Synergistic self-calibration strategy based on nano-cornucopia MOFs for accurate HER-2 detection in precision breast cancer diagnosis.

Background: Testing for human epidermal growth factor receptor-2 (HER-2) is routinely performed after breast cancer diagnosis by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). FISH is more accurate and reproducible, often considered the gold standard, and a more reliable predictor of a patient's response to HER-2 directed therapies in clinical practice. The growth factor receptor-bound protein-7 gene (GRB7) encodes a multi-domain signal transduction molecule located in close proximity to HER-2 on chromo some 17q11-12 and has been shown to be an independent adverse prognostic marker in breast cancer. Methods: We performed western blotting analysis of protein extracts from 563 annotated frozen breast tumors, collected from 1988 - 1998. GRB7 and HER-2 bands were assigned low or high values compared to specific protein controls, and tubulin bands. HER-2 FISH was performed on a subset of these tumors using an FDA approved Path Vysion kit on 4 μm frozen sections. HER-2 and chromosome 17 centromere (CEP17) signals were enumerated according to ASCO/CAP guidelines. Results: Sixty-six tumors (11.5%) over-expressed HER-2 and GRB7 proteins by Western analysis, 66 (11.5%) over-expressed HER-2 but not GRB7, and 30 (5.5%) over-expressed GRB7 but not HER-2. All 32 tumors (32/32) submitted to FISH analysis that over-expressed both HER-2 and GRB7 proteins demonstrated HER-2 gene amplification, while only one out of thirty-five (1/35) tumors that over-expressed HER-2 without GRB7 protein over-expression were HER-2 amplified. Interestingly, 7 of 28 tumors over-expressing GRB7 but not HER-2 protein were amplified for the HER-2 gene. Clinical laboratory HER-2 IHC testing confirmed the absence of HER-2 protein over-expression in all 7 of these tumors. Conclusions: Our study demonstrates that about ten percent of human breast cancers that test positive with FISH for HER-2 gene amplification do not over-express HER-2 protein, which may influence sensitivity to HER-2 directed therapy. All tumors that show false positive HER-2 gene amplification over-express GRB7 protein, suggesting the presence of GRB7 driven gene amplification in these tumors. Given that the HER-2 FISH probe contains GRB7 sequences, it is possible that HER-2 FISH false positivity may reflect GRB7 gene amplification. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-03.

Background: The human epidermal growth factor receptor-2 (HER2) is a member of a family of transmembrane tyrosine kinase receptors that play an important role in regulated normal cell growth and differentiation. The over-expression of HER2 in a subset of 15-20% of invasive breast cancers has an important bearing on prognosis, as HER2-positive tumors are associated with an aggressive clinical course and poor outcome. Targeting HER2-overexpression has been shown to be a remarkably effective therapeutic modality; however testing of tumor samples to assess the HER2 status of the patient's breast cancer is required. Clinical assays to assess the HER2 status in patients being considered for targeted therapy include immunohistochemistry (IHC), which detects protein over-expression, or fluorescence in situ hybridization (FISH), which detects gene amplification. Both the IHC and FISH methodologies have limitations. Given that the target of the currently approved drugs is the receptor protein, novel detections systems that could more accurately and quantitatively detect HER2 protein in clinical samples over a broad dynamic range would be advantageous and may be clinically helpful. Material and Methods: A novel detection technology using streptavidin-coated Phosphor Integrated Dot fluorescent nanoparticles (PID) has been developed that can be visualized by fluorescence microscopy and used for quantitative immunofluorescence detection of protein in clinical samples using computer assisted image analysis. In the current study, PID- nanoparticles were used to analyze HER2 protein expression in breast cancer cell lines and 120 well characterized breast cancer samples. These results have been compared with HER2 IHC and HER2 FISH analysis. Results: The expression levels of HER2 protein from 8 breast cancer cell lines was evaluated by antibody-binding capacity with FACS analysis. Formalin fixed paraffin embedded cell pellets for these cell lines were prepared and used for quantitative HER2 analysis by PID. The PID score/cell for each of these cell lines showed a strong linear correlation with antibody-binding capacity sites/cell by FACS analysis (R2 = 0.94). For the 120 breast cancer samples, PID score/cell was measured and compared against HER2 IHC membrane intensity measure by image analysis (Aperio) and HER2 FISH results. The HER2 PID score/cell showed a correlation coefficient of R2=0.72 versus the average HER2 copy number per cell by FISH, compared with a correlation coefficient of R2=0.41 for HER2 IHC membrane intensity measured by Aperio. For the HER2/CEP17 ratio, the correlation coefficient for the PID score/cell was R2=0.79 compared with a correlation coefficient of R2=0.32 for the HER2 IHC membrane intensity. Conclusions: PID-nanoparticles demonstrate great potential for the quantitative measurement of protein of clinical interest in routine clinical samples with morphologic confirmation of the tissue being studied. Further studies looking for PID-score thresholds for HER2 gene amplification and correlations with clinical outcome data are warranted and ongoing. Citation Format: Hicks DG, Goda H, Zhai H, Okada H, McMahon L, Sullivan N, Tang P, Nakano Y. HER2 expression in clinical breast cancer samples: A novel detection methodology for HER2 protein quantitation using fluorescent nanoparticles [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-10.

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK.S100A14,one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens,andthere is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding.Moreover,we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.

The response of human epidermal growth factor receptor2 (HER2)- positive breast cancer (BC) patients to anti-HER2 targeted therapy is significant. However, the response is not uniform and a proportion of HER2-positive patients do not respond. This study aims to identify predictors of response in the neoadjuvant treatment and to assess the discordance rate of HER2 status between pre- and post-treatment specimens in HER2-positive BC patients. The study group comprised 500 BC patients treated with neoadjuvant chemotherapy (NACT) and/or neoadjuvant anti-HER2 therapy and surgery who had tumours that were 3+ or 2+ with HER2 immunohistochemistry (IHC). HER2 IHC 2+ tumours were classified into five groups by fluorescence in situ hybridisation (FISH) according to the 2018 ASCO/CAP guidelines of which Groups 1, 2 and 3 were considered HER2 amplified. Pathological complete response (pCR) was more frequent in HER2 IHC 3+ tumours than in HER2 IHC 2+/HER2 amplified tumours, when either in receipt of NACT alone (38% versus 13%; p = 0.22) or neoadjuvant anti-HER2 therapy (52% versus 20%; p < 0.001). Multivariate logistic regression analysis showed that HER2 IHC 3+ and histological grade 3 were independent predictors of pCR following neoadjuvant anti-HER2 therapy. In the HER2 IHC 2+/HER2 amplified tumours or ASCO/CAP FISH Group 1 alone, ER-negativity was an independent predictor of pCR following NACT and/or neoadjuvant anti-HER2 therapy. In the current study, 22% of HER2-positive tumours became HER2-negative by IHC and FISH following neoadjuvant treatment, the majority (74%) HER2 IHC 2+/HER2 amplified tumours. Repeat HER2 testing after neoadjuvant treatment should therefore be considered.

Human epidermal growth factor receptor‐2 (HER‐2) overexpression in breast tumor tissues is associated with a poor prognosis but may benefit from treatment with trastuzumab. The extracellular domain (ECD) of HER‐2 can be measured in serum and which has been a new inspection item in clinical laboratory of several hospitals. However, whether serum HER‐2 ECD can be a marker of HER‐2 status in tumor tissues still confused clinicians. This study is a retrospective observation to explore the correlation between serum HER‐2 ECD shedding and tissue HER‐2 status in breast cancer patients. Meanwhile, we will further uncover the potential clinical significance of serum HER‐2 ECD detection. A total of 545 unselected breast cancer patients from Fudan University Shanghai Cancer Center were enrolled in this study. At primary diagnosis without any treatment, serum HER‐2 ECD was measured on ADVIA Centaur assay; meanwhile, tissue HER‐2 from core needle biopsy was tested through immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). We showed that serum HER‐2 ECD concentration was related to tissue HER‐2 status. Nevertheless, 36.9% of patients with tissue HER‐2 overexpression had low levels of HER‐2 ECD shedding (<15 ng/mL) in serum. Here, we demonstrated that HER‐2 ECD shedding was also associated with protein expression and alpha‐secretase activity of a disintegrin and metalloproteinase 10 (ADAM10) using tumor tissues and cell lines. Progression‐free survival (PFS) data from breast cancer patients in TNM phase II and III with tissue HER‐2 IHC 3+ were analyzed using Kaplan‐Meier plotter. The patients with serum HER‐2 ECD above 15 ng/mL had lower progression‐free survival than those with serum HER‐2 ECD <15 ng/mL. Thus, serum HER‐2 ECD could be a biomarker to identify the subgroup of poorer outcome among HER‐2 overexpression breast cancer patients. Inhibition of ADAM10 activity may have potential therapeutic benefit for this most aggressive tumor subgroup.

Purpose: Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor2 (HER2) have been reported to lead to resistance to HER2-targeted therapies in HER2-positive breast cancer, while activating mutations of HER2 have been described in HER2-negative breast cancer. The prevalence, clinicopathological characteristics, and phenotypes of HER2 mutations are not well established, thus we sought to describe the HER2 mutation profile of Chinese breast cancer patients. Methods: DNA samples were gathered from breast cancer patients undergoing neoadjuvant (N=102) or adjuvant therapy (N=498) at Fudan University Shanghai Cancer Center between January 1, 2006 and December 31, 2012. Sanger sequencing was performed to analyze all exons of HER2 to identify somatic mutations. To determine the phenotypes of novel HER2 mutations, in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments were conducted. Results: 10 HER2 somatic mutations were observed in 17 patients (17/600, 2.83%). 7 novel HER2 mutations were uncovered, 4 in the transmembrane domain and 3 in the kinase domain. Kinase domain mutations L768S and V773L were detected in HER2-negative tumors, while K753E was found in HER2-positive disease. In vitro kinase assays found that L768S and V773L exhibited a significant increase of tyrosine kinase-specific activity, while Western blots showed that L768S and V773L strongly increased phosphorylation of all signaling proteins in both MCF10A and MCF7cell lines, indicating that they were activating mutations. In Matrigel cultures, L768S and V773L formed acini when seeded in vehicle, but maintained spherical morphology when seeded in culture containing trastuzumab. The addition of lapatinib in Matrigel culture inhibited the growth of all except K753E, which was successfully inhibited by neratinib. Similarly, L768S, V773L and K753E increased the number of cell colonies formed in soft agar, trastuzumab and lapatinib treatment decreased the number of colonies formed by L768S and V773L, but only neratinib could inhibit the colony growth of K753E. Xenograft showed L768S and V773L displayed a more rapid growth, while K753E showed resistance to lapatinib in vivo. MCF10A cells bearing K753E mutation were found to be resistant to lapatinib (IC50&amp;gt;10,000 nmol/L), but could be inhibited by neratinib, though requiring a relatively higher dosage (IC50 of 32 nmol/L) than HER2 WT (IC50 of 480 nmol/L for lapatinib, &amp;lt;2 nmol/L for neratinib) and other HER2 mutations. Meanwhile, clinical follow-up showed that the 2 patients with K753E mutation who received adjuvant trastuzumab treatment presented with either brain or bone metastasis, in their 3rd and 5th year after initial cancer diagnosis, suggesting K753E mutation may have a role in trastuzumab resistance as well. Conclusions: HER2 somatic mutations were found in 2.83% of patients in this study. HER2-positive tumors harboring certain HER2 kinase domain resistance mutations may not benefit from trastuzumab or lapatinib treatment, and neratinib may offer an alternative treatment option for these patients. HER2-negative disease with activating mutations may benefit from HER2-targeted therapies, and may be of interest in prospective clinical trials. Citation Format: Wen-Jia Zuo, Yi-Zhou Jiang, Ke-Da Yu, Zhi-Ming Shao. Activating HER2 mutations promote oncogenesis and resistance to HER2-targeted therapies in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-04-04.

Breast cancer with positive hormone receptor (HR) and human epidermal growth factor receptor-2 (HER2) is a special subgroup with different clinical features and survival, especially the endocrine therapy resistance. The main purpose of the study is to find the potential markers to predict the survival and endocrine therapy resistance of patients with HR+ /HER2+ breast cancer. Surveillance, Epidemiology, and End Results (SEER) database was used to collect patients' clinical information and tumor features including age, tumor size, grade, stage and long-term survival; the BioPortal for Cancer Genomics (https://cbioportal.org) was used to download the gene data for specific patient group; clusteranalysesofgeneexpression were conducted through the DAVID Bioinformatics Resources 6.8 software. All of the included patients were diagnosed as HR positive breast cancer, but the PR positive rates were more common in HER2- group and also the ER+ /PR+ disease. Patients in HR+ /HER2+ group were more likely to present as stage III-IV and grade III disease. Among HR+ /HER2+ patients, 68.6% received chemotherapy, while only 28.9% in HR+ /HER2- group received chemotherapy (P < 0.0001). The survival of HR+ /HER2+ group was poorer. From TCGA database, series genes which were differed between HR+ /HER2+ and HR+ /HER2- were screened out that related to ERBB2 closely: IKZF3, LASP1, CDK12, MLLT6, and RARA. The first three candidate genes were associated with patients' survival, especially in patients who received hormone therapies. This study analyzed the clinical characteristics and survival of patients with HR+/HER2+ breast cancer as a special subgroup. ERBB2, IKZF3, LASP1, and CDK12 were the potential markers of the resistance of endocrine therapy, and they will provide new strategies for clinicians.

Background: Breast cancer constitutes a significant proportion of malignancies among the female population, accounting for approximately 25% of all cancer cases on a global scale. Human epidermal growth factor receptor-2 (HER2) is the more reliable marker in breast cancer diagnosis. Objective: This study investigates the potential of soluble salivary HER2 in breast cancer diagnosis and explores its correlation with demographic and hormonal factors. Materials and Methods: A total of 45 subjects were selected and divided into three groups: Group A: healthy patients (n = 15), Group B: HER2-negative breast cancer patients (n = 15), and Group C: HER2-positive breast cancer patients (n = 15). Patients’ saliva was collected, and assessment of salivary soluble HER2 was performed by using an enzyme-linked immunosorbent assay kit. Results: Salivary HER2 levels were 32.3 pg/ml in healthy group, 43.2 pg/ml in HER2-negative group, and 147.8 pg/ml in HER2-positive group. On evaluating the risk factors associated with the salivary HER2 levels, increased age (P = 0.007), positive family history (P = 0.006), patients with the habit of tobacco chewing (P = 0.001), and patients with no breastfeeding history (P = 0.001) showed a statistically significant result. Conclusion: Salivary HER2 levels were higher in both HER2-positive and -negative breast cancer groups compared to controls, indicating its potential as a noninvasive diagnostic tool for breast cancer screening.

Increased serum human epidermal growth factor receptor 2 levels have been found in metastatic breast cancer patients and are correlated with human epidermal growth factor receptor 2 overexpression in tumor cells. However, the prevalence of serum human epidermal growth factor receptor 2 in gastric cancer patients has not been elucidated. We retrospectively analyzed formalin-fixed paraffin-embedded tumor tissues and serum samples from 96 advanced gastric cancer patients. Human epidermal growth factor receptor 2 expression and gene amplification in tumor cells were determined by immunohistochemistry and fluorescence in situ hybridization. Serum human epidermal growth factor receptor 2 levels were measured using a chemiluminescent immunoassay. Human epidermal growth factor receptor 2 positivity in tumor cells was defined as immunohistochemistry 2+ with fluorescence in situ hybridization positive or immunohistochemistry 3+ with any fluorescence in situ hybridization results. All tissue samples and serum samples were successfully measured. Nineteen patients (20%) were human epidermal growth factor receptor 2-positive in tumor cells. The median serum human epidermal growth factor receptor 2 level was 9.3 ng/ml (range, 5.0-332.4 ng/ml), and serum human epidermal growth factor receptor 2 levels were significantly separated according to human epidermal growth factor receptor 2 status in tumor cells (P < 0.0001, Wilcoxon's rank sum test); median serum human epidermal growth factor receptor 2 levels in human epidermal growth factor receptor 2-negative patients and -positive patients were 8.9 (range, 5.0-20.5) and 24.0 (range, 9.7-332.4), respectively. There were 15 serum human epidermal growth factor receptor 2-positive patients (16%) using a cutoff value of 15 ng/ml. The sensitivity and the specificity of serum human epidermal growth factor receptor 2 with respect to human epidermal growth factor receptor 2 positivity in tumor cells were 53 and 94%, respectively. Serum human epidermal growth factor receptor 2 measurements cannot be substituted for tissue human epidermal growth factor receptor 2 diagnosis in advanced gastric cancer patients. However, serum human epidermal growth factor receptor 2 levels are associated with human epidermal growth factor receptor 2 overexpression in tumor cells. Further investigations of clinical significance of serum human epidermal growth factor receptor 2 as a predictive marker and a therapy-monitoring marker are warranted.

How breast cancer subtypes should affect treatment decisions for breast cancer patients with brain metastases is unclear. We analyzed local brain metastases treatments and their outcomes according to subtype in patients with breast cancer and brain metastases. We reviewed records and database information for women treated at the National Kyushu Cancer Center between 2001 and 2010. Patients were divided into three breast cancer subtype groups: Luminal (estrogen receptor positive and/or progesterone receptor positive, but human epidermal growth factor receptor 2 negative); human epidermal growth factor receptor 2 positive and triple negative (estrogen receptor negative, progesterone receptor negative and human epidermal growth factor receptor 2 negative). Of 524 advanced breast cancer patients, we reviewed 65 (12%) with brain metastases and records showing estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 status, as well as outcome data; there were 26 (40%) Luminal, 26 (40%) had human epidermal growth factor receptor 2 and 13 (20%) had triple negative subtypes. There was no statistical difference in the number of brain metastases among subtypes; however, rates of stereotactic radiosurgery or surgery for brain metastases differed significantly by subtype (human epidermal growth factor receptor 2: 81%, Luminal: 42% and triple negative: 47%; P = 0.03). Patients having the human epidermal growth factor receptor 2 subtype, a performance status of ≤1 and ≤4 brain metastases, who underwent systemic therapy after brain metastases and underwent stereotactic radiosurgery or surgery, were predicted to have longer overall survival after brain metastases. Multivariate analysis demonstrated that not having systemic therapy and not having the human epidermal growth factor receptor 2 subtype were independent factors associated with an increased risk of death (hazard ratio 2.4, 95% confidence interval 1.01-5.6; P = 0.05 and hazard ratio 2.9, 95% confidence interval 1.5-5.8; P = 0.003, respectively). Our study showed that local brain treatments and prognosis differed by subtype in breast cancer patients with brain metastases.

Human epidermal growth factor receptor 2 (HER2) gene amplification is a major therapeutic target in breast cancer, and has been introduced as a predictive biomarker to identify patients who may benefit from therapy with anti-human epidermal growth factor receptor 2 agents. Human epidermal growth factor receptor 2 somatic mutations have been reported in patients without human epidermal growth factor receptor 2 gene amplification. Since these are activating mutations, these patients may also benefit from human epidermal growth factor receptor 2-targeted drugs. In this study, we searched for human epidermal growth factor receptor 2 mutations in a group of 286 Japanese breast cancer patients with human epidermal growth factor receptor 2-negative tumors. The activating mutations of human epidermal growth factor receptor 2 identified were analyzed by direct Sanger sequencing of two major areas: the extracellular domain at 309-310 and the kinase domain between 755 and 781. Two tumors were found to have a human epidermal growth factor receptor 2 somatic mutation; one with I767M mutation and another with D769Y. No mutation was observed in the extracellular domain. One of these patients with human epidermal growth factor receptor 2 mutation recurred early with liver metastasis. Better knowledge of human epidermal growth factor receptor 2 mutation status will help us to choose personalized molecular targeted therapy for use in human epidermal growth factor receptor 2-negative Japanese breast cancer patients.

DNA framework-controlled Poly(MOFs) as a visual platform for diagnosis of HER2-positive breast cancer

Introduction: Human epidermal growth factor receptor-2 (HER-2) low expression breast malignant tumors have become a research hotspot in recent years, but it is still unclear whether HER-2 low expression represents a special subtype of breast cancer. However, this molecular type requires more effective treatment regimens in the neoadjuvant therapy stage. Methods: This study enrolled breast cancer patients who were treated at Harbin Medical University Cancer Hospital with neoadjuvant treatment between October 2011 and May 2019 and was a single-center retrospective study. Results: A total of 1,053 breast cancer patients who received preoperative therapy, including 279 (26%) HER-2 low expression patients, were included in this retrospective study. The HER-2 low expression group had a higher proportion of patients under 50 years old than the other two molecular subtype groups (p = 0.047, 62.0% vs. 57.2% and 52.5%), and the percentage of patients with Ki67 index above 15% was lower than that in HER-2-negative and HER-2-positive patients (p < 0.001, 50.2% vs. 63.6% and 71.5%). Most of the patients with HER-2 low expression were hormone receptor (HR) positive (p < 0.001, 85.7% vs. 60.4% and 36.0%), and their pathologic complete response (pCR) rate after neoadjuvant therapy was significantly lower than that of HER-2-negative and HER-2-positive patients (p < 0.001, 5.7% vs. 11.8% and 20.5%). The results of the subgroup analysis showed HR-positive patients with HER-2 low expression had a lower pCR rate (p < 0.001, 4.6% vs. 14.6%) and objective response rate (p = 0.001, 77.8% vs. 91.0%) than HER-2-positive patients and had no significant difference in these rates compared to HER-2-negative patients. There were no significant differences in overall survival (OS) and disease-free survival (DFS) up to 67 months (the median follow-up time) among HER-2 low, HER-2-negative, and HER-2-positive patients. The results of Cox hazard proportional showed that the Ki67 index and T stage (T3) were independent influencing factors for DFS. In terms of OS, Ki67 index, P53, T stage, and objective response were independent influencing factors for OS in HER-2 low expression patients. Conclusions: In general, further studies are needed to confirm that HER-2 low expression is a special breast cancer molecular subtype. The efficacy of neoadjuvant therapy in patients with HER-2 low expression is relatively poor, and the efficacy of neoadjuvant therapy can predict the prognosis of patients with HER-2 low expression.

Brain metastases remain a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Unlike HER2-amplified breast tumors growing in extra-cranial locations, brain metastases do not respond well to HER2 inhibitors and are often the reason for treatment failure. One of the major challenges in studying brain metastases is the lack of preclinical models. We developed a HER2-amplified mouse model of brain metastasis using an orthotopic xenograft of BT474 cells in mice. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib failed to contain brain metastatic tumor growth. Based on previous findings from our laboratory suggesting a role of vascular endothelial growth factor (VEGF) in the resistance of HER2-overexpressing breast cancer brain metastases to trastuzumab, we combined HER2 inhibitors with the anti-VEGFR2 antibody DC101. The combination of either trastuzumab and DC101 or lapatinib and DC101 significantly slowed metastatic tumor growth in the brain, and resulted in a striking improvement in overall survival. The benefit is due largely to an anti-angiogenic effect. The combination of anti-HER2 and anti-VEGFR2 therapy reduced both the total and functional microvascular density in the brain metastatic tumors. Moreover, tumor tissues under combination therapy showed a marked increase in necrosis. Preclinical and clinical evidence suggest that the combination of trastuzumab and lapatinib is superior to either agent alone – though this has never been tested in the brain metastatic setting. We consistently observed increased phosphorylation of HER2 in breast tumor cells growing in the brain compared with the mammary fat pad. In addition, while short-term lapatinib treatment significantly reduced HER2 activation in the brain, it could do so only to the level of that observed in the untreated mammary fat pad - and this effect disappeared over time. We hypothesized that more pronounced HER2 inhibition would be beneficial to these brain metastases with increased HER2 activation. We show here a significant growth delay with the combination of the two HER2 inhibitors compared with monotherapy. Moreover, we found a dramatic brain metastatic tumor growth delay in mice treated with both HER2 inhibitors, trastuzumab and lapatinib, and DC101. The triple combination prolonged overall survival 5 times longer than control-treated mice. Brain metastasis from breast cancer is considered the “final frontier” of breast cancer research and treatment. Our findings support the clinical development of a three-drug regimen of trastuzumab, lapatinib and a VEGF pathway inhibitor for the treatment of HER2-amplified breast cancer brain metastases. While the anti-VEGF antibody bevacizumab in combination with trastuzumab and chemotherapy has shown some promise in HER2-positive metastatic breast cancer patient, there are no data on its efficacy in the context of brain metastases. A clinical trial is now recruiting patients to evaluate the efficacy of bevacizumab in breast cancer patients with active brain metastases, including its combination with trastuzumab in patients with HER2-positive disease. This trial may provide clinical evidence for the approach presented here. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-12-03.

B-cell lymphoma-2 (Bcl-2) is one of the most important anti-apoptotic genes. Although Bcl-2 promotes tumor cell survival in vitro, previous studies have shown conflicting results regarding the association between Bcl-2 and breast cancer survival. The aim of this study was to assess the prognostic significance of Bcl-2 according to the molecular tumor subtype in primary invasive breast cancer patients. The relationship between immunohistochemical Bcl-2 expression and overall survival was analyzed in 2399 primary invasive breast cancer patients treated by curative surgery. Patients were classified into four subtypes based on hormone receptor (HR) and human epidermal growth factor receptor-2 (HER2) status: HR+/HER2-, HR+/HER2+, HR-/HER2+, and HR-/HER2-. A total of 1304 patients (54.4%) had Bcl-2 positive (+) tumors by immunohistochemistry. Bcl-2 (+) tumors were significantly associated with a younger age (<50years), early stage, lower grade, positive expression of HR, and negative expression of HER2. In the HR+/HER2- group, patients with Bcl-2 (+) tumors showed a significantly better prognosis (p<0.001). In contrast, there was no significant prognostic effect of Bcl-2 expression in other subtypes. In multivariate analysis, Bcl-2 positivity remained an independent, favorable prognostic factor in the HR+/HER2- subtype (hazard ratio, 0.609; 95% confidence interval, 0.424-0.874; p<0.007). The prognostic significance of Bcl-2 expression differed according to the molecular subtype of breast cancer. The expression of Bcl-2 was an independent, favorable prognostic factor in breast cancer patients with the HR+/HER2- subtype.