Ancestral Clone Research Articles

Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates.

Abstract LB-341: Evolving functional heterogeneity in B-acute lymphoblastic leukemia

Abstract Current cancer therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells. However, recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. In over 50% of patients with relapsed acute lymphoblastic leukemia (ALL), the genetic clones present at relapse are not the dominant clone present at diagnosis, but have evolved from a minor or ancestral clone (Mullighan et al., Science, 2008). Previous work has shown that this subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells (L-IC) capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics. Patient samples were subjected to whole exome sequencing (WES), SNP analysis and RNA sequencing. Diagnosis-specific, relapse-specific, and shared variants at both clonal and subclonal frequencies were identified. Limiting dilution analysis by transplantation of CD19+ leukemic blasts into 870 immune-deficient mice (xenografts) identified no significant trend in enrichment in L-IC frequency between paired patient samples with a median frequency of 1 in 2691. Despite similar frequencies of L-IC, functional differences within identically sourced patient xenografts were observed, including increased leukemic dissemination of relapse cells to distal sites such as the central nervous system (CNS), differences in engraftment levels and differences in immunophenotypes. Targeted-sequencing and copy number analysis of the xenografts, in comparison to the patient sample from which they were derived, has uncovered clonal variation and the unequivocal identification of minor subclones ancestral to the relapse in xenografts transplanted with the diagnostic sample from 8 patients. Some of these subclones are rare and were not captured through standard WES analysis of the patient samples, highlighting the value of xenografting to functionally identify and viably isolate subclones for further study. Interrogation of the therapeutic responses of the ‘relapse-like’ diagnosis subclones in secondary xenografts displayed differential resistance to standard chemotherapeutic agents (vincristine and L-asparaginase) pre-existing in the patient diagnosis samples prior to treatment. Furthermore, investigation of different sites of leukemic infiltration in the xenografts provided evidence of distinct clonal selection in the CNS, a known site of disease relapse, in comparison to the bone marrow. Using this data we can begin to draw the evolutionary paths to relapse. We have shown evidence that minor subclones at diagnosis, ancestral to the relapsing clone, possess functional advantages over other diagnostic clones. Overall, this work provides a substantial advance in connecting genetic diversity to functional consequences, thereby furthering our understanding of the heterogeneity identified in B-ALL and its contributions to therapy failure and disease recurrence. Citation Format: Stephanie M. Dobson, Robert Vanner, Esmé Waanders, Olga I. Gan, Jessica McLeod, Ildiko Grandal, Debbie Payne-Turner, Michael Edmonson, Zhaohui Gu, Xioatu Ma, Yiping Fan, Pankaj Gupta, Sagi Abelson, Michael Rusch, Ying Shao, Scott Olsen, Geoffrey Neale, John Easton, Cynthia J. Guidos, Jayne S. Danska, Jinghui Zhang, Mark D. Minden, Charles G. Mullighan, John E. Dick. Evolving functional heterogeneity in B-acute lymphoblastic leukemia. [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 LB-341.

Abstract 2381: A multiancestral model of colorectal cancer: in vivo evidence that early heterogeneity contributes to cancer progression

Abstract Background: Intratumoral heterogeneity has been linked to tumor progression and chemotherapy resistance in the clinic. Recent models of colorectal tumor evolution indicate that heterogeneity likely arises early during the first few cell divisions and is maintained in a non-Darwinian fashion with discrete clones coevolving. However, how this heterogeneity arises is not fully understood. We propose that some cancers are derived from multiple unique ancestors, and that discrete clones enhance establishment, growth, progression, and resistance to therapy. Methods: Mice carrying the Min allele of Apc and expressing a constitutively active form of PI3K in a subset of colonic epithelial cells develop multiple adenomas and adenocarcinomas in the colon. Cell lineage tracing and fluorescent endoscopy were used to follow the progeny of individual founding cells through establishment, growth, progression, and response to targeted therapy. Cell sorting and 3D tumor spheroid co-culture were used to further examine the growth behavior and treatment response in vitro. Results: Nearly half (44%; 30/68) of the tumors were derived from at least two ancestral clones. The presence of multiple clones was associated with an increased likelihood of a tumor becoming invasive (p = 0.006). Moreover, each clone was more invasive within multi-ancestral tumors than within their homotypic counterparts, indicating that the increased invasion is shared among clones rather than owing to a single dominant clone (p = 0.05). Additionally, the presence of both clones appeared to protect susceptible clones from targeted therapy. In vitro experiments demonstrated that co-evolved clones adopted similar growth patterns, whereas independently evolved clones did not. Conclusions: Taken together, these data strongly indicate that distinct tumor founding cells and their coevolving progeny can contribute to tumor establishment and moreover can enhance growth and survival during tumor progression and response to therapy. Citation Format: Alyssa A. Leystra, Brook Luers, Junbo Son, Chelsie K. Sievers, Amanda M. Wisinger, Alexander R. Schwartz, Christopher D. Zahm, Kristina A. Matkowskyj, Dawn M. Albrecht, Linda Clipson, Dustin A. Deming, Michael A. Newton, Richard B. Halberg. A multiancestral model of colorectal cancer: in vivo evidence that early heterogeneity contributes to cancer progression. [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 2381.

High genotypic diversity among methicillin-resistant Staphylococcus pseudintermedius isolated from canine infections in Denmark.

BackgroundMethicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged globally in companion animals in the last decade. In Europe, the multidrug-resistant sequence type (ST)71 is widespread, but recently other clones have appeared. The objective of this study was to examine genotypic diversity and antimicrobial resistance of clinical MRSP isolates obtained from dogs, including dogs sampled on multiple occasions, in Denmark over a six-year period. For that purpose a total of 46 clinical MRSP isolates obtained from 36 dogs between 2009 and 2014 were subjected to antimicrobial susceptibility testing, multilocus-sequence typing (MLST) and SCCmec typing.ResultsTwenty-three sequence types were identified with ST71, mostly associated with SCCmec II-III, as the most common occurring in 13 dogs. Among the remaining 33 isolates, 19 belonged to clonal complex (CC)258 comprising ST258-SCCmec IV and its single- and double-locus variants. These were susceptible to 4–7 of the 22 antibiotics tested, whereas CC71 isolates were susceptible to only 2–5 antibiotics. Clone-specific differences were especially pronounced for fluoroquinolones and aminoglycosides with most CC71 isolates being resistant and almost all CC258 isolates being susceptible. Sixteen of the 19 CC258 isolates had oxacillin MICs of 0.5 g/L, whereas MICs for CC71 isolates were consistently above 4 g/L. Four of five dogs representing multiple isolates had distinct STs on different sampling events.ConclusionsThe overall genotypic diversity of MRSP is high in Denmark indicating multiple acquisitions of SCCmec into distinct clones, and mutational evolution, which appears to be particularly rapid for certain ancestral clones such as ST258. ST71-SCCmec II-III is the most common MRSP lineage and is typically multidrug-resistant. CC258-SCCmec IV isolates, which emerged in Denmark since 2012, display susceptibility to a wider range of antimicrobials. The isolation of distinct STs in individual dogs over time suggests repeated exposure or short-term genetic evolution of MRSP clones within patients.

Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells

Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution.

Abstract A25: Evolving functional heterogeneity in B-acute lymphoblastic leukemia

Abstract Despite high survival rates for children with acute lymphoblastic leukemia (ALL), only 40% of adult patients will achieve long-term disease-free survival, and relapses in both pediatric and adult ALL are often fatal. B-ALL leukemic blasts exhibit considerable subclonal genomic diversity. In 50% of patients, the clones present at relapse are not the dominant clone at diagnosis, but have evolved from a minor or ancestral clone. B-ALL subclonal diversity also exists in leukemia-initiating cells (L-IC) that are functionally capable of initiating xenografts. In order to investigate the clonal evolution during disease progression and link genetic diversity with functional characteristics, including differentiation, migratory properties, L-IC frequency and therapy resistance, we performed in depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics. Time to relapse ranged from 6 to 97 months. Patient samples were subjected to whole exome/genome sequencing, SNP analysis and RNA sequencing. Diagnosis-specific, relapse-specific and shared variants at both clonal and subclonal frequencies were identified. Limiting dilution analysis by transplantation of CD19+ leukemic blasts into immune-deficient mice (xenografts) identified no significant trend in enrichment in L-IC frequency between paired patient samples with a median frequency of 1 in 2691. Despite similar frequencies of L-IC, functional differences within identically sourced patient xenografts were observed, including increased leukemic dissemination of relapse cells to the spleen and/or central nervous system, differences in engraftment levels and differences in immunophenotypes. Copy number analysis and ongoing variant sequencing of the xenografts, in comparison to the patient sample from which they were derived, has uncovered clonal variation and the outgrowth of ‘relapse-like’ subclones in xenografts transplanted with the diagnostic sample. Interrogation of the therapeutic response of these subclones in secondary xenografts displayed evidence of resistance to standard chemotherapeutic agents (vincristine and L-asparaginase). Therefore we have shown evidence that relapse subclones/ ancestral clones present at diagnosis possess functional advantages over other diagnostic clones. Overall, this work will provide further understanding of the heterogeneity identified in B-ALL and how it contributes to lymphoid leukemogenesis, therapy failure, and disease recurrence. Citation Format: Stephanie M. Dobson, Robert Vanner, Esmé Waanders, Jessica McLeod, Olga I. Gan, Zhaohui Gu, Debbie Payne-Turner, Xiaotu Ma, Yiping Fan, Pankaj Gupta, Michael Rusch, John Easton, Cynthia J. Guidos, Jayne S. Danska, Jinghui Zhang, Mark D. Minden, Charles G. Mullighan, John E. Dick. Evolving functional heterogeneity in B-acute lymphoblastic leukemia. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A25.

Abstract PR08: Evidence for extra-ovarian origin and peritoneal metastases that precede ovarian carcinomas in high-grade serous ovarian cancer.

Abstract High-grade serous (HGS) ovarian cancer is the most lethal form of epithelial ovarian cancer because it represents the most common histologic subtype and usually present at an advanced stage. Due to its advanced stage presentation, HGS ovarian cancer has been described as arising de novo from undefined precursor lesions that progress rapidly to advanced disease. To gain insights into clonal heterogeneity and progression to distant metastases, we analyzed single nucleotide variations (SNVs) from nine regions of carcinomas from four patients with HGS ovarian cancer. Three primary sites, three metastases from omentum, and three metastases from bowel were collected from each patient and subjected to RNA sequencing and SNV analysis. Although a modest number (23.62%) of SNVs are shared within the same anatomic site, only limited number (1.26%) of SNVs are shared among all 9 sites, suggesting a high degree of intratumor heterogeneity in these patients. Truncal mutations, such as TP53 mutations, are shared among all sites and observed at high allele frequencies. In addition, mutations in genes associated with epigenetic modifiers, such as SETD2, CHD8, KDM5A, SMARCA1 and ARID5B are observed as truncal mutations in these patients. Phylogenetic tree reconstruction of tumors from various sites suggests a concurrent ovarian and peritoneal metastases in one patient and peritoneal metastases preceding the ovarian carcinomas in three other patients. Finally, subclone inference from multi-region sequencing indicates the presence of multiple subclones at each site in these patients. Dominant subclones, present at metastases, are often ancestral clones in the clonal hierarchy, further providing evidence that peritoneal spreading occurs early in the clonal expansion. Collectively, these results provide evidence for early peritoneal spreading that often precedes ovarian carcinomas in HGS ovarian cancer. This abstract is also presented as Poster B44. Citation Format: Jeremy Chien, Viji Shridhar, Andrea Mariani. Evidence for extra-ovarian origin and peritoneal metastases that precede ovarian carcinomas in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR08.

The triploid East African Highland Banana (EAHB) genepool is genetically uniform arising from a single ancestral clone that underwent population expansion by vegetative propagation.

All East African Highland Banana varieties are genetically uniform having arisen from a single clone introduced to Africa. East African Highland bananas (EAHBs) are a subgroup of triploid (AAA genome) bananas of importance to food security in the Great Lakes region of Africa. Little is known about their genetic variation, population structure and evolutionary history. Ninety phenotypically diverse EAHB cultivars were genotyped at 100 SSR microsatellite markers to investigate population genetic diversity, the correlation of genetic variability with morphological classes, and evolutionary origins since introduction to Africa. Population-level statistics were compared to those for plantain (AAB) and dessert (AAA) cultivars representing other M. acuminata subgroups. EAHBs displayed minimal genetic variation and are largely genetically uniform, irrespective of whether they were derived from the distinct Ugandan or Kenyan germplasm collections. No association was observed between EAHB genetic diversity and currently employed morphological taxonomic systems for EAHB germplasm. Population size dynamics indicated that triploid EAHBs arose as a single hybridization event, which generated a genetic bottleneck during foundation of the EAHB genepool. As EAHB triploids are sterile, subsequent asexual vegetative propagation of EAHBs allowed a recent rapid expansion in population size. This provided a basis for emergence of genetically near-isogenic somatic mutants selected across farmers and environments in East Africa over the past 2000 years since EAHBs were first introduced to the African continent.

Sequential Molecular Characterization Based Delineation of Potential Driver Aberrations in ACUTE Myeloid Leukemia Following Myelodysplastic Syndrome

Abstract Background: Myelodysplastic syndrome (MDS) patients exhibit an increased risk of progression towards secondary acute myeloid leukemia (sAML) with poor prognosis. The mechanisms underlying transformation from MDS to sAML are largely unknown. Aims: To identify genetic lesions associated with transformation in AML [copy number alterations (CNA), chromothripsis, uniparental disomies (UPD), and gene mutations], we performed genome-wide SNP array 6.0 profiling and mutational screening of 9 AML associated genes (RUNX1, TP53, NPM1, NF1, FLT3, ASXL1, DNMT3A, IDH1 and IDH2) in 42 paired MDS/sAML samples including 5 relapsed AML samples (rAML) [WHO MDS categories: RA (n=5), RCMD (n=12), RAEB-I (n=10), RAEB-II (n=8), 5q- (n=1), CMML-1 (n=1), CMML-2 (n=1), unclassifiable (n=4)]. In addition, whole exome sequencing (WES) was performed in 10 selected cases. Results: In total, 26 (62%) patients acquired genomic aberrations [CNAs and/or UPDs] at diagnosis of sAML as compared to the corresponding MDS sample. Most frequent numerical aberrations were trisomy 8 (n=7) and monosomy 7 (n=4); recurrent submicroscopic losses were identified at 17q11.2 (n=6, encompassing NF1), 17p13 (n=2, TP53), 12p13 (n=2, ETV6), and 21q22.12 (n=2, RUNX1); biallelic 17q11.2 and 21q22.12 losses were detected in one sAML each. Of note, all chromosome 5 alterations (n=11) were already present in MDS. Six (14%) cases acquired UPDs affecting the regions 1p, 11q, 13q, 20q, and 21q (n=2). Paired sequencing of candidate genes in 1p, 13q, and 21q delineated UPD related homozygous mutation patterns for pre-existing heterozygous NRAS, FLT3 -ITD, and RUNX1 mutations in sAML. Acquired AML associated gene mutations were identified in 11 (26%) patients with RUNX1 mutations being the most frequent. Sequential sequencing revealed mutation incidences in MDS and sAML as follows: RUNX1, 17% and 26%; TP53, 19% and 21%; ASXL1, 14% and 17%; DNMT3A, 12% and 14%; NF1, 10% and 14%; NPM1, 7% each; FLT3, 2% and 5%; IDH2, 2% and 5%; and IDH1, 2% each, respectively. While in the NPM1 mutated sAML cases the mutation was already present in the MDS sample (RCMD, n=2; RAEB-I, n=1), all cases transformed quickly within 5 to 6 months into AML compared to NPM1 wildtype MDS patients with a median time to transformation of 14 months. Other variables predicting inferior transformation free survival were biallelic TP53 alteration (n=5; p=.006), chromothripsis (n=4; p=.03), and presence of >4 CNAs (n=10; p=.04). Additional analysis of 5 rAML samples displayed aberration patterns closely related to the sAML in 4 cases; only one rAML seems to be evolved from an ancestral pre-MDS clone. In selected cases with available material (n=10) WES identified an average of 3.5 additional mutations per sAML. These included not only mutations in genes known to be associated with transformation (like e.g. NRAS, RUNX1, ETV6,and FLT3) but also in potential novel driver genes such as e.g. AFF1, NSD1, and STAG2. In accordance, the mutational pattern was not only significantly enriched for genes known to be associated with AML, but also for genes located in commonly gained regions of tumor genomes. Summary/Conclusions: Over two thirds (71%) of MDS cases acquired additional genetic abnormalities during progression to sAML as detected by SNP profiling and conventional Sanger sequencing; WES suggests that in all cases additional mutations are acquired. The genetic spectrum of our cohort demonstrates a multistep process for leukemogenesis with mutations and genomic aberrations occurring over time. Deletion 5q and mutations in DNMT3A and ASXL1 seem to be early events usually already found in MDS, and UPDs and alterations of RUNX1 and NF1 rather late events acquired in sAML. Ongoing extensive molecular analyses will further unravel the genetic aberrations involved in this multistep process. Supported by: FP7 NGS-PTL project. LB and KD contributed equally. Disclosures Schlenk: Pfizer: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Teva: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Arog: Honoraria, Research Funding.

Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse

AbstractAcute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Abstract IA25: Targeting genetic heterogeneity in multiple myeloma through immune activation.

Abstract IA25: Targeting genetic heterogeneity in multiple myeloma through immune activation.

Abstract IA39: The genetic basis of diffuse large B cell lymphoma.

Abstract Diffuse large B cell lymphoma (DLBCL), the most common form of human lymphoma, is a heterogeneous neoplasm comprising multiple genetic, phenotypic and clinical subtypes, approximately 50% of which are incurable. These tumors may arise de novo or from the transformation of more indolent lymphomas, as observed in 30-40% of follicular lymphoma (FL) cases. Over the past decade, the introduction of next-generation sequencing technologies combined with genome-wide copy number analysis has allowed to comprehensively define the genomic landscape of this disease, leading to the identification of multiple genes and signaling pathways that are dysregulated by genetic lesions and represent potential targets for diagnosis and therapy. In addition to perturbations in the transcriptional control of apoptosis, differentiation and DNA damage responses or NF-κB activation, these efforts revealed the frequent targeting of genes implicated in chromatin remodeling and immune recognition/surveillance. In particular, one of the most commonly disrupted programs in both de novo DLBCL, FL and transformed FL (tFL) comprises genes encoding histone/chromatin modifying enzymes, including methyltransferases (MLL2, EZH2) and acetyltransferases (CREBBP, EP300), which may play a central role during malignant transformation. Interestingly, sequential analysis of tumor samples during transformation of FL to tFL indicates that inactivating mutations of CREBBP and MLL2 are acquired early in the evolutionary history of a common mutated ancestral clone. The disruption of these genes by genetic alterations may thus contribute to malignant transformation by shaping the epigenetic landscape of the cancer cell as well as by perturbing specific biological programs, in part through the altered balance between acetylation-mediated activation of the p53 tumor suppressor and inactivation of the BCL6 proto-oncogene. This talk will cover recent advances in understanding the genetic basis of DLBCL, as revealed by in vitro and in vivo studies. Citation Format: Laura Pasqualucci. The genetic basis of diffuse large B cell lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA39.

Abstract NG02: Polyclonal and heterogeneous resistance to targeted therapy in leukemia

Abstract NG02: Polyclonal and heterogeneous resistance to targeted therapy in leukemia

Differentiation of a free-living alga into forms with ecto- and endosymbiotic associations with heterotrophic organisms in a 5-year microcosm culture

The ecological mechanisms underlying the diversification of autotrophic species into endosymbiotic lifestyles and the ways in which the evolution of endosymbiotic species is ecologically and evolutionarily affected by sister lineages/lines that are adapted to extra-host environments remain unclear. In this paper, we investigated a differentiation process of algal species in which an endosymbiotic type was differentiated phenotypically from a free-living ancestral clone, by using an experimental model called the CET microcosm, which contains a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila) cultured together without an external resource supply for over 5 years. We then analyzed the algal diversification process by comparing algal phenotypic properties regarding cell-aggregate formation and their effects on the survival of Tetrahymena (using a clone isolated on day 2668) in the absence of bacteria. We examined 13 Micractinium clones, including both ancestral and derived clones isolated from long-term (day 1819–1847) CET microcosm cultures. The results revealed that the free-living ancestral algal strain diversified in sympatry into an aggregate-forming type that associates with E. coli, and a non-aggregate-forming type that associates with Tetrahymena. Furthermore, a competition experiment revealed that the endosymbiotic (non-aggregate-forming) type was less adapted to the extracellular environment than the aggregate-forming type. This result suggests that severe competition with a nonendosymbiotic sister line in the extra-host environment favors the host-benefiting phenotype in an endosymbiotic line, because such symbionts can enhance the longevity of the host, thereby enabling the survival and reproduction of the symbiont within the host.

Tracking clonal diversity in metastatic prostate cancer progression.

193 Background: Genomic heterogeneity has been observed in a number of tumor types including prostate cancer. However, how subclonal tumor diversity changes during metastasis and progression to lethality remains unexplored. Large scale genomic analyses have reported the most prevalent somatic aberrations associated with the dominant clone of the tumor without permitting an analysis of subclonal complexity or how this complexity impinges on metastatic potential or resistance to treatment. Methods: To understand and track the evolution of lethal prostate cancer from initial therapy to end stage metastases, we performed longitudinal and multiregional sampling of tumors from 7 patients with lethal prostate cancer. We performed whole-genome sequencing, RNA sequencing, and SNP profiling. Computational approaches were used to reconstruct the genetic relationships and evolution of the tumors. These evolutionary tree reconstructions allowed us to observe the dynamics of chromoplexy and mutational processes along specific branches of tumor evolution. To refine the genetic landscape and spatial connections between subclones, we employed deep, targeted re-sequencing of variant loci in the original sequenced samples, in additional FFPE sites sampled from the organ confined tumors, and from blood. Results: We show that while all primary and metastatic prostate tumors share a single ancestral clone, metastases arise from subclones present at minor frequencies in the primary tumor. We reveal that individual metastases comprise mixtures of subclones indicative of intra-metastatic heterogeneity. We provide evidence for cross-metastatic site seeding and dynamic remolding of subclonal mixtures in response to therapy suggesting a distinct metastatic hierarchy. Ultra-deep sequencing of end-stage blood reveals the presence of diverse subclones with metastatic potential derived from various stages in the evolution of the tumor. Conclusions: Our results demonstrate unexpected complexity in the origins of both primary and metastatic prostate cancer, with distinct implications for treatment of advanced disease.

Tumor evolution and intratumor heterogeneity of an epithelial ovarian cancer investigated using next-generation sequencing.

BackgroundThe extent to which metastatic tumors further evolve by accumulating additional mutations is unclear and has yet to be addressed extensively using next-generation sequencing of high-grade serous ovarian cancer.MethodsEleven spatially separated tumor samples from the primary tumor and associated metastatic sites and two normal samples were obtained from a Stage IIIC ovarian cancer patient during cytoreductive surgery prior to chemotherapy. Whole exome sequencing and copy number analysis were performed. Omental exomes were sequenced with a high depth of coverage to thoroughly explore the variants in metastatic lesions. Somatic mutations were further validated by ultra-deep targeted sequencing to sort out false positives and false negatives. Based on the somatic mutations and copy number variation profiles, a phylogenetic tree was generated to explore the evolutionary relationship among tumor samples.ResultsOnly 6% of the somatic mutations were present in every sample of a given case with TP53 as the only known mutant gene consistently present in all samples. Two non-spatial clusters of primary tumors (cluster P1 and P2), and a cluster of metastatic regions (cluster M) were identified. The patterns of mutations indicate that cluster P1 and P2 diverged in the early phase of tumorigenesis, and that metastatic cluster M originated from the common ancestral clone of cluster P1 with few somatic mutations and copy number variations.ConclusionsAlthough a high level of intratumor heterogeneity was evident in high-grade serous ovarian cancer, our results suggest that transcoelomic metastasis arises with little accumulation of somatic mutations and copy number alterations in this patient.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1077-4) contains supplementary material, which is available to authorized users.

ARID1A Is Recurrently and Significantly Mutated in Follicular Lymphoma (FL) and Impairs DNA Repair Efficiency

FL is the most common indolent nodal lymphoma and considered incurable for the majority of patients. Virtually all patients eventually develop resistant disease, and up to 45% histologic transformation (tFL) with poor outcome. Alterations associated with progression to tFL include increased copy number variations (e.g. CDKN2A deletions, MYC amplification), acquisition of additional translocations (e.g. MYC or BCL6), and higher mutation load (including TP53). However the underlying molecular mechanisms promoting this genomic instability remain elusive. Interestingly, histologic transformation occurs at a remarkably constant rate of ~3% per year implying a rather stochastic process.We previously described convergent evolution for disruptive ARID1A mutations in FLs of a donor and the recipient patient following an allogeneic bone marrow transplantation (Weigert, Canc Discovery 2012). The independent acquisition of ARID1Aloss in two separate hosts after transplantation of an identical FL ancestor clone suggested (i) a later acquired event in the molecular ontogeny and (ii) functional relevance.ARID1A promotes the formation of SWI/SNF nucleosome remodeling complexes containing BRG1 or BRM, which catalyze disruption of DNA-histone contacts, thereby controlling chromatin condensation and DNA accessibility. Nucleosome-remodelling has been linked to DNA damage response, including nucleotide and base excision repair, homologous recombination and non-homologous end-joining (NHEJ). We thus hypothesized that ARID1Ahaplodeficiency might contribute to genetic and genomic instability in FL.In a cohort of 305 FL specimens, we identified 44 ARID1A mutations in 43 patients (14%). The majority of these mutations were disruptive (66%; 19 nonsense and 10 frame shift mutations), 6 cases harbored splice site mutations. MutSigCV analysis (Lawrence, Nature 2013) indicated that ARID1A was significantly mutated in FL, i.e. more often than expected by chance given background mutation processes. Consistent with our hypothesis, the number of non-synonymous mutations in 104 targeted genes was higher in ARID1A mutated FLs (5.6+0.34, mean+sem) compared to ARID1A wild-type (wt) FLs (4.7+0.12; p=0.0099), and similar compared to 17 FLs with TP53 mutations (5.6+0.12). Of note, ARID1A and TP53 mutations were mutually exclusive.For functional studies, we confirmed deleterious ARID1A alterations in 6 lymphoma cell lines (Namalwa, Karpas422, SUPHD-1, SU-DHL5, WSU-FSCCL, U-OH1). Immunoblotting showed decreased ARID1A protein expression in all 6 lines compared to 5 ARID1A wt lymphomas. Tet-induced re-expression of ARID1A in two tested ARID1A mutant cell lines (Namalwa and SUP-HD1) reduced cell proliferation compared to tet-induced control (LacZ), but had no effect in ARID1A wt OCI-Ly1.As a proof-of-principle experiment we assayed the impact of ARID1A loss on DNA double strand break (DSB) repair efficiency in genetically well defined mouse embryo fibroblasts (MEFs), avoiding uncontrolled bias introduced by the genetic and genomic complexity in lymphoma cell lines. Early passage MEFs from either wt or conditional ARID1A knock-out mice (ARID1Af/f; Gao, PNAS 2008) were transiently transfected or retrovirally transduced with self-excising Cre-recombinase (Silver&Livingston, Mol Cell 2001) or control, and irradiated with 2 Gy after 36 hrs. Cre-induced ARID1A loss was confirmed by Western blot. To assay NHEJ, the predominate DSB-repair pathway used by cells in G1 phase, we stained cells for γH2A.X and 53BP1. In 3 independent and blinded experiments, evaluation of >100 cells per condition demonstrated significantly more double-positive cells (>6 foci/cell) in ARID1A-/- MEFs (43+14%) compared to both ARID1Af/fMEFs (9+1%) and Cre-exposed wt MEFs (15+5%) at 16 hrs post irradiation, whereas there was no significant difference for ARID1Af/for wt MEFs with or without Cre at baseline (all <10%), and 1 hr post irradiation (all >50%). This data indicates that ARID1A loss slows the repair kinetics of NHEJ and delays the clearance of DSB.We conclude that ARID1A is recurrently and significantly mutated in FL. Most mutations are disruptive leading to functionally relevant ARID1A protein haplodeficiency. ARID1A loss impairs NHEJ, which might sensitize tumors to DNA damaging agents and irradiation, but also contribute to genetic and genomic instability promoting histologic transformation. DisclosuresNo relevant conflicts of interest to declare.

Association between the cytogenetic profile of tumor cells and response to preoperative radiochemotherapy in locally advanced rectal cancer.

Neoadjuvant radiochemotherapy to locally advanced rectal carcinoma patients has proven efficient in a high percentage of cases. Despite this, some patients show nonresponse or even disease progression. Recent studies suggest that different genetic alterations may be associated with sensitivity versus resistance of rectal cancer tumor cells to neoadjuvant therapy. We investigated the relationship between intratumoral pathways of clonal evolution as assessed by interphase fluorescence in situ hybridization (51 different probes) and response to neoadjuvant radiochemotherapy, evaluated by Dworak criteria in 45 rectal cancer tumors before (n = 45) and after (n = 31) treatment. Losses of chromosomes 1p (44%), 8p (53%), 17p (47%), and 18q (38%) and gains of 1q (49%) and 13q (75%) as well as amplification of 8q (38%) and 20q (47%) chromosomal regions were those specific alterations found at higher frequencies. Significant association (P < 0.05) was found between alteration of 1p, 1q, 11p, 12p, and 17p chromosomal regions and degree of response to neoadjuvant therapy. A clear association was observed between cytogenetic profile of the ancestral tumor cell clone and response to radiochemotherapy; cases presenting with del(17p) showed a poor response to neoadjuvant treatment (P = 0.03), whereas presence of del(1p) was more frequently observed in responder patients (P = 0.0002). Moreover, a significantly higher number of copies of chromosomes 8q (P = 0.004), 13q (P = 0.003), and 20q (P = 0.002) were found after therapy versus paired pretreatment rectal cancer samples. Our results point out the existence of an association between tumor cytogenetics and response to neoadjuvant therapy in locally advanced rectal cancer. Further studies in larger series of patients are necessary to confirm our results.

GE-15 * CLONAL EVOLUTION AND INTRATUMORAL HETEROGENEITY OF LOW-GRADE GLIOMA GENOMES

Low-grade gliomas frequently recur after surgical resection and may undergo malignant progression to a higher grade with a significantly worse prognosis. Understanding the origin and evolution of recurrences is critical for effectively treating residual disease to delay or prevent recurrence. Here, we extend previous work by sequencing the exomes of over 30 initial low-grade gliomas and their patient-matched recurrences to reconstruct the patterns of clonal evolution. We also sequence multiple spatially distinct samples from both initial low-grade and recurrent high-grade tumors to extensively dissect intratumoral heterogeneity. We identify a broad spectrum of genetic relatedness within tumor pairs, with the unexpected loss of canonical driver mutations upon recurrence. Therefore, resected portions of initial tumors can differ dramatically from tumorigenic portions of adjacent residual disease. The identification of common ancestral clones additionally reveals the order in which key driver mutations are acquired, allowing for a model of low-grade astrocytoma gliomagenesis. We have also previously shown that the treatment of low-grade gliomas with temozolomide induces hypermutation and alters the function of key cancer genes and their cognate pathways, potentially driving malignant progression. We extend that finding with an analysis of the evolution and intratumoral heterogeneity of additional hypermutated glioblastoma recurrences. These evolutionary trajectories have immediate ramifications for the use of mutagenic chemotherapies such as temozolomide, and for personalizing therapy to eliminate residual disease.

Isolation, identification and molecular characterization of Mycobacterium bovis from tuberculin-positive cattle in Hamedan province of Iran

IntroductionTuberculosis (TB) remains a major cause of death in many countries. Bovine tuberculosis caused by Mycobacterium bovis is one of the key members of the Mycobacterium tuberculosis complex. Current methods need long incubation times, which is in contrast to the necessity for rapid and accurate identification and isolation. Molecular techniques, such as RFLP fingerprinting, for accurate identification and differentiation of M. tuberculosis isolates are a more desirable approach. Materials and methodsIn a 12-month study plan, which began in November 2011, lymph nodes obtained from 92 samples of dairy cattle yielded tuberculin-positive marks that were collected from 10 different regions of the province; 43 isolates were identified by acid-fast culture. Using biochemical tests along with IS6110 specific fragment search, colonies were proved to belong to M. tuberculosis complex and more precisely M. bovis strains. PVUII enzymatic digestion of their DNA was followed by Southern blotting and hybridization with DR and PGRS markers. Finally, RFLP fragment hybridized with substrate BCIP and NBT was carried out, and their results were evaluated by GelproAnalyser software. ResultsAll 43 isolates out of the 92 samples that were analyzed by RFLP with two probes of DR and PGRS, interestingly, proved the circulation of an identical strain of M. bovis throughout the whole province. Discussion and conclusionAssessing the genomic patterns of all the strains and comparing the results with previous studies conducted in the area revealed that, while they represent the circulation of a common ancestral clone, as a preliminary successful achievement in industrial and semi-industrial dairy farms located in the zone of the tuberculosis control program, they still have a long way to go to meet the ultimate eradication goals.