensitivity. Putative drivers were detected in over 90% of clinical samples, showing high sensitivity. The Canine Oncopanel is suitable to map mutation profiles and identify putative driver mutations across common and rare cancer types in dogs. The data generated by the Canine Oncopanel presents a rich resource of putative oncogenic driver mutations and potential clinically relevant markers, paving the way for personalized diagnostics and precision medicine in canine oncology.

Molecular subtypes in canine hemangiosarcoma reveal similarities with human angiosarcoma.

Angiosarcoma (AS) is a rare neoplasm with limited treatment options and a poor survival rate. Development of effective therapies is hindered by the rarity of this disease. Dogs spontaneously develop hemangiosarcoma (HSA), a common, histologically similar neoplasm. Metastatic disease occurs rapidly and despite chemotherapy, most dogs die several months after diagnosis. These features suggest that HSA might provide a tractable model to test experimental therapies in clinical trials. We previously reported whole exome sequencing of 20 HSA cases. Here we report development of a NGS targeted resequencing panel to detect driver mutations in HSA and other canine tumors. We validated the panel by resequencing the original 20 cases and sequenced 30 additional cases. Overall, we identified potential driver mutations in over 90% of the cases, including well-documented (in human cancers) oncogenic mutations in PIK3CA (46%), PTEN (6%), PLCG1(4%), and TP53 (66%), as well as previously undetected recurrent activating mutations in NRAS (24%). The driver role of these mutations is further demonstrated by augmented downstream signaling crucial to tumor growth. The recurrent, mutually exclusive mutation patterns suggest distinct molecular subtypes of HSA. Driver mutations in some subtypes closely resemble those seen in some AS cases, including NRAS, PLCG1, PIK3CA and TP53. Furthermore, activation of the MAPK and PI3K pathways appear to be key oncogenic mechanisms in both species. Together, these observations suggest that dogs with spontaneous HSA could serve as a useful model for testing the efficacy of targeted therapies, some of which could potentially be of therapeutic value in AS.

Actionable mutations in canine hemangiosarcoma.

BACKGROUND: Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. METHODS: Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. RESULTS AND CONCLUSIONS: Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect.

Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.

Clinical Utility of Next-Generation Sequencing for Oncogenic Mutations in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation.

To determine the association of somatic mutations in acute myeloid leukemia (AML) with risk of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT), we retrospectively studied pre-transplantation genetic profiles obtained from next-generation sequencing of 26 genes in 112 adult patients with AML who underwent alloHSCT. Univariable and multivariable regression analyses were used to assess the association between the presence of a pathogenic mutation and risk of relapse after alloHSCT. Eighty-six percent (96 of 112) of patients had at least 1 pathogenic mutation. Mutations in TP53, WT1, and FLT3-internal tandem duplication (ITD) were associated with an increased risk of relapse after alloHSCT (adjusted hazard ratio [aHR], 2.90; P = .009; aHR, 2.51; P= .02; and aHR, 1.83; P = .07, respectively). DNMT3A mutation in the absence of FLT3-ITD and NPM1 mutations was associated with a lower relapse risk (aHR, .22; P = .04). Comparison of pre-alloHSCT and post-alloHSCT genetic profiles showed clonal evolution in 6 of 6 patients, including acquisition of actionable mutations in 4 patients. In summary, genetic profiling is useful for assessing relapse risk in patients with AML undergoing alloHSCT and may identify patients in need of strategies to reduce this risk. Clonal evolution is present at post-alloHSCT relapse and repeat genetic profiling may uncover acquired actionable mutations.

Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing.

Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two FLT3 internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.

RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.

Using "residual" FNA rinse and body fluid specimens for next-generation sequencing: An institutional experience.

BACKGROUND: Tissue specimens are typically considered optimal for molecular testing; however, in the current era of personalized medicine, cytopathology specimens are increasingly recognized as potential sources for molecular testing. This is often accomplished by using cell block specimens and/or fine-needle aspiration (FNA) smear preparations. In this study, the authors investigated the feasibility, performance, and quality of "residual" FNA rinse and body effusion fluids used for next-generation sequencing (NGS). METHODS: Sequence data were generated from 17 malignancies in 16 patients from 13 FNA (10 lymph nodes, 1 lung, and 2 bone lesions) and 4 effusion (3 pleural and 1 pericardial) specimens. Malignancies included carcinomas (lung, breast, ovarian, and unknown primary), melanoma, and myeloma. Paired NGS testing was performed in 7 patients who had surgical biopsy or cell block specimens available. Routinely processed residual FNA rinse material and body fluids were used for DNA extraction and NGS (targeted gene panel). RESULTS: NGS was successfully performed on all 17 specimens. A significant amount of DNA was obtained from the residual FNA rinse (176.3 ng/µL) compared with the paired cell block slides (10.6 ng/µL). Two of the 10 lung adenocarcinomas (20%) demonstrated epidermal growth factor receptor (EGFR) mutations, including 1 leucine-to-arginine substitution at codon 858 (L858R) in exon 21 and 1 codon 2235_2249 deletion (resulting in an in-frame deletion of 5 amino acids from position 746 to 750 [glutamic acid, leucine, arginine, glutamic acid, and alanine]; E746_A750del) in exon 19. Three KRAS [Kirsten rat sarcoma viral oncogene homolog] mutations, 1 BRAF (v-Raf murine sarcoma viral oncogene homolog B1) mutation, and 1 NRAS (neuroblastoma RAS viral oncogene homolog) mutation were identified in the remaining lung adenocarcinomas. Patients who underwent paired testing demonstrated 100% concordant mutations. CONCLUSIONS: Targeted NGS can be performed on residual FNA rinse and body fluid specimens. This approach is particularly important when a paucicellular cell block or biopsy specimen is encountered. Cancer Cytopathol 2016;124:324-29. © 2015 American Cancer Society.

Off-Target V(D)J Recombination Drives Lymphomagenesis and Is Escalated by Loss of the Rag2 C Terminus.

Genome-wide analysis of thymic lymphomas from Tp53(-/-) mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification.

RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of 'alternative NHEJ'.

DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

An unbiased method for detection of genome-wide off-target effects in cell lines treated with zinc finger nucleases.

We describe a method for detecting and validating genomic aberrations arising from cell lines exposed to zinc finger nucleases (ZFNs), an important reagent used for targeted genome modifications. This method makes use of cloned cell lines, an approach that adds power when testing variables that may affect gene correction efficiency and evaluating potential side effects on a genome-wide scale. After cell treatment, the genomic DNA isolation method, as described, is ideal for high-resolution array comparative genomic hybridization (aCGH) and quantitative PCR. Guidelines for aCGH analysis and calling significant copy number variations (CNVs) for validation by qPCR are also discussed. Using this method, we describe a novel ZFN-associated chromosome 4 copy number variation (CNV) attributable to a predicted ZFN off-target cleavage site found within the CNV.

V(D)J Recombination: Mechanism, Errors, and Fidelity.

V(D)J recombination, the mechanism responsible for generating antigen receptor diversity, has the potential to generate aberrant DNA rearrangements in developing lymphocytes. Indeed, the recombinase has been implicated in several different kinds of errors leading to oncogenic transformation. Here we review the basic aspects of V(D)J recombination, mechanisms underlying aberrant DNA rearrangements, and the types of aberrant events uncovered in recent genomewide analyses of lymphoid neoplasms.

Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage.

DNA double-strand breaks (DSBs) are common lesions that continually threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including cell death. Misrepair is also fraught with danger, especially inappropriate end-joining events, which commonly underlie oncogenic transformation and can scramble the genome. Canonically, cells employ two basic mechanisms to repair DSBs: homologous recombination (HR) and the classical nonhomologous end-joining pathway (cNHEJ). More recent experiments identified a highly error-prone NHEJ pathway, termed alternative NHEJ (aNHEJ), which operates in both cNHEJ-proficient and cNHEJ-deficient cells. aNHEJ is now recognized to catalyze many genome rearrangements, some leading to oncogenic transformation. Here, we review the mechanisms of cNHEJ and aNHEJ, their interconnections with the DNA damage response (DDR), and the mechanisms used to determine which of the three DSB repair pathways is used to heal a particular DSB. We briefly review recent clinical applications involving NHEJ and NHEJ inhibitors.

RAG2's acidic hinge restricts repair-pathway choice and promotes genomic stability.

V(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high-fidelity classical nonhomologous end-joining (cNHEJ) machinery. Previous studies implicated the recombination-activating gene (RAG)/DNA postcleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here, we report that RAG2's "acidic hinge," previously of unknown function, is critical for several key steps. Mutations that reduce the hinge's negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge's negative charge, permit aNHEJ, and diminish genomic integrity.

The RAG2 C-terminus and ATM protect genome integrity by controlling antigen receptor gene cleavage.

Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of leukaemia and lymphoma. Nonetheless, mistakes can happen, leading to the generation of aberrant rearrangements, such as Tcra/d-Igh inter-locus translocations that are a hallmark of ataxia telangiectasia-mutated (ATM) deficiency. Current evidence indicates that these translocations arise from the persistence of unrepaired breaks converging at different stages of thymocyte differentiation. Here we show that a defect in feedback control of RAG2 activity gives rise to bi-locus breaks and damage on Tcra/d and Igh in the same T cell at the same developmental stage, which provides a direct mechanism for generating these inter-locus rearrangements. Both the RAG2 C-terminus and ATM prevent bi-locus RAG-mediated cleavage through modulation of three-dimensional conformation (higher-order loops) and nuclear organization of the two loci. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability.

A streamlined method for detecting structural variants in cancer genomes by short read paired-end sequencing.

Defining the architecture of a specific cancer genome, including its structural variants, is essential for understanding tumor biology, mechanisms of oncogenesis, and for designing effective personalized therapies. Short read paired-end sequencing is currently the most sensitive method for detecting somatic mutations that arise during tumor development. However, mapping structural variants using this method leads to a large number of false positive calls, mostly due to the repetitive nature of the genome and the difficulty of assigning correct mapping positions to short reads. This study describes a method to efficiently identify large tumor-specific deletions, inversions, duplications and translocations from low coverage data using SVDetect or BreakDancer software and a set of novel filtering procedures designed to reduce false positive calls. Applying our method to a spontaneous T cell lymphoma arising in a core RAG2/p53-deficient mouse, we identified 40 validated tumor-specific structural rearrangements supported by as few as 2 independent read pairs.

The RAG2 C terminus suppresses genomic instability and lymphomagenesis.

Misrepair of DNA double-strand breaks produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans and in mice. Defects in DNA damage response factors such as ataxia telangiectasia mutated (ATM) protein and combined deficiencies in classical non-homologous end joining and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis. Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical non-homologous end joining for proper repair, roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 carboxy (C) terminus, although dispensable for recombination, is critical for maintaining genomic stability. Thymocytes from 'core' Rag2 homozygotes (Rag2(c/c) mice) show dramatic disruption of Tcrα/δ locus integrity. Furthermore, all Rag2(c/c) p53(-/-) mice, unlike Rag1(c/c) p53(-/-) and p53(-/-) animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications and deletions involving the Tcrα/δ and Igh loci. We also find these features in lymphomas from Atm(-/-) mice. We show that, like ATM-deficiency, core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar 'end release/end persistence' mechanisms underlie genomic instability and lymphomagenesis in Rag2(c/c) p53(-/-) and Atm(-/-) mice.

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways.

V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC's ability to restrict repair pathway choice.

Base flipping in V(D)J recombination: insights into the mechanism of hairpin formation, the 12/23 rule, and the coordination of double-strand breaks.

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position -1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex.

Recent insights into the formation of RAG-induced chromosomal translocations.

Chromosomal translocations are found in many types of tumors, where they may be either a cause or a result of malignant transformation. In lymphoid neoplasms, however, it is dear that pathogenesis is initiated by any of a number of recurrent DNA rearrangements. These particular translocations typically place an oncogene under the regulatory control of an Ig or TCR gene promoter, dysregulating cell growth, differentiation, or apoptosis. Given that physiological DNA rearrangements (V(D)J and class switch recombination) are integral to lymphocyte development, it is critical to understand how genomic stability is maintained during these processes. Recent advances in our understanding of DNA damage signaling and repair have provided clues to the kinds of mechanisms that lead to V(D)J-mediated translocations. In turn, investigations into the regulation of V(D)J joining have illuminated a formerly obscure pathway of DNA repair known as alternative NHEJ, which is error-prone and frequently involved in translocations. In this chapter we consider recent advances in our understanding of the functions of the RAG proteins, RAG interactions with DNA repair pathways, damage signaling and chromosome biology, all of which shed light on how mistakes at different stages of V(D)J recombination might lead to leukemias and lymphomas.