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Supplementary Figures S1-S5. Supplementary Tables S1-S5. Figure S1. Comparison of SNV mutation signatures across histological and molecular subtypes. Figure S2. Treatment timelines and radiographic outcomes. Figure S3. Mutation signature stability metrics and comparisons to known signatures. Figure S4. Structural variant mutation sigantures. Figure S5. Comparison of mutation burden between aging-driven and non-aging-driven breast tumours. Table S1. Kruskal-Wallis tests. Table S2. HRDetect prediction test metrics. Table S3. Area under the curve of HRD signatures in platinum response prediction. Table S4. Logistic regression model odds ratios of clinical improvement on platinum. Table S5. Accession IDs of genome datasets submitted to the European Genome-Phenome Archive.

Table S5: Data from this study have been submitted to the European Genome-Phenome Archive (EGA) (www.ebi.ac.uk/ega/home) under the study accession number EGAS00001001159. This table provides the accession numbers for each dataset included in this study.

Purpose: Recent studies have identified mutation signatures of homologous recombination deficiency (HRD) in over 20% of breast cancers, as well as pancreatic, ovarian, and gastric cancers. There is an urgent need to understand the clinical implications of HRD signatures. Whereas BRCA1/2 mutations confer sensitivity to platinum-based chemotherapies, it is not yet clear whether mutation signatures can independently predict platinum response.Experimental Design: In this observational study, we sequenced tumor whole genomes (100× depth) and matched normals (60×) of 93 advanced-stage breast cancers (33 platinum-treated). We computed a published metric called HRDetect, independently trained to predict BRCA1/2 status, and assessed its capacity to predict outcomes on platinum-based chemotherapies. Clinical endpoints were overall survival (OS), total duration on platinum-based therapy (TDT), and radiographic evidence of clinical improvement (CI).Results: HRDetect predicted BRCA1/2 status with an area under the curve (AUC) of 0.94 and optimal threshold of 0.7. Elevated HRDetect was also significantly associated with CI on platinum-based therapy (AUC = 0.89; P = 0.006) with the same optimal threshold, even after adjusting for BRCA1/2 mutation status and treatment timing. HRDetect scores over 0.7 were associated with a 3-month extended median TDT (P = 0.0003) and 1.3-year extended median OS (P = 0.04).Conclusions: Our findings not only independently validate HRDetect, but also provide the first evidence of its association with platinum response in advanced breast cancer. We demonstrate that HRD mutation signatures may offer clinically relevant information independently of BRCA1/2 mutation status and hope this work will guide the development of clinical trials. Clin Cancer Res; 23(24); 7521–30. ©2017 AACR.

Supplementary Figure 1 from Targeting YB-1 in HER-2 Overexpressing Breast Cancer Cells Induces Apoptosis via the mTOR/STAT3 Pathway and Suppresses Tumor Growth in Mice

The Y-box binding protein-1 (YB-1) is a transcription/translation factor that is highly expressed in primary breast tumors where it is consistently associated with poor survival. It induces human epidermal growth factor receptor (her-2) along with its dimerization partner egfr by directly binding to their promoters. In addition to promoting growth by inducing receptor tyrosine kinases, YB-1 also protects cells against apoptosis through mechanisms that have not been fully revealed. Given this, we addressed whether YB-1 might be an eventual therapeutic target for breast cancer by inhibiting it with small interfering RNAs in vitro and in vivo. Inhibiting YB-1 suppressed the growth of six of seven breast cancer cell lines that had amplified her-2 or were triple negative. Importantly, targeting YB-1 induced apoptosis in BT474-m1 and Au565 breast cancer cells known to have her-2 amplifications. The potential role of signal transducers and activators of transcription 3 (STAT3) was pursued to address the underlying mechanism for YB-1–mediated survival. Inhibition of YB-1 decreased P-STAT3S727 but not P-STAT3Y705 or total STAT3. This was accompanied by decreased P-ERK1/2T202/Y204, P-mTORS2448, and total mammalian target of rapamycin mTOR. Furthering the role of STAT3 in these cells, we show that knocking it down recapitulated the induction of apoptosis. Alternatively, constitutively active P-STAT3 rescued YB-1–induced apoptosis. Finally, targeting YB-1 with 2 different siRNAs remarkably suppressed tumor cell growth in soft agar by >90% and delayed tumorigenesis in nude mice. We conclude that HER-2 overexpressing as well as triple-negative breast cancer cells are YB-1 dependent, suggesting it may be a good therapeutic target for these exceptionally aggressive tumors. [Cancer Res 2008;68(21):8661–6]

Supplementary Figure 2 from Targeting YB-1 in HER-2 Overexpressing Breast Cancer Cells Induces Apoptosis via the mTOR/STAT3 Pathway and Suppresses Tumor Growth in Mice

Cancer genomes exhibit extensive chromosomal copy number changes and structural variation, yet how allele specific alterations drive cancer genome evolution remains unclear. Here, through application of a new computational approach we report allele specific copy number alterations in 11,097 single cell whole genomes from genetically engineered mammary epithelial cells and 21,852 cells from high grade serous ovarian and triple negative breast cancers. Resolving single cell copy number profiles to individual alleles uncovered genomic background distributions of gains, losses and loss of heterozygosity, yielding evidence of positive selection of specific chromosomal alterations. In addition specific genomic loci in maternal and paternal alleles were commonly found to be altered in parallel with convergent phenotypic transcriptional effects. Finally we show that haplotype specific alterations trace the cyclical etiology of high level amplifications and reveal clonal haplotype decomposition of complex structures. Together, our results illuminate how allele and haplotype specific alterations, here determined across thousands of single cell cancer genomes, impact the etiology and evolution of structural variations in human tumours.

Structural genome alterations are determinants of cancer ontogeny and therapeutic response. While bulk genome sequencing has enabled delineation of structural variation (SV) mutational processes which generate patterns of DNA damage, we have little understanding of how these processes lead to cell-to-cell variations which underlie selection and rates of accrual of different genomic lesions. We analysed 309 high grade serous ovarian and triple negative breast cancer genomes to determine their mutational processes, selecting 22 from which we sequenced >22,000 single cell whole genomes across a spectrum of mutational processes. We show that distinct patterns of cell-to-cell variation in aneuploidy, copy number alteration (CNA) and segment length occur in homologous recombination deficiency (HRD) and fold-back inversion (FBI) phenotypes. Widespread aneuploidy through induction of HRD throughBRCA1andBRCA2inactivation was mirrored by continuous whole genome duplication in HRD tumours, contrasted with early ploidy fixation in FBI. FBI tumours exhibited copy number distributions skewed towards gains, widespread clone-specific variation in amplitude of high-level amplifications, often impacting oncogenes, and break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). SSVs were consistent with a CNA-based molecular clock reflecting a continual and distributed process across clones within tumours. These observations reveal previously obscured genome plasticity and evolutionary properties with implications for cancer evolution, therapeutic targeting and response.

How cell-to-cell copy number alterations that underpin genomic instability

Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.

Tumour fitness landscapes underpin selection in cancer, impacting etiology, evolution and response to treatment. Progress in defining fitness landscapes has been impeded by a lack of timeseries perturbation experiments over realistic intervals at single cell resolution. We studied the nature of clonal dynamics induced by genetic and pharmacologic perturbation with a quantitative fitness model developed to ascribe quantitative selective coefficients to individual cancer clones, enable prediction of clone-specific growth potential, and forecast competitive clonal dynamics over time. We applied the model to serial single cell genome (>60,000 cells) and transcriptome (>58,000 cells) experiments ranging from 10 months to 2.5 years in duration. We found that genetic perturbation ofTP53in epithelial cell lines induces multiple forms of copy number alteration that confer increased fitness to clonal populations with measurable consequences on gene expression. In patient derived xenografts, predicted selective coefficients accurately forecasted clonal competition dynamics, that were validated with timeseries sampling of experimentally engineered mixtures of low and high fitness clones. In cisplatin-treated patient derived xenografts, the fitness landscape was inverted in a time-dependent manner, whereby a drug resistant clone emerged from a phylogenetic lineage of low fitness clones, and high fitness clones were eradicated. Moreover, clonal selection mediated reversible drug response early in the selection process, whereas late dynamics in genomically fixed clones were associated with transcriptional plasticity on a fixed clonal genotype. Together, our findings outline causal mechanisms with implication for interpreting how mutations and multi-faceted drug resistance mechanisms shape the etiology and cellular fitness of human cancers.

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

BackgroundSingle-cell RNA sequencing (scRNAseq) is a powerful tool for studying complex biological systems, such as tumour heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumour tissues and patient-derived mouse xenografts for scRNAseq, are not well understood. Here, we used low temperature (6°C) protease and collagenase (37°C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNAseq dataset comprising 128,481 cells from patient cancer tissues, patient-derived breast cancer xenografts and cancer cell lines.ResultsWe observe substantial variation in standard quality control (QC) metrics of cell viability across conditions and tissues. From FACS sorted populations gated for cell viability, we identify a sub-population of dead cells that would pass standard data filtering practices, and quantify the extent to which their transcriptomes differ from live cells. We identify a further subpopulation of transcriptomically “dying” cells that exhibit up-regulation of MHC class I transcripts, in contrast with live and fully dead cells. From the contrast between tissue protease dissociation at 37°C or 6°C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, includingFOSandJUN, induced by collagenase (37°C), which are minimized by dissociation with a cold active protease (6°C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. We observe that the yield of cancer and non-cancer cell types varies between tissues and dissociation methods.ConclusionsThe method and conditions of tumour dissociation influence cell yield and transcriptome state and are both tissue and cell type dependent. Interpretation of stress pathway expression differences in cancer single cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with identification of such effects in dissociated scRNA-seq experiments.

Purpose: As we have published expertise in breast cancer xenograft models and clonal dynamics, our aim was to explore rates of engraftment based on type of tissue for attempted xenografting (primary vs relapsed/metastatic disease) and clinical breast biomarker subtype. Methods: Tissue from patients (pts) enrolled in a locally advanced/metastatic study and a breast tumour tissue repository (ie. resectable primaries) between Sept. 2008 and July 2015 underwent xenografting using NodScid/IL2rgKO (NSG) mice. Xenografts were passaged when tumour volume reached 1 cm3. Mice with no engraftment after 12 months (mos) were sacrificed. Pt charts were reviewed to determine biomarker status (hormone receptor [HR], HER2), date and type of tissue collection for xenografting. Prediction of successful engraftment based on tissue type and biomarker status was performed using nominal logistic regression. Results: A total of 70 tissue samples with known engraftment status were included in the analysis: 51 from primary breast tumour, 10 from relapsed disease (dz) with ≤ 1 line of therapy in the advanced setting and 9 from relapsed dz with > 1 line of therapy in the advanced setting. Tumours from pts treated with > 1 line of therapy were more likely to engraft compared to primary or recurrent dz with ≤ 1 line of therapy (89%, 35%, and 40% respectively; p=.008). HR- primary tumours were more likely to engraft compared to HR+ primary tumours: 71% of HR-/HER2- (triple negative) and 67% of HR-/HER2+ tumours versus 4% of HR+/HER2- and 38% of HR+/HER2+ tumours; p Conclusion: This preliminary study highlights potential differences in successful xenoengraftment based on biomarker status at diagnosis and type of tissue, primary vs relapsed tumour, the latter suggesting that the underlying biology of primary or first relapsed recurrent disease is distinct from more refractory disease, and warrants further exploration. This work is ongoing. (Funded by CBCRA, BCCF) Engraftment of primary tumour vs relapsed disease Primary tumour (N=52) N, (%)Recurrent disease and ≤ 1 line of Rx in advanced setting (N=10) N, (%)Recurrent disease and > 1 line of Rx in advanced setting (N=9) N, (%)Engraftment Yes18 (35)4 (40)8 (89)HR-/HER2-10 (55)1 (25)2 (25)HR-/HER2+4 (22)1 (25)1 (13)HR+/HER2+3 (17)00HR+/HER2-1 (6)2 (50)5 (62)Engraftment No33 (65)6 (60)1 (11)HR-/HER2-4 (12)00HR-/HER2+2 (6)1 (17)0HR+/HER2+5 (15)00HR+/HER2-22 (67)5 (83)1 (100) Citation Format: den Brok W-l, Chia S, Kalloger S, Bates C, Aparicio S, Mar C, Gelmon K, Eirew P. Rates of successful engraftment in breast cancer xenograft models based on tissue type: Primary vs relapsed disease [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-10.

SummaryEssential features of cancer tissue cellular heterogeneity such as negatively selected genome topologies, sub-clonal mutation patterns and genome replication states can only effectively be studied by sequencing single-cell genomes at scale and high fidelity. Using an amplification-free single-cell genome sequencing approach implemented on commodity hardware (DLP+) coupled with a cloud-based computational platform, we define a resource of 40,000 single-cell genomes characterized by their genome states, across a wide range of tissue types and conditions. We show that shallow sequencing across thousands of genomes permits reconstruction of clonal genomes to single nucleotide resolution through aggregation analysis of cells sharing higher order genome structure. From large-scale population analysis over thousands of cells, we identify rare cells exhibiting mitotic mis-segregation of whole chromosomes. We observe that tissue derived scWGS libraries exhibit lower rates of whole chromosome anueploidy than cell lines, and loss of p53 results in a shift in event type, but not overall prevalence in breast epithelium. Finally, we demonstrate that the replication states of genomes can be identified, allowing the number and proportion of replicating cells, as well as the chromosomal pattern of replication to be unambiguously identified in single-cell genome sequencing experiments. The combined annotated resource and approach provide a re-implementable large scale platform for studying lineages and tissue heterogeneity.

Summary Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations., Graphical Abstract, Highlights • Scaled method and resource of > 50K single-cell whole genomes from diverse cell types • Clonal merging can resolve clone specific mutations to single-nucleotide level • Image analysis of single cells permits correlation of morphology and genome features • Clonal replication states and rare aneuploidy patterns of single cells measured, A high-throughput method for amplication-free single-cell whole-genome sequencing can be scaled up to analyze tens of thousands of cells from different tissues and clinical sample types and identifies replication states, aneuploidies, and subclonal mutations.

Somatic evolution of malignant cells produces tumors composed of multiple clonal populations, distinguished in part by rearrangements and copy number changes affecting chromosomal segments. Whole genome sequencing mixes the signals of sampled populations, diluting the signals of clone-specific aberrations, and complicating estimation of clone-specific genotypes. We introduce ReMixT, a method to unmix tumor and contaminating normal signals and jointly predict mixture proportions, clone-specific segment copy number, and clone specificity of breakpoints. ReMixT is free, open-source software and is available at http://bitbucket.org/dranew/remixt. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1267-2) contains supplementary material, which is available to authorized users.

Summary Human breast cancer cells are known to activate adjacent “normal-like” cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor β. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention., Graphical Abstract, Highlights • Alterations to the breast tissue extend as far as 6 cm away from the primary tumors • The matching contralateral non-tumor-bearing breast tissue remains unaltered • Tumor-adjacent breast tissue contained significantly diminished progenitor pool • Extending surgical margins may not be effective in reducing risk of tumor recurrence, Because breast-conserving surgeries are popular among breast cancer patients, studying the “normal” tissue remaining after the surgery is important for understanding how this tissue is primed to promote the growth of residual tumor cells. In this article, Raouf and colleagues show that tumor-adjacent breast tissue contains altered fibroblasts that diminish normal progenitor proliferation while augmenting breast cancer cell growth.

Purpose: Recent studies have identified mutation signatures of homologous recombination deficiency (HRD) in over 20% of breast cancers, as well as pancreatic, ovarian, and gastric cancers. There is an urgent need to understand the clinical implications of HRD signatures. Whereas BRCA1/2 mutations confer sensitivity to platinum-based chemotherapies, it is not yet clear whether mutation signatures can independently predict platinum response.Experimental Design: In this observational study, we sequenced tumor whole genomes (100× depth) and matched normals (60×) of 93 advanced-stage breast cancers (33 platinum-treated). We computed a published metric called HRDetect, independently trained to predict BRCA1/2 status, and assessed its capacity to predict outcomes on platinum-based chemotherapies. Clinical endpoints were overall survival (OS), total duration on platinum-based therapy (TDT), and radiographic evidence of clinical improvement (CI).Results: HRDetect predicted BRCA1/2 status with an area under the curve (AUC) of 0.94 and optimal threshold of 0.7. Elevated HRDetect was also significantly associated with CI on platinum-based therapy (AUC = 0.89; P = 0.006) with the same optimal threshold, even after adjusting for BRCA1/2 mutation status and treatment timing. HRDetect scores over 0.7 were associated with a 3-month extended median TDT (P = 0.0003) and 1.3-year extended median OS (P = 0.04).Conclusions: Our findings not only independently validate HRDetect, but also provide the first evidence of its association with platinum response in advanced breast cancer. We demonstrate that HRD mutation signatures may offer clinically relevant information independently of BRCA1/2 mutation status and hope this work will guide the development of clinical trials. Clin Cancer Res; 23(24); 7521–30. ©2017 AACR.

Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting. We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized OncoGenomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequencing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homozygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscape of an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level.

SummaryCellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.

BACKGROUND Triple-negative breast cancers (TNBC) do not represent a single disease subgroup and are often aggressive breast cancers with poor prognoses. Unlike estrogen/progesterone receptor and HER2 (human epidermal growth factor receptor 2) breast cancers, which are responsive to targeted treatments, there is no effective targeted therapy for TNBC, although approximately 50% of patients respond to conventional chemotherapies, including taxanes, anthracyclines, cyclophosphamide, and platinum salts. CONTENT Genomic studies have helped clarify some of the possible disease groupings that make up TNBC. We discuss the findings, including copy number–transcriptome analysis, whole genome sequencing, and exome sequencing, in terms of the biological properties and phenotypes that make up the constellation of TNBC. The relationships between subgroups defined by transcriptome and genome analysis are discussed. SUMMARY TNBC is not a uniform molecular or disease entity but a constellation of variably well-defined biological properties whose relationship to each other is not understood. There is good support for the existence of a basal expression subtype, p53 mutated, high–genomic instability subtype of TNBC. This should be considered a distinct TNBC subtype. Other subtypes with variable degrees of supporting evidence exist within the nonbasal/p53wt (wild-type p53) TNBC, including a group of TNBC with PI3K (phosphoinositide 3-kinase) pathway activation that have better overall prognosis than the basal TNBC. Consistent molecular phenotyping of TNBC by whole genome sequencing, transcriptomics, and functional studies with patient-derived tumor xenograft models will be essential components in clinical and biological studies as means of resolving this heterogeneity.

We report a case of early-onset pancreatic ductal adenocarcinoma in a patient harboring biallelic MUTYH germline mutations, whose tumor featured somatic mutational signatures consistent with defective MUTYH-mediated base excision repair and the associated driver KRAS transversion mutation p.Gly12Cys. Analysis of an additional 730 advanced cancer cases (N = 731) was undertaken to determine whether the mutational signatures were also present in tumors from germline MUTYH heterozygote carriers or if instead the signatures were only seen in those with biallelic loss of function. We identified two patients with breast cancer each carrying a pathogenic germline MUTYH variant with a somatic MUTYH copy loss leading to the germline variant being homozygous in the tumor and demonstrating the same somatic signatures. Our results suggest that monoallelic inactivation of MUTYH is not sufficient for C:G>A:T transversion signatures previously linked to MUTYH deficiency to arise (N = 9), but that biallelic complete loss of MUTYH function can cause such signatures to arise even in tumors not classically seen in MUTYH-associated polyposis (N = 3). Although defective MUTYH is not the only determinant of these signatures, MUTYH germline variants may be present in a subset of patients with tumors demonstrating elevated somatic signatures possibly suggestive of MUTYH deficiency (e.g., COSMIC Signature 18, SigProfiler SBS18/SBS36, SignatureAnalyzer SBS18/SBS36).

Basal-like breast cancers (BLBCs) are aggressive tumors with high relapse rates and poor survival. We recently reported that >70% of primary BLBCs express the oncogenic transcription/translation factor Y-box binding protein-1 (YB-1) and silencing it with small interfering RNAs (siRNAs) attenuates the growth of BLBC cell lines. To understand the basis of these earlier findings, we profiled YB-1:DNA complexes by chromatin immunoprecipitation (ChIP)-on-chip. Several tumor growth-promoting genes such as MET, CD44, CD49f, WNT and NOTCH family members were identified. In addition, YB-1 and MET are coordinately expressed in BLBC cell lines, as well as in normal human mammary progenitor cells. MET was confirmed to be a YB-1 target through traditional ChIP and gel-shift assays. More specifically, YB-1 binds to -1018 bp on the MET promoter. Silencing YB-1 with siRNA decreased MET promoter activity, transcripts, as well as protein levels and signaling. Conversely, expressing wild-type YB-1 or a constitutively active mutant YB-1 (D102) increased MET expression. Finally, silencing YB-1 or MET attenuated anchorage-independent growth of BLBC cell lines. Together, these findings implicate MET as a target of YB-1 that work in concert to promote BLBC growth.

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels, which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks, the gels contain bilayered mammary epithelial structures, including luminal and myoepithelial cells, their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations.

Clonal assays offer a powerful approach to dissecting the many events involved in the generation and maintenance of complex tissues from an undifferentiated stem cell pool. The application of such quantitative functional methodologies to studies of the hematopoietic system have been key to defining the hierarchy of progenitor subsets that reflect an irreversible stepwise process of lineage restriction. Recent studies now suggest that a similar paradigm applies to the normal mammary gland. The adult mouse mammary gland maintains a population of stem cells that generate biologically distinct and physically separable subpopulations of mammary epithelial progenitors which, in turn, generate terminally differentiated cells. Suggestive parallels between mouse and human mammary cells point to the likelihood that a similarly structured multi-step differentiation program characterizes the mammary gland from both species.

Whole-genome and transcriptome sequencing were performed to identify potential therapeutic strategies in the absence of viable treatment options for a patient initially diagnosed with vulvar adenocarcinoma. Genomic events were prioritized by comparison against variant distributions in the TCGA pan-cancer data set and complemented with detailed transcriptome sequencing and copy-number analysis. These findings were considered against published scientific literature in order to evaluate the functional effects of potentially relevant genomic events. Analysis of the transcriptome against a background of 27 TCGA cancer types led to reclassification of the tumor as a primary HER2+ mammary-like adenocarcinoma of the vulva. This revised diagnosis was subsequently confirmed by follow-up immunohistochemistry for a mammary-like adenocarcinoma. The patient was treated with chemotherapy and targeted therapies for HER2+ breast cancer. The detailed pathology and genomic findings of this case are presented herein.

Background: Homologous recombination deficiency (HRD) is common in cancer - germline BRCA1 & BRCA2 mutations account for 5-10% of breast cancers and confer 85% lifetime risk. HRD cancers exhibit genomic instability and sensitivity to platinum-based therapy and PARP inhibitors. While not all causes of HRD are known, recent sequencing efforts have revealed genome-wide somatic mutation signatures that characterize the HRD genomic instability phenotype, also known as “BRCA-ness”. This provides a promising new assay to predict sensitivity to platinum-based therapy. Here, we integrate two whole-genome sequencing metrics to assess their association with therapeutic outcomes in a breast cancer cohort. Methods: Whole-genome sequencing of 47 breast cancer tumors (100x coverage) and matched normals (60x) was performed on an Illumina HiSeq. Alignment, assembly, SNV calling, and loss of heterozygosity (LOH) detection were performed with BWA, ABySS, Strelka, and APOLLOH respectively. SNV signatures were deciphered by non-negative matrix factorization with Monte Carlo resampling. An HRD score comprised of LOH, telomeric allelic imbalance (TAI), and large scale transition (LST) counts was computed. Clinical endpoints were obtained by retrospective review of treatment and imaging reports. Analysis is ongoing in an independent validation cohort of 62 sequenced cases. Results: The HRD-linked SNV signature was significantly associated with radiographic clinical response (CR) to platinum-based therapy (p=0.015). Logistic regression demonstrated a 59% improved odds of CR to platinum-based therapy per 1000 somatic SNVs attributed to HRD (odds ratio 1.16-2.50). Tumors carried up to 10,246 such SNVs and all patients with CR were among the top quartile. The LOH-TAI-LST score was correlated with SNV signature (r=0.6, p=7×10-6) and associated with CR (p=0.025). Notably, elevated HRD signatures associated with CR were identified in tumors with wild-type BRCA1/BRCA2 or variants of unknown significance. Tumors with above median HRD signatures were associated with a 69-day longer time to treatment failure and an 18% daily decreased probability of treatment failure per 1000 HRD-attributed SNVs (hazard ratio 0.71-0.95, p = 0.007). Discussion: We found that HRD mutation signatures are associated with clinical response and longer time to treatment failure with platinum-based therapy. While similar benefits were observed in patients with somatic bi-allelic loss of BRCA1/BRCA2, such cases are less common (8% of our cohort) compared to those with elevated HRD signature. Thus, mutation signature methods may identify patients who stand to benefit from platinum-based therapy missed by BRCA screening alone. Citation Format: Eric Y. Zhao, Yaoqing Shen, Erin Pleasance, Katayoon Kasaian, Martin R. Jones, Carolyn Ch'ng, Caralyn Reisle, Peter Eirew, Karen Mungall, Nina Thiessen, Yussanne Ma, Alexandra Fok, Andrew J. Mungall, Yongjun Zhao, Richard Moore, Diego Villa, Tamara Shenkier, Caroline Lohrisch, Stephen Chia, Stephen Yip, Karen Gelmon, Howard Lim, Sophie Sun, Kasmintan A. Schrader, Sean Young, Aly Karsan, Robyn Roscoe, Janessa Laskin, Marco A. Marra, Steven J. Jones. Breast cancer whole genomes link homologous recombination deficiency (HRD) with therapeutic outcomes [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 2473. doi:10.1158/1538-7445.AM2017-2473

Genomic and phenotypic analyses indicate extensive intra- as well as intertumoral heterogeneity in primary human malignant cell populations despite their clonal origin. Cellular DNA barcoding offers a powerful and unbiased alternative to track the number and size of multiple subclones within a single human tumour xenograft and their response to continued in vivo passaging. Using this approach we find clone-initiating cell frequencies that vary from ~1/10 to ~1/10,000 cells transplanted for two human breast cancer cell lines and breast cancer xenografts derived from three different patients. For the cell lines, these frequencies are negatively affected in transplants of more than 20,000 cells. Serial transplants reveal five clonal growth patterns (unchanging, expanding, diminishing, fluctuating or of delayed onset), whose predominance is highly variable both between and within original samples. This study thus demonstrates the high growth potential and diverse growth properties of xenografted human breast cancer cells., Cancer cells within the same tumour are heterogeneous in their tumorigenic potential, differentiation status and sensitivity to treatments. Here Nguyen et al. use a sensitive DNA barcoding method to characterize the diversity of clonal growth behaviour within human breast tumours.

Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

Background: In the absence of defined tumor molecular subtypes and validated predictive markers, PDAC has been largely treated as a single disease. Recent studies of molecular subtyping in PDAC [1-4] reveal a complex mutational landscape with data suggesting the presence of genomic and gene expression signatures that may have prognostic and therapeutic significance. However, most of these PDAC datasets consisted of resected tumors, cell lines or xenografts and lack data on metastatic tumors. The aim of this study is to evaluate gene signatures using whole genome sequencing (WGS) and transcriptome (RNA-Seq) data from metastatic biopsy samples in patients with advanced PDAC. Methods: Patients with incurable advanced cancers undergo fresh tumor biopsies for indepth WGS and RNA sequencing as part of an ongoing prospective study (NCT02155621). DNA and RNA extraction and library construction were performed according to standard protocols. Paired-end reads were generated on an Illumina HiSeq2500 sequencer. RNA-Seq expression values were converted into centile expression ranks against the TCGA PDAC dataset. Centile distributions for genes in published signatures were compared by pairwise Wilcoxon-Mann-Whitney tests using one-sided p=0.1 as the significance cutoff. Survival analysis was performed using the Kaplan-Meier method. Results: Molecular data is available for 12 patients with metastatic PDAC; median age 63 years, 6 males, 8 with de novo metastatic disease (67%). 10 tumor samples (83%) were obtained from liver biopsies; average tumor content was 41% (range 24-51%). The average number of structural variants per sample was 125 (range 40-271). Rearrangement-based subtypes [3] were distributed as follows: stable (n=3), locally rearranged (n=1), scattered (n=7) and unstable (n=1). 1 patient harbored a germline BRCA1 185delAG founder mutation but had a stable genotype. Gene expression analysis for classical and basal subtypes similar to those recently described [4] identified 3 and 7 patients with classical and basal expression patterns respectively. Gene signatures were undetermined for 2 patients, where no significant difference in expression of classical or basal signature genes was noted. At median follow-up of 16.7 months, 8 patients had died. Median overall survival was 19.1 vs 7 months in patients with classical and basal subtypes respectively (p=0.078). Conclusion: Despite small patient numbers, gene expression analysis demonstrated the presence of distinct signatures in metastatic PDAC, with a trend towards worse outcomes for patients with a basal expression subtype. Future challenges include prospective validation in larger cohorts, standardization of RNA data acquisition and analysis, and better definition of prognostic and predictive signatures that may be of clinical utility in metastatic PDAC. References 1. Collison E, et al. Nat Med. 2011 [PMID: 21460848] 2. Biankin A, et al. Nature. 2012 [PMID: 23103869] 3. Waddell N, et al. Nature. 2015 [PMID: 25719666] 4. Moffitt R, et al. Nat Genet. 2015 [PMID: 26343385] Citation Format: Hui-li Wong, Joanna M. Karasinska, Martin Jones, Peter Eirew, Kasmintan Schrader, Howard Lim, Yaoqing Shen, Steven Jones, Stephen Yip, Janessa Laskin, Marco Marra, David F. Schaeffer, Daniel J. Renouf.{Authors}. Gene expression analysis demonstrates prognostic subtypes in metastatic pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B81.

Deep-genome and single-cell sequencing analyses of patient-derived breast cancer xenografts reveal extensive, dynamic and reproducible changes in intra-tumoral mutational clonal composition on engraftment and serial propagation. Xenograft transplantation of primary human cancer cells into mice provides valuable models in which to study mechanisms underlying tumorigenesis, drug response and resistance. This study demonstrates that clonal evolution resembling that seen in human tumours also occurs on engraftment and during subsequent passaging of breast tumours in immunodeficient mice. In addition, similar clonal expansion patterns emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. These findings suggest that patient-derived xenografts may be useful for studying patient-specific tumour characteristics such as the response to drugs tailored to specific genomic alterations. Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution1,2, underpinning important emergent features such as drug resistance and metastasis3,4,5,6,7. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours8,9,10. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (

Mouse fetal mammary cells display greater regenerative activity than do adult mammary cells when stimulated to proliferate in a new system that supports the production of transplantable mammary stem cells ex vivo., Many normal adult tissues contain rare stem cells with extensive self-maintaining regenerative potential. During development, the stem cells of the hematopoietic and neural systems undergo intrinsically specified changes in their self-renewal potential. In the mouse, mammary stem cells with transplantable regenerative activity are first detectable a few days before birth. They share some phenotypic properties with their adult counterparts but are enriched in a subpopulation that displays a distinct gene expression profile. Here we show that fetal mammary epithelial cells have a greater direct and inducible growth potential than their adult counterparts. The latter feature is revealed in a novel culture system that enables large numbers of in vitro clonogenic progenitors as well as mammary stem cells with serially transplantable activity to be produced within 7 days from single fetal or adult input cells. We further show that these responses are highly dependent on novel factors produced by fibroblasts. These findings provide new avenues for elucidating mechanisms that regulate normal mammary epithelial stem cell properties at the single-cell level, how these change during development, and how their perturbation may contribute to transformation., Author Summary Many adult tissues are maintained by a rare subset of undifferentiated stem cells that can self-renew and give rise to specialized daughter cells that have a more limited regenerative ability. The recent identification of cells in the fetal and adult mammary gland that display the properties of stem cells provides a foundation for investigating their self-renewal and differentiation control. We now show that these stem cell properties can be elicited from single mouse mammary cells placed in 3D cultures if novel factors produced by fibroblasts are present. Moreover, a comparison of the clonal outputs of fetal and adult mammary cells in this in vitro system shows that the fetal mammary cells have superior regenerative activity relative to their adult counterparts. The ability to activate and quantify the regenerative capacity of single mouse mammary epithelial cells in vitro sets the stage for further investigations of the timing and mechanisms that alter their stem cell properties during development, the potential relevance of these events to other normal epithelial tissues, and how these processes might be involved in the genesis of breast cancer.

Biallelic pathogenic germline variants in the DNA repair glycosylase, MUTYH, cause MUTYH-associated polyposis, characterised by an increased susceptibility to colorectal adenomas and carcinomas secondary to defective base excision repair. We report a patient diagnosed with Stage IIB distal pancreatic ductal adenocarcinoma (PDAC) at the age of 45 years. Prior colonoscopy and gastroscopy noted three colonic tubular adenomas and a gastric fundic gland polyp. The patient was consented to whole genome and transcriptome sequencing of the PDAC and matched normal blood DNA through the British Columbia Personalized Onco-Genomics (POG) program. Analysis of germline and somatic variants including single nucleotide variants, copy number determination, loss of heterozygosity detection and mutational signatures was undertaken. Expression fold-changes were calculated against Illumina BodyMap pancreatic tissue averages and compared against The Cancer Genome Atlas PDAC cases. Germline analysis revealed biallelic mutations in the MUTYH gene. In light of this patient's personal and family history of adenomatous colon polyps, clinic-initiated panel testing of 14 cancer susceptibility genes, including MUTYH, via Illumina sequencing with reflex Sanger confirmation revealed the same biallelic MUTYH changes. Analysis of the patient's PDAC revealed a base excision repair pathway signature, demonstrated by an increased frequency of C:G>A:T transversions, consistent with deficient MUTYH activity. This is the first association of germline MUTYH biallelic pathogenic variants with PDAC and provides evidence of the contribution of aberrant MUTYH function to the genomic landscape of a PDAC. Detection of the base excision repair mutational signature may be a sensitive way to screen tumors for aberrant MUTYH function that can reveal potential germline MUTYH-related cancer susceptibility, and allow inference of pathogenicity of detected MUTYH variants, which may have cancer prevention and therapeutic implications. Citation Format: Kasmintan A. Schrader, Carolyn Chu’ng, Eric Zhao, Hui-li Wong, Yaoqing Shen, Martin Jones, Tom Thomson, Howard Lim, Sean Young, Carol Cremin, Robert Holt, Peter Eirew, Joanna Karasinska, Jacquie Schein, Yongjun Zhao, Andy Mungall, Richard Moore, Yussanne Ma, Alexandra Fok, Robyn Roscoe, Stephen Yip, Gillian Mitchell, Aly Karsan, Steven Jones, David Schaeffer, Janessa Laskin, Marco Marra, Daniel Renouf. Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis. [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 5226.

Purpose: We have expertise in breast cancer xenograft models and have previously published data on clonal dynamics. Our aim was to explore clinical characteristics of those patients (pts) whose breast cancer tumours engrafted versus those that did not. Methods: Tissue from pts enrolled in a locally advanced/metastatic (MBC) study and a breast tumour tissue repository between Sept. 2008 and July 2014 underwent xenografting using NodScid/IL2rgKO (NSG) mice. Xenografts were passaged when tumour volume reached 1 cm3. Mice were sacrificed if no engraftment by 12 months (mos). Pt charts were reviewed to determine biomarker status (hormone receptor [HR], HER2), grade, LVI, time free from disease/progression, and pathologic complete response (pCR) for pts receiving neoadjuvant therapy. Results: A total of 64 pts had known xenograft status: 32 engrafters, 32 non-engrafters. Biomarker status did not predict likelihood of engraftment (p = .0695) and is shown in Table 1 along with site/timing of biopsy for tissue engraftment within biomarker groups. When HER2+ cases are excluded from analysis, the HR-/HER2- phenotype yields a 72% probability of engraftment compared to 39% for the HR+/HER2- group (p = .0418). For engrafters, 22/32 (68%) of pts had or went on to have relapsed or de novo MBC. For non-engrafters, 7/32 (22%) had or went on to have relapsed disease (p = .0004). Grade and LVI did not predict engraftment (p = .1806 and p = .8657 respectively). No grade 1 tumours engrafted (n = 3). There were 25 pts who received neoadjuvant chemotherapy: 14 engrafters, 11 non-engrafters. None of the engrafters achieved a pCR; 3 non-engrafters achieved a pCR. Median time to engraftment was 5 mos for pts with relapsed/advanced disease vs 9.8 mos for pts who did not relapse however, treatment was variable. Conclusion: This preliminary study highlights potential differences in clinical characteristics of engrafters vs non-engrafters in breast cancer xenograft models and warrants further exploration. (Funded by CBCRA, BCCF) TABLE 1.Biomarker status, tissue site/type in attempted xenografts.Engrafter (N = 32) N, (%)Non-engrafter (N = 32) N, (%)HR+/HER2-14 (44)22 (69)Tissue from:Primary tumour419Recurrence23Advanced dz on therapy80HR-/HER2-13 (40)5 (16)Tissue from:Primary tumour114Recurrence21Advanced dz on therapy00HER2+/HR+0 (0)2 (9)Tissue from:Primary tumour02Recurrence00Advanced dz on therapy00HER2+/HR-5 (16)3 (9)Tissue from:Primary tumour42Recurrence01Advanced dz on therapy10 Citation Format: Wendie D. den Brok, Stephen Chia, Cherie Bates, Steve Kalloger, Samuel Aparicio, Mar Mar, Karen Gelmon, Peter Eirew. Clinical characteristics of breast cancer xenograft models. [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 638.

Hydrogen peroxides (H2O2) are known to activate multiple cell signaling pathways but the mechanisms involved and how they are differentially regulated in specific normal mammary cell types is unknown. The luminal progenitor (LP) fraction of cells of the normal human mammary gland are of particular interest in this regard because, compared to the basal cells (BCs), these cells consume more O2, sustain higher levels of ROS, and are more resistant to H2O2 levels by virtue of their repertoire of enzymes that reduce both ROS and oxidized nucleotide products of ROS. However, these features of normal human LPs are also accompanied by their accumulation of more DNA damage. Here we examine the idea that the greater tolerance of LPs to ROS may be associated with a previously unknown intracellular signaling role of ROS in these cells. Using an optimized quantitative twin-photon and confocal-reflectance imaging system, we have found that the size of the spherical 3D structures produced in Matrigel cultures by freshly isolated, FACS-purified normal human LPs is increased in the presence of exogenous H2O2 at concentrations that are toxic to BCs. In addition to LPs, co-purified non-clonogenic luminal cells (LCs) display elevated levels of peroxiredoxin-1 peroxidase, a negative regulator of H2O2 action, as compared to BCs. Both of the luminal cell types (but not BCs) also showed tyrosine phosphorylation of peroxiredoxin-1 peroxidase (a biomarker of H2O2 action) when exposed for 10 minutes to exogenous H2O2, but LPs only showed marked inactivation of peroxiredoxin-1 in the absence of an external stimulus. Western blot analysis revealed a parallel and dramatic H2O2-induced pan-tyrosine phosphorylation response selectively in both luminal subsets, and their analysis at the single cell level by mass cytometry using a CyTOF identified multiple activated signaling intermediates. From FACS, immunohistochemistry, Western blot, microarray and RNA-Seq data analyses, we identified cKIT as the most differentially and highly expressed (albeit trypsin-sensitive) tyrosine kinase in LPs. Epigenetic analysis of the cKIT promoter showed it to be in an ‘open’ state exclusively in human LPs, and H2O2 treatment alone was sufficient to rapidly activate auto-phosphorylation of the cKIT Y719 residue, a site known to bind and thereby lead to the activation of PI3 kinase. Taken together, these findings reveal a new, ligand-independent function of a cell surface receptor in mediating a potent, lineage-specific signaling function of H2O2 that, in normal human mammary cells influences cell growth. Citation Format: Nagarajan Kannan, Kingsley Shih, Yifei Dong, Peter Eirew, David Knapp, Davide Pellacani, Hequn Wang, Haishan Zeng, Connie Eaves. Human mammary luminal progenitor cells use cKIT-H2O2 interactions to regulate their growth. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A23.

285 Background: In the absence of defined tumor molecular subtypes and validated predictive markers, PDAC has been largely treated as a single disease. Recent studies of molecular subtyping in PDAC reveal a complex mutational landscape with data suggesting the presence of genomic and gene expression signatures that may have prognostic and therapeutic significance. These studies predominantly focused on resected PDAC and lack data on metastatic tumors. We aim to explore the clinical utility of whole genome sequencing (WGS) and transcriptome analysis from metastatic biopsy samples in patients (pts) with advanced PDAC. Methods: Pts with incurable advanced cancers undergo tumor biopsy for in-depth WGS and RNA sequencing (RNASeq) as part of an ongoing prospective study (NCT02155621). Comprehensive bioinformatics analysis is performed to identify somatic cancer aberrations, gene expression changes and cellular pathway abnormalities. Here we describe clinical and molecular data on the subset of pts with advanced PDAC. Results: Sixteen PDAC pts have been enrolled; median age 59 years, 8 males (50%), 10 with de novo metastases (63%). Full WGS and RNASeq were completed in 11 pts (1 failed biopsy, 4 had insufficient tumor). KRAS codon 12 and TP53 mutations were present in all but one pt. CDKN2A and SMAD4 were also frequently altered (7 and 4 pts respectively). Gene expression analysis for classical and basal subtypes similar to those recently described (PMID 26343385) identified 3 and 6 pts with classical and basal expression patterns respectively, and 2 pts with mixed expression. Overall survival (OS) was significantly worse for the basal subtype vs all others (median OS 7 vs. 13.9 months (ms), p = 0.017). When separated into 3 subtypes a significant difference was still noted (median OS 7 ms in basal, 19.2 ms in classical and 11.8 ms in mixed subtype, p = 0.032). Conclusions: WGS analysis demonstrated a similar mutation pattern to that described in resectable PDAC, with no novel actionable mutations identified. Gene expression analysis demonstrated the presence of distinct gene expression signatures significantly associated with outcome, despite small pt numbers. These results need to be validated prospectively in larger cohorts. Clinical trial information: NCT02155621.

Elevated aldehyde dehydrogenase (ALDH) expression/activity has been identified as an important biomarker of primitive cells in various normal and malignant human tissues. Here we examined the level and type of ALDH expression and activity in different subsets of phenotypically and functionally defined normal human mammary cells. We find that the most primitive human mammary stem and progenitor cell types with bilineage differentiation potential show low ALDH activity but undergo a marked, selective, and transient upregulation of ALDH activity at the point of commitment to the luminal lineage. This mirrors a corresponding change in transcripts and protein levels of ALDH1A3, an enzyme involved in retinoic acid synthesis and the most highly expressed ALDH gene in normal human mammary tissue. In contrast, ALDH1A1 is expressed at low levels in all mammary epithelial cells. These findings raise interesting questions about the reported association of ALDH activity with breast cancer stem cells and breast cancer prognosis. Disclosure of potential conflicts of interest is found at the end of this article.

Methods to identify and enumerate primitive, and typically rare, undifferentiated cells in normal tissue using functional endpoints are powerful tools for acquiring insights into the mechanisms that regulate normal tissue stem cell turnover and differentiation. In this paper, we describe a xenotransplantation-based protocol that allows mammary stem cells with in vivo tissue regenerative properties to be specifically detected and quantified among the heterogeneous cell populations obtained from dissociated normal human mammary tissue. This methodology involves implanting a collagen gel containing the test cells in combination with supportive fibroblasts under the kidney capsule of highly immune-deficient, hormone-supplemented mice and then, 4 weeks later, searching for regenerated human cells with in vitro clonogenic activity. Quantification of the input human mammary stem cells is achieved using standard limiting dilution transplant approaches. This approach circumvents the need to modify the mouse mammary fat pad, and is objective, rapid (∼5 weeks) and economical to perform.

Background: In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow’s milk using gene transfer. Methods and Findings: We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human b-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. Conclusions: These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted “panel” sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate “drivers.” Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.

Background Breast cancer is a complex disease with clinical, pathological, and molecular heterogeneity. Recent studies have identified several subtypes of breast cancers driven by specific molecular pathways that can be inhibited by targeted drugs. We studied the feasibility of using molecular data from whole genome and transcriptome sequencing of breast cancers to guide treatment. Methods Patients were consented as part of the Personalized Onco-Genomics (POG) study at the British Columbia Cancer Agency. Fresh tumor, blood for normal DNA, and archival tumor were collected. Tissue and blood samples were sequenced using the Ion Torrent AmpliSeq panel, followed by comprehensive DNA and RNA sequencing. In-depth bioinformatic analyses were performed. Somatic mutations, copy number changes, structural variants and gene expression were characterized. Results from genomic analyses were reviewed in a multi-disciplinary team. Results From August 2012 to October 2014, tissue samples from 30 patients with advanced stage breast cancer were analyzed. The median age at diagnosis was 53 years (range 32-75). The median number of lines of cytotoxic therapy prior to sequencing was 2 (range 0-10). All cases were invasive ductal carcinoma; 63% were ER+ and HER2-; 27% triple negative; 7% ER+ and HER2+; and 3% ER- and HER2+. The most frequently mutated genes were TP53 (67%), PI3KCA (23%), ESR1 (20%), ATM (10%), ARID1A (10%), and BRCA1/2 (10%). Somatic mutations in other genes of interest including HER2, PARP1, NF1, BAP1, PTEN, NOTCH1, were also identified. Molecular data were informative for patient care and/or actionable to guide treatment in 57% (17/30) of cases. Conclusion The use of whole genome sequencing technology to identify valuable molecular information to guide personalized breast cancer treatment is feasible. Further studies are warranted to evaluate the usefulness of genome-wide sequencing of breast cancers in clinical practice. Citation Format: Sophie Sun, Karen A. Gelmon, Stephen Chia, Caroline Lohrisch, Tamara Shenkier, Diego Villa, Yaoqing Shen, Martin Jones, Erin Pleasance, Katayoon Kasaian, Peter Eirew, Sreeja Leelakumari, Yusanne Ma, Tony Ng, Stephen Yip, Steven JM Jones, Marco A. Marra, Janessa J. Laskin. Personalized oncogenomics in advanced stage breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1122. doi:10.1158/1538-7445.AM2015-1122

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.

The Personalized Oncogenomics initiative at the British Columbia Cancer Agency aims to identify tumor-specific therapeutic targets in cancer patients with late stage disease who have failed standard therapy. Comprehensive profiling of individual patients' tumor(s) at the DNA and RNA level allows for characterization of altered pathways and hence identification of therapeutics designed to specifically target them. Data for each individual study included whole genome and transcriptome sequence of the fresh biopsy and whole genome sequence of patient's blood. When tissues were available, transcriptome of a matched normal sample and genome of the commonly formalin-fixed paraffin-embedded primary tumor were also sequenced. All sequencing experiments were performed on Illumina machines. Genomic data was examined, depending on the case, for germline and/or somatic mutations. These included single nucleotide variants, small insertions and deletions and copy number variations. All sequence data were assembled de novo in order to identify rearrangements causing gene fusions; transcriptome data also revealed allelic expression of variants and provided a profile for the entire transcribed genome. Differential abundance estimation was run against a rich repository of publicly available data from The Cancer Genome Atlas project and transcriptome datasets available in-house. The variants and pathways were then mapped to drug databases as well as clinical trial records. This was followed by an extensive literature search for evidence of drug combinations, drug-drug interactions and efficacy of a drug for a particular cancer type, especially those not recognized as the approved disease group for the drug under consideration. The project has sequenced 50 patients; the average length of time between acquiring the biopsy and delivering a report to clinical oncologists was 37 days. Bioinformatic analysis of the sequence data led to identification of informative or actionable targets in up to 80% of cases. The findings were not restricted to target identification but also led to change of diagnosis, treatment and characterization of tumor evolution. De novo assembly of the data in a non-small cell lung cancer patient led to the identification of the well-characterized EML4-ALK oncogenic fusion which had been missed through the use of clinically approved fluorescence in situ hybridization test. Analysis of two separate malignant masses in another patient revealed two divergent and unique tumors. Two different therapeutic were prescribed in order to target these; this led to the disappearance of both tumors and disease stabilization for 7 months. Through the design of an efficient and automated bioinformatics pipeline, individual patient's tumor specimen(s) were profiled in a clinically relevant time frame. This in turn enabled the delivery of targeted therapies and disease stabilization in patients who had no remaining standard therapeutic options. Citation Format: Katayoon Kasaian, Yaoqing Shen, Sreeja Leelakumari, Peter Eirew, Yvonne Y. Li, Erin Pleasance, Richard Corbett, Karen L. Mungall, Jacquie Schein, Andrew J. Mungall, Yongjun Zhao, Richard A. Moore, Stephen Yip, Karen Gelmon, Howard Lim, Daniel Renouf, Robyn Roscoe, Yussanne Ma, Marco A. Marra, Janessa Laskin, Steven JM Jones. Bioinformatic analyses approaches for personalized oncogenomics. [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 5340. doi:10.1158/1538-7445.AM2014-5340