Intratumoral Heterogeneity In Breast Cancer Research Articles

Abstract PL-01: Breast Cancer Evolution, Immune Evasion and Metastasis Driven by Chromosomal Instability

Abstract Chromosomal instability (CIN) is a poor prognostic feature with high prevalence in breast cancer and is responsible for driving Somatic Copy Number Aberrations and Intratumour Heterogeneity (ITH) within subclones during breast cancer evolution. We have observed that SCNAs are enriched in metastatic samples, including gains in chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. However, there have thus far been no prospective, comprehensive studies of intratumor heterogeneity in breast cancer. We will present data from TRACERx Breast/SCANDARE a prospective, multicentre study of triple-negative breast cancer (TNBC) in which multi-region biopsies have been collected from primary TNBC prior to neoadjuvant treatment, and at longitudinal time points including surgery and relapse. We find prevalent intratumor heterogeneity in somatic mutations and SCNAs in the primary tumor prior to treatment. We have leveraged the rich clinical annotation in this cohort to correlate intratumor heterogeneity scores with key clinical outcomes, including treatment response (pathological complete response to neoadjuvant chemotherapy) and survival. We find that CIN manifests as oncogenic amplification on extrachromosomal DNA (ecDNA), which plays a pivotal role in driving drug resistance and tumor evolution. Through analysis of Whole Genome Sequencing data from 2936 breast tumors from the Genomics England breast cancer cohort, we have identified focal amplifications driven by ecDNA in 46.4% of HER2+ breast cancers. EcDNA were enriched in metastatic tumors and was associated with poor clinical outcomes. In addition to oncogenic amplifications such as HER2 derived from ecDNA during cancer evolution, ecDNAs contained immunomodulatory genes associated with reduced T cell infiltration. Further Immune-modulation by CIN results from loss of heterozygosity of the HLA locus (HLA LOH) and is a prevalent subclonal event in breast cancer. CIN is further drives haploid LOH, resulting in the high prevalence of whole genome doubling and further CIN in TNBC. Tumors are heterogeneous compositions of distinct clones with different compliments of SCNAs and varying levels of fitness, hence measuring the proliferation of individual clones to predict future evolutionary outcomes is likely to be critical. We have developed SPRINTER (Single-cell Proliferation Rate Inference in Non-homogeneous Tumours through Evolutionary Routes), a novel computational method to measure proliferation rates in individual tumour clones using single-cell whole-genome DNA sequencing data. We applied SPRINTER to 42,009 triple negative breast cancer cells, demonstrating significant proliferative heterogeneity between distinct tumor clones. Importantly, we show a correlation between higher proliferation rates and increased rates of somatic variants, suggesting proliferation links to clone evolvability. Taken together, these complimentary analyses provide evidence for the importance of CIN in driving ITH, selection, metastasis and immune evasion and provide a framework to determine recurrent evolutionary patterns in breast cancer evolution. Citation Format: C. Swanton. Breast Cancer Evolution, Immune Evasion and Metastasis Driven by Chromosomal Instability [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PL-01.

Phenotypic heterogeneity drives differential disease outcome in a mouse model of triple negative breast cancer.

The triple negative breast cancer (TNBC) subtype is one of the most aggressive forms of breast cancer that has poor clinical outcome and is an unmet clinical challenge. Accumulating evidence suggests that intratumoral heterogeneity or the presence of phenotypically distinct cell populations within a tumor play a crucial role in chemoresistance, tumor progression and metastasis. An increased understanding of the molecular regulators of intratumoral heterogeneity is crucial to the development of effective therapeutic strategies in TNBC. To this end, we used an unbiased approach to identify a molecular mediator of intratumoral heterogeneity in breast cancer by isolating two tumor cell populations (T1 and T2) from the 4T1 TNBC model. Phenotypic characterization revealed that the cells are different in terms of their morphology, proliferation and self-renewal ability in vitro as well as primary tumor formation and metastatic potential in vivo. Bioinformatic analysis followed by Kaplan Meier survival analysis in TNBC patients identified Metastasis associated colon cancer 1 (Macc1) as one of the top candidate genes mediating the aggressive phenotype in the T1 tumor cells. The role of Macc1 in regulating the proliferative phenotype was validated and taken forward in a therapeutic context with Lovastatin, a small molecule transcriptional inhibitor of Macc1 to target the T1 cell population. This study increases our understanding of the molecular underpinnings of intratumoral heterogeneity in breast cancer that is critical to improve the treatment of women currently living with the highly aggressive TNBC subtype.

HER2 Intratumoral Heterogeneity in Breast Cancer, an Evolving Concept.

Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is associated with an adverse prognosis. The introduction of anti-HER2 targeted therapy has dramatically improved the clinical outcomes of patients with HER2-positive breast cancer. Unfortunately, a significant number of patients eventually relapse and develop distant metastasis. HER2 intratumoral heterogeneity (ITH) has been reported to be associated with poor prognosis in patients with anti-HER2 targeted therapies and was proposed to be a potential mechanism for anti-HER2 resistance. In this review, we described the current definition, common types of HER2 ITH in breast cancer, the challenge in interpretation of HER2 status in cases showing ITH and the clinical applications of anti-HER2 agents in breast cancer showing heterogeneous HER2 expression. Digital image analysis has emerged as an objective and reproducible scoring method and its role in the assessment of HER2 status with ITH remains to be demonstrated.

Transcriptomic intratumor heterogeneity of breast cancer patient-derived organoids may reflect the unique biological features of the tumor of origin

BackgroundThe intratumor heterogeneity (ITH) of cancer cells plays an important role in breast cancer resistance and recurrence. To develop better therapeutic strategies, it is necessary to understand the molecular mechanisms underlying ITH and their functional significance. Patient-derived organoids (PDOs) have recently been utilized in cancer research. They can also be used to study ITH as cancer cell diversity is thought to be maintained within the organoid line. However, no reports investigated intratumor transcriptomic heterogeneity in organoids derived from patients with breast cancer. This study aimed to investigate transcriptomic ITH in breast cancer PDOs.MethodsWe established PDO lines from ten patients with breast cancer and performed single-cell transcriptomic analysis. First, we clustered cancer cells for each PDO using the Seurat package. Then, we defined and compared the cluster-specific gene signature (ClustGS) corresponding to each cell cluster in each PDO.ResultsCancer cells were clustered into 3–6 cell populations with distinct cellular states in each PDO line. We identified 38 clusters with ClustGS in 10 PDO lines and used Jaccard similarity index to compare the similarity of these signatures. We found that 29 signatures could be categorized into 7 shared meta-ClustGSs, such as those related to the cell cycle or epithelial–mesenchymal transition, and 9 signatures were unique to single PDO lines. These unique cell populations appeared to represent the characteristics of the original tumors derived from patients.ConclusionsWe confirmed the existence of transcriptomic ITH in breast cancer PDOs. Some cellular states were commonly observed in multiple PDOs, whereas others were specific to single PDO lines. The combination of these shared and unique cellular states formed the ITH of each PDO.

APPLICATIONS OF TRANSCRIPTOMICS METHODS IN BREAST CANCER

Gene expression studies using cDNA microarrays have revolutionized the breast cancer subclassification. The application of RNA sequencing technologies has revealed additional complexity of breast cancer. The integrated genomic/transcriptomic analysis of breast tumors (METABRIC) and the Cancer Genome Atlas (TCGA) with clinical follow-up data shaped the molecular landscape of breast cancer the last decade. High-throughput RNA sequencing technologies have shown that large proportions of mammalian transcriptome include regulatory non-protein-coding RNAs (ncRNAs) besides protein-coding RNAs. Role of ncRNAs have been well documented in breast cancer. Using RNA sequencing and probe-based transcriptome analyses, several studies, including ours, have emphasized the role alternative splicing events in breast cancer progression. We have demonstrated that Epithelial Splicing Regulatory Protein 1 (ESRP1), a splicing factor, is associated with poor prognosis and endocrine resistance in human ER-positive (ER+) breast tumors. These studies have further implicated that aberrant regulation of alternative splicing plays an important role in all hallmarks of cancer and provide novel strategies for the therapeutics in breast cancer. In addition to bulk sequencing, deconvolution methods such as CIBERSORT was useful to understand the role of individual cells in cancer progression. Using TCGA and METABRIC cohorts, we identified immune-subtypes with better outcomes in triple negative breast cancer (TNBCs). Recently, single cell RNA-seq (sc-RNAseq) and spatial RNA-seq technologies have enabled analysis of single tumor cell phenotypes and intratumor heterogeneity. The application of these technologies to formalin-fixed paraffin-embedded (FFPE) tumor samples with clinical outcomes will present a valuable resource for understanding the inter and intra-tumor heterogeneity in breast cancer.

Identification of Breast Cancer Immune Subtypes by Analyzing Bulk Tumor and Single Cell Transcriptomes.

Background: The histological and molecular classification of breast cancer (BC) is being used in the clinical management of this disease. However, subtyping of BC based on the tumor immune microenvironment (TIME) remains insufficiently explored, although such investigation may provide new insights into intratumor heterogeneity in BC and potential clinical implications for BC immunotherapy. Methods: Based on the enrichment scores of 28 immune cell types, we performed clustering analysis of transcriptomic data to identify immune-specific subtypes of BC using six different datasets, including five bulk tumor datasets and one single-cell dataset. We further analyzed the molecular and clinical features of these subtypes. Results: Consistently in the six datasets, we identified three BC subtypes: BC-ImH, BC-ImM, and BC-ImL, which had high, medium, and low immune signature scores, respectively. BC-ImH displayed a significantly better survival prognosis than BC-ImL. Triple-negative BC (TNBC) and human epidermal growth factor receptor-2-positive (HER2+) BC were likely to have the highest proportion in BC-ImH and the lowest proportion in BC-ImL. In contrast, hormone receptor-positive (HR+) BC had the highest proportion in BC-ImL and the lowest proportion in BC-ImH. Furthermore, BC-ImH had the highest tumor mutation burden (TMB) and predicted neoantigens, while BC-ImL had the highest somatic copy number alteration (SCNA) scores. It is consistent with that TMB and SCNA correlate positively and negatively with anti-tumor immune response, respectively. TP53 had the highest mutation rate in BC-ImH and the lowest mutation rate in BC-ImL, supporting that TP53 mutations promote anti-tumor immune response in BC. In contrast, PIK3CA displayed the highest mutation rate in BC-ImM, while GATA3 had the highest mutation rate in BC-ImL. Besides immune pathways, many oncogenic pathways were upregulated in BC-ImH, including ErbB, MAPK, VEGF, and Wnt signaling pathways; the activities of these pathways correlated positively with immune signature scores in BC. Conclusions: The tumors with the strong immune response (“hot” tumors) have better clinical outcomes than the tumors with the weak immune response (“cold” tumors) in BC. TNBC and HER2+ BC are more immunogenic, while HR + BC is less immunogenic. Certain HER2+ or HR + BC patients could be propitious to immunotherapy in addition to TNBC.

Integration of Multitargeted Polymer-Based Contrast Agents with Photoacoustic Computed Tomography: An Imaging Technique to Visualize Breast Cancer Intratumor Heterogeneity.

One of the primary challenges in breast cancer diagnosis and treatment is intratumor heterogeneity (ITH), i.e., the coexistence of different genetically and epigenetically distinct malignant cells within the same tumor. Thus, the identification of ITH is critical for designing better treatments and hence to increase patient survival rates. Herein, we report a noninvasive hybrid imaging technology that integrates multitargeted and multiplexed patchy polymeric photoacoustic contrast agents (MTMPPPCAs) with single-impulse panoramic photoacoustic computed tomography (SIP-PACT). The target specificity ability of MTMPPPCAs to distinguish estrogen and progesterone receptor-positive breast tumors was demonstrated through both fluorescence and photoacoustic measurements and validated by tissue pathology analysis. This work provides the proof-of-concept of the MTMPPPCAs/SIP-PACT system to identify ITH in nonmetastatic tumors, with both high molecular specificity and real-time detection capability.

Methodological Advancements for Investigating Intra-tumoral Heterogeneity in Breast Cancer at the Bench and Bedside

There is a major need to overcome therapeutic resistance and metastasis that eventually arises in many breast cancer patients. Therapy resistant and metastatic tumors are increasingly recognized to possess intra-tumoral heterogeneity (ITH), a diversity of cells within an individual tumor. First hypothesized in the 1970s, the possibility that this complex ITH may endow tumors with adaptability and evolvability to metastasize and evade therapies is now supported by multiple lines of evidence. Our understanding of ITH has been driven by recent methodological advances including next-generation sequencing, computational modeling, lineage tracing, single-cell technologies, and multiplexed in situ approaches. These have been applied across a range of specimens, including patient tumor biopsies, liquid biopsies, cultured cell lines, and mouse models. In this review, we discuss these approaches and how they have deepened our understanding of the mechanistic origins of ITH amongst tumor cells, including stem cell-like differentiation hierarchies and Darwinian evolution, and the functional role for ITH in breast cancer progression. While ITH presents a challenge for combating tumor evolution, in-depth analyses of ITH in clinical biopsies and laboratory models hold promise to elucidate therapeutic strategies that should ultimately improve outcomes for breast cancer patients.

Antibody-drug conjugates in breast cancer: the chemotherapy of the future?

Antibody-drug conjugates (ADCs) represent an interesting new class of anticancer agents, capable of exploiting the specificity of monoclonal antibodies toward cellular-antigens for a targeted release of potent cytotoxic drugs, with a potential increased activity and reduced toxicity compared with traditional chemotherapies. The aim of this article is to review the efficacy and safety of ADCs in breast cancer. Following the approval of T-DM1 both in early and advanced human epidermal growth factor receptor 2 (HER2)-positive breast cancer, novel anti-HER2 ADCs have been investigated. Some of these compounds, such as the recently FDA-approved trastuzumab deruxtecan, have shown relevant activity in T-DM1-pretreated patients, possibly thanks to the so-called bystander effect, namely the ability to exert cytotoxic activity also against antigen-negative cells. Such feature allows to overcome the HER2 intratumoral heterogeneity in breast cancer and could explain in the preliminary activity demonstrated also in HER2-low breast cancers. However, several ADCs targeting other cancer-associated antigens than HER2 are under development, representing a promising strategy for the treatment of triple-negative tumors, exemplified by the encouraging results of sacituzumab govitecan. ADCs are innovative and effective therapeutic drugs in breast cancer. Research efforts are ongoing to identify novel targets and combination with other treatment modalities, particularly with immunotherapy, to further improve patients' outcomes.

Single-Cell Analysis Reveals a Preexisting Drug-Resistant Subpopulation in the Luminal Breast Cancer Subtype.

Drug resistance is a major obstacle in the treatment of breast cancer. Surviving cells lead to tumor recurrence and metastasis, which remains the main cause of cancer-related mortality. Breast cancer is also highly heterogeneous, which hinders the identification of individual cells with the capacity to survive anticancer treatment. To address this, we performed extensive single-cell gene-expression profiling of the luminal-type breast cancer cell line MCF7 and its derivatives, including docetaxel-resistant cells. Upregulation of epithelial-to-mesenchymal transition and stemness-related genes and downregulation of cell-cycle-related genes, which were mainly regulated by LEF1, were observed in the drug-resistant cells. Interestingly, a small number of cells in the parental population exhibited a gene-expression profile similar to that of the drug-resistant cells, indicating that the untreated parental cells already contained a rare subpopulation of stem-like cells with an inherent predisposition toward docetaxel resistance. Our data suggest that during chemotherapy, this population may be positively selected, leading to treatment failure. SIGNIFICANCE: This study highlights the role of breast cancer intratumor heterogeneity in drug resistance at a single-cell level.

Abstract 140: Comprehensive analysis of immune evasion in breast cancer by single-cell RNA-seq

Abstract The tumor microenvironment is composed of numerous cell types, including tumor, immune and stromal cells. Cancer cells interact with the tumor microenvironment to suppress anticancer immunity. In this study, we molecularly dissected the tumor microenvironment of breast cancer by single-cell RNA-seq. The breast tumor microenvironment was profiled by analyzing the single-cell transcriptomes of 52,163 cells from the tumor tissues of 15 breast cancer patients. The tumor cells and immune cells from individual patients were analyzed simultaneously at the single-cell level. Among more than 40,000 tumor cells, we identified distinct cell clusters with different molecular subtypes and gene signatures in the same tumor, which revealed intra-tumor heterogeneity in breast cancer. We comprehensively profiled the functional state of T cells, macrophages, B cells, CAFs in breast cancer tumor microenvironment, which highlight the immune cell and CAF diversity in the breast cancer TME. Moreover, we analyzed intercellular communication in the breast cancer TME using a dataset of human ligand receptor pairs, which showed distinct cell interaction models across tumors. The evolution of the tumor microenvironment during clinical treatment was dissected by comparing tumor microenvironment of breast cancer pre- and post-chemotherapy. This study investigated the diversity of the cell types in the tumor microenvironment and provided information on the mechanisms of escape from clearance by immune cells in breast cancer, which could be a beneficial resource for immunotherapy development. Citation Format: Jianhua Yin, Hanlin Zhou, Chen Yan, Ruqian lv, Ting Wang, Chenyang He, Chen Huang, Yong Hou, Shida Zhu, Ling Wang, Michael Dean, Kui Wu, Hong Hu, Guibo Li. Comprehensive analysis of immune evasion in breast cancer by single-cell RNA-seq [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 140.

Abstract 5152: EMT cells increase the metastatic potential of neighboring carcinoma cells via non-canonical activation of GLI signaling through secretion of VEGF-C

Abstract Breast cancer is a devastating disease that claims around 50 million lives per year worldwide, and almost all of these deaths result from metastatic disease rather than from primary tumor burden. Recent studies clearly demonstrate that there is significant intratumor heterogeneity in breast cancer, and that this heterogeneity contributes to malignant progression. Our laboratories previously demonstrated that breast cancer cells that had undergone an oncogenic epithelial-to-mesenchymal transition (EMT) could increase metastasis of neighboring cells via activation of non-canonical GLI transcription factor activity in a paracrine manner that is dependent on Six1 expression in the EMT cells. However, the mechanism by which these Six1-expressing EMT cells activate GLI signaling thereby imparting aggressive properties on neighboring cells remained unknown. Herein we describe the novel discovery that VEGF-C, which is transcriptionally upregulated by Six1, mediates paracrine non-canonical activation of GLI and resultant enhancement of metastatic properties in cells that do not express Six1. Our data demonstrate that VEGF-C is upregulated in HMLER-Snail1 cells, MCF7-Six1 cells and in Met-1 cells endogenously expressing Six1, in a manner that depends on Six1 expression, and that VEGF-C is secreted into the conditioned media (CM) of these cells. Inhibition of VEGF-C in the HMLER-Snail1, MCF7-Six1, and the Met-1 models abrogates paracrine GLI activation and attenuates non-cell autonomous induction of proliferation, migration, and invasion in vitro. In vivo, we show that cells expressing Six1 can enhance the growth and metastasis of those not expressing Six1, and studies will be discussed that examine whether VEGF-C inhibition in Six1 expressing cells disrupts the crosstalk and inhibits non-cell autonomous induction of metastasis by Six1. Finally, by interrogating the TCGA dataset we find that VEGF-C and GLI1 expression significantly positively correlate in human breast cancer encompassing all molecular subtypes. Taken together, these data suggest that VEGF-C secretion may be a novel and conserved paracrine means by which EMT cells activate GLI in neighboring tumor cells that do not express these EMT-inducing transcription factors (TF), ultimately enhancing overall metastasis of heterogenous breast tumors. Citation Format: Deguang Kong, Deepika Neelakantan, Hengbo Zhou, Michael T. Lewis, Heide L. Ford. EMT cells increase the metastatic potential of neighboring carcinoma cells via non-canonical activation of GLI signaling through secretion of VEGF-C [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 5152.

Identification of ERBB Pathway-Activated Cells in Triple-Negative Breast Cancer.

Intratumor heterogeneity within a single tumor mass is one of the hallmarks of malignancy and has been reported in various tumor types. The molecular characterization of intratumor heterogeneity in breast cancer is a significant challenge for effective treatment. Using single-cell RNA sequencing (RNA-seq) data from a public resource, an ERBB pathway activated triple-negative cell population was identified. The differential expression of three subtyping marker genes (ERBB2, ESR1, and PGR) was not changed in the bulk RNA-seq data, but the single-cell transcriptomes showed intratumor heterogeneity. This result shows that ERBB signaling is activated using an indirect route and that the molecular subtype is changed on a single-cell level. Our data propose a different view on breast cancer subtypes, clarifying much confusion in this field and contributing to precision medicine.

Abstract P6-04-02: Evaluation of breast cancer intratumor heterogeneity and its implications to the therapeutic agents with organoids subclones

Abstract Background: Breast cancer is the second most common cancer in women, accounting for 15.3% new cancer cases and 6.7% cancer death in 2018. Chemotherapy, target therapy and endocrine therapy are main strategies for breast cancer treatment after surgery to prevent cancer recurrence and metastasis. The choice of these strategies is mainly based on the molecular and pathological markers as well as experience from clinicians. However, due to the heterogeneity of intra-tumor and also between different patients, it is hard for clinicians to make the best therapeutic regimen to target all the cancer sub-population at current stage. Developing more precise and personalized chemotherapy strategy for individual patient would be of great importance. Cancer organoids, established based on in vitro 3-D culture techniques, were demonstrated to largely retain the biological characteristics of tumors from patients, and made it possible to assess the responses to various strategies for individual patient. Comparing with other cancer type such as colon cancer, breast cancer organoids study was relatively immature. This study was designed to efficiently establish breast cancer organoids from clinical patients and further test the sensitivity of these organoids to therapeutic agents. Methods: Fresh breast cancer tissues from patients were dissected into small pieces and subjected to enzyme digestion to prepare a single cell suspension. Organoids were established from the single cell suspension in Matrigel and modified PDTO culture medium. Organoid clones derived from various cancer cells were heterogeneous, which were isolated with pipette tips to establish breast cancer organoids subclones. First line chemotherapy drugs such as Docetaxel, Adriamycin, and Fluorouracil, target therapy agents and endocrine therapy drug such as PD-991, Neratinib and Afimoxifene were used to treat each organoid clones. Cell survival rate was measured by CellTiter-Glo Kit and drug sensitivity was assessed by IC50 value. Results: 23 organoid subclones from 5 patients were successfully established. Drug response (effect) varied to individual organoid subclones from the same patient. Some subclones showed totally resistance while others were sensitive or parcial sensitive to indicated therapeutic agents, indicating the heterogeneity of breast cancer and different intra-tumor subpopulation have distinct drug response. The heterogeneity, especially the resistant subclones put forward to explain the acquired drug resistance and relapse in clinical practice. Conclusion:Breast cancer organoids are good in vitro models for drug sensitivity screening, which would assist clinicians make better chemotherapy strategy to achieve precise and personalized medication for patients. Moreover, organoid models would also be an ideal platform to help dissect the underlying mechanism of cancer heterogeneity induced drug resistance. Citation Format: Zhang B, Xu X, Cai H, Sun Z. Evaluation of breast cancer intratumor heterogeneity and its implications to the therapeutic agents with organoids subclones [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-04-02.

Intratumoral Heterogeneity in Breast Cancer: A Case Report and Molecular Discussion

The emerging era of personalized medicine makes it increasingly important to consider intratumoral heterogeneity, which has been found in some breast cancer cases. However, its identification criteria, form of reporting, and subsequent effects on the clinical course of this disease remain controversial and not fully defined. Here, we report and discuss a case of breast invasive ductal adenocarcinoma with substantial intratumoral heterogeneity, discrepancy between Her2/neu immunostaining and in situ hybridization, and disparity between estrogen receptor status before and after neoadjuvant therapy.

Intra-tumor molecular heterogeneity in breast cancer: definitions of measures and association with distant recurrence-free survival.

The purpose of the study was to define quantitative measures of intra-tumor heterogeneity in breast cancer based on histopathology data gathered from multiple samples on individual patients and determine their association with distant recurrence-free survival (DRFS). We collected data from 971 invasive breast cancers, from 1st January 2000 to 23rd March 2014, that underwent repeat tumor sampling at our institution. We defined and calculated 31 measures of intra-tumor heterogeneity including ER, PR, and HER2 immunohistochemistry (IHC), proliferation, EGFR IHC, grade, and histology. For each heterogeneity measure, Cox proportional hazards models were used to determine whether patients with heterogeneous disease had different distant recurrence-free survival (DRFS) than those with homogeneous disease. The presence of heterogeneity in ER percentage staining was prognostic of reduced DRFS with a hazard ratio of 4.26 (95% CI 2.22-8.18, p < 0.00002). It remained significant after controlling for the ER status itself (p < 0.00062) and for patients that had chemotherapy (p < 0.00032). Most of the heterogeneity measures did not show any association with DRFS despite the considerable sample size. Intra-tumor heterogeneity of ER receptor status may be a predictor of patient DRFS. Histopathologic data from multiple tissue samples may offer a view of tumor heterogeneity and assess recurrence risk.

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

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

Abstract P5-06-02: Ex vivo CSC assays for personalized testing of drug susceptibility in advanced breast cancer

Abstract In the developing area of personalized medicine, targeted therapies are mainly based on genomic characterization of each tumor, and is currently proposed as promising strategies for advanced breast cancer (ABC). Despite the promises of advanced genome sequencing, many patients still fail therapy, resulting in disease progression, recurrence, and metastases. Cancer stem cells (CSCs) concept illustrates the non-genetic intrinsic resistance, recapitulates tumor heterogeneity that creates hierarchically organized tumor tissues where a subpopulation of self-renewing cancer stem cells (CSCs) sustains the long- term clonal maintenance of the neoplasm. Evidences indicate that CSCs survive many commonly employed cancer therapeutics. Patient-derived tumor xenograft (PDXs) models recapitulate tumor complexity and heterogeneity at cellular and molecular level. We aimed to specifically address the therapeutic sensitivity in ABC, by using an ex vivo assay based on PDX prospective collection, fully characterized for genomic alterations. In this work, we aim at defining for each tumor the best therapy to target breast cancer intratumor heterogeneity, the CSC component. For that, we defined a panel of 44 FDA-approved compounds used for cancer treatment, including breast and other types of cancer, cancer stem cell drugs, chemo or targeted therapies. For each drug, we screened the differential sensitivity of the bulk tumor cells and the CSC components for 12 PDX models using an ex vivo screening approach on short term culture. To assess intra tumor heterogeneity, we set up an original dual strategy: for the bulk cells, an ex vivo assay based on IC50, and for breast CSC component a miniaturized Aldefluor assay. First, we demonstrate that bulk cells and CSCs sensitivity may be dissociated for the same drug in the same PDX models. Then, we observed that whereas bulk cell sensitivity may be correlated to tumor genomic abnormalities, CSC drug sensitivity seems not to follow the rule.CSC are selectively sensitive to specific compounds. We are exploring the pathways that sustain this selective sensitivity in the CSCs components. We are currently identifying targets using mass spectrometry in CSCs and bulk cells.Then, we validated the hits predicted from ex vivo screening assays by in vivo treatment of using PDX models for the selected drugs, and in a patient with ABC. In that work, we demonstrated that CSCs display different sensitivity profiles than bulk cells to the same agents, irrespective to their genomic background and are identifying the CSC specific targets. Here, we propose a new model of precision medicine based on ex vivo CSC assays for personalized testing of drug susceptibility in advanced breast cancer. Citation Format: Charafe-Jauffret E, Wicinski J, Cabaud O, Lopez M, Audebert S, Adelaide J, Chaffanet M, Guille A, Goncalves A, Bertucci F, Birnbaum D, Ginestier C. Ex vivo CSC assays for personalized testing of drug susceptibility in advanced breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-06-02.

Intra-tumor heterogeneity in breast cancer has limited impact on transcriptomic-based molecular profiling

BackgroundTranscriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics.MethodsIn this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications.ResultsMolecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications.ConclusionsOur results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.

The Assessment of Estrogen Receptor Status and Its Intratumoral Heterogeneity in Patients With Breast Cancer by Using 18F-Fluoroestradiol PET/CT.

The aim of this study was to investigate the clinical value of F-fluoroestradiol (F-FES) PET/CT in the assessment of the estrogen receptor (ER) and its intratumoral heterogeneity in breast cancer patients. Forty-six female patients (50 lesions) with histologically confirmed invasive breast cancer who underwent both F-FES and F-FDG PET/CT in our center were retrospectively included. All the patients enrolled were scheduled to undergo biopsy. The F-FES and FDG uptakes were compared with pathological features (tumor size, ER, progesterone receptor, human epidermal growth factor receptor 2, and Ki67%). The optimal threshold to discriminate ER-positive and ER-negative lesions was determined by receiver operating characteristic curve analysis. Furthermore, we observed the intratumoral heterogeneity by a heterogeneity index (SUVmax/SUVmean) and compared the results with the Chang-Gung Image Texture Analysis. There was good agreement between F-FES uptake and ER, progesterone receptor, and human epidermal growth factor receptor 2 expression (P < 0.001), and the use of SUVmean instead of SUVmax can provide a slightly better correlation. The optimal threshold for F-FES PET/CT to discriminate between ER-positive and ER-negative lesions, as determined by receiver operating characteristic curve analysis, was an SUVmax of 1.82 (sensitivity = 88.2% and specificity = 87.5%) and SUVmean of 1.21 (sensitivity = 85.3% and specificity = 93.7). Our simplified heterogeneity index-FES can easily observe ER heterogeneity. In addition, our results suggested that recurrent/metastatic patients and lesions located other than breast might have greater heterogeneity. F-FES PET/CT is a feasible, noninvasive method for assessing ER expression in breast cancer patients. Because intratumoral heterogeneity exists, F-FES PET/CT might better reflect the ER expression, especially in metastatic patients after treatment, thus assisting in making individualized treatment decisions.