Outcome In HER2 Research Articles

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

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

An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models.

IntroductionThe human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance.MethodsUsing a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs.ResultsThe signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings.ConclusionsMolecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.

Rac-specific guanine nucleotide exchange factor DOCK1 is a critical regulator of HER2-mediated breast cancer metastasis

Progression of solid tumors to the metastatic stage is accountable for the majority of cancer-related deaths. Further understanding of the molecular mechanisms governing metastasis is essential for the development of antimetastatic regimens. Here, we aimed to identify Rac activators that could promote metastasis downstream of human epithelial growth factor receptor 2 (HER2). We investigated if Dedicator of Cytokinesis 1 (DOCK1), based on its evolutionarily conserved role in receptor tyrosine kinases (RTKs)-mediated Rac activation and cell invasion, could be a regulator of metastasis. We report that high expression of DOCK1 in HER2(+) and basal breast cancer subtypes inversely correlates with human patients' survival. Mechanistically, DOCK1 interacts with HER2 and promotes HER2-induced Rac activation and cell migration. To gain further insight, we developed a HER2 breast cancer mouse model with mammary-gland-specific inactivation of DOCK1. In this in vivo model, a significant decrease in tumor growth and metastasis in lungs was found in animals where DOCK1 is inactivated. Furthermore, we found that DOCK1 is required for maximal activation of two HER2 effectors, c-JUN and STAT3. Using an unbiased gene profiling approach, we identified a mammary tumor DOCK1-associated gene signature enriched for genes implicated in response to IFN type I. This analysis revealed a unique set of genes, including Receptor Transporter Protein 4 (RTP4) and STAT1, for which the expression levels can be used to independently predict breast cancer outcome in HER2(+) patients. Our work demonstrates DOCK1-Rac signaling as an HER2 effector pathway essential for HER2-mediated breast cancer progression to metastasis and offers a therapeutic opportunity to limit the spread of metastatic breast cancers.

Abstract 2387: Activation of Inhibin A signaling is associated with a basal-like HER2 subtype and resistance to lapatinib in breast cancer cell lines.

Abstract HER2 amplification occurs in ∼20% of all breast cancers, and is associated with poor prognosis. Targeted therapeutics such as trastuzumab and lapatinib have been developed and have improved outcomes for patients with HER2+ disease. However, a significant proportion of patients have tumors that either develop resistance or simply fail to respond to these therapies at all. To better understand the mechanism of resistance, we screened a panel of 22 HER2+ breast cancer cell lines with lapatinib. Of these, 6 showed significant resistance, failing to reach 50% growth inhibition (GI50) even at 5uM, the highest dose tested. The resistant lines were all more basal-like in expression patterns, and were more responsive to MEK inhibitors than their luminal-like counterparts. These two patterns of expression are similar to the different HER2 subtypes recently identified by the TCGA project. Differential expression analysis utilizing t-tests with Benjamini-Hochberg correction for multiple comparisons identified approximately 150 transcripts that were more highly expressed in resistant cell lines. We performed high-throughput screening using siRNA knockdown of the target genes in combination with lapatinib treatment. Staining for markers of proliferation (Ki67), apoptosis (cleaved PARP), and activity of the PI3K-AKT pathway showed that silencing of inhibin A resulted in a decrease in the ratio of proliferative to apoptotic cells in the resistant cell lines 21-NT and 21-PT. Concurrent with this decrease was a down-regulation of both p-MAPK and p-AKT. GO analysis revealed a strong enrichment for Inhibin A signaling amongst the 150 resistance associated transcripts. Mining of tumor databases revealed that there was a trend between high levels of Inhibin A expression and poor outcome in HER2 positive tumors (p=0.08). These data suggest that activation of Inhibin A signaling is associated with a more basal like subclass of HER2+ tumors, resistance to HER2 targeted therapeutics such as lapatinib, and that corresponding over-expression of Inhibin A is associated with poor outcome in HER2+ patients. Citation Format: James E. Korkola, Juha Rantala, Nora Bayani, Laura Heiser, Nicholas Wang, Obi Griffith, Joe Gray. Activation of Inhibin A signaling is associated with a basal-like HER2 subtype and resistance to lapatinib in breast cancer cell lines. [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 2387. doi:10.1158/1538-7445.AM2013-2387

Abstract S1-5: PIK3CA mutations are linked to PgR expression: a Tamoxifen Exemestane Adjuvant Multinational (TEAM) pathology study.

Abstract Background: PIK3CA is mutated in about 26% of breast cancers (BC) and is the most frequently mutated gene in (BC). Almost 95% of mutations occur in exons 9 (E9) or 20 (E20). PIK3CA mutations may be associated with increased survival in endocrine-treated patients; however the impact of mutations in E9 vs E20 is not clear. We assessed 10 common PIK3CA mutations (95% of all mutations), in ER-positive (+ve) samples from the TEAM pathology study (n = ∼4500), and determined the impact of PIK3CA mutations on survival. We report an interim analysis of 1969 TEAM cases. Methods: DNA was extracted from formalin-fixed paraffin embedded sections. Mutational analyses were performed on 25 mutations in 6 genes (PIK3CAx10, Akt1x1, KRASx5, HRASx3, NRASx2 & BRAFx4), using Sequenom MassArray. Results: Mutations were found in PIK3CA: 37.5%; Akt1: 3.3%; KRAS: 0.3%; and BRAF: 0.1% of cases. No mutations were found in HRAS or NRAS (n = 1969). 90% of PIK3CA mutations were located in E9 and E20. Outcome data was available for 1958/1969 patients. Patients whose tumours contained any PIK3CA mutations (n = 739) were at lower risk of distant metastasis, Hazard ratio=0.86 (0.67–1.11), when compared to those without PIK3CA mutations (n = 1219); although this difference was not statistically significant (Cox Regression; p = 0.24). PIK3CA mutations were significantly more frequent in HER2-negative (−ve) (39%) than in HER2+ve samples (25%) (p = 0.001), without evidence that PIK3CA mutations differentially impacted outcome in HER2+ve vs HER2-ve patients. A positive correlation was demonstrated between PIK3CA mutations and PgR Allred score (p = 0.002) but not ER Allred score (p = 0.37). With increasing PgR Allred score increase there is an increased frequency of mutations in E20 but not E9 (Table 1). Discussion: This study indicates a higher percentage of PIK3CA mutations in ER+ve BC samples than previously demonstrated, either for BC as a whole or for ER+ve cases, suggesting that in ER+ve early BC, PIK3CA mutations are more common than previously reported. Furthermore, increased PIK3CA mutation frequency is significantly associated with increasing PgR Allred score and this appears solely due to increased numbers of patients with E20 mutations further complicating the analysis of the impact of PIK3CA mutations in BC. This may explain current uncertainty regarding the impact of PIK3CA mutations in E9 vs E20 with respect to clinical outcome. Whilst we were unable to show a significant impact on outcome in patients whose tumours contained PIK3CA mutations, we believe the complex relationship between PgR expression (good prognosis indicator) and PI3K mutations requires further exploration in the full dataset using interaction techniques adjusting for the impact of PgR on outcome. Mutational analysis and correlation with clinical outcome data for the remaining ∼2500 DNA samples, along with the existing data for 1969 patients, will be presented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S1-5.

Abstract P5-10-02: High serum levels of miR-19a are associated with poor outcome in metastatic inflammatory breast cancer

Abstract Background: Increased expression of miR-19a plays an important role in tumor progression by regulating angiogenesis and apoptosis. As miRs can be a valuable diagnostic and prognostic serum biomarker, we assessed if high serum levels of miR-19a were associated with the shorter overall survival (OS) of patients with metastatic inflammatory breast cancer (MIBC) and metastatic non-inflammatory breast cancer (MNIBC). Methods: We analyzed 27 MNIBC (10 HER2 normal [HER2−] and 17 HER2 amplified [HER2+]), 37 MIBC (18 HER2− and 19 HER2+) patients, and 30 healthy donors (HDs). Patients' sera were collected before starting a new line of treatment. MiR-19a levels were measured in patients' sera and cell lines (MCF-7, SKBR3, MA-231, KPL-4, SUM-149) by qRT-PCR to assess differences in expression levels between IBC and non-IBC. MiR-192 and U6 snRNA were used to normalize miR-19a expression levels in serum and cell lines, respectively. Fold-changes in expression of miR-19a were calculated using the 2−DCt method. Mann-Whitney U test, ROC curve analysis, Kaplan-Meier, and Student's t-test were used for statistical analysis. Results: Median levels of miR-19a were higher in sera of both MNIBC and MIBC patients than in HDs (p = .001, p < 001, respectively). Median serum level of miR-19a was higher in MIBC than in MNIBC (p = .013). ROC curve analysis revealed that miR-19a could differentiate MNIBC from MIBC (AUC = 0.683; p = 0.013). With a cutoff value of 1.62 set by ROC, the sensitivity, specificity and positive predictive value (PPV) were 56.7%, 85.2%, and 84.0%, respectively. The triple receptor negative (TN) IBC cell line SUM-149 had the highest level of miR-19a and it was higher than the non-IBC: MDA-231 (TN; p = .031); SKBR3 (HER2+; p = .021); and MCF-7 (ER/PR+; p = .003). KPL-4 (IBC, HER2+) had the second highest miR-19a levels after SUM-149. MIBC patients had a shorter median OS than MNIBC patients (18.8 vs 27.8 months; p = .041; range, 0.7–40.3 months; median follow-up 19.5 months). MIBC patients with HER2− tumor (n = 11) and high serum levels of miR-19a (>1.62) had a shorter median OS than MNIBC patients with HER2− tumors (n = 9) and low serum level of miR-19a (<1.62) (16.15 vs 33.45 months; p = .025). There was no difference in the OS between HER2+ MIBC patients with high miR-19a (> 1.62) and HER2+ MNIBC patients with low miR-19a (<1.62). Discussion: In this pilot study, miR-19a may be a potential biomarker for inflammatory breast cancer, as significantly higher miR-19a levels were present in the sera of MIBC compared with MNIBC as well as in IBC compared with non-IBC cell lines. Furthermore, miR-19a may be predictive of OS, as HER2− MIBC patients with high serum levels of miR-19a had shorter OS than HER2− MNIBC patients with low serum levels of miR-19a. Our data suggest that high expression of miR-19a: is a feature of IBC tumor cells; can be detected in the serum; may favor tumor progression and predict poor outcome of HER2− MNIBC patient. A larger cohort of patients is required to confirm these observations and examine predictive potential. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-02.

Correlation of quantitative p95HER2, HER2, and HER3 protein expression with pathologic complete response (pCR) in HER2-positive breast cancer patients (pts) treated with neoadjuvant chemotherapy and trastuzumab.

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<0.0001) and was significantly associated with higher H2T levels (p=0.02) and lower H3T levels (p=0.04). In ER- subjects (N=24), no difference in H2T levels was observed between pCR vs non-pCR groups (median H2T=111.5 vs 150.5, respectively; p=0.721). However, within the ER+ group (N=21), H2T levels were significantly higher in the pCR group vs non-pCR group (median H2T=254 vs 37.3; p=0.024). Using multivariate logistic regression, increasing log(H2T) (p = 0.012), ER-negativity (p = 0.027) and low p95 (p = 0.074) were found to correlate or trend with pCR. Conclusions: pCR was significantly associated with high H2T, particularly in ER+ HER2+ breast cancer pts who received NEO therapy with T + chemotherapy. Lower H3T was also associated with pCR. A trend towards pCR was seen in tumors with low p95. These data suggest that quantitative H2T, H3T and p95 may provide additional information on response to T-based regimens in breast cancer stratified by ER status. Additional investigation into the relationship between quantitative H2T, p95 and H3T expression and T response in the NEO setting in larger cohorts is warranted.

Correlation of quantitative p95HER2 and HER2 protein expression with pathologic complete response (pCR) in HER2-positive breast cancer patients (pts) treated with neoadjuvant (NEO) trastuzumab-containing therapy.

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<0.0001) and was significantly associated with higher H2T levels (p=0.02). In ER- subjects (N=24), no difference in H2T levels was observed between pCR vs non-pCR groups [median H2T=111.5 (IQR 63.4-162.2) vs 150.5 (IQR 43 – 226.2), respectively; p=0.721]. However, within the ER+ group (N=21), H2T levels were significantly higher in the pCR group vs non-pCR group [median H2T=254 (IQR 181.5-584.5) vs 37.3 (IQR 16.4-89); p=0.024]. Using multivariate logistic regression, increasing log(H2T) (p = 0.011), ER-negativity (p = 0.027) and low p95 (p = 0.074) were found to correlate or trend with pCR. Conclusions: pCR was significantly associated with high H2T expression in ER+ HER2+ breast cancer pts who received NEO therapy with T + chemotherapy. A trend towards pCR was seen in tumors that had low p95. These data suggest that quantitative H2T and p95 may provide additional information on response to T-based regimens in breast cancer, particularly ER+ breast cancer. Additional investigation into the possible relationship between quantitative levels of HER2 and p95 expression and T response in the NEO setting in larger cohorts is warranted.

Susceptibility of HER2+ breast cancer cells to poly (ADP-ribose) polymerase (PARP) inhibition independent of an inherent DNA repair defect.

621 Background: HER2 overexpression in breast cancer confers increased tumor aggressiveness. Targeted therapy against HER2 has improved outcomes, but resistance and disease progression ultimately occurs, thus necessitating novel therapeutic strategies. PARP inhibitors target homologous recombination (HR) deficient tumors, such as the BRCA-associated breast and ovarian cancers. In this study, we report unexpected susceptibility of HER2+ breast cancer cells to PARP inhibition alone independent of an inherent HR deficiency. Methods: Cell viability was measured using colony formation and ATPLite assays. Tumor growth delay was assessed in vivo in mice bearing breast cancer xenografts. Proteins were detected by immunoblotting. HR was assayed using radiation (IR)-induced Rad51 foci or a GFP-based HR assay. NFκB activity was measured using a NFκB-driven luciferase assay. Results: Surprisingly, PARP inhibition with ABT-888 alone reduced the colony forming ability and cell viability of the HER2+ breast cancer cell lines BT474, SKBR3, MDA-MB361, and HCC1954 (~70 – 99% reduction at 10μM). This susceptibility did not correlate with an inherent HR deficit. Interestingly, HER2 overexpression itself may be one mechanism of susceptibility to ABT-888 as evidenced by increased cellular cytotoxicity and in vivo tumor growth delay of MCF7 cells stably expressing HER2. Further dissection of the mechanism revealed that NFκB transcriptional activity was significantly inhibited by ABT-888 (>95%) which corresponded with reduced levels of phosphorylated p65 and total IKKα, and a concomitant increase in IkBα. Furthermore, overexpression of p65 abrogated cellular sensitivity to ABT-888. Conclusions: HER2+ breast cancer cells are highly susceptible to PARP inhibition despite being HR proficient. This may be, in part, due to inhibition of NFκB. Further investigation of whether the addition of PARP inhibition to HER2 targeted therapy will delay the onset of resistance to therapy is warranted to potentially improve outcomes in HER2+ breast cancer patients.

VEGF polymorphism may be associated with overall survival in lapatinib-treated breast cancer patients with brain metastases.

10532 Background: Lapatinib is a dual HER2/EGFR tyrosine kinase inhibitor (TKI) approved in combination with capecitabine or letrozole for patients with HER2+ metastatic breast cancer (MBC). Consistent with TKI therapies, patient response is variable and suggestive of additional determinants of sensitivity and resistance. This exploratory pharmacogenetic study sought to identify germline genetic variants that associate with lapatinib treatment outcomes in HER2+ MBC patients. Methods: Fifty five functional single nucleotide polymorphisms (SNPs) in 24 candidate genes were evaluated in a subset of MBC patients participating in two clinical trials: EGF105084: lapatinib monotherapy in HER2+ MBC patients with recurrent brain metastases following trastuzumab and cranial radiotherapy (n=120) and EGF104900: lapatinib plus trastuzumab (n=92) and lapatinib monotherapy (n=103) in HER2+ MBC patients with disease progression following trastuzumab. Testing for associations of SNPs with progression free survival (PFS) and overall survival (OS) during lapatinib treatment was performed using Cox proportional hazards methods, with covariate adjustment. Markers were considered to be significantly associated if they achieved a predefined multiple testing threshold of p<0.0003. Results: No SNPs were significantly associated with PFS in either study. A SNP in VEGFA (rs3025039, 936C>T) was significantly associated with improved OS (p=0.0002) for the T allele carriers, with an allelic hazard ratio of 0.21 (0.08-0.52) in EGF105084. The association was not seen in EGF104900. This SNP is located in the 3’ UTR gene region, the T allele is associated with lower serum VEGFA levels and reduced breast cancer risk [Krippl et al, 2003, Int J Cancer 106: 468; Kataoka et al, 2006, Cancer Epidemiol Biomarkers Prev, 15:1148]. Conclusions: A germline variant in VEGFA may be associated with survival outcome in MBC patients with brain metastases who are treated with lapatinib. This may represent activation of VEGF angiogenic pathways to overcome HER2 inhibition in patients carrying the higher expression genotype. These associations are considered exploratory and require confirmation in an independent dataset.

S3-3: Association of PTEN Loss and PIK3CA Mutations on Outcome in HER2+ Metastatic Breast Cancer Patients Treated with First-Line Lapatinib Plus Paclitaxel or Paclitaxel Alone.

Abstract Background Identifying molecular determinants underpinning response and resistance to anti-cancer therapies is essential to selecting optimal treatment for patients (pts). The PI3K pathway is frequently deregulated in cancer. In preclinical models deregulated PI3K has been shown to enhance the survival of breast cancer cells and confer resistance to chemotherapy and HER2−directed agents. This exploratory study evaluated the impact PIK3CA mutations and PTEN loss had on the efficacy of paclitaxel alone (P) and in combination with lapatinib (L+P) in HER2−positive metastatic breast cancer (MBC) pts enrolled in study EGF104535, results of which demonstrated that pts treated with the combination of L+P derived an absolute improvement in median overall survival (OS) of 7.3.months (median OS=27.8 months in L+P compared with 20.5 months in P). Method Baseline tissue from primary breast tumor or metastatic site was obtained in the form of formalin-fixed, paraffin-embedded material. Of the total study population (n=444), 389 pts had tissue available for biomarker analyses, 277 of whom provided written informed consent for optional tumor genetics (i.e., PIK3CA analysis). PTEN was evaluated on whole sections by immunohistochemistry (rabbit monoclonal antibody); pathology review and image analysis were performed, producing an ordinal score and optical density (OD). Qiagen/DxS assay was used in assessing PIK3CA mutation status. Logistic regression and Cox-proportional hazard models were used in analyses of biomarkers with efficacy endpoints. Results Evaluable results for PTEN and PIK3CA were available for 91% (354/389) and 62% (171/274) of pts, respectively. In the overall population, 24% (65/274) had tumors harboring PIK3CA mutations (E542K, E545K/D, H1047R); 13% (49/389) had absence of PTEN expression. Survival outcome appeared to be independent of PTEN expression (P >0.2). PIK3CA mutations were significantly associated with worse OS (HR=1.88; 95% CI=1.26, 2.80; P=0.002). PFS was significantly improved in pts treated with L+P compared with P in both PTEN strata (loss/expression; P<0.001). In pts stratified by PIK3CA status, treatment with L+P reduced the risk of progression compared with P in the wildtype strata (N=106; HR=0.43; 95% CI=0.28, 0.65; P<0.0001); in the mutation strata, PFS was not significantly reduced with the addition of L (N=65; HR=0.70; 95% CI= 0.42, 1.17; P=0.179). Conclusions In this exploratory study, PTEN was not prognostic whereas PIK3CA mutations appear to be an adverse prognostic feature in HER2+ MBC. PTEN expression may not be predictive as both strata derived significant benefit with L+P treatment. PFS benefit from L+P was significant in pts with PIK3CA wildtype tumors whereas the PFS benefit was not significant in the PIK3CA mutation strata. Additional analyses are ongoing and will be presented. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S3-3.

S3-2: Neoadjuvant Chemotherapy Adapted by Interim Response Improves Overall Survival of Primary Breast Cancer Patients – Results of the GeparTrio Trial.

Abstract Background: The GeparTrio phase III trial investigated the concept of interim response-adapted neoadjuvant chemotherapy. Patients with an early response after 2 cycles chemotherapy were considered highly chemo-sensitive and randomized to additional 2 chemotherapy cycles compared to standard treatment. Patients with no early response were considered less chemo-sensitive and randomized to continue with a non-cross-resistant chemotherapy or with standard chemotherapy. Pathological complete response (pCR) rates were different between responders and non-responders but not between the randomized arms (von Minckwitz G, et al JNCI 2008+2008; Huober et al. BCRT 2010). We report here on the results of the secondary endpoints: disease-free (DFS) and overall survival (OS). Patients and Methods: 2072 patients with operable or locally advanced breast cancer were treated with 2 cycles TAC (docetaxel, doxorubicin cyclophosphamide) before interim response assessment. Responders were randomized to additional TACx4 (N=704) or TACx6 (N=686) and non-responders to TACx4 (N=321) or NXx4 (vinorelbine, capecitabine) (N=301). None of the HER2+ patients received Trastuzumab. Endocrine treatment was given postoperatively to ER+ and/or PgR+ patients. We observed 480 recurrences and 302 deaths during median 62 months of follow up. Results: Patients receiving the experimental treatments (TACx8 or TACx2-NXx4) showed a longer DFS (HR 0.71; 95%CI 0.60−0.86, p<0.001) and longer OS (HR 0.79; 95%CI 0.63−0.99, p=0.048) compared to patients receiving standard TACx6 treatment. Treatment effects on DFS were restricted to patients with luminal A (p=0.003), luminal B (HER2−) (p=0.006) and luminal B(HER2+) (p=0.04) tumors. Experimental treatments did not improve outcome in HER2+(non-luminal) (p=1.0) and triple-negative (p=0.5) tumors. Responders showed a significant longer DFS (HR 0.79; 95%CI 0.62−0.97), p=0.026) and a trend towards a longer OS (HR 0.76; 95%CI 0.57−1.01, p=0.061) if they were treated with TACx8 compared to TACx6. Non-responders showed a longer DFS (HR 0.6; 95%CI 0.43−0.82, p=0.001) but not OS (HR 0.85; 95%CI 0.57−1.27, p=0.4) when treated with TACx2-NXx4 compared to TACx6. Results according to phenotypes of responders and non-responders were comparable to the overall comparison. In general, patients with a pCR showed a better DFS if they had triple-negative (p<0.0001), HER2+(non-luminal) (p<0.0001) or luminal B(HER2−) (p=0.004), but not if they had luminal A (p=0.66) or luminal B (HER2+) (0.67) disease. Conclusion: Adapting neoadjuvant chemotherapy according to interim response leads to better DFS and OS and represents therefore a unique advantage over adjuvant treatment. The investigated strategies to improve standard chemotherapy were most effective in the luminal A and B phenotypes. These phenotypes are usually considered less chemo-sensitive and pCR is not a prognostic factor. This might explain why the observed survival advantages could not be predicted by pCR. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S3-2.

P1-12-15: Adjuvant Trastuzumab Effect on HER2−Positive Breast Cancers According to Hormonal Receptor (HR) Status: Crosstalk between ER and EGFR/HER2 Pathway May Prevent Trastuzumab from Improving Outcomes in HER2−Positive and HR-Positive Breast Cancers.

Abstract Background: Crosstalk between growth factor receptor, especially the EGFR/HER2 pathway, and ER pathways has been associated with endocrine resistance. Thus, combination therapy targeting both ER and EGFR/HER2 signaling to block the crosstalk between these pathways and eliminate escape routes have been proven effective in both preclinical and clinical models. Anti-HER2 directed therapy has been reported to restore hormone sensitivity in HER2−positive breast cancers. Adding trastuzumab to conventional treatment has been a standard treatment of choice in HER2−positive breast cancer irrespective of hormonal receptor (HR) status. The purpose of the study is to evaluate adding effect of 1 year of trastuzumab to conventional adjuvant treatment in patients with HER2−positive breast cancer who received surgery according to HR status. Patients and Methods: We retrospectively analyzed the clinicopathologic characteristics and outcomes of 618 postoperative HER2−positive breast cancer patients between 2001 and 2008 at the Samsung Medical Center. Most of HER2−positive patients in our institute were treated with 1 year of trastuzumab as a part of adjuvant therapy since 2007 (post-trastuzumab era) compared with 2000–2006 (pre-trastuzumab era). Clinical outcomes including recurrence-free survival (RFS) were analyzed between pre-trastuzumab and post-trastuzumab era according to HR status. We performed Cox regression multivariate analysis for relapse using variables from univariate analysis by log-rank test for relapse. Clinical presentations and clinicopathologic characteristics were evaluated at the time of recurrence between both eras. Results: The median age at diagnosis was 46 years (range, 22–79). During the median 60.0 months of follow-up, the 5-year recurrence rate was 20.2%. The 618 patients were divided into two groups (patients who received (n=175) and did not receive (n=443) adjuvant trastuzumab). Recurrence rate was much lower in post-trastuzumab era than in pre-trastuzumab era (13.6% vs. 32.3%, p<0.0001). Improving outcomes due to adding trastuzumab in patients with HER2+ve/HR-ve patients showed significant benefit from trastuzumab throughout the follow-up period (p=0.004). However, this improving effect appeared not to be consistent with statistical significance in HER2+ve/HR+ve patients (p=0.135). The analyses were performed according to quantitative ER Allred scores in HER2+ve/ER+ve patients, the effect of adding trastuzumab appeared to be mitigated as time over without any statistical significance (p=0.975). Young age (≤35) (hazard ratio (HR) 2.4, p<0.0001), trastuzumab use (HR 0.4, p=0.001), and node positivity (HR 2.8, p<0.0001) were identified as independent prognostic factors for recurrence in Cox-regression multivariate analysis. Limiting to HER2+ve/ER+ve patients, the statistical significances of trastuzumab use as independent factors were not maintained in Cox-regression models (p=0.074 for trastuzumab use). Conclusion: Cross-talk between ER and EGFR/HER2 pathways may mitigate trastuzumab effect in HER2+ve/ER+ve breast cancers. Further study is warranted. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-15.

S1-4: Retrospective Analysis of Study EGF30008 by Mass-Spectrometry Based Serum Assay (VeriStrat®).

Abstract Background: In EGF30008 the addition of lapatinib (La) to first-line treatment with letrozole (Le) significantly improved progression-free survival (PFS) in patients (pts) with hormone receptor positive (HR+), HER2 positive (HER2+) metastatic breast cancer (hazard ratio (HR) = 0.71; p=0.019), but not in HR+, HER2 negative (HER2−) pts. This study assessed the ability of VeriStrat (VS), a mass-spectrometry blood-based pretreatment assay correlating with clinical outcome from EGFR-TKI therapy, to stratify pt outcome in EGF30008. The VS assay assigns VS Good (Good), VS Poor (Poor), or Indeterminate labels to a serum sample based on a specific 8-peak signature in the mass-spectra (Taguchi F et al., JNCI 2007). Methods: Blinded to clinical data, pretreatment serum was analyzed using standard VS procedurei. Statistical analyses were performed using Mantel-Haenszel and Cox proportional hazards methods; correlations were evaluated using Fisher's exact and χ2 tests. Results: Of the 1286 pts randomized (HER2+=219; HER2−=952; HER2 unknown=115), 1163 pts had serum available; a VS label was assigned to 1046 pts (961 Good; 80 Poor; 5 Indeterminate); 117 were not evaluable (hemolyzed). In the overall population there was no significant difference in PFS between Good and Poor groups within the Le+La arm (p=0.53). In contrast, PFS of the Good group was longer than that of the Poor group within the Le only arm (HR=0.36, p<0.001) and comparable with that of Good pts within the Le+La arm (median PFS 10.8 mo vs 11.0 mo). An interaction test showed significantly different HRs for PFS in Good vs Poor between treatment arms (p=0.002). In the HER2+ population both Good (n= 169) and Poor (n= 13) groups received significant PFS benefit from addition of La to Le: in Good HR=0.71, p=0.046, medians 3.0 mo (Le) vs 8.0 mo (Le+La); in Poor HR=0.17, p=0.02, medians 2.3 mo (Le) vs 8.6 mo (Le+La). In the HER2− population median PFS for the Poor group (n=58) increased from 3.1 to 11.0 mo (HR=0.57, p=0.068) with addition of La to Le, whereas the Good group (n=702) received no significant benefit, median 13.6 mo on Le and 13.8 mo on Le+La (PFS HR= 0.85, p=0.09). In multivariate analysis VS remained independently significant and predictive of differential treatment effect in the overall population. No significant correlation of VS classification with HER2 status (p=0.35) or prior adjuvant hormonal therapy (p=0.33) was found. VeriStrat was also not significantly correlated with baseline HER2 ECD levels (low <15 ng/ml vs. high ≥15 ng/ml), p = 0.23, or ER expression (H-score:lower quartile:<160 vs. rest), p=0.18. Conclusions: In the overall population VeriStrat predicted PFS in Le alone but not in the Le+La combination, with a significant differential treatment effect. VeriStrat identified a subset of pts with inferior PFS on Le therapy. Adding La to Le improved outcome in HER2+ pts, and interestingly suggested an improvement in the VS Poor subset of HER2− pts. HER2− VS Good pts do not gain benefit with the addition of La. No correlation between VS and investigated predictors of benefit from the addition of La to Le in HER2− pts was observed. If these results can be validated in a prospective study, the addition of La to Le may be a potential treatment option for HER2− HR+ VS poor pts. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S1-4.

Management of early-stage breast cancer: Are we headed in the right direction?

284 Background: The understanding of breast cancer (BC) as a heterogeneous disease consisting of distinct subtypes based on variation in expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) has led to more personalized treatment. We postulated that with increased adoption of chemotherapy and targeted therapy for HER2 positive patients, the outcomes of ER/PR+, HER2- and of HER2+ subtypes of breast cancer would be similar. Methods: A chart review was performed of female patients >18 years old seen by a medical oncologist at an academic cancer centre in Toronto, Canada, between January 1, 2005 and December 31, 2006, for stage I-III invasive breast cancer. Clinical features, 5-year overall (OS) and relapse-free survival (RFS) of three BC subtypes were compared: ER/PR+, HER2- (hormone receptor positive, HR), HER2+ (HER2), and ER/PR-, HER2- (triple negative, TN). Results: Of 870 patient charts reviewed, 525 were analysed. There were 341 HR, 101 HER2 and 83 TN patients. TN patients were younger (p<0.001), and had higher stage (p<0.001) and higher histological grade tumors (p<0.001). The 5-year RFS and OS were: HR: 88.2% and 96.6%, HER2: 76.7% and 92%, TN: 79.8% and 83.9%. Chemotherapy was used in 41.1%, 84.2% and 83.1% of HR, HER2 and TN patients. Anthracycline plus taxane regimens were used in 48.6%, 52.9% and 68.1% of HR, HER2 and TN patients, respectively. Among HER2 patients, only 74.3% received trastuzumab. The 5-year RFS and OS for HER2 patients who received trastuzumab were 79.9% and 91.8%, and for those who did not: 69% and 91.6%. Conclusions: HR+ patients have an excellent outcome. Despite significant improvement in outcomes of HER2+ patients with early stage breast cancer, they still remain at higher risk of recurrence along with TN patients. Trastuzumab was underutilized among HER2+ patients in this cohort, which may have contributed to decreased 5 year RFS. Ongoing prospective follow up of early BC outcomes by breast cancer subtypes is important.

5045 POSTER Outcomes of HER2+ Metastatic Breast Cancer (MBC) Patients (PTS) Treated With Continuous Inhibition of HER2 Activity: a Single Institution Study

5045 POSTER Outcomes of HER2+ Metastatic Breast Cancer (MBC) Patients (PTS) Treated With Continuous Inhibition of HER2 Activity: a Single Institution Study

Observational study to evaluate the pattern of trastuzumab (T) use and survival outcomes in HER2-positive (HER2+) early breast cancer (EBC): Regional Southern Italy experience.

e11043 Background: HER-2 have shown to be over-expressed in 20-25 % of primary breast cancer, and is associated with poor prognosis. Several large, randomized studies have reported a significant reduction in the risk of recurrence when trastuzumab is administered with chemotherapy in EBC HER2+ cancer. The aim of this study is to identify the pattern of use of T and survival outcomes in HER2+ EBC in Sicily. Methods: This observational, multicenter, retrospective study was conducted in 11 oncology centers in Sicily during 2006-2010. Inclusion criteria: age ≥18 years, surgery for early breast cancer, diagnosis of invasive disease, HER2-positivity as defined by current guidelines, and treatment with T. Results: 569 patients (pts) were included. Surgical outcomes: quadrantectomy 58.1%, mastectomy 33.7% and tumorectomy 8.2%. Tumor stage: T1 50.6%, T2 37.9% and T3 11.5%. Histology: invasive ductal carcinoma 94.7%. Histological Grade: G3 55.6%. Node status: N0 52.6%, N1 27.3%, N2 12.5%, N3 6.7% and Nx 0.9%. Hormonal status: estrogen receptor positive 60.1% and progesterone receptor positive 47.3%. Proliferation: Ki67 ≥10% in 82.4%. Neoadjuvant and adjuvant T was administered in 11.9% and 93.4% pts, respectively. Sequential administration of T after adjuvant chemotherapy was the most common schedule. Cytotoxic agents used: anthracyclines 76.04%, taxanes 33.02% and both agents in combination 26.8%. Relapses were observed in 5.6% pts. The main metastatic sites were: liver 18.75%, bone 12.5%, lung 12.75%, brain 12.75%, locoregional and contralateral 12.5% and lymph nodes 9.4%. N positivity and hormone receptor negativity are significantly associated to relapse. Cardiac toxicity (LVEF reduction >10%) was observed in 3.5% pts. DFS at 25 months of FU was similar to that of HERA DFS at 23.5 months of FU. Conclusions: Our study confirms the efficacy and safety of T in early HER2+ breast cancer, although includes a better prognostic population. These data indicate a satisfactory level of adherence to the international guidelines in the management in HER-2 positive EBC, showing a significant improvement in DFS when T was added to adjuvant chemotherapy.

Immunologic profiling and clinical outcome in HER2+ breast cancer patients treated in a neoadjuvant phase II study: A step forward to understand trastuzumab activity.

e11083 Background: A phase II study was performed to assess the role of patients’ immune profile in the activity of a trastuzumab (T) based, non anthracycline containing neoadjuvant chemotherapy (NC) regimen in breast cancer (BC) patients (pts). Methods: Newly diagnosed T2-3 HER2+ BC pts received a 12 week NC with weekly paclitaxel (P) and T, which was continued for further 12 weeks before surgery in the absence of progression (PRO). Further 12 weeks of the same therapy was planned after surgery and then T alone to 1 year of treatment ± hormonal therapy and radiotherapy. In case of PRO, T was discontinued and anthracyclines were planned. Blood samples were collected at diagnosis and every 3 months for 1 year then yearly. The % of NK cells, T cells and Treg cells was evaluated by flow cytometry, and serum levels of 9 cytokines were assessed by SearchLight multiplex array technology. In vitro T-mediated, antibody-dependent cell cytotoxicity (ADCC) was also assessed using patients’ PBMCs. Results: From July 2006 32 pts were enrolled, 31 were evaluable (1 ongoing). Pathological complete response (pCR) was reported in 15 pts (48%, 95% CI: 31-66%), objective overall clinical response was observed in 30 pts (97%, 95% CI: 91-100%). No cardiotoxicity occurred. In 3 cases (9.7%) PRO occurred at 26.3 months median follow-up. At diagnosis, pts showed an immune profile similar to that of healthy women, whereas higher numbers of Treg cells (p=0.02), NK cells (p=0.03), lower T cell numbers (p<0.01) and lower amounts of serum IL-2, IL-6, and IL-8 were found in a concomitant control group of untreated HER2- pts. Notably, study pts achieving pCR presented higher efficiency of basal T-mediated ADCC compared to pts with partial responses (p=0.05). The treatment induced a progressive increase in the number of NK cells and in the efficiency of T-mediated ADCC. Conclusions: NC with P and T induces high rates of pCR with no cardiotoxicity. This clinical efficacy is favored by the retained immunological proficiency of HER2+ pts, who may benefit from the immunomodulating properties of T started early. Whether T-mediated ADCC is significant also for event-free survival is under evaluation.

Multi-epitopic anti-tumor T cell responses and efficient Trastuzumab-mediated ADCC promote a favorable clinical outcome in HER2+ breast cancer patients after neoadjuvant chemotherapy (165.8)

Abstract The ability of drugs such as Trastuzumab and taxanes to act through immunomediated and/or immunomodulatory mechanisms suggest that anti-tumor immune responses may influence the clinical outcome of breast cancer patients. We have enumerated circulating CD8+ T cells specific for a broad spectrum of tumor-associated antigens (her2, muc-1, mammaglobin-A, trag-3, survivin, bcl-xL) in 13 patients with locally advanced breast cancer (6 HER2+ and 7 HER2-). At diagnosis, increased numbers of CD8+ T cells specific for all 13 HLA-A*0201 epitopes were observed in the whole series, with a markedly higher extent in HER2+ cases. This subset of patients retained an immunological profile similar to that of healthy women, whereas HER2- cases showed decreased circulating CD3+ T lymphocytes, higher numbers of regulatory T cells and alterations in serum cytokine levels (decreased IL-2, IL-6, and IL-8). Notably, patients with complete pathologic response (pCR), unlike those showing partial responses, retained high numbers of epitope-specific CD8+ T cells throughout the neoadjuvant chemotherapy (NC) treatment, particularly versus survivin and mammaglobin-A epitopes. Moreover, a higher efficacy of Trastuzumab-mediated ADCC in vitro correlated with the induction of pCR in HER2+ cases. These findings emphasize the crucial role of host anti-tumor T cell responses in promoting a favourable clinical outcome in the setting of neoadjuvant chemotherapy for breast cancer, particularly in HER2+ cases.

Stereotactic Radiosurgery for Breast Cancer Brain Metastases: Improved Outcomes in Her2 Amplified Patients

Stereotactic Radiosurgery for Breast Cancer Brain Metastases: Improved Outcomes in Her2 Amplified Patients