Increase In HER2 Research Articles

The Prospective role of lapatinib as an adjuvant therapy in prevalent cancers: Insights from in silico analysis targeting EGFR and HER2

IntroductionVarious pieces of evidence suggest an elevation in the levels of EGFR and HER2 in different cancers leading to the proliferation, invasion, and metastasis of cancer cells. In this study, we conducted a comprehensive investigation into the expression alterations of these two receptors in various cancers using in silico data. In addition, we investigated the therapeutic potential of lapatinib as an inhibitor of these receptors in various cancer types. MethodsRNAseq data for prevalent cancers were downloaded from The Cancer Genome Atlas (TCGA). After initial preprocessing, expression changes of HER2, EGFR, and candidate genes—identified based on their association with EGFR and HER2 signaling pathways—were examined. Human protein atlas data were utilized to assess the protein expression of HER2 and EGFR. GSE129254 was employed to identify molecular pathways and candidate genes associated with lapatinib. The protein-protein interaction network was used to identify lapatinib-influenced hub genes. Clinical data for common cancers were used to investigate the correlation between the expression of candidate genes and patients' mortality rates by Cox regression test. ResultsThe findings clearly indicated a significant increase in the expression levels of HER2 and EGFR in cancers such as kidney, lung, breast, bladder, pancreas, head and neck, stomach, and endometrial, both at the mRNA and protein levels (p-value <0.01). Additionally, more than 30 % of samples in some cancers showed a twofold increase in HER2 or EGFR expression. The analysis of GSE129254 data revealed that lapatinib reduces the expression of numerous genes associated with cell proliferation. METTL1, LYAR, LTV1, CCND1, NOP2, and DDX21 were identified as hub genes related to the effect of lapatinib. Our results demonstrated that many hub genes exhibited elevated expression in candidate cancers, and the upregulation of some of them was correlated with poor prognosis. ConclusionOur results indicate an upregulation in the expression levels of HER2 and EGFR in certain common cancers, suggesting that lapatinib, in addition to breast cancer, could be considered for the treatment of these cancers. Furthermore, we demonstrated that some genes with increased expression in prevalent cancers and associated with poor prognosis have the potential to be modulated by lapatinib.

Evolution of neoadjuvant therapy for breast cancer regimens over 12 years and pathologic response rates according to tumor subtypes and clinical stage: A single-center retrospective study.

Given the evolution of neoadjuvant therapy (NAT) for breast cancer, this study aimed to analyze trends in NAT regimens over time and patients' pathological responses, tumor stages, and subtypes. Data were analyzed for 548 patients with cT1-4N0-3M0 breast cancer who received NAT at Shandong Cancer Hospital between 2011 and 2022. The 12-year study period was divided into six 2-year periods termed P1 to P6. From P1 to P6, the proportion of stage II patients treated with NAT increased from 6.4% to 33.8% compared with same-stage operable breast cancer (r = 0.228, P < 0.001), while the proportion of the full-course group increased from 50.0% to 99.0% (r = 0.354, P < 0.001). The pathologic complete remission (pCR) rate in the full-course group increased from 30.8% to 54.6% (r = 0.248, P < 0.001). In the full-course human epidermal growth factor receptor-2 positive (HER2+) group, the proportion of chemotherapy combined with inhibition therapy increased from 33.3% to 100% (r = 0.530, P < 0.001). Furthermore, dual inhibition therapy increased from 0 to 98.9%. The proportion of the nonanthracycline group (dual inhibition) increased from 56.0% at P5 to 76.6% at P6 (r = 0.190, P = 0.042). In the full-course Triple-Negative Breast Cancer (TNBC) group, the proportion of platinum therapy increased from 0 to 41.9% (r = 0.324, P < 0.001) and immune drugs increased from 0 to 53.2% (r = 0.500, P < 0.001). Overall, the results indicate an increasing proportion of patients receiving NAT therapy over time. Furthermore, there were increases in HER2 + patients receiving inhibition therapy (especially dual inhibition) and TNBC patients receiving platinum and immune therapy as part of NAT. Notably, these changes were associated with improved outcomes.

ECOG-ACRIN E2197: Comparison of HER2 gene expression by RT-PCR across all HER2 immunohistochemistry groups with recurrence analysis.

515 Background: Accurate assessment of HER2-low [immunohistochemical (IHC) score of 1+ or 2+ and in-situ hybridization negative] breast cancer is clinically relevant following DESTINY-Breast04. In hormone receptor positive breast cancers, Oncotype DX assay Recurrence Score results are widely used to guide adjuvant chemotherapy selection, and standardized quantitative HER2 mRNA reference-normalized expression levels by RT-PCR are included in patient reports. Previously, a high degree of concordance between central IHC and quantitative HER2 by RT-PCR was demonstrated in E2197. Here, we compare quantitative HER2 gene expression using Oncotype DX assay within all IHC subgroups and risk of recurrence in E2197. Methods: Data analyzed were from a case-control sample from E2197 (no adjuvant anti-HER2 therapy) of 755 patients. Central IHC for HER2 used duplicate 1.0 mm microarrays (HercepTest, Dako) and scored per 2007 ASCO/CAP guidelines. Based on quantitative reference-normalized RT-PCR measures of HER2 expression, cases were assigned: positive ≥11.5 units, equivocal ≥10.7 to &lt;11.5 units, and negative &lt;10.7 units (1 unit increase represents ~2-fold increase in RNA). Spearman’s rank correlation was used to measure strength of association between the 2 methods. Cox proportional hazards regression was used to evaluate the association between quantitative HER2 expression and recurrence (defined as invasive breast cancer in local, regional, or distant sites). Analyses were weighted to account for study design. Results: 48% were IHC 0, 11% were IHC 1+, 23% were IHC 2+, and 18% were IHC 3+. 85% were HER2 negative, 3% equivocal, and 11% positive by RT-PCR. While there was moderately strong correlation between HER2 IHC and HER2 gene expression by RT-PCR [Spearman’s rho: 0.63 (95% CI: 0.59, 0.67); p&lt;0.001], there were wide and overlapping ranges of HER2 gene expression across all IHC categories with almost identical medians and inter-quartile ranges for HER2 IHC 1+ and 2+ (n=248; see table). An exploratory analysis showed 86% of IHC 0, 97% of IHC 1+, and 99% of IHC 2+ had HER2 &gt;8 units. After controlling for baseline clinicopathologic features (age, hormone receptor status, grade, size, and nodal status), continuous quantitative HER2 expression was associated with time to recurrence in HER2 IHC 0 to 2+ [HR 1.17 per 1-unit increase in HER2 (95% CI 1.01, 1.35; p=0.036)]. Conclusions: There was a wide range of quantitative HER2 gene expression using Oncotype DX assay across all IHC categories; continuous HER2 expression is independently associated with recurrence. Quantitative HER2 expression may be useful for identification of HER2-low breast cancer. Further studies are needed. [Table: see text]

Molecular and Immunohistochemical Alterations in Breast Cancer Patients in Upper Egypt.

Breast cancer (BC) plays a major public health in Egyptian woman. In Upper Egypt, there is an increase in incidence of BC compared to other Egyptian areas. Triple-negative BC, estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-neu-negative, is a high-risk BC that lacks the benefit of specific therapy that targets these proteins. Accurate determination of Caveolin-1(Cav-1), Caveolin-2 (Cav-2) and HER-2/neu status have become of major clinical significance in BC by focusing about its role as a tumor marker for response to different therapies. The present study was performed on 73 female BC patients in the South Egypt Cancer Institute. Blood samples were used for Cav-1, Cav-2, and HER-2/neu genes amplification and expression. In addition, immunohistological analysis of mammaglobin, GATA3, ER, PR, and HER-2/neu was done. There was a statistically significant association between Cav-1, 2 and HER-2/neu genes expression and the age of patients (P< 0.001). There are increase in the level of Cav-1, 2 and increase in HER-2/neu mRNA expression in groups treated with chemotherapy and group treated with both chemotherapy and radiotherapy compared to each group baseline level of genes mRNA expression before treatment. On the contrary, the group treated with chemotherapy, radiotherapy and hormonal therapy revealed increase on the level of Cav-1, 2 and HER-2/neu mRNA expression when compared with their baseline for the same patients before treatment. Noninvasive molecular biomarkers such as Cav-1 and Cav-2 have been proposed for use in the diagnosis and prognosis for women with BC.

Hepatitis B virus X protein increases LASP1 SUMOylation to stabilize HER2 and facilitate hepatocarcinogenesis

The hepatitis B virus (HBV) X protein (HBX), a viral macromolecule, plays a vital role in the development of HBV-related hepatocellular carcinoma (HCC). Increased expression of HER2 is linked to HBV infection, and HBX is responsible for HER2 upregulation in HCC. Nevertheless, the underlying molecular mechanisms are not yet fully understood. In the study, we discovered that HBX promoted HER2 expression to facilitate the sensitization of the insulin signaling pathway and enhance the growth and migration of HCC cells. Mechanistically, the viral protein enhanced the stability of HER2 by preventing its ubiquitination-mediated proteasomal degradation through LASP1, which could bind to HER2. Furthermore, increased SUMOylation of LASP1 contributed to the upregulation of HER2 and the interaction of LASP1 with HER2. In addition, RANBP2 and RANGAP1 were found to interact with LASP1 and promote SUMOylation of LASP1 to upregulate HER2 expression in HBX-associated hepatoma cells. In summary, our work provides a novel insight into hepatocarcinogenesis mediated by HBX and estimates the detailed mechanisms related to the increase in HER2 regulated by the viral protein, which might help provide a theoretical basis for identifying novel targets for HBV-positive HCC treatment.

Neoadjuvant chemotherapy use trends among older women with breast cancer: 2010-2017.

This study assessed chemotherapy use trends before (neoadjuvant chemotherapy [NAC]) or after surgery (adjuvant chemotherapy [AdC]) among older women with breast cancer and examined factors related to NAC receipt. Women (> 65years) diagnosed with stage I-III breast cancer during 2010-2017 who received NAC or AdC were identified from the Surveillance, Epidemiology, and End Results (SEER)-Medicare database. All patients were stratified into six strata based on subtype (hormone receptor-positive/human epidermal growth factor receptor 2-negative [HR + /HER2-], HER2 + , and triple-negative breast cancer [TNBC]) and stage (I-II and III). Cochran-Armitage tests were performed to test temporal trends of NAC use in each stratum. Multivariable logistic regression analyses were performed to identify factors (sociodemographic and clinical) related to NAC use. Among included older (mean ± standard deviation: 72.3 ± 5.2years) women (N = 8,495) with stage I-III breast cancer, NAC use increased from 11.7% (2010) to 32.6% (2017). Significant increases in NAC were found in all strata (p < .0001) with more substantial increases in HER2 + disease and TNBC compared to HR + /HER2- disease. Multivariable logistic regressions identified the youngest age category (66-69years) and later stage as significant (p < 0.05) predictors of NAC receipt in most strata, in addition to diagnosis year. Similar to the overall breast cancer population, NAC use increased among a population of older women. NAC was received by most patients with stage III HER2 + disease or TNBC in more recent years and was more common among younger elderly women and those in stage III.

Human Serum Albumin and HER2-Binding Affibody Fusion Proteins for Targeted Delivery of Fatty Acid-Modified Molecules and Therapy.

Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (ZHER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of ZHER2:342 has limited its pharmaceutical application. Human serum albumin (HSA) and ZHER2:342 fusion protein may not only extend the serum half-life of ZHER2:342 but also preserve the biological function of HSA to bind and transport fatty acids, which can be used to deliver fatty acid-modified therapeutics to HER2-positive cancer cells. Two HSA and ZHER2:342 fusion proteins, one with a single ZHER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and another with two tandem copies of ZHER2:342 fused to the C terminus of HSA (rHSA-(ZHER2)2), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)2, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)2 treatment has been observed. HSA fusion proteins are easy and economical to express, purify, and formulate. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified FITC could be efficiently taken up by cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.

Abstract B16: Potassium channel activators inhibit migration of breast cancer cells by hijacking the WNT signaling pathway

Abstract Potassium ion channels are transmembrane associated proteins that play fundamental role in several biological processes including cell proliferation by controlling potassium ion flow across the membrane. Interestingly, the Kv11.1 potassium channel has been found expressed in a large variety of cancers of different histogenesis including breast cancers. However, very little is known about the role of this channels in cancer biology. We recently demonstrated that stimulation of Kv11.1 activity with the pharmacological Kv11.1 activators (e.g. NS1643) induces irreversible growth arrest in several breast cancer cell types, and this G1 cell cycle arrest was maintained after the chemical activator was removed. Kv11.1 activator-induced growth arrest was associated with increased expression of tumor suppressor including p21Waf and p16INK4A, reduced protein level of breast tumor markers such as cyclin E2, and augmented beta galactosidase activity. These outcomes are hallmarks of senescent cells that exist in an irreversible non-proliferative state. In the current work we have further studied the role of KV11.1 stimulation on migration of breast cancer cells. In the current study we report that activation of Kv11.1 by small molecule NS1643 inhibits migration of various cancer cells irrespective of their biomarker status. Using a wound healing and chamber migration assay we observed that NS1643 inhibits migration of triple negative (MDA-MB-231), Her2+ (SKBR3) and as well as ER+ (MCF7) cell lines. Following the molecular mechanisms of NS1643 dependent inhibition of migration, we observed that, the nuclear translocation of β-catenin and subsequently Wnt-3a mediated β-catenin/TCF promoter reporter activity was inhibited when treated with NS1643, suggesting that, activation of Kv11.1 with NS 1643 inhibited the Wnt3a/β-catenin pathway. Consistent with the β-catenin nuclear activity it was observed that phosphorylation of β-catenin (ser 552/656) was significantly decreased following treatment with NS1643. Interestingly however it was observed that the total β-cateninin levels were differentially regulated in breast cancer cell subtypes. We observed that β-catenin levels were increase in Her2+ and triple negative cell lines while decreased in ER+ cell lines following KV11.1 stimulation by NS 1643. Furthermore using immunoprecipitation and confocal microscopy we observed that, in E-cadherin expressing breast cancer cell lines, NS1643 was able confer a mesenchymal to epithelial transformation by promoting the sequestration of β-catenin and E-cadherin complex at the plasma membrane. These results indicate that, activation of Kv11.1 in breast cancer cell lines could be a novel therapeutic approach for inhibition of breast cancer cell migration. Note: This abstract was not presented at the conference. Citation Format: Gentile Saverio, Vidhya Rao, Mathew Perez-Neut. Potassium channel activators inhibit migration of breast cancer cells by hijacking the WNT signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B16.

Expression analysis of human epidermal growth factor receptor type 2 transcripts in breast cancer cohort and its association with clinical features.

Increased expression of human epidermal growth factor receptor type 2 (HER2) is significantly associated with poor prognosis in breast cancer patients. However, data on HER2 at transcript levels in Pakistani mammary tumor affected females is still limited. In the current study, HER2 transcripts were explored in breast cancer cohort and correlated with various clinical parameters. Freshly excised tumors along with adjacent normal background tissues of 94 patients were collected at the time of surgery and immediately stored in RNAlater ® solution. Clinical data for these samples (disease stage, grade, age, and menopausal status) was also retrieved after a subsequent follow-up. Isolation of RNA and cDNA synthesis was done using an already established protocol. HER2 expression was evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR) technique while β-actin was used as an internal control. In the given cohort, 31 (33%) patients were found positive for HER2. These tumors showed a pronounced increase in HER2 as compared to controls (P = 0.0004). Interestingly, the significant relevance of high HER2 mRNA among moderately differentiated tumor tissues in comparison to controls was also observed (P = 0.02). A significant association of HER2 levels with premenopausal status was also reported. Based on these findings, early screening of HER2 using qRT-PCR should be incorporated for breast cancer patients of Pakistani population diagnosis.

Molecular Targeting of Her-2/neu Protein Is Not Recommended as an Adjuvant Therapy in Oral Squamous Cell Carcinoma and Oral Lichen Planus.

Target therapy against molecular markers of EGFR family has been recently used in the treatment protocol of several diseases. However, the role of Her-2/neu in OSCC is a matter of controversy and more studies are required to clarify the role of Her-2/neu in OSCC. We aimed to investigate the role of Her-2/neu in the process of carcinogenesis in oral epithelium as ELP is a premalignant and OSCC is a malignant lesion, but RLP shows no evidence of premalignancy and malignancy.To our Knowledge, this is the first study comparing Her-2/neu expression in erosive lichen planus (ELP), reticular lichen planus (RLP), and oral squamous cell carcinoma (OSCC). 60 samples, including 20 cases of RLP, 20 cases of ELP, and 20 cases of OSCC were evaluated immunohistochemically in this study. Our findings showed that there was not a significant increase in Her-2/neu expression from RLP to ELP and from ELP to OSCC. Therefore, Her-2/neu had no role in differentiating between RLP, ELP and OSCC and this protein does not contribute to the process of carcinogenesis in the oral epithelium. The lack of Her-2/neu overexpression indicates that molecular targeting of Her-2/neu protein is not recommended as an adjuvant therapy in OSCC and OLP.

Histone deacetylase inhibitor entinostat in combination with a retinoid downregulates HER2 and reduces the tumor initiating cell population in aromatase inhibitor-resistant breast cancer.

Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.

Combined HER2 analysis of biopsies and surgical specimens to optimize detection of trastuzumab-eligible patients in eso-gastric adenocarcinoma: a GERCOR study

BackgroundHER2 is overexpressed in 10 to 20% of gastro-esophageal adenocarcinoma (GE-ADK), and is a target for trastuzumab in metastatic patients. We conducted a study to compare HER2 expression between diagnostic biopsies (DBs) and surgical specimens (SSs) of GE-ADK, and to determine the influence of non-trastuzumab containing neoadjuvant chemotherapy (NAC) on this expression. Patients and methodsPathological specimens from biopsies of 228 patients operated on between 2004 and 2011 were collected. Two cohorts treated (n = 141) or not (n = 87) with a NAC were constituted. Two blind independent pathological HER2 analyses on DB and on SS were carried out using immunohistochemistry (IHC) and colorimetric in situ hybridization (CISH). HER-2 overexpression (HER2+) was defined by a score 3+ in IHC, or 2+ with a positive CISH test, according to the specific HER2 scoring guidelines for GE-ADK. ResultsPaired HER2 status could be determined for 218 out of the 228 patients (95.6%). HER2+ rates were 13.3% on DB (29/218) and 14.7% on SS (32/218). HER2+ tumors were mainly cardial or esophageal adenocarcinomas, with a well-differentiated, intestinal histological type. HER2 status differed between DB and SS in 6% of cases. When DB analyses were added to SS analyses, the relative increase in HER2+ cases was 13.5% (17.1% for patients with NAC and 23.5% for patients with histological response to NAC, versus 7.1% for patients without NAC, P = 0.4, NS). Differences between DB and SS HER2 expression could be explained by intratumoral heterogeneity and by a HER2 expression decrease in SS after NAC in responding patients possibly due to a higher chemosensitivity of HER2-positive clones. ConclusionThe determination of HER2 status on DB provides results that complete those obtained with SS. Combining the analysis of DB and of SS enables to optimize the selection of trastuzumab-eligible patients in case of metastatic relapse, and particularly in previously NAC-responding patients.

Mechanisms of primary AI resistance in ER-positive HER2-negative breast cancer.

e11519 Background: Current approaches of neoadjuvant endocrine therapy (NAET) are focusing on biomarker analysis of the diagnostic specimens however inadequate response occurs in 5-10% of patients treated. It has been reported hormone resistance may predict Ki67 level after treatment than ΔKi67. Much less is known about the difference between Ki67 increases and still high level of Ki67 after NAET. A pilot study was conducted to investigate the differences of biomarker changes in short-term NAET (letrozole vs. exemestane). Differences of resistant mechanisms between Ki67 increases and Ki67 still high expression and between two AIs were studied. Methods: Totally 116 samples from 58 postmenopausal women with ER positive HER2 negative stage 1-3 breast cancers comparing LET and EXE were analyzed with pre- and post-treatment mRNA expression of multiple biomarkers using QuantiGene Assay. Each of 29 patients took LET or EXE until the surgery (median 22 days). Compared with Ki67 expression of pre/post treatment, we classified resistance into 2 groups, Ki67 increase after treatment (INC) or Ki67 still high expression (equivalent over 14% in IHC) except those in INC (HIG). Results: Clinical characteristics were well balanced between the two treatment arms. Median % changes of Ki67 were -59% in both LET and EXE. Six cases (10%) were in INC (3/LET and 3/EXE). Among 10 cases of still high Ki67 expression, 6 (10%) were in HIG except 4 cases of Ki67 increases (3/LET and 3/EXE). All 6 cases in INC had increases of ERβ, MAPK, PIK3CA, ALDH1, NFkB, and RANKL and decrease of ERα. Five out of 6 in INC had an increase of HER2 and a decrease of PR. While changes in HIG were various, a nonsignificant trend to predict resistance was found. However all 3 cases whose HER2 increased in HIG had also increases of MAPK and PIK3CA. All 3 receiving EXE had increases of PR. Conclusions: Although both LET and EXE had significant decreases of Ki67, no significant difference was found between two. Both had 20% of primary resistance in terms of Ki67. Tumors which Ki67 increase seem to activate various signaling pathway, which mechanism is class effect of AI. What Ki67 is still high supposed to be nonclass effect.

Comparison of HER2 expression in paired biopsies and surgical specimen of gastric and esophageal adenocarcinoma: Impact of neoadjuvant chemotherapy.

4069 Background: HER2 is overexpressed in 10 to 20% of gastro-esophageal adenocarcinoma (GE-ADK), and is a target for trastuzumab in metastatic patients. We conducted a study to compare HER2 expression between diagnostic biopsies (DB) and surgical specimens (SS) of GE-ADK, and to determine the influence of non-trastuzumab containing neoadjuvant chemotherapy (NAC) on this expression. Methods: Pathological specimens from biopsies of 228 patients operated on with a curative aim in two French hospitals between 2004 and 2011 were collected. Two cohorts treated (n=141) or not (n=87) with a NAC were constituted. Two blind independent pathological HER2 analyses on DB and on SS were performed using IHC and CISH. HER-2 overexpression (HER2 +) was defined by a score 3+ in IHC, or 2+ with a positive CISH test, and according to the specific HER2 scoring guidelines for GE-ADK. Results: Paired HER2 status could be determined for 218 out of the 228 patients (95.6%). HER2 + rates were 13.3% on DB (29/218) and 14.7% on SS (32/218). HER2 + tumors were mainly cardial or esophageal adenocarcinomas, with a well-differentiated, intestinal histological type. HER2 status differed between DB and SS in 13 patients (6%). When DB analyses were added to SS analyses, 5 additional patients were HER2 +, the relative increase in HER2 + cases being 13.5% (17.1% for patients with NAC versus 7.1% for patients without NAC, p=0.4, NS). Differences between DB and SS HER2 expression could be explained by the intratumoral heterogeneity and also by a possible HER2 expression decrease in SS after NAC. Conclusions: The determination of HER2 status on DB provides results that complete those obtained with SS. Combining the analysis of DB and of SS enables to optimize the selection of trastuzumab-eligible patients in case of metastatic relapse, especially in patients previously treated with NAC. [Table: see text]

Abstract P3-04-02: Protein pathway activation mapping of I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with HR+/HER2− tumors

Abstract Background: Profiling protein signaling activation in cancer is critical as these proteins represent targets for new molecular therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane- based chemotherapy that longitudinally collected biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens (T2, 1–4 days post-chemotherapy). Reverse Phase Protein Microarray technology was used to quantitatively measure the activation of 39 and 100 key signaling proteins in the T1 and T2 biopsies, respectively, including numerous drug targets for Phase I-III and FDA-cleared therapeutics. Associations between protein activation and response to chemotherapy (RCB 0/1 vs 2/3) and relapse-free survival (RFS) were evaluated for the HR+/HER2− patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p &amp;lt; 0.05; Cox proportional hazard model, likelihood ratio p &amp;lt; 0.05. Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of HER family signaling and downstream AKT activation in tumors from patients who do not respond (RCB2/3) and/or whom recurred. Increased ERBB2 and ERBB4 levels were observed at T1 and T2, respectively, from patients who did not respond to neoadjuvant therapy. Dynamic changes in ERBB3 and AKT phosphorylation/activation were revealed between the T1 and T2 time points, which were associated with recurrence. Increased phosphorylation of MARCKS, a known PKC substrate, was seen in the T1 biopsy in the non-response cohort. Conclusions: Our analysis revealed activation/increased expression of HER family proteins along with downstream AKT activation in HR+/HER2− patients who have poor clinical response. These events, if validated, point to potential treatment options that could be rationally considered for non-responding HR+/HER2− patients with a number of investigational agents that target ERBB3 and AKT signaling in the clinic today. Increasing HER2 protein expression in a HER2− cohort may appear contradictory, however these findings may point to important subtle increases in HER2 that have important clinical considerations. Given the known clinical benefit from HER2 directed therapy in HER2− patients seen in other studies, our findings may have clinical relevance if validated. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with HR+/HER2− tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-02.

Abstract 328: The mitotic kinase Aurora-A promotes breast cancer metastases by inducing a CD24low/- basal-like stem cell phenotype

Abstract In this study we demonstrate that gain of HER-2/Neu expression in human breast cancer xenografts harboring constitutive active Raf-1 induces Aurora-A over-expression that ultimately drives the transition from a luminal to a highly invasive basal-like phenotype. Over-expression of Aurora-A was induced by phosphorylation and protein stabilization by HER-2/Neu rather than increased Aurora-A gene expression. Moreover, Aurora-A stabilization induced an increase of HER-2/Neu expression in breast cancer cells defining a novel positive feed-back loop between Aurora-A and HER-2/Neu oncogenic signaling pathways. Basal-like tumor cells over-expressing Aurora-A and HER-2/Neu underwent to loss of CD24 surface receptor, expressed epithelial to mesenchymal transition (EMT) markers and acquired stem cell-like properties such as mammospheres formation and self-renewal. Pharmacologic inhibition of HER-2/Neu signaling pathway with lapatinib in cancer cells over-expressing Aurora-A demonstrated that Aurora-A was downstream to HER-2/Neu in the development of EMT and stemness. To determine whether Aurora-A induced a cancer stem cell-like phenotype through development of chromosomal instability and tumor cell phenotypic heterogeneity, we performed a SKY analysis on the breast cancer cell lines employed in this study. Our results demonstrate that invasive breast cancer cells harboring a basal-like phenotype did not develop higher chromosomal instability compared to luminal non-invasive cancer cells, signifying that Aurora-A has a direct effect on the development of basal-like breast cancer cells with stemness properties. Importantly, pharmacologic inhibition of Aurora-A kinase activity with the small molecule MLN8237 restored CD24 expression and reversed EMT leading to the re-establishment of a luminal phenotype and suppression of distant metastases. Our findings implicate for the first time the mitotic kinase Aurora-A in the development and maintenance of CD24 negative basal-like tumor stem cells responsible for breast cancer recurrence and progression. These observations have also important implications for the development of molecular targeted therapies to inhibit Aurora-A kinase activity and stemness improving the overall survival of breast cancer patients. 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 328. doi:1538-7445.AM2012-328

Dual EGFR/HER2 Inhibition Sensitizes Prostate Cancer Cells to Androgen Withdrawal by Suppressing ErbB3

Patients with recurrent prostate cancer are commonly treated with androgen withdrawal therapy (AWT); however, almost all patients eventually progress to castration resistant prostate cancer (CRPC), indicating failure of AWT to eliminate androgen-sensitive prostate cancer. The overall goal of these studies is to determine whether dual inhibition of the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and HER2 would prolong the effectiveness of this treatment in prostate cancer. We used androgen-dependent LNCaP cells and its CRPC sublines LNCaP-AI and C4-2. Additional data were collected in pRNS-1-1 cells stably expressing a mutant androgen receptor (AR-T877A), and in nude mice harboring CWR22 tumors. Studies utilized EGFR inhibitors erlotinib and AG1478, and HER2 inhibitors trastuzumab and AG879. Dual EGFR/HER2 inhibition induced apoptosis selectively in androgen-sensitive prostate cancer cells undergoing AWT, but not in the presence of androgens, or in CRPC cells. We show that AWT alone failed to induce significant apoptosis in androgen-dependent cells, due to AWT-induced increase in HER2 and ErbB3, which promoted survival by increasing Akt phosphorylation. AWT-induced ErbB3 stabilized the AR and stimulated PSA, while it was inactivated only by inhibition of both its dimerization partners EGFR and HER2 (prostate cancer cells do not express ErbB4); but not the inhibition of any one receptor alone, explaining the success of dual EGFR/HER2 inhibition in sensitizing androgen-dependent cells to AWT. The effectiveness of the inhibitors in suppressing growth correlated with its ability to prevent Akt phosphorylation. These studies indicate that dual EGFR/HER2 inhibition, administered together with AWT, sensitize prostate cancer cells to apoptosis during AWT.

Abstract P4-02-02: Epidermal Growth Factor Receptors (ErbB/HER) and the Ligand Neuregulin 1 (NRG1) Increase in Breast Tumors during Short Time Endocrine Treatment

Abstract Background: Cross-talk between estrogen receptor (ER) and growth factor signaling pathways has been suggested to play a role in development of resistance to endocrine treatment with tamoxifen or aromatase inhibitors (AIs). While HER-2/neu amplification has been associated with lack of endocrine sensitivity in ER+ breast cancer, recent data have suggestedanti-HER-2 therapy to enhance efficacy of AIs in a subset of HER-2/neu non-amplified breast cancers [1], and we found HER-2/neu upregulation in tumors responding to therapy with AIs [2]. Here, we aimed at exploring the effect of treatment with AIs on other components of the HER family, including NRG1, recently reported to be subject to silencing through promoter methylation in breast cancers [3]. Methods: Tumor biopsies were collected from 60 ER + breast cancers before and after 3 months treatment with the AIs, Anatrozole or Letrozole. Tumor biopsies after 2 weeks of treatment were available for 39 of the patients. RNA was extracted from the tumors and mRNA expression analysis of HER-1, HER-2/neu, HER-3, HER-4 and NRG1 were performed with gene-specific primers and probes by real-time PCR. Changes in mRNA expression during treatment were analyzed by Wilcoxon Sign Rank tests, and correlation between expression groups by Spearman correlation. Results: Among HER-2/neu non-amplified tumors (n=56), we observed a significant increase in mRNA expression for HER-1 (40/50 tumors; mean increase 2.0-fold) and NRG1 (38/54 tumors; mean increase 2.9-fold) during treatment independent of treatment response (P&amp;lt;0.001 for both). A non-significant increase in Her-2/neu was observed in 28/54 tumors (18/38 responders). Interestingly, individual alterations in tumor HER-2/neu correlated negatively to change in HER-1 (R=-0.414, p=0.001). Data on DNA methylation at the NRG1 gene will be presented. While no statistical significant increase or decrease in HER-3 or -4 were observed, individual variations in these two parameters were statistically correlated among individual tumors (R=0.621, P&amp;lt;0.001). Conclusions: Estrogen suppression with use of AIs significantly increased intratumor expression levels of HER-1 and NRG1 in HER-2/neu non-amplified tumors, while HER-2/neu increased in a subset of tumors. These findings suggest extensive cross-talks between estrogen stimulation and the HER system, indicating potential mechanisms of therapy resistance. The increase in NRG1 during treatment with AIs may represent a cellular response that leads to changes in its tumor suppressor activity. These findings merits further explorations. 1. Johnston, S., et al., Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone receptor-positive metastatic breast cancer. J Clin Oncol, 2009. 27(33): p. 5538-46. 2. Flageng, M.H., et al., Nuclear receptor co-activators and HER-2/neu are upregulated in breast cancer patients during neo-adjuvant treatment with aromatase inhibitors. Br J Cancer, 2009. 101(8): p. 1253-60. 3. Chua, Y.L., et al., The NRG1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene. Oncogene, 2009. 28(46): p. 4041-52. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-02.

Lapatinib Restores Hormone Sensitivity with Differential Effects on Estrogen Receptor Signaling in Cell Models of Human Epidermal Growth Factor Receptor 2–Negative Breast Cancer with Acquired Endocrine Resistance

Acquired endocrine resistance in estrogen receptor (ER)alpha+/human epidermal growth factor receptor 2-negative (HER2-) breast cancer has been associated with modest adaptive increases in HER2, although exactly how aberrant HER2 signaling affects the ERalpha pathway is poorly understood. We investigated (a) whether the epidermal growth factor receptor/HER2 inhibitor lapatinib could restore endocrine responsiveness in cell models of acquired endocrine resistance with modest increases in HER2, and (b) the nature of ERalpha-HER2 cross-talk in this process. Combination growth studies, ERalpha transcription, immunoblot, and gene expression assays were conducted in two models of acquired resistance to (a) estrogen deprivation (long-term estrogen-deprived cells) and (b) tamoxifen (long-term tamoxifen-treated cells), and in hormone sensitive controls. Changes in ERalpha, PgR, and HER2 were assessed in samples from patients treated with tamoxifen. Both cell models of acquired endocrine resistance showed modest adaptive upregulation in HER2, and lapatinib restored endocrine sensitivity in both. The effect of lapatinib on ERalpha signaling varied markedly depending on the nature of the HER2/ERalpha cross-talk. In long-term estrogen-deprived cells characterized by enhanced ERalpha function, lapatinib suppressed ERalpha genomic activity (as measured by pERSer118, ERalpha transcriptional activity, and PGR gene expression). In contrast, in long-term tamoxifen-treated cells with reduced ERalpha activation, lapatinib reactivated ERalpha genomic function. Twenty percent of tamoxifen-resistant patients relapsed with modest increases in HER2 and either suppressed or enhanced ERalpha/PgR expression. Aberrant GFR signaling can augment or suppress ERalpha function. Regardless, interrupting the HER2/ERalpha cross-talk with lapatinib can restore endocrine sensitivity and should be investigated as a therapeutic strategy in combination with endocrine therapy in ERalpha+/HER2- patients with acquired endocrine resistance.

Changes in the Dynamic Relationship between ER and HER2 in Paired Breast Cancer (BC) Biopsies from Patients (Pts) with Recurrent Tamoxifen-Resistant Breast Cancer.

Abstract Background: Paired biopsies at diagnosis and relapse on tamoxifen (TAM) provide information regarding adaptive changes associated with endocrine resistance. We previously reported that conversion to a HER2+ phenotype occurs infrequently1, however modest increases in HER2 may be relevant in the context of TAM resistance. Also while TAM resistance in general is associated with a fall in mean ER and PgR levels, this masks changes in individual pts.Aim: To determine whether a subset of HER2 negative BC demonstrated modest increases in HER2 by IHC and FISH, and if so whether this correlated with changes in ER and PgR expression in individual pts.Methods: Triplicate TMAs were constructed of paired primary and recurrent tumor samples from pts relapsing after adjuvant TAM. TMAs were stained for ER and HER2, whole sections for PgR. HER2 amplification was assessed by FISH.Results: Paired samples from primary and TAM-resistant recurrences were available on 53 ER+ and/or PgR+ pts. 10% had a HER2+ primary (IHC3+ and FISH+). 19% (10/53) showed increasing HER2 in the relapsed tumor (0 to 1+ in 5 pts, 1+ to 2+ in 2 pts and 0/1+ to 3+ in 3 pts). The 3 pts converting to 3+ also became FISH+. 71% (38/53) had stable or decreasing HER2. DFI was 50.3 months in the subset with stable or decreasing HER2, 40.2 in the subset with increasing HER2, and 35.2 in the HER2+ subset (p=NS). We next compared changes in ER and PgR in the 3 subsets. Among pts with stable or decreasing HER2 (N=38), there was a significant decrease in mean H-scores for both ER (p=0.001) and PgR (p=0.002) at relapse (Fig 1); the majority recurred with reduced ER (76%) or PgR levels (84%). In contrast, among pts with increasing HER2, there was no change in mean ER or PgR levels (Fig 2A); however when considering changes in individual pts, there was a balanced split between tumors relapsing with up- vs. down-regulation in ER/PgR (Fig 2B,C). In the HER2 positive subset, 100% of the pts relapsed with suppressed ER and PgR.Conclusion: In an updated series of TAM-resistant pts, we show that while conversions to HER2 3+/FISH+ remain rare (5%), modest increases in HER2 are more frequent (19%). TAM resistance in general is associated with decreasing ER and PgR. However, among pts with adaptive increases in HER2, TAM resistance is associated with either down- or up-regulation in ER expression and function. This is consistent with data from acquired endocrine resistance models we previously reported2,3.1Gutierrez et al, JCO 20052Pancholi et al, ERC 2008,3 Martin et al, JBC 2003 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 704.