Search

The HER2 gene, which encodes the growth factor receptor HER2, is amplified and HER2 is overexpressed in 25 to 30 percent of breast cancers, increasing the aggressiveness of the tumor.We evaluated the efficacy and safety of trastuzumab, a recombinant monoclonal antibody against HER2, in women with metastatic breast cancer that overexpressed HER2. We randomly assigned 234 patients to receive standard chemotherapy alone and 235 patients to receive standard chemotherapy plus trastuzumab. Patients who had not previously received adjuvant (postoperative) therapy with an anthracycline were treated with doxorubicin (or epirubicin in the case of 36 women) and cyclophosphamide alone (138 women) or with trastuzumab (143 women). Patients who had previously received adjuvant anthracycline were treated with paclitaxel alone (96 women) or paclitaxel with trastuzumab (92 women).The addition of trastuzumab to chemotherapy was associated with a longer time to disease progression (median, 7.4 vs. 4.6 months; P0.001), a higher rate of objective response (50 percent vs. 32 percent, P0.001), a longer duration of response (median, 9.1 vs. 6.1 months; P0.001), a lower rate of death at 1 year (22 percent vs. 33 percent, P=0.008), longer survival (median survival, 25.1 vs. 20.3 months; P=0.01), and a 20 percent reduction in the risk of death. The most important adverse event was cardiac dysfunction of New York Heart Association class III or IV, which occurred in 27 percent of the group given an anthracycline, cyclophosphamide, and trastuzumab; 8 percent of the group given an anthracycline and cyclophosphamide alone; 13 percent of the group given paclitaxel and trastuzumab; and 1 percent of the group given paclitaxel alone. Although the cardiotoxicity was potentially severe and, in some cases, life-threatening, the symptoms generally improved with standard medical management.Trastuzumab increases the clinical benefit of first-line chemotherapy in metastatic breast cancer that overexpresses HER2.

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 (p0.05 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 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.

Our purpose was to: (a) study the in vitro chemosensitivity of primary epithelial ovarian cancer to drug combinations with cisplatin (CDDP), carboplatin (CBDCA), paclitaxel (PTX), epirubicin (EPI), or cyclophosphamide (CTX) utilizing the ATP tumorchemosensitivity assay (ATP-TCA); (b) correlate the test results with clinical response in patients with FIGO stage III ovarian cancer; and (c) analyze the most useful parameters for interpretation of test results.CBDCA/CTX, CBDCA/PTX, CDDP/PTX, and EPI/PTX were tested in 93 fresh human primary epithelial ovarian cancer specimens. Correlations of in vitro drug sensitivity/resistance and clinical response were performed in 38 patients with FIGO stage III disease utilizing Fisher's exact test and by comparison of progression-free (PFS) and overall survival (OS) between those testing as sensitive or resistant. A progression-free interval of more than 12 months following surgery was classified as clinical response. ATP-TCA results were analyzed using the median effective dose, area under the curve, or a defined sensitivity index.Evaluable test results were achieved in 83 of 93 patients (89%). EPI/PTX had the highest in vitro activity (P0.001). In the clinical correlation, 29 of 38 patients (76%) were classified as in vitro sensitive (sensitivity index [SI]250) and 9 patients as in vitro resistant (SI250). The SI was superior for interpretation of test results. Patients testing as chemosensitive had a significantly longer mean PFS (28.5 vs 12.6 months, P = 0.033) and OS (46.1 vs 17.6, P = 0.03) compared to those patients predicted to be resistant. The assay demonstrated a sensitivity, specificity, and positive and negative predictive value of 95, 44, 66, and 89%, respectively (Fisher's exact test, P = 0. 007).The observed in vitro efficacy of EPI/PTX in primary epithelial ovarian cancer specimens warrants further clinical evaluation. The high evaluability rate and the observed correlation with PFS and OS, within the limitations of a nonrandomized study, support the use of the ATP chemosensitivity assay in future prospective assay-directed trials.

Proto oncogenes represent a family of genes which when activated have been shown to play a role in the pathogenesis of a number of malignancies in vertebrate species including humans.1,2 In physiologic states these same genes are known to play a role in the normal cell growth control and differentiation. The HER2/neu gene is a member of the Type I receptor tyrosine kinase (RTK) group which is one of the subfamilies in the proto oncogene family and encodes a 185kD surface membrane receptor protein. This gene has been localized to chromosome 17q21,1 and the encoded protein is expressed in a wide variety of tissues including the skin, oral mucera, breast, ovary, endometrium, lung, liver, pancreas, small and large bowel, kidney, bladder and the central nervous system as well as some connective tissues.4–5 The exact physiological role of the HER2/neu protein in these tissues is not completely understood, but like other proto oncogenes it is believed to play an important signaling role in cellular proliferation and differentiation processes. Current data suggests that it forms hetero-dimers with other members of the RTKI family (such as HER 1, HER3 and HER4 in response to various ligands known as heregulins.6–8 Activation of HER2/neu results in an increase its kinase activity which in turn initiates signal transduction resulting in either cellular proliferation and/or differentiation, depending on the ligand as well as the conditions.9–14 In human breast cancers, a non-inherited alteration occurs in this gene in 25–30% of cases.

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach comparesin vivotumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

Amplification of the HER-2/neu (c-erbB-2) gene resulting in overexpression of the p185HER-2 growth factor receptor occurs in approximately 25% of early stage breast cancers. HER-2/neu has been established as an important independent prognostic factor in early stage breast cancer in large cohorts of patients and in cohorts with very long (30 year) follow-up duration. New data are emerging to suggest that HER-2/neu may be useful not only as a prognostic factor but also as a predictive marker for projecting response to chemotherapeutics, antiestrogens, and therapeutic anti-HER-2/neu monoclonal antibodies. In this review we highlight recent data on HER-2/neu as a predictive marker of response to breast cancer therapy and discuss the clinical implications of this information. The difficulty in comparing results from different data sets due to the wide variety of reagents and technologies used to detect HER-2/neu amplification/overexpression in clinical specimens is also discussed. Finally, we report results from experimental models of HER-2/neu overexpression which have been used in an effort to understand the relationship between HER-2/neu and response to chemotherapeutics and antiestrogens in breast cancer.

Gene alterations play an important role in the origin and pathogenesis of human breast cancer. Three broad categories of gene changes that appear to contribute to tumor progression include tumor suppressor genes, repair-mutator genes, and oncogenes (1). Inherited defects in some somatic genes appear to be responsible for development of hereditary and familial breast cancer, estimated at 1% and 5%, respectively, of all breast cancer cases. Identified alterations include mutations in tumor suppressor genes such as p53 in Li-Fraumeni syndrome (2, 3). Mutations in the BRCA1 gene at chromosome 17q21 have been documented in familial breast cancer (4). A separate locus on chromosome 13q13, the BRCA2 gene, was also associated with familial breast cancer (5,6). Recent studies suggest that BRCA1 may represent a repair-mutator gene, a gene responsible for maintaining the fidelity of DNA duplication (7). The failure of gene surveillance can result in further alterations in gene function and thereby increase the mutation rate of other genes. Presumably, tumor suppressor genes and oncogenes would be prominent targets of faulty repair-mutator genes (1, 7).

HER-2 oncogene encodes a transmembrane growth factor receptor that is overexpressed in 25-30% of patients with primary breast and ovarian cancer. A murine monoclonal antibody, 4D5, to the extracellular domain of HER-2 receptor elicits cytostatic growth inhibition of tumor cells overexpressing HER-2 protein, but clinical use of this antibody is limited by genesis of human anti-mouse antibodies. To avoid this problem, a recombinant humanized 4D5 monoclonal antibody (rhuMAb HER-2) was developed and tested using a human tumor xenograft model. Human breast and ovarian cancer cells which overexpress HER-2 were inhibited in vivo by the rhuMAb HER-2 antibody. Tumor growth relative to control was reduced at all doses of antibody tested, and the magnitude of growth inhibition was directly related to dose of rhuMAb HER-2. Tumor growth resumed on termination of antibody therapy, indicating a cytostatic effect. To elicit a cytotoxic response, human breast tumor xenografts were treated with a combination of antibody and antitumor drugs, cisplatin or doxorubicin. The combination of antibody with either cisplatin or doxorubicin resulted in significantly greater growth inhibition, with the cisplatin combination demonstrating a greater response. In addition, therapy with cisplatin and antireceptor antibody elicited complete tumor remissions after 2-3 cycles of therapy. The schedule of administration of anti-receptor antibody and cisplatin was critical for occurrence of antibody-induced potentiation in cisplatin cytotoxicity. Enhanced killing of tumor cells was found only if antibody and drug were given in close temporal proximity. Since interference with DNA repair pathways may contribute to this receptor-enhanced chemosensitivity, repair of cisplatin-damaged reporter DNA (pCMV-beta) was determined in human breast cells. As in studies of antibody-enhanced cisplatin cytotoxicity in vivo, treatment with rhuMAb HER-2 blocked the repair of cisplatin-damaged DNA only if the antibody was administered in close temporal proximity to transfection of the drug-exposed reporter DNA. An alternative measure of DNA repair, unscheduled DNA synthesis, was also assessed. Treatment with either cisplatin or doxorubicin led to an increase in unscheduled DNA synthesis that was reduced by combined therapy with antireceptor antibody specific to HER-2-overexpressing breast cancer cells. Using a direct measure of DNA repair, therapy of HER-2-overexpressing cells with rhuMAb HER-2 also blocked the removal of cisplatin-induced DNA adducts. Expression of p21/WAF1, an important mediator of DNA repair, was disrupted in breast cancer cells with HER-2 overexpression, but not in control cells, after treatment with HER-2 antibody, thus suggesting cross-communication between the HER-2 signaling and DNA repair pathways. These data demonstrate an in vivo antiproliferative effect of rhuMAb HER-2 on tumors that overexpress HER-2 receptor and a therapeutic advantage in the administration of the antireceptor antibody in combination with chemotherapeutic agents.

PURPOSE To determine the toxicity, pharmacokinetics, response rate, and response duration of intravenous (i.v.) administration of recombinant, humanized anti-p185HER2 monoclonal antibody (rhuMAb HER2) plus cisplatin (CDDP) in a phase II, open-label, multicenter clinical trial for patients with HER2/neu-overexpressing metastatic breast cancer. PATIENTS AND METHODS The study population consisted of extensively pretreated advanced breast cancer patients with HER2/neu overexpression and disease progression during standard chemotherapy. Patients received a loading dose of rhuMAb HER2 (250 mg i.v.) on day 0, followed by weekly doses of 100 mg i.v. for 9 weeks. Patients received CDDP (75 mg/m2) on days 1, 29, and 57. RESULTS Of 37 patients assessable for response, nine (24.3%) achieved a PR, nine (24.3%) had a minor response or stable disease, and disease progression occurred in 19 (51.3%). The median response duration was 5.3 months (range, 1.6-18). Grade III or IV toxicity was observed in 22 of 39 patients (56%). The toxicity profile reflected that expected from CDDP alone with the most common toxicities being cytopenias (n = 10), nausea/vomiting (n = 9), and asthenia (n = 5). Mean pharmacokinetic parameters of rhuMAb HER2 were unaltered by coadministration of CDDP. CONCLUSION The use of rhuMAb HER2 in combination with CDDP in patients with HER2/neu-overexpressing metastatic breast cancer results in objective clinical response rates higher than those reported previously for CDDP alone, or rhuMAb HER2 alone. In addition, the combination results in no apparent increase in toxicity. Finally, the pharmacology of rhuMAb HER2 was unaffected by coadministration with CDDP.

As a step in the design of directed toxins, aimed at cells that overexpress HER receptors, particularly breast carcinoma cells, we studied the properties of a chimera of diphtheria toxin (DT) and heregulin beta1. The EGF-like growth hormone heregulin is a ligand for the HER3 and HER4 receptors and their heterodimers with HER2. The 60-residue EGF-like domain (hrg) of heregulin elicits a biological response and binds to these receptors primarily through its N terminus. We tested a fusion protein in which hrg replaces the C-terminal receptor-binding domain of DT (DT(389)hrg) and an alternative design in which this domain is fused to the N terminus of DT(389). Of those two constructs, the N-terminal fusion was not active as a directed toxin but elicited a growth response. The C-terminal fusion of hrg to DT(389) yielded a functional toxin and showed cell line specific cytotoxicity that is consistent with heregulin specificity. The binding of hrg to its cognate receptor is not impaired as shown by receptor activation, direct binding, and competition with free hrg. Cytotoxicity is dependent on high-affinity binding of DT(389)hrg to HER3 and HER4 receptors and is not mediated by HER2 overexpression alone. For those cell lines exhibiting high-affinity binding sites, the level of cytotoxicity correlates with the rate of internalization. Thus DT(389)hrg chimeras offer a possible avenue toward directed toxins against cells that overexpress HER receptors.

Allelic deletions of chromosome 18q are reported to be common in prostate and colorectal cancers, suggesting that one or more tumor suppressor genes on 18q are involved in the genesis of these neoplasms. The DPC4 gene, a recently identified candidate tumor suppressor in 18q21, was examined for evidence of inactivation in prostatic carcinomas, and results compared to those of a parallel analysis of colorectal carcinomas, for which DPC4 mutation has been reported in approximately 10% of cases. In this study, only three (10%) of 29 informative primary prostate cancers showed allelic loss of chromosome 18q21 markers, and no point mutations or deletions of DPC4 were detected in the complete set of 45 primary or metastatic cases. In contrast, five (56%) of nine primary colorectal tumors displayed allelic loss of 18q markers and in one of these a somatically acquired G-->T missense mutation was found in exon 1. Of twelve colorectal tumor cell lines, one showed a G-->C missense mutation in exon 8 and two had partial homozygous deletions that would likely abrogate gene function. These data suggest that DPC4 is rarely if ever mutated during prostatic oncogenesis, whereas inactivation of this gene may contribute to the genesis of a subset of colorectal carcinomas.

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

521 Background: The addition of trastuzumab to chemotherapy improves responses to therapy and extends survival among patients with metastatic HER2 breast cancer. Several mechanisms have been proposed for the activity of this combination therapy. Trastuzumab, specifically, is thought to activate NK cells and blunt HER2 signaling. Prior work from us has shown that combination trastuzumab and chemotherapy induces HER2-specific antibodies which correlate with response to therapy. Despite that, it remains unclear whether the immunity that was induced was due to complexing of non-tumor derived HER2 or antigen derived from the tumor site. In the present work, we addressed this question by assessing if combination therapy induced epitope spreading to tumor antigens other than HER2. Methods: Pre-and post-treatment sera were obtained from 56 women enrolled in 2 NCCTG clinical trials, N0337 and 98-32-52. IgG antibodies to HER2 intracellular domain (HER2), p53, IGFBP2, CEA and tetanus toxoid (TT) were examined using ELISAs. Sera from an age-matched group (N=56) of controls and 12 patients treated in the adjuvant setting were also examined. Results: Prior to therapy, metastatic patients had higher IgG levels (≥ 2-fold) to p53 and HER2 but not CEA, IGFBP2 and TT, relative to the controls. Similarly, adjuvant patients had elevated IgGs to multiple tumor antigens prior to therapy, relative to controls. Following therapy, levels of IgG to IGFBP2, HER2, and p53 increased in 81% of metastatic patients, with mean increases of 3.2 (±0.6 sem), 6.2 (±2.7) and 2.7 (±0.7) fold, respectively (p

Background: The antibody-drug conjugate T-DM1 retains the mechanisms of action of trastuzumab, including HER2 targeting and interruption of HER2 signaling, and provides a means of delivering the cytotoxic agent DM1 directly to HER2-positive tumors. In the EMILIA study, T-DM1 demonstrated a statistically significant progression-free and overall survival (PFS, OS) benefit with less toxicity vs capecitabine plus lapatinib (XL) in patients (pts) with previously treated HER2-positive MBC. Activating mutations of PIK3CA may lead to resistance to currently available HER2-directed therapies. The relationship between treatment efficacy and tumor HER2 mRNA expression or PIK3CA mutation status was examined in pts from EMILIA. Methods: Tumor tissue collected for HER2 testing was also used for HER2 mRNA analysis by qRT-PCR and for PIK3CA assessment (with additional consent), using a PIK3CA Mutation Detection Kit. PFS and OS were analyzed for each BM subgroup using the Kaplan-Meier method and a Cox regression model. Results: Median mRNA concentration ratios and PIK3CA mutation frequency were similar across treatment arms and consistent with data previously reported. T-DM1 demonstrated superior PFS and OS vs XL in all BM subgroups. The hazard ratio of OS was less for pts with high (>median) vs low tumor HER2 mRNA levels. For XL-treated pts, PIK3CA mutations were associated with shorter median PFS and OS; PIK3CA mutations did not significantly affect T-DM1 treatment outcomes. Efficacy by BM Subgroup in EMILIA PFS T-DM1 XL Hazard ratioa 95% CI n Median PFS (months) n Median PFS (months) All patients 495 9.6 496 6.4 0.66 (0.56, 0.78) HER2 mRNA concentration ratio ≤Median 230 8.2 204 6.4 0.64 (0.50, 0.82) >Median 197 10.6 235 6.9 0.65 (0.50, 0.85) PIK3CA mutation status Mutated 40 10.9 39 4.3 0.45 (0.25, 0.82) Wild type 93 9.8 87 6.4 0.74 (0.50, 1.10) OS T-DM1 XL Hazard ratio a 95% CI n Median OS (months) n Median OS (months) All patients 495 30.9 496 25.1 0.70 (0.56, 0.87) HER2 mRNA concentration ratio ≤Median 230 26.5 204 23.7 0.80 (0.59, 1.09) >Median 197 34.1 235 24.8 0.53 (0.37, 0.76) PIK3CA mutation status Mutated 40 NE 39 17.3 0.26 (0.12, 0.57) Wild type 93 NE 87 27.8 0.68 (0.40, 1.15) aHazard ratios are based on unstratified analyses. CI, confidence interval; HER, human epidermal growth factor receptor; NE, not estimable; OS, overall survival; PIK3CA, phosphatidylinositide 3-kinase catalytic subunit α; PFS; progression-free survival; T-DM1, trastuzumab emtansine; XL, capecitabine + lapatinib Conclusions: Pts in all BM subgroups analyzed to date had longer PFS and OS with T-DM1 vs XL. Pts with tumors expressing high HER2 mRNA levels derived even greater OS benefit from T-DM1. XL-treated pts with PIK3CA mutations had worse outcomes than those with wild type PIK3CA. T-DM1-treated pts with PIK3CA mutations had a similar treatment benefit as those without, suggesting that the unique mechanism of action of T-DM1 may overcome PIK3CA mutation resistance. Citation Format: José Baselga, Sunil Verma, Jungsil Ro, Jens Huober, Ellie Guardino, Liang Fang, Steven Olsen, Gail Lewis Phillips, Sanne de Haas, Mark Pegram. Relationship between tumor biomarkers (BM) and efficacy in EMILIA, a phase III study of trastuzumab emtansine (T-DM1) in HER2-positive metastatic breast cancer (MBC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-63. doi:10.1158/1538-7445.AM2013-LB-63

Strong epidemiological data supports breast cancer (BC) as the second leading cause of cancer death among US African American (AA) women, with a 20% greater mortality rate than that in Caucasians (Cauc). Collection of similar BC incidence and mortality data for Eastern Africa is limited, however, it is known that BC presents as advanced-stage disease, comprised mainly of poorly differentiated cancers that are less likely to be hormone responsive. Our combined investigative team continues to analyze a cohort of indigenous Kenyan BC patients, in order to begin to understand biological similarities and differences between BC in native African and AA patients. Previous related gene expression studies conducted in a Triple Negative Breast Cancer (TNBC) stage-matched multi-ethnic US cohort suggested differential expression patterns across ethnicities. We are extending this focus to similar studies involving native African samples. Archived BC pathology samples (FFPE) were either obtained from University of Miami or from a native African tumor bank (Kenya). Forty-seven BC samples were contributed from Kenya, and re-analyzed (in Miami) for ER/PR/ Her2Neu status, resulting in confirmation of 29 Kenyan TNBC cases. 10 um sections were cut from each tumor FFPE block, and following RNA isolation and cDNA preparation, hybridized to the Almac breast-enriched gene expression array (Breast Cancer DSA Research Tool). After quality control assessment of array data, a total of 60,856 gene/probes were analyzed by RMA; normalized, log transformed to the median, and compared on GeneSpring® analytical software. Data sets were separated by further analysis based on node status [(N0-Kenyan-7, AA-10); (Mixed Node-Kenyan-16, AA-7)]. Unpaired student's T-test was performed on all subsets and the resulting p-values were corrected for Multiple Testing using Benjamin-Hochberg. Differentially expressed genes/probes were extracted for each subset using p-value < 0.01 and fold change of > 1.5. These probe/gene lists were further analyzed using GeneGo pathway analysis and enriched pathways were identified. Comparisons of transcriptional differences between Kenyan and AA TNBC, regardless of node status, suggest expression alterations in several key pathways, including EMT Transition, Cytoskeleton Remodeling, Immune Response and Epigenetic Control. These preliminary results are being validated by QPCR and other methodologies. In conclusion, we have demonstrated that high quality RNA can be extracted from archived FFPE tumor samples, including samples from East Africa. This observation raises the possibility of future genomic analyses using archived samples. We are intrigued by our study observations, which suggest that inherent gene expression differences exist between Kenyan and AA TNBC samples in previously-recognized pathways important in onocogenesis. Citation Format: Lisa L. Baumbach-Reardon, Julie Getz, Mary Ellen Ahearn, Carmen Gomez, Peter Bird, John Carpten, Mark Pegram. Comparison of transcriptional signatures in US African American and Kenyan TNBC samples identifies differential expression in key oncogenic pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2013-4023

Introduction: Strong epidemiological data supports breast cancer (BC) as the second leading cause of cancer death among African-American (AA) women, with a 20% greater mortality rate than that in Caucasians (Cauc). Collection of similar BC incidence and mortality data for Eastern Africa is limited, with the Nairobi Cancer Registry serving as the primary data collection source. However, it is known that in this African region, BC presents as an advanced-stage disease, comprised mainly of poorly differentiated cancers that are less likely to be hormone responsive. Objectives: Our combined investigative team is studying a cohort of indigenous Kenyan breast cancer patients, in order to begin to understand biological similarities and differences between BC in native African and US African patients. Recent gene expression studies conducted by our group suggest differential expression patterns in Triple Negative Breast Cancer (TNBC) across a stage-matched multi-ethnic US cohort. We are extending this focus to similar studies involving native African samples. Methods: Archived BC pathology samples (FFPE) were either obtained from University of Miami (C. Gomez; M. Jorda) or from a native African tumor bank (P. A. Bird, Kijabe). Forty-seven BC samples were contributed from Kenya, and re-analyzed (in Miami) for ER/PR/ Her2Neu status, resulting in confirmation of 29 Kenyan TNBC cases. As expected, a high percentage of the Kenyan tumors were advanced stage and high grade. These Kenyan TNBC cases, as well as US AA TNBC cases from Miami, were analyzed by gene expression studies. 10 um sections were cut from each tumor FFPE block, and following RNA isolation and cDNA preparation, hybridized to the Almac breast-enriched gene expression array (Breast Cancer DSA Research Tool). After quality control assessment of array data, unpaired student's T-test was performed between Kenyan and AA samples, and resulting p-values were corrected for Multiple Testing using Benjamin-Hochberg. Results: Three sets of differentially expressed genes/probes were extracted using different thresholds: Stringent (P-value ≤ 0.01 & Fold Change ≥ 2.0; 1013 probes); Less Stringent (P-value ≤ 0.02 & Fold Change ≥ 2.0; 1669 probes); and Most Significant (P-value ≤ 0.001 & Fold Change ≥ 1.5,136 probes), the latter of which generated hierarchical clustering of the most significant differentially expressed genes. These probe/gene lists were further analyzed using GeneGo pathway analysis. Initial analyses point to possible gene expression differences within several key pathways, including signal transduction in the AKT signaling pathway. Further data set analyses are being performed, including consideration of additional tumor characteristics. The latest data interpretations will be presented. Conclusions: This study represents the first direct comparison of gene expression in TNBC specimens across US AA and Kenyan East Africans. These results will be compared with data from our previous ethnic cohort studies. We will strive to correlate all experimental data with available clinical data, and determine possible correlations between genomic signatures, clinical tumor characteristics, and demographic information among and across ethnic groups. Ultimately, this work will contribute to our further biologic understanding of BC across different ethnicities, and thus, development of new preventive, predictive and therapeutic measures. This abstract is also presented as Poster B67. Citation Format: Lisa Baumbach, Mark Pegram, Carmen Gomez, Biju Issac, Jennifer Clarke, Merce Jorda, Mary Ellen Ahearn, John Carpten, Peter Bird. Investigation of transcriptome differences in breast cancer tissues from African American and East African patients with triple-negative breast cancer. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PR03.

137 Background: Elevated p95HER2 [HER2-M611-CTF (carboxy-terminal-fragment) also known as p95] expression has been correlated with poor outcomes in HER2+ pts with metastatic breast cancer treated with trastuzumab (T); however, limited data is available on the correlation between p95 and pCR to T in the neoadjuvant (NEO) setting, where p95 was measured by immunohistochemistry. The current study aims to determine whether quantitative p95, HER3 and HER2 expression correlated with pCR in pts treated with T + chemotherapy in the NEO setting. Methods: pCR data and quantitative HER2 (H2T), p95, and HER3 (H3T) results by HERmark/VeraTag assays were available in 45 patient cases with pre-therapy, formalin-fixed, paraffin-embedded breast tumors. pCR was defined as the absence of invasive disease in the breast. Quantitative biomarker data were correlated with pCR according to previously published or presented biomarker cutoffs. Results: The overall pCR rate was 46.7% (ER+: 14.3% vs. ER-: 75%; p

Background Tumor EGFR and KRas mutations are both predictive and prognostic biomarkers in patients with advanced NSCLC. We analyzed the correlation between these biomarkers and treatment outcomes in a phase III trial of 3rd/4th line sorafenib in patients with NSCLC. Methods The global, randomized, placebo-controlled MISSION trial enrolled 703 patients with advanced relapsed/refractory NSCLC of predominantly non-squamous histology. The primary study endpoint was overall survival (OS). EGFR and KRas mutations were analyzed in archival tumor samples and in circulating tumor DNA isolated from plasma. Results Tumor and/or plasma mutation data were available from 347 patients (49%). EGFR and KRas mutations were detected in 89 (26%) and 68 (20%) patients, respectively, and were well balanced between treatment arms. Analysis of the interaction between EGFR mutation status and treatment effect on survival suggested that patients with EGFR mutations (mEGFR) benefitted from sorafenib, while those with wild-type EGFR (wtEGFR) did not (p = 0.023). Median OS was two-fold longer in mEGFR patients receiving sorafenib versus placebo (423 vs 197 days, HR 0.48, p = 0.002). There was no significant difference in OS between patients with wtEGFR receiving sorafenib or placebo (253 vs 256 days, HR 0.92, p = 0.559). An interaction was also seen between EGFR mutation status and the sorafenib effect on PFS (p = 0.015). Patients with mEGFR treated with sorafenib had better outcomes compared to placebo based on Cox regression analysis (HR 0.27, p Conclusion Post-hoc analyses of efficacy outcomes in MISSION suggest that advanced NSCLC patients with EGFR mutations may derive a survival benefit from receiving 3rd/4th line sorafenib. These results must be interpreted with caution due to the small, non-representative nature of the genetic biomarker subpopulation analyzed in this trial. Further prospective investigation may be warranted. Disclosure T.S.K. Mok: Honoraria: AstraZeneca, Roche, Eli Lilly, Merck Serono, Eisai, BMS, BeiGene, AVEO, Pfizer, Taiho, Boehringer Ingelheim, and GSK Biologicals Speaker: Astrazeneca, Roche, Eli Lilly, Boehringer Ingelheim, and Merck Serono Research funding: Astrazeneca. L. Paz-Ares: Dr. Paz-Ares has received honoraria from Bayer HealthCare Pharmaceuticals, Lilly, Roche and Pfizer. Y. Wu: Dr. Wu has received lecture fees from Roche, AstraZeneca, Eli Lilly, and Pfizer. V. Hirsh: Member of the steering committee for the MISSION trial. C. Lathia: Dr. Lathia is an employee of Bayer HealthCare. T.J. Ong: Dr. Ong is an employee of, and owns shares in, Bayer HealthCare. C. Pena: Dr. Pena is an employee of Bayer HealthCare. All other authors have declared no conflicts of interest.

LBA1 Background: T-DM1 is an antibody-drug conjugate comprising T, a stable linker, and the potent cytotoxic agent DM1; it incorporates the antitumor activities of T and the HER2-targeted delivery of DM1. Methods: EMILIA is a randomized study of T-DM1 vs XL, the only approved combination for T-refractory HER2+ MBC. Patients (pts) received T-DM1 (3.6 mg/kg IV q3w) or X (1000 mg/m2 PO bid, days 1–14 q3w) + L (1,250 mg PO daily) until progressive disease (PD) or unmanageable toxicity. Pts had confirmed HER2+ MBC (IHC3+ and/or FISH+), and prior therapy with T and a taxane. Primary end points were PFS by independent review, OS and safety. An interim OS analysis (efficacy boundary: HR= 0.617; p=0.0003) was planned at the time of the final PFS analysis. Results: 991 pts were enrolled; 978 received treatment. Median (med) durations of follow-up were 12.9 (T-DM1) and 12.4 (XL) months (mo). Baseline demographics, prior therapy and disease characteristics were balanced. There was a significant improvement in PFS favoring T-DM1 (med 9.6 vs 6.4 mo; HR=0.650 [95% CI, 0.549–0.771]; p

608 Background: Elevated p95 [HER2-M611-CTF (carboxy-terminal-fragment) also known as p110 or p95HER2] expression has been correlated with poor outcomes in HER2+ pts with metastatic breast cancer treated with trastuzumab (T); however, limited data have been presented on the correlation between p95 and pCR to T in the NEO setting, where p95 was measured by immunohistochemistry. In the current study, we sought to determine whether quantitative p95 and HER2 expression correlated with pCR in pts treated with T + chemotherapy in the NEO setting. Methods: HER2 expression (H2T) was quantified by HERmark in 47 breast tumors using formalin-fixed, paraffin-embedded sections. Tissue remained in 40 cases to measure p95 by VeraTag and compare to a previously published cutoff (Clin Cancer Res 16:4226, 2010). pCR data were available for 45 cases. pCR was defined as the absence of invasive disease in the breast. Results: The overall pCR rate was 46.7% (ER+: 14.3% vs. ER-: 75%; Wilcoxon rank p

Disparities in breast cancer (BC) stage of presentation and survival are well documented in patients of different ethnicities. These differences are undoubtedly due to a combination of extrinsic and intrinsic factors. Our group is focused on the genetic contributions to these disparities. Recent observations by our group and others suggest not only the involvement of specific genes contributing to BC risk in African-American (AA) women, but that multiple distinct ethnic-specific gene expression patterns exist in both matched cancerous and normal breast tissue samples. We report the first results of a parallel study which is addressing an unanswered question: are there ethnic-specific differences in the transcriptome of normal breast tissue? We are completing gene expression profiling studies in normal breast FFPE samples from 25 AA and 25 Caucasian (Cauc) women with no history of breast cancer. Laser capture micro-dissection (LCM) is used to further identify and extract epithelial vs stromal cells from each FFPE slide for use in RNA expression profiling. RNA isolation and hybridization occurs in collaboration with Almac Diagnostics (Dx) using their proprietary Breast Cancer DSA array. Gene expression data analyses involves multiple statistical packages developed for DNA microarray studies, and is focusing on comparison of gene expression profiles across four study groups- AA vs Cauc samples and epithelial vs stroma cells. For baseline comparison, a subset of this study group (10 AA and 10 Cauc samples non-LCM dissected) has also been analyzed. Results from these non-LCM dissected samples displayed expression differences between AA and Cauc in 333 known genes. Applying the stringency criteria of a fold change of two or greater and a statistical p-value of 0.01, reduced the significant gene list to 61 genes. Initial pathway analyses indicate these genes are involved in cell adhesion, G-protein signaling, apoptosis and survival pathways, and cytoskeleton remodeling - all critical pathways for cell survival. These initial results are being validated in the LCM-dissected samples, and our latest results will be presented. Results from analyses of these normal samples will be compared to our previous results obtained in matched tumor-normal triple negative AA and Cauc breast cancer samples (as well as publicly available data sets). Combined analysis of tumor and normal breast expression (transcriptome) data will help to better understand the possible significance of gene expression differences in breast tissue between ethnic groups. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4196. doi:1538-7445.AM2012-4196

Disparities in breast cancer stage of presentation and survival rates exist in patients of different ethnicities. These differences are undoubtedly a result of a combination of factors, including socio-economic, lifestyle, tumor characteristics and inherent factors, such as genetic composition. Our group remains focused on analyzing genetic/genomic contributions to these disparities, with the ultimate goal of increased biological understanding, leading ultimately to individualized, ethnic-specific diagnostic and therapeutic approaches. We have previously reported ethnicspecific expression patterns in matched tumor and adjacent samples from a cohort of triple negative breast cancer (BC) samples. Here we report results from a parallel study focusing on gene expression profiling in a multi-ethnic collection of normal breast tissues. Study samples were cut from FFPE (formalin fixed paraffin-embedded tissue) blocks saved from local reduction mammoplasty cases [5 Caucasians (CAU); 7 African-American (AA); and 4 Hispanics (HIS) women with no personal or family BC history]. These were sent to Almac Diagnostics for RNA isolation, cDNA preparation, and hybridization of cDNAs to a cancer focused gene expression array (Xcel) containing 110,961 probes, representing 19,905 unique known genes. Arrays were quantile normalized and log transformed to the median of all samples. The probes were filtered to remove variation within each ethnic group to ≤ 0.5 SD, while the variation between the three groups was maintained at ≥ 0.2 SD. Samples which had < 90 % similarity based on ‘Pearson correlation’ were removed from subsequent analyses. This removed three AA, 2 CAU and 1 HIS samples. Finally, the filtered subset of 68,145 probes common to the three ethnic groups was then compared on GeneSpring® analytical software. A mean centered Principal Component Analysis (PCA) was performed on the filtered subset of normalized data. In addition, a one way ANOVA between the three ethnic groups identified 1884 significantly differentially expressed probes representing 237 unknown and 1647 known genes (P ≤ 0.05; Benjamin-Hochberg multiple testing corrected). Post-hoc analysis of the 1884 significant probes identified 207 probes significantly differentially expressed between CAU and AA; 1863 probes were found to be differentially expressed between HIS and CAU, and 1873 probes were significantly differentially expressed between HIS and AA. These results suggests that the HIS group/samples have a unique gene signature in comparison to the other ethnic groups analyzed. When the differentially expressed gene list across ethnicities is reduced to those genes with 1.5 fold change, the number of statistically-significant differentially-expressed genes across ethnicities decreases dramatically (7 AA vs. Cau; approx 600 AA vs HIS; 600 CAU vs His), suggesting a much more select group of differentially-expressed genes in normal breast tissue across these ethnic groups. Since the overall sample size is small, we are continuing to validate these data on a larger set of samples. In addition, we are performing laser capture micro-disssection of these samples, to compare gene expression patterns in stroma vs. epithelial. We will present our latest study results, including pathway analysis of statistically-significant differentially-expressed genes across the three ethnic groups. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B39.

TPS116 Background: Despite advances in the treatment of HER2 overexpressed MBC, nearly all patients develop progressive disease and innovative well-tolerated HER2-directed therapies are needed. T-DM1 is a unique antibody-drug conjugate that combines the antitumor activities of trastuzumab with intracellular delivery of the highly potent cytotoxic agent, DM1 (derivative of maytansine). In two phase II studies (TDM4258g; TDM4374g) single-agent T-DM1 3.6 mg/kg IV q3w showed substantial efficacy in patients with HER2-positive MBC who had received prior chemotherapy and ≥1 HER2-directed therapy for MBC (Burris JCO 2010; Krop ESMO 2010) providing strong rationale to assess T-DM1 in the phase III setting. In addition, comparable response rates and improved safety relative to trastuzumab and docetaxel were reported in a randomized phase II study of T-DM1 in first-line MBC (Perez ESMO 2010). Methods: EMILIA (TDM4370g, registry #NCT00829166) is an international, phase III study evaluating the safety and efficacy of...

Disparities in breast cancer stage of presentation and survival rates exist in patients of different ethnicities. These differences are undoubtedly a result of a combination of factors, including socio-economic, lifestyle, tumor characteristics and inherent factors, such as genetic composition. Our group is analyzing the genetic contributions to these disparities, with the goal to increase understanding of the underlying biology, leading ultimately to individualized, ethnic-specific diagnostic and therapeutic approaches. Here we report our results of a recently completed study focusing on gene expression profiling in a multi-ethnic cohort of triple negative breast cancer patients. We analyzed breast cancer and self-matched normal tissue samples from 10 African-American (AA), 10 Hispanic (His), and 10 non-Hispanic white (Caucasian) patients from south Florida. Study samples were cut from FFPE (Formalin Fixed Paraffin-embedded tissue) blocks marked by pathology as normal vs. tumor tissue, and sent to Almac Diagnostics for RNA isolation, cDNA preparation, and hybridization of tumor/normal cDNAs to a breast cancer focused gene expression array (Breast Cancer DSA Research Tool). From the Breast Cancer DSA arrays data, a two-way ANOVA (disease state and ethnicity) was used to identify transcripts with a p-value less than 0.01. Data QC indicated that samples clustered well with respect to ethnicity and adjacent normal vs. tumor tissue. We have identified ethnic-specific expression patterns in the matched normal and tumor samples. Initial pathway analysis using MetaCore Program shows that a number of genes related to the DNA repair pathway are differentially expressed across the ethnicities. In a set of ten DNA repair/cell cycle genes, the direction of change (increased or decreased expression) was the same for all three ethnic groups, however, the level of change differed greatly between ethnic groups. Fold change in this set of ten genes ranged from -6.54 to +5.53 with all being greater than 2 fold change in at least one ethnic group. Additional pathway analysis and validation of these results is ongoing. In follow up to this study, we have initiated parallel analysis in normal tissue samples (reduction mammoplasty) samples from AA and Cau non-cancer patients. Combined analysis of tumor and normal expression data will help to better understand the possible significance of gene expression differences in breast tissue between ethnic groups. These studies have important implications for addressing BC health disparities, as well as tailored approaches to prediction, prevention and treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5602. doi:10.1158/1538-7445.AM2011-5602

The full-length p185 ERBB2 (HER2) receptor tyrosine kinase is a validated target for breast cancer (BC) therapy. It is now appreciated that a portion of ERBB2+ BC expresses truncated isoforms of the receptor, including the membrane localized forms p95 and p110. Importantly, t-ERBB2s lack the extracellular domain epitope of trastuzumab and act as a resistance mechanism to therapy. Data from transgenic mouse models implicate expression of t-ERBB2s in tumor formation and metastasis. Furthermore, human clinical studies have revealed that expression of t-ERBB2s correlates with poor patient outcome, increased nodal involvement, and increased metastasis. In this study, we used partially transformed breast epithelial cells (HMLEs) to extend the studies of t-ERBB2 expression in human cells. Stable expression of cDNAs coding for p110 t-ERBB2 led to increased migration (79 cells/ well versus 39, p = 0.04), and invasion (583 cell/ well vs. 290) p = 0.03) of p110 versus p185 expressing HMLE cells, respectively. Importantly, expression of p110 t-ERBB2, but not other isoforms, led to xenograft formation by HMLE cells in NOD/SCID mice (p110, 10 of 12 possible xenografts formed, p185, no xenografts formed). Accurate clinical profiling of t-ERBB2 expression may aid in selection of ERBB2-targeted therapy. To this end, we used a novel proximity-mediated immunoassay (CoPIA) to detect and quantify t-ERBB2 receptors in patient samples. Analysis of a cohort of 74 primary breast tumors revealed that 52% of breast tumor samples with a high ERBB2-IHC score (3+) contained t-ERBB2, with 32% containing phosphorylated t-ERBB2. In several samples t-ERBB2 accounted for 10-20% of the total ERBB2 receptor population in tumor cells. Finally, fine-needle aspirates of ERBB2+ metastases also revealed expression and phosphorylation of t-ERBB2s. In addition to its role in prognosis and therapeutic guidance, p110 t-ERBB2 represents an attractive target for therapy. We have generated several mouse monoclonal antibodies targeting the N-termini of p110 t-ERBB2s. Binding of p110 on the surface of living cells was assayed by flow cytometry, and the biological activity of these antibodies is currently being assessed in vitro and in vivo. Together, these data imply a functional role for the p110 t-ERBB2 isoform in tumorigenesis, invasion, and migration. Furthermore, expression of t-ERBB2 in the majority of IHC 3+ tumor samples reveals a significant incidence of t-ERBB2 in human cancers. Targeting of p110 t-ERBB2 may allow for novel therapeutic intervention in ERBB2+ primary and metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1211. doi:10.1158/1538-7445.AM2011-1211

ErbB2 gene amplification occurs in approximately 20% of all breast cancers and is associated with an aggressive subtype with poor clinical prognosis. Acquired resistance to erbB2-targeted therapies is a common occurance even in patients who show an initial clinical response to trastuzumab or lapatinib-based regimens. Optimal treatment for relapsed disease following erbB2 targeted therapies has not been defined. Therefore, we sought to evaluate whether cytotoxic agents have cross-resistance to lapatinib in cell lines that have acquired resistance to lapatinib. Here we established a model of acquired lapatinib resistance derived from erbB2 overexpressing breast cancer cell lines, BT474 and SKBR3. Lapatinib resistant cell lines, BTLapR and SKLapR, were generated by growth in the continuous presence of 2.6 uM lapatinib (peak plasma concentration in human subjects) resulting in IC50 of 4.72 uM (BTLapR) and 0.083 uM (BT474) (P IC 50 of Significant Drugs on Lapatinib Resistant BT474 cells Previous studies have shown that resistance to lapatinib can switch cell survival signaling from the erbB2 to ER pathway, and that ER signaling can stimulate beta III tubulin expression. We therefore hypothesize the differential responses to microtubule inhibitors in BTLapR and SKLapR might be explained by the fact that BT474 has both estrogen (ER) and progesterone receptor (PR), but SKBR3 express neither. Moreover, beta-III tubulin is known to be a key resistance mechanism of microtubule inhibition. Western blot analysis of baseline protein expression in both acquired lapatinib-resistant and parental cells indicates that ER signaling is involved in stimulating beta III tubulin and is linked to the development of resistance to microtubule inhibitors. These findings point to a potential mechanism to address cross resistance between lapatinib and microtubule-interacting agents, which could have potential clinical implications when considering salvage therapies following lapatinib treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-05.

Background: Cancer is caused by mutations in oncogenes and tumor suppressor genes resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Methods: In order to identify functionally important tumor suppressor genes we have conducted the first human whole genome in vivo RNA interference (RNAi) screen. Partially transformed human mammary epithelial cells (HMLEs), which do not form tumors in immunodeficient mice, were infected with the Expression Arrest™ GIPZ lentiviral shRNA library consisting of 62,000 shRNAs targeting the whole human genome, and injected into the mammary fat pad of immunodeficient mice. shRNAs that silenced tumor suppressor genes fully transformed the mammary epithelial cells resulting in tumor formation. Candidate tumor suppressor genes were identified by PCR amplification and sequencing of tumor integrated shRNAs. For validation, candidate tumor suppressor genes were silenced in HMLEs and ectopically expressed in fully transformed breast cancer cells. The effect of modifying gene expression on the transformed phenotype was assessed using soft agar colony formation assays. Clinical significance was determined by comparing expression in normal and cancerous human breast tissue using Oncomine Research. Results and Discussion: Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). Silencing LIFR expression with multiple shRNA constructs fully transformed human mammary epithelial cells resulting in enhanced colony formation in soft agar (P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-02.

586 Background: We previously conducted a phase II study to evaluate a NEO regimen of docetaxel/carboplatin/trastuzumab (TCH) in 48 pts with clinical stage II-IIIB HER2 overexpressing breast cancer. The pCR rate was 42%. The current study sought to retrospectively investigate the correlation between total HER2 protein expression (H2T) and HER2 homodimers (H2D), by a novel quantitative HER2 assay, in pre-therapy tumor specimens from this trial and pCR and percent change in tumor size (ΔTS) following NEO ddTCH therapy. Methods: Pre-therapy formalin-fixed, paraffin-embedded (FFPE) tissue sections with ≥10 mm2 tumor area were available from 17 pts enrolled in the phase II NEO ddTCH study. H2T and H2D in these samples were quantified by the HERmark assay. Correlations between quantitative H2T and H2D measurements and pCR and ΔTS (as measured by longest tumor dimension) pre- versus post-therapy were assessed using the Mann-Whitney test and Spearman correlation, respectively. Results: All pts were previously ass...

e13074 Background: cSrc is overexpressed/activated in ∼70% of primary human BC. Src phosphorylates p27 reducing its inhibitory function toward cyclin E-Cdk2, thereby facilitating p27 degradation. A...

Background: Overexpression of HER2/neu is associated with upregulation of VEGF in human breast cancer cells in vitro. In xenograft models, superior efficacy is observed when anti-HER2 antibody trastuzumab (T) is given in combination with anti-VEGF antibody bevacizumab (B). Results from a phase I study of T + B (Breast Cancer Res Treat. 88:S124, 2004) were sufficiently positive to proceed with phase II testing. The objectives of this phase II study were to more fully determine clinical efficacy and safety of T + B.Methods: Women with normal cardiac function and bidimensionally measurable, HER2-amplified (FISH), metastatic or locally recurrent unresectable breast cancer were eligible. Patients (pts) were excluded if they had received chemotherapy in the metastatic setting, had CNS metastases, proteinuria, >3 different organ sites of metastasis, >50% parenchymal liver metastasis, or symptomatic lung metastases. Prior adjuvant T was allowed if it was discontinued ≥ 1 year before study entry. B (10mg/kg q 2 weeks) + T (4mg/kg loading, then 2mg/kg weekly) IV was given, per the results of phase I testing (above).Results: From 12/2004 to 4/2007, 50 pts were enrolled at 19 US sites. Pt characteristics: median age 58, prior breast surgery-38 (76%), prior radiation-22 (44%), prior (neo)adjuvant chemotherapy-26 (52%), prior anthracyclines-25 (50%), prior taxanes-20 (40%), prior endocrine therapy–23 (46%), visceral metastasis-36 (72%). A median of 6.25 cycles (range 1-34) were administered to pts. Drug-related adverse events (AEs) (all Grade (Gr) 3/4 and any events reported in >10% of patients, NCI-CTC v.2) included hypertension (N= 30: 18 Gr 3/4, 12 Gr 1/2), fever/chills/infusion reaction (N=18: 2 Gr 3/4, 16 Gr 1/2), headache (N=17: 2 Gr 3/4, 15 Gr 1/2), epistaxis (N=17: all Gr 1/2), fatigue (N=15: 1 Gr 3, 14 Gr 1/2), proteinuria (N=12: 4 Gr 3/4, 8 Gr 1/2), AST /ALT increase (N=12, all Gr 1/2), diarrhea (N=10: 1 Gr 3, 9 Gr 1/2), edema (N=6, all Gr 1/2), nail changes (N=6, all Gr 1/2), dyspnea (N=5: 2 Gr 3/4, 3 Gr 1/2), leucopenia (N=5: Gr 3/4=2, Gr 1/2=3), inflammatory demyelination (N=1, Gr 3), hyperglycemia (N=1, Gr 3), hyponatremia (N=1, Gr 3) and irregular menses (N=1, Gr 3). One pt developed a perforated ulcer and 4 weeks later died of sepsis (Gr 5). One Gr 4 cardiac AE was reported at the end of cycle 2 in a pt who had previously received doxorubicin. In addition, there were 9 Gr 2 and 9 Gr 1 cardiac AEs (all asymptomatic). Objective clinical responses (WHO criteria) were documented in 24 pts (48%), including 2 CRs. 15 pts (30%) had stable disease, 6 of which lasted ≥ 6 mos (12%) for a clinical benefit rate of 60%. No pts remain on active study treatment. The median time to progression (TTP) was 9.2 mos (95% CI: 5.4-20.5). No difference in TTP was detected between hormone receptor negative and positive tumor types. Median overall survival was 43.8 months (95% CI: 40.6,NA).Conclusions: These are the final results for the first phase II trial of 2 humanized antibodies given in combination to human subjects. The results show that T + B is clinically feasible and very active despite the absence of chemotherapy in HER2+ advanced breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6094.

Background:Objective clinical response to trastuzumab monotherapy in erbB2-amplified first line metastatic breast cancer is 34% (Vogel, et al., JCO 20: 719-26, 2002). Amongst patients who respond, most develop resistance (defined by disease progression on trastuzumab). One proposed mechanism of trastuzumab resistance is proteolytic cleavage of erbB2 receptor from its full-length (p185) form into truncated, constitutively active p95. Increased expression of p95erbB2 correlates with increased nodal involvement and poor clinical outcome. Because p95erbB2 lacks the trastuzumab binding epitope, expression may designate patients who would be suitable for treatment with erbB2 kinase inhibitors.Materials and Methods:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in several cell types via retroviral vector. Additionally, an intracellular form of p95erbB2 that arises via alternative translation and an intracellular p95erbB2 construct containing two copies of a nuclear localization sequence were also expressed. Expression and proper subcellular localization of constructs were confirmed by cell fractionation, western blot analysis and confocal microscopy. Transformation of human mammary epithelial (HMEC) and NIH3T3 cells by p185erbB2 and p95erbB2 isoforms was evaluated by anchorage independent growth using a quantitative fluorescent soft agar assay, and effects on migration and invasion of these cells were investigated by wound-healing and transwell assays. Cells transfected with oncogenic Ras or empty vector were used as positive and negative controls in these experiments.Results and Discussion:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in HMEC and NIH3T3 cells, and were correctly directed to the cell membrane; nuclear targeted intracellular p95erbB2 was correctly localized to the cell nucleus. Both p185 erbB2 and membrane-bound truncated p95erbB2 were sufficient to transform HMEC cells as compared to empty vector control transfected cells [mean fluorescence intensity empty vector control 2480 ± 464 (1 standard deviation); mean fluorescence intensity p185erbB2 9208 ± 2528, p= 0.0106; mean fluorescence intensity p95erbB2 6615 ± 1588, p= 0.0124)] as was the positive control oncogenic Ras (mean fluorescence intensity 4350 ± 433, p=0.0069). Interestingly, nuclear-targeted p95erbB2 was also sufficient to transform HMEC cells (mean fluorescence intensity 6492 ± 818, p=0.0018). These data support the hypothesis that truncated p95erbB2 species may be major pathogenic drivers in erbB2-amplified cancers. P95erbB2 therefore represents an attractive target for diagnosis and treatment of erbB2+ breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3136.

Background: FFPE tumor samples present a technical challenge for gene expression profiling studies. Newer technologies are resulting in quality data and new insights from these archived tissues. TNBC is a sub-type of BC characterized by a lack of erbB2 gene amplification and estrogen and progesterone receptor expression. This clinically-defined BC sub-type carries a poor prognosis, is insensitive to hormonal or HER-2 targeted therapeutic agents, and has different incidence among ethnic groups. A better understanding of the genetic and molecular mechanisms underlying TNBC is critical to improving clinic outcomes and developing individualized therapies.Study Objective: We demonstrate the utility of FFPE samples in obtaining consistent, reproducible data from gene expression arrays, and apply this technology to the identification of differentially expressed genes between TNBC and normal breast tissue that are common or unique among selected ethnic groups.Methods: RNA isolation and labeled cDNA preparation from freshly cut FFPE blocks (marked by a pathologist as to normal vs. tumor tissue) was performed using the NuGEN™ WT-Ovation™ FFPE RNA Amplification System. Hybridization of tumor and normal cDNAs occurred to a breast cancer focused gene expression array (Breast Cancer DSA Research Tool, Almac Diagnostics Inc). Each patient' sample served as it's own control (tumor vs. normal). In total, 75 FFPE samples were profiled. The quality of each DSA chip was assessed on parameters selected automatically from GCOS report files per chip using MATLAB script based web application developed by Almac Dx. Data pre-processing used the Resolver Error Model. All parameters including Raw Q, Background, Scaling Factor and all controls met quality criteria set by Affymetrix and Almac Dx SOPs. Hybridization results were assessed using Principal Component Analysis and Clustering Analysis in Rosetta Resolver Gene Expression Data Analysis System 7.1 to identify potential outliers, contamination, or intra-tumor heterogeneity.Results: A Sign Agreement Matrix of FFPE and fresh frozen tissue samples during validation of the Breast Cancer DSA demonstrated that 98% of the probesets showed the same direction of fold change [p Spearman(FC)=0.84]. The FFPE samples had an average present call ∼ 43%, and more than 90% of the FFPE samples had present call rates greater than 25%. QC analysis demonstrated that the Almac Breast Cancer DSA was able to clearly and consistently separate tumor samples from normal in FFPE tissues and to identify samples of quality or integrity issues. Application of this methodology to analyses of differentially expressed transcripts between cancer and normal tissue samples across ethnicities in the TNBC samples, detected 1350 differentially expressed genes in the African-American group, 1220 genes in the Caucasian group and 1226 genes in the White Hispanic group. We also observed certain subtle ethnic-specific expression patterns across these three ethnic groups.Summary: The above-described methodology can be used to reliably measure gene expression in FFPE breast samples. Our study results are being validated in a larger data set. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6125.

1019 Background: Elevated baseline sECD (BsECD) correlates weakly with HER2 overexpression (HER2+). HR+ MBC. Lapatinib benefit was reported in pts with HER2+ tumors irrespective of BsECD status; BsECD status did not predict benefit for HER2- negative (HER2-ve) tumors. sECD levels and their predictive and prognostic value for progression-free survival (PFS) were further examined in a randomized, phase III trial of letrozole ± lapatinib. Methods: Pts (n=1286) with HR+ MBC were randomized to letrozole with placebo (P) or lapatinib (L). HER2 status was evaluated by FISH or IHC in archived tissue. sECD was measured by ELISA at baseline (n=1102 available samples), and every 4 wk. Pts were considered sECD+ if serum sECD was >15 ng/ml. Results: BsECD was positive in 14% (125/894) and 42% (87/208) of pts with HER2-ve and HER2+ tumors, respectively. Correlation between BsECD and FISH was weak but significant in HER2+ pts (R=0.35, p [Table: see text] [Table: see text]

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #46 Background: Cross-talk between growth factor and steroid receptor pathways has been implicated in endocrine resistance in breast cancer. Combining EGFR/HER2-targeted therapy with aromatase inhibitors for HR+ postmenopausal breast cancer may enhance endocrine responsiveness and delay the onset of resistance. [EGF30008][1] is a double-blind, placebo-controlled, first-line phase III trial to explore the benefit of letrozole with/without lapatinib, an oral dual tyrosine-kinase inhibitor of HER2 and EGFR, in patients (pts) with HR+ postmenopausal MBC. Methods: 1286 postmenopausal women with HR+ untreated MBC from 212 international centers were randomized to once daily treatment with letrozole 2.5 mg and lapatinib 1500 mg or letrozole and placebo. Pts were stratified according to presence of visceral vs bone only disease and time since completion of prior adjuvant endocrine tamoxifen therapy (

Abstract #2024 Background and Rational: Ethnic-specific disparities in breast cancer (BC) stage of presentation and survival rates are well documented. To further investigate possible ethnic-specific genetic contributions to these disparities, we are completing gene expression profiling studies in a multi-ethnic cohort consisting of thirty “Triple Negative” BC patients [10 each African-American (AA), Hispanic (His) and non-Hispanic white (Cauc) women] matched for age of diagnosis and hormone receptor status. The overall study aim is an increased understanding of the biological basis of ethnic-specific BC disparities, leading ultimately to individualized, ethnic-specific diagnostic and therapeutic approaches. Two immediate study goals are to demonstrate the utility of FFPE samples in obtaining consistent, reproducible data from gene expression arrays, and secondly, to identify differentially expressed genes between tumor and normal tissue that are common or unique among the three ethnic groups. Methods: Pathology specimens were freshly cut from FFPE blocks and marked by a pathologist as to normal vs. tumor tissue. RNA isolation, labeled cDNA preparation, and hybridization of tumor and normal cDNAs to a breast cancer focused gene expression microarray (Breast Cancer DSA Research Tool) was performed by Almac Diagnostics. Each patient was self-matched (tumor vs. normal tissue) for gene expression studies. Results: Using 36 matched tumor and normal FFPE samples from 18 patients, approximately 17516 transcripts were detected on the Breast Cancer DSA with intensity significantly greater than background. For normal and tumor tissue samples, 9399 and 10,296 transcripts respectively, were detected in all three ethnic groups. Importantly, a subset of transcripts (hundreds to one thousand) was detected in only one or two ethnic groups. Using two-way ANOVA (disease state and ethnicity), a subset of 6479 transcripts was identified with p-value less than 0.01 in the statistical test and was selected and further used in data quality control. Data QC indicated that patient samples clustered well with respect to both ethnicity and normal versus tumor tissue. Additional analytical methods included K-means 2-Dimensional clustering and Principal Component Analysis. From these analyses, we identified ethnic-specific expression patterns in the matched normal and tumor tissue samples. We are completing these studies by increasing sample size and matching for stage of diagnosis, mapping clusters of differentially-expressed genes in pathway analysis, and validation by real-time PCR. In the longer term, DNA copy number variation (CNV) and chromosomal alterations will be investigated by high density arrays. Summary: These preliminary analyses shows that high quality gene expression data can be generated from FFPE samples, and that ethnic specific gene expression differences can be detected in tumor and matched normal breast tissue samples across ethnic groups. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2024.

Abstract #3146 Background: Although serum extracellular domain (sECD) HER2 levels can be easily measured by enzyme immunoassays, the potential clinical utility of HER2 extracellular domain protein shed in the serum is not established. High serum protein levels may be indicative of metastatic disease to the liver or perhaps greater tumor burden. EGF30008 is a double-blind, placebo-controlled phase III trial evaluating the effect of letrozole with/without lapatinib in previously untreated postmenopausal women with hormone receptor-positive metastatic breast cancer. Levels of sECD HER2 were collected on all 1,286 women at baseline and every 4 weeks on study including at disease progression. Methods: Baseline serum levels and tumor characteristics were collated in blinded fashion for all 1,286 patients in the trial (data cut off 12May08). Patients with baseline serum protein levels ≥ 15 ng/mL were defined as sECD HER2+. Correlations were assessed between baseline serum protein levels and baseline liver disease and bone-only disease and total tumor burden (as defined by number of metastatic sites). Chi-square test was applied to assess the correlation between serum protein levels and liver disease or bone-only disease. Wilcoxon rank-sum test using normal approximation was used to compare total tumor burden and baseline protein levels. Results: Among sECD HER2+ patients, 42% had at least one liver metastasis, whereas 20% of sECD HER2-negative patients had at least one liver metastasis (P < 0.0001). No correlation exists between baseline serum protein level and bone-only disease (P = 0.64). Patients who were sECD HER2+ had a greater total tumor burden (mean metastatic sites = 5.4; standard deviation = 3.2; median = 5) compared with patients who were sECD HER2-negative (mean metastatic sites = 4.4; standard deviation = 3.1; and median = 4; P < 0.0001). Conclusion: High baseline sECD HER2 strongly correlates with tumor burden. There was also a significant correlation between sECD and liver metastases, but no correlation at all with bone-only disease. These analyses will be repeated after the database is finalized and unblinded. Our analysis is limited to detection of the number of metastatic sites and is not sensitive to the individual sizes of metastases. In addition, another potential explanation for the low sensitivity of HER2 detection by sECD is that small metastatic deposits that are HER2+ in situ do not shed enough sECD into the serum to be detected by the assay. Conversely, some very large HER2-negative tumors do in fact shed measurable sECD if the tumor bulk is very high, even though in situ they may be nonamplified. A future analysis will take the sum of the longest diameters of all known lesions and compare it to sECD HER2 levels. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3146.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #3147 Background: The extracellular domain (ECD) of the HER2 protein is cleaved by metalloproteases and shed into the serum. Serum ECD (sECD) HER2 can be detected by ELISA and may have prognostic/predictive associations in patients (pts) with breast cancer. Although use of sECD HER2 as a surrogate for HER2 overexpression in hormone receptor-positive (HR+) metastatic breast cancer (MBC) is controversial, seroconversion can occur at disease relapse and may reflect a change in tumor HER2 status that is associated with a poorer prognosis. [EGF30008][1] is a double-blind, placebo-controlled, first-line, phase III trial of letrozole with/without lapatinib in 1,286 postmenopausal women with HR+ MBC. We report baseline sECD HER2 at study entry on all pts and the association with original tumor HER2 status. Methods: Serum samples were collected at study entry and every 4 weeks on study, including at disease progression. Patients were defined as sECD HER2+ if baseline sECD HER2 was ≥15 ng/mL. HER2 status was evaluated in tumor tissue by both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), using conventional cutoffs. To assess the correlation between baseline sECD HER2 and tumor tissue HER2 status, odds ratios (OR) with 95% confidence intervals (CI) and sensitivity/specificity (tumor tissue HER2 status as standard) were calculated. Data were from a blinded cutoff on 12May08. These analyses will be repeated after the database is finalized and unblinded. Results: Mean age was 63 ± 9.8 years. All pts had a HR+ original tumor, and 217 (17%) pts were confirmed as HER2+ by IHC or FISH. Median time from primary diagnosis to MBC was 3.3 years (range 0 - 33.9 yrs). Baseline sECD HER2 levels ranged from 2.5 to > 560 ng/mL (mean 21 ± 60 ng/mL) with a bimodal distribution consistent with previously published data. The mean was 62 ng/mL (range 2.8 to > 560 ng/mL) for HER2+ pts and 12 ng/mL (range 2.5 to 113.1 ng/mL) for HER2-negative pts. The OR between sECD HER2 at study entry and HER2 status of the original tumor was 4.5 (95% CI: 3.2, 6.2) with 42% sensitivity and 86% specificity. C onclusions: This OR suggests some correlation exists between baseline sECD HER2 and original tumor HER2 status. We cannot assess the rate of seroconversion that may have occurred from primary diagnosis until MBC at study entry. The bimodal distribution suggests that a clear cohort of pts exists at the time of relapse/study entry who are positive for sECD HER2. Analyses are evaluating whether subsequent changes in sECD HER2 levels occur over time during therapy, whether seroconversion predicts for a shorter time to relapse during letrozole monotherapy, and whether any differences exist in sECD HER2 levels over time for pts treated with letrozole plus lapatinib are ongoing. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3147. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=EGF30008&atom=%2Fcanres%2F69%2F2_Supplement%2F3147.atom

2562 Background: This dose-escalation study defined a maximum tolerated dose (MTD) and pharmacokinetics (PK) of PR-104. In preclinical studies PR-104 is converted systemically to the corresponding ...

14049 Background: IGF-IR signaling interferes with the growth inhibitory action of T in BrCa cell lines and may play a role in clinical resistance to T. We studied the effect of IGF-IR inhibition on T resistance. Methods: HER2 and IGF-IR protein levels were determined by ELISA. T growth inhibition of HER2+ BrCa cell lines was measured by cell counting. HER2 and IGF-IR activity were determined by immunoprecipitation with HER2 and IGF-IR antibodies, followed by western blotting with phospho-tyrosine antibody. The effects of IGF- I/IGF-IR inhibition on proliferation and response to T were investigated. Results: No significant correlation was found between HER2, phospho-HER2 or IGF-IR levels and response to T in 12 HER+ BrCa lines. However, two T-conditioned cell lines show resistance to T (BT474 - 2.6 fold; SKBR3 - 1.8 fold) and have higher levels of phospho-IGF-IR compared to parental cell lines. Neither IGFBP3 nor IGF-IR antibody aIR3 significantly inhibited the growth of T-conditioned cell lines or enhanced the anti-proliferative effects of T. IGF-IR siRNA decreased IGF-IR protein levels and inhibited proliferation (29.8 ± 9.3 %) of T-conditioned SKBR3 cells. IGF-IR siRNA also enhanced T growth inhibition of T-conditioned SKBR3 cells (47.7 ± 13.7 % compared to 17.7 ± 14.8 % for T alone). The small molecule IGF-IR tyrosine kinase inhibitor (TKI), NVP-AEW541, inhibited growth of parental and T-conditioned SKBR3 cells and enhanced response to T (Table). Conclusions: Increased activation of IGF-IR may play a role in resistance to T. Inhibition of IGF-I ligand binding does not appear to alter IGF-IR mediated T-resistance. However, reducing IGF-IR protein levels or blocking IGF-IR TK activity improves response to T in resistant cells. Thus, the combination of IGF-IR TKIs, such as NVP-AEW541, with T may be beneficial for T-resistant HER2+ BrCa. [Table: see text] [Table: see text]

LBA1008 Background: Based on preclinical synergism between docetaxel (T), carboplatin (C) and trastuzumab (H), BCIRG conducted a phase III trial in HER2-positive MBC to evaluate efficacy and safety of H in combination with T or TC. Methods: 263 patients (pts) with HER2 FISH+ MBC were randomized to TH (H with T 100mg/m2) or TCH (H with T 75mg/m2 and C AUC=6). Chemotherapy was given every 3 weeks (q3w) for 8 cycles with weekly H at 2mg/kg (loading dose of 4 mg/kg) followed by H q3w at 6 mg/kg until progression. Pts were stratified by centre and prior (neo) adjuvant taxane chemotherapy. Primary endpoint was Time To disease Progression (TTP). Secondary endpoints include overall survival, response rate, duration of response (DR), clinical benefit (CB) and safety. Results: 131 pts were treated in each arm Pt characteristics were well balanced in both groups. A first efficacy analysis was conducted at 204 events. There was no significant difference between TH and TCH in median TTP (11.1 vs 10.4 mos, p=0.57), ORR (73% in both arms), DR (10.7 vs 9.4 mos) and CB (67% in both arms). At 39 months of median follow-up, median overall survival was 36.40 and 36.57 months in TH and TCH arms respectively. More patients on TCH received the max number of chemotherapy cycles, and numerically fewer patients on TCH discontinued treatment as a result of non hematological toxicity. The most common gr 3/4 toxicities were: Neutropenic infection that was 16.8% vs 9.2% respectively for TH and TCH, thrombocytopenia (2% vs 15%), asthenia (5% vs 12%), anemia (5% vs 11%), and diarrhea (2% vs 10%). Two pts died (1.5%) due to sepsis in TCH. Absolute LVEF decline > 15 % were seen in 5.5 % vs 6.7 % of pts. One pt (0.8%) had a symptomatic CHF in TH arm. Conclusion: Both TH (T 100) and TCH (T 75) were highly effective treatment regimens in women having HER2-positive MBC, demonstrating high response rates, median TTP > 10 months, and median overall survival > 36 months in both TH and TCH. Cardiac toxicity was no significant problem with either treatment. [Table: see text]

2533 Background: MUC-1 glycoprotein establishes a molecular barrier at the epithelial surface, and engages in morphogenetic signal transduction. A 20 amino acid MUC-1 core tandem repeat is aberrantly glycosylated in >90% of epithelial tumors, exposing peptide sequence PDTRP. Humanized IgG1 κ monoclonal antibody R1550 binds PDTRP (Kd∼1nM) and induces potent antibody-dependent cellular cytotoxicity. Methods: R1550 clinical tolerability and PK was determined in advanced, PDTRP-expressing (by IHC), breast cancer patients progressing after anthracyclines and taxanes. Key eligibility: informed consent, WHO PS 0–1, life expectancy >4 mos, non-inflammatory histology, and no CNS metastasis. R1550 was infused IV over 1–3 hrs: cohort 1 (N = 3) 1 mg/kg; cohort 2 (N = 9) 3 mg/kg; cohort 3 (N = 6) 9 mg/kg; cohort 4 (N=3) 16 mg/kg, with repeat dosing every 1–2 weeks (patient-individualized PK assessment) until disease progression. Plasma [R1550] was measured by quantitative ELISA. PK parameters were analyzed by a non-compartmental model, and a 2-compartment population PK model. Results: 21 patients (mean age 54, median # of prior chemotherapy agents = 4) have been dosed. Safety data (n=18): R1550 is generally well tolerated with fatigue (28%), gastrointestinal symptoms (22%), skin rash (17%), pyrexia (11%), and elevated liver function tests (11%) as the most frequently reported adverse events (AEs). Two grade 4 AEs were reported: elevated AST, and hyperglycemia, both in cohort 2. Neither have been observed in expanded or subsequent cohorts. Median number of doses received per patient = 3 (range, 3–4), 4 (1–13) and 6 (4–21), in cohorts 1, 2, and 3, respectively. No anti-humanized antibodies were detected. PK parameters of cohorts 1–3 are shown below. Conclusions: R1550 is well tolerated, with MTD exceeding 9 mg/kg. PK parameters using non-compartmental, and 2-compartment population PK models are in agreement. R1550 half-life suggests weekly dosing. Plasma exposure and dose relationships are linear within 1–9 mg/kg dose range. Accrual to cohort 4 is ongoing. [Table: see text] [Table: see text]

10515 Background: A phase II clinical trial was conducted to study the safety and efficacy of neoadjuvant docetaxel/carboplatin (T/C) with or without trastuzumab (H) in women with stage III breast cancer. Methods: Forty-eight of 75 planned primary breast cancer patients (T3 or T4, any N, M0), age between 18 and 80 have been enrolled. Four cycles of T (75 mg/m2) + C (AUC 6) were given every 3 weeks preoperatively. Patients with HER-2 amplified tumors (FISH +) were randomized to receive either weekly concurrent H or T/C alone preoperatively and T/C plus H postoperatively. Tumors were assessed clinically at baseline and after neoadjuvant therapy. Cardiac assessment consisted of medical history, EKG and LVEF (by echocardiogram or MUGA) at baseline and at the end of neoadjuvant chemotherapy. Results: Available data from 45 of 48 enrolled patients showed 49% (22 cases) with complete clinical response, with 54.5% being HER-2 (+) (12 cases). Stable disease was seen in one patient who was HER-2 (−) (2.2%). Of 37 with complete pathology verification, 11 (29.7%) showed pathologic complete response (pCR) of the primary tumor with 5 cases being HER-2 (+). Of the 22 HER-2 (+) cases that completed neoadjuvant treatment, 11 received T/C/H and 11 received T/C. pCR was noted in 36.4% of the T/C/H group and 9% of the T/C group. LVEF data is available from 43 patients during the neoadjuvant phase, showing 18.6% (8 cases) with decrease of ≥ 10% (5 patients in the T/C arm and 3 patients in the T/C/H arm), although none had cardiac symptoms or LVEF below the normal limit. Conclusions: T/C ± H is clinically active in patients with locally advanced breast cancer including a 30% pCR rate. The cardiotoxicity rates were comparable between patients who received T/C and T/C/H. No significant financial relationships to disclose.

LBA516 Background: Based on preclinical synergism between docetaxel (T), carboplatin (C) and trastuzumab (H), BCIRG conducted a phase III trial for women with HER2+ MBC to evaluate efficacy and safety of H in combination with T or TC. Methods: 263 patients (pts) with HER2 FISH+ MBC were randomized to TH, (H with T 100 mg/m2) or TCH, (H with T 75 mg/m2 and C AUC=6). Chemo was given q3 wks for 8 cycles with wkly H at 2 mg/kg (loading dose of 4 mg/kg), followed by H q3 wks at 6 mg/kg until progression. Pts were stratified by centre and prior (neo) adjuvant taxane chemotherapy. Primary endpoint was TTP with 80% power (0.05 significance) to detect a 50% improvement in median TTP between the 2 arms. Secondary endpoints include overall survival, response rate, duration of response (DR), clinical benefit (CB) and safety. Results: 131 pts were treated in each arm. Pt characteristics were well balanced in both groups. Importantly, only 52% of pts received C at the protocol specified dose (RDI > 0.9). Efficacy analysis was conducted at 204 events. There was no significant difference between TH and TCH in median TTP (11.1 vs 10.4 mos, p = 0.57), ORR (73% in both arms), DR (10.7 vs 9.4 mos) and CB (67% in both arms). The most common gr 3/4 toxicities were: infection (44% vs 30%), neutropenic infection (22% vs 12%), thrombocytopenia (2% vs 15%), febrile neutropenia (12% vs 13%) asthenia (5% vs 12%), anemia (5% vs 11%), and diarrhea (2% vs 9%). Two pts died (1.5%) due to sepsis in TCH. Absolute LVEF decline > 15% were seen in 5.5% vs 6.7% of pts. One pt (0.8%) had a symptomatic CHF in TH arm Conclusions: The already effective TH regimen does not benefit from the addition of C, when the T dose in TH is 100 mg/m2 and 75 mg/m2 in TCH, in women with HER2+ MBC. No significant financial relationships to disclose.

563 Background: Autocrine and paracrine signaling involving VEGFR and PDGFR are implicated in angiogenesis and proliferation of BC. SU11248, an oral multitargeted TKI with both antiangiogenic and anittumor activity, inhibits VEGF, PDGF, KIT, and FLT3 receptor TKs. Therefore, SU11248 was studied in pts with previously treated MBC. Methods: Eligible pts had histologically confirmed MBC and had received prior chemotherapy regimens that must have included treatment with an anthracycline and taxane; measurable disease was required. Pts were treated with SU11248, 50 mg daily, in repeating 6-wk cycles of 4 wks of dosing followed by 2 wks without treatment. The primary endpoint was ORR defined by RECIST. Sample size was calculated using Simon’s Minimax 2-stage design to identify a clinically meaningful ORR ≥15%. The first stage will enroll 38 pts, with a second cohort of 25 to be enrolled if >2/38 pts achieve confirmed PRs. Results: The study has enrolled 37/38 pts in the first stage. Preliminary data are availab...

5051 Background: Pertuzumab (P), a humanized HER2 antibody, represents a new class of targeted agents called HER dimerization inhibitors (HDIs) that inhibit dimerization of HER2 with EGFR, HER3 and...