Search

Understanding how standard-of-care drug treatments affect tumor intrinsic biology and microenvironment is critical for elucidating drug resistance mechanisms and developing better combination therapies as well as new therapies. To characterize the effects of neoadjuvant chemotherapy (NAC) on the genome, transcriptome and tumor infiltrating leukocytes (TILs), we have conducted whole exome and whole transcriptome sequencing of a large longitudinal breast cancer cohort consisting of 146 cases and 281 paired tumor samples. In total, 52 (38%) patients achieved pathologic complete response (pCR) while 85 patients (62%) had residual disease with standard chemotherapy regimen. Tumor biopsies were collected for each patient at three time points – pre-treatment, three weeks after the first cycle of anthracycline and cyclophosphamide (AC) and at the time of surgery after 3 more cycles of AC followed by 4 cycles of taxane or taxane plus Herceptin in case of HER2+ subtype. We detected 5,955 protein-altering somatic mutations affecting 4,414 genes in pretreatment samples and 502 acquired mutations in surgery samples affecting 477 genes including 19recurrently mutated genes such as TP53 and NOTCH1. Across all subtypes, 4,346 genes were differentially expressed (DE) following NAC treatment and significantly enriched in pathways such as cell cycle, ER signaling, PI3K/mTOR, immune and metabolism. Expression-based virtual microdissection analysis indicated that NAC treatment induced an increase in the fractions of stromal and adjacent normal tissue compartment, consistent with observed reduction in tumor cellularity. To assess the NAC induced changes in the molecular landscape of these tumors, we compared molecular features including gene expression signatures, mutation prevalence and copy number alteration between three time points while adjusting for confounding effects of molecular subtype and tumor cellularity. We found that NAC induced dynamic changes in gene expression signatures associated with proliferation and immunomodulatory treatment response. We further validated the observed pattern of change in TILs through histopathology and digital imaging analyses. In pretreatment tumors, 116 genes were DE between patients with pCR vs. those with residual disease with significant enrichment in immune/inflammatory pathways. Further, pre-treatment TIL levels were found to be significantly associated with pCR, echoing previous reports in breast cancers that implicated anti-tumor immunity in mediating the efficacy of chemotherapies. Our analyses also revealed associations between NAC response and baseline genomic attributes such as genomic alterations that affect DNA damage repair pathways. Taken together, these results suggest that NAC induced a multitude of changes on the genomic landscape and immune microenvironment of breast cancers, some of which point to combination strategies with immunomodulatory therapies and therapies that target DNA damage repair. Citation Format: Kan Z, Lal S, Ding Y, Lee JE, Lee S-H, Lee SK, Yu JH, Choi Y-l, Kim SW, Nam SJ, Kim J-Y, Ram S, Powell E, Ching K, Cho SY, Bonato V, Deng S, Park W-Y, Rejto P, Bienkowska J, Park Y-H. Neoadjuvant chemotherapy alters the genomic landscape and immune microenvironment of breast cancers [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 PD5-08.

BackgroundIntratumoural heterogeneity is a poor prognostic feature in triple-negative breast cancer (TNBC) and other high-grade malignancies. It is caused by genomic instability and phenotypic plasticity, but how these features co-evolve during tumour development remains unclear. SOX10 is a transcription factor, neural crest stem cell (NCSC) specifier and candidate mediator of cancer-associated phenotypic plasticity.MethodsUsing immunophenotyping, we investigated the expression of SOX10 in normal human breast tissue and breast cancer (n=21 cosmetic breast reduction and 1,860 tumour samples with clinical annotation). We then defined the context and evolution of its expression in TNBC compared to 21 other malignancies using systems-level transcriptomics.ResultsSOX10 was detected in nuclei of normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In breast cancer, nuclear SOX10 predicted poor outcome amongst cross-sectional (log-rank p=0.0015, hazard ratio 2.02, n=224) and metaplastic (log-rank p=0.04, n=66) TNBCs. Systems-level transcriptional network analysis identified a core module in SOX10’s normal mammary epithelial transcription program that is rewired to NCSC genes in TNBC. Reprogramming was proportional to DNA damage and genome-wide promoter hypomethylation, particularly at CpG island shores. Using a novel network analysis pipeline, we found that NCSC-like transcriptional reprogramming is also strongly associated with promoter hypomethylation in other SOX10+ malignancies: glioma and melanoma.ConclusionsWe propose that cancer-associated genome hypomethylation simulates the open chromatin landscape of more primitive cell states, and that on this relatively unrestricted background, SOX10 recreates its ancestral gene regulatory circuits by default. These findings provide new insights about the basis of intratumoural heterogeneity and resurrection of developmental phenotypes in cancer; and highlight the potential for therapeutics that limit chromatin remodelling.

To elucidate the effects of neoadjuvant chemotherapy (NAC), we conduct whole transcriptome profiling coupled with histopathology analyses of a longitudinal breast cancer cohort of 146 patients including 110 pairs of serial tumor biopsies collected before treatment, after the first cycle of treatment and at the time of surgery. Here, we show that cytotoxic chemotherapies induce dynamic changes in the tumor immune microenvironment that vary by subtype and pathologic response. Just one cycle of treatment induces an immune stimulatory microenvironment harboring more tumor infiltrating lymphocytes (TILs) and up-regulation of inflammatory signatures predictive of response to anti-PD1 therapies while residual tumors are immune suppressed at end-of-treatment compared to the baseline. Increases in TILs and CD8+ T cell proportions in response to NAC are independently associated with pathologic complete response. Further, on-treatment immune response is more predictive of treatment outcome than immune features in paired baseline samples although these are strongly correlated., Neoadjuvant chemotherapy is a therapeutic option for the treatment of breast cancer. Here, the authors characterize changes in the gene expression profiles and immune microenvironment in serial breast cancer biopsies taken before, during and after neoadjuvant chemotherapy.

Breast cancer metastasis to gynaecological organs is an understudied pattern of tumour spread. We explored clinico‐pathological and molecular features of these metastases to better understand whether this pattern of dissemination is organotropic or a consequence of wider metastatic dissemination. Primary and metastatic tumours from 54 breast cancer patients with gynaecological metastases were analysed using immunohistochemistry, DNA copy‐number profiling, and targeted sequencing of 386 cancer‐related genes. The median age of primary tumour diagnosis amongst patients with gynaecological metastases was significantly younger compared to a general breast cancer population (46.5 versus 60 years; p < 0.0001). Median age at metastatic diagnosis was 54.4, time to progression was 4.8 years (range 0–20 years), and survival following a diagnosis of metastasis was 1.95 years (range 0–18 years). Patients had an average of five involved sites (most frequently ovary, fallopian tube, omentum/peritoneum), with fewer instances of spread to the lungs, liver, or brain. Invasive lobular histology and luminal A‐like phenotype were over‐represented in this group (42.8 and 87.5%, respectively) and most patients had involved axillary lymph nodes (p < 0.001). Primary tumours frequently co‐expressed oestrogen receptor cofactors (GATA3, FOXA1) and harboured amplifications at 8p12, 8q24, and 11q13. In terms of phenotype conversion, oestrogen receptor status was generally maintained in metastases, FOXA1 increased, and expression of progesterone receptor, androgen receptor, and GATA3 decreased. ESR1 and novel AR mutations were identified. Metastasis to gynaecological organs is a complication frequently affecting young women with invasive lobular carcinoma and luminal A‐like breast cancer, and hence may be driven by sustained hormonal signalling. Molecular analyses reveal a spectrum of factors that could contribute to de novo or acquired resistance to therapy and disease progression.

Background: Invasive lobular carcinoma (ILC) is the most commonly diagnosed special histological type of breast cancer, accounting for up to 15% of all cases. Accumulating data suggests the biology and clinical features of ILC differ to those of the more commonly diagnosed Invasive Carcinoma of No Special Type (IC-NST), including evidence of there being an overall worse long term outcome associated with ILC. Several large-scale molecular profiling studies have recently been published regarding ILC, highlighting molecular heterogeneity and important drivers of tumour behaviour. We hypothesize that an integrative analysis of gene expression and DNA copy number data will identify novel drivers, and prognostic and predictive biomarkers of lobular phenotype. Methods: Gene expression and copy number data from ILC tumours profiled in-house (n=25), METABRIC (n=125) and TCGA (n=145) were assembled and analysed. Integration of genome and transcriptome data was performed using two methods. Firstly, by Spearman correlation with a meta-analysis by combining gene level correlation coefficients from each study using a random effects model, weighting each study with inverse variance. Secondly using ANOVA, followed by combining gene level p-values across studies using Stouffers Z-score. Disease specific survival was examined at an individual gene level and using all genes simultaneously (via a Cox Boost analysis) to identify gene expression changes that are associated with poor outcome. Results: DNA copy number profiling identified recurrent gains (1q, 8q, 16p), losses (11q, 16q) and amplifications (1q32, 8p12-p11.2, 11q13). 11q13 amplifications were prevalent at a higher frequency in patients with poor outcome compared to patients with better outcome. The integrative analysis identified 1928 candidate genes whose expression was associated with gene copy number; as expected being enriched from genome regions highlighted above. One hundred and sixty of these genes were of prognostic significance in the METABRIC ILC cohort, several are known cancer drivers (e.g. PBX1, CCND1) and five new candidates are being investigated as novel prognostic biomarkers by immunohistochemistry. Conclusions: An integrated molecular analysis of ILC has identified a large number of gene expression changes that are dictated by gene copy number in ILC. Some of these are expected to influence tumour behavior and to highlight aggressive cancers with poor prognosis. Citation Format: Lal S, McCart Reed A, Nones K, Wockner L, Song S, Lakhani S, Simpson P. An integrated molecular analysis of invasive lobular carcinoma [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 P1-05-16.

The Homologous Recombination (HR) pathway is crucial for the repair of DNA double-strand breaks (DSBs) generated during DNA replication. Defects in HR repair have been linked to the initiation and development of a wide variety of human malignancies, and exploited in chemical, radiological and targeted therapies. In this study, we performed a personalised pathway analysis independently for four large sporadic breast cancer cohorts to investigate the status of HR pathway dysregulation in individual sporadic breast tumours, its association with HR repair deficiency and its impact on tumour characteristics. Specifically, we first manually curated a list of HR genes according to our recent review on this pathway (Liu et al., 2014), and then applied a personalised pathway analysis method named Pathifier (Drier et al., 2013) on the expression levels of the curated genes to obtain an HR score quantifying HR pathway dysregulation in individual tumours. Based on the score, we observed a great diversity in HR dysregulation between and within gene expression-based breast cancer subtypes, and by using two published HR-defect signatures, we found HR pathway dysregulation reflects HR repair deficiency. Furthermore, we identified a novel association between HR pathway dysregulation and chromosomal instability (CIN) in sporadic breast cancer. Although CIN has long been considered as a hallmark of most solid tumours, with recent extensive studies highlighting its importance in tumour evolution and drug resistance, the molecular basis of CIN in sporadic cancers remains poorly understood. Our results imply that HR pathway dysregulation might contribute to CIN in sporadic breast cancer.

The use of molecular signatures to add value to standard clinical and pathological parameters has impacted clinical practice in many cancer types, but perhaps most notably in the breast cancer field. This is, in part, due to the considerable complexity of the disease at the clinical, morphological and molecular levels. The adoption of molecular profiling of DNA, RNA and protein continues to reveal important differences in the intrinsic biology between molecular subtypes and has begun to impact the way patients are managed. Several bioinformatic tools have been developed using DNA or RNA-based signatures to stratify the disease into biologically and/or clinically meaningful subgroups. Here, we review the approaches that have been used to develop gene expression signatures into currently available diagnostic assays (e.g., OncotypeDX® and Mammaprint®), plus we describe the latest work on genome sequencing, the methodologies used in the discovery process of mutational signatures, and the potential of these signatures to impact the clinic.

The molecular bases underlying neoadjuvant chemotherapy (NAC) response are poorly understood. To elucidate the effects of NAC on breast tumor biology and its association with clinical outcome, we have conducted WES and RNA-Seq profiling of a longitudinal breast cancer (BC) cohort consisting of 146 cases (281 tumors, 109 pairs), including 55 (38%) that achieved pathologic complete responses (pCR) and 91 (62%) that harbored residual diseases at time of surgery. Tumor biopsies were collected for each patient at three time points - pre-treatment, three weeks after the first cycle of anthracycline and cyclophosphamide (AC) and at the time of surgery, after 3 more cycles of AC followed by 4 cycles of taxane. In addition to somatic mutations and copy number alterations, we also derived a comprehensive set of genomic and molecular features for each tumor including chromosomal instability, loss-of-heterozygosity, mutation burden, mutation signatures and expression signatures for oncogenic signaling pathways and immune cell subsets. Virtual microdissection analysis inferred 14 factors that represent distinct tissue compartment including a tumor infiltrating lymphocyte (TIL) factor and revealed that initial NAC treatment increased stromal and adjacent normal tissue fractions while reducing tumor cellularity. NAC also induced dynamic changes in immune gene expressions over time, a pattern that was validated through detecting and quantifying the density of TILs from H&E images. To investigate NAC induced changes in tumor intrinsic biology we classified tumors into five oncogenic cellular states on a reference Onco-GPS map defined by transcriptional signatures from breast cancer cell lines. We observed that, as a result of NAC treatment, tumors often change from one oncogenic state to another, transiently upregulating an EMT program that appears to mediate drug resistance and increase the likelihood of residual disease. Multiple regression and multivariate analyses were performed to identify predictive biomarkers of pCR status while adjusting for BC subtypes and tumor purity. We found that ER+ subtype and estrogen response signature but not Ki-67 were independently associated with NAC response. Within TNBC, the immunomodulatory subtype was enriched in responders while the basal-like subtype had the poorest response. Pretreatment TIL and changes in TIL level over time were independently associated with NAC response, implicating anti-tumor immunity in mediating the efficacy of chemotherapies. Through multi-omics characterization of longitudinally paired tumor biopsies, we have revealed dynamic changes in the tumor molecular states in BC patients undergoing NAC treatment, identified molecular markers of treatment outcome and derived insights into the mechanism of action as well as resistance to an important class of therapy. Citation Format: Samir Lal, Ying Ding, Jeong Eon Lee, Soo-Hyeon Lee, Se Kyung Lee, Jae-Yong Nam, Jong Han Yu, Yoon-la Choi, Seok Won Kim, Seok Jin Nam, Ji-Yeon Kim, Sripad Ram, Eric Powell, Keith A. Ching, Pablo Tamayo, William Kim, Huwate Yeerna, Soo Youn Cho, Vinicius Bonato, Shibing Deng, Jinho Kim, Hyuntae Shin, Woong-Yang Park, Paul A. Rejto, Jadwiga Bienkowska, Yeon-Hee Park, Zhengyan Kan. Multi-omics profiling of breast cancers during neoadjuvant chemotherapy identified distinct molecular changes and biomarkers associated with clinical response [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 1684.

The authors regret an error made in this review relating to mRNA-based gene signatures. A revised Table 1 is shown below. The authors would like to apologise for any inconvenience caused.

Copy number alterations (CNAs) are thought to account for 85% of the variation in gene expression observed among breast tumours. The expression of cis-associated genes is impacted by CNAs occurring at proximal loci of these genes, whereas the expression of trans-associated genes is impacted by CNAs occurring at distal loci. While a majority of these CNA-driven genes responsible for breast tumourigenesis are cis-associated, trans-associated genes are thought to further abet the development of cancer and influence disease outcomes in patients. Here we present a network-based approach that integrates copy-number and expression profiles to identify putative cis- and trans-associated genes in breast cancer pathogenesis. We validate these cis- and trans-associated genes by employing them to subtype a large cohort of breast tumours obtained from the METABRIC consortium, and demonstrate that these genes accurately reconstruct the ten subtypes of breast cancer. We observe that individual breast cancer subtypes are driven by distinct sets of cis- and trans-associated genes. Among the cis-associated genes, we recover several known drivers of breast cancer (e.g. CCND1, ERRB2, MDM2 and ZNF703) and some novel putative drivers (e.g. BRF2 and SF3B3). siRNA-mediated knockdown of BRF2 across a panel of breast cancer cell lines showed significant reduction specifically in cell proliferation in HER2+ lines, thereby indicating that BRF2 could be a context-dependent oncogene and potentially targetable in these lines. Among the trans-associated genes, we identify modules of immune-response (CD2, CD19, CD38 and CD79B), mitotic/cell-cycle kinases (e.g. AURKB, MELK, PLK1 and TTK), and DNA-damage response genes (e.g. RFC4 and FEN1)., Comment: 23 pages, 2 tables, 7 figures