Citations

Abstract Ki-67 proliferation marker-based response monitoring will be prospectively tested in the ALternate Treatment for ER positive NeoAdjuvant TrEatment (ALTERNATE) trial. Tumor Ki-67 will be analyzed after one month of neoadjuvant anastrozole, fulvestrant or the combination to detect early resistance (Ki-67 >10%) for triage to alternate treatment (chemotherapy/investigational agent). Ki-67 will be repeated on the surgical specimen after 24 weeks to detect PEPI-0 cases (path Stage 0/1/2A and Ki-67 ≤2.7%), which will be managed without adjuvant chemotherapy. A CLIA-approved Ki-67 assay was required by CTEP/FDA. FDA reviewed-components included the CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody, the Benchmark XT platform and the VENTANA iScan Coreo scanner. The validation process was as follows: Step 1 was a TMA-based bridging study between a SP6-based research assay to the 30-9 CLIA assay using visual point counting (VPC); Step 2 trained a scoring SOP using Companion Algorithm Ki-67 (30-9) image analysis software with triage to VPC when certain observations were flagged; Step 3 was an independent test of the SOP using relevant patient outcome-associated samples from the P024 and POL neoadjuvant endocrine studies with two independent pathology analyses. STEP 1: Bridging Research to CLIA. Using a 28-case TMA, and VPC, the intra-class correlation (ICC) for agreement measure of Ki-67 10% cut-point (CP) between research Ki-67 and CLIA Ki-67 was 0.96 (CI 0.92-0.98). The Cohen's simple kappa for agreement measure was 0.78 (CI 0.55-1.0). STEP 2: Scanner-based Algorithm training. An SOP for the image analysis was developed by reviewing discordant cases in two independent reads at the 10% and 2.7% CP in a training set (N+ disease from P024 N = 61). Histology features that led to inaccurate scoring from the image analysis included: streaming /fascicular tumor cells, a large amount of intervening stromal cells, heavy immune cell infiltration, sparse scattered tumor cells, faint staining, and plump stromal cells. These cases were flagged for VPC. The final Ki-67 scoring SOP used image analysis with triage to VPC when necessary (required in 16% of cases). STEP 3: Scoring Algorithm Validation. The Ki-67 SOP was run on T1/2 N0 cases from the P024 trial N = 58. The% positive agreement for the combined scanner and VPC approach using the Ki-67 2.7% CP was 87.5% (95% CI 61.7- 98.5%); percent negative agreement 88.9% (95% CI: 65.3-98.6%). Minor discordance did not affect the ability to predict very similar survival outcomes (Log Rank P = 0.044 and P = 0.055). The data for the 10% CP was similar (N = 66), the percentage positive agreement was 100%; percent negative agreement 93.55% (95% CI: 78.58-99.21%). The survival predictions were again similar (P = 0.0001 and P = 0.0002). The FDA ruled the use of Ki-67 in the ALTERNATE trial is “no significant risk” because previous clinical trial data indicated the majority of patients with Stage 0/1/2A disease post neoadjuvant endocrine therapy already do not receive chemotherapy. Thus the use of Ki-67 reduces the risk of under-treatment of patients with low stage disease but aggressive biology. CTEP accepted these data on assay concordance and performance and the ALTERNATE trial will proceed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-10.

Abstract Background: Retrospective studies suggest that primary breast surgery is associated with improved overall survival (OS) in Stage IV breast cancer. TBCRC 013 is a multi-center prospective registry study evaluating the role of surgery for the primary tumor in Stage IV disease. Methods: From 7/09 - 4/12, 128 eligible pts from 14 sites were enrolled in two cohorts (A: Stage IV with intact primary tumor (n = 112); B: metastases (mets) within 3 months of primary surgery (n = 16)). Baseline patient and tumor characteristics, and surgery of the primary were correlated with 2yr overall survival (OS) using log rank, Kaplan Meier and Cox regression for all patients. Comparisons were also made between cohorts A and B and within cohort A stratified by response to systemic therapy. Responders included those with any response (partial,complete) or stable disease at distant sites. Results: Median pt age was 52yrs(21-79) and median primary tumor size 3.2cm(0.7-15). Phenotypes were ER+ 106 (83%), HER2neg 91(71%), and triple neg 10(8%). 60(47%) had bone-only mets. The only significant baseline difference between cohorts A and B was the presence of palpable nodes (A:62% vs B:0%, p<0.001). There were no differences in age, tumor size, ECOG scores, ER or HER2 status, number or sites of mets between cohorts A and B. At a median follow-up of 25 mo (95%CI, 23-31), 2yr OS for all pts is 86% (80-92) and is superior in cohort B as compared to A, (100% vs 84%, p = 0.03). On multivariate analysis (MVA) including all pts, only surgery, ER and HER2 positivity were associated with improved OS (table). Among 112 pts with an intact primary (Arm A), 17(15%) were non-responsive (NR) to systemic therapy, 3(18%) of whom required palliative surgery at a median time of 10.4mos. 2yr OS among NR was 33% (15-69) as compared to 94% (88-99) in Arm A responders (R) (p<0.001). ER positivity was the only baseline difference between NR (65%) and R (88%) within Arm A (p = 0.02). Among 94(85%) Arm A responders, 39(41%) had elective surgery (median 8.4 mos, 95%CI:6.9-10.3) and only 1 of the remaining 47(2%) required palliation. When limited to Arm A responders, elective surgery was not associated with improved 2yr OS (surgery vs not: 94% vs 92%, p = 0.5). Median time to progression following elective surgery was 12 mos (95%CI,8-19mos). When cohort B (n = 16) and cohort A surgery groups (n = 39) are combined, surgery of the primary tumor is associated with improved 2yr OS (96% vs 74%, p = 0.002). Conclusions: In this prospective registry study, patients diagnosed with mets within 3 months of primary breast surgery have an improved 2yr OS. When all pts having surgery (A + B) are examined, surgery is associated with improved OS on MVA, but when limited to Arm A responders, elective surgery does not improve OS. Given that the number of Arm A responders is small, results from the prospective randomized trial will be needed to address this question. These data also demonstrate that the need for surgical palliation of the primary tumor is uncommon in the modern era. Multivariate Analysis for OSN = 128HR Death (95%CI)pvalueSurgery0.28 (0.10-0.74)0.01ER positivity0.28 (0.10-0.72)0.01HER2 positivity0.21 (0.06-0.74)0.01MVA included: age, tumor size, ECOG, ER, PR, HER2, met sites, and surgery Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-18-09.

Abstract Background: Hsp90 is a molecular chaperone protein required for the stabilization and activation of many proteins, referred to as Hsp90 ‘clients’, such as HER2, HIF1-a, EGFR, ER, PI3K, AKT, P53 and VEGFR. The drug candidate, ganetespib is a novel triazolone inhibitor of Hsp90, with over 700 patients treated to date. Ganetespib has shown activity in preclinical models of HER2+, ER+/PR+ and TNBC. Early clinical trials documented ganetespib single agent activity in heavily pretreated HER2+ and TNBC patients. Ganetespib has been well tolerated in clinical trials with a favorable safety profile. This efficacy-screening study is designed to provide further evidence of ganetespib activity and identify potentially predictive biomarkers in metastatic breast cancer (BC). Methods: The ENCHANT-1 Trial is an international, first-line 2-cohort Phase 2 study in BC patients: Cohort A, HER2 amplified (n = 35), and Cohort B, TNBC (n = 35). Patients who present with previously untreated metastatic disease are eligible for treatment with ganetespib at 150 mg/m2 twice weekly on 3 out of 4 wks, for a total of up to 12 wks. Primary endpoint: ORR assessed using RECIST1.1 criteria. Key secondary endpoints include metabolic response as assessed by PET/CT at wk 3 utilizing modified EORTC criteria. Disease progression (PD) at wk 3 by PET imaging indicates discontinuation of study therapy, and is performed to quickly offer patients with metabolic PD a standard of care treatment. The study is designed as Simon 2-stage requiring at least one OR in 15 patients for the respective cohort to expand to 35 patients. A Steering Committee is established to oversee the overall study and review the interim results. Results: The study was initiated in 23 centers globally. At the time of submission, a total of 17 patients had been enrolled; TNBC (n = 15) and HER2 (n = 2). Here we report the interim analysis in the TNBC cohort. The median age was 54 years (range 30 -77) with ECOG PS 0 (n = 7/15). Most patients (n = 9) presented with de novo metastatic disease. 5 patients were not evaluable for PET assessment (3 had not yet reached wk 3 and 2 withdrawn before wk 3 for clinical progression), and 9 patients were not evaluable for objective response at wk 6 (3 withdrawn before or at wk 3 for clinical progression and 6 had not yet reached wk 6 evaluation). In the 10 patients with evaluable PET imaging, 9 patients achieved metabolic (m) response (2 mPR, 4 mSD with dominant tumor shrinkage and 3 SD) and one patient with mPD. In the 6 patients evaluable for OR at wk 6, one patient achieved PR, 2 SD and 3 PD. Treatment with ganetespib was well tolerated; the most common AEs were mild or moderate diarrhea (8/15, 53%), fatigue (5/15, 33%), decreased appetite (4/15, 27%), insomnia (4/15, 27%), and nausea (4/15, 27%). Conclusion: Ganetespib single agent was generally well tolerated and showed anti-tumor activity TNBC patients as early as 3 weeks following treatment. PET seems to be a good tool to screen antitumor activity of new agents in early settings rather that in heavily pretreated patients. The TNBC cohort has met the protocol criteria for proceeding to stage 2. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-23.

Abstract Background: HER2 overexpression or amplification, occuring in ∼15-20% of breast cancers (BC), is associated with a poor prognosis. Trastuzumab (T) and pertuzumab (P) are humanized monoclonal antibodies that bind to different HER2 epitopes, inhibiting HER2 signaling. It was previously shown that first-line (1L) treatment of patients (pts) with HER2-positive metastatic breast cancer (MBC) with vinorelbine (V)+T had similar efficacy to docetaxel+T, but with fewer adverse events (AEs) (HERNATA). In a recent study, P+T+docetaxel significantly improved progression-free survival (PFS) and overall survival (OS) compared with T+docetaxel in pts with HER2-positive 1L MBC (CLEOPATRA). The objective of the VELVET study is to investigate the efficacy and safety of P+T+V for 1L treatment of HER2-positive MBC. Methods: This is a multicenter, open-label, single-arm, two-cohort, Phase II study. The recruitment target is 105 pts per cohort. Pts in Cohort 1 receive P+T+V as separate infusions. Pts in Cohort 2 receive P+T from a single infusion bag followed by V. The initial dose of P is 840 mg, followed by 420 mg q3w; the initial dose of T is 8 mg/kg, followed by 6 mg/kg q3w; V is administered at 25 mg/m2 in Cycle 1, followed by 30-35 mg/m2, on Days 1 and 8 of each cycle, q3w. Pts must have HER2-positive MBC or locally advanced BC (LABC) and a baseline left ventricular ejection fraction (LVEF) of ≥55%. Previous treatment with systemic nonhormonal anticancer therapy in the metastatic setting is not allowed. The primary endpoint is independently assessed overall response rate. Secondary endpoints include PFS, OS, and safety. Results: Interim safety data are presented for Cohort 1; 106 pts were enrolled. Median age at screening was 56 years. Median interval between initial BC diagnosis and enrollment was 2.6 years. 33% of pts had Stage IV BC at initial BC diagnosis. At diagnosis of advanced disease, 13% and 87% of pts had LABC and MBC, respectively. 54% of pts had previously received chemotherapy, including taxane (38%) and anthracycline (36%). 41% had prior T exposure. A median of 6 cycles of P, T, and V was received at the time of this analysis. An AE overview is shown in the table. Pts, n(%)N = 106Any AE102(96)Grade 338(36)Grade 417(16)Grade 52(2)aAny serious AE25(24)Most frequent (≥4 pts) grade ≥3 AEs Neutropeniab21(20)Febrile neutropenia6(6)Leukopenia5(5)Asthenia4(4)Constipation4(4)Diarrhea4(4)a Myocardial infarction and septic shock; b Covers ‘neutropenia’ and ‘neutrophil count decreased’ Grade ≥3 neutropenia/febrile neutropenia was experienced by 8% of pts in Cycle 1 and by 14% of pts in Cycle 2; this proportion decreased during subsequent cycles. There was no overall decrease in mean LVEF from baseline. Conclusions: The combination of P+T+V was well tolerated; no new safety signals were observed. The incidences of grade ≥3 neutropenia, febrile neutropenia, and leukopenia were lower than those previously observed in pts with HER2-positive MBC treated with 1L P+T+docetaxel in CLEOPATRA or those treated with T+V in HERNATA; however, it should be noted that the treatment period in these two studies was longer. Efficacy data for Cohorts 1 and 2 will be reported at the end of the study. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-10.

Abstract Background: The potential for exploiting BRCA deficiencies with DNA repair inhibitors has both pre-clinical and clinical support. ABT-888 (veliparib), a DNA repair inhibitor initially thought to target Poly(ADP-Ribose) Polymerases (PARP), has demonstrated in vitro inhibition of BRCA1 and BRCA2 deficient mouse embryonic stell cells, with a larger effect on BRCA1 cells. We report on the pre-planned interim analysis of the efficacy of single agent veliparib in patients with either BRCA1 or BRCA2-associated stage IV breast cancer. Methods: BRCA 1 or 2 carrier patients with stage IV breast cancer, with measurable disease, without prior exposure to a PARP inhibitor or a platinum compound in the metastatic setting, were eligible. Velapirib was administered orally, at doses of 400 mg twice daily. Dose adjustments based on toxicity were permitted. Patients progressing on velapirib alone received carboplatin at an AUC of 5, IV, given Q 21 days, and velapirib 150 mg twice daily (the maximum tolerated dose [MTD] of the combination from our completed Phase I study: J Clin Oncol 30, 2012 [suppl; abstr 1024]). Patients were to be accrued from 7 NCI NO1- supported consortia. Initially 10 patients were to be accrued to each stratum (BRCA1 and BRCA2) to provide evidence of single agent activity. If there was sufficient activity to warrant consideration of velapirib as single agent therapy (defined as 2 or more confirmed partial [PR] or better responses out of 10 per stratum), an additional 12 patients would be accrued per stratum. Results: 20 evaluable patients (11 BRCA1 and 9 BRCA2 [1 in screening]) have been accrued, the majority with lung or liver as visceral metastatic sites of disease. Median age (range) is 46 (29-68) years. Tumors from 9 patients were hormone receptor positive. BRCA1 cohort: 4 of 11 patients are off treatment at a median of 2 months (1-4); 1 patient stopped velapirib due to toxicity (grade 2 rash/pruritus, grade 2 vomiting), 3 stopped for progressive disease (one with an unconfirmed PR). Seven patients are still on single agent veliparib with 1 unconfirmed PR, and 1 patient with two evaluations showing stable disease. BRCA2 cohort: 2 patients are off treatment at 2 months for progressive disease, 7 are still on treatment with 1 confirmed PR, and 3 unconfirmed PRs. Data on patients receiving combination of velapirib and carboplatin after progression is too early. Treatment-related toxicity is being updated and has so far been reported from 14 patients: 1 patient had grade 3 fatigue, 1 patient with liver metastasis had both grade 3 alanine aminotransferase elevation and grade 3 abdominal pain. Grade 2 toxicities occurring in more than 1 patient included nausea/vomiting (6 patients), chills (2 patients), and fatigue (2 patients). Conclusion: Velapirib has single agent activity in both BRCA1 and BRCA2-associated stage IV breast cancer patients, and is well-tolerated. Mature response, treatment, and toxicity data will be presented. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-05.

Abstract BACKGROUND: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis in patients with breast cancer. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A in breast cancer cells will lead to increased sensitivity to chemotherapy. RESULTS: Increased Aurora-A expression, as assessed by IHC staining, in patients with locally advanced disease was associated with worse prognosis. We show that in ER+ cell line, MCF7, activating MAPK pathway through overexpression of Raf1 leads to increase in Aurora-A activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to untransfected cells in MTT assays. This effect is even more exacerbated with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by specific inhibitor, Alisertib. This was further explored in triple negative (MDA-MB-231) or Her-2/neu expressing (SUM149) cell lines. These cells express higher levels of Aurora-A. Inhibition of Aurora-A activity in combination with paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are looking at a role of Aurora-A in activating SMAD5 and its role in resistance to chemotherapy. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing. CONCLUSIONS: Inhibition of Aurora-A by a specific inhibitor, MLN8237, resulted in decreased SMAD5 expression and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be very useful in developing more effective treatments for breast cancer patients in neo-adjuvant, adjuvant and metastatic settings. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-07.

Abstract Background- The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. Studies of cell-free DNA in the peripheral blood of breast cancer patients suggest that methylated DNA markers in serum or plasma could be used for detection of advanced disease, monitoring of therapeutic response, and for early detection of disease recurrence. Methods- A genome-wide serum DNA methylome array (Illumina HumanMethylation27 BeadChip) analysis was conducted on cell-free circulating DNA in serum from women with stage IV recurrent breast cancer, and 232 key CpG loci were identified. Methylation for this panel of 10 gene loci was evaluated using our newly developed cMethDNA assay to detect miniscule amounts of methylated DNA in Training and Test sets of sera from a total of 112 women (n = 55 normal, n = 57 metastatic breast cancer). The clinical sensitivity and specificity of the assay, along with technical reproducibility, was determined. To evaluate the concordance of DNA methylation patterns, the 10 gene panel was tested on 22 DNA sets of primary tumor, metastases and serum from the same patient. Finally, the ability of cMethDNA to monitor response to therapy was evaluated in 28 patients with metastatic disease. Results- A normal laboratory threshold of 7 cumulative methylation units was set and assay parameters were locked, based on Receiver Operating Characteristic (ROC) analyses of DNA from 300 ul of patient sera in the Training set (normal, n = 28; cancer, n = 24; 92% sensitivity, 96% specificity, and AUC = 0.950). Evaluation of the Test set of patient sera (normal, n = 27; cancer n = 33) resulted in detection of metastatic breast cancer with 91% sensitivity, 100% specificity, and AUC = 0.994 (0.984-1.005, p<0.0001). Reproducibility of the cMethDNA assay increased with copy number; with the highest variation at 50 copies (CV = 29.1%) and the lowest at 3,200 copies (CV = 2.5%) of methylated DNA. The test was shown to be operator independent (ICC = 0.99). Evaluation of concordance between primary and disseminated tumor methylation showed that the methylation pattern from any given individual is highly conserved between serum, primary tissue and their metastases, and poorly conserved between different individuals. cMethDNA analysis of 28 patients before and after initiation of therapy showed a decrease in cumulative methylation in women with stable/responsive disease and a correlation with disease progression free survival (p<0.0001). Conclusion- Together, our data suggest that the cMethDNA test 1) can detect tumor DNA shed into blood, 2) reflect the methylation alterations typical of the primary tumor and its metastatic lesions, and 3) reflect response to treatment after chemotherapy. Next, we will test the clinical utility of cMethDNA in independent clinical trial sample sets where it's complementary and independent roles will be examined against CA15.3 and CTC assays. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-01.

Abstract Background: Based upon the association between pathologic response and disease free survival, the neoadjuvant setting is increasingly being used for drug development. NGS has identified unique and recurrent genetic alterations in breast cancer (BC) that are potentially targetable; however, the clinical implications are mostly unknown. We developed a prospective neoadjuvant study (BEAUTY) in high risk BC patients (pts) using weekly T followed by anthracycline-based chemo wherein percutaneous tumor biopsies (PTB) are obtained before/during/after chemo for NGS and PDX. Our goal is to identify novel biomarkers/pathways and develop PDX to test new therapeutic approaches. Methods: Pts underwent PTB at baseline and after 12 wks of T. Response to T was defined based upon 12 week Ki-67: responder (<15%) vs non-responder (≥15%). Pts with histologic response and absence of invasive BC at 12 wks were classified as responders. NGS was performed using PTB/blood DNA (exome) and PTB (RNA seq). MRI response was classified using RECIST criteria. NGS data were used to identify somatic copy number variants (cnvs) and expressed single nucleotide variants (eSNVs). Non-SCID mice (estrogen supplemented) were implanted ≤ 30 minutes with PTB samples. Results: Of the first 78 pts, 44 have completed T. Here we focus on 18 pts with either triple negative or luminal B BC. Clinical characteristics according to Ki-67 response are shown in Table 1. Comparison of genomic alterations in BEAUTY pts with TCGA identified a greater overlap with copy number gains (73%) compared to deletions (40%), along with similar observations of mutations in TP53, PTEN, RYR2, and AKT1 genes. Association analysis of CNVs and eSNVs between responders/non-responders identified 33 genes (predominantly located in chromosomes 1, 8, 13) and 580 eSNVs (corresponding to 497 genes) with a p < 0.05. Differential gene expression (DGE) analysis of responders/non-responders identified 198 genes with a p-value < 0.05. Integrated analysis of 539 genes (CNVs, eSNVs and DGE) identified pathways such as TGF-beta, Jak-Stat, WNT and NOTCH signalling. PDX take rate was 44% [triple negative (6/10); Luminal B (2/8)]. PDX growth rate was significantly associated with clinical baseline Ki-67 (p = 0.00014). Conclusion: This is the first prospective study to demonstrate the feasibility of using PTB to obtain both NGS data and PDX in the neoadjuvant setting. PDX take rate is associated with BC subtype and baseline Ki-67. Studies are ongoing to 1) validate genes/pathways associated with treatment response in subsequent BEAUTY pts; 2) genomically characterize and assess PDX in vivo response to T and 3) Use NGS data to prioritize new drugs/drug combinations in PDX. Funded by Mayo Clinic Center for Individualized Medicine and MC Cancer Center. Clinical CharacteristicsOverallResponders: 12 week Ki-67 < 15% (n = 9)Non-Responders: 12 week Ki-67 ≥ 15% (n = 9)Median Age495345T stage T2/T314 (78%)7 (78%)7 (78%)Node Positive8 (44%)4 (44%)4 (44%)Triple negative10 (56%)6 (67%)4 (44%)Luminal B8 (44%)3 (33%)5 (56%)Ki-67 after 12 Weeks of T Median 5% (0-11%)Median 35% (17-60%)Complete/Partial MRI Response after T 6 (67%)2 (22%) Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-10.

Abstract Background: Epidermal Growth Factor Receptor (EGFR, HER1) is known to heterodimerize with HER2 and may modulate the effectiveness of trastuzumab or lapatinib. Historically, it has been difficult to measure, but recently we reported a new, standardized, quantitative immunofluorescence assay and a novel EGFR antibody which showed that high levels of EGFR were associated with decreased benefit in the concurrent arm of the North Central Cancer Treatment Group (NCCTG/Alliance) N9831 trial (Rimm et al, SABCS 2012). Here we assess its value in the neo-adjuvant setting in the NeoALTTO trial. Methods: NeoALTTO randomized 455 breast cancer patients to neoadjuvant trastuzumab, lapatinib or both and showed benefit in both single anti-HER2 therapy arms, and the combination therapy showing about twice as much benefit as either single drug arm. We used the previously described AQUA method of quantitative immunofluorescence to measure EGFR levels using the D38B1 antibody on between 4 and 140 fields of view per slide on 353 specimens. The averaged EGFR score was standardized to the absolute concentration using cell lines and western blots and the 13 ng/ug total protein established in the N9831 trial was tested to determine predictive value for pathological complete response (pCR) defined as ypT0/is. Results: In NeoALTTO, 19% of the patients had EGFR levels above 13 ng/ug, compared to 16% in NCCTG N9831. The pCR rate was 35.3% in the patients with low EGFR, compared with 29.8% for high EGFR but this difference was not statistically significant. However, when adjusted for treatment and hormone receptor (HR) status, high EGFR is statistically significantly associated with lower response rate(p = 0.038). Continuous analysis measures were not significant. The effect on overall response at 6 weeks was also weakly significant when adjusting for treatment and HR status (p = 0.06). Surprisingly, for HR negative patients in the lapatinib only arm, the pCR rate in the low EGFR group was 47.2% compared with 16.0% in the high group. While this difference is statistically significant (p = 0.014), it should be interpreted cautiously because of the small numbers of patients and the multiple tests performed. Conclusions: High expression of EGFR by the AQUA platform appears to be associated with decreased pCR rate from HER2 targeted therapy in the neoadjuvant setting. This observation is consistent with NCCTG N9831. Although underpowered, high EGFR levels predicted decreased pCR rate from lapatinib, the opposite of that anticipated for this small molecule dual EGFR/HER2 inhibitor. Further studies are required to determine the clinical utility of this assay and the biological mechanisms underlying these observations. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-09.

Abstract Background: Circulating Tumor cells (CTCs) are prognostic at baseline and first follow-up in patients with metastatic breast cancer (MBC). Using the most commonly used assay (CellSearch®), we have previously reported the ability to detect apoptotic vs. non-apoptotic CTCs in patients with MBC. However, there has been concern regarding the performance of the CellSearch® assay in patients with triple negative (TN) MBC. We hypothesized that CellSearch® is an effective assay in patients with TN MBC, and that CTC-apoptosis might further separate prognosis. Therefore, we studied CTCs in patients with TN MBC who participated in a prospective randomized phase II trial testing for activity of tigatuzumab (TIG) in combination with nanoparticle albumin-bound paclitaxel (nab-PAC) conducted by the Translational Breast Cancer Research Consortium (overall results reported by Forero A., et al, ASCO 2013). Methods: Whole blood (WB) was drawn into a CellSave tube at baseline, day 15, and day 29 and CTC counts were determined using the CXC CellSearch® kit. Apoptosis was characterized by staining with a monoclonal antibody that detects a neo-epitope on fragmented cytokeratin (M-30) and independently by visual inspection (nucleic condensation and/or fragmentation, as well as granular cytokeratin). Association between levels of CTCs and CTC-apoptosis with the overall response rate (ORR) and progression free survival (PFS) at baseline, day 15, and day 29 was assessed using logistic regression, Kaplan Meier curves, and Cox proportional hazards modeling. Results: Of the 60 patients entered into the trial, 52 were evaluable for CTCs. Of these, 19/52 (36.5%), 14/52 (26.9%), and 13/49 (26.5%) had elevated CTCs (≥5CTC/7.5 ml WB) at baseline, day 15, and day 29, respectively. Patients with elevated CTCs at each time point had worse PFS than patients with low or no CTCs. Hazard rates (HR) at baseline, day 15, and day 29 were 2.38 (95% CI: 1.27-4.45, p = 0.007), 2.87 (95% CI: 1.46-5.66, p = 0.002), and 3.40 (95% CI: 1.68-6.89, p = 0.001), respectively. The odds of overall response for those who had elevated CTCs compared to those who did not at baseline, day 15, and day 29, were 0.25 (95% CI: 0.073-0.81, p = 0.024), 0.18 (95% CI: 0.04-0.67, p = 0.014), and 0.06 (95% CI: 0.01-0.28, p = 0.001), respectively. There was no apparent prognostic effect comparing the degree of CTC-apoptosis vs. non-apoptosis. Conclusions: Similar to observations in other intrinsic subgroups, CTCs were detected in a large fraction of TN MBC patients at baseline using CellSearch® assay, and reductions in CTC levels reflected response. In these homogenously prospectively enrolled TN MBC patients, regardless of treatment, CTCs are prognostic at baseline, day 15, and day 29. It does not appear that analysis of CTC-apoptosis is prognostic. Supported by Susan G. Komen for the Cure, Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP), Triple Negative Breast Cancer Foundation, The AVON Foundation, and The Breast Cancer Research Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-01.