Response In Triple-negative Breast Cancer Research Articles

42O - Profiles of genome complexity identify HORMAD1 as a driver of homologous recombination deficiency and platinum therapy response in triple-negative breast cancer

A. Grigoriadis1, D. Weekes1, J.A. Watkins1, P. Gazinska1, J. Frankum2, C. Lord2, A. Ashworth2, J.M. Ford3, M. Telli3, A. Tutt4 Breakthrough Breast Cancer Research Unit, King’s College London Guy’s Hospital, London, UK Cancer Genomics and Therapeutics, Institute of Cancer Research ICR, London, UK Division of Oncology, Stanford University School of Medicine, Stanford, CA, USA Breakthrough Breast Cancer Research Unit, King’s College London School of Medicine, London, UK

Novel role of PELP1 in regulating chemotherapy response in mutant p53-expressing triple negative breast cancer cells.

Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, occurs in younger women and is associated with poor prognosis. Gain-of-function mutations in TP53 are a frequent occurrence in TNBC and have been demonstrated to repress apoptosis and up-regulate cell cycle progression. Even though TNBC responds to initial chemotherapy, resistance to chemotherapy develops and is a major clinical problem. Tumor recurrence eventually occurs and most patients die from their disease. An urgent need exists to identify molecular-targeted therapies that can enhance chemotherapy response. In the present study, we report that targeting PELP1, an oncogenic co-regulator molecule, could enhance the chemotherapeutic response of TNBC through the inhibition of cell cycle progression and activation of apoptosis. We demonstrate that PELP1 interacts with MTp53, regulates its recruitment, and alters epigenetic marks at the target gene promoters. PELP1 knockdown reduced MTp53 target gene expression, resulting in decreased cell survival and increased apoptosis upon genotoxic stress. Mechanistic studies revealed that PELP1 depletion contributes to increased stability of E2F1, a transcription factor that regulates both cell cycle and apoptosis in a context-dependent manner. Further, PELP1 regulates E2F1 stability in a KDM1A-dependent manner, and PELP1 phosphorylation at the S1033 residue plays an important role in mediating its oncogenic functions in TNBC cells. Accordingly, depletion of PELP1 increased the expression of E2F1 target genes and reduced TNBC cell survival in response to genotoxic agents. PELP1 phosphorylation was significantly greater in the TNBC tumors than in the other subtypes of breast cancer and in the normal tissues. These findings suggest that PELP1 is an important molecular target in TNBC, and that PELP1-targeted therapies may enhance response to chemotherapies.

BRCA1-IRIS inactivation overcomes paclitaxel resistance in triple negative breast cancers.

IntroductionIntrinsic or acquired chemoresistance is a major problem in oncology. Although highly responsive to chemotherapies such as paclitaxel, most triple negative breast cancer (TNBC) patients develop chemoresistance. Here we investigate the role of BRCA1-IRIS as a novel treatment target for TNBCs and their paclitaxel-resistant recurrences.MethodsWe analyzed the response of BRCA1-IRIS overexpressing normal mammary cells or established TNBC cells silenced from BRCA1-IRIS to paclitaxel in vitro and in vivo. We analyzed BRCA1-IRIS downstream signaling pathways in relation to paclitaxel treatment. We also analyzed a large cohort of breast tumor samples for BRCA1-IRIS, Forkhead box class O3a (FOXO3a) and survivin expression. Finally, we analyzed the effect of BRCA1-IRIS silencing or inactivation on TNBCs formation, maintenance and response to paclitaxel in an orthotopic model.ResultsWe show that low concentrations of paclitaxel triggers BRCA1-IRIS expression in vitro and in vivo, and that BRCA1-IRIS activates two autocrine signaling loops (epidermal growth factor (EGF)/EGF receptor 1 (EGFR)-EGF receptor 2 (ErbB2) and neurogulin 1 (NRG1)/ErbB2-EGF receptor 3 (ErbB3), which enhances protein kinase B (AKT) and thus survivin expression/activation through promoting FOXO3a degradation. This signaling pathway is intact in TNBCs endogenously overexpressing BRCA1-IRIS. These events trigger the intrinsic and acquired paclitaxel resistance phenotype known for BRCA1-IRIS-overexpressing TNBCs. Inactivating BRCA1-IRIS signaling using a novel inhibitory mimetic peptide inactivates these autocrine loops, AKT and survivin activity/expression, in part by restoring FOXO3a expression, and sensitizes TNBC cells to low paclitaxel concentrations in vitro and in vivo. Finally, we show BRCA1-IRIS and survivin overexpression is correlated with lack of FOXO3a expression in a large cohort of primary tumor samples, and that BRCA1-IRIS overexpression-induced signature is associated with decreased disease free survival in heavily treated estrogen receptor alpha-negative patients.ConclusionsIn addition to driving TNBC tumor formation, BRCA1-IRIS overexpression drives their intrinsic and acquired paclitaxel resistance, partly by activating autocrine signaling loops EGF/EGFR-ErbB2 and NRG1/ErbB2-ErbB3. These loops activate AKT, causing FOXO3a degradation and survivin overexpression. Taken together, this underscores the need for BRCA1-IRIS-specific therapy and strongly suggests that BRCA1-IRIS and/or signaling loops activated by it could be rational therapeutic targets for advanced TNBCs.

Abstract 550: Genomic instability and telomere characteristics as predictive biomarkers of therapeutic response in triple-negative breast cancer

Abstract Background. Triple negative breast cancers (TNBC) represent about 15 % of invasive breast carcinomas. These tumors are identified in clinical practice according to their “triple negative” immunophenotype (estrogen receptor, progesterone receptor and HER2 negative). TNBC show aggressive behavior and an unusual sarcoma-like pattern of metastasis. Identification of predictive biomarkers is critical to optimal therapeutic management of these tumors. TNBC are characterized by increased genomic instability. These tumors present complex pattern of numerous low-amplitude genomic gains and losses as well as shorter telomeres. Aim. Identification of predictive biomarkers of response to neoadjuvant chemotherapy in TNBC by evaluating telomeric parameters and genomic instability. Methods. We studied 38 TNBC patients treated with neoadjuvant chemotherapy according to the TVA protocol (FEC 100, docetaxel, panitumumab). DNA and RNA were extracted from snap-frozen tumor samples obtained before neoadjuvant therapy. The pathological response to treatment was evaluated according to Chevallier's and Sataloff's classifications and correlated with aCGH profiles (8x60K, Agilent) and telomere characteristics, including telomere length (qPCR) and expression (qRT-PCR) of telomerase (hTERT) and shelterin complex genes (TRF1, TRF2, POT1, TPP1, RAP1 and TIN2). Results. Telomere shortening and high hTERT expression were found in more aggressive tumors according to TNM classification (P< 0.04), and hTERT expression was also highly correlated with an increased tumor size (P= 0.0058). Short telomeres were strongly associated with incomplete histological response to neoadjuvant therapy according to Chevallier's (P= 0.0004) and Sataloff's classifications, (P= 0.0007). hTERT expression was significantly increased in cases with incomplete response (P< 0.04). Among shelterin complex genes, TRF2 and TPP1 overexpression was found in more aggressive TNBC (according to TNM classification and SBR grading, P< 0.05), but there was no correlation with pathological response. aCGH data indicated that genomic instability in non-responders was mainly represented by genomic gains. In particular, cases with incomplete therapeutic responses showed significantly larger size of gains than cases with complete responses. (P= 0.0089). Short telomeres were also associated with larger gains of genomic material (P= 0.0067). Of note, telomere shortening has been previously described as one of the mechanisms of genomic gains and amplifications. Conclusions. Telomere shortening and hTERT overexpression were observed in resistant TNBC. Genomic profile of non-responding tumors was characterized by larger genomic gains. Telomere length, hTERT expression and genomic instability profiles may represent predictive biomarkers of response to neoadjuvant chemotherapy in TNBC. Citation Format: Mathilde Gay-bellile, Nina Radosevic-Robin, Eleonore Eymard-Pierre, Fabrice Kwiatkowski, Marie-Mélanie Dauplat, Maud Privat, Catherine Abrial, Patricia Combes, Gwendoline Soler, Yves-Jean Bignon, Jean-Marc Nabholtz, Philippe Vago, Frédérique Penault-Llorca, Andreï Tchirkov. Genomic instability and telomere characteristics as predictive biomarkers of therapeutic response in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2014-550

Discovery of Biomarkers Predictive of GSI Response in Triple-Negative Breast Cancer and Adenoid Cystic Carcinoma

Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant. NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.

Abstract 1819: Heterogeneity of triple-negative breast cancer response to neoadjuvant treatment: tumor EGFR, HER3 and MET expressions can provide clues for therapy tailoring

Abstract Background: We have recently demonstrated (AACR 2013, abstract 4669) that antibody (Ab)-based EGFR targeting, sequentially combined with cytotoxic agents, can achieve pathologic complete response in almost 50% of triple-negative breast cancer (TNBC) patients (pts). However, the pts left with a residual tumor (RT), need a modification of treatment approach. We investigated whether expression of EGFR and two of its major cooperators, HER3 and MET, can offer a simple and practical base for neoadjuvant therapy (NAT) improvement in TNBC. Methods: The study included pts with a post-NAT RT in 2 pilot clinical trials of anti-EGFR Abs in TNBC, conducted from 2009 to 2011 by our team. One trial combined panitumumab with standard fluorouracil-epirubicin-cyclophosphamide and docetaxel (P-FEC-T) while another applied cetuximab with docetaxel (C-T). EGFR, HER3 and MET were assessed by immunohistochemistry (IHC) on pre- and post-NAT BC tissue. Expressions were evaluated by Quick score (% of positive cells x stain intensity as 0, 0.5, 1, 1.5, 2, 2.5 or 3; score range 0-300). At least 30 points of difference qualified the post-NAT score as changed, compared to the pre-NAT level. Results: 44 pts were eligible (P-FEC-T: 26 pts, C-T: 18 pts). Using 150 (the middle of the score range) as cut-off, we separated the tumors in each study on pre-NAT EGFR-high (score ≥ 150) and EGFR-low (score < 150). The P-FEC-T trial had 13 EGFR-high tumors (median score 220) and 13 EGFR-low ones (median score 0). After NAT, the pre-NAT EGFR-high BC most frequently showed EGFR decrease (9/13), HER3 increase (6/10) and MET decrease (7/12). However, the pre-NAT EGFR-low tumors showed no change in EGFR (7/13) and HER3 (6/11) post-NAT, but most of them increased MET (7/11). The C-T trial had 7 EGFR-high (median score 210) and 11 EGFR-low cases (median score 70). The EGFR-high BC from that trial showed a similar pattern of score changes as the same group from the P-FEC-T trial: frequent EGFR decrease (4/7), HER3 increase (5/7) and MET decrease (4/7). The EGFR-low BC were more heterogeneous than in P-FEC-T: most frequently EGFR, HER3 and MET were increased (4/11, 5/11, 5/11 respectively) or unchanged (4/11, 4/11, 3/11 resp.). Conclusion: Our results indicate that: a) there is a TNBC subgroup with relatively high EGFR expression (score ≥ 150, median around 200) which resist to anti-EGFR treatment mainly by downregulating EGFR and upregulating HER3; b) TNBC with low EGFR expression (score < 150) are a heterogeneous subgroup, which will frequently upregulate HER3 and/or MET after exposure to the anti-EGFR agents. The IHC assessment of EGFR, HER3 and MET before and on-NAT can help improving TNBC treatment by introduction of currently available anti-EGFR/HER3, anti-HER3 and anti-MET agents. Citation Format: Nina Radosevic-Robin, Catherine Abrial, Marie-Melanie Dauplat, Matthieu Roche, Anne Cayre, Maud Privat, Fabrice Kwiatkowski, Nassera Chalabi, Marie-Ange Mouret-Reynier, Yves-Jean Bignon, Philippe Chollet, Jean-Marc Nabholtz, Frederique Penault-Llorca. Heterogeneity of triple-negative breast cancer response to neoadjuvant treatment: tumor EGFR, HER3 and MET expressions can provide clues for therapy tailoring. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1819. doi:10.1158/1538-7445.AM2014-1819

Emerging Prognostic and Predictive Biomarkers for Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is associated with a poor prognosis especially in the advanced setting and continues to pose a challenge for oncologists worldwide. Several biomarkers for this disease have been described. Recently the heterogeneity of TNBC has changed our perspective toward this disease. Differential response of TNBC to chemotherapy, poor prognosis of patients with residual disease following neoadjuvant chemotherapy, and the immunogenicity underlying TNBC make the management of this disease even more complex. Multiple signaling pathways and dynamic pathway programming are the hallmarks of TNBC. Newer trials for this disease therefore, need to be innovative and include biomarker enrichment and adaptive designs. This article highlights the emerging prognostic and predictive biomarkers for TNBC and discusses possible targets for future therapeutic strategies.

Analysis of circulating tumor cells for monitoring treatment response in triple-negative breast cancer.

e22017 Background: The University of Washington Center for Cancer Innovation is intensively studying triple-negative breast cancer (TNBC) at the individual patient level. We used a high-recovery rare cell analysis system to count and characterize circulating tumor cells (CTCs) from advanced TNBC patients treated with cis-platinum. Methods: Two patients have been studied to date. Blood was collected from patients prior to treatment and regularly thereafter. Density-based enrichment of blood cells was performed using the AccuCyte tube, float and collector system. Collected cells were processed and applied to microscopic slides. Fluorescently labeled antibodies to cytokeratin, CD45 and EpCAM, and a nuclear dye were applied to samples using an automated slide stainer. Slides were scanned on a digital microscope and candidate CTCs identified using image analysis software. CTCs were verified by appropriate morphology and expression of epithelial and nuclear stains without CD45 expression. Other antibodies used to characterize cells included Her2, EGFR, and Ki-67. Results: CTC levels before cis-platinum treatment in a patient with extensive bone-only metastatic disease were extremely high (1,500 cells/mL) and she was thrombocytopenic and short of breath. One week after treatment CTC levels spiked (5670 cells/mL) and high numbers of non-nucleated cells and fragments were observed. CTC counts dropped exponentially after the spike to 21 cells/mL after 3 months. CTC clusters and Ki-67 positive cells have decreased during therapy. Clinical response to treatment has paralleled the reduction in CTC levels, with elimination of oxygen therapy and normalization of platelet count. In the second patient, CTCs have decreased from approximately 4 cells/mL to 0.5 over 3 months with generalized reduction in size and number of positive lymph nodes as measured by imaging. Conclusions: Analysis of CTCs may provide a non-invasive measure of cancer progression/response and the molecular evolution of tumor cells in TNBC.

Association of increased tumor-infiltrating lymphocytes (TILs) with immunomodulatory (IM) triple-negative breast cancer (TNBC) subtype and response to neoadjuvant platinum-based therapy in PrECOG0105.

1000^ Background: Increased TILs are prognostic and predictive of therapy response in TNBC. PrECOG 0105, a neoadjuvant trial of carboplatin, gemcitabine and iniparib, enrolled 80 pts with clinical ...

Abstract P1-08-34: Is it possible to predict the efficacy of a combination of cetuximab plus docetaxel in patients with operable, triple negative breast cancer (TNBC)? Final biomarker results from a phase II neoadjuvant trial

Abstract Background: TNBC is a heterogenous group of tumors for some of which the Epithelial Growth Factor Receptor pathway (EGFR) may play an important role. We evaluated the efficacy and toxicity of an anti-EGFR antibody (cetuximab) combined with docetaxel, which were given to the TNBC patients (pts) in the neoadjuvant setting. A biomarker analysis accompanied this trial, aiming to identify predictors of response. Methods: 35 patients with stage II-IIIA TN breast disease were prospectively included in this multicentre pilot study. Systemic therapy (ST) consisted of 6 cycles of docetaxel (100 mg/m2) each 3 weeks, in combination with weekly cetuximab (first dose 400mg/m2 then 250 mg/m2/week) for 6 cycles. All patients underwent surgery at completion of ST. Patient characteristics : mean age 48 [28-67]; TNM: T1: 3%, T2: 73%, T3: 24%; N0: 61%, N1-N2: 39%; mean tumor size 40 mm [15-100]; SBR: grade III: 73%, grade II: 27%. The median number of cycles was for docetaxel 6 [1-6] and for cetuximab 15 [1-18]. Pathological complete response (pCR) rate was 24% according to the Chevallier and Sataloff classifications; 28% if we consider breast pCR only. Overall clinical response rate was 57% (22% CR). Conservative surgery was performed in 75% of cases. The main side effect was skin toxicity: grade II: 39%, grade III: 36%, grade IV: 3%. Other side effects were: neutropenia grade IV: 12.7%, febrile neutropenia: 1.3%, hand-foot syndrome grade III: 3%, grade II: 3%, ungueal toxicity grade III: 3%, grade II: 33%. Paraffin-embedded and frozen samples were systematically collected before and after ST for biomarker studies. Germinal BRCA1 mutations and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki67, p53 and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TIL) were evaluated by immunohistochemistry. Results: The biomarker analysis was interpretable on the samples from 21 pts (3 pCR and 18 non-pCR). We applied the ROC curve to identify the best cut-off value for Ki67, EGFR, MET, cytokeratin 5/6 and 8/18, p53, ALDH1, PTEN, P-cadherin and the FOXP3+ or CD8+ TIL counts. None of these biomarkers was predictive of pCR except for the CD8+/FOXP3+ TIL count ratio. pCR rate was higher in the pts with the ratio equal or higher than 2.75 than in the others (43% versus 0%, p = 0.047). BRCA1 mutations were detected in 16% of pts. PI3K and EGFR somatic mutations were observed in 1 and 3 patients, respectively. The presence of the mutations was not predictive of pCR. Conclusion: Similarly to the previously reported trial by our group (SABCS 2012, abstract 1081), the immune component of the tumor microenvironment plays a very important role in the TNBC response to cytotoxic therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-34.

Dynamic contrast-enhanced MRI-based biomarkers of therapeutic response in triple-negative breast cancer

To predict the response of breast cancer patients to neoadjuvant chemotherapy (NAC) using features derived from dynamic contrast-enhanced (DCE) MRI. 60 patients with triple-negative early-stage breast cancer receiving NAC were evaluated. Features assessed included clinical data, patterns of tumor response to treatment determined by DCE-MRI, MRI breast imaging-reporting and data system descriptors, and quantitative lesion kinetic texture derived from the gray-level co-occurrence matrix (GLCM). All features except for patterns of response were derived before chemotherapy; GLCM features were determined before and after chemotherapy. Treatment response was defined by the presence of residual invasive tumor and/or positive lymph nodes after chemotherapy. Statistical modeling was performed using Lasso logistic regression. Pre-chemotherapy imaging features predicted all measures of response except for residual tumor. Feature sets varied in effectiveness at predicting different definitions of treatment response, but in general, pre-chemotherapy imaging features were able to predict pathological complete response with area under the curve (AUC)=0.68, residual lymph node metastases with AUC=0.84 and residual tumor with lymph node metastases with AUC=0.83. Imaging features assessed after chemotherapy yielded significantly improved model performance over those assessed before chemotherapy for predicting residual tumor, but no other outcomes. DCE-MRI features can be used to predict whether triple-negative breast cancer patients will respond to NAC. Models such as the ones presented could help to identify patients not likely to respond to treatment and to direct them towards alternative therapies.

Roles of p53 and p16 in triple-negative breast cancer

SUMMARY p53 and p16 are both tumor suppressors. This article summarizes the current investigation status on their roles and interaction in triple-negative breast cancer (TNBC) to explore their potential as prognostic, predictive and therapeutic targets. There is significant evidence implicating that p53 mutation as a driver plays an important role in tumorigenesis and progression of TNBC. p53 mutations are more frequently observed in TNBCs compared with ER-positive breast cancers. The presence of a p53 mutation is associated with poorer prognosis in TNBC patients. The status of p53 protein expression divides TNBCs into two biologically distinct subgroups (p53-positive vs p53-negative); p53-positive tumors have more aggressive behavior. p53 has clinical prognostic utility and is a promising target candidate for directed therapy for TNBC patients. p16 appears to play a significant role in tumorigenesis of TNBC through the Rb/p16 dysfunction pathway. A high level of p16 protein expression is associated with aggressive behavior and tumor proliferation of TNBC. The status of p16 expression may have potential predictive value for TNBC response to chemotherapy; further investigation is needed to support the proposal. p16 expression has been strongly correlated with p53 expression in TNBC, suggesting interactions between the two mutations in tumorigenesis. It has been proposed that the two tumor suppressors appear to be involved in early events of TNBC formation with subsequent interplay resulting in their mutual inactivation and, ultimately, leading to a proliferative environment.

Next-generation sequencing to find predictors for chemotherapy response in triple-negative breast cancer (TNBC).

1010 Background: In TNBC initial response to chemotherapy is often favorable, but relapse and chemotherapy resistance frequently occur in advanced disease. Hence there is an urgent need for targeted treatments in this breast cancer subtype. Methods: To identify biomarkers of chemotherapy resistance and putative directed treatment targets, we performed next generation sequencing (NGS) of 2,000 genes implicated in oncogenesis. DNA from 31 pre-treatment biopsies and matched normal blood was sequenced. Biopsies were derived from patients scheduled to receive neoadjuvant chemotherapy with doxorubicin/cyclophosphamide. For the analysis, the tumors were divided in responders and non-responders, depending on whether or not a pathological complete remission (pCR) was achieved. Two definitions of pCR were employed: either the complete absence of infiltrating tumor cells in the breast (n=18) or a pCR of both the breast and lymph nodes (n=15). Tumors with partial or no responses were grouped in the non-responder category. Results: As a positive control, we verified that all patients with a known germline BRCA1 mutation (n=8) could be detected by the NGS analysis. In further analyses, we focused only on somatic mutations. Overall, the mutation rate was slightly higher in the non-responders (per tumor, average=12, range=[3-25]) than in the responders (average=8, range=[3-19]) (p=0.17). The analysis of individual genes did not reveal significant differences between responders and non-responders. However, pathway analysis showed that mutations in phosphatidylinositol signaling (e.g., PIK3CA, CALML5) were significantly more frequent in the non-responders, with mutations present in 10/13 non-responders and 2/18 responders (adjusted p=0.013). The chemokine and integrin signaling pathways also revealed a significantly higher mutation rate in the non-responders. Conclusions: Mutations in genes of the phosphatidylinositol pathway occur frequently in TNBCs that do not achieve a pCR on a neoadjuvant chemotherapy regimen consisting of doxorubicin and cyclophosphamide. After validation, alternative chemotherapy regimens and targeted agents should be investigated for these tumors.

Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple-negative breast cancer (TNBC): The immune and IGFR pathways.

1058 Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC definition by immunohistochemistry (IHC). In a phase II neoadjuvant clinical trial targeting EGFR in TNBC, we investigated various biomarkers to better identify an EGFR-sensitive population for potential further regimen development. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm (20-120); invasive ductal carcinoma: 96.7%; SBR grade III: 71.7%; complete pathological response (pCR) rate: 55.3% and 46.8% (according to Sataloff’s and Chevallier’s classifications, respectively). Paraffin-embedded and frozen tumor samples were collected before and after ST for biologic studies. Germinal BRCA1 mutations, and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs) were evaluated by IHC. Results: High CD8+ TILs was response-predictive (p=3.4.10-6). Tumor FOXP3 expression and high FOXP3 TILs tended to be predictive. High IGF-1R expressors responded better than low expressors (p = 0.028). Comparison of EGFR, IGF-1R and Her3 in biopsies versus surgical samples showed reduced EGFR levels in non-responders (p = 0.037), while Her3 (p = 0.049) and IGF-1R (p = 0.08) increased. Sequencing revealed BRCA1 mutations in 10% of pts. No difference of response (pCR) was observed between mutated patients and others. Somatic mutations of PI3K were observed in 6 pts. No mutations were observed in BRAF, KRAS, or EGFR. Conclusions: The CD8+ TIL count seems to predict the response to panitumumab. Tumor FOXP3 expression and high FOXP3 TILs also tended to be predictive. Tumor levels of IGF-1R seem to play a determinant role in TNBC response to anti-EGFR antibodies, in concordance with our observations in a head-and-neck cancer cohort. Clinical trial information: NCT00933517.

Abstract 4669: Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways.

Abstract Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC definition by immunohistochemistry (IHC) (Nielsen, 2004). In a phase II neoadjuvant clinical trial targeting EGFR in TNBC, we investigated various biomarkers to better identify an EGFR-sensitive population for potential further regimen development. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm (20-120); invasive ductal carcinoma: 96.7%; SBR grade III: 71.7%; complete pathological response (pCR) rate: 46.8% (Chevallier's classification). Paraffin-embedded and frozen tumor samples were collected before and after ST for biologic studies. Germinal BRCA1 mutations, and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs) were evaluated by IHC. Biopsies and surgical samples were analysed using Affimetrix arrays. Results : By univariate analysis, high CD8+ TILs was response-predictive (pCR rates: CD8 TILs: 84% high vs 10% low; p=3.4.10−6). Tumor FOXP3 expression and high FOXP3 TILs tended to be predictive (pCR rates: tumor FOXP3: 77% positive vs 36% negative, p=0.07; FOXP3 TILs: 66% high vs 37% low, p= 0.08). High IGF-1R expressors responded better than low expressors (80% vs 33%, respectively, p=0.028). As could be expected, a positive correlation between tumor FOXP3 expression and FOXP3 TILs was found (p = 2.7.10−4, r = 0.56). Surprisingly, a positive correlation was found between FOXP3 TILs and CD8+ TILs (p = 2.10−5, r = 0.59) and between tumor FOXP3 and CD8+ TILs (p = 6.3.10−3, r = 0.43). Comparison of EGFR, IGF-1R and Her3 in biopsies versus surgical samples showed reduced EGFR levels in non-responders (p = 0.037), while Her3 (p = 0.049) and IGF-1R (p = 0.08) increased. Sequencing revealed BRCA1 mutations in 10% of pts. No difference of response (pCR) was observed between mutated patients and others (p=0.91). Somatic mutations of PI3K were observed in 6 pts. No mutations were observed in BRAF, KRAS, or EGFR. Analysis of Affymetrix arrays for gene expression is underway and will be presented. Conclusions : Interestingly, the CD8+ TIL count seems to predict the response to panitumumab. Tumor FOXP3 expression and high FOXP3 TILs also tended to be predictive. Tumor levels of IGF-1R seem to play a determinant role in TNBC response to anti-EGFR antibodies, in concordance with our observations in a head-and-neck cancer cohort (Clin Canc Res 2012). Confirmatory and mechanistic studies of those biomarkers are warranted. Citation Format: Frederique Penault-llorca, Catherine Abrial, Marie-Melanie Dauplat, Maud Privat, Nancy Uhrhammer, Alexis Desrichard, Yannick Bidet, Nina Radosevic-Robin, Anne Cayre, Cecile Aube, Fabrice Kwiatkowski, Nassera Chalabi, Yves-Jean Bignon, Philippe Chollet, Jean-Marc Nabholtz. Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4669. doi:10.1158/1538-7445.AM2013-4669

Abstract 506: Targeting Y-box binding protein-1 (YB-1) overcomes drug resistance in triple-negative breast cancer.

Abstract Despite advances in treating breast cancer, disease recurrence rates remain high and secondary tumors are often resistant to chemotherapy and incurable. Currently, the treatment for triple-negative breast cancer (TNBC) relies upon conventional chemotherapeutics as there are no targeted therapies available. Although these tumors initially respond well, they paradoxically have the highest relapse rates. Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor abundantly expressed in TNBC (∼70% of patients). It is activated predominantly by phosphorylation via p90 ribosomal S6 kinase (RSK). Once activated it up-regulates the tumor-initiating-cell (TIC) marker CD44 and induces a TIC phenotype[1]. Due to their inherent drug-resistance, TICs survive chemotherapy and go on to drive relapse[2]. We recently identified RSK2 is critical to the survival of TNBC[3]. Inhibiting RSK2 blocks activation of YB-1 and induces apoptosis in TNBC, including CD44+/CD24- cells. Interestingly, inducing YB-1 is sufficient for transformation of human mammary epithelial cells into TNBC. Moreover, P-YB-1S102 is associated with poor overall survival (P<0.001) and relapse (P<0.001) in a cohort of 1057 patients with invasive breast cancer. Taken together, these data indicate YB-1 is important both in the genesis and maintenance of TNBC and implicate it as a key mediator of drug-resistance and relapse within this subtype. Residual cells after paclitaxel or epirubicin have increased P-RSKS221/7, P-YB-1S102 and CD44 and retain mammosphere-forming capability. Cells expressing high P-YB-1S102 exclusively proliferate in the presence of these chemotherapies. Inhibiting YB-1 suppresses growth and induces apoptosis in residual cells and in CD44+/CD24–sorted cells. Pre-emptive knockdown of YB-1 prevents induction of CD44 and sensitizes cells to treatment with paclitaxel. Moreover, blocking RSK/YB-1 signaling suppresses growth and induces cell death in primary TNBC cells (x43) derived from a patient that eventually relapsed. To investigate the role of RSK/YB-1 signaling in bona fide drug-resistant cells, we created paclitaxel-resistant SUM149 cells (SUM149-PTXR) which are cultured in 2 nM paclitaxel (PTX); a concentration that kills >90% of paclitaxel-naive SUM149’s (SUM149-DMSO) by 72 hrs. These cells express increased P-RSKS221/7 and P-YB-1S102 without alterations in total protein levels. Despite their resistance to paclitaxel, these cells remain sensitive to RSK/YB-1 signaling inhibition. We are currently comparing the response of TNBC either with or without YB-1 to paclitaxel in an in vivo orthotopic xenograft model. Collectively, these data indicate that inhibiting RSK/YB-1 signaling can overcome drug resistance and potentially reduce relapse in TNBC. 1.To K, Fotovati A et al. (2010) 2.Li X, Lewis MT et al. (2008) 3.Stratford A, Reipas K et al. (2012) Citation Format: Kristen Reipas, Anna Stratford, Arezoo Astanehe, Kaiji Hu, Sandra Dunn. Targeting Y-box binding protein-1 (YB-1) overcomes drug resistance in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2013-506

Abstract A103: Investigation of novel therapeutic and diagnostic strategies for the management of triple-negative breast cancer

Abstract Approximately 10-24% of invasive breast cancers (BCs) are characterized as ‘triple-negative’ breast cancer (TNBC), a specific BC subtype that lacks expression of the estrogen and progesterone receptors, as well as that of Her2 over expression. TNBC is one of the most clinically aggressive forms of BC, exhibiting elevated rates of metastasis, disease recurrence, and poor overall survival. Metastatic TNBCs rapidly progress through multiple lines of chemotherapy and currently there are no targeted therapies approved for the treatment of TNBC highlighting the importance of investigating novel druggable targets for this highly aggressive disease. Previously, we demonstrated that the heterologous expression of a constitutively active (CST) c-Abl, a non-receptor tyrosine kinase, mutant morphologically and phenotypically reverted late-stage murine BC cells both in vitro and in vivo. We further showed that CST-Abl enhanced cytostatic activity in part by elevating p21 expression levels thereby preventing TNBC tumorigenesis when implanted into the mammary fat pads of mice. Additionally, we found c-Abl expression to associate with invasive BC patient's response to docetaxel treatment through Oncomine in silico data. Recently the natural product, Securinine, has been shown to mediate an anti-cancer response through modulating p53 family member expression in leukemic and colorectal carcinoma cells. We have shown a strong induction of c-Abl expression in response to Securinine administration in highly aggressive late stage murine BC cells suggestive of its involvement in the mechanism of Securinine-mediated cell death. Our studies tested the hypothesis that c-Abl is a biomarker for a TNBC response to docetaxel and furthermore targeted activation of c-Abl utilizing DPH, a novel pharmacological allosteric c-Abl activator, will inhibit the tumorigenicity of TNBCs in vitro. We additionally hypothesized that Securinine would be an effective novel therapy for the treatment of TNBCs. We found DPH to inhibit the proliferation of highly aggressive 4T1 murine metastatic TNBC cells in a 3D-organotypic culture model. In addition, DPH suppressed the up-regulation of β3-integrin, a robust marker of epithelial to mesenchymal transitions (EMT), in response to TGF-β, in normal murine mammary gland (NMuMG) cells. Through phalloidin staining, DPH was shown to partially blunt a morphological EMT, induced by TGF-β, in NMuMGs and enhanced a basal epithelial morphology that was reminiscent of CST-Abl activity. Furthermore, we have recapitulated that DPH is specific to the kinase activity of c-Abl. Utilizing cell lines expressing a lentiviral shRNA construct targeting c-Abl in dormant and metastatic D2 hyperaveolar nodule (D2.HAN) cells, we have shown through clonogenic assays and 3D-organotypic culturing models, that the loss of c-Abl expression directly correlates with a blunted response to docetaxel. Finally, Securinine was found to strongly induce p73 expression in both dormant and metastatic TNBCs and suppressed their proliferation in 3D-culture, but did not have an effect on their normal counterparts. These innovative findings suggest that c-Abl expression associates with a TNBC response to docetaxel and Securinine and that its targeted activation could lead to an effective treatment for TNBCs. Citation Format: Chevaun Danielle Morrison, William Schiemann. Investigation of novel therapeutic and diagnostic strategies for the management of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A103.

A DNA Repair BRCA1 Estrogen Receptor and Targeted Therapy in Breast Cancer

BRCA1 is a key mediator of DNA repair pathways and participates in the maintenance of the genomic integrity of cells. The control of DNA damage repair mechanisms by BRCA1 is of great interest since molecular defects in this pathway may reflect a predictive value in terms of a cell’s sensitivity to DNA damaging agents or anticancer drugs. BRCA1 has been found to exhibit a hormone-dependent pattern of expression in breast cells. Wild-type BRCA1 is required for the inhibition of the growth of breast tumor cells in response to the pure steroidal ERα antagonist fulvestrant. Also a loss of BRCA1-mediated transcriptional activation of ERα expression results in increased resistance to ERα antagonists. Platinum-based drugs, poly(ADP-ribose) polymerase (PARP) inhibitors, and their combination are currently included in chemotherapy regimens for breast cancer. Preclinical and clinical studies in a BRCA1-defective setting have recently indicated a rationale for the use of these compounds against hereditary breast cancers. Initial findings indicate that neoadjuvant use of cisplatin results in high rates of complete pathological response in patients with breast cancer who have BRCA1 mutations. Cisplatin produces a better response in triple-negative breast cancer (TNBC) than in non-TNBC diseases in both the neoadjuvant and adjuvant settings. This implies that TNBC cells may harbor a dysfunctional BRCA1 repair pathway.

Thymosin beta 15A (TMSB15A) is a predictor of chemotherapy response in triple-negative breast cancer

Background:Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed.Methods:Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts (n=86 and n=55). Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n=212, and GeparQuattro, n=383).Results:A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts (P<0.0001; P<0.0042). In the GeparTrio FFPE training cohort, a significant linear correlation between TMSB15A expression and pCR was apparent in triple-negative breast cancer (TNBC) (n=61, P=0.040). A cutoff point was then defined that divided TNBC into a low and a high expression group (pCR rate 16.0% vs 47.2%). Both linear correlation of TMSB15A mRNA levels (P=0.017) and the pre-defined cutoff point were validated in 134 TNBC from GeparQuattro (pCR rate 36.8% vs 17.0%, P=0.020). No significant predictive capacity was observed in luminal carcinomas from GeparTrio and GeparQuattro.Conclusion:In TNBC, TMSB15A gene expression analysis might help to select patients with a high chance for pCR after NACT.

Imaging of triple-negative breast cancer

Although triple-negative breast cancer (TNBC) has been studied extensively in the oncology and pathology literature, there are few reports on imaging features. Emerging data suggest that imaging features of TNBC are substantially different from other primary breast cancer immunotypes. In this work, we reviewed multi-modality imaging features of primary TNBC with emphasis on the appropriate niche for each technology in diagnosis, staging and management. TNBC lacks the typical suspicious mammographic features of breast cancer; namely irregular mass shape, spiculated margins and associated suspicious calcifications. Therefore, mammography alone is usually a sub-optimal tool for its initial diagnostic evaluation. Ultrasound has a much higher sensitivity, although its diagnostic capability may be impaired by associated benign features encountered in 21%-41% of TNBC lesions. Magnetic resonance imaging (MRI) consistently demonstrates the presence of all TNBC with a higher level of accuracy compared with other tumour sub-types, and provides a reliable baseline for neoadjuvant chemotherapy (NAC) follow-up. Preliminary studies also suggest that MRI may predict complete NAC response in TNBC more sensitively than other methods. [(18)F]2-fluoro-2-deoxy-D-glucose positron emission tomography ((18)F-FDG-PET) has a higher sensitivity for TNBC than estrogen-positive and human epidermal growth factor receptor 2-positive tumours, and axillary lymph node metastases are detected with more accuracy compared with other tumour molecular sub-types, although low FDG uptake of breast cancers in general may limit the routine clinical use of (18)F-FDG-PET in the initial diagnosis of TNBC patients and NAC follow-up.