HER2 Breast Cancer Patients Research Articles

Abstract P6-09-02: Improved clinical outcomes associated with vitamin D supplementation during adjuvant chemotherapy in patients with HER2+ non-metastatic breast cancer

Abstract Background Vitamin D (VitD) supplementation decreases the risk of osteoporotic fractures in the elderly; however, its extraskeletal benefits, especially in the prevention and treatment of breast cancer, are less well-established. Many studies have shown an association of low VitD levels with higher cancer incidence, including breast cancer and poorer outcomes, but whether this association merely reflects a selection bias to healthier lifestyles, remains an area of controversy. We hypothesized that women with more aggressive HER2+ breast cancer would have improved clinical outcomes while on VitD supplements. Methods We performed a retrospective review of all patients (n = 300) given trastuzumab chemotherapy between 2006 and 2012 at UM/SCCC. We identified two groups of patients for comparison - those who received VitD supplementation (VD) during adjuvant chemotherapy (n = 130) or none (NVD) during adjuvant chemotherapy (n = 123). Patients who lacked sufficient records to clarify VitD supplement use, men, patients with de-novo-metastatic breast cancer, bilateral breast cancers, and patients without follow-up were excluded. Five-year disease-free survival (DFS) and overall survival (OS) were calculated. Univariate and multivariate analyses were performed using a Cox proportional hazards (CPH) model to evaluate the relationship between VD supplementation and death. Results The median age at diagnosis was 54 and 50 in the VD and NVD groups. In the VD group, the average VitD dose was 10,890 IU/wk, and the baseline and post-25-H VitD serum level was 35 and 41ng/ml, respectively. Descriptive analysis of the VD and NVD groups were as follows: postmenopausal (55.4%, 43.9%), tumor <2cm (42.3%, 36.6%), no lymph node involvement (42.3%, 36.6%), LVI (46.4%, 33.3%), high nuclear grade (60%, 61.5%), HR+ (66.2%, 54.5%), African American race (4.6%, 9.8%), and BMI>30 at end of chemotherapy (26.2%, 31.7%). At a median follow-up of 31 and 23 months, the estimated five-year DFS (69.4% vs. 44.7%, p = 0.009) and OS (97.5% vs. 85.6%, p = <0.0001) were significantly superior in the VD group versus the NVD group. These differences remained significant after adjustment for age, ethnicity, menopausal state, tumor size, node positivity, LVI, high-grade tumor, HR+, and BMI>30. Analysis showed an interaction between OS and ethnicity (African American = 0.008) and node positivity (p = 0.02) and near-significance for LVI (p = 0.07). Despite those confounding variables, VitD use remained significantly associated with improved OS (p = 0.003) and had a HR or 0.10 with a 95% CI of 0.02-0.45. Conclusion Our study suggests that Vitamin D supplementation in non-metastatic HER2 breast cancer patients is associated with improved DFS and OS, and the relationship remains significant after adjusting for potential confounding factors. It is unclear whether vitamin D supplementation might have pre-selected for HER2+ breast cancers with more favorable prognosis or synergized with anti-HER2 therapy. To our knowledge, this is the first study reporting improved outcomes associated with relatively high dose Vitamin D supplementation in the HER2+ breast cancer population. Further research is warranted to define the role of Vitamin D in breast cancer treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-02.

Abstract P4-13-04: Autologous dendritic cells vaccines combined with neoadjuvant chemotherapy increase total pCR in stages II-III non-overexpressing HER2 breast cancer patients and induce phenotypic changes in peripheral blood

Abstract Background Based on the synergistic effect between immuno- and chemotherapy (CT), we have elaborated an autologous vaccine with dendritic cells loaded with patients´ own tumor antigens (lysate), and we have already demonstrated that the addition of the vaccines to a standard neoadjuvant CT schedule has increased total pCR (breast+ axilla) in stages II-III non-overexpressing HER2 breast cancer patients (Santisteban M, SABCS 2012). Both cohorts, the control (C) and the vaccinated (V) were well balanced related to demographic characteristics. Toxicity has been similar in both the C and the V cohorts. Moreover, we have analyzed the phenotypic changes in peripheral blood induced by the vaccine and its correlation with pathologic responses. Indeed, we have studied if the amount of lysate used to load the dendritic cells or the total dendritic cell numbers received by the patients in the first five doses (before surgery) is correlated with pCR Methods Twenty-eight patients with stage II-III HER2 negative breast cancer have started on sequential neoadjuvant CT based on dose dense antracyclines (E 100mg/m2 and C 600 mgr/m2) x4 cycles plus GM-CSF followed by taxanes (DOC 75-100 mgr/m2) x4 cycles plus vaccination. The C historic cohort was composed of thirty patients who received the same treatment except for the absence of the vaccines. Vaccine calendar was started after the 4th EC and alternated with DOC and as maintenance up to a maximum of a 2 year-period. The first 5 vaccines were administered before breast surgery. Changes in different lymphocytes populations were measured in peripheral blood of patients at different points by flow cytometry (absolute cell counts). To date, twenty-one patients have both determinations of lymphocyte subpopulations before the 1st and the 6th vaccine. Paired samples t-tests and Fisher exact were used Results pCR was superior in the V cohort (24% versus 3.3%, p = 0.04). Lymphocyte subpopulations were measured in peripheral blood (cells/uL) and a stimulation of the immune system was found after the 5 vaccines schedule at the time of surgery as follows: NK (p<0.001), T cytotoxic CD8 (p = 0.018), T helper CD4 (p = 0.04), CD19 (p = 0.001), HLADRCD8 (p = 0.007), CD16CD8 (p = 0.003), HLADRCD4 (p<0.001), CD16CD4 (p = 0.008) and T regulators lymphocytes (p = 0.004). We did not find any differences among CD57CD8 (p = 0.17), CD56CD8 (p = 0.11), CD57CD4 (p = 0.45) and CD56CD4 (p = 0.65). We neither see correlation among the amount of lysate to load dendritic cells and the tpCR (p = 0.09) nor the amount of dendritic cells (summatory of 5 vaccines) administered intradermally and the pCR (p = 0.59) Conclusions Immunotherapy added to standard neoadjuvant CT could improve total pCR in stage II-III non-overexpressing HER2 breast cancer patients. After 5 doses of vaccination plus chemotherapy, we can observe phenotypic changes in peripheral blood: some immune system subpopulations increased statistically after the treatment in vaccinated patients. Neither the amount of lysate nor the number of dendritic cells used in the five first vaccines significantly correlated with the pRC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-13-04.

Abstract B123: Analysis of the associations of FC gamma receptor polymorphisms (FCGR3A, FCGR2A, and FCGR2B) with outcome in HER2+ breast cancer patients treated with trastuzumab in NCCTG (Alliance) trial N9831

Abstract Background: In the adjuvant setting, HER2 breast cancer patients treated with trastuzumab along with chemotherapy have superior survival compared to patients treated with chemotherapy alone (Perez EA, et al. J Clin Oncol. 2013 31:2115-22). Functional evidence suggests a role for the FC gamma receptor genes in antibody response. Mice deficient in the FC gamma receptor genes show significantly reduced antitumor effect. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab and a polymorphism in FCGR2B (I232T, T allele) is associated with impaired negative regulatory activity. Association of FC gamma receptor polymorphisms with clinical response among trastuzumab-treated patients is equivocal with both positive (Musolino A, et al. J Clin Oncol. 2008 26:1789-96) and negative associations (Hurvitz SA, et al. Clin Cancer Res. 2012 18:3478-86), a lack of sufficiently powered clinical samples and a lack of cohorts with clearly demonstrated survival benefit to trastuzumab containing regimens. Methods: Genomic DNA was analyzed for FcyRIIIa, FcyRIIa, and FcyRIIb polymorphisms (FCGR3A V158F, FCGR2A H131R and FCGR2B I232T) using Taqman SNP real-time PCR assays (Applied Biosystems) in 1,336 patients from the N9831 clinical trial of adjuvant trastuzumab in HER2+ breast cancer. 1325 patients were evaluable. Patients in Arm A (N=419) received chemotherapy only. Patients in Arms B (N=469) and C (N= 437) were treated with chemotherapy and trastuzumab (sequentially in Arm B and concurrently in Arm C). We performed Kaplan-Meier analyses of disease free survival (DFS) by arm and genotype for each polymorphism. Results: There were only minor differences in demographics and tumor features when comparing the subset with DNA to other N9831 participants who did not provide DNA. Patients who provided DNA in Arms B and C demonstrated superior DFS relative to patients in Arm A who provided DNA (p<0.001). Genotype distributions in the N9831 population for FCGR2A and FCGR3A polymorphisms were in Hardy-Weinberg equilibrium, p>0.05 and FCGR2B, p>0.01. In contrast to previous reports in the metastatic setting, we did not find that trastuzumab-treated patients who had the FCGR3A 158 V and/or FCGR2A 131 H genotypes demonstrated superior DFS to those without those genotypes. Similarly, we did not see an effect of FCGR2B I232T with the exception that I/I patients (p<0.001) apparently derived more benefit, in terms of DFS, from trastuzumab than did T carriers (p=0.81). However, 5-year DFS was not different between these two groups, I/I and T carriers within Arms B/C. Conclusions: Our analysis failed to reveal strong associations between FCGR genotypes and outcome in patients with HER2+ breast cancer treated with trastuzumab. While the present data do not preclude a functional role for FC gamma receptors in the clinical activity of trastuzumab, the results suggest that these naturally occurring polymorphisms may have minor (if any) impact in the adjuvant setting, which is consistent with previous findings by Hurvitz and colleagues. Citation Format: Mark Pegram, Rebecca M. Olson, Nadine Norton, Kathleen Tenner, Karla L. Ballman, Raphael Clynes, Keith L. Knutson, Edith A. Perez. Analysis of the associations of FC gamma receptor polymorphisms (FCGR3A, FCGR2A, and FCGR2B) with outcome in HER2+ breast cancer patients treated with trastuzumab in NCCTG (Alliance) trial N9831. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B123.

C-Myc and Her2 cooperate to drive a stem-like phenotype with poor prognosis in breast cancer

The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial-mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2(+) breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2(+) breast cancer.

Autocrine/paracrine mechanism of interleukin-17B receptor promotes breast tumorigenesis through NF-κB-mediated antiapoptotic pathway

Gain of function of membrane receptor was a good strategy exploited by cancer cells to benefit own growth and survival. Overexpression of HER2 has been found to serve as a target for developing trastuzumab to treat 20-25% of breast cancer. However, little or none of the other membrane receptor was found to be useful as a potential target for breast cancer treatment since then. Here, we showed that amplified signaling of interleukin-17 receptor B (IL-17RB) and its ligand IL-17B promoted tumorigenicity in breast cancer cells and impeded acinus formation in immortalized normal mammary epithelial cells. External signal transmitted through IL-17RB activated nuclear factor-κB to upregulate antiapoptotic factor Bcl-2 and induced etoposide resistance. Elevated expression of IL-17RB had a stronger correlation with poor prognosis than HER2 in breast cancer patients. Interestingly, breast cancer patients with high expression of IL-17RB and HER2 had the shortest survival rate. Depletion of IL-17RB in trastuzumab-resistant breast cancer cells significantly reduced their tumorigenic activity, suggesting that IL-17RB and HER2 have an independent role in breast carcinogenesis. Furthermore, treatment with antibodies specifically against IL-17RB or IL-17B effectively attenuated tumorigenicity of breast cancer cells. These results suggest that the amplified IL-17RB/IL-17B signaling pathways may serve as a therapeutic target for developing treatment to manage IL-17RB-associated breast cancer.

Exosomal pMHC-I complex targets T cell-based vaccine to directly stimulate CTL responses leading to antitumor immunity in transgenic FVBneuN and HLA-A2/HER2 mice and eradicating trastuzumab-resistant tumor in athymic nude mice

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.

Generation of adaptive HER2-specific immunity in HER2 breast cancer patients by addition of trastuzumab to chemotherapy in the adjuvant setting: NCCTG (Alliance) study N9831.

522 Background: In the adjuvant setting, patients with HER2 breast cancer treated with trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induces HER2-specific antibodies which correlate with response to therapy in patients with HER2+ metastatic breast cancer. It remained unclear from those studies, however, whether the HER2-specific immunity played a role and if antibody immunity was associated with improved disease free survival in the adjuvant setting. In the present study, we addressed these questions by analyzing sera samples from a subset of patients enrolled in the NCCTG adjuvant trial, N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively. Methods: Pre-and post-treatment initiation sera were obtained from 50 women enrolled in N9831 (22 Arm A; 14 Arm B, and 14 Arm C). Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and presented as an index (>0.2 was considered a positive response). Results: Prior to therapy, across all three arms, N9831 patients had similar mean HER2 IgG levels (0.19 units in Arm A, 0.14 in Arm B, and 0.23 in Arm C, P=0.85). Following treatment, the mean levels of antibodies increased in Arm B to 0.35 units and in Arm C to 0.56 units and were higher (p<0.001) than in Arm A where levels did not increase. The proportion of patients who demonstrated antibody immunity increased by 9% in Arm A, 50% in Arm B and 28% in Arm C (P=0.026). Although the event rate was low in this cohort, Cox modeling suggested that larger increases in antibodies were associated with improved disease free survival (HR=0.23; p=0.04). Conclusions: These results show that the increased antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant and results from the inclusion of trastuzumab. The findings may have important implications for improving treatment outcomes in patients treated with trastuzumab.

Association of Human Epidermal Growth Factor Receptor 2 with Radiotherapy Resistance in Patients with T1N0M0 Breast Cancer

PurposePreclinical studies have shown that human epidermal growth factor receptor 2 (HER2) status is associated with resistance to radiotherapy (RT). In this study, we evaluated the overall survival of a T1N0M0 breast cancer cohort in Korea according to the use of RT and the HER2 status.MethodsWe analyzed data collected from 11,552 patients with invasive breast cancer who were enrolled in the Korean Breast Cancer Society Registration Program between 1999 and 2007. Data on the TNM stage, estrogen receptor status, progesterone receptor status, HER2 status, operation method, and the use of RT were analyzed.ResultsThe median follow-up period was 51 months. A significant improvement in overall survival after RT was observed only in the HER2(-) group. In this group, the 10-year overall survival rate was 95.5% for patients who did not receive RT and 96.3% for patients who received RT (p=0.037). In contrast, in the HER2(+) group, RT was not associated with a survival benefit (p=0.887). Multivariate analysis showed that RT was significantly associated with a reduction in mortality in the HER2(-) group (hazard ratio, 0.738; 95% confidence interval, 0.549-0.993; p=0.045).ConclusionWe found that postoperative RT was not associated with a survival benefit in HER2(+) breast cancer patients, suggesting that HER2(+) breast cancers could be RT resistant.

Clinical role of detecting circulating tumor cells

Using ultrasensitive molecular or immunological/cytometric approaches, identification of circulating tumor cells (CTC) thus far undetectable at primary diagnosis even by high resolution imaging technologies is feasible. Results from several clinical studies revealed that in breast cancer patients with metastatic disease positivity for CTC is associated with worse prognosis. Thus, information obtained from CTC research might support clinicians in assessing individual prognosis, stratifying patients at risk to systemic adjuvant anti-cancer therapies, early response evaluation and monitoring therapeutic efficacy. At present, the CellSearchTM system combining automated capturing of CTC, immunostaining and image analysis is the only standardized technology approved by the FDA for the detection of CTC in patients with metastatic breast, colorectal and prostate cancer [1–3]. However, evidence has emerged that most currently used CTC detection methods still lack sensitivity or specificity to find all CTC especially those that have lost epithelial features, e.g. in the course of epithelial-mesenchymal transition [4–7]. Therefore special emphasis is placed to the development of innovative new strategies with the capacity to track additional CTC and to identify those with the potential to initiate metastasis. In this context, discovery of approaches enabling further phenotypical and molecular characterisation of CTC, e.g., analysis of single cell whole genomes, next generation sequencing and gene expression analysis on CTC is pivotal particularly in view of the development of new personalized targeting therapies. It is also worth mentioning that detection and characterization of CTC within translational research projects has entered clinical trials also enrolling patients with early breast cancer. First promising results on CTC detection especially within treatment regimens including targeted therapy, e.g., trastuzumab directed against HER2 in breast cancer patients receiving neoadjuvant or adjuvant therapy have been published recently [8–11]. Furthermore, characterization of the HER2 status of CTC might help identifying additional patients profiting from an anti-HER2 treatment and getting insights into therapy-induced selection of CTC.

P2-11-09: EGFR Overexpression in Triple Negative Breast Cancer (TNBC) and Its Association with the Prognosis.

Abstract Objective: The aim of this study is to investigate EGFR expression in Triple Negative Breast Cancer (TNBC), and to find the relationship between EGFR overexpression and prognosis of TNBC, further to clarify the significance of EGFR in TNBC and provide valuable information for TNBC therapy. Methods: 42 triple ***negandection ***ssitive breast cancer patients(studying group) and 40 HER2(3+) breast cancer patients(controling group) who underwent surgery from January 2000 to December 2005 were analyzed. 82 cases of paraffin-embedded specimens were detected by Immunohistochemistry(IHC), fluorescence in situ hybridization(FISH) and polymerase chain reaction(PCR) to investigate the overexpression, amplification and mutation of EGFR gene. The distant-free survival(DFS) and overall survival(OS) of these patients were used to investigate the relationship between EGFR overexpression and the prognosis of TNBC. Results: 34(43.9%) EGFR overexpression was observed in all cases, while gene amplification was only 7(9.1%) cases. No EGFR gene mutation was found in all cases. Overexpression of EGFR occurring in 57.1% patients in TNBC group and 25.0% patients in HER2 group, and we didn't found any correlation between EGFR overexpression and clinicopathology. 50(61.0%) patients relapsed (TNBC 28,HER2 22) and 27(32.9%) patients died(TNBC 18,HER2 9) were observed during the more than 5 years follow-up. The 5-year DFS was 57.1% and 77.5% respectively, the 5-year OS was 71.4% and 87.5% in TNBC and HER2 groups. In TNBC group, the survival of the EGFR-overexpressing group patients was significantly lower than that of the non-EGFR-overexpressing group patients (p=0.018 for DFS, p=0.026 for OS); In HER2 group, no statistical difference was found (p=0.079 for DFS, p=0.055 for OS). Conclusions: This study showed that EGFR overexpression increased significantly in TNBC patients, which was no correlation with their clinico-pathological data. EGFR gene amplification was much less frequent than its overexpression. It suggested that EGFR gene amplification may not be the unique mechanism of EGFR overexpression in TNBC. There may be other possible mechanisms and pathways that cause EGFR overexpression. In addition, it may suggest that gefitinib therapy is useless in TNBC patients because we did not find any mutations in the tested exons of TNBC. EGFR overexpression may associate with a poor outcome of TNBC patients which suggest it could be a significant prognostic factor for TNBC patients. EGFR may play important role for molecular-targeting therapy of TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-09.

Abstract 3633: eEF2 in acquired lapatinib resistance in HER2 positive breast cancer cells

Abstract Approximately 25% of breast cancers overexpress HER2. Although HER2 targeted therapies, trastuzumab and more recently lapatinib, have improved prognosis for HER2 breast cancer patients not all HER2 positive tumors respond to HER2 targeted therapies. Using phosphoproteomic profiling, we examined mechanisms of resistance in an in vitro model of acquired lapatinib resistance. SKBR3 cells were conditioned in lapatinib containing media (200 nM) for six months, after which time the resulting SKBR3-L cells exhibited significantly reduced growth inhibition (27.7 ± 0.1 %) when treated with lapatinib (1 µM) treatment compared to parental SKBR3 (90.7 ± 0.2 %) (p<0.01). Using phosphoproteomic profiling, eukaryotic elongation factor 2 (eEF2) was identified as a phosphoprotein which was significantly altered in the SKBR3-L cells compared to the parental SKBR3 cells. One form of phospho-eEF2 was up-regulated and 6 forms were down-regulated in SKBR3-L compared to SKBR3. eEF2 is a phosphoprotein with an essential role in protein synthesis. Phosphorylation of eEF2 at Thr56 and Thr58 by eEF2 kinase inhibits eEF2 activity. eEF2 kinase activity is regulated by the mTOR pathway, via phosphorylation by p70S6 kinase. To examine the role of eEF2 in lapatinib resistance, levels of phospho-eEF2, eEF2, phospho-p70S6K and p70S6K, were examined in a panel of HER2 positive breast cancer cell lines following 24 hour treatment with lapatinib (1 µM). Treatment with lapatinib resulted in a decrease in phospho-p70S6K and an increase in phospho-eEF2, with no change in the levels of total protein, in lapatinib sensitive cell lines. In order to further examine the role the mTOR pathway and eEF2, plays in acquired resistance to lapatinib, the effect of rapamycin, alone and in combination with lapatinib, was investigated in SKBR3 par and SKBR3-L cells. Combined treatment with lapatinib (200 nM) and rapamycin (2 nM) results in similar inhibition of growth (64.5 ± 3.3 %) in SKBR3-L cells as lapatinib alone in SKBR3 parental cells (62.5 ± 0.6 %). In conclusion, response to lapatinib is associated with decreased eEF2 activity in HER2 positive cell lines. Furthermore, inhibition of mTOR signaling, which causes inhibition of eEF2 activity, appears to restore sensitivity to lapatinib in our model of acquired lapatinib resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3633.

Abstract ED4-2: Functional imaging: Getting beyond FDG-PET

Abstract Molecular imaging of metabolism and drug targets in the tumor may well be of additional value in the treatment of cancer patients. It can potentially be used for screening, ((pre) operative) staging, restaging and guiding targeted therapies. Molecular imaging is still in its infancy. It can be performed with various imaging modalities. Tracers used for this purpose can be labeled with either a contrast agent for magnetic resonance imaging (MRI), a fluorescent dye for optical imaging, or a radioactive nuclide for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging. We will focus on PET and SPECT imaging given their sensitivity and available results. Most data are gathered on the visualization of general processes such as glucose metabolism and DNA synthesis. The PET-tracer [18F]fluorodeoxyglucose (FDG) is a glucose analogue and is used to visualize glucose metabolism, which is often increased in cancer cells. Increased DNA synthesis can be imaged with the pyrimidine analogue PET-tracer [18F]fluoro-L-thymidine (FLT), where FLT tumor uptake reflects the proliferation rate of cancer cells. With the increasing knowledge of the molecular processes that drive cancer and the introduction of the molecular targeted drugs, interest has been raised to image characteristics of tumor cells such as hormone receptors, growth factors and growth factor receptors. Molecular imaging of these characteristics can provide serial non-invasive information about the status of these characteristics within the tumor and its micro-environment and across lesions in the entire body when whole body imaging is performed. This is of interest as these characteristics can change over time and vary across lesions. Molecular imaging of the ER with the PET tracer 16-α-[18F]fluoro-17-β-estradiol (FES) has shown a good correlation between FES tumor uptake and ER density. This tracer is currently being tested for various applications in clinical trials. The field of visualizing growth factors and growth factor receptors is evolving rapidly. We imaged HER2 in breast cancer patients with the SPECT tracer 111In-trastuzumab and recently also with the PET tracer 89Zr-trastuzumab. With 111In-trastuzumab SPECT we showed that in 13 out of 15 patients with metastatic HER2 overexpressing disease more lesions were detected than with conventional staging procedures. Additionally the PET tracer 89Zr-trastuzumab shows excellent, quantifiable, and specific tumor uptake. In addition, we have developed 111In-bevacizumab for SPECT and 89Zr-bevacizumab for PET-imaging of vascular endothelial growth factor (VEGF) activity as an angiogenic marker. In melanoma patients small tumor lesions proved to be clearly visible with 111In-bevacizumab. Apart from using these techniques for staging and determination of drug targets, it is possible to evaluate modulation of these targets by drugs. Both HER2 and VEGF are downregulated by inhibitors of the heat shock protein (HSP) 90 and imaging of their expression was preclinically in vivo an excellent readout for the early molecular tumor response to HSP90 inhibitors. This is currently translated to clinical trials. Lastly, tracers for the receptors EGFR, IGF-1R, and the ligand TGFβ and are under development. Supported by grants from Dutch Cancer Society, Pink Ribbon and Pink Ribbon Gala. Citation Information: Clin Cancer Res 2010;16(7 Suppl):ED4-2

Cyclooxygenase-2 predicts adverse effects of tamoxifen: a possible mechanism of role for nuclear HER2 in breast cancer patients

Cyclooxygenase-2 (COX-2) is associated with breast tumour progression. Clinical and molecular studies implicate human epidermal growth factor receptor 2 (HER2) in the regulation of COX-2 expression. Recent reports raise the possibility that HER2 could mediate these effects through direct transcriptional mechanisms. The relationship between HER2 and COX-2 was investigated in a cohort of breast cancer patients with or without endocrine treatment. A tissue microarray comprising tumours from 560 patients with 10-year follow-up was analysed for HER2, ERK1/2, polyoma enhancer activator 3 (PEA3) and COX-2 expression. Subcellular localisation of HER2 was assessed by immunofluorescence and confocal microscopy. Expression of markers examined was analysed in relation to classic clinicopathological parameters and disease-free survival in the presence and absence of tamoxifen. COX-2 expression associated with both membranous and nuclear expression of HER2 (P=0.0033 and P<0.00001 respectively). No association was detected between COX-2 and either ERK1/2 or PEA3 (P=0.7 and P=0.3 respectively). None of the markers were found to be independently prognostic. Membrane HER2, nuclear HER2 and COX-2, however, were all found to predict poor disease-free survival in patients on endocrine treatment (P=0.0017, P=0.0003 and P=0.0202 respectively). Moreover, patients who were positive for COX-2 predicted adverse effects of tamoxifen (P=0.0427). These clinical ex vivo data are consistent with molecular observations that HER2 can regulate COX-2 expression through direct transcriptional mechanisms. COX-2 expression correlates with disease progression on endocrine treatment. This study supports a role for COX-2 as a predictor of adverse effects of tamoxifen in breast cancer patients.

Breast cancer: The upgraded role of HER-3 and HER-4

Breast cancer is the most common cancer in women and the ErbB receptor family holds crucial role in its pathogenesis. Among them, epidermal growth factor receptor and HER-2 are the most studied members and their overexpression has been associated with aggressive clinical behaviour. These data were further strengthened by the clinical success of trastuzumab, a monoclonal antibody against HER-2 in breast cancer patients with HER-2 overexpression and/or amplification. However, trastuzumab failure in some patients may partly be attributed to co-expression of other ErbB receptors. Herein, we provide updated views regarding the role of HER-3 and HER-4 in breast cancer. Accumulated evidence implies that these receptors should be considered more than heterodimerisation partners. Their expression profile might be useful in predicting responsiveness to current treatment options, while new strategies targeting their ligands and downstream effectors are being developed.

P185HER2 Overexpression in Human Breast Cancer Using Molecular and Immunohistochemical Methods

With the successful clinical trials of the engineered antibody Herceptin(tm) (in advanced-stage breast cancer) and adriamycin-based chemotherapy regimens (in the adjuvant setting), the need to detect pi85HER2 overexpression or associated amplification of the coding gene HER2 in breast cancer patients is escalating. Twenty to 30% of breast carcinomas have overexpression of p185HEK2. This condition correlates with poor patient prognosis and predicts response to chemotherapy in lymph node-positive patients. In this study we compare quantisation of p185HER2 in breast cancer at the gene and protein levels using differential polymerase chain reaction (PCR) and immunohistochemistiy, respectively. To assign HER2 gene copy numbers, a calibration curve was constructed using normal breast epithelia and breast carcinoma cell lines having known dosages of amplified HER2. We found corresponding molecular and immunohistochemical results in 85% of the 13 paraffin-embedded breast carcinoma cases examined. Two cases were found to have minimum gene amplification but marked p185HER2 overexpression, suggesting an alternative mechanism to overexpression such as transcriptional activation. although the differential PCR assay exhibits saturation approaching 20 HER2 gene copies, this may not be clinically significant because the immunohistochemical assay also appears to saturate in this gene copy number range.