HER2 mRNA Expression Research Articles

RNA-based determination of ESR1 and HER2 expression and response to neoadjuvant chemotherapy

Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2- tumors was 7.3%, 8.0% and 8.6%; for ESR1-/HER2- tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the Kaplan-Meier analysis in GeparTrio patients with ESR1+/HER2- tumors had the best prognosis, compared with ESR1-/HER2- and ESR1-/HER2+ tumors [disease-free survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

HER2 and ESR1 mRNA expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer

IntroductionRecent data suggest that benefit from trastuzumab and chemotherapy might be related to expression of HER2 and estrogen receptor (ESR1). Therefore, we investigated HER2 and ESR1 mRNA levels in core biopsies of HER2-positive breast carcinomas from patients treated within the neoadjuvant GeparQuattro trial.MethodsHER2 levels were centrally analyzed by immunohistochemistry (IHC), silver in situ hybridization (SISH) and qRT-PCR in 217 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) core biopsies. All tumors had been HER2-positive by local pathology and had been treated with neoadjuvant trastuzumab/ chemotherapy in GeparQuattro.ResultsOnly 73% of the tumors (158 of 217) were centrally HER2-positive (cHER2-positive) by IHC/SISH, with cHER2-positive tumors showing a significantly higher pCR rate (46.8% vs. 20.3%, P <0.0005). HER2 status by qRT-PCR showed a concordance of 88.5% with the central IHC/SISH status, with a low pCR rate in those tumors that were HER2-negative by mRNA analysis (21.1% vs. 49.6%, P <0.0005). The level of HER2 mRNA expression was linked to response rate in ESR1-positive tumors, but not in ESR1-negative tumors. HER2 mRNA expression was significantly associated with pCR in the HER2-positive/ESR1-positive tumors (P = 0.004), but not in HER2-positive/ESR1-negative tumors.ConclusionsOnly patients with cHER2-positive tumors - irrespective of the method used - have an increased pCR rate with trastuzumab plus chemotherapy. In patients with cHER2-negative tumors the pCR rate is comparable to the pCR rate in the non-trastuzumab treated HER-negative population. Response to trastuzumab is correlated to HER2 mRNA levels only in ESR1-positive tumors. This study adds further evidence to the different biology of both subsets within the HER2-positive group.Introduction The human epidermal growth factor receptor 2 (HER2) is the prototype of a predictive biomarker for targeted treatment [1-8]. International initiatives have established the combination of immunohistochemistry (IHC) and in situ hybridization as the current gold standard [9,10]. As an additional approach determination of HER2 mRNA expression is technically feasible in formalin-fixed paraffin-embedded (FFPE) tissue [11-13]. Crosstalk between the estrogen receptor (ER) and the HER2 pathway has been suggested based on cell culture and animal models [14]. Consequently, the 2011 St Gallen panel has pointed out that HER2-positive tumors should be divided into two groups based on expression of the ER [15].A retrospective analysis of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 study has suggested that mRNA levels of HER2 and ESR1 might be relevant for the degree of benefit from adjuvant trastuzumab. By subpopulation treatment effect pattern plot (STEPP) analysis in ER-positive tumors, benefit from trastuzumab was shown to be restricted to those with higher levels of HER2 mRNA (S Paik, personal communication, results summarized in [15]).In our study we evaluated this hypothesis in the neoadjuvant setting in a cohort of 217 patients from the neoadjuvant GeparQuattro trial [5]. All patients had been HER2- positive by local pathology assessment and had received 24 to 36 weeks of neoadjuvant trastuzumab plus an anthracycline/taxane-based chemotherapy. For central evaluation we used three different methods, HER2 IHC, and HER2 silver in situ hybridization (SISH), as well as measurement of HER2 mRNA by quantitative real-time (qRT)-PCR [11].The primary objective of this analysis was to investigate if pathological complete response (pCR) rate in HER2-positive breast cancer would depend on the level of HER2 mRNA expression, with a separate analysis for HR-positive and -negative tumors. Central evaluation of the HER2 status showed that 27% of the tumors with HER2 overexpression by local pathology were HER2-negative. This enabled us to compare response rates in patients with HER2-positive and -negative tumors as a secondary objective.

Anticancer Activity of Pristimerin in Epidermal Growth Factor Receptor 2-Positive SKBR3 Human Breast Cancer Cells

Pristimerin is a naturally occurring triterpenoid that causes cytotoxicity in several cancer cell lines. However, the mechanism of action for the cytotoxic effect of pristimerin has not been unexplored. The purpose of this study was to investigate the effect of pristimerin on cytotoxicity using the epidermal growth factor receptor 2 (HER2)-positive SKBR3 human breast cancer cell line. Pristimerin inhibited proliferation in dose- and time-dependent manners in cells. We found it to be effective for suppressing HER2 protein and mRNA expression. Fatty acid synthase (FASN) expression and FASN activity were downregulated by pristimerin. Adding of exogenous palmitate, the end product of de novo fatty acid synthesis, reduced the proliferation activity of pristimerin. The changes in HER2 and FASN expression induced by pristimerin altered the levels of Akt and mitogen-activated protein kinase (MAPK) phosphorylation (Erk1/2, p38, and c-Jun N-terminal kinase (JNK)). Pristimerin lowered the levels of phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets such as phosphoprotein 70 ribosomal protein S6 kinase and 4E binding protein1. Pristimerin inhibited migration and invasion of cells, and co-treatment with the mTOR inhibitor rapamycin additionally suppressed these activities. Pristimerin-induced apoptosis was evaluated using Western blotting for caspase-3, -8, -9, and poly (ADP-ribose) polymerase expression and flow cytometric analysis for propidium iodide labeling. These results suggest that pristimerin is a novel HER2-downregulated compound that is able to decrease fatty acid synthase and modulate the Akt, MAPK, and mTOR signaling pathways to influence metastasis and apoptosis. Pristimerin may be further evaluated as a chemotherapeutic agent for HER2-positive breast cancers.

A randomized phase II study evaluating the combination of carboplatin-based chemotherapy with pertuzumab versus carboplatin-based therapy alone in patients with relapsed, platinum-sensitive ovarian cancer

Pertuzumab, a humanized monoclonal antibody targeting human epidermal growth factor receptor (HER)-mediated signalling, has shown activity in ovarian cancer in preclinical models and in the clinic. This randomized phase II study evaluated efficacy and safety of pertuzumab in combination with carboplatin-based chemotherapy in patients with platinum-sensitive, recurrent advanced ovarian cancer. Patients were randomized to receive six cycles of chemotherapy (carboplatin and either paclitaxel (Taxol) or gemcitabine) with or without pertuzumab. The primary end point was progression-free survival (PFS) as determined by Response Evaluation Criteria in Solid Tumors and/or by CA 125 measurements. Secondary end points evaluated the response rate, safety profile, duration of response, time to progression and overall survival for both treatment arms. A total of 149 patients received either chemotherapy with pertuzumab (arm A, n=74) or chemotherapy alone (arm B, n=75). There was no significant difference either in median PFS or in the secondary end points between the two arms. No differences were seen in an exploratory biomarker analysis of HER3 mRNA expression between the two arms. Pertuzumab was well tolerated, with no increase in cardiac adverse events compared with chemotherapy alone. The addition of pertuzumab to carboplatin-based chemotherapy did not substantially prolong PFS in unselected patients with platinum-sensitive ovarian cancer.

Alterations in epidermal growth factor receptors 1 and 2 in esophageal squamous cell carcinomas

BackgroundEsophageal squamous cell carcinoma (ESCC) shows a 5-year survival rate below 10%, demonstrating the urgency in improving its treatment. Alterations in epidermal growth factor receptors are closely related to malignancy transformation in a number of tumors and recent successful targeted therapies have been directed to these molecules. Therefore, in this study, we analyzed the expression of EGFR and HER2 and evaluated EGFR mutation profile as well as the presence of mutations in hotspots of KRAS and BRAF in ESCC patients.MethodsWe performed RT-qPCR, immunohistochemistry and Fluorescent in situ hybridization to determine EGFR and HER2 expression in ESCC patients, and direct sequencing and PCR-RFLP for mutations and polymorphism analysis.ResultsOur results showed an increased EGFR mRNA expression in tumors compared to surrounding tissue (p <0.05), with 11% of the cases presenting at least a four-fold difference between tumor and paired adjacent mucosa. EGFR protein overexpression was present only in 4% of the cases. The median expression of HER2 mRNA was not different between tumors and adjacent mucosa. Still, 7% of the tumors presented at least a 25-fold higher expression of this gene when compared to its paired counterpart. Immunohistochemical analysis revealed that 21% of the tumors were positive for HER2 (scores 2+ and 3+), although only 3+ tumors presented amplification of this gene. Mutation analysis for EGFR (exons 18-21), KRAS (codons 12 and 13) and BRAF (V600E) showed no mutations in any of the hotspots of these genes in almost 100 patients analyzed. EGFR presented synonymous polymorphisms at codon 836 (C>T) in 2.1% of the patients, and at codon 787 (G>A) in 79.2% of the cases. This last polymorphism was also evaluated in 304 healthy controls, which presented a similar frequency (73.7%) in comparison with ESCC patients. The absence of mutations of EGFR, KRAS and BRAF as well as the overexpression of EGFR and HER2 in less than 10% of the patients suggest that this signaling pathway is altered in only a small proportion of patients with ESCC.ConclusionHER receptors target therapies may have the potential to be effective in only a minor fraction of patients with ESCC.

Rs12976445 variant in the pri-miR-125a correlates with a lower level of hsa-miR-125a and ERBB2 overexpression in breast cancer patients

Expression of MIR125A is diminished in breast tumors, however the reason for the hsa-mir-125a decrease in the cancer is not known. HER2 is encoded by ERBB2, a target for hsa-miR-125a which interacts with the 3′UTR of ERBB2 mRNA. The present study reveals that a polymorphism (rs12976445) within the pri-miR-125a sequence correlates with the amount of mature hsa-miR-125a in breast tumor samples. miRNA, RNA and DNA were extracted from breast cancer samples obtained from 26 patients. Following immunohistological evaluation of the samples, the ERBB2, PGR and ESR1 mRNA profiles were also analyzed using real-time PCR. Genomic DNA was sequenced using MIR125A flanking primers. PCR products were analyzed using a BaeGI restriction enzyme specific to the rs12976445 variant. The rs12976445 variant (C/T and C/C) correlated with a lower level of hsa-miR-125a in comparison with the T/T variant. The expression of HER2 mRNA was increased in tumors with the rs12976445 variant (C/T and C/C) compared with T/T. We conclude that rs12976445 may be a potential prognostic marker of HER2 expression in breast cancer. Its predictive value on the efficacy of trastuzumab treatment in patients with HER2-positive breast cancer warrants further study.

In situ analysis of HER2 mRNA in gastric carcinoma: comparison with fluorescence in situ hybridization, dual-color silver in situ hybridization, and immunohistochemistry

The importance of anti-HER2 therapy has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. In the present study, we evaluated HER2 mRNA expression using a new mRNA in situ detection technique called RNAscope in 211 cases of formalin-fixed, paraffin-embedded gastric carcinoma. In addition, we compared the results with the conventional methods of immunohistochemistry, fluorescence in situ hybridization, and dual-color silver in situ hybridization. RNA in situ hybridization (in situ hybridization) showed that 162 cases (76.8%) were score 0, 5 cases (2.4%) were score 1, 10 cases (4.7%) were score 2, 13 cases (6.2%) were score 3, and 21 cases (10.0%) were score 4. HER2 transcription levels were found to be significantly related to pT class, pN class, and tumor recurrence. mRNA expression was well correlated with protein overexpression and gene amplification; 20 cases out of 23 with DNA amplification showed a score of 4 in RNA in situ hybridization (P < .001). Three cases showed false negative and one case showed false positive results by in situ hybridization. More studies are needed to determine whether the in situ hybridization method can identify additional patients that may benefit from anti-HER2 therapy or exclude those who may be resistant to anti-HER2 therapy.

226P - Exploratory Analysis of the Relationship Between Her2 Expression (BY QRT-PCR) and Efficacy with First-Line Trastuzumab Emtansine (T-DM1) Vs Trastuzumab Plus Docetaxel (HT) in a Randomized Phase 2 Study of Patients (PTS) with HER2-Positive MBC

ABSTRACT Background In TDM4450g (NCT00679341), 137 pts with HER2-positive MBC and no prior treatment for metastatic disease were randomized to T-DM1 or HT. Median progression-free survival (PFS) was 14.2 vs 9.2 mo, respectively (HR = 0.594); objective response rates (ORR) were 64.2% and 58.0%, respectively (Hurvitz 2011). In exploratory analyses of single-arm phase 2 MBC studies of T-DM1, greater expression of HER2 mRNA (assessed by qRT-PCR) was associated with greater ORR and longer PFS (Burris 2011; Krop 2012, in press). We now report a retrospective exploratory analysis of the relationship between qRT-PCR HER2 expression and efficacy outcomes in pts receiving T-DM1 or HT in TDM4450g. Table: 226P Median pre-treatment HER2 levels, concentration ratio units (range) HT T-DM1 (n = 61) 8.7 (0.5-105.0) (n = 55) 10.3 (0.4-103.0) Median PFS, months HER2 expression HT T-DM1 Hazard ratio relative to HT (95% CI) Low (n = 32) 9.8 (n = 26) 10.6 0.85 (0.44-1.67) High (n = 29) 8.8 (n = 29) Not reached 0.39 (0.18-0.85) ORR, % HER2 expression HT T-DM1 Odds ratio relative to HT (95% CI) Low (n = 31) 58.1 (n = 26) 53.8 0.84 (0.30-2.41) High (n = 29) 65.5 (n = 29) 72.4 1.58 (0.50-4.98) Methods Pts were randomized 1:1 to T-DM1 3.6 mg/kg q3w or H 6 mg/kg q3w (8 kg/mg in cycle 1) + T 75 or 100 mg/m2 q3w and treated until disease progression. HER2 expression was measured by qRT-PCR in archival tissue samples from all randomized pts. Efficacy outcomes included investigator-assessed PFS and ORR. The analysis results presented here are based on the clinical data with a median follow-up of ∼14 mo in each arm. Results In total, 122 tumor samples from 137 randomized pts were available for analysis of pre-treatment HER2 by qRT-PCR, resulting in a valid result for 116 pts (6 samples were below the limit of quantification). Efficacy outcomes by low ( Conclusions These results from an exploratory analysis suggest that the magnitude of efficacy from T-DM1 vs HT may correlate with HER2 expression levels (by qRT-PCR); this effect appears to be most pronounced for PFS. Analyses exploring the relationship between these findings and baseline characteristics are planned. Similar analyses are planned for ongoing phase 3 studies of T-DM1. Disclosure S. Hurvitz: SA Hurvitz has received support for study-related travel from Roche and research funding for her institution from Roche. L.C. Amler: Dr. Amler is employed by Genentech and has stock ownership in F. Hoffmann-La Roche. V. Ng: Dr. Ng is employed by Genentech and has stock ownership in F. Hoffmann-La Roche. E. Guardino: Dr. Guardino is employed by Genentech and has stock ownership in F. Hoffmann-La Roche. L. Gianni: L Gianni has had compensated consultant/advisory relationships with Genentech/Roche, GlaxoSmithKline and Pfizer. All other authors have declared no conflicts of interest.

Steroid receptor coactivators, HER-2 and HER-3 expression is stimulated by tamoxifen treatment in DMBA-induced breast cancer

BackgroundSteroid receptor coactivators (SRCs) may modulate estrogen receptor (ER) activity and the response to endocrine treatment in breast cancer, in part through interaction with growth factor receptor signaling pathways. In the present study the effects of tamoxifen treatment on the expression of SRCs and human epidermal growth factor receptors (HERs) were examined in an animal model of ER positive breast cancer.MethodsSprague-Dawley rats with DMBA-induced breast cancer were randomized to 14 days of oral tamoxifen 40 mg/kg bodyweight/day or vehicle only (controls). Tumors were measured throughout the study period. Blood samples and tumor tissue were collected at sacrifice and tamoxifen and its main metabolites were quantified using LC-MS/MS. The gene expression in tumor of SRC-1, SRC-2/transcription intermediary factor-2 (TIF-2), SRC-3/amplified in breast cancer 1 (AIB1), ER, HER-1, -2, -3 and HER-4, as well as the transcription factor Ets-2, was measured by real-time RT-PCR. Protein levels were further assessed by Western blotting.ResultsTamoxifen and its main metabolites were detected at high concentrations in serum and accumulated in tumor tissue in up to tenfolds the concentration in serum. Mean tumor volume/rat decreased in the tamoxifen treated group, but continued to increase in controls. The mRNA expression levels of SRC-1 (P = 0.035), SRC-2/TIF-2 (P = 0.002), HER-2 (P = 0.035) and HER-3 (P = 0.006) were significantly higher in tamoxifen treated tumors compared to controls, and the results were confirmed at the protein level using Western blotting. SRC-3/AIB1 protein was also higher in tamoxifen treated tumors. SRC-1 and SRC-2/TIF-2 mRNA levels were positively correlated with each other and with HER-2 (P ≤ 0.001), and the HER-2 mRNA expression correlated with the levels of the other three HER family members (P < 0.05). Furthermore, SRC-3/AIB1 and HER-4 were positively correlated with each other and Ets-2 (P < 0.001).ConclusionsThe expression of SRCs and HER-2 and -3 is stimulated by tamoxifen treatment in DMBA-induced breast cancer. Stimulation and positive correlation of coactivators and HERs may represent an early response to endocrine treatment. The role of SRCs and HER-2 and -3 should be further studied in order to evaluate their effects on response to long-term tamoxifen treatment.

A short-term neoadjuvant pilot study comparing exemestane and letrozole in postmenopausal women with estrogen receptor-positive HER2-negative breast cancer.

591 Background: Current approaches of neoadjuvant endocrine therapy (NAET) are focusing on biomarker analysis of the diagnostic specimens. One of the important things in neoadjuvant treatment is to determine the in-vivo responsiveness to the agent. Little is known regarding biomarker changes in specific agents and the association with biomarker changes and treatment duration. A pilot study was conducted to investigate the differences of biomarker changes in short-term NAET (letrozole vs. exemestane) and also the difference in treatment durations of each AI. Methods: Totally 120 samples from 60 postmenopausal women with ER positive HER2 negative stage 1-3 breast cancers in NAET comparing letrozole (LET) and exemestane (EXE) were analyzed with pre- and post-treatment mRNA expression of ERα, ERβ, PR, HER2, Ki67, PIK3CA, and MAPK using QuantiGene Assay. Thirty-one patients took letrozole 2.5mg daily and 29 did exemestane 25mg daily until the surgery (median duration 22 days). Statistical tests were two-sided. Results: Clinical characteristics were well balanced between the two treatment arms. Median % changes of Ki67 were -58%, ERα -44%, ERβ +93%, and PR -69% in LET and Ki67 -59%, ERα -30%, ERβ +135%, PR -72% in EXE. There were no significant differences of these marker changes between two agents. Dividing into 4 groups in treatment duration, 10-14 days (q1), 15-21 days (q2), 22-28 days (q3), and over 29 days (q4), LET had 2 peaks in Ki67 decreases (-69% (q1), -28% (q2), -78% (q3), -59% (q4)), whereas EXE had a peak in q2 (-62%, -77%, -49%, -26%). Ki67 increasing cases including primary resistance and tachyphylaxis were 5 (16%) in LET (3 in q2 and 2 in q4) and 3 (10%) in EXE (one of each in q2-4). All 8 cases had increases of ERβ, PIK3CA, and MAPK. Conclusions: Although both LET and EXE for short duration had significantly decreased Ki67 mRNA level irrespective of treatment duration, no significant differences was found between two agents. Tumor resistance with AIs may be associated with the other pathways.

Extracellular matrix and breast cancer cells' response to trastuzumab: The role of glycosaminoglycans, heparanases, and syndecan-1.

10568 Background: Trastuzumab is an antibody anti-epidermal growth factor 2 receptor (HER2), which improves disease-free and overall survival in HER2 positive breast cancer. Nevertheless, many patients become resistant to this treatment. Heparanase (HPSE) is an enzyme that is responsible for removal of heparan sulfate (HS) chains from proteoglycans, generating free oligosaccharides that modulate many physiopathological functions, including tumor developing. We have analyzed whether some extracellular matrix components influence trastuzumab efficacy. Methods: Heparanase-1 (HPSE-1) overexpression effect was analyzed using MCF7 cells stable transfected with HPSE-1 cDNA (MCF7-HPSE-1). HPSE-1, HPSE-2, Syndecan-1 (Syn-1) and HER2 expression, HPSE-1 activity and cell viability were evaluated in different breast cancer cells treated or not with trastuzumab. The glycosaminoglycans synthesis and shedding were also evaluated. Trastuzumab and HS binding were analyzed by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). Results: MCF7 transfected with HPSE-1 cDNA becomes completely resistant to trastuzumab. HS affinity by Trastuzumab was then tested, showing that they bind in high levels and this binding is necessary to antibody activity. In MCF7 cells, trastuzumab decreases HPSE-1, HPSE-2, HER2 and Syn-1 mRNA expression, while in MCF7-HPSE-1 the antibody increases the expression of these molecules. Conclusions: Our results have demonstrated that an ideal concentration of HS in cell surface, regulated by trastuzumab, is necessary to its action, beyond HER2 high levels. High HS concentration at cell surface enhances the antibody amount disposable to interact with HER2 in cell surface, determining breast cancer cells susceptibility to trastuzumab. These new insights could be useful when devising strategies for overcoming trastuzumab resistance in HER2 positive cancers. Supported by FAPESP, CNPq, CAPES.

Impact of expression levels of mRNA HER2 and ESR1 on the pathologic complete remission (pCR) rate after neoadjuvant treatment with anthracycline-taxane containing chemotherapy in combination with trastuzumab in the GeparQuattro trial.

523 Background: Recent data suggest that benefit from (neo)adjuvant trastuzumab might be related to expression of HER2 and estrogen receptor (ESR1). We investigated mRNA levels of HER2 and ESR1 in core biopsies in HER2 positive tumors of the GeparQuattro trial and compared it to response to neoadjuvant treatment. Methods: In the GeparQuattro trial 445 HER2+ pts were included based on local testing and received neoadjuvant trastuzumab and chemotherapy (either 4 x EC →4 x docetaxel (D) or 4 x EC → 4 x D/capecitabine (C) or 4 x EC →4x D →4 X C). In 217 available pretherapeutic core biopsies HER2 levels were analysed by IHC, SISH and quantitative RT-PCR using predefined cutoffs; pCR was defined as ypT0is;ypN0. Results: Only 73% of the tumors (158 of 217) were centrally HER2 positive (cHER2+) by IHC/SISH, while 59 tumors (27%) were centrally HER2 negative (cHER2-). As all pts had received neoadjuvant trastuzumab, this gave us the possibility to evaluate response of HER2- tumors to trastuzumab. The pCR rate of cHER2+ tumors was significantly increased (46.8%, p&lt;0.0005) compared to cHER2-, who had a pCR rate of only 20.3%. Similar results were obtained if HER2 positivity was determined by RT-PCR (pCR rate 50% vs. 17.4%, p&lt;0.0005). Assessment of HER2 status by RT-PCR showed a concordance of 86.2% with the IHC/SISH status. In uni- and multivariate logistic regression analysis of cHER2+ cases including continuous HER2 and ESR1 mRNA levels the HER2 mRNA expression was significantly associated with prediction for a pCR in (univ.: OR 1.43, 95% CI 1.11-1.83, p=0.005; multiv.: OR 1.42, 95% CI 1.11-1.83, p=0.006). In the cHER2+ tumors, the pCR rate was similar for ESR1 mRNA positive (47.4%) and ESR1 negative tumors (46.3%). Conclusions: Only pts with cHER2+ tumors independently of the method used have an additional benefit in terms of a pCR from adding trastuzumab to chemotherapy. In cHER2-negative patients the pCR rate is comparable to the pCR rate in the non trastuzumab treated population. Increasing HER2 mRNA levels were associated with a better response to trastuzumab based treatment in cHER2 + tumors.

Abstract 315: Potential Regulation of Intimal Hyperplasia by ADAM12 and Polo-Like Kinase-1 in Human Saphenous Vein Grafts in Coronary Artery Bypass Patients

Proliferation and migration of smooth muscle cells (SMCs) are the key events in the development of intimal hyperplasia, causing coronary artery bypass graft (CABG) failure. Although internal mammary artery (IMA) is comparatively immune to restenosis, saphenous veins (SV) are most commonly used bypass conduits. The underlying mechanisms that makes SV graft more prone to restenosis are not known as yet. The matrix metalloproteinases (MMPs) and kinases may be potential contributing factors in uncontrolled proliferation, migration and invasion of the graft conduit. In this study, we compared SMCs cultured from SV with IMA of the same patients undergoing CABG. The mRNA expression of polo-like kinases (PLKs), HER-2, ADAM 12L (long-form) and ADAM12S (short-form) were quantified by real-time PCR. The mRNA expression of cell proliferation related protein Ki-67 was measured in both conduits. Migration rate was determined using scratch assay. The mRNA expression of PLK-1, PLK-3, PLK-4, HER-2, ADAM 12L and ADAM12S were significantly higher in human SV SMCs compared to IMA SMCs. Also, SV SMCs showed higher proliferation and migration than IMA SMCs, which may be associated with increased expression of PLKs, ADAM 12 and HER-2. The protease domain of ADAM12 can cleave ECM substrates and cause migration. PLK1 is overexpressed in a variety of pathophysiological conditions and its expression correlates with cellular progression. In contrast, PLK-2 mRNA expression was higher in IMA SMCs compared to SV SMCs. The kinase activity of PLK-2 inhibits oncogenic transformation. Thus, overexpression of PLK 1 and ADAM12 in human SV bypass conduit may cause uncontrolled proliferation, migration and invasion leading to its increased incidence of intimal hyperplasia and vein graft failure in CABG patients.

CK19, CK20, EGFR and HER2 Status of Circulating Tumor Cells in Patients with Breast Cancer

The major cause of death in breast cancer patients is metastasis. Various biomarkers have been used for the early detection of circulating tumor cells in the peripheral blood of breast cancer patients. The aims of the current study were to analyze circulating tumor cells in the blood of breast cancer patients by investigating EGFR, CK19, CK20 and HER2 expression profiles and to evaluate their prognostic importance. CK19, CK20 and EGFR gene expression profiles were evaluated in the blood samples of 84 female patients with primary invasive ductal breast cancer and 20 healthy female volunteers using SYBR green-based real-time qPCR assays. HER2 expression analyses were conducted in 46 patients who had an HER2-positive primary tumor and in 30 healthy women to determine the cutoff level of positivity. The positive rates of CK20, EGFR, CK19 and HER2 mRNA expression in the peripheral blood were 28.57% (24/84), 20.23% (17/84), 5.95% (5/84) and 2.17% (1/46), respectively. The high positive ratio of CK20 mRNA expression in the peripheral blood of breast cancer was identified for the first time in the current study. Significant differences were identified in CK20 expression status and several clinical parameters related with aggressiveness of tumors using a binary logistic regression analysis. Higher CK20-positive levels were observed in patients who had lymph node metastasis and advanced-grade primary tumors, which were estrogen receptor-negative. We have demonstrated that CK20 may be a novel biomarker that is useful to identify circulating tumor cells and predict breast cancer progression. The results suggest that the investigation of CK20 mRNA with other biomarkers in the peripheral blood of breast cancer patients may be useful to monitor the presence of disseminated tumor cells in the blood circulation and to predict the prognosis of breast cancer.

HER2 and TOP2A in high-risk early breast cancer patients treated with adjuvant epirubicin-based dose-dense sequential chemotherapy

BackgroundHER2 and TOP2A parameters (gene status, mRNA and protein expression) have individually been associated with the outcome of patients treated with anthracyclines. The aim of this study was to comprehensively evaluate the prognostic/predictive significance of the above parameters in early, high-risk breast cancer patients treated with epirubicin-based, dose-dense sequential adjuvant chemotherapy.MethodsIn a series of 352 breast carcinoma tissues from patients that had been post-operatively treated with epirubicin-CMF with or without paclitaxel, we assessed HER2 and TOP2A gene status (chromogenic in situ hybridization), mRNA expression (quantitative reverse transcription PCR), as well as HER2 and TopoIIa protein expression (immunohistochemistry).ResultsHER2 and TOP2A amplification did not share the same effects on their downstream molecules, with consistent patterns observed in HER2 mRNA and protein expression according to HER2 amplification (all parameters strongly inter-related, p values < 0.001), but inconsistent patterns in the case of TOP2A. TOP2A gene amplification (7% of all cases) was not related to TOP2A mRNA and TopoIIa protein expression, while TOP2A mRNA and TopoIIa protein were strongly related to each other (p < 0.001). Hence, TOP2A amplified tumors did not correspond to tumors with high TOP2A mRNA or TopoIIa protein expression, while the latter were characterized by high Ki67 scores (p = 0.003 and p < 0.001, respectively). Multivariate analysis adjusted for nodal involvement, hormone receptor status, Ki67 score and HER2/TOP2A parameters revealed HER2/TOP2A co-amplification (21.2% of HER2 amplified tumors) as an independent favorable prognostic factor for DFS (HR = 0.13, 95% CI: 0.02-0.96, p = 0.046); in contrast, increased HER2/TOP2A mRNA co-expression was identified as an independent adverse prognostic factor for both DFS (HR = 2.41, 95% CI: 1.31-4.42, p = 0.005) and OS (HR = 2.83, 95% CI: 1.42-5.63, p = 0.003), while high TOP2A mRNA expression was an independent adverse prognostic factor for OS (HR = 2.06, 95% CI: 1.23-3.46, p = 0.006). None of the parameters tested was associated with response to paclitaxel.ConclusionsThis study confirms the favorable prognostic value of HER2/TOP2A co-amplification and the adverse prognostic value of high TOP2A mRNA expression extending it to the adjuvant treatment setting in early high-risk breast cancer. The strong adverse prognostic impact of high HER2/TOP2A mRNA co-expression needs further validation in studies designed to evaluate markers predictive for anthracyclines.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12611000506998.

P1-07-01: Comparison of Four HER2 Testing Methods in the Detection of HER2−Positive Breast Cancer: Results from the FinHer Study Cohort.

Abstract Background: Accurate assessment of the HER2 status is essential for identifying patients who may benefit from HER2 targeted therapy. The current methods, immunohistochemistry (IHC) and in situ hybridization (ISH), determine HER2 status semi-quantitatively as positive (+), equivocal (+/−) and negative (−) with predefined cutoff values. Recent studies have suggested that current HER2 cutoffs may not be optimal for all clinical settings of HER2 targeted therapy. In a small subset of adjuvant NCCTG N9831 patients confirmed as HER2−normal by round-robin review of HER2 testing, trastuzumab benefit was observed (Perez et al, SABCS 2010). Quantification of HER2 as continuous variable may enable a more accurate optimization of HER2 cutoffs for various HER2 targeted therapies. In this study, we measured continuous HER2 protein expression by the HERmark™ assay and continuous mRNA expression by quantitative real time polymerase chain reaction (qPCR), and compared these results with central IHC and central chromogenic in situ hybridization (CISH) results of FinHer. Methods: Total HER2 protein expression (H2T) was quantified using the HERmark assay as previously described (Huang et al. Am J Clin Pathol 2010;134:303). HER2 mRNA expression (H2N) was measured by qPCR as previously published (Noske et al. Br Cancer Res Treat 2011;126:109). The results of H2T and H2N as continuous variables and as predefined categories were compared with central CISH results from FinHer (Joensuu et al, N Engl J Med 2006;354), and central IHC retesting. Results: H2T in 899 evaluable samples described a continuum of 0.4 to 721.2 (relative HERmark unit); while H2N in 915 evaluable samples showed a continuum of 31.4 to 42.8 (delta-Ct). Significant correlation between H2T and H2N as continuous variable was found (R2= 0.56, P&amp;lt; .0001). Paired method comparison was performed for samples with valid results in any two of the four testing methods. Overall concordance of H2T and H2N with predefined categories (+, +/−, -) was 81%, and concordance of (+) and (−) subsets was 95% when (+/−) cases (H2T 11%; H2N 6%) were excluded. Overall concordance of central IHC and H2T categories (+, +/−, -) was 75%, and concordance of (+) and (−) subsets was 96% when (+/−) cases (IHC 16%; H2T 11%) were excluded. Overall concordance of IHC and H2N categories (+, +/−, -) was 84%, and concordance of (+) and (−) subsets was 99% when (+/−) cases (IHC 16%; H2N 6%) were excluded. Concordance of central CISH (+, -) with H2T and H2N categories (+, -) was 89% and 91%, respectively, when (+/−) cases were excluded from H2T (13%) and H2N (8%), respectively. Conclusions: All four methods identified HER2−positive breast cancers. The discordance rate between the methods tested was approximately 10 to 20% despite careful delineation of cancerous tissue in the sample and analysis of adjacent tumor sections. No combination of assays could be identified with concordance rate &amp;gt;95% when the equivocal subsets were included in comparisons. Exclusion of the equivocal subsets (about 10% of samples) yielded high concordance rates of approximately 95% or higher. H2T and H2N showed comparable continuous distribution patterns and significant concordance with standard HER2 status by central IHC and CISH. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-01.

P2-12-04: RACGAP1 mRNA Assay Outperforms Ki-67 as a Proliferation Marker in the FinHer Study Cohort.

Abstract Background: Molecular subtyping of breast cancer has become an integral part of standard evaluation of breast cancer patients. Their assessment requires combining data from analyses on ER, PR, HER2 and cell proliferation markers. However, their immunohistochemical (IHC) testing carries an up to 20% risk of erroneous results. Similarly, assessment of cell proliferation by Ki-67 staining is hampered by lack of standardization of laboratory methods and agreement on cut-offs. Here we tested the prognostic value of objective quantitation of ESR1, PgR, HER2 and the proliferation markers RACGAP1 using RT-qPCR and compared the results with local and central IHC assessments. Methods: RNA was extracted from FFPE tumor tissue of 917 patients who participated in the FinHer trial. ESR1, PgR, HER2 and RACGAP1 mRNA expression were measured using RT-qPCR. The molecular subtypes (luminal, HER2−enriched and triple-negative) were determined. Prognostic significance of proliferation markers was assessed using univariate and multivariate analyses. The RT-qPCR results were compared with local and central IHC results. Results: HER2 mRNA showed a bimodal distribution with 197 (21.4%) out of the 917 tumors being above the predefined cut-off. HER2 mRNA expression increased in parallel with HER protein expression. Overall concordance of HER2 mRNA testing with central IHC and CISH was good, while local IHC testing suffered higher false positive rates. RACGAP1 mRNA expression was the greater the higher the histological grade. ESR1 and PgR mRNA correlated negatively with the histological grade (r=-0.38 and r=-0.33; p&amp;lt;0.0001), whereas HER2 and RACGAP1 mRNA were correlated positively (r=0.10 and r=0.49; p=0.002 and p&amp;lt;0.0001, respectively). RACGAP1 mRNA was negatively associated with ESR1 and PgR mRNA (r=-0.17 and r=-0.26, respectively; p&amp;lt;0.0001 for each). Molecular subtypes determined by RT-qPCR using predefined cut-off values were highly prognostic for overall survival (OS) (p&amp;lt;0.001). The 5-year OS rate for patients with luminal cancer was 94% and 86% for HER2−enriched cancer and 84% for triple-negative cancer. In the subset of luminal tumors, high expression of RACGAP1 identified a population of patients who were at a high risk of death (5-year OS 82% versus 95%; p&amp;lt;0.0001). In a multivariate analysis RACGAP1 mRNA expression, nodal status and chemotherapy type were independent prognostic factors, whereas IHC of ER, PgR, Ki-67 and histological grade were not significant. Conclusions: Molecular subtyping of breast cancer by RT-qPCR using RNA isolated from FFPE tissue proved successfully in this large patient cohort. RACGAP1 mRNA expression distinguished high and low risk luminal breast cancers. In a multivariate analysis mRNA-based molecular markers outperformed the immunohistochemical markers ER, PgR and Ki-67. Of note, quantitative assessment of the proliferation marker RACGAP1 was superior to semiquantitative assessment of Ki-67 from routine FFPE tissues using IHC. We conclude that quantitative assessment of ESR1, PgR, HER2 and RACGAP1 mRNA by RT-qPCR is a robust and reproducible method to assess these key tumor biological factors from archival FFPE tumor tissue. RACGAP1 is novel cell proliferation marker in breast cancer that warrants further validation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-04.

S5-1: Neoadjuvant Pertuzumab (P) and Trastuzumab (H): Biomarker Analyses of a 4-Arm Randomized Phase II Study (NeoSphere) in Patients (pts) with HER2−Positive Breast Cancer (BC).

Abstract Background: NeoSphere showed that P with H and docetaxel (T) had a significantly higher pCR rate (45.8%) compared with TH (29%) or other P combinations (HP, TP) (Gianni, SABCS 2010). HER2+/ER+ and HER2+/ER− BC have different patterns of gene expression, and treatment response seems to be driven by different biologic pathways (Bianchini, ASCO 2011). This effect was also seen in NeoSphere. A broad panel of biomarkers (BMs) was assessed in tumor specimens and in sera to characterize the molecular profile of the ITT population and to explore BMs with different treatments and in different subsets of pts. Methods: Core biopsies and sera from 387/417 pts collected at baseline were available for BM analyses. HER2, all its truncated forms, HER3, IGF1R, PTEN, and pAKT were assessed by IHC and a modified H-score was derived per each marker. HER1, HER2, HER3, amphiregulin (AREG) and betacellulin mRNA expression in tumor tissue was assessed by qRT-PCR. C-myc was assessed by FISH. Mutational analyses of 8 hot spots within PIK3CA on tumor DNA was done via TaqMan-PCR. ELISA was used to detect HER2 extracellular domain (ECD), AREG, EGF, and TGFα in serum. Treatment interaction test was performed to test for association between BMs and pCR. In the analysis, median values were used as cutoffs, except for PTEN, PIK3CA mutation, and c-myc amplification, where cutoffs were applied following a biologic rationale. Results: In the overall population, high HER2 membrane protein expression measured by modified H-score was associated with a significantly higher pCR rate in the THP arm (p=0.001), with a large benefit from combination of P with TH (OR=3.74, CI 80%, 2.21−6.34; p=0.0009). The cutoff derived from NeoSphere has no immediate clinical applicability. A decreased likelihood of pCR was detected for pts with PI3K mutations in all 4 arms in a preliminary analysis limited to about 70% of cases. A final analysis will be presented. A significantly different distribution of BM expression in the ER+ vs ER− subgroups was found for 10 of 16 BMs (FDR&amp;lt;0.05). Low levels of IGF1R were found to be associated with higher pCR with combination of P with TH in the ER− (OR=4.40, CI 80%, 2.17−8.9; p=0.006) but not ER+ subgroup. High HER2 mRNA expression was associated with higher pCR rate in the HP arm in ER− (p=0.021) but not ER+ (p=0.74) tumors. Data on the full BM analysis including those on the ratio between HER2 ECD and intracellular domain (p95) will be presented. The levels measured for AREG and betacellulin were generally very low (less than the limits of variability for these assays), hence further analysis was not felt meaningful. Summary: The level of HER2 membrane protein expression was associated with increased benefit from combination of P with TH. The PI3K mutational status appears to have a relevant role in defining the likelihood of response with all treatments. The different treatment effects according to ER status are reflected in a different molecular BM profile in ER+ vs ER− tumors, with the IGF1R membrane score being correlated with ER− tumors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S5-1.

P4-16-02: Problems with Identifying Bone Metastasis-Specific Genes without Considering Biological Differences between ER-Positive and ER-Negative Breast Cancers.

Abstract Background: Bone metastasis-specific genes in breast cancer have been reported without considering the significant differences in ER status between bone and non-bone metastases. The aims of this study were to validate genes that had been reported as bone metastasis-specific genes using our data set and to identify bone metastasis-specific genes on the basis of biological differences between ERpositive and ER-negative breast cancers. Patients and Methods: We used Affymetrix GeneChip arrays to analyze tumor samples obtained from 365 primary invasive breast cancer patients who underwent surgery from 1999 to 2008. We excluded patients with HER2−positive breast cancer (normalized HER2 mRNA expression [probe set 216836_s_at] &amp;gt; 12.54). We classified the samples into 3 cohorts according to first metastatic site: bone, non-bone, or no metastasis. Differential expression of genes between bone and non-bone cohorts that were differentially expressed was identified using the Cox proportional hazards model, and gene sets was assessed using gene-set analysis. Results: Of the 365 patients, 34 (9.3%) were included in the bone cohort and 32 (8.8%) in the non-bone cohort. Two hundred fourteen (58.6%) had ER-positive and 151 (41.4%) had ER-negative breast cancer. First, we performed gene-set analysis using 5 gene sets that had been reported to be associated with bone metastasis. One gene set, which had been detected using an ER-negative breast cancer cell line, was validated as predicting bone metastasis in ER-positive breast cancer. None of the 5 gene sets predicted bone metastasis in ER-negative breast cancer. We then determined the levels of individual genes associated with bone metastasis by ER status using all 16,712 probe sets filtered by average gene expression level. When we analyzed all patients without any stratification by ER status, as in previous studies, 592 probe sets were significantly overexpressed in the bone cohort compared with the non-bone cohort, with a false discovery rate of ≤0.05. However, when we analyzed ER-positive and ER-negative breast cancers separately, no genes were found with significant differences between bone and non-bone cohorts. Finally, we used 2,246 functionally annotated gene sets assembled from Gene Ontology to examine possible biological differences between bone and non-bone cohorts. In the bone cohort, 151 and 125 gene sets were significantly overexpressed in ER-positive and ER-negative breast cancers, respectively (P ≤ 0.05). Ppathways related to Cellular growth and proliferation, and intracellular and second-messenger signaling were overexpressed in ER-positive breast cancer, whereas pathways related to nuclear receptor and cytokine signaling were overexpressed in ER-negative breast cancer. Most bone-metastasis-related pathways were different in ER-positive and ER-negative breast cancers (91.4% and 89.6% of the gene sets, respectively). Discussion: No genes were found that can predict bone metastasis. ER-positive and ER-negative breast cancers have different biological potentials for bone metastasis. Therefore, we need to assess the prediction model of bone metastasis based on the biological features for each ER status separately. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-16-02.

Correlation of expression of Survivin, BCRP and HER-2 genes with therapeutic response of TE regimen neoadjuvant chemotherapy in breast cancer patients

To study the changes of expression of Survivin mRNA, BCRP mRNA and HER-2 mRNA in breast cancer after TE regimen neoadjuvant chemotherapy, and to find biological markers to predict the efficiency of TE regimen neoadjuvant chemotherapy. The gene expressions were detected by RT-PCR from 56 breast cancer patients before and after TE regimen neoadjuvant chemotherapy (docetaxel and epirubicin). The relationships between these gene expressions and chemotherapy responses were analyzed. The overall response rate to neoadjuvant chemotherapy was 71.4%, including 8.9% (5/56) with complete response and 62.5% (35/56) with partial response. Pathological complete response was found in 4 cases (7.1%). Stable disease and progression of disease were 23.2% (13/56) and 5.4% (3/56), respectively. The expression of Survivin mRNA after neoadjuvant chemotherapy was 35.7% (20/56), significantly lower than 60.7% (34/56) before neoadjuvant chemotherapy (P = 0.008). The expression of BCRP mRNA after neoadjuvant chemotherapy was 19.6%, significantly lower than 37.5% before neoadjuvant chemotherapy (P = 0.036). The positive rate of HER-2 mRNA expression was 41.1% before the chemotherapy, and reduced to 21.4% after the chemotherapy (P = 0.025). The effective rates of the single positive expression of Survivin mRNA or BCRP mRNA were both lower than that of negative expression (P < 0.05). The level of HER-2 mRNA expression alone was not significantly associated with the effective rate of chemotherapy (P = 0.144). When the expression of all Survivin mRNA, BCRP mRNA and HER-2 mRNA were negative, the effective rate of neoadjuvant chemotherapy was higher than that in patients with positive expression (P = 0.003). The level of Survivin mRNA expression was not significantly associated with BCRP mRNA and HER-2 mRNA (P > 0.05). The expression of Survivin in combination with BCRP and HER-2 is associated with clinical response to TE neoadjuvant chemotherapy in breast cancer, and can be used as predictive biomarkers for chemosensitivity of TE regimen neoadjuvant chemotherapy for breast cancer.