Absence Of HER2 Amplification Research Articles

Evaluating antitumor activity and response determinants to trastuzumab deruxtecan in pediatric solid tumors.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a >30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

Abstract PO2-18-08: Identification of novel genetic/therapeutic vulnerabilities in breast cancer

Abstract Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women worldwide. Triple negative breast cancer (TNBC) is an aggressive subtype that is defined by the lack of expression of estrogen and progesterone receptors in the absence of HER2 amplification or overexpression. TNBC represents 15-20% of all breast cancers and occurs more frequently in young premenopausal women. TNBC often ends with a poor clinical outcome due to high histological grade, rapid recurrence in many patients combined with few treatment options. These realities highlight the urgent need to identify novel therapeutics vulnerabilities that can be leveraged to improve TNBC patient outcomes. Methods: We utilized the publicly available DepMap and Kaplan Meier datasets to identify essential genes that were unique to TNBC cells and whose expression levels were associated with worse patient outcomes. siRNA based approaches were used to confirm the relevance of top hits in mediating cancer cell phenotypes including viability, cell-cycle profiles and induction of apoptosis among others. A novel drug currently being developed was identified for the top hit in these studies and was subsequently employed in multiple in vitro approaches to characterize the mechanisms of action in TNBC as well as initial efficacy studies using clinically relevant ex vivo patient derived models. Results: Using the DepMap database, we interrogated genome-wide sgRNA knockout screens conducted in over 40 breast cancer cell lines to identify specific genes whose expression was required for cell viability in TNBC cells, but not other breast cancer cell lines. This endeavor identified 6 genes (CTPS1, HUS1, PRKRA, RAD1, RAD9A and RHOA) that were preferentially essential in TNBC cells. Further profiling of these genes revealed that CTPS1 expression is higher in cancer relative to normal tissue and that its expression is further upregulated in chemotherapy- and PARPi-resistant models. Knockdown of CTPS1 in TNBC cells was shown to decrease cell proliferation and viability with no effects observed in estrogen receptor+ cell lines. Using a first-in-class, highly selective and orally bioavailable CTPS1 inhibitor (STP938) developed by Step-Pharma, we identified IC50s in the low nM range for many TNBC cell lines, including cell models that are resistant to existing standard-of-care therapies. Inhibition of CTPS1 was shown to arrest cells in S phase. Conclusions: Our studies have identified CTPS1 as an essential gene in TNBC. CTPS1 encodes an enzyme responsible for the catalytic conversion of UTP (uridine triphosphate) to CTP (cytidine triphosphate), an essential step in the biosynthesis of nucleic acids. These findings are timely in light of the recent and ongoing development of the first-in-class CTPS1 inhibitor, STP938, for which first-in-human studies are underway for refractory B- and T-cell lymphomas. Based on our initial studies using this drug, we provide pre-clinical evidence of its efficacy in multiple models of primary and advanced TNBC. Efforts are currently underway to move this work toward a clinical trial for TNBC patients. Citation Format: Xiyin Wang, Michael Emch, Lauren Voll, Rebecca Russell, Esther Rodman, Philip Beer, John Hawse. Identification of novel genetic/therapeutic vulnerabilities in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-08.

Prognostic value of tumor-infiltrating lymphocytes (TILS) and their association with clinicopathological features in breast cancer: A retrospective study involving 53 cases

ObjectiveQuantitative analysis of tumor-infiltrating lymphocytes (TILs) is currently considered as a prognostic factor in several malignant tumors. The aim of our study was to assess the prognostic value of TILs in breast cancers and its association with other clinicopathological prognostic factors in non-specific type (NST) breast carcinoma in Tunisian population. MethodsRetrospective study included 53 women with NST breast carcinoma. The data were collected over a period of 13 months with a follow-up of 40 months for all the patients. The TILs were evaluated according to the 2014 recommendations of the international working group on TILs. ResultsTILs level was between 3% and 60% with mean of 21%. Ten patients had lymphocyte predominant breast cancer (LPBC). Statistical analysis had shown that the TILs level ≤50% was associated with the presence of vascular emboli and the absence of HER2 amplification. Inflammatory-type carcinoma and HER2 amplification significantly worsened OS. Presence of vascular emboli, lymph node metastases, inflammatory type of carcinoma, TILs ≤50%, and absence of estrogen receptors (ER) were associated with reduced RFS. In multivariate analysis, the presence of vascular emboli was an independent factor for OS. TILs ≤50%, inflammatory type of carcinoma and presence of vascular emboli were independent risk factors for RFS. ConclusionThis Tunisian pilot study showed higher level of TILs in NST breast carcinomas is associated with improved RFS. The therapeutic implications will benefit from multiple research studies including ours on the predictive value of TILs for neoadjuvant or adjuvant treatment.

Identification of Hub Genes and Pathways of Triple Negative Breast Cancer by Expression Profiles Analysis.

PurposeTriple negative breast cancer (TNBC) is an intrinsic subtype of breast cancer with a poor prognosis, characterized by a lack of ER and PR expression and the absence of HER2 amplification. The aim of this study is to characterize hub genes (key genes in the molecular interaction network) expression in TNBC, which may serve as prognostic predictors for TNBC treatment.MethodsFour transcriptome microarray datasets GSE27447, GSE39004, GSE43358 and GSE45827 were obtained from the Gene Expression Omnibus (GEO) database, and R package limma and RobustRankAggreg were employed to identify common differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted by DAVID and KOBAS database. Thereafter, protein–protein interaction (PPI) network was constructed according to STRING online database. Functional modules and hub genes were screened by MCODE and cytohubba plug-ins, and the Cancer Genome Atlas (TCGA) survival analysis and qRT-PCR were utilized to validate the expression of these hub genes on TNBC.ResultsA total of 134 DEGs were identified by differential expression analysis, consisting of 88 up- and 46 down-regulated genes. GO and KEGG analyses showed that the terms and pathways enriched were mainly associated with cell adhesion, tumorigenesis and cellular immunity. From the PPI network, we identified six hub genes, including CD3D, CD3E, CD3G, FYN, GRAP2 and ITK. Survival analysis and the qRT-PCR results confirmed the robustness of the identified hub genes.ConclusionThis study provides a new insight into the understanding of the molecular mechanisms associated with TNBC and suggested that the hub genes may serve as prognostic predictors.

Abstract P6-03-16: Role of autophagy in CD24-mediated TNBC drug resistance

Abstract Background: Triple-negative breast cancer (TNBC) is defined by lack of estrogen and progesterone receptors and absence of HER2 amplification. Compared with other breast cancer subtypes, TNBC is more aggressive with more frequent metastasis especially involving visceral and central nervous system. Currently, the only available standard adjuvant treatment is chemotherapy. However, mortality rate remains high despite chemotherapy. To improve treatment, it is crucial to develop novel strategies to predict and overcome drug resistance. Our pervious study showed that CD44/CD24 expressions were associated with drug sensitivity and CD44+/CD24+ TNBC was shown to be resistant to docetaxel. In this study, we aimed to investigate the regulatory mechanism involved in CD24-mediated taxane resistance in TNBC. Methods: In this study, we used flow cytometry to determine the CD24 expressions in 7 TNBC cell lines. Western blot was used to demonstrate the levels of CD24 and LC3B-II (a marker for cellular autophagy activity) expressions in TNBC cell lines. CD24 expressions in TNBC cell lines were manipulated by target-specific shRNA and gene overexpression approaches. Cell apoptosis assay was used to determine docetaxel drug sensitivity in cells with manipulated CD24 expression. Using the same assay, autophagy inhibitors were used to further evaluate the involvement of autophagy in CD24-mediated drug resistance. To investigate the possible regulatory mechanism, autophagy related signaling pathway Akt/mTOR was studied in CD24 modified TNBC cells. Results: In this study, the relationship between CD24 expression and autophagy activities in TNBC was evaluated. LC3B-II protein expression was used to determinate autophagy activities. We have shown that TNBC cell lines with high CD24 expressions are associated with increased LC3B-II expression. Suppression of autophagy in CD24 high TNBC cells improves drug sensitivities toward docetaxel. We further demonstrated that knocking down CD24 in TNBC cells suppressed LC3B-II expression and improved docetaxel sensitivity. In addition, gene overexpression of CD24was found to enhance docetaxel resistance in TNBC cells. Our study has also shown that docetaxel increases the activation of Akt/mTOR signaling pathway in drug-resistant TNBC cells. Knocking down CD24 increased Akt/mTOR activation, and decreased the expression of PP2A and LC3B-II in TNBC. Conclusions: CD24 expression is associated with docetaxel sensitivity and autophagy activity in TNBC cells. Drug sensitivity of CD44+CD24+/high TNBC cells is regulated through autophagy related signaling pathway. Taken together, CD24 mediated TNBC drug resistance is at least partially through autophagy activity. Citation Format: Lihong Huo, Xinyu Deng, Xiang Huang, Helena R Chang. Role of autophagy in CD24-mediated TNBC drug resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-16.

Fam-] trastuzumab deruxtecan, antitumor activity is dependent on HER2 expression level rather than on HER2 amplification.

Therapies targeted to human epidermal growth factor receptor 2 (HER2) have proven effective against tumors positive for HER2 amplification, but there is an unmet clinical need for the treatment of tumors that express HER2 protein in the absence of HER2 amplification. [fam-] trastuzumab deruxtecan (DS-8201a) is a novel antibody-drug conjugate composed of the anti-HER2 antibody and the topoisomerase I inhibitor, an exatecan derivative. It has shown efficacy against tumors that express HER2 and is currently under evaluation in clinical trials. We here show that the antitumor activity of [fam-] trastuzumab deruxtecan is dependent on the expression level of HER2 protein in colorectal cancer (CRC) cell lines negative for HER2 amplification. We established isogenic CRC cell lines that express various levels of HER2 protein in the absence of HER2 amplification, and we found that cells that express HER2 at a high level were sensitive to [fam-] trastuzumab deruxtecan but not to conventional HER2-targeted therapies. Furthermore, [fam-] trastuzumab deruxtecan manifested a bystander killing effect both in vitro and in vivo, with cells essentially negative for HER2 expression also being killed in the presence of HER2-expressing cells, an effect that has the potential to overcome heterogeneity of HER2 expression in CRC tumors. Our results thus suggest that [fam-] trastuzumab deruxtecan warrants further study as a potential treatment for CRC tumors that express HER2 protein in the absence of HER2 amplification.

Investigating the use of Chk1 Inhibitors for Triple‐Negative Breast Cancer

Triple‐negative breast cancer (TNBC) accounts for 15–20% of all diagnosed breast cancers. It is characterized by lack of expression of estrogen receptor and progesterone receptor and an absence of HER2 amplification. TNBC is aggressive, highly metastatic, and ~50% of patients do not respond to first line chemotherapy. TNBC frequently harbors mutations in TP53, suggesting that therapies that exploit this vulnerability may be an effective treatment option. In particular, p53‐deficient cells are hypothesized to be highly sensitive to inhibitors of checkpoint kinase 1 (Chk1) due to checkpoint abrogation. We evaluated the efficacy of two clinically relevant Chk1 inhibitors, LY2606368 (LY) and SCH900776 (SCH), to elucidate the reasons for differing cellular response to the two drugs and evaluate the potential for synergy of each drug in combination with different DNA damaging agents. We hypothesized that both agents would synergize with gemcitabine regardless of p53 status, but would only synergize in combination with cisplatin in p53‐deficient cells. We tested the effects of LY and SCH alone and in combination with cisplatin or gemcitabine on cell viability, cell proliferation, and relevant signaling pathways in a TNBC cell line. Our data suggest that the differential cellular response to the two drugs is mainly due to a difference in potency. LY, but not SCH, preferentially sensitizes p53‐deficient cells to cisplatin. Notably, both LY and SCH synergize with gemcitabine regardless of p53 status, suggesting that Chk1 inhibitors + gemcitabine might be effective in both p53‐proficient and p53‐deficient backgrounds. Further studies are necessary to more fully evaluate the synergistic potential between cisplatin and LY or SCH, to characterize the sensitivity of cells carrying mutant p53, and to verify these results in vivo.Support or Funding InformationKomen Foundation; Cancer Prevention & Research Institute of Texas

Abstract SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance

Abstract SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance

Investigating the Use of Targeted Therapies for Triple‐Negative Breast Cancer

Triple‐negative breast cancer (TNBC) accounts for 15‐20% of all diagnosed breast cancers. It is characterized by lack of expression of estrogen receptor and progesterone receptor and an absence of HER2 amplification. TNBC is aggressive, highly metastatic, and resistant to current chemotherapies. TNBC frequently harbors mutations in TP53, suggesting that therapies that exploit this vulnerability may be an effective treatment option. In particular, p53‐deficient cells are hypothesized to be highly sensitive to inhibitors of checkpoint kinase 1 (Chk1) due to checkpoint abrogation. We have evaluated the use of mammosphere cultures as an in vitro system for testing the sensitivity of TNBC tumor cells to chemotherapy and targeted therapy. We hypothesized that mammospheres would display higher levels of chemo‐resistance than adherent cells, due to their ability to enrich for tumor initiating cells (TICs), which have been shown to display chemo‐resistance. However, we reasoned that molecularly targeted therapies should maintain their effect in TICs, since the pathways that are targeted would remain vulnerable. To test these hypotheses, we evaluated the effect of cisplatin (DNA damaging agent, chemotherapy) and LY2606368 (inhibitor of Chk1, targeted therapy) on the viability of TNBC cells. Our data suggest that the TIC population of a tumor may retain its sensitivity to targeted therapy despite resistance to cytotoxic chemotherapy. Further studies are necessary to determine if targeted therapy can impair TIC self‐renewal capacity, to evaluate combination therapies, and to verify these results in vivo.Funding: Komen Foundation; Cancer Prevention & Research Institute of Texas

Positive expression of insulin-like growth factor-1 receptor is associated with a positive hormone receptor status and a favorable prognosis in breast cancer.

PurposeInsulin-like growth factor 1 receptor (IGF-1R) is commonly expressed in primary breast cancers. Understanding the role of IGF-1R signaling in the different subtypes of breast cancer is important because each subtype has a different outcome and requires different treatment modalities. However, the precise biological significance of IGF-1R expression in cancer cells is still unclear. In this study, we examined the expression of IGF-1R in the different molecular subtypes of breast cancer. The effects of IGF-1R expression on the survival rates and outcomes of breast cancer were also examined.MethodsIGF-1R expression was evaluated immunohistochemically in tissue microarray blocks constructed from 1,198 invasive breast cancer samples collected from six medical institutions. IGF-1R expression was interpreted according to the human epidermal growth factor receptor 2 (HER2)/neu immunohistochemistry scoring system. Scores of 2+ and 3+ were considered positive.ResultsPositive IGF-1R expression was observed in 65.4% of invasive breast cancer samples. IGF-1R expression was detected in all cancer subtypes (luminal A, 84.4%; luminal B, 75.9%; HER2, 21.2%; triple-negative, 46.6%) and was found to be associated with a positive hormone receptor status and the absence of HER2 amplification (p<0.001). Positive IGF-1R expression was significantly associated with high survival rates (p=0.014). However, a multivariate analysis revealed that the expression levels of IGF-1R did not achieve statistical significance. In the triple-negative cancer subtype, IGF-1R expression was found to be associated with a lower disease-free survival rate (p=0.031).ConclusionPositive IGF-1R expression is associated with a favorable prognosis in breast cancer. IGF-1R is frequently expressed in the luminal A/B subtypes of breast cancer, and its expression is related to the hormone receptor status.

Abstract 912: Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease.

Abstract Effective therapies for women with low-HER2 expressing breast cancer remain a significant unmet clinical need. We have detected significant phosphorylated-HER and HER2:HER3 dimer expression in clinical breast cancer samples without HER2 amplification suggesting that HER signaling may play a role in these tumors. It is our hypothesis that ligand-driven signaling is the major route of HER family activation in the absence of HER2 amplification. AZD8931 is an orally bioavailable and highly selective small molecule inhibitor of EGFR, HER2 and HER3 signaling, which shows greatest potency when HER signaling is ligand-driven(1). In a range of low HER2 breast cancer cell lines, AZD8931 inhibited heregulin-driven proliferation (GI50 range 0.05 to 0.38μM) HER2:HER3 signaling, HER3:PI3K interaction and downstream signaling. In vivo, twice-daily oral dosing of AZD8931 showed significant monotherapy anti-tumor efficacy in a xenograft model of low HER2 breast cancer (MDA-MB-175VII) at well-tolerated doses (90% TGI at 12.5 mg/kg/bid). We further evaluated the activity of AZD8931 in combination with paclitaxel, a commonly used standard of care chemotherapy for patients with advanced breast cancer expressing low levels of HER2. Pre-clinically, the combination of AZD8931 with paclitaxel showed at least additive activity in vitro in a range of breast cell lines and additive efficacy in vivo in a BT474 (high HER2) xenograft model at well tolerated doses (AZD8931 12.5 mg/kg/bid TGI 63%; paclitaxel 7.5mg/kg/qw TGI 41%; combination TGI 95%). Combination work in the low HER2 xenograft model is ongoing. These data demonstrate that AZD8931 inhibits ligand-driven HER family receptor activation, receptor dimerization, PI3K interaction and downstream signaling leading to anti-tumor activity in vivo. These data also support the potential clinical utility of AZD8931 for the therapeutic treatment of low HER2 expressing breast cancers in combination with paclitaxel. 1 Hickinson et al. Clin.Cancer Res (2010) 16:1159-69. Citation Format: Gayle Marshall, Susan Ashton, Georgina Speake, Celina D'Cruz, Michael Grondine, Cath Trigwell, Graham Bigley, Garry Beran, Katy Lynaugh, Teresa C. Klinowska. Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease. [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 912. doi:10.1158/1538-7445.AM2013-912

HER2 Drives Luminal Breast Cancer Stem Cells in the Absence of HER2 Amplification: Implications for Efficacy of Adjuvant Trastuzumab

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)), HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. ©2012 AACR.

Abstract P3-04-07: Comparison of breast tumors with HER2 amplification and polysomy 17

Abstract Background: Approximately 20% of invasive breast cancers overexpress the human epidermal growth factor receptor, Her2, and are associated with poor prognosis including decreased relapse-free and overall survival. Overexpression of the Her2 protein is primarily due to amplification of the HER2 oncogene on chromosome 17, and these patients are typically treated with targeted therapies such as trastuzumab or lapatinib. However, increased HER2 copies may also result from chromosome 17 polysomy in which the entire chromosome has been duplicated one or more times. The clinical benefit of treating patients with polysomy 17 in the absence of HER2 amplification with targeted therapy remains unclear. Herein, we compared the clinical characteristics as well as the gene expression profiles of breast tumors with increased HER2 copy numbers due to HER2 amplification or chromosome 17 polysomy. Methods: Patients for this study were enrolled in the Clinical Breast Care Project. HER2 and CEP17 copy numbers were determined using the PathVysion HER-2 DNA Probe Kit (Abbott Laboratories). Only those patients with at least 4 HER2 copies per cell were included in the analysis; those with a HER2/CEP17 ratio &amp;lt;2.2 were considered polysomic (N = 29) while those with a ratio &amp;gt;2.2 (N = 44) were considered amplified. Microarray data were generated on HG U133A 2.0 arrays (Affymetrix) for those samples with tissue available (14 polysomy and 18 amplified cases) and analyzed using Partek Genomics Suite. Results: Tumor grade and size did not differ between polysomic and amplified samples. Although amplified cases tended to be younger at diagnosis and less likely to be diagnosed with Stage I tumors, this difference did not reach statistical significance. HER2 amplified tumors were more likely to be ER negative (P = 0.027) and have lymph node metastases (P = 0.028) than polysomic tumors. Comparison of polysomic and amplified cases by expression microarray identified 67 genes that were differentially expressed (P &amp;lt; 0.01; fold change &amp;gt;2) between the two groups. Thirty-nine genes were more highly expressed and 28 genes more lowly expressed in tumors with HER2 gene amplification when compared to tumors from patients with chromosome 17 polysomy. Discussion: ER and lymph node status of breast tumors from women with increased HER2 copy number differed depending on whether the increased copy number was due to HER2 gene amplification or conversely, polysomy of chromosome 17. Likewise, gene expression profiles also differed depending on whether increased HER2 was due to gene amplification or chromosomal gain. Those genes with significant levels of differential expression between these two groups are involved in cell cycle control, cell adhesion and migration, cell differentiation, cytoskeleton organization, signal transduction, protein synthesis and degradation, and ion transport. Interestingly, the top 4 most significant genes with higher expression in the polysomic samples, including TUBG1, CPD, BLMH, and JUP, are all located on 17q. Currently, it is not clear whether treatment with targeted Her2 therapy is beneficial in patients with chromosome 17 polysomy. However, the genes identified here may represent novel targets for developing new treatments that are effective in these women and require further study. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-07.

Abstract B13: High-risk patients found affected with breast cancer during a multimodality screening program: Triple negative versus non-triple negative breast cancers

Abstract Rationale and purpose: Triple negative breast cancers (TNBCs), characterized by lack of hormone receptors' expression in absence of HER2 amplification, partially overlap with the basal-like subtype, with frequent occurrence in BRCA1 mutation carriers (BRCA1+). TNBCs are often associated with earlier onset, interval cancer diagnosis, larger size and aggressive clinical course with peak risk of recurrence at 1-3 years and increased mortality rate in the first 5 years. Thus, when screening women at high risk of breast cancer, special attention should be paid to patient outcome for TNBCs in comparison with non-TNBCs. Our aim was to compare TNBCs and non-TNBCs diagnosed during a prospective, non-randomized, multimodality screening study – including clinical breast examination (CBE), mammography, ultrasound (US) and magnetic resonance imaging (MRI) – on women at familial/genetic high risk of breast cancer conducted in 18 centres from June 2000 to March 2008 (ISS-HIBCRIT-1; Sardanelli F et al, Invest Radiol 2011). Methods: Comparisons were performed using Mann-Whitney U, Fisher exact and χ2 tests. Results: Among the 44 patients diagnosed with invasive cancers, 14 (31%) were TNBCs and 30 (69%) non-TNBCs, the former being 13 invasive ductal (IDC) and 1 atypical medullary carcinoma, the latter also including 15/30 lobular subtypes and/or DCIS component (p=0.005). Of the 14 TNBCs, 10 (71%) were found in BRCA1+, 2 (14%) in BRCA2+ and 2 (14%) in BRCA-untested women with strong family history of breast/ovarian cancer; the same data for 30 non-TNBCs were 9 (30%), 6 (20%) and 15 (50%) respectively (p=0.028). We had only three interval cancers, all TNBCs. The median age at diagnosis was 49 years (range 36-62) for TNBCs and 53 years (range 35-72) for non-TNBCs (p=n.s). TNBCs presented a higher rate (11/14, 79%) of pathological grade 3 IDCs compared with non-TNBCs (8/30, 27%) (p=0.002). The mean tumor size was 1.6 cm for TNBCs and 1.2 cm for non-TNBCs (p=n.s). Nodal status was negative in 12/14 (86%) TNBCs and in only 16/30 (53%) non-TNBCs (p=0.038). MRI similarly outperformed CBE, mammography and US in both TNBCs and non-TNBCs. Clinical course and survival could be monitored for 40/44 patients (91%), 13 TNBCs and 27 non-TNBCs, with a follow-up of 5.8 and 6.3 years (p=n.s) respectively. The rate of disease-free patients for over 5 years was 8/13 (62%; mean disease-free interval 7.0 years, range 5.0-8.0) for TNBCs and 17/27 (63%; mean 7.2 years, range 5.2-9.9) for non-TNBCs. Death due to BC occurred for 2/13 TNBC (15%, at 3.5 and 4.2 years) and 3/27 non-TNBC patients (11%; at 2.0, 4.9 and 5.7 years). The rate of locoregional relapse was 1/13 (8%, at 4.4 years) and 5/27 (19%; mean time of 5.0 years, range 2.4-7.0) respectively. Distant recurrence was reported for only 2 non-TNBC patients. Conclusion: TNBCs showed stronger association with BRCA1+ status, lower rate of lobular subtypes or DCIS component, and less frequent nodal involvement. Despite a more frequent pathological grade 3 and the tendency to be diagnosed as interval cancers, under the current treatment protocols TNBCs showed relapse and BC-related death rates and over-5-year disease-free intervals similar to those of non-TNBCs. These data provide outcome evidence supporting the value of entering women at high risk of TNBC (in particular BRCA1+) in intensive screening programs including MRI. Citation Format: Franca Podo, Filippo Santoro, Siranoush Manoukian, Clelia de Giacomi, Laura Cortesi, Lorenzo Preda, Stefano Corcione, Francesco Sardanelli. High-risk patients found affected with breast cancer during a multimodality screening program: Triple negative versus non-triple negative breast cancers. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B13.

HER2 Expression in Breast Cancer With Nonamplified HER2 and Gains of Chromosome 17 Centromere

Gains of chromosome 17 centromere (CEP17) may be accompanied by gains of chromosome 17q. To evaluate the effect of CEP17 gains (CEP17>3 copies per tumor nucleus) on the expression of the HER2 gene, which is located on chromosome 17q12-21.32, we analyzed HER2 amplification and expression in breast carcinomas with and without CEP17 gains. We isolated tumor nuclei from frozen tissues of 37 breast carcinomas for analysis of the HER2 gene and CEP17 by fluorescence in situ hybridization. HER2 expression was detected by immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded sections of the corresponding tumors. Tumors with amplified HER2 as determined by both HER2 copy number and HER2/CEP17 ratio were detected in 29.7% (11/37). CEP17 gains were significantly associated with HER2 amplification (P=0.005) but not associated with estrogen receptor status, tumor grade, and lymph node status (P>0.05). In contrast, HER2 amplification was significantly associated with estrogen receptor negativity (P=0.020) but not with tumor grade and lymph node status (P>0.05). IHC analysis was performed in 7 HER2-amplified tumors and all of these were IHC 3+, which were used as positive controls. Among HER2-non-amplified tumors with CEP17 gains, only 1 tumor (1/8, 12.5%) was IHC 3+. However, none of the HER2-non-amplified tumors without CEP17 gains was IHC 3+. In HER2-non-amplified tumors, there was no significant association between HER2 protein expression as detected by IHC and CEP17 or HER2 copy number (P=0.999, P=0.785, respectively). These findings indicate that in the absence of HER2 amplification, CEP17 gains do not have a significant effect on HER2 protein expression.

ESR1 gene amplification and protein expression in 946 patients with resected breast cancer: A Hellenic Cooperative Oncology Group (HeCOG) translational research study.

592 Background: Discrepant data have been reported on the incidence of ESR1 gene amplification in breast cancer, its correlation to clinicopathologic characteristics and its impact on prognosis. Methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 946 patients participating in two adjuvant chemotherapy phase III trials (HE10/97 and HE10/00) were centrally assessed in tissue microarrays by immunohistochemistry (IHC) for estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2). FISH was also performed for HER2, TOP2A and ESR1 using commercially available dual (for ESR1) and triple (for HER2 and TOP2A) hybridization probes. Results: The majority of patients had &gt;T2 (69%), node-positive, ER-positive (&gt;1% stained cells, 73%) tumors managed with resection, chemotherapy and hormonotherapy (76%). Only 38 tumors (4.0%) had ESR1 gene amplification (ESR1/CEN6 ratio &gt;2) and 514 (54.3%) ESR1 gene gain (1&lt;ratio&lt;2). The number of ESR1 gene copies was 3-4 in 251 (26.5%) and 5-10 in 42 (4.4%) of cases. HER2 and TOP2A gene amplification was seen in 234 (25.3%) and 101 (10.9%) of tumors, respectively. We studied the immunohistochemical expression of ER protein by evaluating the percentage of stained cells, the Allred score (0-2, 26.8%; 3-6, 62.5%; 7-8, 10.7% of tumors) and the semiquantitative H-Score (50-100, 13.8%; 101-200, 36.8%; 201-300, 15% of tumors). ESR1 gene amplification was significantly associated with taxane therapy, age &gt;50, postmenopausal status, grade III-IV, absence of HER2 amplification and ER protein expression (p&lt;0.05). At a median follow-up of 92 months, univariate Cox regression analysis showed that ER protein expression, but not ESR1 gene status, was a predictor of favorable outcome. In multivariate analysis, tumor size &gt;5 cm, &gt;4 involved nodes and negative/low ER protein expression by Allred score were independent adverse prognostic factors. Conclusions: Our data showed a rather low incidence of ESR1 gene amplification and failed to confirm its prognostic/predictive utility. ESR1 mRNA expression data will be presented at the meeting.

Abstract 3470: HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: Implications for efficacy of adjuvant trastuzumab

Abstract Although current breast treatment guidelines limit the use of HER2 blocking agents such as trastuzumab to women whose tumors display HER2 gene amplification, recent retrospective analysis by Paik et al., have suggested that a wider group of patients may benefit from this therapy. Utilizing breast cancer cell lines, mouse xenograft models and primary human tumors and metastatic tissues, we provide a potential molecular explanation for these unanticipated clinical finding by demonstrating that HER2 is selectively expressed in the cancer stem cell (CSC) population in luminal ER+ breast cancers that do not display HER2 amplification. Furthermore, we demonstrate that the effects of trastuzumab on mouse tumor xenografts are highly dependent on the timing of administration. While the effects of trastuzumab on established tumors are limited to those with HER2 amplification, when this HER2 blocker is given immediately after tumor inoculation simulating an adjuvant setting, it significantly inhibits the growth of luminal tumors in the absence of HER2 amplification. In addition, HER2 expression is increased in luminal tumors propagated in mouse tibias as compared to primary tumors grown in mammary fat pads. Utilizing co-culture experiments with human osteocytes, we provide evidence that this regulated expression of HER2 is mediated by RANK ligand present in the bone microenvironment. The clinical relevance of these studies is demonstrated in breast cancer tissues by the co-expression of HER2 and CSC marker ALDH1 in a subset of cells which are selectively found at the invasive tumor front in luminal breast cancers. Recapitulating the mouse xenograft studies, we demonstrate increased expression of HER2 in bone metastasis compared to matched primary tumors in 8 patients with luminal breast cancer. Our results have significant clinical implications since they suggest that the benefits of adjuvant HER2 targeting therapies may extend to patients with luminal breast cancers who are currently classified as HER2-negative. Furthermore, these results support the cancer stem cell hypothesis and emphasize the clinical importance of targeting cancer stem cells in the adjuvant setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3470. doi:1538-7445.AM2012-3470

Abstract 2272: Survivin and acetylated Survivin predict outcome in breast cancer and correlate separately with a basal-like and luminal-type expression profile

Abstract Background: Basal-like breast cancer is a highly aggressive breast tumor subtype associated with an adverse prognosis for which biologic therapies are not available. These tumors have a distinct molecular profile characterized by an absence of estrogen receptor expression, absence of HER2 amplification, and positive expression of EGFR and/or cytokeratin (CK) 5/6. The object of this study was to evaluate the expression of the anti-apoptotic protein Survivin in basal-like breast cancer and compare it with other molecular subtypes. Our laboratory recently identified that post-translational modification of Survivin by acetylation (Ac) at Lys-129 regulates its export from the nucleus and therefore its anti-apoptotic function. In an effort to understand the expression profile of Survivin and Ac-Survivin in breast tumors, we developed an antibody to the Ac Lys-129 residue. Methods: 4 μM sections of 238 consecutive grade 3 invasive ductal carcinoma cases, arranged on 15 tissue microarrays were stratified into 67 luminal (ER+), 66 HER2 positive (HER2+), 93 basal-like (ER-, HER2-, CK5/6+ and/or EGFR+), and 12 ER-/HER2-/CK5/6-/EGFR- carcinomas. TMAs were immunostained with a rabbit polyclonal Survivin antibody generated to the Lys-129 acetylated residue, a full-length (FL) rabbit polyclonal Survivin antibody (NB-500-201), or normal rabbit serum control. Tumors were scored semiquantitatively and compared with recurrence-free (RF) and overall survival (OS) and with expression of ER, PR, HER2, CK 5/6, and EGFR. Results: In all breast carcinomas examined, FL-Survivin and Ac-Survivin had a predominantly nuclear localization pattern. Ac-Survivin was also expressed in the nucleus of normal breast epithelium. RF (70%) and OS (90%) for FL-Survivin low tumors was significantly better than for tumors with strong FL-Survivin expression (40% and 70%, respectively) at 10 yrs, p = 0.02. By contrast, strong expression of Ac-Survivin was associated with improved RF (70% vs. 40%) and OS (85% vs. 75%), p = 0.03. Molecular subset analyses showed that FL-Survivin expression was associated with a basal-like expression profile, while Ac-Survivin expression was associated with a luminal-type profile, p &amp;lt;0.001. Conclusions: This is the first study to comprehensively demonstrate Survivin and Ac-Survivin expression in different molecular subtypes of breast cancer. High levels of the FL-Survivin protein are predictive of poor outcome in patients with grade 3 invasive ductal carcinoma and correlate directly with a basal-like phenotype. By contrast, high expression of Ac-Survivin is associated with favorable outcome and preferentially correlates with luminal-type tumors. These data suggest that Survivin has different functions in different types of breast cancer and may be useful as a prognostic marker for these distinct tumor subtypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2272. doi:10.1158/1538-7445.AM2011-2272

HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer.

IntroductionMetastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.MethodsWe used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.ResultsMost brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.ConclusionsDeregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.

The Influence of Polysomy 17 on HER2 Gene and Protein Expression in Adenocarcinoma of the Breast

Breast carcinomas with amplification of HER2 on chromosome 17 are associated with HER2 protein overexpression, adversely affecting prognosis and predicting response to Herceptin therapy. Chromosome 17 polysomy is encountered in assessing HER2 gene status, and its impact on HER2 gene and protein expression remains unclear. This impact was investigated in breast carcinomas identified by fluorescence in situ hybridization (FISH) to have a gain of chromosome 17 (CEP17+; n = 56), using a dual probe assay, which detects HER2 gene copy number and enumerates chromosome 17 (HER2/CEP17; Vysis). Cases were immunostained for HER2 protein (CB-11, Ventana), and scored blinded to FISH. A subgroup was evaluated by isotopic in situ hybridization for HER2 mRNA expression. Controls included ten HER2 amplified and ten nonamplified tumors, eusomic for chromosome 17. Immunohistochemistry (IHC) for HER2 protein was negative (0 or 1+) in 69% (39 of 56), 2+ in 27% (15 of 56), and 3+ in 3% (2 of 56) of CEP17+ cases. The mean CEP17 copy number among the three groups was similar (3.1, 3.0, and 3.1 for IHC 0/1+, 2+, and 3+, respectively). Isotopic in situ hybridization for HER2 mRNA performed on 26 CEP17+ cases (16 IHC 0-1+, 10 IHC 2+ or 3+) showed no increased HER2 mRNA expression (normalized to beta-actin mRNA). The mRNA expression and the IHC staining of the HER2-amplified and nonamplified controls was concordant with their FISH status. These results suggest that chromosome 17 polysomy in the absence of HER2 amplification does not have a significant biologic influence on HER2 gene expression in breast carcinoma.