Clinical Outcome In Breast Cancer Research Articles

Abstract 4832: Overcoming breast cancer chemoresistance and blocking metastasis by targeting RNA-binding protein HuR

Abstract Patients with metastatic breast cancer have a dismal 5-year survival rate of only 24%. Chemoresistance is another contributor to the high mortality of advanced breast cancer. The RNA-binding protein, Hu antigen R (HuR), is overexpressed at all stages of breast cancer development. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. HuR also modulates sensitivity of breast cancer cells to chemotherapy. Taken together, HuR is an emerging target for breast cancer therapy, especially metastatic breast cancer. Using shRNA and CRISPR technologies to modulate HuR expression in breast cancer cells, we found that cells with HuR KD/KO were less invasive and more sensitive to chemotherapy. In an effort to discover small molecules that disrupt the HuR-mRNA interaction and block HuR functions in breast cancer progression, metastasis and chemoresistance, high throughput screening (HTS) was carried out using several chemical libraries (∼23,000 compounds) using fluorescence polarization (FP) assay and identified several initial hits with sub-micromolar inhibitory constants (Ki). Those potential disruptors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit KH-3 and its optimized analogs, specifically shortened HuR target mRNA half-lifes and decreased the level of the encoded proteins. Moreover, those compounds inhibited invasion and restored chemosensitivity. qPCR arrays focusing on specific pathways revealed that HuR inhibitors potently upregulated metastatic suppressors and downregulated genes frequently overexpressed in lung metastases. In animal studies, KH-3 not only powerfully exhibited antitumor efficacy in orthotopic xenografts, but also efficiently blocked experimental metastasis. In conclusion, we identified potent and specific small molecule disrupters of HuR-mRNA interaction for potential anti-metastatic therapy for breast cancer with HuR overexpression. Citation Format: Xiaoqing Wu, Rebecca T. Marquez, Shuang Han, Ke Li, Lan Lan, Yuxiao Guo, Dan A. Dixon, Jeffrey Aubé, Danny R. Welch, Liang Xu. Overcoming breast cancer chemoresistance and blocking metastasis by targeting RNA-binding protein HuR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4832.

Abstract 3152: Breast cancer-derived extracellular vesicles: clinical and prognostic impact

Abstract Background: Extracellular vesicles (EVs) are released from cancer cells into the tumor microenvironment, there participating in intercellular communication by altering recipient cell function. Breast cancer (BC) derived EVs are hypothesized to have an impact on tumor growth, immunosuppression or metastatic development, indicating analytical significance of EV concentrations in BC patients. For this reason, we investigated plasmatic EV numbers from locally advanced, neoadjuvant treated (NACT) BC patients in association with clinical parameters and prognostic impact. Material and methods: Plasmatic extracellular vesicles were isolated using ExoQuick™ precipitation reagent (SBI Inc., Mountain View, CA, USA), according to manufacturer's instructions, from locally advanced BC patients before (n = 142) and after (n = 156) NACT as well as from healthy female controls (n = 16). Subsequently, number and EV particle size were analyzed using ZetaView Laser Scattering Video Microscope (Particle Metrix GmbH, Microtrac, Meerbusch, Germany). Samples were 1:50.000 in PBS pre-diluted to obtain particle concentrations of approx. 1 × 10⁁6 particles per ml. Circulating tumor cells (CTCs) and stem cell-like circulating tumor cells (slCTCs) were evaluated using the AdnaTests BreastCancer, StemCell and EMT, respectively (QIAGEN Hannover GmbH, Germany). Results: EV particle concentrations (mean ± SEM in 10⁁9/ml) were significantly (p < 0.0001) elevated in BC patients (n = 104, respectively) before (2370 ± 170) and after (3524 ± 523) NACT compared to healthy females (90 ± 19). Paired analysis before and after NACT revealed higher EV levels after NACT (p = 0.008). In association studies, high EV numbers before NACT were related to less differentiated carcinomas and to lymph node spread. ROC analysis showed optimal cut-off values of EV levels (i) before NACT: 3540 × 10⁁9/ml (sensitivity: 66.7%; specificity: 78.1%; AUC = 0.706) and (ii) after NACT: 2480 × 10⁁9/ml (sensitivity: 100%; specificity: 44,3%; AUC = 0.654). In Kaplan-Meier analysis, a decreased 3-year PFS was significantly associated with EV particle concentrations after NACT > 2480 × 10⁁9/ml (p = 0.005). A reduced OS of BC patients was significantly associated with (i) EV levels before NACT > 3540 × 10⁁9/ml) (p = 0.001) and (ii) EV levels after NACT > 2480 × 109/ml) (p = 0.003). Decreased EV levels were found in BC patients after NACT with CTCs expressing ERCC1 (p = 0.025) or the stem cell marker ALDH1 (p = 0.004). Conclusion: In conclusion, BC patients showed elevated plasmatic EV levels compared to healthy females, while increased EV levels after NACT might be due to therapeutic effects. The association between EV particle concentrations and clinical parameters as well as their prognostic impact on clinical outcome in BC indicate the importance of EVs as a mediator in the BC tumor microenvironment. Therefore, determination of plasmatic EV particle amount might serve as a biomarker for BC monitoring. Citation Format: Lisa König, Vera Rebmann, Oliver Hoffmann, Ann-Kathrin Bittner, Bettina Wagner, Luis Felipe Santos Manvailer, Sabine Schramm, Agnes Bankfalvi, Bernd Giebel, Rainer Kimmig, Peter A. Horn, Sabine Kasimir-Bauer. Breast cancer-derived extracellular vesicles: clinical and prognostic impact. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3152.

Abstract 855: p53 isoform Ä133p53â triple negative breast cancer and increased relapse with neoadjuvant taxanes

Abstract Background: Studies of TP53 mutation, protein expression and modifications of the p53 network in breast cancer yield a complexity which has so far hindered substantive clinical progress. The effects of p53 mutation and p53 protein expression in breast cancer is dependent on the breast cancer subtype and stressors involved. Since identifying differential expression of p53 isoform mRNAs in breast cancer a decade ago, we have demonstrated associations between individual p53 isoforms (p53â, p53ã, Ä133p53á, Ä133p53â), breast cancer cell behaviour and clinical outcomes. Co-expressed combinations of isoforms may influence canonical p53 (p53á), whether mutant or wild type. Patterns of p53 isoform co-expression may hold the key to understanding p53 functionality, responses to therapy and disease behaviour in breast cancer. Methods: Expression of p53á, p53â, p53ã, Ä133p53á, Ä133p53â, Ä133p53ã by reverse-transcription PCR was analysed for the EORTC 10994 neoadjuvant breast cancer trial. In 469 available primary breast tumors, 223 patients had been randomised to a preoperative anthracycline regimen (fluorouracil, epirubicin, cyclophosphamide [FEC]) and 246 patients to receive a taxane based regimen (three cycles of docetaxel followed by three cycles of docetaxel + epirubicin [T-ET]). TP53 status was assessed by use of the yeast functional assay (FASAY) on biopsy samples that contained at least 20% tumour cells. Breast tumor subtypes: HER2 positive, triple-negative (TNBC), luminal-A, luminal-B (HER2 negative) and luminal-B (HER2 positive) were determined using the pathological markers recommended by the St Gallen breast cancer consensus guidelines. The primary endpoint was progression-free survival (PFS) with 9 years median follow up at the time of analysis. Results: All cancers expressed multiple p53 isoform mRNAs; none expressed canonical p53 mRNA (p53á) alone. There was a strong association between the p53 isoforms with 4 combinations of co-expressed p53 mRNAs predominated. Expression patterns included: (a) 285 tumours co-expressed p53á, p53â, p53ã, Ä133p53á, and Ä133p53ã; (b) 122 tumours co-expressed p53á, p53â, p53ã, Ä133p53á, Ä133p53â and Ä133p53ã (c) in 25 Ä133p53 negative tumors, p53á, p53â and p53ã were co-expressed (d) 22 p53â negative tumors co-expressed p53á, p53ã, Ä133p53á and Ä133p53ã. While no individual p53 isoform expression was associated with PFS, among the 104 TNBC expressing all p53 isoforms (pattern (b), Ä133p53â positive tumors), patients had 11.31 times greater risk of relapse when treated with T-ET than when treated with FEC (95%CI 1.91-66.99, p<0.0075). Conclusions: Corroborating the experimental and animal model data that p53 isoform expression, p53 mutation and p53 protein expression influence the pluripotent nature of p53, patients bearing Ä133p53â positive TNBC may benefit more from treatment with FEC than T-ET in the neoadjuvant setting. Citation Format: Jean-Christophe Bourdon, Alexandra Diot, Valerie Meuray, Leen Slaets, Richard Iggo, Herve Bonnefoi, David Cameron, Alastair M. Thompson. p53 isoform Ä133p53â triple negative breast cancer and increased relapse with neoadjuvant taxanes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 855.

ERCC1 and telomere status in breast tumours treated with neoadjuvant chemotherapy and their association with patient prognosis.

Dysfunctional telomeres and DNA damage repair (DDR) play important roles in cancer progression. Studies have reported correlations between these factors and tumour aggressiveness and clinical outcome in breast cancer. We studied the characteristics of telomeres and expression of ERCC1, a protein involved in a number of DNA repair pathways and in telomere homeostasis, to assess their prognostic value, alone or in combination, in 90 residual breast tumours after treatment with neoadjuvant chemotherapy (NCT). ERCC1 status was investigated at different molecular levels (protein and gene expression and gene copy‐number variations) by immunohistochemistry, qRT‐PCR and quantitative multiplex fluorescent‐PCR (QMF‐PCR). A comprehensive analysis of telomere characteristics was performed using qPCR for telomere length and qRT‐PCR for telomerase (hTERT), tankyrase 1 (TNKS) and shelterin complex (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2) gene expression. Short telomeres, high hTERT and TNKS expression and low ERCC1 protein expression were independently associated with worse survival outcome. Interestingly, ERCC1 gains and losses correlated with worse disease‐free (p = 0.026) and overall (p = 0.043) survival as compared to survival of patients with normal gene copy‐numbers. Unsupervised hierarchical clustering of all ERCC1 and telomere parameters identified four subgroups with distinct prognosis. In particular, a cluster combining low ERCC1, ERCC1 gene alterations, dysfunctional telomeres and high hTERT and a cluster with high TNKS and shelterin expression correlated with poor disease‐free (HR= 5.41, p= 0.0044) and overall survival (HR= 6.01, p= 0.0023) irrespective of tumour stage and grade. This comprehensive study demonstrates that telomere dysfunction and DDR can contribute synergistically to tumour progression and chemoresistance. These parameters are predictors of clinical outcome in breast cancer patients treated with NCT and could be useful clinically as prognostic biomarkers to tailor adjuvant chemotherapy post‐NCT.

Proteomic modulation in breast tumors after metformin exposure: results from a "window of opportunity" trial.

Reverse Phase Protein Array (RPPA) is a high-throughput antibody-based technique to assess cellular protein activity. The goal of this study was to assess protein marker changes by RPPA in tumor tissue from a pre-surgical metformin trial in women with operable breast cancer (BC). In an open-label trial, metformin 1500-mg PO daily was administered prior to resection in 35 non-diabetic patients with stage 0-III BC, body mass index ≥25kg/m2. For RPPA, formalin-fixed paraffin-embedded (FFPE) samples were probed with 160 antibodies. Paired and two-sample t-tests were performed (p≤0.05). Multiple comparisons were adjusted for by fixing the false discovery rate at 25%. We evaluated whether pre- and post-metformin changes of select markers by RPPA were identified by immunohistochemistry (IHC) in these samples. We also assessed for these changes by western blot in metformin-treated BC cell lines. After adjusting for multiple comparisons in the 32 tumors from metformin-treated patients vs. 34 untreated historical controls, 11 proteins were significantly different between cases vs. increases in Raptor, C-Raf, Cyclin B1, Cyclin D1, TRFC, and Syk; and reductions in pMAPKpT202,Y204, JNKpT183,pT185, BadpS112, PKC.alphapS657, and SrcpY416. Cyclin D1 change after metformin by IHC was not observed. In cell lines, reductions in JNKpT183 and BadpS112 were seen, with no change in Cyclin D1 or Raptor. These results suggest that metformin modulates apoptosis/cell cycle, cell signaling, and invasion/motility. These findings should be assessed in larger metformin trials. If confirmed, associations between these changes and BC clinical outcome should be evaluated. CLINICALTRIALS. NCT00930579.

MicroRNA-320a inhibits breast cancer metastasis by targeting metadherin.

Dysregulated microRNAs play important pathological roles in carcinogenesis that are yet to be fully elucidated. This study was performed to investigate the biological functions of microRNA-320a (miR-320a) in breast cancer and the underlying mechanisms. Function analyses for cell proliferation, cell cycle, and cell invasion/migration, were conducted after miR-320a silencing and overexpression. The specific target genes of miR-320a were predicted by TargetScan algorithm and then determined by dual luciferase reporter assay and rescue experiment. The relationship between miR-320a and its target genes was explored in human breast cancer tissues. We found that miR-320a overexpression could inhibit breast cancer invasion and migration abilities in vitro, while miR-320a silencing could enhance that. In addition, miR-320a could suppress activity of 3′-untranslated region luciferase of metadherin (MTDH), a potent oncogene. The rescue experiment revealed that MTDH was a functional target of miR-320a. Moreover, we found that MTDH was negatively correlated with miR-320a expression, and it was related to clinical outcomes of breast cancer. Further xenograft experiment also showed that miR-320a could inhibit breast cancer metastasis in vivo. Our findings clearly demonstrate that miR-320a suppresses breast cancer metastasis by directly inhibiting MTDH expression. The present study provides a new insight into anti-oncogenic roles of miR-320a and suggests that miR-320a/MTDH pathway is a putative therapeutic target in breast cancer.

Disease presentation and clinical outcomes of breast cancer (BC) in black women (BW) 40 yrs and under at a single urban hospital.

e12044Background: BC is the most common cancer in women of all ages; but less common under 40 years (6.6%). Younger BC patients (pts) are known to have Triple negative or Her2neu positive, advanced...

Association of nuclear localization of SATB1 with adverse clinical outcome in breast cancer.

e12029Background: Genome organizer SATB1 has been recently reported to associate with negative prognosis in several cancer forms.However, the role of SATB1 in breast cancer(BC) remains controversial.This study evaluated the expression of SATB1 and its role in predicting prognosis in BC. Methods: The SATB1 expression and its clinical significance were examined in 93 consecutive patients with invasive ductal breast carcinoma using immunohistochemistry. Kaplan-Meier survival analysis was used to estimate the prognostic relevance of SATB1 and the survival difference between groups was assessed by the log-rank test. Univariate and multivariate Cox regression analysis was performed to evaluate difference of all possible factors. Moreover, the sensitivity and specificity of the aforementioned biomarkers were identified by using receiver operating characteristic (ROC) curve. Results: Subcellular staining of SATB1 was heterogeneous, ranging from cytoplasmic to nuclear. SATB1 staining was strongly related to clinic...

Association of gamma-H2AX expression with clinopathologic characteristics and clinical outcomes in breast cancer.

11577Background: Histone H2AX phosphorylation at Ser139 (gamma-H2AX) represents an event of cellular response to DNA double-strand breaks and constitutive expression of gamma-H2AX may reflect intri...

Evaluation of clinical parameters influencing the development of bone metastasis in breast cancer.

BackgroundThe development of metastases is a negative prognostic parameter for the clinical outcome of breast cancer. Bone constitutes the first site of distant metastases for many affected women. The purpose of this retrospective multicentre study was to evaluate if and how different variables such as primary tumour stage, biological and histological subtype, age at primary diagnosis, tumour size, the number of affected lymph nodes as well as grading influence the development of bone-only metastases.MethodsThis retrospective German multicentre study is based on the BRENDA collective and included 9625 patients with primary breast cancer recruited from 1992 to 2008. In this analysis, we investigated a subgroup of 226 patients with bone-only metastases. Association between bone-only relapse and clinico-pathological risk factors was assessed in multivariate models using the tree-building algorithms “exhausted CHAID (Chi-square Automatic Interaction Detectors)” and CART(Classification and Regression Tree), as well as radial basis function networks (RBF-net), feedforward multilayer perceptron networks (MLP) and logistic regression.ResultsMultivariate analysis demonstrated that breast cancer subtypes have the strongest influence on the development of bone-only metastases (χ2 = 28). 29.9 % of patients with luminal A or luminal B (ABC-patients) and 11.4 % with triple negative BC (TNBC) or HER2-overexpressing tumours had bone-only metastases (p < 0.001). Five different mathematical models confirmed this correlation. The second important risk factor is the age at primary diagnosis. Moreover, BC subcategories influence the overall survival from date of metastatic disease of patients with bone-only metastases. Patients with bone-only metastases and TNBC (p < 0.001; HR = 7.47 (95 % CI: 3.52–15.87) or HER2 overexpressing BC (p = 0.007; HR = 3.04 (95 % CI: 1.36–6.80) have the worst outcome compared to patients with luminal A or luminal B tumours and bone-only metastases.ConclusionThe bottom line of different mathematical models is the prior importance of subcategories of breast cancer and the age at primary diagnosis for the appearance of osseous metastases. The primary tumour stage, histological subtype, tumour size, the number of affected lymph nodes, grading and NPI seem to have only a minor influence on the development of bone-only metastases.

Met and its ligand HGF are associated with clinical outcome in breast cancer.

Few biomarkers exist to predict radiotherapy response in breast cancer. In vitro studies suggest a role for Met and its ligand HGF. To study this suggested role, MET and HGF gene copy numbers were determined by droplet digital PCR in tumours from 205 pre-menopausal and 184 post-menopausal patients, both cohorts randomised to receive either chemo- or radiotherapy. MET amplification was found in 8% of the patients in both cohorts and HGF amplification in 7% and 6% of the patients in the pre- and post-menopausal cohort, respectively. Met, phosphorylated Met (pMet), and HGF protein expression was determined by immunohistochemistry in the pre-menopausal cohort. Met, pMet, and HGF was expressed in 33%, 53%, and 49% of the tumours, respectively. MET amplification was associated with increased risk of distant recurrence for patients receiving chemotherapy. For the pre-menopausal patients, expression of cytoplasmic pMet and HGF significantly predicted benefit from radiotherapy in terms of loco-regional recurrence. Similar trends were seen for MET and HGF copy gain. In the post-menopausal cohort, no significant association of benefit from radiotherapy with neither genes nor proteins was found. The present results do not support that inhibition of Met prior to radiotherapy would be favourable for pre-menopausal breast cancer, as previously suggested.

The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ.

Mammary MCF-10A cells seeded on reconstituted basement membrane form spherical structures with a hollow central lumen, termed acini, which are a physiologically relevant model of mammary morphogenesis. Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional protein overexpressed in breast cancer and ductal carcinoma in situ. When present in the nucleus Bag-1 is predictive of clinical outcome in breast cancer. Bag-1 exists as three main isoforms, which are produced by alternative translation initiation from a single mRNA. The long isoform of Bag-1, Bag-1L, contains a nuclear localisation sequence not present in the other isoforms. When present in the nucleus Bag-1L, but not the other Bag-1 isoforms, can interact with and modulate the activities of estrogen-, androgen- and vitamin D-receptors. Overexpression of Bag-1 mRNA in MCF-10A is known to produce acini with luminal filling reminiscent of ductal carcinoma in situ. As this mRNA predominantly overexpresses the short isoform of Bag-1, Bag-1S, we set out to examine whether the nuclear Bag-1L isoform is sufficient to drive premalignant change by developing a Bag-1L-overexpressing MCF-10A model. Two clones differentially overexpressing Bag-1L were grown in two-dimensional (2D) and three-dimensional (3D) cultures and compared with an established model of HER2-driven transformation. In 2D cultures, Bag-1L overexpression reduced proliferation but did not affect growth factor responsiveness or clonogenicity. Acini formed by Bag-1L-overexpressing cells exhibited reduced luminal clearing when compared with controls. An abnormal branching morphology was also observed which correlated with the level of Bag-1L overexpression, suggesting further malignant change. Treatment with Thio-2, a small-molecule inhibitor of Bag-1, reduced the level of branching. In summary, 3D cultures of MCF-10A mammary epithelial cells overexpressing Bag-1L demonstrate a premalignant phenotype with features of ductal carcinoma in situ. Using this model to test the small-molecule Bag-1 inhibitor, Thio-2, reveals its potential to reverse the atypical branched morphology of acini that characterizes this premalignant change.

Prognostic Value of Axillary Nodal Ratio after Neoadjuvant Chemotherapy of Doxorubicin/Cyclophosphamide Followed by Docetaxel in Breast Cancer: A Multicenter Retrospective Cohort Study.

PurposeThe purpose of this study is to investigate the prognostic value of lymph node (LN) ratio (LNR) in patients with breast cancer after neoadjuvant chemotherapy.Materials and MethodsThis retrospective analysis is based on the data of 814 patientswith stage II/III breast cancer treated with four cycles of doxorubicin/cyclophosphamide followed by four cycles of docetaxel before surgery. We evaluated the clinical significance of LNR (3 categories: low 0-0.20 vs. intermediate 0.21-0.65 vs. high 0.66-1.00) using a Cox proportional regression model.ResultsA total of 799 patients underwent breast surgery. Pathologic complete response (pCR, ypT0/isN0) was achieved in 129 patients (16.1%) (hormone receptor [HR] +/human epidermal growth factor receptor 2 [HER2] –, 34/373 [9.1%]; HER2+, 45/210 [21.4%]; triple negative breast cancer, 50/216 [23.1%]). The mean numbers of involved LN and retrieved LN were 2.70 (range, 0 to 42) and 13.98 (range, 1 to 64), respectively. The mean LNR was 0.17 (low, 574 [71.8%]; intermediate, 170 [21.3%]; high, 55 [6.9%]). In univariate analysis, LNR showed significant association with a worse relapse-free survival (3-year relapse-free survival rate 84.8% in low vs. 66.2% in intermediate vs. 54.3% in high; p < 0.001, log-rank test). In multivariate analysis, LNR did not show significant association with recurrence after adjusting for other clinical factors (age, histologic grade, subtype, ypT stage, ypN stage, lymphatic or vascular invasion, and pCR). In subgroup analysis, the LNR system had good prognostic value in HR+/HER2–subtype.ConclusionLNR is not superior to ypN stage in predicting clinical outcome of breast cancer after neoadjuvant chemotherapy. However, the prognostic value of the LNR system in HR+/HER2–patients is notable and worthy of further investigation.

New insights on PI3K/AKT pathway alterations and clinical outcomes in breast cancer.

PI3K/AKT signaling pathway plays an important role in tumorigenesis and regulates critical cellular functions including survival, proliferation and metabolism. PIK3CA mutations and AKT activation by phosphorylation (pAKT) are often detected in many cancers and especially at high frequencies in breast cancer. Mounting data suggest that PIK3CA mutations or pAKT are mostly associated with better or insignificant outcomes in estrogen receptor-positive (ER+) early stage breast cancer and tend to be with worse prognosis in ER- disease. pAKT expression has been identified to predict paclitaxel chemotherapy benefit in node-positive breast cancer. Preclinical and neoadjuvant trial data suggest that PIK3CA alterations confer resistance to HER2-targeted therapy and are associated with lower pathological complete response (pCR) rate in HER2-positive breast cancer. However, recent results from randomized clinical trials of adjuvant and metastatic settings show that patients with mutant and wildtype PIK3CA tumors derived similar benefit from anti-HER2 therapy. This article, with our new insights, aims to decipher the mixed data and discusses the influence of the potential confounding factors in the assessments. We also share our views for validation of PI3K/AKT alterations in relation to clinical outcome in the context of specific breast cancer subtypes and treatment modalities towards further advance of the precision medicine for breast cancer treatment.

Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448.

BackgroundTriple negative breast cancers (TNBC) possess cell dedifferentiation characteristics, carry out activities connate to those of cancer stem cells (CSCs) and are associated with increased metastasis, as well as, poor clinical prognosis. The regulatory mechanism of this highly malignant phenotype is still poorly characterized. Accruing evidence support the role of non-coding RNAs (ncRNAs) as potent regulators of CSC and metastatic gene expression, with their dysregulation implicated in tumorigenesis and disease progression.MethodsIn this study, we investigated TNBC metastasis, metastasis-associated genes and potential inhibitory mechanisms using bioinformatics, tissue microarray analyses, immunoblotting, polymerase chain reaction, loss and gain of gene function assays and comparative analyses of data obtained.ResultsCompared with other breast cancer types, the highly metastatic MDA-MB-231 cells concurrently exhibited increased expression levels of Lysine-specific demethylase 5B protein (KDM5B) and long non-coding RNA (lncRNA), MALAT1, suggesting their functional association. KDM5B-silencing in the TNBC cells correlated with the upregulation of hsa-miR-448 and led to suppression of MALAT1 expression with decreased migration, invasion and clonogenic capacity in vitro, as well as, poor survival in vivo. This projects MALAT1 as a mediator of KDM5B oncogenic potential and highlights the critical role of this microRNA, lncRNA and histone demethylase in cancer cell motility and metastatic colonization. Increased expression of KDM5B correlating with disease progression and poor clinical outcome in breast cancer was reversed by hsa-miR-448.ConclusionsOur findings demonstrate the critical role of KDM5B and its negative regulator hsa-miR-448 in TNBC metastasis and progression. Hsa-miR-448 disrupting KDM5B-MALAT1 signalling axis and associated activities in TNBC cells, projects it as a putative therapeutic factor for selective eradication of TNBC cells.Graphical abstractKDM5B, MALAT1 and hsa-miR-448 are active looped components of the epigenetic poculo mortis in aggressive breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2108-5) contains supplementary material, which is available to authorized users.

Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology-Genomic Integration Analysis.

BackgroundThe intra-tumor diversity of cancer cells is under intense investigation; however, little is known about the heterogeneity of the tumor microenvironment that is key to cancer progression and evolution. We aimed to assess the degree of microenvironmental heterogeneity in breast cancer and correlate this with genomic and clinical parameters.Methods and FindingsWe developed a quantitative measure of microenvironmental heterogeneity along three spatial dimensions (3-D) in solid tumors, termed the tumor ecosystem diversity index (EDI), using fully automated histology image analysis coupled with statistical measures commonly used in ecology. This measure was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set. In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types. However, this association was not observed in low-grade (grade 1 and 2) breast cancers. The prognostic value of EDI was superior to known prognostic factors and was enhanced with the addition of TP53 mutation status (multivariate analysis test set, p = 9 × 10−4, hazard ratio = 1.47, 95% CI 1.17–1.84; validation set, p = 0.0011, hazard ratio = 1.78, 95% CI 1.26–2.52). Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. Limitations of this study include the number of cell types included in the model, that EDI has prognostic value only in grade 3 tumors, and that our spatial heterogeneity measure was dependent on spatial scale and tumor size.ConclusionsTo our knowledge, this is the first study to couple unbiased measures of microenvironmental heterogeneity with genomic alterations to predict breast cancer clinical outcome. We propose a clinically relevant role of microenvironmental heterogeneity for advanced breast tumors, and highlight that ecological statistics can be translated into medical advances for identifying a new type of biomarker and, furthermore, for understanding the synergistic interplay of microenvironmental heterogeneity with genomic alterations in cancer cells.

Abstract P5-08-47: Clinical outcome of pathological T1N0 breast cancer according to the hormone receptor and HER2 status and adjuvant therapy

Abstract Background: The outcome of T1N0 breast cancer is relatively good, however, a subpopulation of this stage has a high population of relapse. It is important to clarify the factors associated with the outcome in order to determine the adequate adjuvant systemic therapy for T1N0 breast cancer. Aims: To investigate the prognosis of pathological T1N0 (pT1N0) breast cancer by receptor (estrogen receptor; ER, progesterone receptor; PgR, human epidermal growth factor receptor 2; HER2) status and adjuvant systemic therapy, and thus to identify the factors associated with the outcome. Methods: Among 2164 women who underwent surgery for primary breast cancer in our department, 925 had pathological T1N0 tumors (130 T1a, 234 T1b and 561 T1c). The associations between clinicopathological characteristics, adjuvant therapy and relapse-free survival (RFS) were examined Results: Of 908 patients with known hormone receptor (HR; ER and PgR) and HER2 status, 675, 79, 64 and 90 had HR+/HER2-, HR+/HER2+, HR-/HER2+ and HR-/HER2- (triple negative; TN) tumors, respectively. The prognosis of patients with T1c tumors was significantly poorer than that of the patients with T1a and T1b tumors (5-year DFS of T1a, T1b and T1c: 97.5%, 97.9%, 94.5%, p=0.0201). HR+/HER2- subtype was significantly associated with better prognosis than other subtypes (5-year DFS: 96.9% vs. 93.6%, p=0.0194). Patients younger than 40 year old or older than 74 years old had significantly shorter PFS (p=0.0039). Lymphovessel invasion (ly), high histological grade (HG2, 3) were associated with poor outcome in all cohort (p=0.0026, p=0.0356). In HR-positive tumors, ly, high HG and omission of the adjuvant endocrine therapy were associated with shorter RFS (p=0.0009, p=0.0306 and p=0.0016, respectively). Adjuvant chemotherapy was not associated with RFS regardless of HR and HER2 status. However, in T1c with nuclear grade (NG) 3 tumors, use of the adjuvant chemotherapy was associated better prognosis. The prognosis of patients with HER2+ tumors was not significantly different among patients with or without trastuzumab in this cohort. Conclusion: In spite of the excellent prognosis of pT1N0 breast cancer, adjuvant endocrine therapy is important for patients with HR+ tumors. However, the use of adjuvant chemotherapy or trastuzumab did not significantly improve the prognosis. Thus, the indication of chemotherapy or anti-HER2 therapy should be determined in consideration of the several prognostic factors for pT1N0 breast cancer. Citation Format: Tokunaga E, Akiyoshi S, Koga C, Nakamura Y, Taguchi K, Ishida M, Ohno S. Clinical outcome of pathological T1N0 breast cancer according to the hormone receptor and HER2 status and adjuvant therapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-47.

Abstract P3-07-32: Tumour infiltrating lymphocyte (TIL) and chemokine gene signature predicts for benefit of anthracycline-containing chemotherapy in breast cancer patients

Abstract Background: The contribution of immune cells has long been appreciated in tumour development and disease progression; however, their translational potential as cancer-associated prognostic and predictive markers was only recently recognized. High densities of tumour-infiltrating lymphocytes (TILs) correlate with improved clinical outcome in breast cancer; whether TILs also predict anthracycline benefit in all, or only a particular subgroup, of breast cancer patients remains largely unknown. Furthermore, since identification of TILs is generally based on H&amp;E staining, it has not previously been possible to evaluate relative contribution of distinct T-cell types, and B cells, to patient outcome. Methods: We assessed 290 patient samples from the BR9601 adjuvant breast cancer trial for the capacity of TIL contexture to predict for anthracycline (E-CMF) benefit over CMF. We immunoprofiled patient samples on the Nanostring platform to gain insight into the impact of lymphocyte populations predicting for anthracycline benefit. Our immunoprofiling panel included 38 genes representing TIL-gene signatures and chemokines that may be responsible for recruiting TILs to the tumour site. Results: The analyses revealed two important findings. First, refinement of the 38-gene panel resulted in the generation of a novel 9-gene signature that includes cytotoxic T lymphocytes (CTL) and chemokine genes. Low CTL gene expression correlated with ER+ expression while high expression correlated with ER- expression (p&amp;lt;0.0001), consistent with the notion that high TIL densities are predominantly observed in non-luminal breast cancers. Second, in an univariate Cox regression analysis, this 9-gene signature was a predictive biomarker of anthracycline benefit with respect to breast-cancer specific OS (HR: 0.371, 95%CI 0.158-0.868, p=0.022) and DRFS (HR: 0.395, 95%CI 0.172-0.907, p=0.028); this effect was no longer significant after adjustment for other prognostic factors (OS HR: 0.437, 95%CI 0.166-1.150, p=0.094; DRFS HR: 0.488, 95%CI 0.185-1.287, p=0.147). Conclusion: This study highlights the significance of assessing the entire tumour since TILs, tumour and stromal cells collectively engage in a complex interplay that contributes to disease development and progression. Importantly, it reveals that not only CTLs but also chemokines may be clinically relevant and should be validated as potential biomarkers of anthracycline benefit and as therapeutic targets. Citation Format: Braunstein M, Yao C, Lyttle N, Liao L, Boutros PC, Twelves CJ, Bartlett JMS, Spears M. Tumour infiltrating lymphocyte (TIL) and chemokine gene signature predicts for benefit of anthracycline-containing chemotherapy in breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-32.

Abstract PD1-04: Association of aspirin and clinical outcomes in patients with invasive breast cancer

Abstract Long-term low-dose aspirin use has been observed to reduce the risk of colorectal, breast and other cancers. The most prominent effect has been in colorectal cancer, in which large-scale meta-analyses have shown that there is an approximately 20% relative risk reduction in participants who took aspirin for four or more years. The role of long-term NSAID use in breast cancer risk is less clear although preliminary observational case-control studies suggest an association between aspirin use and reduced incidence of hormone receptor-positive breast cancers though no clear evidence exists to support a clear mortality benefit among patients with a history of prior NSAID use as opposed to those who do not. To investigate whether a history of aspirin use is associated with improved clinical outcome in breast cancer, we examined the pattern of aspirin use, cancer pathology and overall survival of over 1000 patients diagnosed with and treated for invasive breast cancer at our institution, for whom long-term follow up was available. A history of aspirin use for at least a period of 30 days prior to breast cancer diagnosis was reported in nearly 14% of individuals. Aspirin use was associated with being older than the age of 50 at diagnosis (79.8% vs 66.5%; Fisher's Exact Test (P &amp;lt; 3.2x10-3) and being of African American race (49.1% vs 28.7%; P &amp;lt; 3.4x10-2), when compared to those who have not used aspirin. Aspirin use correlated with prognostic factors that are known to be associated with poor outcomes. They include axilla node positive disease (44.5% vs 27.0%, p&amp;lt; 0.032), evidence of lymphovascular invasion (24.7% vs 15.4%, p&amp;lt; 0.049), Her2-neu positive disease (&amp;lt;0.0083). In contrast to prior retrospective case-control studies, no significant association between aspirin use and hormone receptor positive disease was noted for either ER (p=0.19) or PR(+) receptor status (p=0.12). Finally, we examined if aspirin use prior to breast cancer diagnosis has any impact on disease outcome. Over a median follow up of 60.0 months, univariate analysis using cox proportional hazard modeling demonstrated that the use of low-dose aspirin prior to the diagnosis of breast cancer was associated with an increased all-cause mortality when compared to patients without aspirin use prior to cancer diagnosis (HR=3.084, 95% CI=1.961 to 4.848). On multivariate analysis, we found that recent history of aspirin use was significantly associated with a worse overall survival (HR 2.65; 95%CI 1.37 -5.12, P &amp;lt; 3.77 x 10-3), when controlled for other prognostic factors including receptor status, tumor size, tumor grade, number of positive regional lymph nodes, positive margins, as well as race and age at diagnosis. This is the first study to report on the association of aspirin use with breast cancer outcomes in a large patient cohort treated at a single institution. Although aspirin in breast and cancers has been associated with reduced cancer incidence, a history of aspirin use prior to breast cancer diagnosis does not appear to be protective or associated with improve clinical outcomes or survival among breast cancer patients. Ongoing efforts are examining the mechanism underlying this association. Citation Format: Li YR, Steel L, Carrigan E, Tchou J. Association of aspirin and clinical outcomes in patients with invasive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD1-04.

Abstract P6-07-01: Multigene profiling to identify clinically relevant actionable mutations in breast cancer: An Indian study

Abstract Background: Numerous chemotherapeutic agents are available against breast cancer (BC), but a vast majority of patients diagnosed with this disease still develop treatment resistance and eventually succumb to disease. It remains an unmet need to identify specific molecular defects against which targeted therapy are available for improving clinical outcomes in BC. Our study aims to identify frequent hotspot mutations in BCs and determine their clinical impact. Methods: 200 women with BC(early diagnosed and/or metastatic) aged 26-75 yrs (median age 50.5yrs) diagnosed at HCG from April 2013-15 were consented to be profiled by targeted deep sequencing for hotspot mutations in 48 cancer-related genes using Illumina's TSCAP panel and MiSeq technology in an IRB-approved prospective study in a CLIA compliant laboratory. All the cases had pathology review for stage, histological type, hormonal status and Ki67. The average coverage across 220 hot spots was greater than 1000X. Data was processed using Strand Avadis NGS™. Mutations identified in the tumor were assessed for 'actionability' i.e. response to therapy and impact on prognosis. Results: Somatic variants were detected in 75% of cases with direct impact on therapy or prognosis. Genetic aberrations were identified in PI3K/AKT/ mTOR signalling pathway in substantial fraction (27%) of breast cancer cases, out of which 17% had PIK3CA activating mutations,13 and 5 cases had PTEN and AKT deletions or truncating mutations respectively. Aberration in this pathway was more prevalent in HR+ve (53%) and HER2-ve including TNBC (61%) than in HR+/HER2+ve tumors (10.6%) of IDC histology. However, no correlation was found with stage and Ki67 index of the tumor. Notably 80% of BC cases presented with liver metastasis at the time of diagnosis were detected with PIK3CA mutation indicating its role as a surrogate marker of organ specific metastasis. PIK3CA was found to be co mutated with p53 in 16 cases (9%) of which 4 cases showed npCR post NACT. Also disruptive and non-disruptive mutations in TP53 alone were found in 25% of BC, varying widely among different histologies. A follow up of few cases showed shorter PFS and poor outcome in resected BC treated with NACT indicating its robust prognostic value in NACT setting. Furthermore, two patients were detected with cKIT mutations indicating sensitivity to imatinib and therefore enrolled on a clinical trial. The other variants were found in RB1(n=8),Her2 (n=2),FGFR amplification(n=1), KRAS(n=2),NRAS(n=3)CDH1(n=1),FBXW7(n=2) and EGFR(n=1).All these variants detected indicated resistance to conventional therapy and suggested sensitivity to available targeted therapy, either approved or in clinical trials. The response and outcome are being monitored in about 20 (10%) patients who have been enrolled in clinical trials and receiving mutation specific targeted therapy. Conclusions: This study confirms the utility of multigene profiling in early diagnosed and advanced BC patients, to stratify them on their molecular profile who could potentially benefit from targeted therapy. Prospective studies and randomized clinical trials are ongoing to confirm the independent prognostic and therapeutic value of the mutations in a larger cohort of Indian population. Citation Format: Ghosh M, Sheela ML, Choudhury S, Bahadur U, Patil S, Satheesh CT, Murugan K, Nayak R, Sridhar PS, Rao N, Mahesh B, Shashidhara HP, Krishnamoorthy N, Gupta V, Sankaran S, Subramanian K, Ajaikumar BS. Multigene profiling to identify clinically relevant actionable mutations in breast cancer: An Indian study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-01.