Different trajectories of polyploidization shape the genomic landscape of the Brettanomyces bruxellensis yeast species

Polyploidization events are observed across the tree of life and occur in many fungi, plant, and animal species. During evolution, polyploidy is thought to be an important source of speciation and tumorigenesis. However, the origin of polyploid populations is not always clear, and little is known about the precise nature and structure of their complex genome. Using a long-read sequencing strategy, we sequenced 71 strains from the Brettanomyces bruxellensis yeast species, which is found in anthropized environments (e.g., beer, contaminant of wine, kombucha, and ethanol production) and characterized by several polyploid subpopulations. To reconstruct the polyploid genomes, we phased them by using different strategies and found that each subpopulation had a unique polyploidization history with distinct trajectories. The polyploid genomes contain either genetically closely related (with a genetic divergence <1%) or diverged copies (>3%), indicating auto- as well as allopolyploidization events. These latest events have occurred independently with a specific and unique donor in each of the polyploid subpopulations and exclude the known Brettanomyces sister species as possible donors. Finally, loss of heterozygosity events has shaped the structure of these polyploid genomes and underline their dynamics. Overall, our study highlights the multiplicity of the trajectories leading to polyploid genomes within the same species.

Aborting meiosis allows recombination in sterile diploid yeast hybrids

Hybrids between diverged lineages contain novel genetic combinations but an impaired meiosis often makes them evolutionary dead ends. Here, we explore to what extent an aborted meiosis followed by a return-to-growth (RTG) promotes recombination across a panel of 20 Saccharomyces cerevisiae and S. paradoxus diploid hybrids with different genomic structures and levels of sterility. Genome analyses of 275 clones reveal that RTG promotes recombination and generates extensive regions of loss-of-heterozygosity in sterile hybrids with either a defective meiosis or a heavily rearranged karyotype, whereas RTG recombination is reduced by high sequence divergence between parental subgenomes. The RTG recombination preferentially arises in regions with low local heterozygosity and near meiotic recombination hotspots. The loss-of-heterozygosity has a profound impact on sexual and asexual fitness, and enables genetic mapping of phenotypic differences in sterile lineages where linkage analysis would fail. We propose that RTG gives sterile yeast hybrids access to a natural route for genome recombination and adaptation.

Founding Events and the Maintenance of Genetic Diversity in Reintroduced Populations

<p>As habitat loss, introduced predators, and disease epidemics threaten species worldwide, translocation provides one of the most powerful tools for species conservation. However, reintroduced populations of threatened species are often founded by a small number of individuals (typically 30 in New Zealand) and generally have low success rates. The loss of genetic diversity combined with inbreeding depression in a small reintroduced population could reduce the probability of establishment and persistence. Effective management of genetic diversity is therefore central to the success of reintroduced populations in both the short- and long-term. Using population modelling and empirical data from source and reintroduced populations of skinks and tuatara, I examined factors that influence inbreeding dynamics and the long-term maintenance of genetic diversity in translocated populations. The translocation of gravid females aided in increasing the effective population size after reintroduction. Models showed that supplementation of reintroduced populations reduced the loss of heterozygosity over 10 generations in species with low reproductive output, but not for species with higher output. Harvesting from a reintroduced population for a second-order translocation accelerated the loss of heterozygosity in species with low intrinsic rates of population growth. Male reproductive skew also accelerated the loss of genetic diversity over 10 generations, but the effect was only significant when the population size was small. Further, when populations at opposite ends of a species' historic range are disproportionately vulnerable to extinction and background inbreeding is high, genetic differentiation among populations may be an artefact of an historic genetic gradient coupled with rapid genetic drift. In these situations, marked genetic differences should not preclude hybridising populations to mitigate the risks of inbreeding after reintroduction. These results improve translocation planning for many species by offering guidelines for maximising genetic diversity in founder groups and managing populations to improve the long-term maintenance of diversity. For example, founder groups should be larger than 30 for reintroductions of species with low reproductive output, high mortality rates after release, highly polygynous mating systems, and high levels of background inbreeding. This study also provides a basis for the development of more complex models of losses of genetic diversity after translocation and how genetic drift may affect the long-term persistence of these valuable populations.</p>

Founding Events and the Maintenance of Genetic Diversity in Reintroduced Populations

<p>As habitat loss, introduced predators, and disease epidemics threaten species worldwide, translocation provides one of the most powerful tools for species conservation. However, reintroduced populations of threatened species are often founded by a small number of individuals (typically 30 in New Zealand) and generally have low success rates. The loss of genetic diversity combined with inbreeding depression in a small reintroduced population could reduce the probability of establishment and persistence. Effective management of genetic diversity is therefore central to the success of reintroduced populations in both the short- and long-term. Using population modelling and empirical data from source and reintroduced populations of skinks and tuatara, I examined factors that influence inbreeding dynamics and the long-term maintenance of genetic diversity in translocated populations. The translocation of gravid females aided in increasing the effective population size after reintroduction. Models showed that supplementation of reintroduced populations reduced the loss of heterozygosity over 10 generations in species with low reproductive output, but not for species with higher output. Harvesting from a reintroduced population for a second-order translocation accelerated the loss of heterozygosity in species with low intrinsic rates of population growth. Male reproductive skew also accelerated the loss of genetic diversity over 10 generations, but the effect was only significant when the population size was small. Further, when populations at opposite ends of a species' historic range are disproportionately vulnerable to extinction and background inbreeding is high, genetic differentiation among populations may be an artefact of an historic genetic gradient coupled with rapid genetic drift. In these situations, marked genetic differences should not preclude hybridising populations to mitigate the risks of inbreeding after reintroduction. These results improve translocation planning for many species by offering guidelines for maximising genetic diversity in founder groups and managing populations to improve the long-term maintenance of diversity. For example, founder groups should be larger than 30 for reintroductions of species with low reproductive output, high mortality rates after release, highly polygynous mating systems, and high levels of background inbreeding. This study also provides a basis for the development of more complex models of losses of genetic diversity after translocation and how genetic drift may affect the long-term persistence of these valuable populations.</p>

Copy-Neutral Loss-of-Heterozygosity Adds Diagnostic and Prognostic Information to the Molecular Profiling of Hematological Malignancies

Background: Copy-neutral loss-of-heterozygosity (CN-LOH) - not detectable by chromosome banding analysis - is gaining importance as a prognostic factor and can either cause the duplication of an activating mutation in an oncogene, the deletion of a tumor suppressor gene or the gain/loss of specific methylated regions. However, examination for possible CN-LOH in hematological diagnostics is at present not routinely performed and, hence, data regarding the occurrence of CN-LOH across different entities as well as the association of relevant genes is limited. Aim: (1) Frequency assessment of CN-LOH by target enrichment sequencing (TES) in a diagnostic setting, (2) evaluation of whole genome sequencing (WGS) data to estimate the prevalence of CN-LOH in a larger cohort, to pinpoint relevant genes for CN-LOHs with so far unknown associations, and to determine cross-entity variability. Patients and Methods: 1196 patients (507 female, 689 male, median age: 66 years), sent between 04/2021-07/2021 for diagnostic work-up, were analyzed by TES with a median coverage of 1765x for the gene panel and 52x for the CNV spike-in panel (IDT, Coralville, IA). Amplification-free WGS libraries of 3851 different patients were sequenced with a median coverage of 102x. Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner). Cnvkit (v 0.9.9) was used to call copy number variations (CNVs) and CN-LOH for TES and HadoopCNV (Yang et al. 2017) was used to call CN-LOH for WGS. Results: 1196 patients were analyzed by TES. For 10% of the patients at least one CN-LOH event was detected without any association to age or gender but a slightly higher incidence in myeloid compared to lymphoid neoplasms (10% vs 6%). In 14 patients, CN-LOH affected more than one chromosome arm. CN-LOH occurred most frequently in 4q (n = 15), 7q (n = 16), 9p (n = 25) and 11q (n = 10). As expected, 4q CN-LOH co-occurred with high variant allele frequencies (VAF) of TET2. Based on WGS data, 4q CN-LOH occurred predominately in AML (35%), CMML (22%), and MDS (20%). In rare cases, 4q CN-LOH was associated with FBXW7 variants in T-ALL. 7q CN-LOH occurred nearly exclusively in myeloid neoplasms (95%) and was associated with high VAFs in EZH2 in 69% of TES and 82% of WGS cases. CUX1 variants with high VAFs were detected in 80% (TES) and 45% (WGS) of the remaining cases, respectively. The well-known 9p CN-LOH led to JAK2V617F homozygosity in all myeloid neoplasms and occurred most often in MPNs. In T-ALL, regions of 9p CN-LOH harbored CDKN2A/B deletions. 11q CN-LOH occurred more often in myeloid than lymphoid neoplasms (79% vs 21%) and was associated with CBL variants in 61% and KMT2A-PTD in 19% of the cases. In contrast, ATM was the relevant gene in all lymphoid cases with 11q CN-LOH. CN-LOH in 11p was detected less frequently and only in 25% of cases an association with WT1 variants could be identified. Our WGS data confirmed the known associations between 1p CN-LOH and high allele burden in MPL, CSF3R and NRAS, 2p CN-LOH and DNMT3A variants, 13q CN-LOH and FLT3-ITD, the near exclusive occurrence of 16p CN-LOH in follicular lymphoma (FL, 98%) with high CREBBP-mutant allele burden , 17p CN-LOH and TP53 homozygosity, and the exclusive occurrence of 21q CN-LOH in AML and its association with RUNX1 mutations. Besides, 12q CN-LOH was associated with KMT2D in FL, with SH2B3 in MDS/MPN overlaps and in rare cases with KDM2B. For 17q CN-LOH the relevant gene was not unequivocally identifiable with high mutant allele variants in SRSF2, STAT5B, and NF1. 18q CN-LOH was a very rare event but consistently associated with a high VAF of MBD2, which presumably influences cell proliferation (Cheng et al. 2018). 19q CN-LOH was mostly (63%) associated with a high VAF of CEBPA variants, except for patients with hairy cell leukemia: in these cases nonsense mutations in CIC (VAF &gt; 90%) were detected. CN-LOH in 22q was more common in myeloid malignancies (65% vs 35%) and associated with PRR14L mutations in the majority of myeloid cases (62%). Of note, this association occurred neither in AML samples nor in lymphoid neoplasms. No recurrent mutations were found for 6p and 14q CN-LOHs. For all other chromosomes, CN-LOH events were very rare. Conclusions: By using a CNV spike-in panel, TES adds additional diagnostic and prognostic information by enabling simultaneous detection of selected gene mutations and genome-wide CNVs, as well as CN-LOH, without increase in sequencing costs and turn-around times. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

The Genomic Landscape of Waldenström Macroglobulinemia Reveals Sustained Germinal Center Activity and Late-Developing Copy Number Aberrations

The genomic landscape of Waldenström Macroglobulinemia (WM) is characterized by recurrent somatic mutations in MYD88, with a lower incidence of mutations affecting CXCR4, ARID1A, CD79B and the NFKB signaling pathway (Hunter et. al. Blood 2014). We aimed to characterize the relationship between single base substitutions (SBS), mutational signatures, copy number aberrations (CNA) and structural variants (SV) in WM. We performed whole genome sequencing (WGS) on 14 primary samples from WM patients at various clinical stages, including IgM monoclonal gammopathy (n=1), smoldering (n=5), newly diagnosed (n=7) and relapsed WM (n=1). We identified a median of 2806 clonal SBS per sample (IQR 1870-3079), and 12/14 (85%) samples harbored MYD88 mutations. To investigate which mutational processes are involved in shaping the genomic landscape of WM we performed a mutational signature analysis. Four previously reported SBS signatures were detected: SBS1 and SBS5 (aging), SBS9 (germinal center; GC) and SBS8, with the contribution of age-related signatures SBS1/SBS5 being directly correlated with age at presentation (R 2=0.44, p=0.014). The GC signature SBS9 demonstrated sustained GC activity, as evidenced by the same proportion of mutations attributable to SBS9 at both the clonal and subclonal level (24%). At the immunoglobulin loci, we observed evidence of clustered SBS84 (AID), reflecting somatic hypermutation, with SBS84 accounting for 30% of signature contribution from subclonal mutations. Overall, these data suggest that, similarly to MM and other hematological malignancies, the interaction between WM and the GC is sustained over time. We have previously demonstrated that SV and complex events are critical in the pathogenesis and clinical outcomes of multiple myeloma. In contrast, in this WM WGS cohort, we found a low prevalence of complex SV, with no chromothripsis detected, and a single chromoplexy event found in 3 patients (21%), all of whom had progressed to symptomatic WM. To explore WM CNA features in a larger cohort, we examined the WGS data together with 38 MYD88-mutated WM samples for which targeted sequencing was available (MSK-IMPACT-Heme 400 gene panel). In this combined dataset (n=52), GISTIC analysis identified significantly deleted regions at 6q16.1, 7q34, 17p13.1 (TP53) and 21q22.2, along with significant amplification at 6p22.1 (HLA-A). To better characterize the HLA loci using the loss of heterozygosity in human leukocyte antigen (LOHHLA) tool (McGranahan et. al. Cell 2017) we found the presence of HLA-specific loss of heterozygosity in 1 sample, while 4 samples had HLA CN &gt;2.5 (all from patients who progressed to symptomatic WM). CNA analysis demonstrated that while some samples harbored typical CNA features, others had minimal changes, with MGUS / smoldering WM samples having less CNA compared with those who progressed to symptomatic WM. The 2 MYD88 wild type WGS contained a clonal gain affecting chromosome 12, which is typically an early event in chronic lymphocytic leukemia. Molecular time analysis (the corrected ratio between duplicated and non-duplicated clonal mutations within large chromosomal gains [Maura et al. Nat Comm 2019]) demonstrated that these 2 chromosomal gain events occurred early in cancer development (relative timing &lt;0.5), while multiple other CNA changes occurred later in the disease course (timing &gt;0.5) and tended to be subclonal. This data suggests that, while MYD88-mutations are central to WM clone establishment and can be observed in precursor disease, CNA may contribute to later phases and disease progression. In summary, WGS in WM allows the demonstration that germinal center activity is sustained over time. CNA in WM are not random in distribution, with specific loci being significantly amplified or deleted, and a potential role for HLA CNA. In contrast to MYD88 mutations, which are carried by stable precursor patients, the subclonal status and late molecular time of most CNA changes suggest a late role in cancer progression. Disclosures Kastritis: Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Diamond: Sanofi: Honoraria; Medscape: Honoraria. Kazandjian: Arcellx: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy. Dogan: Roche: Consultancy, Research Funding; Seattle Genetics: Consultancy; EUSA Pharma: Consultancy; Peer View: Honoraria; Takeda: Consultancy, Research Funding; Physicians' Education Resource: Honoraria. Lesokhin: Trillium Therapeutics: Consultancy; Serametrix, Inc: Patents & Royalties; Genetech: Research Funding; Iteos: Consultancy; bristol myers squibb: Research Funding; Behringer Ingelheim: Honoraria; pfizer: Consultancy, Research Funding; Janssen: Honoraria, Research Funding. Landgren: Janssen: Other: IDMC; Janssen: Honoraria; Celgene: Research Funding; Amgen: Honoraria; Janssen: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Palomba: Rheos: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; Notch: Honoraria, Other: Stock; Priothera: Honoraria; Kite: Consultancy; Novartis: Consultancy; Magenta: Honoraria; WindMIL: Honoraria; BeiGene: Consultancy; Juno: Patents & Royalties. Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Takeda: Honoraria; BeiGene: Honoraria.

122 A powerful, precise targeting system controlled by tumor deletions transforms CEA and MSLN CAR-T cells into tumor-selective agents

BackgroundMesothelin (MSLN) and carcinoembryonic antigen (CEA) are classic tumor-associated antigens that are expressed in many solid tumors including the majority of lung, colorectal and pancreatic cancers. However, both MSLN and CEA are also expressed in vital normal organs. This normal expression creates risk of serious inflammation for CEA- or MSLN-directed therapeutics. To date all active CEA- or MSLN-targeted investigational therapeutics have been toxic when administered systemically.MethodsWe have developed a safety mechanism to protect normal tissues without abrogating sensitivity of cytotoxic T cells directed at MLSN(+) or CEA(+) tumors in a subset of patients with defined loss of heterozygosity (LOH) in their tumors (figure 1). This dual-receptor (Tmod< sup >TM</sup >) system exploits common LOH at the HLA locus in cancer cells, allowing T cells to recognize the difference between tumor and normal tissue.1 2 T cells engineered with specific Tmod constructs contain: (i) a MSLN- or CEA-activated CAR; and, (ii) an inhibitory receptor gated by HLA-A*02. HLA-A*02 binding blocks T cell cytotoxicity, even in the presence of MSLN or CEA. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When HLA-A*02 is absent from tumors selected for LOH, the CARs are predicted to mediate potent killing of the A*02(-) malignant cells.ResultsThe Tmod system robustly protects surrogate normal cells even in mixed-cell populations in vitro while mediating robust cytotoxicity of tumor cells in xenograft models (see example in figure 2). The MSLN CAR can also be paired with other blockers, supporting scalability of the approach to patients beyond HLA-A*02 heterozygotes.Abstract 122 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cells.Abstract 122 Figure 2Bioluminescence measurements show the average difference between the size of the MSLN(+)A*02(+) ‘normal’ graft compared to the MSLN(+)A*02(-) tumor graft on the two flanks of mice after T cell infusion. Both tumor and normal grafts are destroyed by CAR-Ts (CAR-3 and M5 benchmark) while the MSLN Tmod cells kill the tumor but not the normal graft.ConclusionsThe Tmod mechanism may provide an alternative route to leverage solid-tumor antigens such as MSLN and CEA in safer, more effective ways than previously possible.ReferencesHamburger AE, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.

Germline Testing Data Validate Inferences of Mutational Status for Variants Detected From Tumor-Only Sequencing

PURPOSE Pathogenic germline variants (PGVs) in cancer susceptibility genes are usually identified through germline testing of DNA from blood or saliva: their detection can affect treatment options and potential risk-reduction strategies for patient relatives. PGV can also be identified in tumor sequencing assays, which, when performed without patient-matched normal specimens, render determination of variants' germline or somatic origin critical. METHODS Tumor-only sequencing data from 1,608 patients were retrospectively analyzed to infer germline versus somatic status of variants using an information-theoretic, gene-independent approach. Loss of heterozygosity was also determined. Predicted mutational models were compared with clinical germline testing results. Statistical measures were computed to evaluate performance. RESULTS Tumor-only sequencing detected 3,988 variants across 70 cancer susceptibility genes for which germline testing data were available. We imputed germline versus somatic status for > 75% of all detected variants, with a sensitivity of 65%, specificity of 88%, and overall accuracy of 86% for pathogenic variants. False omission rate was 3%, signifying minimal error in misclassifying true PGV. A higher portion of PGV in known hereditary tumor suppressors were found to be retained with loss of heterozygosity in the tumor specimens (72%) compared with variants of uncertain significance (58%). CONCLUSION Analyzing tumor-only data in the context of specimens' tumor cell content allows precise, systematic exclusion of somatic variants and suggests a balance between type 1 and 2 errors for identification of patients with candidate PGV for standard germline testing. Although technical or systematic errors in measuring variant allele frequency could result in incorrect inference, misestimation of specimen purity could result in inferring somatic variants as germline in somatically mutated tumor suppressor genes. A user-friendly bioinformatics application facilitates objective analysis of tumor-only data in clinical settings.

Whole-genome sequencing of Vero E6 (C1008) and comparative analysis of four Vero cell sublines

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of sublines have been established from the original cell line, which display different characteristics. In this study, we determined the whole-genome sequence of Vero E6 (C1008) and performed comparative analysis among Vero JCRB 0111, Vero CCL-81, Vero 76 and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that all sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we focused on Vero E6-specific/enriched variants and identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoV-2, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 contained the allele with isoleucine, resulting from the loss of one of the X chromosomes.