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Background: The risk of distant recurrence gene signature, MammaPrint (MP), together with the molecular subtyping gene signature, BluePrint (BP), stratifies breast tumors into Luminal A, Luminal B, HER2, and Basal subtypes, independent of immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH) expression. In the Neoadjuvant Breast Registry Symphony Trial (NBRST), MP and BP identified patients likely to respond to neoadjuvant treatment with higher accuracy compared to conventional methods. Here, we report 5-year follow up (FU) data in breast cancer (BC) patients from the NBRST registry with discordant clinical and genomic subtyping.Methods: This prospective study enrolled 1072 early-stage BC patients from 2009-2014 who received MP and BP testing. Patients received neoadjuvant therapy following standard of care and consented to 5 years post-surgery FU. IHC determined hormone receptor (HR) status, including ER and PR, and IHC and/or FISH determined HER2 status. Median FU for distant metastasis free survival (DMFS) and overall survival (OS) was 4.6 and 5 years, respectively. Differences in DMFS and OS was assessed by Kaplan Meier analysis and log-rank test.Results: Overall, BP reclassified 22% of tumors into different molecular subtypes compared to IHC/FISH (Table). BP reclassified 17% of ER+HER2- tumors as BP Basal, with higher pathological complete response (pCR) rates compared to ER+/BP Luminal tumors (36% vs. 4%). ER+/BP Basal patients had similar pCR rates as triple negative BC (TNBC)/BP Basal patients (36% vs. 37%) following neoadjuvant treatment, and pCR correlated with improved survival outcomes. The 5-year DMFS and OS probabilities were lower in ER+/BP Basal patients compared to TNBC/BP Basal patients and were substantially lower compared to ER+/BP Luminal patients (P < 0.001). There were 106 HR-HER2+ patients, of whom BP reclassified 23.6% to Basal and 2.8% as Luminal B; the remaining 73.6% were confirmed HER2 by BP. The 5-year DMFS and OS probabilities were worse in HER2+/BP Basal patients compared to HER2+/BP HER2 patients. Of 142 triple positive (TP, ER+PR+HER2+) patients, BP classified 55% as Luminal, 39% as HER2, and 6% as Basal, with higher pCR rates observed in BP Basal and BP HER2 tumors compared to BP Luminal. The 5-year DMFS and OS probabilities were substantially lower in TP/BP Basal patients compared to TP/BP HER2 and TP/BP Luminal patients (P < 0.05 and P < 0.04). Of clinical HER2+ patients (HR+ or HR-) that received pertuzumab, patients that reclassified as BP Basal had worse OS compared to BP HER2 patients (P < 0.04).Conclusion: ER+HER2- and HER2+ patients that reclassified as BP Basal are more likely to achieve pCR and have improved survival, demonstrating the clinical utility of BP in the neoadjuvant setting. These patients may benefit from optimized chemotherapy used for TNBC, including novel emerging treatments such as PD-1 and PARP1 inhibitors, in addition to HER2-targeted therapy. Furthermore, HER2+ tumors that were confirmed HER2 by BP may have high response rates to regimens containing TDM-1. Lastly, BP identified a subgroup of triple positive BC patients, who reclassified as BP Luminal, that may avoid overtreatment. Overall, molecular subtyping using MP and BP is more accurate in stratifying patients and predicting treatment responses and 5-year disease outcomes than conventional methods and thus, facilitates successful treatment decisions. Clinical subtypeFrequency of BP classificationBluePrint subtypepCR%5-yr DMFS (95% CI)5-yr OS (95% CI)TNBC (n=236)0.42% (1/236)Luminal A100% (1/1)N/AN/A2.54% (6/236)Luminal B16.67% (1/6)N/AN/A1.27% (3/236)HER233.33% (1/3)N/AN/A95.76% (226/236)Basal36.73% (83/226)100% (pCR)100% (pCR)60.5% (50.5-69.1)(non-PCR)64.3%(52.1-71.2) (non-PCR)ER+HER2- (n=520)28.84% (152/520)Luminal A1.97% (3/152)91.1% (84.0-95.2)94.6% (88.3-97.6)52.37% (276/520)Luminal B5.43% (15/276)75.2% (69.0-80.4)84.5% (79.0-88.7)1.33% (7/520)HER214.29% (1/7)N/AN/A17.46% (92/520)Basal35.9%(33/92)84.1% (67.8-92.5) (pCR)86.3% (70.1-94.1) (pCR)54.6% (42.0-65.5) (non-pCR)57% (43.7-68.2) (non-pCR)HR-HER2+ (n=106)2.83% (3/106)Luminal B66.67% (2/3)100%100%73.59% (78/106)HER269% (54/78)82.8% (69.9-90.5)88.6% (76.0-94.8)23.58% (25/106)Basal40% (10/25)79.0% (52.5-91.7)79.0% (52.5-91.7)Triple Positive (n=142)12.68% (18/142)Luminal A22.22% (4/18)88.8% (76.5-94.8)94.5% (83.8-98.2)42.25% (60/142)Luminal B11.67%(7/60)38.73% (55/142)HER244.44% (24/55)87.5% (72.0-94.7)97.9% (83.8-98.2)6.34% (9/142)Basal55.56%(5/9)62.5% (22.9-86.1)70.0% (22.5-91.8)HER2+ (HR+ or HR-)treated with pertuzumab (n=105)28.6%(30/105)Luminal37% (11/30)84.7% (63.8-94.1)92.2%(71.8-98.0)57%(60/105)HER282% (49/60)91.4% (78.3-96.8)92.9%(79.2-97.7)14%(15/105)Basal40% (6/15)66.0%(31.1-86.3)64.0% (29.1-85.1) Citation Format: Pat Whitworth, James Pellicane, Jr, Paul Baron, Peter Beitsch, Laura Lee, Michael Rotkis, Angela Mislowsky, Carrie Dul, Charles Nash, Bichlien Nguyen, Mary Murray, Paul Richards, Mark Gittleman, Stephanie Akbari, Shiyu Wang, Andrea Menicucci, Erin B Yoder, Lisa Blumencranz, William Audeh. Molecular subtyping by BluePrint improves prediction of treatment responses and survival outcomes in patients with discordant clinical and genomic classification [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-04.

Background: MammaPrint (MP) is used to identify breast cancer (BC) patients who can safely forego adjuvant chemotherapy. MP combined with the BluePrint (BP) molecular subtyping signature identifies BC subtypes with distinct therapeutic response rates and survival outcomes. In the Neoadjuvant Breast Symphony Trial (NBRST), MP and BP (MP/BP) predicted rates of pathologic complete response to neoadjuvant chemotherapy (NCT) and partial response to neoadjuvant endocrine therapy (NET). Here, we report 5-year overall survival (OS) and distant metastasis-free survival (DMFS) in patients from the NBRST registry according to MP/BP molecular classification. Methods: The NBRST trial (NCT01479101) prospectively enrolled 1072 patients from 2011 to 2014, who received MP and BP testing. Patients were assigned to receive NCT or NET according to NCCN guidelines and consented to 5 years post-surgery follow-up (FU). Clinical outcomes were available for 913 patients from 67 US institutions. Median FU for OS and DMFS was 5 and 4.6 years, respectively. Tumors classified by MP as High Risk (HR) or Low Risk (LR) were further stratified into four molecular subtypes by BP: Luminal A, Luminal B, HER2, and Basal. Differences in OS and DMFS at 3 and 5 years were assessed by Kaplan Meier analysis and log-rank test. Results: MP results from neoadjuvant patients (N=913) classified 16% of tumors as MP LR and 84% as MP HR. MP and BP classified 15.7% (143/913) of tumors as Luminal A, 32.5% (297/913) as Luminal B, 17.1% (156/913) as HER2, and 34.7% (317/913) as Basal. The 5-year OS and DMFS probabilities were significantly lower in HR compared to LR patients (p < 0.001 for OS and DMFS), and lowest in Basal and Luminal B compared to Luminal A and HER2 subtypes (p < 0.001 for OS and DMFS). Most DMFS events in BP Basal tumors occurred within the first 3 years. Of 841 patients that received NCT with or without HER2-targeted therapy, 12.2% (103/841) were LR and 87.8% (738/841) were HR. MP and BP classified 11.9% (100/841) of these patients as Luminal A, 32.6% (274/841) as Luminal B, 8.3% (154/841) as HER2 subtype, and 37.2% (313/841) as Basal. The 5-year OS and DMFS probabilities were lowest in HR, Basal or Luminal B patients (p < 0.001). In 59 patients who received NET alone, 5-year OS and DMFS were significantly worse in HR patients that had Luminal B or HER2 tumors compared to LR Luminal A patients. In the 39 patients with Luminal A tumors, response to NET at the time of surgery was: 46.2% partial response, 41.0% stable disease, 5.1% progressive disease, 2.6% not reported. Five year DMFS in patients with Luminal A tumors treated with NCT or NET was not significantly different (p=0.67).Conclusions: MammaPrint remained prognostic in BC patients undergoing neoadjuvant therapy. Long -term prognosis was excellent in LR groups who received NCT or NET alone. MP and BP can accurately classify patients into specific subtypes with distinct OS and DMFS outcomes at five years, with BP Basals having the worst outcomes, followed by Luminal B, HER2, and Luminal A subtypes. BP Basal patients had the highest frequency of events within the first 3 years post-surgery, suggesting a genomic risk timeline distinct from other BP subtypes and a potential benefit from a secondary therapeutic immediately post-surgery. Additionally, Luminal A patients had a very low risk of progressive disease while on NET alone prior to surgery, with similar DMFS outcomes to Luminal A-types who received NCT. Number of patientsObserved events% at 5 year (95% CI)p-valueAll patients - MammaPrint Risk GroupOS913134p Citation Format: Pat Whitworth, James V Pellicane, Jr., Paul Baron, Peter Beitsch, Laura Lee, Michael Rotkis, Angela Mislowsky, Carrie Dul, Charles Nash, Bichlien Nguyen, Mary Murray, Paul Richards, Mark Gittleman, Stephanie Akbari, Shiyu Wang, Erin B Yoder, Andrea Menicucci, Lisa Blumencranz, William Audeh, NBRST Investigators Group. 5-year outcomes in the NBRST trial: Preoperative MammaPrint and BluePrint breast cancer subtype is associated with neoadjuvant treatment response and survival [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-01.

BACKGROUND: ExteNET showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, significantly improves 2-year invasive disease-free survival after trastuzumab-based adjuvant therapy in women with HER2-positive breast cancer. We report updated efficacy outcomes from a protocol-defined 5-year follow-up sensitivity analysis and long-term toxicity findings. METHODS: In this ongoing randomised, double-blind, placebo-controlled, phase 3 trial, eligible women aged 18 years or older (≥20 years in Japan) with stage 1-3c (modified to stage 2-3c in February, 2010) operable breast cancer, who had completed neoadjuvant and adjuvant chemotherapy plus trastuzumab with no evidence of disease recurrence or metastatic disease at study entry. Patients who were eligible patients were randomly assigned (1:1) via permuted blocks stratified according to hormone receptor status (hormone receptor-positive vs hormone receptor-negative), nodal status (0 vs 1-3 vs or ≥4 positive nodes), and trastuzumab adjuvant regimen (given sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system, to receive 1 year of oral neratinib 240 mg/day or matching placebo. Treatment was given continuously for 1 year, unless disease recurrence or new breast cancer, intolerable adverse events, or consent withdrawal occurred. Patients, investigators, and trial funder were masked to treatment allocation. The predefined endpoint of the 5-year analysis was invasive disease-free survival, analysed by intention to treat. ExteNET is registered with ClinicalTrials.gov, number NCT00878709, and is closed to new participants. FINDINGS: Between July 9, 2009, and Oct 24, 2011, 2840 eligible women with early HER2-positive breast cancer were recruited from community-based and academic institutions in 40 countries and randomly assigned to receive neratinib (n=1420) or placebo (n=1420). After a median follow-up of 5·2 years (IQR 2·1-5·3), patients in the neratinib group had significantly fewer invasive disease-free survival events than those in the placebo group (116 vs 163 events; stratified hazard ratio 0·73, 95% CI 0·57-0·92, p=0·0083). The 5-year invasive disease-free survival was 90·2% (95% CI 88·3-91·8) in the neratinib group and 87·7% (85·7-89·4) in the placebo group. Without diarrhoea prophylaxis, the most common grade 3-4 adverse events in the neratinib group, compared with the placebo group, were diarrhoea (561 [40%] grade 3 and one [BACKGROUND: ExteNET showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, significantly improves 2-year invasive disease-free survival after trastuzumab-based adjuvant therapy in women with HER2-positive breast cancer. We report updated efficacy outcomes from a protocol-defined 5-year follow-up sensitivity analysis and long-term toxicity findings.METHODS: In this ongoing randomised, double-blind, placebo-controlled, phase 3 trial, eligible women aged 18 years or older (≥20 years in Japan) with stage 1-3c (modified to stage 2-3c in February, 2010) operable breast cancer, who had completed neoadjuvant and adjuvant chemotherapy plus trastuzumab with no evidence of disease recurrence or metastatic disease at study entry. Patients who were eligible patients were randomly assigned (1:1) via permuted blocks stratified according to hormone receptor status (hormone receptor-positive vs hormone receptor-negative), nodal status (0 vs 1-3 vs or ≥4 positive nodes), and trastuzumab adjuvant regimen (given sequentially vs concurrently with chemotherapy), then implemented centrally via an interactive voice and web-response system, to receive 1 year of oral neratinib 240 mg/day or matching placebo. Treatment was given continuously for 1 year, unless disease recurrence or new breast cancer, intolerable adverse events, or consent withdrawal occurred. Patients, investigators, and trial funder were masked to treatment allocation. The predefined endpoint of the 5-year analysis was invasive disease-free survival, analysed by intention to treat. ExteNET is registered with ClinicalTrials.gov, number NCT00878709, and is closed to new participants.FINDINGS: Between July 9, 2009, and Oct 24, 2011, 2840 eligible women with early HER2-positive breast cancer were recruited from community-based and academic institutions in 40 countries and randomly assigned to receive neratinib (n=1420) or placebo (n=1420). After a median follow-up of 5·2 years (IQR 2·1-5·3), patients in the neratinib group had significantly fewer invasive disease-free survival events than those in the placebo group (116 vs 163 events; stratified hazard ratio 0·73, 95% CI 0·57-0·92, p=0·0083). The 5-year invasive disease-free survival was 90·2% (95% CI 88·3-91·8) in the neratinib group and 87·7% (85·7-89·4) in the placebo group. Without diarrhoea prophylaxis, the most common grade 3-4 adverse events in the neratinib group, compared with the placebo group, were diarrhoea (561 [40%] grade 3 and one [INTERPRETATION: At the 5-year follow-up, 1 year of extended adjuvant therapy with neratinib, administered after chemotherapy and trastuzumab, significantly reduced the proportion of clinically relevant breast cancer relapses-ie, those that might lead to death, such as distant and locoregional relapses outside the preserved breast-without increasing the risk of long-term toxicity. An analysis of overall survival is planned after 248 events.FUNDING: Wyeth, Pfizer, and Puma Biotechnology.

Purpose The purpose of the NBRST study is to compare a multigene classifier to conventional immunohistochemistry (IHC)/fluorescence in situ hybridization (FISH) subtyping to predict chemosensitivity as defined by pathological complete response (pCR) or endocrine sensitivity as defined by partial response. Methods The study includes women with histologically proven breast cancer, who will receive neoadjuvant chemotherapy (NCT) or neoadjuvant endocrine therapy. BluePrint in combination with MammaPrint classifies patients into four molecular subgroups: Luminal A, Luminal B, HER2, and Basal. Results A total of 426 patients had definitive surgery. Thirty-seven of 211 (18 %) IHC/FISH hormone receptor (HR)+/HER2− patients were reclassified by Blueprint as Basal (n = 35) or HER2 (n = 2). Fifty-three of 123 (43 %) IHC/FISH HER2+ patients were reclassified as Luminal (n = 36) or Basal (n = 17). Four of 92 (4 %) IHC/FISH triple-negative (TN) patients were reclassified as Luminal (n = 2) or HER2 (n = 2). NCT pCR rates were 2 % in Luminal A and 7 % Luminal B patients versus 10 % pCR in IHC/FISH HR+/HER2− patients. The NCT pCR rate was 53 % in BluePrint HER2 patients. This is significantly superior (p = 0.047) to the pCR rate in IHC/FISH HER2+ patients (38 %). The pCR rate of 36 of 75 IHC/FISH HER2+/HR+ patients reclassified as BPLuminal is 3 %. NCT pCR for BluePrint Basal patients was 49 of 140 (35 %), comparable to the 34 of 92 pCR rate (37 %) in IHC/FISH TN patients. Conclusions BluePrint molecular subtyping reclassifies 22 % (94/426) of tumors, reassigning more responsive patients to the HER2 and Basal categories while reassigning less responsive patients to the Luminal category. These findings suggest that compared with IHC/FISH, BluePrint more accurately identifies patients likely to respond (or not respond) to NCT.

Microelectrode arrays can be ideal tools for probing a host of biological interactions. Yet in spite of their general utility, their use is still limited by our ability to construct complex molecular surfaces that can spatially isolate molecules in specific locations proximal to specific electrodes. With this in mind, we have been developing electrochemically based tools that allow for the placement of molecules by specific electrodes in an array and then probe the binding of those molecules with biological targets in “real-time”. Yet while those methods are very useful, they have limits. They enable the analysis of smaller libraries, but are not as effective if one wants to probe a receptor with even a medium size library. The problem is that the tools developed to date require a library to be synthesized at a remote site and then transfer that library to the array one molecule at a time. For larger libraries, this is time consuming and cumbersome. A better strategy would be to build the library directly on the array. The approach to building a molecular library typically involves a diversification strategy like the one shown in Scheme 1. In the Scheme, P1-P4 represent protecting groups that can be orthogonally removed. But what are the orthogonal protecting groups that can be removed site-selectively on a microelectrode array so that such a strategy can be used to synthesize more complex molecular surfaces? Furthermore, are the current surfaces used on the arrays compatible with such an intensive synthetic effort? In the talk to be given, we will describe initial findings that begin to address these questions as we push for the development of new methods that will allow for the greater community to take full advantage of what microelectrode arrays can offer. Figure 1

1101 Background: Miller, et al demonstrated the combination of bevacizumab and paclitaxel has significant activity in metastatic breast cancer. Because paclitaxel albumin bound (PAB) has been shown to have less toxicity, a better tumor delivery and possibly better response for metastatic breast cancer, we combined it with bevacizumab (B) to treat women with metastatic breast cancer. Methods: This is a retrospective analysis. Billing records from March 2005 through December 2006 were reviewed to obtain all patients treated consecutively with a combination of PAB (80–125mg/m2 days 1,8,15 or 170–200 mg/m2 every 14 days on a 28 day cycle) and B (10mg/kg every 14 days). A total of 40 women were identified. A minimum of two courses were given. All women had received a minimum of 3 prior chemotherapy regimens including anthracyclines 34/40 and taxanes 35/40. Patients were monitored for response using RECIST criteria based on PE, and PET/CT imaging. Six women with bone only disease were monitored with PET, CT/MRI and tumor markers. All response data were confirmed by independent review. Results: 20 women had objective responses to the PAB/B regimen (3CRs and 17PRs) for an overall response rate of 50%. Another seven women had stable disease (SD) for a mean duration of 212 days. Thirteen women progressed. The mean time to progression for the responders was 132 days. Toxicity was acceptable with fatigue (9 gr 2), neuropathy (4 gr 2, 1 gr 3), anemia (2 gr 2, 2 gr 3), and hypertension (3 gr 2) being the most common complaints. Two patients were discontinued due to a possible CNS hemorrhage into a metastatic brain lesion. There were no other treatment discontinuations due to non-tolerance Conclusions: In our limited series of consecutive woman with advanced, heavily pretreated metastatic breast cancer treated with PAB (Abraxane) and bevacizumab (Avastin), we saw a 50% objective response rate (3CR, 17PR). The regimen was well tolerated with acceptable toxicity. Another seven women had stable disease for an average duration of >200days giving an objective response rate + SD of 67%. No significant financial relationships to disclose.

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, inter-laboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.

Purpose. To compare breast cancer subtyping with the three centrally assessed microarray-based assays BluePrint, MammaPrint, and TargetPrint with locally assessed clinical subtyping using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Methods. BluePrint, MammaPrint, and TargetPrint were all performed on fresh tumor samples. Microarray analysis was performed at Agendia Laboratories, blinded for clinical and pathological data. IHC/FISH assessments were performed according to local practice at each institution; estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) assessments were performed on 132 samples, and Ki-67 on 79 samples. Results. The concordance between BluePrint and IHC/ FISH subtyping was 94 % for the Luminal-type, 95 % for the HER2-type, and 94 % for the Basal-type subgroups. The concordance of BluePrint with subtyping using mRNA single gene readout (TargetPrint) was 96 % for the Luminal-type, 97 % for the HER2-type, and 98 % for the Basal-type subgroups. The concordance for substratification into Luminal A and B using MammaPrint and Ki-67 was 68 %. The concordance between TargetPrint and IHC/FISH was 97 % for ER, 80 % for PR, and 95 % for HER2. Conclusions. The implementation of multigene assays such as TargetPrint, BluePrint, and MammaPrint may improve the clinical management of breast cancer patients. High discordance between Luminal A and B substratification based on MammaPrint versus locally assessed Ki67 or grade indicates that chemotherapy decisions should not be based on the basis of Ki-67 readout or tumor grade alone. TargetPrint serves as a second opinion for those local pathology settings where high-quality standardization is harder to maintain.

Twenty-nine chemotherapy-naive patients with primary hepatocellular carcinoma were treated with oral beta-all trans-retinoic acid (retinoic acid, TRA 50 mg/m2 t.i.d.) on a 3-week on/one week off schedule until progression or grade 3 or 4 toxicity. Eligibility requirements allowed abnormal liver function tests as long as the creatinine and bilirubin levels were normal. No responses were seen and the median survival was four months. Grade 3 side effects occurred in II patients and grade 4 in four and included a wide range of toxicities. The results indicate that oral TRA is ineffective against primary hepatocellular carcinoma and suggest that dose-modification of this retinoid may be required in patients with significant malignant hepatic involvement.

Background: The FDA-approved MammaPrint 70 gene profile is performed on a diagnostic multi-gene array. This platform enables additional gene expression profiles to be analyzed simultaneously on one tumor specimen. One such gene expression profile is BluePrint, an 80 gene molecular subtyping profile that discriminates between three distinctive subtypes; Basal-type, Luminal-type, and HER2-type. By combining MammaPrint and BluePrint, patients can be stratified into the following subgroups: Luminal-type/MammaPrint Low Risk; Luminal-type/MammaPrint High Risk; HER2-type and Basal-type. Marked differences are observed in response to neo-adjuvant treatment in groups stratified by MammaPrint and BluePrint [Glück et al. ASCO 2012]. Trial design: NBRST is a prospective observational, case-only study linking MammaPrint and BluePrint to neo-adjuvant treatment response and Distant Metastases-Free Survival (DMFS) and Relapse-Free Survival (RFS). MammaPrint and BluePrint are assessed on fresh or formalin-fixed, paraffin-embedded core needle biopsy samples. Treatment is at the discretion of the physician, adhering to NCCN approved regimens or a recognized alternative. Eligibility: The study will include women aged 18–90 with histologically proven breast cancer, who have started or are scheduled to start neo-adjuvant chemotherapy or neo-adjuvant hormonal therapy, after successful MammaPrint and BluePrint assessment, and who provide written informed consent. Additional inclusion criteria are that women must have had no excisional biopsy or axillary dissection, no confirmed distant metastatic disease, and no prior therapy for the treatment of breast cancer. Objectives: The main objectives of this registry study are to: 1. Measure chemosensitivity (as defined by pathological complete response [pCR]) or endocrine sensitivity (as defined by decrease in longest tumor diameter or RCB1) in the molecular subgroups as determined by combining MammaPrint and BluePrint results.2. Compare local IHC and FISH results with TargetPrint results and BluePrint results.3. Document impact of MammaPrint, TargetPrint and BluePrint results on treatment decisions.4. Assess the 2–3 and 5-year DMFS and RFS for the different molecular subgroups. Statistical methods: Comparison of response rates between different molecular subgroups will be conducted using the Pearson Chi-square test. Accrual: The trial started in August 2011 and there are currently 36 participating sites in the US. To date (June 06, 2012), 158 of the planned 400 patients have been enrolled.Clinical trial registry number: NCT01479101. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-4-03.

Background: MammaPrint (MP) is a powerful predictor of disease outcome in early stage breast cancer. In addition, TargetPrint (TP), a quality approved microarray-based test that measures the mRNA expression level of ER, PR and HER2 and an 80 gene expression Molecular Subtyping profile BluePrint (BP) were developed. In the present study, MP, BP and TP were measured in a prospective U.S. breast cancer patient cohort. Methods: MP results were evaluated in fresh tumor samples from 141 breast cancer patients (T1-4N0-2; median age 62 [35-97 yr]) collected by core needle biopsy or from a surgical specimen between July 2008 and February 2011. We compared treatment advice as recommended by NCCN guidelines and classification according to MP. In addition, we compared IHC/FISHER, PR and HER2 assessments with TP. The MP and BP results were used to subtype the patients into molecular subgroups. Results: For the group of patients (n=69) for which NCCN recommends the use of a multi-gene signature for determining chemotherapy treatment recommendations, 50 patients were classified as High Risk and 19 as Low Risk by MP. Comparison of TP with IHC/FISH indicated a concordance of 98% for ER, 91% for PR, and 94% for HER2. For a subgroup of 63 patients combined MP and BP results were available; 22 patients were Luminal-type/MP Low Risk, 31 patients were Luminal-type/MP High risk, 1 patient was Her2-type/MP Low Risk, 2 patients were Her2-type/MP High Risk and 7 patients were Basal-type/MP High Risk. Conclusion:The multi-gene signature MammaPrint, as well as BluePrint and TargetPrint provides additional information for treatment guidance. By combining MammaPrint with the BluePrint molecular subtyping profile, specific groups of patients can be recognized that are at high risk of recurrence and that would possibly benefit from specific treatment. This study shows that TargetPrint provides high quality second opinion for local IHC/FISH assessment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-04-06.

11 Background: Differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and is 98% concordant with centrally assessed ER as presented by Viale et al, SABCS 2011. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures. Methods: Fresh tumor samples (core needle biopsies or surgical) were collected for 831 patients diagnosed with breast cancer stage I to IV (Feb 2008 - Jan 2011) from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts with TargetPrint. Discordant cases were centrally reviewed for IHC/FISH assessment. Results: Of the 831 samples, IHC assessment was unknown for 4 ER/ PR samples; HER2 was unknown for 12 samples. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 83% for PR and 94% for HER2. In this study, 3% of all IHC ER positive samples were classified negative by microarray, and 11% of IHC PR positive samples were classified negative by microarray. For HER2, 4% of IHC/FISH HER2 positive samples were classified negative by microarray and 2% of IHC/FISH HER2 negative samples were classified positive by microarray. Most notably, all available 5 ER IHC negative/TargetPrint positive samples turned out to be positive with central re-assessment. HER2 IHC2+ samples with discordant classifications for TargetPrint and local assessment are currently being reviewed for FISH/SISH assessment. Conclusions: Microarray based readout of ER, PR and HER2 status using TargetPrint is fairly comparable to local IHC and FISH analysis in 827 analyzed samples in various hospitals worldwide. However, re-assessment of discordant cases–especially IHC ER-/TargetPrint ER+ cases- confirms TargetPrint to be a useful high quality second opinion for local IHC/FISH assessment.

10554 Background: Differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and is 98% concordant with centrally assessed ER as presented by Viale et al, SABCS 2011. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures. Methods: Fresh tumor samples (core needle biopsies or surgical) were collected for 831 patients diagnosed with breast cancer stage I to IV (Feb 2008 - Jan 2011) from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts with TargetPrint. Discordant cases were centrally reviewed for IHC/FISH assessment. Results: Of the 831 samples, IHC assessment was unknown for 4 ER/ PR samples; HER2 was unknown for 12 samples. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 83% for PR and 94% for HER2. In this study, 3% of all IHC ER positive samples were classified negative by microarray, and 11% of IHC PR positive samples were classified negative by microarray. For HER2, 4% of IHC/FISH HER2 positive samples were classified negative by microarray and 2% of IHC/FISH HER2 negative samples were classified positive by microarray. Most notably, all available 5 ER IHC negative/TargetPrint positive samples turned out to be positive with central re-assessment. HER2 IHC2+ samples with discordant classifications for TargetPrint and local assessment are currently being reviewed for FISH/SISH assessment. Conclusions: Microarray based readout of ER, PR and HER2 status using TargetPrint is fairly comparable to local IHC and FISH analysis in 827 analyzed samples in various hospitals worldwide. However, re-assessment of discordant cases –especially IHC ER-/TargetPrint ER+ cases- confirms TargetPrint to be a useful high quality second opinion for local IHC/FISH assessment.

47 Background: MammaPrint (MP) is a powerful predictor of disease outcome in early stage breast cancer. In addition, TargetPrint (TP), a microarray-based test that measures the mRNA expression level of ER, PR and HER2 and an 80 gene expression Molecular Subtyping profile BluePrint (BP) were developed. In the present study, MP, BP and TP were measured in a prospective U.S. breast cancer patient cohort. Methods: MP results were evaluated in fresh tumor samples from 127 breast cancer patients (T1-4N0-2; median age 62 [39-97 yr]) collected by core needle biopsy or from a surgical specimen between July 2008 and January 2011. We compared treatment advice as recommended by NCCN guidelines and classification according to MP. In addition, we compared IHC/FISH ER, PR and HER2 assessments with TP. The MP and BP results were used to subtype the patients into molecular subgroups. Results: For the group of patients (n=59) for which NCCN recommends the use of a multi-gene signature for determining chemotherapy treatment recommendations, 42 patients were classified as High Risk and 17 as Low Risk by MP. Comparison of TP with IHC/FISH indicated a concordance of 98% for ER, 94% for PR, and 98% for HER2. For a subgroup of 53 patients combined MP and BP results were available; 18 patients were Luminal-type/MP Low Risk, 27 patients were Luminal-type/MP High risk, 1 patient was Her2-type/MP Low Risk, 1 patient was Her2-type/MP High Risk and 6 patients were Basal-type/MP High Risk. Conclusions: Adding the multi-gene signature MammaPrint, as well as BluePrint and TargetPrint provides additional information for treatment guidance. By combining MammaPrint with the BluePrint molecular subtyping profile, specific groups of patients can be recognized that are at high risk of recurrence and that would possibly benefit from specific treatment. This study shows that TargetPrint provides high quality second opinion for local IHC/FISH assessment.

e11094 Background: The MammaPrint 70-gene expression profile (MP) is a powerful predictor of disease outcome in early stage breast cancer. In addition, TargetPrint (TP), a microarray-based test tha...