Citations

Abstract We have recently reported that in patients with HER2-positive breast cancer, neoadjuvant targeted therapy with lapatinib and trastuzumab to more completely block the HER receptor layer, combined with endocrine therapy (in ER-positive tumors) and without chemotherapy led to a substantial 27% pathologic complete response (pCR) rate in the breast. Activation of downstream signaling pathways may lead to resistance to therapies targeting the HER pathway receptors. Aberrant activation of the PI3K pathway via decreased levels of PTEN and/or the presence of activating PIK3CA mutations has been implicated in resistance to targeted anti-HER2 therapy, but results of clinical trials are all confounded by the co-administration of chemotherapy and are inconsistent. We sought to clarify the role of these variables in predicting pCR, a surrogate for long-term outcome, in patients treated with potent targeted therapy alone in a prospective Phase II neoadjuvant trial in patients with HER2-positive breast cancer. Patients with large tumors (median 6 cm) were given 12 weeks of lapatinib plus trastuzumab followed by surgery (Rimawi et al. JCO, 2013). Serial tissue biopsies were obtained from study participants. For this study, we focused on baseline pre-treatment characteristics. PTEN protein levels were measured by IHC and scored using the H-score. PIK3CA mutations were identified on extracted DNA using multiplex PCR with targeted next generation sequencing (the Ion Torrent 50-gene cancer mutation panel). Of 64 evaluable patients, tissue was available on 59 for PTEN IHC, and sufficient DNA was available on 33 for the mutation panel. PTEN median H-score was 100 (range 0-300). PTEN status when dichotomized by the median was correlated with pCR (32% in high PTEN vs. 9% in low PTEN, p = 0.04). Activating PIK3CA mutations were identified in 12 out of 33 tumors (36%; 3 mutations in the helical and 9 in the catalytic domain) and were independent of ER status. None of the patients whose tumors harbored a PIK3CA mutation achieved pCR (p = 0.06). There was no association between PTEN status and PIK3CA mutation suggesting they are independent variables (p = 0.44). When PIK3CA mutations were considered together with PTEN status, there were 31 cases with data on both. The overall pCR rate in this cohort was 16% (lower than pCR rate observed in the overall trial). However, 0/17 cases (0%) with a mutation and/or PTEN low expression (<100 H score) had a pCR compared to 5/14 cases (36%) with PI3KCA wild type and high PTEN levels (p = 0.01). We conclude that PI3K pathway activation downstream of HER2 as a result of either low PTEN or activating PIK3CA mutation results in resistance to the combination of lapatinib and trastuzumab. This is the first report on patient tissue samples from a neoadjuvant trial using the combination of lapatinib and trastuzumab without chemotherapy. If validated in a larger cohort, our findings suggest that patients with HER2 positive tumors and who also harbor aberrant downstream PI3K pathway activation may benefit from the addition of PI3K/Akt/mTOR inhibitors to potent HER2 blockade. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-2.

Abstract Background: Estrogen receptor beta (ERβ), unlike ERα, classically functions as a tumor suppressor in vitro. However, ERβ's biological functions in vivo and predictive/prognostic value in breast cancer are controversial. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in the following 3 cohorts: 1) a cohort with all breast cancer subtypes (n = 182), 2) a prospective NCCTG adjuvant tamoxifen trial for postmenopausal women with ERα positive breast cancer with long-term follow-up (n = 198) and 3) a cohort of 80 triple negative breast cancers (TNBCs). To elucidate the biological functions of ERβ in breast cancer, novel ERβ expressing MCF7 and MDA-MB-231 cell lines were developed and characterized using multiple techniques and were examined for responsiveness to various ERβ targeted therapies. Results: About one-third of all breast tumors, regardless of sub-type, were shown to express nuclear ERβ and this expression was independent of ERα or HER2. In the NCCTG 89-30-52 cohort, breast cancer recurrence rates were significantly associated with ERβ protein expression with 10 year recurrence rates of 25%, 15% and 4% for zero, low or high levels of ERβ expression respectively. Interestingly, in TNBCs, nuclear ERβ was expressed at intermediate or high levels in 24% of tumors. In the triple negative MDA-MB-231 cell line, expression of ERβ led to inhibition of proliferation and induction of apoptosis in response to estrogen and multiple ERβ specific agonists. Conversely, these same treatments induced proliferation of ERβ-expressing MCF7 cells which endogenously express ERα. However, ERβ expression sensitized MCF7 cells to the anti-proliferative effects of anti-estrogens. Microarray analysis and RT-PCR profiling of MDA-MB-231-ERβ cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors which function as tumor suppressors, and potently inhibited canonical TGFβ signaling. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells suppressed the proliferation rates and inhibited TGFβ signaling in other TNBC cell lines, effects that were completely reversed following the depletion of cystatins from the conditioned media. Conclusions: These data demonstrate that ERβ is expressed in a substantial proportion of breast cancers and may have value as a predictive and/or prognostic biomarker. Therapeutic targeting of ERβ may have clinical benefit in multiple breast cancer sub-types; however, the specific drug of choice may vary based on ERα status. Specifically, we have demonstrated that ERβ expression in ERα+ MCF7 cells sensitizes them to the effectiveness of anti-estrogens, an observation that was confirmed in women enrolled in a prospective adjuvant tamoxifen trial. In TNBCs, where targeted therapies are lacking, our data suggest that targeting ERβ with either estrogen or ERβ specific agonists will elicit anti-tumor effects through the induction of cystatins and inhibition of TGFβ signaling resulting in tumor regression and improved patient outcomes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-03.

Abstract Purpose/Objective: The landmark ACOSOG Z0011 trial established that axillary lymph node dissection (ALND) is unnecessary in breast cancer patients with one or two positive sentinel lymph nodes (SLN) who undergo lumpectomy, radiation therapy (RT) and systemic therapy. At a median follow-up of 6.3 years there were no differences in overall survival or local-regional recurrences between patients receiving ALND or SLN biopsy alone. The protocol required whole breast RT using standard tangential fields, but the extent of RT coverage of the regional nodes in these patients has not previously been described. We sought to ascertain this information. Methods: The RT data on the ACOSOG Z0011 trial was collected from case report forms completed by enrolling providers 18 months after enrollment. The response options on the forms included breast, supraclavicular, & other. We reviewed these data and, in 2011-2013, we attempted to identify and contact all treating radiation oncology practices to request detailed radiation treatment records for central review. RT data was sent to the Quality Assurance Review Center (QARC) and reviewed by two independent radiation oncologists, blinded regarding the arm to which the patients were randomized. Results: Of the 891 patients enrolled on Z0011, 35 withdrew consent prior to surgery, leaving 856 who were analyzed in the primary report on trial outcomes.1 Among the 605 of these patients with a completed adjuvant radiation therapy form, 89% were noted to receive whole breast RT. Of these, 89 patients (15%) were recorded as also receiving treatment to the supraclavicular region. Detailed RT records were received on 228 patients: 104/389 (26.7%) and 124/404 (30.7%) on the ALND and SLND arms, respectively. Of the 228 patients, 185 patients (81.1%) received tangent-only treatment. Among these 185 patients, there was sufficient data to evaluate tangent field height in 142 (76.8%). High tangent RT fields (with cranial tangent border within 2 cm of the humeral head) were used in 52.6% (40/76) patients randomized to the ALND arm and 50% (33/66) patients randomized to the SLND arm. Of the 228 patients with records reviewed, 43 (18.9%) received directed regional nodal RT using ≥3 fields: 22 in the ALND and 21 in the SLND arm. Those receiving directed nodal RT tended to have greater nodal involvement (p<0.001). Conclusions: Most patients treated on the Z0011 trial received tangential field RT alone, and some received no radiotherapy at all. Some patients received directed nodal irradiation via a third field. In a subgroup for whom detailed RT records were available, the highest rates of directed nodal irradiation were among those with multiple nodes involved. As survival rates and local-regional recurrences were similar in the study arms, no conclusions can be drawn from this analysis on whether this additional radiation treatment was necessary or beneficial. 1JAMA 2011; 305 (6): 569-575. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-19.

Abstract Background: Endoxifen (Endx) is being developed as a drug for patients with estrogen receptor (ER)+ breast cancer (BC). We previously demonstrated that Endx was superior to TAM in both a letrozole (L) sensitive and resistant MCF7/AC1 model. Tamoxifen (TAM) and its most commonly studied metabolite, 4-hydroxy TAM (4HT), are known to activate critical ER co-regulators such as SRC3, which are oncogenic, and contribute to acquired and de novo endocrine resistance. While both Endx and 4HT are more potent inhibitors of genomic ERα functions compared to TAM, the effects of Endx on non-genomic signaling have not been characterized and its efficacy as an anti-cancer agent compared to RAD001 in AI resistant BC is unknown. Methods: We evaluated the 1 hour in vitro effects of TAM, 4HT, Endx, and L, in terms of activation of multiple signaling cascades using an MCF7/AC1 L-resistant cells and long term estrogen deprived (LTED) cells (MDA 361 and ZR75). Protein Docking studies (shape screening, pharmacophore modeling and ligand-protein docking) for oncogenic kinases, and their associated IC50's were determined for TAM and Endx. The in vivo activity of Endx (50 mg/kg, oral) was determined in MCF/AC1 L-resistant xenograft tumors and compared with L control (10 ug/day, sc), Exemestane (E) (250 ug/d, sc) and E+RAD001 (2.5 mg/kg/d, oral). Results: In MCF7/AC1 L-resistant cells, 1 hr treatment with either TAM (100 nM or 5 uM) or 4HT (100 nM or 5 uM) phosphorylated SRC3 (AIB1) and resulted in downstream activation (phosphorylation) of IGF1R, PDK1, AKT, and p70S6K. In contrast, Endx (100 nM and 5 uM) did not phosphorylate SRC3 nor any downstream kinases but resulted in a dose dependent decrease in AKT phosphorylation. Similar findings were observed in LTED cells MDA 361 and ZR75. Docking studies identified a significant decrease in the binding energy (>2 kcal/mol) of Endx compared to TAM for PKC and PI3K and subsequent kinase profiling demonstrated that Endx more potently inhibits PKCβ1 (IC50 357nM) and PI3KΔ (2.5uM) compared to TAM (IC50 4.8μM and 6.2μM respectively). Therefore, we evaluated the in vivo anti-tumor activity of Endx with E and E+RAD001 in MCF7 L-resistant xenograft tumors. After 9 weeks, the% change in tumor volume relative to baseline was significantly lower for both Endx (median -34%, p = 0.0002) and E+RAD001 (median +1.5%, p = 0.0002) as compared to L controls (median +301%). In contrast, E alone was non-significantly lower (median +172%, (p = 0.09). No significant differences were observed comparing Endx and E+RAD001 (p = 0.56). Conclusions: Endx inhibits PKCβ1 and PI3KΔ at concentrations that are pharmacologically relevant, and unlike TAM and 4HT, does not activate SRC3 nor induce oncogenic signaling pathways in AI resistant cells. Importantly, Endx elicits substantial anti-tumor effects in AI resistant models that are similar to the current standard of care of E+RAD001. These data suggest that Endx may be superior to TAM for the treatment of ER+ BC and may result in clinical benefit for patients with AI resistant disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-08.

Abstract Background: Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We and others have previously demonstrated that endoxifen is the most active tamoxifen metabolite responsible for eliciting the anti-cancer effects of this drug and that endoxifen concentrations are an important factor with regard to tamoxifen efficacy. These studies have led to the development of endoxifen as a novel anti-breast cancer drug for which phase I clinical trials are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast gene expression profiles were compared to that of estrogen, tamoxifen, raloxifene and lasofoxifene by microarray and RT-PCR analyses in both estrogen receptor alpha (ERα) and ERβ expressing cell lines. The in vivo effects of an anti-cancer dose of endoxifen (50mg/kg/day) on the skeleton were first analyzed in 3-month-old ovariectomized C57BL/6 mice using Dual-energy X-ray absorptiometry, peripheral Quantitative Computed Tomography, micro-Computed Tomography and histomorphometry. In a second set of studies, a pre-clinical rat model was used to determine the effects of endoxifen (10mg/kg/day) on the skeleton in both a pre- and post-menopausal setting. Results: Endoxifen treatment of ERα and ERβ expressing mouse osteoblast cells led to dramatically different gene expression profiles when compared to that of estrogen and other anti-estrogens. In ovariectomized mice, daily administration of endoxifen led to significant increases in bone mineral density and content throughout the skeleton relative to vehicle control treated animals. The numbers and activity of both osteoblasts and osteoclasts were also found to be significantly higher in endoxifen treated mice. In the pre-clinical model system, endoxifen treatment of 4 month-old ovariectomized Sprague-Dawley rats significantly protected against bone loss following estrogen depletion primarily due to suppression of osteoclast mediated bone resorption. Importantly, in sham operated rats (thus retaining ovarian function), endoxifen treatment enhanced bone volume and trabecular thickness and did not suppress osteoclast activity. Conclusions: These data are the first to examine the effects of the novel breast cancer therapy, endoxifen, on bone and reveal that the molecular mechanisms of action of this compound are substantially different than that of other SERMs. Endoxifen was shown to protect against bone loss following estrogen depletion in both mice and rats and interestingly, enhanced bone mass in ovary intact rats, an observation that is in stark contrast to the known effects of tamoxifen which induces bone loss in the “pre-menopausal” setting. These studies suggest that endoxifen may have superior bone-beneficial effects compared to tamoxifen, and if efficacy is confirmed in later phase trials, endoxifen may represent a better drug of choice for a sub-set of breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-01.

Abstract Background: Imaging biomarkers of cellular division offer promise as non-invasive measures of tumor response. 3’-deoxy-3’[18F]-fluorothymidine ([18F]-FLT) positron emission tomography (PET) imaging generally correlates with pathology-based measurements of cancer proliferation, especially the Ki67 score. Though clinical studies have associated changes in [18F]-FLT uptake with therapeutic response, clinical studies validating its ability to assess cell proliferation are comparatively lacking. The goal of this study was to determine quantitative relationships between [18F]-FLT compared with molecular and cellular metrics of proliferation during treatment for locally advanced breast cancer (LABC). Methods: Baseline [18F]−FLT-PET scans were obtained prior to the initiation of chemotherapy for LABC from patients enrolled at several academic oncology study sites. MRI scans, and transmission CT scans were obtained. Core needle biopsies were obtained to determine Ki-67 indices using immuno-histochemistry and to assess an mRNA signature based measurement of proliferation. Prospectively specified quantitative relationships between PET, Ki67 immunohistochemistry and the mRNA signature were evaluated using image-matched tumor specimens. Correlations between volumetric MRI changes and pathologic responses were evaluated in a post-hoc exploratory analysis. Results: Motivated by the hypothesis that effective chemotherapies should decrease tumor cell proliferation, FLT-PET was compared with biomarkers of proliferation including Ki67 and the mRNA signature during neoadjuvant treatment for LABC. [18F]-FLT correlated both with the Ki67 labeling index (SUVmean r = 0.53) and with the proliferation signature (SUVmean r = 0.7), validating the principle of thymidine analogue imaging. However, variability in the [18F]-FLT PET and tumor cell proliferation measures likely contributed to correlations less than pre-specified target values considered appropriate for clinical use (r > 0.78). Moreover, none of the proliferation biomarkers predicted pathologic complete responses at the end of neoadjuvant therapy ∼16 weeks after the 3 week response scan. In contrast, an evaluation of change in tumor volume measured by MRI after 3 weeks of therapy confirmed its superior ability to predict pCR and tumor re-staging. Conclusion: With large numbers of cancer drugs entering therapeutic pipelines, early efficacy measures remain critical for drug development. The 3-4 month neoadjuvant treatment paradigm for LABC offers unique opportunities for drug evaluation. Functional imaging using [18F]-FLT has been advanced as an assessor of cellular proliferation, potentially offering a non-invasive approach to response evaluation. While [18F]-FLT generally correlated with proliferation, its lack of association with patient responses likely limit its clinical utility. On the other hand, the predictive value of MRI offers unique opportunities for future trial designs and confirms previous reports (1). 1. N. M. Hylton et al., Locally advanced breast cancer: MR imaging for prediction of response to neoadjuvant chemotherapy. Radiology 263, 663 (2012). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-01-11.

Abstract Background: The Phase III trial (NCT00337103) compared eribulin (E) with capecitabine (C) in patients (pts) with metastatic breast cancer (MBC) in the 1st-, 2nd-, and 3rd-line setting. Median overall survival (OS) was 15.9 and 14.5 months (HR 0.88; 95% CI 0.77, 1.00; P = 0.056) and median progression-free survival (PFS) was 4.1 and 4.2 months (HR 1.08; 95% CI 0.93, 1.25; P = 0.30) for E and C, respectively. This analysis compares toxicity and efficacy of E and C in relation to age. Material and methods: In this post-hoc exploratory analysis, the effect of age on the incidence of adverse events (AEs), OS, PFS, and objective response rate (ORR) with E and C were analyzed for two age groups: ≤65 years (E, n = 468; C, n = 491) and >65 years (E, n = 86; C, n = 57). For OS and PFS, analyses were stratified by HER2 and geographic region. Results: With increasing age, the proportion of pts with worse performance status (PS ≥1: 54.5% vs 69.2% for ≤65 and >65 years, respectively), ER+ (47.4% vs 57.3%), and PgR+ MBC (41.3% vs 45.5%) increased, and the proportion with triple-negative MBC decreased (26.6% vs 20.3%). With both treatments, AEs were reported in a higher proportion of pts in the older age group, this becoming more apparent for grade 3+ AEs (E: 64.6% vs 70.2%, and C: 45.0% vs 54.4% for ≤65 and >65 respectively). With E, there was a trend for increased incidence of grade 3/4 neutropenia (45.0% vs 50.0%) and leukopenia (13.7% vs 22.6%) but, in contrast, decreased peripheral sensory neuropathy (3.9% vs 1.2%) with increasing age. For C, there was a trend for increased palmar-plantar erythrodysethesia syndrome (total: 44.4% vs 50.9%; grade 3/4: 14.1% vs 17.5%), and grade 3/4 fatigue (1.8% vs 7.0%) and diarrhea (4.7% vs 10.5%) with increasing age; emesis and nausea were similar for both age groups. Dose adjustments due to AEs with E were slightly higher in the older age group: withdrawals 7.4% vs 10.7%; dose reductions 31.1% vs 36.9%; and dose delays 30.9% vs 36.9%. With C, there was a trend for an increased incidence of withdrawals (9.2% vs 21.1%) and dose delays (34.2% vs 49.1%) due to AEs with increasing age: the incidence of dose reductions was slightly higher in the older age group (31.3% vs 36.8%). In an unadjusted analysis, a trend for improved OS with E vs C was observed in both subgroups (≤65 years: median 15.8 vs 14.5 months; HR 0.90; 95% CI 0.78, 1.04; P = 0.16, and >65 years: median 18.4 vs 14.1 months; HR 0.74; 95% CI 0.50, 1.12; P = 0.16). PFS and ORR for E and C were: median PFS: E, 4.0 and 5.4 months; C, 4.2 and 5.9 months; ORR: E, 10.9% and 11.6%; C, 11.6% and 10.5%, in the ≤65 and >65 groups respectively. Conclusions: This exploratory and unadjusted analysis suggests a trend for improved OS with E in both younger and older pts with MBC. With both treatments there was a suggestion that AEs were reported in a higher proportion of pts in the older age group, this becoming more apparent for grade 3+ events. Specifically, these data suggest an increased incidence of grade 3/4 diarrhea, dose delays, and study withdrawal due to AEs in pts treated with C, and potentially suggest that with E there may be less difference between the AE profile in younger vs older pts than with C. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-13-04.

Abstract Background A Phase III trial (Study 301; NCT00337103) in MBC comparing eribulin (E) with capecitabine (C) showed a trend for improved overall survival (OS) (hazard ratio [HR] 0.88; 95% CI 0.77, 1.00; P = 0.056) but not progression-free survival (PFS) (HR 1.08; 95% CI 0.93, 1.25; P = 0.30) with E. In order to investigate this apparent discordance, post-hoc analyses assessed the effect of PPT and events defining disease progression on OS. Methods Pts with locally advanced or MBC who had received an anthracycline and a taxane (≤2 prior chemotherapy regimens for advanced disease), were randomized to E (554) or C (548). Co-primary endpoints were OS and PFS. The impact of PPT on OS was assessed by analysis of exploratory PPT subgroups. Analyses of OS sensitivity censoring at time of PPT, and OS adjusted by PPT as a time-dependent covariate in a Cox regression model, were performed. The relationship between OS and progression events (appearance of a “new” lesion or metastasis [NM]; increase in size of pre-existing lesions [IPEL]; or other [death, clinical progression, or censored]) was investigated using Cox regression. Results PPT was received by 70% and 62% of E and C pts, respectively. For E pts, there were no significant OS differences by PPT (Table 1). Excluding 56 pts (22 E, 34 C) who received anti-HER2 therapy as PPT from the intent-to-treat OS analysis, OS was longer with E (HR 0.86; 95% CI 0.76, 0.99; nominal P = 0.03). OS sensitivity analysis censoring at time of PPT was consistent with the overall study results (HR 0.82; 95% CI 0.65, 1.03). Assessment of the overall effect of PPT, adjusting by PPT as a time-dependent covariate in a Cox regression model, showed that OS was longer with E (HR 0.85; 95% CI 0.75, 0.97; nominal P = 0.02). Progression due to NM or IPEL occurred in 271 and 147 of E pts, and 285 and 129 of C pts, respectively. Median OS was similar between arms in pts with IPEL and longer with E vs C in pts with NM (Table 2). Pts who progressed due to NM were at higher risk of death (HR 2.12; 95% CI 1.84, 2.43; nominal P<0.01). Conclusions Treatment with C or any other PPT after progression on E did not account for the trend in OS benefit with E observed in the primary analysis. Pts who progressed with NM had a worse prognosis than those with IPEL. The appearance of NM was highly correlated with OS, and the apparent discordance between PFS and OS seems to be related to these different progression events. Tab 1: OS by PPT after EPPTAll ptsC as 1stOther than C as 1stC at any timeOther than C at any timeNoneN554221169275115164Median OS, months (95% CI)15.9 (15.2, 17.6)18.3 (15.8, 20.9)19.9 (17.6, 24.0)19.6 (17.6, 21.5)18.0 (15.4, 22.7)7.4 (6.2, 9.1) Tab 2: OS by progression event Progression event NMIPELOther ECECECN271285147129136134Median OS, months (95% CI)15.5 (14.2, 17.5)12.9 (11.3, 14.5)17.4 (14.4, 19.7)17.4 (15.3, 20.9)16.7 (14.8, 24.2)15.5 (11.7, 18.3)HR (95% CI)0.81 (0.68, 0.97)1.13 (0.87, 1.46)0.78 (0.59, 1.03)Nominal P -value0.020.350.08Time to NM or death, months (95% CI)5.8 (5.2, 6.5)5.2 (4.3, 5.9)NANAHR (95% CI)0.90 (0.77, 1.05)NANANominal P-value0.17NANA Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-13-03.

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.