Subclonal Cell Populations Research Articles

Abstract A132: Characterization of intratumoral heterogeneity in drug sensitivity and non-cell autonomous mechanisms of drug resistance using subclonal cell populations derived from a single breast cancer cell line

Abstract Intratumoral heterogeneity impacts both tumor progression and drug sensitivity. While intratumoral genetic heterogeneity has been extensively described, there is still little understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the intra tumor dynamics of different cell populations during anti-cancer compound treatment, and the role of non-cell autonomous mechanism in drug resistance. The objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer, identify interactions between clonal population influencing drug sensitivity, and use this to model the dynamics of sub populations in a tumor during anti-cancer compound treatment. We have characterized 31 subclonal cell populations (SCPs) that were generated by single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic and molecular heterogeneity with respect to morphology, proliferation rate, RNA expression and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation in the SCPs that expressed the greatest range of sensitivity (determined by IC50). To investigate whether interactions between clonal population can influence drug sensitivity heterogeneously responsive subclonal cell lines were transduced with lentiviruses encoding for the H2B-GFP or H2B-RFP fusion protein and then cocultured in the absence or presence of different compounds. We observed shifts in drug sensitivity for SCPs that were cocultured with differentially responsive clones. Examples include SCPs 19 and 27 two highly sensitive populations that were protected by the presence of less sensitive SCPs when under gemcitabine treatment, resulting in IC50-values that were 20-fold higher then when cultured alone. RNA-sequencing and RPPA analysis will be used to characterize this non-cell autonomous mechanism of resistance and the relationship with identified transcriptional programs or genes which expression correlates with drug sensitivity. We are also generating and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations and drug sensitivity within heterogenous mixtures. Moreover, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combination treatments or schedules. Citation Format: Chandler M Friend, Joan S Brugge, Hendrik J Kuiken, Steven M Corsello, Christopher C Mader. Characterization of intratumoral heterogeneity in drug sensitivity and non-cell autonomous mechanisms of drug resistance using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A132. doi:10.1158/1535-7163.TARG-19-A132

Abstract CN06-01: Lineage plasticity and the neuroendocrine phenotype as a resistance mechanism in prostate cancer

Abstract Loss of androgen receptor (AR) signaling dependence occurs in approximately 15-20% of treatment resistant prostate cancers, and this may manifest clinically as transformation from a prostate adenocarcinoma histology to a castration resistant neuroendocrine prostate cancer (CRPC-NE). The diagnosis of CRPC-NE currently relies on a metastatic tumor biopsy, which is invasive for patients and often challenging to diagnose due to tumor heterogeneity. By studying whole exome sequencing and whole genome bisulfite sequencing of metastatic tumor biopsies and matched cell free DNA (cfDNA), we identified distinct genomic and epigenomic features of CRPC-NE and patterns of tumor evolution that occur during clinical progression and treatment resistance. Loss of RB1 and TP53 are enriched in CRPC-NE compared with castration resistant prostate adenocarcinoma, as are significant changes in DNA methylation, which are detectable by cfDNA. There was significantly higher concordance between cfDNA and biopsy tissue genomic alterations in CRPC-NE patients compared to castration resistant adenocarcinoma, supporting greater intra-individual genomic consistency across metastases. cfDNA and serial tumor biopsies allowed for the tracking of dynamic clonal and subclonal tumor cell populations as patients progressed and identified CRPC-NE alterations sometimes prior to the development of clinical features. CRPC-NE appears to arise clonally from a prostate adenocarcinoma precursor likely through a dynamic process of clonal selection and trans-differentiation that occurs during resistance to AR-directed therapies. In addition to loss of AR expression and canonical AR signaling, we identified a dysregulation of key pathways in CRPC-NE including loss of Notch signaling, reactivation of developmental programs, and gain of neuronal/neuroendocrine programs including critical lineage determining transcription factors (LDTFs) such as ASCL1, MYCN, BRN2, pointing to novel biomarkers and potential targets for CRPC-NE. Patient-derived organoids and xenografts were used to interrogate the timing by which genomic and epigenomic changes and LDTFs contribute to lineage plasticity and the development of CRPC-NE as a resistance mechanism in prostate cancer. Citation Format: Himisha Beltran, Sheng-Yu Ku, Vincenza Conteduca, Alessandro Romanel, Loredana Puca, Michael Sigouros, Juan Miguel Mosquera, Scott T. Tagawa, Andrea Sboner, Olivier Elemento, David Goodrich, David Rickman, Amina Zoubeidi, Francesca Demichelis. Lineage plasticity and the neuroendocrine phenotype as a resistance mechanism in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN06-01. doi:10.1158/1535-7163.TARG-19-CN06-01

Dissecting Heterogeneity of Tumor Cells and Their Microenvironment in Refractory Multiple Myeloma

Introduction: Despite significant improvements in therapy during the last decade, most multiple myeloma (MM) patients develop refractory disease over time. Treatment of refractory MM is a major challenge, likely due to the still poorly characterized inter- and intratumor heterogeneity at this stage of the disease, and the complex interplay of MM cells with the microenvironment (ME). In particular, there is an urgent need to unravel how these features of MM are linked to molecular mechanisms of drug resistance. Methods: We resolved the cellular composition, underlying transcriptional inter- and intra-patient heterogeneity and molecular treatment response of relapsed/refractory MM by single cell RNA sequencing (scRNA-seq). Using droplet-based microfluidics, ~230,000 single cell gene expression profiles from bone marrow (BM) aspirates of 21 patients sorted into CD138+and CD138- fractions were acquired, allowing for a comprehensive analysis of both MM cells as well as their ME. Patients had a median of 4 prior lines of therapy including both a proteasome inhibitor and an immunomodulator and were refractory to their immediate prior line of therapy at time of sampling. In addition, paired samples before either pomalidomide- or carfilzomib-based therapies were analyzed for 16/21 patients. Genomic aberrations in individual patients were mapped by interphase fluorescence in situ hybridization. Cells were clustered and CD138+ MM subtypes as well as immune cell-types of the ME were identified from their single cell transcriptomes and a copy number variation (CNV) analysis. As a reference for non-malignant cells and to construct a developmental B-cell trajectory the Human Cell Atlas BM scRNA-seq reference dataset was used. To characterize interactions of MM cells with their ME, the correlated expression of ligand-receptor pairs was exploited. Results: The analysis of inter- and intra-tumor heterogeneity of molecular MM subgroups revealed distinct transcriptome signatures with contributions that could be assigned to differences in heavy and light chain immunoglobulin expression as well as known genomic alterations, including t(11;14), t(4;14) and hyperdiploidy. MM cells from individual patients largely maintained a plasma cell specific gene expression profile but a partial loss of plasma cell identity was detected based on mapping to a developmental B-cell trajectory. It was characterized by the upregulation of subgroup transcriptome signatures associated with earlier stages of B-cell development in almost 50% of patients, such as a pre-B or mature B cell-like phenotype. Within individual samples, subclonal MM cell populations with specific gene expression programs were resolved based on the CNV analysis and included those characterized by expression of the immune-activator CD27 and the modulator of WNT signaling FRZB. The analysis of longitudinally collected samples revealed both changes in the cell subtype cluster structure as well as drug-specific adaptation of gene expression programs in distinct subpopulations persisting or emerging at relapse. These profile changes were characterized by e.g. downregulation of Myc target genes upon pomalidomide treatment or induction of heat shock proteins under carfilzomib. Within the ME of refractory MM patients, we observed that the fraction of B cells and CD4+ T cells was strongly reduced while CD14+ and CD16+ monocytes as well as dendritic cells expanded. Notably, the immune checkpoint protein PD-1H (aka VISTA) that inhibits T cell activation was highly expressed in cell types from the myeloid compartment in contrast to healthy donors. Further, a ligand-receptor analysis revealed that MM cells displayed the strongest interactions with monocytes, which were mediated by MIF, BAFF and other cytokines. Conclusions: Our study demonstrates the value of scRNA-seq analysis for identifying crucial transcriptome features that classify refractory MM subtypes and their evolution in response to treatment including regulation of drug resistance associated signaling pathways. Our data suggest that refractory MM cells shape the myeloid compartment in the BM to generate an immune suppressive ME. Understanding the evolution of MM cell heterogeneity and the bone marrow milieu in refractory disease will lead to novel treatment approaches and eventually improve patient outcome. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:John-Hopkins University: Research Funding; Molecular Partners: Research Funding; Amgen: Consultancy, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding.

MNGI-09. MENINGIOMA WITH MULTIPLE DRIVERS: GENOMIC LANDSCAPE AND CLINICAL CORRELATIONS

Abstract BACKGROUND Previous studies have established several meningioma genomic subgroups, defined by the mutually exclusive genomic drivers. However, we distinguished a small subset of meningiomas simultaneously harboring multiple drivers from different genomic subgroups, thus referred to as “multiple-driver meningiomas”, that has not been previously investigated. We aimed to characterize the genomic landscape and clinical features of multiple-driver meningiomas. METHODS We identified 42 cases of multiple-driver meningiomas from a cohort of 3,016 cases screened for genomic drivers via molecular inversion probe sequencing (MIPS) and/or whole-exome sequencing (WES) previously in our laboratory. All driver mutations were validated via Sanger sequencing and chromosome 22 loss was accessed with qPCR. Clinical information was collected and genome-to-clinical correlations were statistically analyzed. We selected 10 multiple-driver meningioma cases for tumor-normal WES for further characterization of genomic architecture. RESULTS Multiple-driver meningiomas were significantly enriched for NF2 alteration (p< 0.001), had significantly higher WHO grade (p= 0.005) and proportion of recurrent tumors (p= 0.007) when compared with “single-driver meningiomas”, i.e. harboring driver alterations from a single genomic subgroup. Among female cases, those with multiple drivers were nearly four times more likely to be high-grade (II or III) compared to single-driver female samples (48.6% vs. 12.6%, p< 0.001). Among tumors harboring NF2 alteration, multiple-driver meningiomas demonstrated skull base predilection compared with single-driver meningiomas (50.0% vs. 27.9%, p= 0.005). WES analysis of 10 multiple-driver meningiomas identified 1p and/or 14q loss in 60% of cases. Clonality analysis revealed the presence of sub-clonal cell populations in 9 cases with all driver mutations clustered into the founding clone in 7 cases, suggesting their acquisition in the early phase of tumor development. CONCLUSION Multiple-driver meningiomas demonstrate distinct genomic and clinical features that distinguish them as clinically aggressive. They frequently occur in surgically challenging skull base locations, and are more prevalent among high-grade and recurrent cases.

A general approach for detecting expressed mutations in AML cells using single cell RNA-sequencing

Virtually all tumors are genetically heterogeneous, containing mutationally-defined subclonal cell populations that often have distinct phenotypes. Single-cell RNA-sequencing has revealed that a variety of tumors are also transcriptionally heterogeneous, but the relationship between expression heterogeneity and subclonal architecture is unclear. Here, we address this question in the context of Acute Myeloid Leukemia (AML) by integrating whole genome sequencing with single-cell RNA-sequencing (using the 10x Genomics Chromium Single Cell 5’ Gene Expression workflow). Applying this approach to five cryopreserved AML samples, we identify hundreds to thousands of cells containing tumor-specific mutations in each case, and use the results to distinguish AML cells (including normal-karyotype AML cells) from normal cells, identify expression signatures associated with subclonal mutations, and find cell surface markers that could be used to purify subclones for further study. This integrative approach for connecting genotype to phenotype is broadly applicable to any sample that is phenotypically and genetically heterogeneous.

Abstract 3775: Characterization of intratumoral heterogeneity in drug sensitivity and modeling of drug combination effects using subclonal cell populations derived from a single breast cancer cell line

Abstract Intratumoral heterogeneity can have profound effects on tumor progression and drug resistance. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the dynamics of tumor cell subpopulations during treatment with anti-cancer agents and non-cell autonomous mechanisms of drug resistance. The overall objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer and to model and evaluate the effect of drug combinations on the dynamics of subpopulations within heterogeneous mixtures. We have characterized 31 subclonal cell populations (SCPs) that were generated through single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable heterogeneity with respect to morphology, proliferation rate, colony formation in soft agar and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation, including GPX4 inhibitor ML-210 and gemcitabine, to which the SCPs displayed 12-fold and 150-fold differences in the EC50, respectively. The results of these validation experiments were used to identify transcriptional programs and individual genes which expression correlates with drug sensitivity. We have generated and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. To test the effect of individual drugs and combinations, as well as different treatment schedules on heterogeneous populations, we will examine the dynamics of individual SCPs within mixed cultures. To this end, we have transduced 22 SCPs with CloneTracer lentiviruses, each encoding for a unique DNA barcode. The distribution of these unique barcodes can be determined by next generation sequencing. Previously, we have found that propagation of a 22 SCP mixture results in reproducible changes in the composition of mixed monolayer cultures with approximately half of the SCPs having a relative abundance of 1% each in the mixed population following propagation for four months. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations within heterogenous mixtures. Moreover, using this panel of SCPs, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combinations. Citation Format: Hendrik J. Kuiken, Chandler M. Friend, Steven M. Corsello, Christopher C. Mader, Joan S. Brugge. Characterization of intratumoral heterogeneity in drug sensitivity and modeling of drug combination effects using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3775.

Whole-genome sequencing of human malignant mesothelioma tumours and cell lines.

Pleural mesothelioma is a cancer of serosal surfaces caused by environmental exposure to asbestos. Clinical outcome remains poor and while trials of new treatments are ongoing it remains an understudied cancer. Mesothelioma cell lines can readily be grown from primary tumour and from tumour cells shed into pleural effusion with the latter representing a particularly valuable source of DNA in clinical settings, procurable without the need for additional invasive procedures. However, it is not well understood how accurately patient-derived cultured tumour cells represent the molecular characteristics of their primary tumour. We used whole-genome sequencing of primary tumour and matched cultured cells to comprehensively characterize mutations and structural alterations. Most cases had complex rearranged genomes with evidence of chromoanagenesis and rearrangements reminiscent of chromoplexy. Many of the identified driver mutations were structural, indicating that mesothelioma is often caused by structural alterations and catastrophic genomic events, rather than point mutations. Because the majority of genomic changes detected in tumours were also displayed by the genomes of cultured tumour cells, we conclude that low-passage cultured tumour cells are generally suitable for molecular characterization of mesothelioma and may be particularly useful where tissue samples with high tumour cell content are not available. However, the subclonal compositions of the cell lines did not fully recapitulate the subclonal diversity of the primary tumours. Furthermore, longitudinal acquisition of major alterations in subclonal cell populations was observed after long-term passaging. These two factors define limitations of tumour-derived cell lines as genomic substrate for clinical purposes.

A Systematic Pan-Cancer Analysis of Genetic Heterogeneity Reveals Associations with Epigenetic Modifiers.

Intratumor genetic heterogeneity (ITH) is the main obstacle to effective cancer treatment and a major mechanism of drug resistance. It results from the continuous evolution of different clones of a tumor over time. However, the molecular features underlying the emergence of genetically-distinct subclonal cell populations remain elusive. Here, we conducted an exhaustive characterization of ITH across 2807 tumor samples from 16 cancer types. Integration of ITH scores and somatic variants detected in each tumor sample revealed that mutations in epigenetic modifier genes are associated with higher ITH levels. In particular, genes that regulate genome-wide histone and DNA methylation emerged as being determinant of high ITH. Indeed, the knockout of histone methyltransferase SETD2 or DNA methyltransferase DNMT3A using the CRISPR/Cas9 system on cancer cells led to significant expansion of genetically-distinct clones and culminated in highly heterogeneous cell populations. The ITH scores observed in knockout cells recapitulated the heterogeneity levels observed in patient tumor samples and correlated with a better mitochondrial bioenergetic performance under stress conditions. Our work provides new insights into tumor development, and discloses new drivers of ITH, which may be useful as either predictive biomarkers or therapeutic targets to improve cancer treatment.

Simulating genetically heterozygous genomes in the tumour tissue according to its clonal evolution history

Tumours contain multiple, genetically diverse subclonal populations of cells that have evolved from a single progenitor population. Currently, next-generation sequencing (NGS) and the third generation sequencing (TGS) have recently allowed us to develop algorithms to quantitatively dissect the extent of heterogeneity within a tumour, resolve cancer evolution history and identify the somatic variations and aneuploidy events with subclonal frequency. However, existing tumour NGS data has no ground truth annotation to validate all these NGS based tumour analysis algorithms. To benchmark these algorithms, a powerful tumour genome simulation tool which could simulate all the distinct subclonal genomes with diverse aneuploidy events and somatic variations according to the given tumour evolution history is in need. We provide a simulation package, Pysubsim-tree, which could simulate the tumour genomes according to their evolution history defined by the somatic variations and aneuploidy events. Pysubsim-tree is free, open source, available at: https://github.com/dustincys/pysubsimtree.

PDTM-09. DIFFUSE INTRINSIC PONTINE GLIOMA AND PEDIATRIC GLIOBLASTOMA DERIVED-EXOSOMES HAVE SPECIFIC ONCOGENIC SIGNATURES

Diffuse intrinsic pontine glioma (DIPG) and pediatric glioblastoma (pGBM) are heterogeneous brain tumors characterized by different anatomical and molecular subgroups and the presence of genetically and phenotypically distinct subclonal cell populations. It is recognized that exosomes mediate cross-talk among tumor cells. We hypothesize that there are different exosome-mediated paracrine signaling promoting tumour progression in DIPG and pGBM. Our aim was to determine the specific DIPG and pGBM-derived exosome oncogenic signatures. We used a panel of fifteen patient primary-derived cell lines, which included nine DIPG (seven H3.3 K27M, one H3.3 K27M/ACVR1 and one H3.1 K27M/ACVR1), one diffuse midline glioma H3.3 K27M and three GBM (one H3.3 G34R and two histone WT). Conditioned medium was collected from cells maintained under stem-cell culture condition, adherent on laminin and/or as neurospheres (NS), and exosomes harvested through serial centrifugations. Electron microscopy demonstrated that the isolated microvescicles are exosomes sized between 50–80 nm. DIPG derived-exosomes appeared to have a variable cargo of total protein (µg)/106 cells, which was higher than for pGBM-exosomes. Proteomic analysis revealed that proteins associated with vesicle docking, exocytosis and synaptic transmission were exclusively enriched in pontine-derived exosomes, while cell-cell and cell-matrix interaction proteinswere exclusive tohemispheric ones. Proteins in common to the two locations were involved in metabolism and energy pathways. Interestingly, principle component analysis on the different molecular subgroups suggests that ACVR1 may be not implicated in the exosomal proteomic signature. Exosomal miRNA profile appeared to be driven by the two main histone mutated subgroups H3.3 K27M and H3.1 K27M with the latter overexpressing hypoxia and angiogenic-associated miRNAs, leading to distinct oncogenic programs with different specific potential therapeutic targets. This study aimed to development new diagnostic/prognostic tools for DIPG and pGBM patients. Further investigations are aimed to identify new therapeutic strategies to inhibit the cross-talk among glioma subpopulations.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells.

The failure to develop effective therapies for paediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively map assess the extent to which this was present in these tumours through subclonal genomic analyses, and to determine whether distinct tumour subpopulations may interact to promote tumorigenesis by generating novel subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced cases revealed multiple tumour subclones, spatially and temporally co-existing in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations which we propose co-operate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, confer an abrogated DNA repair pathway and increased invasion/migration of neighbouring cells, in vitro and in vivo, through chemokine signalling and modulation of integrins. These data indicate that even rare tumour subpopulations may exert profound effects on tumorigenesis as a whole, and may represent a novel avenue for therapeutic development. Unravelling the mechanisms of subclonal diversity and communication in pGBM/DIPG will be an important step to overcoming barriers to effective treatments.

Incorporating Genomics Into the Care of Patients With Advanced Breast Cancer.

Metastatic breast cancer is a very heterogeneous disease. Recent advances in genomic sequencing have revealed genetic diversity between patients and across distinct subclonal cell populations within the same patient that may evolve across metastatic tumor sites and during treatment. With the increasing availability of commercial and laboratory-developed tests that can detect genomic alterations from patient tumor and blood samples, translating this knowledge into improved clinical care remains a challenge. The goals of this review are to outline the clinical relevance of tumor genomic heterogeneity and clonal evolution, to help clinicians understand how to interpret genomic testing reports, and to provide an overview of recurrent genomic alterations that may be relevant for clinical trials with investigational drug treatments.

Abstract IA15: Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers

Abstract Personalized cancer medicine approaches, inhibiting kinases in tumors driven by defined genomic alterations, have demonstrated striking efficacy in many cancer types. However, the development of drug resistance is a major limitation to the efficacy of targeted therapies in oncology. Identifying and understanding the molecular mechanisms driving resistance may foster opportunities to develop therapeutic strategies to overcome resistance. For example, in the ~10% of colorectal cancer patients whose tumors harbor BRAF V600 mutations, we have found that feedback reactivation of MAPK signaling, often mediated by EGFR, underlies the relative insensitivity of these cancers to RAF inhibitors. The development of targeted therapy combination strategies to block feedback reactivation of MAPK signaling has led to marked improvements in response rates for these patients from ~5% to &amp;gt;30% over the past few years. However, in patients who respond to these targeted combinations, the rapid development of acquired resistance still limits clinical benefit. Acquired resistance in BRAF mutant colorectal cancer and in other molecularly defined tumor types can be marked by the development of extensive molecular heterogeneity due to the selection of sub-clonal tumor cell populations, capable of growing under drug pressures, which poses significant diagnostic and therapeutic challenges. We will present data demonstrating how a single-lesion biopsy at disease progression to diagnose the mechanism of acquired resistance can vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient, and may fail to detect the existence of distinct but important resistance mechanisms that can drive mixed or lesion-specific responses and treatment failure to subsequent targeted therapy. By contrast, liquid biopsy approaches analyzing circulating tumor DNA have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent therapies on specific sub-clonal tumor cell populations. These findings highlight the critical role of tumor heterogeneity in driving therapeutic resistance and how convergent targeted therapy strategies blocking common signaling nodes capable of overcoming multiple resistance mechanisms may be needed. Citation Format: Ryan B. Corcoran. Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr IA15.

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.

Abstract IA23: Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers

Abstract Personalized cancer medicine approaches, inhibiting kinases in tumors driven by defined genomic alterations, have demonstrated striking efficacy in many cancer types. However, the development of drug resistance is a major limitation to the efficacy of targeted therapies in oncology. Identifying and understanding the molecular mechanisms driving resistance may foster opportunities to develop therapeutic strategies to overcome resistance. For example, in the ~10% of colorectal cancer patients whose tumors harbor BRAF V600 mutations, we have found that feedback reactivation of MAPK signaling, often mediated by EGFR, underlies the relative insensitivity of these cancers to RAF inhibitors. The development of targeted therapy combination strategies to block feedback reactivation of MAPK signaling has led to marked improvements in response rates for these patients from ~5% to &amp;gt;30% over the past few years. However, in patients who respond to these targeted combinations, the rapid development of acquired resistance still limits clinical benefit. Acquired resistance in BRAF mutant colorectal cancer and in other molecularly-defined tumor types can be marked by the development of extensive molecular heterogeneity due to the selection of sub-clonal tumor cell populations, capable of growing under drug pressures, which poses significant diagnostic and therapeutic challenges. We will present data demonstrating how a single-lesion biopsy at disease progression to diagnose the mechanism of acquired resistance can vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient, and may fail to detect the existence of distinct but important resistance mechanisms that can drive mixed or lesion-specific responses and treatment failure to subsequent targeted therapy. By contrast, liquid biopsy approaches analyzing circulating tumor DNA have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent therapies on specific sub-clonal tumor cell populations. These findings highlight the critical role of tumor heterogeneity in driving therapeutic resistance and how convergent targeted therapy strategies blocking common signaling nodes capable of overcoming multiple resistance mechanisms may be needed. Citation Format: Ryan B. Corcoran. Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA23.

Intratumor Heterogeneity: Novel Approaches for Resolving Genomic Architecture and Clonal Evolution.

High-throughput genomic technologies have revealed a remarkably complex portrait of intratumor heterogeneity in cancer and have shown that tumors evolve through a reiterative process of genetic diversification and clonal selection. This discovery has challenged the classical paradigm of clonal dominance and brought attention to subclonal tumor cell populations that contribute to the cancer phenotype. Dynamic evolutionary models may explain how these populations grow within the ecosystem of tissues, including linear, branching, neutral, and punctuated patterns. Recent evidence in breast cancer favors branching and punctuated evolution driven by genome instability as well as nongenetic sources of heterogeneity, such as epigenetic variation, hierarchal tumor cell organization, and subclonal cell-cell interactions. Resolution of the full mutational landscape of tumors could help reconstruct their phylogenetic trees and trace the subclonal origins of therapeutic resistance, relapsed disease, and distant metastases, the major causes of cancer-related mortality. Real-time assessment of the tumor subclonal architecture, however, remains limited by the high rate of errors produced by most genome-wide sequencing methods as well as the practical difficulties associated with serial tumor genotyping in patients. This review focuses on novel approaches to mitigate these challenges using bulk tumor, liquid biopsies, single-cell analysis, and deep sequencing techniques. The origins of intratumor heterogeneity and the clinical, diagnostic, and therapeutic consequences in breast cancer are also explored. Mol Cancer Res; 15(9); 1127-37. ©2017 AACR.

Trisomy 12 assessment by conventional fluorescence in-situ hybridization (FISH), FISH in suspension (FISH-IS) and laser scanning cytometry (LSC) in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.

Abstract 3000: Defining the subclonal landscape of high-risk neuroblastoma

Abstract We recently showed that relapsed neuroblastoma (NB) tumors harbor an increased somatic mutational burden, enriched for ALK or RAS-MAPK activating lesions, nominating targeted therapeutic strategies for a condition that is largely incurable. We also showed that these mutations can be present in subclonal tumor cell populations at diagnosis, suggesting that they may contribute to acquired chemoradiotherapy resistance. We hypothesize that subclonal mutations in NB oncogenes are common at diagnosis, are biomarkers for treatment failure, and can be targeted in conjunction with standard chemotherapy to improve patient survival. Here, we focus on the first aspect of this broad hypothesis by presenting the subclonal landscape of diagnostic, high-risk neuroblastomas. A meta-analysis of our previously published primary/relapse (N=23), whole exome (N=232) and whole genome (N=159) diagnostic data, and recent sequencing study of 78 diagnostic and 67 relapse cases were used to inform design of a custom amplicon panel for ultra-deep sequencing. The 28-gene panel covers 57 mutations in frequently mutated genes. Here, 250 high-risk primary tumors were sequenced to an average depth of 50,000X. More subclonal mutations were called (mean=36) compared to clonal alterations (mean=3), demonstrating the complex subclonal architecture in these tumors. We validated five clonal ALK mutations we originally discovered and identified 15 additional pathogenic ALK mutations with mutant allele frequencies (MAFs) ranging from 0.03-23%. Known ALK activating mutations were found at codons 1170, 1174, 1196, 1245, 1275 in 15 of 38 diagnostic primary tumors. This 39.5% frequency is much higher than the 14% frequency defined by us using Sanger or other sequencing methods. Improvement of subclonal mutation detection of actionable, relapse-specific driver mutations demonstrates the clinical utility of utilizing this sequencing methodology at diagnosis to enable timely improvement of outcomes for children with high-risk refractory NB. Citation Format: Jo Lynne Harenza, Maura A. Diamond, Derek A. Oldridge, Olivia Padovan-Merher, Pichai Raman, Yael P. Mosse, John M. Maris. Defining the subclonal landscape of high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2017-3000

Integrating liquid biopsies into the management of cancer.

During cancer progression and treatment, multiple subclonal populations of tumour cells compete with one another, with selective pressures leading to the emergence of predominant subclones that replicate and spread most proficiently, and are least susceptible to treatment. At present, the molecular landscapes of solid tumours are established using surgical or biopsy tissue samples. Tissue-based tumour profiles are, however, subject to sampling bias, provide only a snapshot of tumour heterogeneity, and cannot be obtained repeatedly. Genomic profiles of circulating cell-free tumour DNA (ctDNA) have been shown to closely match those of the corresponding tumours, with important implications for both molecular pathology and clinical oncology. Analyses of circulating nucleic acids, commonly referred to as 'liquid biopsies', can be used to monitor response to treatment, assess the emergence of drug resistance, and quantify minimal residual disease. In addition to blood, several other body fluids, such as urine, saliva, pleural effusions, and cerebrospinal fluid, can contain tumour-derived genetic information. The molecular profiles gathered from ctDNA can be further complemented with those obtained through analysis of circulating tumour cells (CTCs), as well as RNA, proteins, and lipids contained within vesicles, such as exosomes. In this Review, we examine how different forms of liquid biopsies can be exploited to guide patient care and should ultimately be integrated into clinical practice, focusing on liquid biopsy of ctDNA - arguably the most clinically advanced approach.