Subclonal Variation Research Articles

Understanding the Temporal Dynamics of Invasive Late Blight Populations in India for Improved Management Practices.

The microbial oomycete pathogen Phytophthora infestans causes severe epidemics of potato late blight in crops globally. Disease management benefits from an understanding of the diversity of pathogen populations. In this study, we explore the dynamics of P. infestans populations in the late blight-potato agro-ecosystem across the Indian subcontinent. Investigations of the macroecological observations at the field level and microbial ecological principles provided insights into future pathogen behavior. We use a comprehensive simple sequence repeat allele dataset to demonstrate that an invasive clonal lineage called EU_13_A2 has dominated populations over 14 years across India, Bangladesh, and Pakistan. Increasing levels of subclonal variation were tracked over time and space, and, for the first time, populations in Asia were also compared with the source populations from Europe. Within India, a regional pathogen population structure was observed with evidence for local migration, cross-border movement between surrounding countries, and introductions via imports. There was also evidence of genetic drift and between-season transmission of more strongly pathogenic subclones with a complete displacement of some subclonal types. The limited introduction of novel genotypes and the use of resistant potato cultivars could contribute to the dominance of the 13_A2 lineage. The insights will contribute to the management of the pathogen in these key global potato production regions.

Emergence of Highly-Plastic B Cell States Cooperates with Early Immune Microenvironment Remodeling to Drive Follicular Lymphomagenesis

Follicular lymphoma (FL) is a prototypical example of B cell lymphoma arising from germinal center (GC) B cells. Recent single cell transcriptomic studies investigating the transcriptional heterogeneity of FL suggest that tumor B cells are functionally diverse with cells acquiring an intermediate phenotype between GC and memory (mem), in line with the premalignant FL ontogeny where BCL2 + memory B cells require multiple re-entries into the GC to facilitate the transformation process. Since FL cells often hijack the epigenetic machinery through mutations in histone modifying proteins - notably KMT2D - we questioned here how epigenetic alterations contribute to the emergence of this intratumoral heterogeneity. We developed a genetically engineered mouse FL-like model (GEMM) carrying Kmt2d loss-of-function in B cells and BCL2 overexpression recapitulating different progression stages from preneoplastic hyperplasia to FL-like tumors. We analyzed 45,541 high-quality single cell transcriptomes and immune repertoires (B cell receptor, BCR and T cell receptor, TCR) from 18 mice including immunized-control mice in the context of physiological GC reaction. We developed a computational workflow on mutant B cells combining supervised and unsupervised metaclustering approaches in order to highlight recurrent gene programs active in preneoplastic hyperplasia and the various stages leading to FL-like tumors. We used inferCNV to assess the association between genetics and transcriptomic heterogeneity and inferred cell-cell interactions to characterize the microenvironmental determinants associated with B cell heterogeneity throughout FL progression. By modeling the GC cycle dynamics and building B cell signatures during a normal immune response, we found that our FL GEMM mirrored the desynchronization of the GC reaction program previously observed in human FL. Single cell expression profiles of Kmt2d-BCL2 mutant cells also showed a reproducible increase in phenotypic heterogeneity during FL progression, manifesting as a continuum of cell states from proliferating GC-like to quiescent mem-like cell states. We defined this GC-to-Mem axis as the major source of intra-tumor transcriptional heterogeneity. Single-cell BCR sequencing revealed the emergence of highly-plastic B cell states during FL evolution, co-existing within individual mice and being largely independent of subclonal genetic variation. To further elucidate the emergence and maintenance of these cell state transitions, we explored the phenotypic and transcriptional changes within the immune microenvironment along disease progression. Significant compositional changes were found, including the expansion of T follicular helper cells and T regulatory T cells together with the loss of naive CD4, CD8 T, and NK cell populations, preceding malignant transformation and creating a (pre)tumoral supportive niche persisting in advanced tumors. Prior remodeling of a premalignant supportive immune niche may be crucial in establishing malignant cell heterogeneity. Ongoing efforts aim to impair tumor-immune interactions driving tumor B cell dynamics and survival. In conclusion, our results provide a high-resolution view of events spanning FL progression and indicate a major role of early TME remodeling in establishing a suitable niche for progression. Early intervention aimed to target the B cell-tumor microenvironment interactions driving intra-tumoral heterogeneity may represent a promising therapeutic avenue against early disease and prevention of FL recurrence.

Characterization of Phytophthora infestans Populations in Cyprus, the Southernmost Potato-Producing European Country.

Cyprus is the southernmost island country of Europe, located in the Mediterranean. Despite its limited area, potato production is considered an integral source of the national agricultural revenue. During 2010-2012, a late blight epidemic period for the country, the population structure of Phytophthora infestans was analyzed via a sample of 539 isolates collected from all of the main potato-cultivating regions of Cyprus. We determined mating type, mefenoxam sensitivity, and genetic polymorphism at 12 simple sequence repeat (SSRs) loci. Although both mating types were detected in the country, a gradual but dynamic shift toward A2 dominance was manifested over time. The pathogen population also demonstrated reduced sensitivity to the phenylamide fungicide, since 96.2% of the tested isolates had high (70.3%) and intermediate (25.9%) resistance to mefenoxam, which suggests that it should be replaced with other active ingredients in local disease management strategies. The genotypic analysis also revealed the predominance of the highly aggressive mefenoxam-insensitive EU_13_A2 lineage across the country, with a frequency of 79.2%. Other samples comprised an older lineage EU_2_A1 (19.5%), a very low proportion of EU_23_A1 (0.37%), and others that did not match any known lineage (0.92%). SSRs data supported triploid genomes among the dominant lineages, and patterns of their asexual population history were also apparent. A high subclonal variation of the 13_A2 population was detected, which suggested introduction events of this widespread genotype to Cyprus from major tuber-exporting countries. Present data indicate the severe impact of inoculum migration to the structure of the local population; thus, current phytosanitary procedures should be reconsidered and possibly attuned. This is the first comprehensive study to elucidate the diversity of P. infestans in Cyprus and could serve as a baseline for future monitoring of this highly adaptive plant pathogen, given that late blight management strategies should be constantly refined according to the traits of the dominant genotypes of P. infestans.

Abstract LB019: Trisicell: Scalable Tumor Phylogeny Reconstruction and Validation Reveals Developmental Origin and Therapeutic Impact of Intratumoral Heterogeneity

Abstract Emerging sets of single-cell sequencing data makes it appealing to apply existing tumor phylogeny reconstruction methods to analyze associated intratumor heterogeneity. Unfortunately, tumor phylogeny inference is an NP-hard problem and existing principled methods typically fail to scale up to handle thousands of cells and mutations observed in emerging single-cell data sets. Even though there are greedy heuristics to build hierarchical clustering of cells and mutations, they suffer from well-documented issues in accuracy. Additionally even when “optimal” solutions are feasible, existing approaches only provide a single “most likely” tree to depict the evolutionary processes that may result in an observed collection of cells and mutations. To make matters worse, the vast majority of single-cell sequencing data sets are transcriptomic and as a result, suffer from considerable variation in coverage across mutational loci. In this paper, we introduce Trisicell, a computational toolkit for scalable tumor phylogeny reconstruction and validation from single-cell genomic, exomic or transcriptomic sequencing data. Trisicell has three components: (i) Trisicell-DnC, a new tumor phylogeny reconstruction method from genotype matrices derived from single-cell data, (ii) Trisicell-ConT a new algorithm for constructing the consensus for two or more tumor phylogenies - which may be built through the use of different data types on the same set of cells, or built through the use of different methods on the same data, and (iii) Trisicell-PF, a new partition function method for assessing the likelihood of any user-defined subtree/set of cells to be seeded by a given set of mutations in the phylogeny. Collectively, these tools provide means of identifying and validating robust portions of a tumor phylogeny, offering the ability to focus on the most important (sub)clones and the genomic alterations that seed the associated clonal expansion. We applied Trisicell to a panel of clonal sublines derived from single-cells of a parental mouse melanoma model on which we performed both whole exome and whole transcriptome sequencing. The tumor phylogenies of the clonal sublines built on exomic and transcriptomic mutations by Trisicell-DnC, were shown by Trisicell-ConT to be highly similar and the subtrees comprised of phenotypically similar clonal sublines were shown to be strongly associated by Trisicell-PF to their seeding mutations. In addition, we applied Trisicell to single-cell whole transcriptome sequencing data from a tumor derived from the same parental melanoma cell line, which was subjected to anti-CTLA-4 immunotherapy. The phylogenies generated from both studies featured distinct subtrees, strongly associated with phenotypes including cell differentiation status, tumor growth and therapeutic response. These results suggest that Trisicell can be used for scalable tumor phylogeny reconstruction and validation through both single-cell and clonal-subline sequencing data, which may reveal strong phenotypic associations. In particular, they suggest that the developmental status and phenotypic intratumoral heterogeneity of melanoma originates from observable subclonal variation. Citation Format: Farid Rashidi Mehrabadi, Salem Malikić, Kerrie L. Marie, Eva Pérez-Guijarro, Erfan Sadeqi Azer, Howard H. Yang, Can Kızılkale, Charli Gruen, Huaitian Liu, Christina Marcelus, Aydın Buluç, Funda Ergün, Maxwell P. Lee, Glenn Merlino, Chi-Ping Day, S. Cenk Sahinalp. Trisicell: Scalable Tumor Phylogeny Reconstruction and Validation Reveals Developmental Origin and Therapeutic Impact of Intratumoral Heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB019.

Abstract B15: Genomic classification and prognosis in rhabdomyosarcoma: A report from the Children’s Oncology Group, the Institute of Cancer Research, and the National Cancer Institute

Abstract Purpose: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We therefore performed a large-scale study through an international consortium to more accurately determine the incidence of driver mutations and their association with clinical outcome. Patients and Method: Formalin-fixed, paraffin-embedded material was collected from patients enrolled on Children’s Oncology Group trials and UK patients enrolled on MMT trials. Pathology was reviewed centrally and extracted DNA was subjected to targeted capture sequencing using a panel of 39 genes previously associated with RMS. Mutations, indels, deletions, gene amplifications, and copy number variation was called using analysis pipelines developed at the NCI. Results: DNA from six hundred and forty-one patients was suitable for analyses. A median of 1 variant call was found per tumor. Mutation of a RAS isoform was found in 29% of all fusion negative cases, mutation of a RAS pathway member was seen in greater than 50% of cases, and 24% had no putative driver mutation identified. BCOR (15%), NF1 (11%), and TP53 (12%) mutations were found at a higher incidence than previously reported. Interestingly, mutations in HRAS were notable in the infant population whereas those in NRAS were enriched in adolescents. Among infants < 1 year, 71% of cases harbored a mutation in HRAS or KRAS. In contrast, mutation of MYOD1 was associated with an older age and a head and neck primary site. Finally, 29% of the evaluated tumors harbored multiple driver mutations consistent with subclonal variation and tumor heterogeneity in fusion-negative RMS. Conclusion: This is the largest genomic characterization of clinically annotated RMS tumors to date and provides genetic features that refine risk stratification and can be incorporated into prospective trials. Citation Format: Jack Shern, Joanna Selfe, Rajesh Patidar, Young Song, Marielle Yohe, Jun Wei, Xinyu Wen, Erin Rudinski, Donald Barkauskas, David Hall, Corinne Linardic, Meriel Jenney, Julia Chisholm, Rebecca Brown, Anna Kelsey, Susanne Gatz, Stephen Skapek, Douglas Hawkins, Janet Shipley, Javed Khan. Genomic classification and prognosis in rhabdomyosarcoma: A report from the Children’s Oncology Group, the Institute of Cancer Research, and the National Cancer Institute [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B15.

Population structure and host range of the potato late blight pathogen Phytophthora infestans in Peru spanning two decades

The genetic diversity of the late blight pathogen Phytophthora infestans infecting cultivated potato and alternative hosts growing in the vicinity of fields in the main potato‐growing areas of the Peruvian Andes was characterized using collections from 1997–2013 as reference. The Peruvian P. infestans population, including previously collected and current isolates, consists of four clonal lineages (EC‐1, US‐1, PE‐3 and PE‐7) that belong to the A1 mating type and have been present in the country for decades. The first report of US‐1 was in isolates collected between 1982 and 1986; meanwhile, EC‐1 and PE‐3 appeared for the first time in isolates from 1992 and PE‐7 was found in 1997. The pathogen has a very broad host range among the solanaceous plants infecting cultivated potato, tomato, pear melon and several wild species. The solanaceous species growing in the vicinity of the potato fields sampled were identified and surveyed for late blight‐like symptoms. Phytophthora infestans was isolated from nine wild species, including three new host species: Solanum zahlbruckneri, Solanum grandidentatum and Iochroma grandiflorum. There was no clear host specialization, but geographical substructuring was found as well as changes in the pathogen populations at the regional level. The clonal lineage EC‐1, which is mostly resistant to metalaxyl, has complex virulence and contains a high level of subclonal variation, continues to dominate the population. Some multilocus genotypes of the EC‐1 lineage were sampled in high frequencies and were found among the previously collected and new samples.

Subclonal Variation of RAG-Mediated Diversification in B Cell Acute Lymphoblastic Leukemia

Background: Recombination-activating gene (RAG) mediates recombination of the immunoglobulin heavy chain variable gene (IGHV) in immature B lymphocytes. Aberrant targeting of RAG to non-IGH sites in B cell acute lymphoblastic leukemia (B-ALL) contributes to the development of driver mutations and clonal evolution (Papaemmanuil et al. Nat. Genet. 2014). This finding suggests that patients whose ALL involves increased RAG-mediated clonal diversification would exhibit more aggressive disease. As an initial step toward addressing this hypothesis, we asked whether the extent of RAG-mediated diversification in a patient's leukemia is consistent across all cells or variable between subclones. To assess variation in RAG-mediated subclone diversification, we interrogated rearranged IGH sequences from diagnostic B-ALL specimens to identify early subclones (SC) and to quantify RAG-derived sub-subclones (SSC) from each. We hypothesized that if RAG activity is a consistent feature of the leukemia, all SC within a single patient will have a comparable extent of SSC evolution. Methods: Amplicon-based IGHV sequencing identified the number of clonal IGH SC and their RAG-derived SSC in 22 pre-treatment adult and pediatric patients with newly-diagnosed B-ALL. Analysis was performed on peripheral blood (PB) for all patients in addition to diagnostic bone marrow (BM) for 16 of the 22 patients studied. Ultra-deep sequencing of IGH utilized 500 ng genomic DNA (representing approximately 80,000 cells/specimen) for the MiSeqDx platform, generating ~300 bp reads surrounding the VDJ junctional region. The NCBI IgBlast sequence analysis tool assigned IGH VDJ identities according to germline reference. Methods for determining subclones (SC) of shared clonal lineage involved classifying reads with a common Jh identity and 6 shared upstream Dh-Jh junctional nucleotides (termed "6N_Jx") according to defined methods (Gawad et al. Blood 2012). Further evolved sub-subclones (SCC) - as evidence of ongoing RAG activity - were defined by unique junctional sequences upstream of the common Dh-Jh junction (termed the "NDN" region). SSC were quantified according to their relative proportions within each SC family. Results: VDJ-rearranged SC families were detected for 20 of the 22 patients studied (median 2/patient; range 1-7); further analysis to assess variation in RAG-mediated diversification was limited to the 18 cases with ≥ 2 SC. In 9 of these 18, numerous evolved SSCs were identified from at least one SC in the specimen. In 4/9, there were starkly distinct levels of RAG-mediated diversity observed between intrapatient SC families, with some clonal precursors giving rise to numerous SSCs (up to 2,200 SSC per SC) while others showed minimal-to-no RAG-derived evolution (Table 1). Fifteen of 16 patients with matched BM and PB diagnostic specimens had detectable SCs. Of these, 73% (11/15) shared the dominant SC, while in 27% (4/15) the dominant SC differed between sites. However, regardless of which SC predominated, the extent of SSC diversification within each SC was preserved between the BM and the PB, with similar evolution patterns observed regardless of disease site. There was no relationship between SC read frequency and number of SSCs. Conclusions: Using deep sequencing of a single IGHV locus, wide variation in the extent of subclone diversification was observed in 4 of 20 patients with B-ALL. These findings indicate that the degree of RAG-mediated heterogeneity in B-ALL can range from minimal to extensive among distinct subclones in a single patient. The data underscore the relevance of single cell investigation of tumor characteristics to improving our understanding of the mechanisms of clonal evolution in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Genetic Structure and Subclonal Variation of Extant and Recent U.S. Lineages of Phytophthora infestans.

The oomycete Phytophthora infestans is an important plant pathogen on potato and tomato crops. We examined the genetic structure of extant 20th and 21st century U.S. lineages of P. infestans and compared them with populations from South America and Mexico to examine genetic relationships and potential sources of lineages. US-23, currently the most prevalent lineage detected in the United States, shared genetic similarity primarily with the BR-1 lineage identified in the 1990s from Bolivia and Brazil. Lineages US-8, US-14, and US-24, predominantly virulent on potato, formed a cluster distinct from other U.S. lineages. Many of the other U.S. lineages shared significant genetic similarity with Mexican populations. The US-1 lineage, dominant in the mid-20th century, clustered with US-1 lineages from Peru. A survey of the presence of RXLR effector PiAVR2 revealed that some lineages carried PiAVR2, its resistance-breaking variant PiAVR2-like, or both. Minimum spanning networks developed from simple sequence repeat genotype datasets from USABlight outbreaks clearly showed the expansion of US-23 over a 6-year time period and geographic substructuring of some lineages in the western United States. Many clonal lineages of P. infestans in the United States have come from introductions from Mexico, but the US-23 and US-1 lineages were most likely introduced from other sources.

Characterisation of Phytophthora infestans Isolates of Potato Crops from Spain

The characterisation of Phytophthora infestans isolates is a key step in both monitoring population changes and designing control strategies. A collection of 57 isolates (33 from 2003 to 2005 and 24 from 2011 to 2014) of the Spanish population of P. infestans from different potato fields was characterised in this study. Mating types A1 and A2 have been identified for the first time in Spain by using CAPs markers. A total of 23 samples were classified as A1 mating type and 34 as A2 type. All isolates had the Ia mitochondrial haplotype with one exception which was IIa. Three clonal lineages of P. infestans were detected: 2_A1, 3_A2 and 13_A2 (Blue 13), where a sub-clonal variation was observed within this lineage (13_A2_1, 13_A2_2, 13_A2_3, 13_A2_4, 13_A2_15, 13_A2_68 and 13_A2_new). Two isolates denominated as ‘Misc’ could not be ascribed to any of the genotypes characterised. These results highlight the spread of pathogen clones across Europe during this period and the risks related to the presence of both P. infestans mating types are discussed.

Abstract 2194: Subclonal variation and evolutionary dynamics of BRAF mutations in cancer

Abstract Tumors have genetically distinct subclones that compete for space and resources and differentially resist efforts to make them extinct. We studied the intratumoral heterogeneity of BRAF mutations across several cancer types. We identified BRAF driver mutations as predominately clonal in some cancer types (e.g. melanoma) and subclonal in others (e.g. lung adenocarcinoma). Clonality corresponded to the amplification of BRAF and prevalence of V600 mutations in each cancer type. We mathematically and experimentally modeled the propagation and selection of tumors containing BRAF mutations and determined that the speed of clonal sweeps were associated with the extent of activation of MAPK signaling pathway and BRAF copy number. Consistent with these findings, tumors with "hard" sweeps were more likely to respond to BRAF and/or MEK inhibitors. Furthermore, some PDX models treated with cytotoxic therapy underwent BRAF mutant subclone expansion over time and this effect is mitigated by inhibitors of BRAF and/or MEK. Treatment strategies based on subclone composition resulted in significantly improved tumor control. Our study uncovers patterns of distinct BRAF clonal evolutionary trajectories and advances therapeutic strategies on the basis of BRAF mutation identity and subclone composition that merit testing in patients. Citation Format: Priyanka Gopal, Elif I. Sarihan, Mohamed E. Abazeed. Subclonal variation and evolutionary dynamics of BRAF mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2194.

CRISPR-Based Targeted Epigenetic Editing Enables Gene Expression Modulation of the Silenced Beta-Galactoside Alpha-2,6-Sialyltransferase 1 in CHO Cells.

Despite great efforts to control and modify gene expression of Chinese Hamster Ovary (CHO) cells by conventional genetic engineering approaches, i.e. overexpression or knockdown/-out, subclonal variation, induced unknown regulatory effects as well as overexpression stress are still a major hurdle for efficient cell line engineering and for unequivocal characterization of gene function. The use of epigenetic modulators - key players in CHO clonal heterogeneity - has only been marginally addressed so far. Here, we present the application of an alternative engineering strategy in CHO cells by utilizing targeted epigenetic editing tools that enable the turning-on or -off of genes without altering the genomic sequence. The present, but silent beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) gene is activated by targeting the catalytic domain (CD) of Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) via deactivated Cas9 (dCas9) to its methylated promoter. Stable upregulation in up to 60% of transfected cells is achieved over a time span of more than 80 days. No difference in growth and recombinant protein productivity is observed between activated and control cultures. Re-silencing by targeted methylation via DNA methyltransferase (DNMT) 3A-CD resulted in an up to 5.4-fold reduction of ST6GAL1 mRNA expression in ST6GAL1 expressing cells. This proof-of-concept demonstrates the feasibility of using epigenetic editing tools to efficiently modulate gene expression and provide a promising complement to conventional genetic engineering in CHO cells.

Large sub-clonal variation in Phytophthora infestans from recent severe late blight epidemics in India

The population structure of the Phytophthora infestans populations that caused the recent 2013–14 late blight epidemic in eastern India (EI) and northeastern India (NEI) was examined. The data provide new baseline information for populations of P. infestans in India. A migrant European 13_A2 genotype was responsible for the 2013–14 epidemic, replacing the existing populations. Mutations have generated substantial sub-clonal variation with 24 multi-locus genotypes (MLGs) found, of which 19 were unique variants not yet reported elsewhere globally. Samples from West Bengal were the most diverse and grouped alongside MLGs found in Europe, the UK and from neighbouring Bangladesh but were not linked directly to most samples from south India. The pathogen population was broadly more aggressive on potato than on tomato and resistant to the fungicide metalaxyl. Pathogen population diversity was higher in regions around the international borders with Bangladesh and Nepal. Overall, the multiple shared MLGs suggested genetic contributions from UK and Europe in addition to a sub-structure based on the geographical location within India. Our data indicate the need for improved phytosanitary procedures and continuous surveillance to prevent the further introduction of aggressive lineages of P. infestans into the country.

Abstract BS3-2: Breast cancer intra-tumor heterogeneity and its clinical implications

Abstract Breast cancer is a known heterogeneous disease with significant inter-tumor differences being manifested on many levels including histology, response to therapy, and patient survival outcomes. Importantly, even within an individual tumor, significant heterogeneity may exist. This intra-tumor heterogeneity (ITH) poses a challenge to understanding the biology of breast cancer, and an open question is what impact does ITH have on the clinical decision making process? Over the past ten years, a rich body of genetic, genomic, and proteomic data has been accumulated concerning the ITH of breast cancers. Much of these data is coming from Massively Parallel Sequencing, and it is yielding a wealth of quantitative data concerning DNA- and RNA-based ITH. This variation can be in the form of somatic mutations, DNA copy number changes, and gene expression differences due to epigenetic differences (i.e. cell state changes) and or microenvironmental influences. Notable recent examples of DNA-based ITH include PIK3CA mutation heterogeneity across different metastatic sites within the same patient with some lesions responding to PI3K inhibitors while others did not. Another key example of ITH are ESR1 mutations that arise within the setting of aromatase inhibitor treatment, where it is sometimes seen that within an individual, multiple different mutations in ESR1 can occur and that confer estrogen independence. Lastly, significant epigenetic ITH can also occur, with a prime example being epithelial tumor cell plasticity, which is often manifested as “state changes”. Examples of this include chemotherapy induced changes that may select for epithelial cells with more mesenchymal features (often referred to as EMT), and which is also typically linked to a more treatment resistant state. Another important concept that is emerging is that it might be better to target the clonal (or trunk) alterations, as opposed to targeting the subclonal variation. The latter, by definition, is not present within all tumor cells, and thus even if that therapy is effective, it will not target all tumor cells. Conversely, if clonal variants (i.e. those present within the founding clone) are targeted, the treatment may be more effective and cause regression and/or complete response. These multiple examples of ITH are biologically important, but the question still remains, what are the clinical implications? These ITH topics will be presented and discussed within the context of how these findings might affect treatment options and decision making. Citation Format: Perou Perou CM. Breast cancer intra-tumor heterogeneity and its clinical implications [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr BS3-2.

Integration of Genomics Into Treatment: Are We There Yet?

Using advances in genetic analysis to segment and direct treatment of multiple myeloma (MM) represents a way of maintaining therapeutic progress. Recent genetic analyses have opened the possibility of enhancing risk stratification approaches and of using different risk and biologic strata as part of clinical trials. The Myeloma Genome Project is a collaborative project that has compiled the largest set of cases with sequencing and have outcome data that are available for stratification purposes. Mutation-targeted treatment of the Ras pathway has been shown to be active in MM, but is compromised by the presence of the subclonal genetic variation typical of myeloma. Going forward, risk and biologically stratified therapy for MM looks to be a promising way of maintaining therapeutic progress, as does precision immunotherapy directed by the cellular context of the bone marrow.

Sequencing thousands of single-cell genomes with combinatorial indexing.

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

Integration of Genomics Into Treatment: Are We There Yet?

Using advances in genetic analysis to segment and direct treatment of multiple myeloma (MM) represents a way of maintaining therapeutic progress. Recent genetic analyses have opened the possibility of enhancing risk stratification approaches and of using different risk and biologic strata as part of clinical trials. The Myeloma Genome Project is a collaborative project that has compiled the largest set of cases with sequencing and have outcome data that are available for stratification purposes. Mutation-targeted treatment of the Ras pathway has been shown to be active in MM, but is compromised by the presence of the subclonal genetic variation typical of myeloma. Going forward, risk and biologically stratified therapy for MM looks to be a promising way of maintaining therapeutic progress, as does precision immunotherapy directed by the cellular context of the bone marrow.

Abstract 167: Inferring subclonal relationships between multiple metastases from rapid autopsy of a single melanoma patient

Abstract Background: Despite recent advances in personalized cancer therapeutics, little impact has been made on curing advanced melanomas over the last several decades. Patients with metastatic melanoma generally have a poor prognosis; survival is limited and typically the 5-year survival rate is less than 15% in patients with metastatic disease. Dysregulation of BRAF signaling has been shown to be one of these key drivers of the pathogenesis of disease in a large subpopulation (∼40%) of patients. Although targeted therapy with BRAF and MEK inhibitors is the mainstay of therapy for recurrent/metastatic melanomas, this treatment modality provides a temporary respite. Often resistant tumor clones(carrying mutations in oncogenes) emerge, followed by a rapid progression, causing the patients to succumb to the disease. As such, in this study, we aim to understand inter- and intra- tumor heterogeneity that leads to resistance to therapy. Methods: We conducted deep exome sequence analysis (250X coverage) of a collection of 8 metastatic melanoma tumor samples collected by rapid autopsy from a patient who became resistant to Vemurafinib despite harboring sensitizing BRAF p. V600E mutations. We used whole exome sequence analysis to detect somatic mutations and copy number variants in every metastatic tumor. To infer subclonal populations present within each metastasis, we applied a combination of clonal inference tools to determine subclonal structures based on somatic point mutations (PyClone) and copy number alterations (TITAN). Subsequently, we applied clustering algorithms to the inferred populations to construct inter-tumor phylogenies. Results: We found that 89% of somatic point mutations were shared across all metastatic sites and were primarily clonal. Conversely, the cellular prevalence inferred from copy number alterations showed a greater degree of subclonal evolution. We found a clear relationship between the physical locations of tumors and shared subclonal patterns, whereby metastases from lung, brain and omentum metastases clustered distinctly from those in rectum, small intestine, peritoneum, diaphragm and mesentery. To look for shared patterns of subclonal variation underlying metastatic disease and resistance to therapy, we are currently extending our analysis to an additional six patients, each with tissues from 7-9 metastatic sites collected by rapid autopsy. Conclusion: Our analysis of multiple metastatic sites from a single patient by deep exome sequence analysis found most somatic mutations were shared across tumor sites and subclonal evolution appears to be driven primarily by acquisition of copy number alterations. Tumors in near proximity shared greater genomic similarity compare to distant sites. Ongoing analysis of additional patients is required to uncover recurrent mechanisms of resistance to targeted therapies in this disease. Citation Format: Soroush Samadian, Prashant Bavi, Dianne Chadwick, Arnavaz Danesh, Mark Dowar, Tiantian Li, Madura Siva, Michael Roehl, Anthony m. Joshua, Trevor J. Pugh. Inferring subclonal relationships between multiple metastases from rapid autopsy of a single melanoma patient. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 167.

Abstract PR06: Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation

Abstract Leukemia progression and relapse are driven by molecularly distinct and often-rare cancer cells called leukemia-propagating cells (LPCs). If LPCs are retained following treatment, they will ultimately initiate relapse disease. Despite the substantial number of genetic lesions that have been identified in relapse samples and the contention that these mutations likely modulate response to therapy, acquired mutations that increase the overall frequency of tumor propagating cells following continued clonal evolution have not been reported in any cancer to date. Here, we have developed a transgenic zebrafish model where single fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) cells are transplanted into genetically identical recipient fish and functionally assessed for differences in leukemia propagating cell frequency, growth, and dexamethasone resistance. While subclonal variation was observed within single cells from the same primary leukemia, a subset of clones continued to evolve genetic lesions and epigenetic modifications to enhance growth and overall LPC frequency. A majority of evolved clones acquired activated AKT signaling, which simultaneously increased the number of leukemia propagating cells through activating the mTORC1 pathway, enhanced growth by stabilizing Myc protein levels, and rendered T-ALL cells resistant to dexamethasone, which was reversed by combined treatment with an AKT inhibitor. These results were confirmed using large-scale transgenic epistasis experiments and limiting-dilution cell transplantation studies. In total, our data suggest that diagnosis clones can stochastically acquire mutations necessary to survive treatment and drive relapse even before a patient begins treatment, with acquired mutations being independently selected based on important cancer phenotypes including elevated growth rate and leukemia propagating potential. Moreover, our work also identifies combination therapies that utilize dexamethasone and AKT inhibitor can kill LPCs in a subset of refractory T-ALL. Finally, these are the first studies performed in any model to follow single cell evolution as it relates to relapse, utilizing in excess of 6,000 transplant recipient animals and opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. This abstract is also presented as Poster A08. Citation Format: Jessica Blackburn, Sali Liu, Kimberly Dobrinski, Sarah Martinez, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR06.

Abstract PR05: Clonal evolution enhances leukemia-propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation

Abstract Leukemia progression and relapse are driven by molecularly distinct and often-rare cancer cells called leukemia-propagating cells (LPCs). If LPCs are retained following treatment, they will ultimately initiate relapse disease. Despite the substantial number of genetic lesions that have been identified in relapse samples and the contention that these mutations likely modulate response to therapy, acquired mutations that increase the overall frequency of tumor propagating cells following continued clonal evolution have not been reported in any cancer to date. Here, we have developed a transgenic zebrafish model where single fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) cells are transplanted into genetically identical recipient fish and functionally assessed for differences in leukemia propagating cell frequency, growth, and dexamethasone resistance. While subclonal variation was observed within single cells from the same primary leukemia, a subset of clones continued to evolve genetic lesions and epigenetic modifications to enhance growth and overall LPC frequency. A majority of evolved clones acquired activated AKT signaling, which simultaneously increased the number of leukemia propagating cells through activating the mTORC1 pathway, enhanced growth by stabilizing Myc protein levels, and rendered T-ALL cells resistant to dexamethasone, which was reversed by combined treatment with an AKT inhibitor. These results were confirmed using large-scale transgenic epistasis experiments and limiting-dilution cell transplantation studies. In total, our data suggest that diagnosis clones can stochastically acquire mutations necessary to survive treatment and drive relapse even before a patient begins treatment, with acquired mutations being independently selected based on important cancer phenotypes including elevated growth rate and leukemia propagating potential. Moreover, our work also identifies combination therapies that utilize dexamethasone and AKT inhibitor can kill LPCs in a subset of refractory T-ALL. Finally, these are the first studies performed in any model to follow single cell evolution as it relates to relapse, utilizing in excess of 6,000 transplant recipient animals and opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. This abstract is also presented as Poster B23. Citation Format: Jessica Blackburn, Sali Liu, Sarah Martinez, Kimberly Dobrinski, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia-propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR05.

Abstract 5185: Redefining the somatic landscape of pancreatic adenocarcinoma

Abstract Efforts to catalogue genomic abnormalities in pancreatic ductal adenocarcinoma (PDAC) have been challenging due to low tumor cellularity which requires deep sequencing to obtain adequate coverage of tumor DNA. To make whole genome sequencing (WGS) feasible with PDAC tumors, we have developed protocols for sequencing enriched tumor material by laser capture microdissection (LCM) from frozen samples and by flow sorting when fresh tissue is available. The enriched samples have a median tumor cellularity of 88% compared to a median cellularity of 12% in the bulk samples. Furthermore, the enrichment methods may also provide a source of normal or reference material, non-tumor cells in the case of LCM and CD45+ blood cells in the case of flow sorting, and eliminate the need for matched blood samples to call somatic variation. In conjunction with low quantity DNA input protocols for library construction, high quality next generation sequencing libraries are readily produced without sacrificing library complexity that enables somatic mutation, copy-number and structural variant detection at modest sequencing depths (∼50x). These methods are applicable to all tumor types where significant stromal contamination hinders somatic mutation detection. In addition to redefining the rates of known somatically altered genes in PDAC, such as CDKN2A and SMAD4, the high cellularity data set has also allowed us to evaluate PDAC tumor heterogeneity with the ability to detect sub-clonal variation that is not masked by non-tumor DNA. Moreover, in 50% of the samples sequenced to date, we observe evidence of punctuated evolution suggesting a catastrophic event during mutagenesis, which may be associated with the clinically observed rapid progression of the disease. The result of enhanced somatic mutation detection is a more comprehensive picture of the PDAC genomic landscape. Citation Format: Andrew M.K. Brown, Faiyaz Notta, Mathieu Lemire, Ilinca Lungu, Robert E. Denroche, Christina Yung, Kristen Geras, Lincoln Stein, John M. Bartlett, Thomas J. Hudson, Michael H.A. Roehrl, Steven Gallinger, John D. McPherson. Redefining the somatic landscape of pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2014-5185