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Background: The current first-line (1L) standard of care (SOC) for human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer (MBC) is trastuzumab (T) plus pertuzumab (P) and a taxane. Despite advances in 1L SOC, most patients (pts) progress during maintenance therapy with T+P. Tucatinib is a tyrosine kinase inhibitor (TKI) approved in combination with T and capecitabine for adults with HER2+ MBC, with and without brain metastases (BM). In HER2CLIMB, the addition of tucatinib significantly prolonged progression-free survival (PFS) and overall survival (OS) in pts with HER2+ MBC and was well tolerated. Adding tucatinib also reduced the risk of disease progression or death in pts with untreated and/or active BM (Murthy et al. 2020, Curigliano et al. 2021). HER2CLIMB-05 investigates whether adding tucatinib to 1L SOC as maintenance therapy will extend PFS while maintaining quality of life (QOL). Methods: HER2CLIMB-05 (NCT05132582) is a phase 3, randomized, double-blind study evaluating tucatinib plus T+P as maintenance therapy for HER2+ MBC. Approximately 650 pts will be enrolled. Eligible pts will have advanced HER2+ disease, no progression on 4–8 cycles of prior 1L SOC, ECOG Performance Status of 0 or 1, and no or asymptomatic BM. Exclusion criteria include prior treatment with anti-HER2 and/or anti-epidermal growth factor receptor TKI (prior SOC for early BC is permitted) or inability to undergo contrast magnetic resonance imaging of the brain. Pts will be randomized 1:1 to receive either tucatinib or placebo twice daily, with T+P once every 21 days. Pts with HR+ disease may receive endocrine therapy. The primary endpoint is investigator-assessed PFS. Secondary endpoints include OS (key endpoint), time to deterioration of health-related QOL, central nervous system PFS, safety, and pharmacokinetic (PK) parameters. PFS and OS will be compared using a 2-sided stratified log-rank test between treatment groups. Time-to-event endpoints will be summarized using the Kaplan–Meier method. PK and safety data will be summarized using descriptive statistics. Enrollment is ongoing in the US, with additional sites planned. Citation Format: Erika Hamilton, Junji Tsurutani, Giuseppe Curigliano, Miguel Martín, Ciara C. O’Sullivan, Joo Hyuk Sohn, Konstantinos Tryfonidis, Libero Santarpia, Shan Yang, Véronique Diéras. Phase 3 study of tucatinib or placebo in combination with trastuzumab and pertuzumab as maintenance therapy for HER2+ metastatic breast cancer (HER2CLIMB-05, trial in progress) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-16-03.

Supplementary Figures Legends. Supplementary Figures S1-S3 Legends

Supplementary Figure S2. Hierarchical clustering of microRNA expression

Purpose: Triple-negative breast cancers (TNBC) are associated with high risk of early tumor recurrence and poor outcome. Common prognostic biomarkers give very restricted predictive information of tumor recurrences in TNBC. Human serum contains stably expressed microRNAs (miRNAs), which have been discovered to predict prognosis in patients with cancer. The purpose of this study was to identify circulating biomarkers able to predict clinical outcome in TNBC.Experimental Design: We performed genome-wide serum miRNA expression and real-time PCR analyses to investigate the ability of miRNAs in predicting tumor relapse in serum samples from 60 primary TNBC. Patients were divided into training and testing cohorts.Results: By Cox regression analysis, we identified a four-miRNA signature (miR-18b, miR-103, miR-107, and miR-652) that predicted tumor relapse and overall survival. This miRNA signature was further validated in an independent cohort of 70 TNBC. A high-risk signature score was developed and significantly associated with tumor recurrence and reduced survival. Multivariate Cox regression models indicated that the risk score based on the four-miRNA signature was an independent prognostic classifier of patients with TNBC.Conclusions: This signature may serve as a minimally invasive predictor of tumor relapse and overall survival for patients with TNBC. This prediction model may ultimately lead to better treatment options for patients with TNBC. Clin Cancer Res; 21(5); 1207–14. ©2014 AACR.

Supplementary Table S2. The 71 microRNAs expressed in at least 75% of the samples in the training cohort

Different kinases are expressed in different clinical subsets of breast cancer. In this study, we assessed kinase expression patterns in different clinical subtypes of breast cancer, evaluated the prognostic and predictive values of kinase metagenes, and investigated their functions in vitro. Four hundred twenty-eight protein kinases in gene expression data were examined from 684 cases of breast cancer and 51 breast cancer cell lines to identify kinase expression patterns. We tested the prognostic value of kinase metagenes in 684 node-negative patients who received no adjuvant therapy and the predictive value in 233 patients who received uniform neoadjuvant chemotherapy. Twelve kinases were overexpressed in estrogen receptor (ER)–positive/human epidermal growth factor receptor 2 (HER2)–negative, 7 in HER2+, and 28 in ER−/HER2− cancers, respectively. We examined the functional role of 22 kinases overexpressed in ER−/HER2− cancers using siRNA. Downregulation of these kinases caused significant subtype-specific inhibition of cell growth in vitro. Two robust kinase clusters, including an immune kinase cluster and a mitosis kinase cluster, were present in all clinical subgroups. High mitosis kinase score was associated with worse prognosis but higher pathologic complete response (pCR) in ER+/HER2− cancers, but not in ER−/HER2− or HER2+ cancers, in univariate and multivariate analyses including other genomic predictors (MammaPrint, genomic grade index, and the 76-gene signature). Conversely, higher immune kinase score was associated with better survival in ER+/HER2− and HER2+ tumors and also predicted higher probability of pCR in HER2+ cancers. Taken together, our results indicate that kinases regulating mitosis and immune functions convey distinct prognostic information that varies by clinical subtype. Cancer Res; 70(21); 8852–62. ©2010 AACR.

Figure S3, Related to Figure 4. A, c-SRC expression in normal (HFF and MCF-10A), luminal-A (MCF-7), LAR-TNBC (MDA-MB-453), and mesenchymal-TNBC (MDA-MB-231, BT-549, and Hs578T) cell lines. Expression was normalized to GAPDH and made relative to c-SRC expression in HFF lines. B-C, Clonogenic assays in Hs578T (B), and MDA-MB-436 (C) TNBC cells after miR-34a transfection and treatment with Dasatnib (left panels) and paclitaxel (right panel) or as a control HeLa cells after dasatinib treatment (C, right panel). D-E, Assessment of miR-34a target genes in MDA-MB-231 cells after 72 hours of dasatinib treatment (D), or miR-34a levels after 72 hours paclitaxel treatment (E). F, miR-34a promoter luciferase assays in MDA-MB-31 cells after dasatinib treatment. G-H, Spearman rank correlation analysis of SRC levels in cell lines described in Figure 1A (G), and in all breast cancer samples in the Metabric dataset (H). I, Comparable decreases in phospho-Tyr416 active c-SRC, non-phospho-Tyr527 c-SRC, and total c-SRC are observed in MDA-MB-231 cells transfected with 15nM miR-34a versus miR-Scr control, as determined by Western blot analysis. J, Schematic highlighting the miR-34a-c-SRC double-negative feedback loop present in MSL TNBC cells, which can be influenced by exogenous addition of miR-34a, or by dasatinib treatment. * Indicates p

Figure S1, Related to Figure 1. A, miRNA microarray data of the top 50 most variant miRNAs across 17 breast cancer cell lines representing either basal/TNBC (top left blue bar) or luminal (top right red bar) breast cancer subgroups. Amongst these miRNAs, miR-34a (red asterisk) was found to be uniquely downregulated in basal lines. B-C, qPCR validation of array data in TNBC and Luminal-A cancer cell lines as compared to normal mammary epithelial lines (CRCs were cultured using the ROCK inhibitor and condition medium from the 3T3 feeder system as previously described(20)). D, Schematic of WebGestalt 2 analysis of Affymetrix microarray data across 19 cell lines. ¬¬miR-34a is one of several miRNAs with target enrichment in the TNBC overexpressed gene set. E, Results of hypergeometric analysis of miR-34a targets using a more stringent context score cutoff of -0.27. F, SRB growth assays on additional TNBC and normal cell lines.

Supplementary Figure 1 from Prognostic and Therapeutic Implications of Distinct Kinase Expression Patterns in Different Subtypes of Breast Cancer

Figure S4, Related to Figure 5. A-F, Further characterization of c-SRC siRNA treatments in TNBC cell lines. A, Crystal violet staining of MDA-MB-231 and Hs578T cells post 15nM si-SRC and si-Neg transfection (Day 6 images, left panel), and quantification of staining abundance is shown on right panels). B, SRB results on Hs578T cell line. C, Analysis of a second siRNA to c-SRC in MDA-MB-231 cells. SRB assays are shown in the left panel and western blot analysis confirming c-SRC knockdown (72 hour post-transfection) is shown on the right panel. D, Crystal violet staining of HFF cells. E, SA-β-gal assays in the indicated cell lines 5 days after 15nM si-SRC transfection. F, miR-34a levels 72 hours after 15nM si-SRC transfection in Hs578T cells. G, Assessment of SRC mRNA levels in the indicated cell lines 72 hours after 15nM si-SRC transfection.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell–like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC. Cancer Res; 76(4); 927–39. ©2015 AACR.

Figure S5, Related to Figure 6. A, Assessment of SRC mRNA levels in MDA-MB-231 SRC-ORF, EMPTY-ORF, or parental cell lines. Data is normalized in RPL19 levels. B, Crystal violet staining assays on day 5 post-transfection confirms the ability of c-SRC to rescue miR-34a-induced anti-tumor growth. C-D, Examples of Pearson correlation analysis indicating MDA-MB-231 cells were not similar to BT-549 and MDA-MB-436 cells, and therefore were not included in the initial K-Means clustering analysis (results shown in D). E, The fold knockdown by miR-34a as compared to the miR-Scr treatments of the indicated genes in Clusters 1-3 in both BT-549 and MDA-MB-436 cells using a 2-fold change cut-off. None of the genes in Cluster 4 were downregulated by miR-34a (data not shown). F, Schematic of the KEGG pathway (hsa04510: Focal Adhesion) with miR-34a downregulated genes highlighted in red. G-H, Represents further analysis of the miR-34a gene signature in breast cancer. G, Indicates correlation analyses of miR-34a target genes in TNBC patients from Metabric data. H, Confirmation of prognostic importance of mIR-34a gene signature using a PROGgeneV2 algorithm on the TCGA data set.

Supplementary Tables 1-11 from Prognostic and Therapeutic Implications of Distinct Kinase Expression Patterns in Different Subtypes of Breast Cancer

Supplementary Materials and Methods and Supplementary Figure Legends

Supplementary Figures 2-5 from Prognostic and Therapeutic Implications of Distinct Kinase Expression Patterns in Different Subtypes of Breast Cancer

Figure S2, Related to Figure 2. A-F, Functional characterization of miR-34a re-introduction in additional TNBC and non-TNBC cell lines by way of Matrigel-invasion (A), soft agar growth (B), and BrdU labeling (C) experiments. For BrdU assays, we observed only a 5-10% accumulation of BrdU-positive cells 4 days post-transfection. SA-β-gal assays on MDA-MB-157 (D), and BT-20 (E, left panel) TNBC cells, as compared to MCF-7 luminal-A cells (E, right panel). F, MCF-7 cells stably transfected with either a miR-34a (miR-34a SP) or a control (Empty SP) reporter/sponge construct was assayed for SA-β-gal activity. Two independent miR-34a SP pools were generated. G, Luciferase reporter assays indicative of miR-34a activity in all TNBC, luminal, and normal cell lines used in this study. H, miRNA levels in MDA-MB-231, MDA-MB-436, and BT-549 TNBC cells or BT-474 and MCF-7 Luminal-A cells 72 hours after 10nM miR-34a or miR-Scr transfection. In TNBC cells miR-34a expressing lines harbored lower levels of miR-17/92 family members as compared to miR-Scr treated lines. * Indicates p

Background: The current first-line (1L) standard of care (SOC) for human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer (MBC) is trastuzumab (T) plus pertuzumab (P) and a taxane. Despite advances in 1L SOC, most patients (pts) progress during maintenance therapy with T+P. Tucatinib is a tyrosine kinase inhibitor (TKI) approved in combination with T and capecitabine for adults with HER2+ MBC, with and without brain metastases (BM). In HER2CLIMB, the addition of tucatinib significantly prolonged progression-free survival (PFS) and overall survival (OS) in pts with HER2+ MBC and was well tolerated. Adding tucatinib also reduced the risk of disease progression or death in pts with untreated and/or active BM (Murthy et al. 2020, Curigliano et al. 2021). HER2CLIMB-05 investigates whether adding tucatinib to 1L SOC as maintenance therapy will extend PFS while maintaining quality of life (QOL). Methods: HER2CLIMB-05 (NCT05132582) is a phase 3, randomized, double-blind study evaluating tucatinib plus T+P as maintenance therapy for HER2+ MBC. Approximately 650 pts will be enrolled. Eligible pts will have advanced HER2+ disease, no progression on 4-8 cycles of prior 1L SOC, ECOG Performance Status of 0 or 1, and no or asymptomatic BM. Exclusion criteria include prior treatment with anti-HER2 and/or anti-epidermal growth factor receptor TKI (prior SOC for early BC is permitted) or inability to undergo contrast magnetic resonance imaging of the brain. Pts will be randomized 1:1 to receive either tucatinib or placebo twice daily, with T+P once every 21 days. Pts with HR+ disease may receive endocrine therapy. The primary endpoint is investigator-assessed PFS. Secondary endpoints include OS (key endpoint), PFS by blinded independent central review (BICR), time to deterioration of health-related QOL, central nervous system PFS, safety, and pharmacokinetic (PK) parameters. PFS and OS will be compared using a 2-sided stratified log-rank test between treatment groups. Time-to-event endpoints will be summarized using the Kaplan-Meier method. PK and safety data will be summarized using descriptive statistics. Enrollment is ongoing in the US, Canada, and several EU and APAC countries, with additional sites planned. This abstract was previously presented at ESMO-BC 2022, FPN (Final Publication Number): 415, by Veronique Dieras (reused with permission). Citation Format: Erika Hamilton, Junji Tsurutani, Joohyuk Sohn, Giuseppe Curigliano, Ciara C. O’Sullivan, Miguel Martín, Konstantinos Tryfonidis, Libero Santarpia, Shan Yang, Veronique Dieras. Phase 3 study of tucatinib or placebo in combination with trastuzumab and pertuzumab as maintenance therapy for HER2+ metastatic breast cancer (HER2CLIMB-05, trial in progress) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT065.

Background. The development of new anti-HER2 therapies for the treatment of HER2-positive breast cancer (BC) is changing the concept of HER2 dichotomization to select and treat this patient population. In addition, different immunotherapies tested in HER2-low BC are gaining continuous interest. However, a comprehensive characterization of HER2 BC subgroups and patients is required to identify the best treatment approach. Materials and Methods. Using 123 BC samples with gene expression and IHC/FISH determined HER2 status we determined cutoff values to identify HER2 positive, HER2 low, and HER2 ultralow cohorts. With the inclusion of hormone receptor (HR) status six clinically relevant cohorts were defined (HR+/HER2+, HR+/HER2-low, HR+/HER2-ultralow, and HR-/HER2+, HR-/HER2-low, HR-/HER2-low). An ingegrated database of 7,624 BC cases were assigned to the six subtypes. Prognosis determination was based on relapse-free survival (RFS), distant-metastasis-free survival (DMFS), and overall survival (OS). Clinical parameters evaluated include MKI67 expression, lymph node status and grade. All together 17 immune signatures resembling immune genes and related activated pathways were tested against the six molecular BC subgroups. Results. We defined a robust cutoff for HER2 expression levels to define six distinct HER2 BC molecular subgroups (>3034 for HER2 positivity and < 1780 for HER2 ultralow). Regardless the HR positivity, the overall distribution of HER2-low, and HER2-ultralow was 23% and 52%, respectively. In the HR+ subgroups the HER2-low showed a better prognosis as compared to the HER2-ultralow and HER2+ (RFS and DMFS P = 0.0048 and 0.0015, respectively) while there was no prognostic effect of HER2 expression in the HR- subgroups. Not surprisingly, an association with higher grade was demonstrated in all HR- as compared to the HR+ subgroups regardless of HER2 status. Overall, all HR- subgroups showed a higher involvement of immune genes as compared to the three HR+ subgroups. Of interest, HER2-low (HR+ and HR-) and HR-/HER2+ showed a significant overlap expression of immune signatures (71%). While the HR+/HER2-ultralow and HR+/HER2+ displayed minimal activation of immune pathways, the HR-/HER2-ultralow was the group most significantly associated with the activation of immune signaling including IFN signaling (67% percent of genes in the panel with altered expression), T cell active cytokines (34% of genes hit), and cytotoxic effector molecules (48% of genes hit). Conclusions. Our study supports a further molecular stratification of breast cancer based on HER2 status. The different tumor-immune background of these BC subgroups highlights that selected patient cohorts may derive benefit from targeted immunotherapy. Citation Format: Gyongyi Munkacsy, Libero Santarpia, Balazs Gyorffy. Immune Signatures Display Subtype-Specific Activation in Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-04.

INTRODUCTION: Dabrafenib plus trametinib was found to have robust antitumor activity in patients with BRAF V600E-mutant metastatic NSCLC (mNSCLC). We report updated survival analysis of a phase 2 study (NCT01336634) with a minimum of 5-year follow-up and updated genomic data.METHODS: Pretreated (cohort B) and treatment-naive (cohort C) patients with BRAF V600E-mutant mNSCLC received dabrafenib 150 mg twice daily and trametinib 2 mg once daily. The primary end point was investigator-assessed overall response rate per Response Evaluation Criteria in Solid Tumors version 1.1. Secondary end points were duration of response, progression-free survival, overall survival, and safety.RESULTS: At data cutoff, for cohorts B (57 patients) and C (36 patients), the median follow-up was 16.6 (range: 0.5-78.5) and 16.3 (range: 0.4-80) months, overall response rate (95% confidence interval [CI]) was 68.4% (54.8-80.1) and 63.9% (46.2-79.2), median progression-free survival (95% CI) was 10.2 (6.9-16.7) and 10.8 (7.0-14.5) months, and median overall survival (95% CI) was 18.2 (14.3-28.6) and 17.3 (12.3-40.2) months, respectively. The 4- and 5-year survival rates were 26% and 19% in pretreated patients and 34% and 22% in treatment-naive patients, respectively. A total of 17 patients (18%) were still alive. The most frequent adverse event was pyrexia (56%). Exploratory genomic analysis indicated that the presence of coexisting genomic alterations might influence clinical outcomes in these patients; however, these results require further investigation.CONCLUSIONS: Dabrafenib plus trametinib therapy was found to have substantial and durable clinical benefit, with a manageable safety profile, in patients with BRAF V600E-mutant mNSCLC, regardless of previous treatment.

Patients with early-stage hormone receptor-positive, human epidermal growth factor receptor 2-negative (HER2−) breast cancer (BC) are typically treated with surgery, followed by adjuvant systemic endocrine therapy with or without adjuvant chemotherapy and radiation therapy. Current guidelines regarding the use of adjuvant systemic therapy depend on clinical and pathological factors, such as the morphological assessment of tumor subtype; histological grade; tumor size; lymphovascular invasion; and lymph node status combined with estrogen receptor, progesterone receptor, and HER2 biomarker profiles assessed using immunohistochemistry and in situ hybridization. Additionally, the prognostic and predictive value of tumor-infiltrating lymphocytes and their composition is emerging as a key marker in triple negative (TNBC) and HER2-enriched molecular breast tumor subtypes. However, all these factors do not necessarily reflect the molecular heterogeneity and complexity of breast cancer. In the last two decades, gene expression signatures or profiling (GEP) tests have been developed to predict the risk of disease recurrence and estimate the potential benefit of receiving adjuvant systemic chemotherapy in patients with luminal breast cancer. GEPs have been utilized to help physicians to refine decision-making process, complementing clinicopathological parameters, and can now be used to classify the risk of recurrence and tailoring personalized treatments. Several clinical trials using GEPs validate the increasing value of such assays in different clinical settings, addressing relevant clinical endpoints. Finally, the recent approval of immune checkpoint inhibitors in TNBC and the increasing use of immunotherapy in different molecular BC populations highlight the opportunity to refine current GEPs by including a variety of immune-related genes that may help to improve predicting drug response and finetune prognosis.

TPS1108 Background: The current first-line (1L) standard of care (SOC) for human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer (MBC) is trastuzumab (T) plus pertuzumab (P) and a taxane. Despite advances in 1L SOC, most patients (pts) progress during maintenance therapy with T+P. Tucatinib is a tyrosine kinase inhibitor (TKI) approved in combination with T and capecitabine for adults with HER2+ MBC, with and without brain metastases (BM). In HER2CLIMB, the addition of tucatinib significantly prolonged progression-free survival (PFS) and overall survival (OS) in pts with HER2+ MBC and was well tolerated. Adding tucatinib also reduced the risk of disease progression or death in pts with untreated and/or active BM (Murthy et al. 2020, Curigliano et al. 2021). HER2CLIMB-05 investigates whether adding tucatinib to 1L SOC as maintenance therapy will extend PFS while maintaining quality of life (QOL). Methods: HER2CLIMB-05 (NCT05132582) is a phase 3, randomized, double-blind study evaluating tucatinib plus T+P as maintenance therapy for HER2+ MBC. Approximately 650 pts will be enrolled. Eligible pts will have advanced HER2+ disease, no progression on 4–8 cycles of prior 1L SOC, ECOG Performance Status of 0 or 1, and no or asymptomatic BM. Exclusion criteria include prior treatment with anti-HER2 and/or anti-epidermal growth factor receptor TKI (prior SOC for early BC is permitted) or inability to undergo contrast magnetic resonance imaging of the brain. Pts will be randomized 1:1 to receive either tucatinib or placebo twice daily, with T+P once every 21 days. Pts with HR+ disease may receive endocrine therapy. The primary endpoint is investigator-assessed PFS. Secondary endpoints include OS (key endpoint), time to deterioration of health-related QOL, central nervous system PFS, safety, and pharmacokinetic (PK) parameters. PFS and OS will be compared using a 2-sided stratified log-rank test between treatment groups. Time-to-event endpoints will be summarized using the Kaplan–Meier method. PK and safety data will be summarized using descriptive statistics. Enrollment is ongoing in the US, with additional sites planned. Clinical trial information: NCT05132582.

Background Sequence variations in coding and non-coding regions of the genome can affect gene expression and signalling pathways, which in turn may influence disease outcome. Methods In this study, we integrated somatic mutations, gene expression and clinical data from 930 breast cancer patients included in the TCGA database. Genes associated with single mutations in molecular breast cancer subtypes were identified by the Mann-Whitney U-test and their prognostic value was evaluated by Kaplan-Meier and Cox regression analyses. Results were confirmed using gene expression profiles from the Metabric data set (n = 1988) and whole-genome sequencing data from the TCGA cohort (n = 117). Results The overall mutation rate in coding and non-coding regions were significantly higher in ER-negative/HER2-negative tumours (P = 2.8E–03 and P = 2.4E–07, respectively). Recurrent sequence variations were identified in non-coding regulatory regions of several cancer-associated genes, including NBPF1, PIK3CA and TP53. After multivariate regression analysis, gene signatures associated with three coding mutations (CDH1, MAP3K1 and TP53) and two non-coding variants (CRTC3 and STAG2) in cancer-related genes predicted prognosis in ER-positive/HER2-negative tumours. Conclusions These findings demonstrate that sequence alterations influence gene expression and oncogenic pathways, possibly affecting the outcome of breast cancer patients. Our data provide potential opportunities to identify non-coding variations with functional and clinical relevance in breast cancer.

Background: The tumor immune microenvironment is formed by many distinct and interacting cell populations, and its composition may predict patient's prognosis and response to therapies. Triple negative breast cancer (TNBC) is a heterogeneous disease in which different genomic subgroups have been described (Lehmann, et al. J Clin Invest.2011). Our aim was to integrate the composition of the tumor immune microenvironment with the molecular TNBC subtypes. Methods: We retrospectively analyzed the composition and the functional orientation of the immune microenvironment of 963 TNBC tumors clustered in 4 main TNBC subgroups (Basal-Like [BL1/2], Immunomodulatory [IM], Mesenchymal/Mesenchymal Stem-Like [MS], Androgen Receptor [AR]) from independent cohorts using transcriptomic profiling. TNBC were stratified based on 9 different immune signatures (Natural Killer [NK], Dendritic Cells [DC], T-Cells [TC], B-Cells [BC], T-Cytotoxic [TC], Interferon [IF], Nuclear Factor-kB [NF-kB], anti-tumor Macrophages [M1] and pro-tumor Macrophages [M2]. We validated our findings using immunohistochemistry. Results: We report that TNBC molecular subgroups and specific microenvironmental immune signatures are highly correlated. The IM was enriched of almost all the immune modules. On overall TNBC population, the TC (P = 0.01), IF (P = 0.007) and M1 (P = 0.001) signatures were associated with good prognosis. The BL subgroup with a good prognosis was characterized by overexpression of genes specific to M1-macrophages (P = 0.004) and TC-lymphocytes (P = 0.01). In contrast, the poor-prognosis MS expresses markers of cells of monocytic origin and significantly associated with the M2 signature (P = 0.01). The MS subgroup also displays an inflammatory and immunosuppressive signature. Pathological examination revealed that the MS subtype is characterized by a high density of pro-tumor macrophages that likely produce chemokines and cytokines which favor tumor-associated inflammation, resulting in a poor prognosis. In contrast, the AR exhibits low immune and inflammatory signatures. Conclusions: The distinct immune orientations of the TNBC molecular subtypes pave the way for tailored immunotherapies. Citation Format: Bottai G, Balazs G, Nagy A, Pusztai L, Szallasi Z, Reis-Filho JS, Santarpia L. Immune signatures define and affect prognosis in triple-negative breast cancer subtypes [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 P1-07-06.

Background: Breast cancer bone metastasis (BCBM)-specific genes have been reported without considering biological differences based on estrogen receptor (ER) status. The aims of this study were to identify BCBM-specific genes using our patient dataset and validate previously reported BCBM-specific genes, and to determine whether ER-status-related biological differences matter in identification of BCBM-specific genes. Methods: We used Affymetrix GeneChips to analyze 365 primary human epidermal growth factor receptor 2 (HER2)-negative invasive breast cancer specimens. Genes that were differentially expressed between patients who developed bone metastasis and those who developed non-bone metastasis were identified using Cox proportional hazards model, and differential expression of gene sets was assessed using gene set analysis. We performed gene set analysis to determine whether biological function associated with bone metastasis were different by ER status using 2,246 functionally annotated gene sets assembled from Gene Ontology data base. Results: Among 16,712 probe sets, 592 were overexpressed in the bone metastasis cohort compared to the non-bone-metastasis cohort (false discovery rate ≤ 0.05). However, no BCBM-specific genes met our significance tests when the cancers were stratified by ER status. In ER-positive and ER-negative breast cancers, 151 and 125 gene sets, respectively, were overexpressed for BCBM and the majority of BCBM-related pathways were different. Of significant gene sets, only 13 gene sets were overlapped between ER-positive and -negative cohorts. Conclusion: ER-positive and ER-negative breast cancers have different biological pathways in BCBM development. We have yet to explore BCBM-related biomarkers and targets considering the biological features associated with BCBM depending on the ER status.

Chronic inflammation associated with obesity is now recognized to be an important condition in promoting carcinogenesis and progression in breast cancer patients, mostly in postmenopausal women with tumors expressing estrogen and progesterone receptors. In obese patients, altered levels of several inflammatory mediators regulating aromatase and estrogen expression are one of the mechanisms responsible of increase breast cancer risk. Growing attention has also been paid to the local adipose inflammation and the role played by macrophages as determinants of breast cancer risk recurrence and prognosis. The inflammation-obesity axis offers different molecular signaling pathways for therapeutic interventions and potential pharmacological targets. The increasing rate of obesity worldwide associated with the recent findings linking inflammation and breast cancer urge further investigation.

The proper validation of prognostic biomarkers is an important clinical issue in breast cancer research. MicroRNAs (miRNAs) have emerged as a new class of promising breast cancer biomarkers. In the present work, we developed an integrated online bioinformatic tool to validate the prognostic relevance of miRNAs in breast cancer. A database was set up by searching the GEO, EGA, TCGA, and PubMed repositories to identify datasets with published miRNA expression and clinical data. Kaplan–Meier survival analysis was performed to validate the prognostic value of a set of 41 previously published survival-associated miRNAs. All together 2178 samples from four independent datasets were integrated into the system including the expression of 1052 distinct human miRNAs. In addition, the web-tool allows for the selection of patients, which can be filtered by receptors status, lymph node involvement, histological grade, and treatments. The complete analysis tool can be accessed online at: www.kmplot.com/mirpower . We used this tool to analyze a large number of deregulated miRNAs associated with breast cancer features and outcome, and confirmed the prognostic value of 26 miRNAs. A significant correlation in three out of four datasets was validated only for miR-29c and miR-101. In summary, we established an integrated platform capable to mine all available miRNA data to perform a survival analysis for the identification and validation of prognostic miRNA markers in breast cancer.

UNLABELLED : Advances in DNA and RNA sequencing revealed substantially greater genomic complexity in breast cancer than simple models of a few driver mutations would suggest. Only very few, recurrent mutations or copy-number variations in cancer-causing genes have been identified. The two most common alterations in breast cancer are TP53 (affecting the majority of triple-negative breast cancers) and PIK3CA (affecting almost half of estrogen receptor-positive cancers) mutations, followed by a long tail of individually rare mutations affecting

9593 Background: The phase II multicenter, open label study, which evaluated efficacy and safety of D+T in pretreated (cohort B) and treatment (tx)-naive (cohort C) pts with BRAF V600E mut metastatic NSCLC. The results of the primary analysis have been reported. Here, we present an updated survival and genomic analysis data for cohorts B and C. Methods: Tx-naïve (n=36) and pretreated (n=57) pts received D 150 mg twice daily + T 2 mg daily. Primary objective: ORR, secondary objectives: PFS, DOR, OS, safety, tolerability and PK of D+T. Tumor samples were centrally tested using a NGS cancer targeted panel (Oncomine Dx Target test, ThermoFisher Scientific). KM curves and Cox regression models were used to evaluate potential associations between baseline genomic landscape and pt efficacy endpoints. Results: As of June 22, 2019, median (m) follow-up was 16.3 mo in tx-naïve pts and 16.6 mo in pretreated pts. mOS was 17.3 mo (95% CI: 12.3, 40.2; 3 yr OS: 40%) and 18.2 mo (95% CI: 14.3, 28.6; 3 yr OS: 33%) with 14/36 and 11/57 pts alive in tx naïve and pretreated pts respectively. Detailed efficacy results are presented in table. 57/62 tumor samples retrieved from 93 pts were centrally confirmed to have BRAF V600E mut; 5 non-confirmed BRAF tumors (3 pts had PR) were positive for c-MET T1010I, KRAS G12V, ALK fusion and 2 JAK3 S493C with mPFS of 13.8 mo while OS was NE due to limited data points. Eleven pts (18%) had concomitant somatic mutations and/or genetic alterations in addition to BRAF V600E mut: 4 had alterations within PI3K pathway4 had concomitant mutations at IDH1 R132X, and 3 pts had additional mutations at BRAF G466V, KRAS G13C and a cMET exon 14 skipping, respectively. Pts whose tumors had concomitant genetic alterations, particularly in PI3K pathway, showed a trend towards decreased PFS and OS. Safety profile was similar to previous reported results. Conclusions: This update of BRF113928 study reported improved and durable OS rates with combination D+T in BRAF V600E mut NSCLC pts. Co-occurring genetic alterations might influence clinical outcomes of such pts. Further validation is ongoing to corroborate current genomic findings. Clinical trial information: NCT01336634 . [Table: see text]

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell–like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC. Cancer Res; 76(4); 927–39. ©2015 AACR.

Patients with primary HER2-positive breast cancer benefit from HER2-targeted therapies. Nevertheless, a significant proportion of these patients die of disease progression due to mechanisms of drug resistance. MicroRNAs (miRNAs) are emerging as critical core regulators of drug resistance that act by modulating the epithelial- to-mesenchymal transition (EMT) and cancer-related immune responses. In this study, we investigated the association between the expression of a specific subset of 14 miRNAs involved in EMT processes and immune functions and the response to neoadjuvant trastuzumab and chemotherapy in 52 patients with HER2-overexpressing breast tumors. The expression of only a single miRNA, miR-21, was significantly associated with residual disease (p = 0.030) and increased after trastuzumab-chemotherapy (p = 0.012). A target prediction analysis coupled with in vitro and in vivo validations revealed that miR-21 levels inversely correlated with the expression of PTEN (rs = −0.502; p = 0.005) and PDCD4 (rs = −0.426; p = 0.019), which differentially influenced the drug sensitivity of HER2-positive breast cancer cells. However, PTEN expression was only marginally associated with residual disease. We further demonstrated that miR-21 was able to affect the response to both trastuzumab and chemotherapy, triggering an IL-6/STAT3/NF-κB-mediated signaling loop and activating the PI3K pathway. Our findings support the ability of miR-21 signaling to sustain EMT and shape the tumor immune microenvironment in HER2-positive breast cancer. Collectively, these data provide a rationale for using miR-21 expression as a biomarker to select trastuzumab-chemotherapy-resistant HER2-positive breast cancer patients who may benefit from treatments containing PI3K inhibitors or immunomodulatory drugs.

The introduction of trastuzumab for anti-HER2 therapy dramatically changed the clinical outcome for HER2 (ERBB2, neu) positive breast cancer patients. Today, patients eligible for trastuzumab are selected using HER2 expression/amplification status of the primary tumor. However, acquired and inherent resistance to anti-HER2 therapy in these patients poses a significant challenge, and better patient stratification will be needed to improve clinical response. Here, we provide a wide-ranging overview of potential biomarkers capable of stratifying patients regarding their response to trastuzumab. These include HER2 amplification, impaired access to the binding site (p95HER2, Δ16HER-2, MUC4), augmented signaling through other ERBB family receptors (HER1, HER3, HER4) and their ligands, activation of HER2 targets by alternate heterodimers (EphA2, IGF-1R, GDF15, MUC1*), signaling triggered by downstream members (PIK3CA, PTEN, SRC, mTOR), altered expression of cell cycle and apoptotic regulators (CDKs, p27(kip1), Bcl-2), hormone receptor status, resistance to antibody-dependent cellular cytotoxicity (FcγR), and altered miRNA expression signatures. Multigenic molecular profile analyses have revealed further genes not directly associated with classical oncogenic pathways. Although numerous biomarkers have shown promise in pre-clinical studies, many have delivered controversial results when evaluated in clinical trials. One of the keys for targeting ERBB2 will be to consider the entire ERBB family and downstream associated pathways responsible for the malignant transformation. The heterogeneity of the disease is likely to represent a significant obstacle to accurately predicting the course of resistance. The future most probably involves the incorporation of multiple biomarkers into a unified predictor enabling selection of patients for superior targeted drug administration.

DNA methylation has a substantial impact on gene expression, affecting the prognosis of breast cancer (BC) patients dependent on molecular subtypes. In this study, we investigated the prognostic relevance of the expression of genes reported as aberrantly methylated, and the link between gene expression and DNA methylation in BC subtypes. The prognostic value of the expression of 144 aberrantly methylated genes was evaluated in ER+/HER2−, HER2+, and ER−/HER2− molecular BC subtypes, in a meta-analysis of two large transcriptomic cohorts of BC patients (n = 1,938 and n = 1,640). The correlation between gene expression and DNA methylation in distinct gene regions was also investigated in an independent dataset of 104 BCs. Survival and Pearson correlation analyses were computed for each gene separately. The expression of 48 genes was significantly associated with BC prognosis (p

Purpose: Triple-negative breast cancers (TNBC) are associated with high risk of early tumor recurrence and poor outcome. Common prognostic biomarkers give very restricted predictive information of tumor recurrences in TNBC. Human serum contains stably expressed microRNAs (miRNAs), which have been discovered to predict prognosis in patients with cancer. The purpose of this study was to identify circulating biomarkers able to predict clinical outcome in TNBC. Experimental Design: We performed genome-wide serum miRNA expression and real-time PCR analyses to investigate the ability of miRNAs in predicting tumor relapse in serum samples from 60 primary TNBC. Patients were divided into training and testing cohorts. Results: By Cox regression analysis, we identified a four-miRNA signature (miR-18b, miR-103, miR-107, and miR-652) that predicted tumor relapse and overall survival. This miRNA signature was further validated in an independent cohort of 70 TNBC. A high-risk signature score was developed and significantly associated with tumor recurrence and reduced survival. Multivariate Cox regression models indicated that the risk score based on the four-miRNA signature was an independent prognostic classifier of patients with TNBC. Conclusions: This signature may serve as a minimally invasive predictor of tumor relapse and overall survival for patients with TNBC. This prediction model may ultimately lead to better treatment options for patients with TNBC. Clin Cancer Res; 21(5); 1207–14. ©2014 AACR.

Luminal B breast cancers (BC) have a more aggressive behavior associated with a higher rate of tumor relapse and worse prognosis compared to luminal A tumors. In this study, we evaluated the involvement of specific epithelial-to-mesenchymal transition- (EMT-) and immune-related pathways in the dissemination of luminal B BC cells. The expression of 42 EMT- and immune-related genes was evaluated in matched sentinel lymph nodes (SLNs) analyzed by the one-step nucleic acid amplification assay (OSNA) and primary tumors of 40 luminal B BC patients by gene array and immunohistochemistry. The results were validated in an independent group of 150 luminal B tumors by immunohistochemistry and immunofluorescence and using gene expression data from 315 luminal B BC patients included in the Metabric dataset. We found that the expression of CXCR4 (p=3.28E−02) and CD163 (p=6.92E−03) was significantly upregulated in SLNs of recurrent luminal B BC patients. Luminal B primary tumors overexpressing CXCR4 were characterized by an increased expression of vimentin and a high content of CD163-positive macrophages. Bioinformatics analysis confirmed the correlation of CXCR4 with CXCL12, VIM, and CD163 expression and LN involvement. Our results suggest that the upregulation of the CXCR4/CXCL12 pathway and the presence of protumor macrophages in the primary tumor and SLNs sustain the aggressiveness of an important subgroup of luminal B BC.

The possibility of correcting defective genes and modulating gene expression through gene therapy has emerged as a promising treatment strategy for breast cancer. Furthermore, the relevance of tumor immune microenvironment in supporting the oncogenic process has paved the way for novel immunomodulatory applications of gene therapy. Areas covered: In this review, the authors describe the most relevant delivery systems, focusing on nonviral vectors, along with the description of the major approaches used to modify target cells, including gene transfer, RNA interference (RNAi), and epigenetic regulation. Furthermore, they highlight innovative therapeutic strategies and the application of gene therapy in clinical trials for breast cancer. Expert opinion: Gene therapy has the potential to impact breast cancer research. Further efforts are required to increase the clinical application of RNAi-based therapeutics, especially in combination with conventional treatments. Innovative strategies, including genome editing and stem cell-based systems, may contribute to translate gene therapy into clinical practice. Immune-based approaches have emerged as an attractive therapeutic opportunity for selected breast cancer patients. However, several challenges need to be addressed before considering gene therapy as an actual option for the treatment of breast cancer.

Breast cancers (BC) carry a complex set of gene mutations that can influence their gene expression and clinical behavior. We aimed to identify genes driven by the TP53 mutation status and assess their clinical relevance in estrogen receptor (ER)-positive and ER-negative BC, and their potential as targets for patients with TP53 mutated tumors. Separate ROC analyses of each gene expression according to TP53 mutation status were performed. The prognostic value of genes with the highest AUC were assessed in a large dataset of untreated, and neoadjuvant chemotherapy treated patients. The mitotic checkpoint gene MPS1 was the most significant gene correlated with TP53 status, and the most significant prognostic marker in all ER-positive BC datasets. MPS1 retained its prognostic value independently from the type of treatment administered. The biological functions of MPS1 were investigated in different BC cell lines. We also assessed the effects of a potent small molecule inhibitor of MPS1, SP600125, alone and in combination with chemotherapy. Consistent with the gene expression profiling and siRNA assays, the inhibition of MPS1 by SP600125 led to a reduction in cell viability and a significant increase in cell death, selectively in TP53-mutated BC cells. Furthermore, the chemical inhibition of MPS1 sensitized BC cells to conventional chemotherapy, particularly taxanes. Our results collectively demonstrate that TP53-correlated kinase MPS1, is a potential therapeutic target in BC patients with TP53 mutated tumors, and that SP600125 warrant further development in future clinical trials.

DNA repair pathways can enable tumor cells to survive DNA damage induced by chemotherapy and thus provide prognostic and/or predictive value. We evaluated Affymetrix gene expression profiles for 145 DNA repair genes in untreated breast cancer (BC) patients (n = 684) and BC patients treated with regimens containing neoadjuvant taxane/anthracycline (n = 294) or anthracycline (n = 210). We independently assessed estrogen receptor (ER)-positive/HER2-negative, HER2-positive, and ER-negative/HER2-negative subgroups for differential expression, bimodal distribution, and the prognostic and predictive value of DNA repair gene expression. Twenty-two genes were consistently overexpressed in ER-negative tumors, and five genes were overexpressed in ER-positive tumors, but no differences in expression were associated with HER2 status. In ER-positive/HER2-negative tumors, the expression of nine genes (BUB1, FANCI, MNAT1, PARP2, PCNA, POLQ, RPA3, TOP2A, and UBE2V2) was associated with poor prognosis, and the expression of one gene (ATM) was associated with good prognosis. Furthermore, the prognostic value of specific genes did not correlate with proliferation. A few genes were associated with chemotherapy response in BC subtypes and treatment-specific manner. In ER-negative/HER2-negative tumors, the MSH2, MSH6, and FAN1 (previously MTMR15) genes were associated with pathological complete response and residual invasive cancer in taxane/anthracycline-treated patients. Conversely, PMS2 expression was associated with residual invasive cancer in treatments using anthracycline as a single agent. In HER2-positive tumors, TOP2A was associated with patient response to anthracyclines but not to taxane/anthracycline regimens. In genes expressed in a bimodal fashion, RECQL4 was significantly associated with clinical outcome. In vitro studies showed that defects in RECQL4 impair homologous recombination, sensitizing BC cells to DNA-damaging agents.

Triple negative breast cancers, which are defined by lack of expression of estrogen, progesterone, or HER2 receptors, represent approximately 15% of all breast cancers, although they account for a much higher proportional of breast cancer mortality. This is due both to their innate aggressive biological characteristics, but also to lack of effective therapies. Conventional chemotherapy is currently the only treatment option, thus there is a critical need to find new and effective targeted therapies in this disease. While investigation of agents such as poly (ADP-ribose) polymerase (PARP) inhibitors and EGFR inhibitors continues, results from recent clinical trials indicate that these therapies are not as active in sporadic triple negative breast cancers as initially hoped. It is important therefore to consider other emerging therapeutic agents. Mutation in p53 is found in the vast majority of triple negative breast cancers, and as such is a target of particular interest. Within this review, several agents with potential activity against aberrant p53 signaling have been considered, as a novel approach to finding an effective targeted therapy for this aggressive breast cancer subtype.

Breast cancer is a heterogeneous tumor type characterized by a complex spectrum of molecular aberrations, resulting in a diverse array of malignant features and clinical outcomes. Deciphering the molecular mechanisms that fuel breast cancer development and act as determinants of aggressiveness is a primary need to improve patient management. Among other alterations, aberrant expression of microRNAs has been found in breast cancer and other human tumors, where they act as either oncogenes or tumor suppressors by virtue of their ability to finely modulate gene expression at the post-transcriptional level. In this study, we describe a new role for miR-181a/b as negative regulators of the DNA damage response in breast cancer, impacting on the expression and activity of the stress-sensor kinase ataxia telangiectasia mutated (ATM). We report that miR-181a and miR-181b were overexpressed in more aggressive breast cancers, and their expression correlates inversely with ATM levels. Moreover we demonstrate that deregulated expression of miR-181a/b determines the sensitivity of triple-negative breast cancer cells to the poly-ADP-ribose-polymerase1 (PARP1) inhibition. These evidences suggest that monitoring the expression of miR-181a/b could be helpful in tailoring more effective treatments based on inhibition of PARP1 in breast and other tumor types.

MicroRNAs (miRNAs) simultaneously modulate different oncogenic networks, establishing a dynamic system of gene expression and pathway regulation. In this study, we analyzed global miRNA and messenger RNA (mRNA) expression profiles of 17 cell lines representing different molecular breast cancer subtypes. Spearman’s rank correlation test was used to evaluate the correlation between miRNA and mRNA expression. Hierarchical clustering and pathway analysis were also performed. Publicly available gene expression profiles (n = 699) and tumor tissues (n = 80) were analyzed to assess the relevance of key miRNA-regulated pathways in human breast cancer. We identified 39 significantly deregulated miRNAs, and the integration between miRNA and mRNA data revealed the importance of immune-related pathways, particularly the Oncostatin M (OSM) signaling, associated with mesenchymal-like breast cancer cells. OSM levels correlated with genes involved in the inflammatory response, epithelial-to-mesenchymal transition (EMT), and epidermal growth factor (EGF) signaling in human estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Our results suggest that the deregulation of specific miRNAs may cooperatively impair immune and EMT pathways. The identification of the OSM inflammatory pathway as an important mediator of EMT in triple-negative breast cancer (TNBC) may provide a novel potential opportunity to improve therapeutic strategies.

Stromal tumor-infiltrating lymphocytes (TILs) are a robust prognostic factor in triple-negative breast cancer (TNBC). However, the clinical significance of TILs may be influenced by the complex landscape of the tumor immune microenvironment. In this study, we aimed to evaluate the composition and the functionality of lymphocytic infiltration and checkpoint receptors in TNBC., SCOPUS: ar.j, info:eu-repo/semantics/published

Background and Aims: MicroRNAs (miRNAs) are short non-coding RNA whose expression levels have diagnostic and prognostic implications in cancer. However, the relevance of aberrantly expressed miRNAs to breast tumour-immune response has not been established. We aimed to assess the association between deregulated miRNAs and tumour immune response in breast molecular cancer subtypes. Methods: One hundred and seventy ductal infiltrating breast carcinomas (BC) (ER+/HER2− n=85, HER2+ n=40, ER−/HER2− n=45) were evaluated for the presence of cellular immune components by IHC assay. CD3, CD20 were used as markers for T lymphocytes infiltration (TIL), and B lymphocytes infiltration (BIL), respectively, whereas CD68 for the presence of tumour-associated macrophage (TAM). Total RNA (including miRNA) was extracted from all tumours and 32 highly specific miRNAs and 276 immune genes were evaluated by array and qRT-PCR assays. We derived an integrated immune miRNA-regulatory gene profile to tease out relationships of these genes as potential targets of specific miRNAs. In vitro experiments on BC cell lines were performed to confirm potential links between deregulated miRNAs and immune genes. Results: Lymphocytes infiltration (LI) was most common in HER2+ and ER−/HER2− BC samples (P < .004 and < .001) although B and T cells components were differently distributed in these 2 BC groups. TAM was a frequent event in ER−/HER2− BC subtype (P < .005). By gene and integration analyses we identified important miRNA immune-modulated target genes associated with prognostic factors and with molecular BC subtypes. We focused our studies on miR-181 and miR-135a that were significantly associated with ER−/HER2− (P < .001) and HER2+ BC (P < .002), respectively. BCs with abnormal expression of miR-181 and miR-135a showed an important association with distinct TIL and TAM components and specific immune genes. Importantly, preclinical data on BC cell lines demonstrated that miR-181 and miR-135a were able to regulate important cell immune-modulators such as SEMA3B and STAT6 signalling pathways, respectively. These 2 cellular pathways in turn were capable to regulate specific cascades of immune gene networks. Conclusion: We identified specific miRNAs associated with immune cellular components and immune-related tumour response in the 3 main molecular BC subtypes (ER+/HER2−, HER2+ and ER−/HER2). Aberrant expression of miR-181 and miR-135a can contribute to BC tumour immune response programs by regulating important gene targets such as SEMA3B and STAT6 signalling pathways in ER−/HER2− and HER2+ tumours, respectively. Modulation of miRNAs activating tumour immune response programs may have important implications for novel therapeutic strategies in BC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-01.

Until now, no data are present in literature about the prototype Th1 chemokine (C–X–C motif) ligand 10 (CXCL10) in anaplastic thyroid cancer (ATC). This study aimed to test in “primary human ATC cells” (ANA) vs “normal thyroid follicular cells” (TFC): (a) CXCL10 secretion basally and after interferon (IFN)-γ and/or tumor necrosis factor (TNF)-α stimulation; (b) peroxisome proliferator-activated receptor (PPAR)-γ activation by thiazolidinediones, rosiglitazone or pioglitazone, on CXCL10 secretion, on proliferation and apoptosis in ANA. We demonstrate that: (a) ANA, but not TFC, produced basally CXCL10, and did so in half of cases; (b) IFN-γ stimulated dose-dependently CXCL10, in ANA and TFC; (c) TNF-α did not induce CXCL10 secretion, in ANA and TFC; (d) IFN-γ + TNF-α induced a synergistic but variable release of CXCL10 in the different ANA preparations, while it was more reproducible in TFC; (e) rosiglitazone action on CXCL10 in ANA was inhibitory in 2/6, stimulatory in 1/6 and nil in 3/6, whereas it was inhibitory in TFC; (f) rosiglitazone inhibition of proliferation in ANA was not associated with the effect on CXCL10; (g) nuclear factor-κB and ERK1/2 were basally activated in ANA, increased by IFN-γ + TNF-α, and rosiglitazone inhibited that activation. On the whole, the present data first show that ANA cells are able to produce CXCL10, basally and under the influence of cytokines. However, the pattern of modulation by IFN-γ, TNF-α or thiazolidinediones is extremely variable, suggesting that the intracellular pathways involved in the chemokine modulation in ATC have different types of deregulation.