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Purpose: Most hormone receptor (HR)+/HER2− breast cancer patients respond unfavorably to neoadjuvant chemotherapy (NAC); however, genomic tests may identify those patients who are likely to benefit. Using the Prosigna assay, we first evaluated the technical performance of core needle biopsy (CNB) tissues. We then determined whether Prosigna risk of relapse (ROR) score and intrinsic subtype predicted response to NAC in HR+/HER2− patients using CNB samples.Experimental Design: Using the NanoString's nCounter Dx analysis system and a development tissue sample set, we established tissue requirements and assay output variance. We then evaluated the concordance in subtype and correlation in ROR between CNBs and corresponding surgical resection specimens (SRS) in a second independent sample set. Finally, we analyzed 180 independent CNB samples from HR+/HER2− patients who were treated with NAC and correlated ROR and intrinsic subtype with pathologic response.Results: Intra- and interbiopsy variabilities were 2.2 and 6.8 ROR units, respectively. Subtype concordance within multiple CNBs was high for the 4- and 3-subtype classifications (k = 0.885 and 0.889, respectively). Correlation in Prosigna ROR score observed between paired CNBs and SRS was high (r ≥ 0.90), and subtype concordance was also high for the 4- and 3-subtype classifications (kappa = 0.81 and 0.91, respectively). Prosigna results obtained from the HR+/HER2− patient samples showed that both ROR (P = 0.047) and intrinsic subtype (OR LumA vs. non-LumA = 0.341, P = 0.037) were significant predictors of response to NAC.Conclusions: Prosigna ROR and intrinsic subtype are readily obtained from CNB samples in normal practice and reliably predict response to NAC in HR+/HER2− patients. Clin Cancer Res; 22(3); 560–6. ©2015 AACR.

Supplementary Figure S1 and Supplementary Tables S1-S2. Supplementary Figure S1. Diagram of tissue block usage for CNB Development Study and Chemo-prediction Validation Study. Supplemental Table S1. Top-10 and bottom-10 Prosigna gene-correlations between paired CNBs and SRS. Supplemental Table S2. Distribution of the Prosigna subtypes within the three main pathology-based groups in 39 independent metastatic samples.

Purpose The Prosigna-PAM50 risk of recurrence (ROR) score has documented clinical utility for the prediction of 10-year distant recurrence (DR). The present study investigated the value of Prosigna-PAM50 for predicting 10-year DR and overall survival after 5 years of endocrine treatment for postmenopausal patients with invasive lobular carcinoma. Patients and Methods Using the Danish Breast Cancer Group database, we identified patients with a diagnosis from 2000 to 2003 of estrogen receptor-positive, human epidermal growth factor receptor 2-negative invasive ductal (n = 1570) or lobular (n = 341) cancer > 20 mm or 1 to 3 positive lymph nodes and applied multivariate Cox models. Results The median follow-up for DR was 9.3 years and for overall survival 15.2 years. Of the 341 lobular and 1570 ductal cases, 140 (41%) and 349 (22%) were classified as low ROR, with a 10-year DR rate of 7.7% (95% confidence interval [CI], 3.7%-13.6%) and 3.5% (95% CI, 1.8%-6.2%), respectively. The 10-year DR rate for the intermediate ROR group for those with lobular cancer was 18% (95% CI, 10.1%-27.9%) compared with 9.7% (95% CI, 6.7%-13.4%) for those with ductal cancer. Luminal B tumors had a significantly worse outcome than luminal A tumors in both lobular (hazard ratio, 1.89; 95% CI, 1.03%-3.45%; P = .04) and ductal (hazard ratio, 3.18; 95% CI, 2.29%-4.43%; P Conclusion Prosigna PAM-50 provides significant prognostic information beyond the clinicopathologic factors in patients with invasive lobular breast cancer. Those with lobular cancer had worse 10-year DR rates compared with those with ductal cancer in the same ROR category. Our results could have an effect on the treatment decisions regarding the addition of chemotherapy for those in the intermediate ROR group.

The DBCG89D trial randomized high-risk early breast cancer patients to adjuvant CMF (cyclophosphamide, methotrexate and fluorouracil) or CEF (cyclophosphamide, epirubicin and fluorouracil). Prosigna assays were performed by researchers with no access to clinical data. Time to distant recurrence (DR) was the primary endpoint, time to recurrence (TR) and overall survival (OS) secondary. Among the 980 Danish patients enrolled, Prosigna results were obtained in 686. Continuous ROR score was associated with DR for CMF (adjusted hazard ratio (HR) 1.20, 95% CI 1.09–1.33), and for CEF (HR 1.04, 95% CI 0.92–1.18), Pinteraction = 0.06. DR was significantly longer in CEF compared to CMF treated patients with Her2-enriched tumors (HR 0.58, 95% CI 0.38–0.86), but not in patients with luminal tumors. Heterogeneity of treatment effect was significant for TR and OS. In this prospective-retrospective analysis, patients with Her2-enriched breast cancer derived substantial benefit from anthracycline chemotherapy whereas anthracyclines are not an essential component of chemotherapy for patients with luminal subtypes. The benefit of CEF vs. CMF correlated with increasing ROR Score.

The Prosigna-PAM50 risk of recurrence (ROR) score has been validated in randomized clinical trials to predict 10-year distant recurrence (DR) in hormone receptor-positive breast cancer. Here, we examine the ability of Prosigna for predicting DR at 10 years in a subgroup of postmenopausal breast cancer patients with special histological subtypes.Using the population based Danish Breast Cancer Group database, follow-up data were collected on all patients diagnosed from 2000 to 2003 with estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2) normal breast cancer who by nationwide guidelines were treated with 5 year of endocrine therapy (N = 2558). Among patients with 1 to 3 positive lymph nodes or a tumor size20 mm, we identified 1570 with invasive ductal carcinoma (IDC) and 89 with special histological subtypes (apocrine, medullary, mucinous, papillary, secretory, tubular, neuroendocrine) who were tested with Prosigna. Fine and Gray models were applied to determine the prognostic value of the Prosigna-PAM50 ROR score for DR special subtypes as compared to IDC.Median follow-up for DR was 9.2 year and for OS 15.2 year. The 10-year DR rate for the special subtypes was 9.2% (95% CI: 4.0% to 17.2%) as compared to 13.7% (95% CI: 11.9% to 15.7%) for IDC. The 10-year OS was 74.2% (95% CI: 63.7% to 82.0%) for the special subtypes and 75.4% (95% CI: 73.2% to 77.4%) for IDC. Prosigna showed a statistical significant association of the continuous ROR score with risk of DR for both IDC and the special subtypes (IDC: p .0001; special subtypes: p = .01).In the present study, we demonstrated that Prosigna-PAM50 continuous ROR score added significant prognostic information for 10-year DR in postmenopausal patients with special subtypes (tumor size20 mm or 1 to 3 positive lymph nodes) and ER-positive, HER2-normal early breast cancer.

Background: The Prosigna (PAM50) risk of recurrence (ROR) score predicts 10yr DR in early breast cancer patients treated with ET alone (level 1 evidence). Invasive lobular breast cancer (ILBC) accounts for 10-15% of all breast cancer histological subtypes. Sporadic ILBC is characterized by somatic CDH1 mutations with loss of E-cadherin and a majority of low proliferative ER positive/HER2 negative tumors consistent with Luminal A subtype. Here we examine the ability of PAM50/ROR to predict 10yr DR in postmenopausal women who following a diagnosis of HR+ early ILBC were allocated to 5yr of ET alone. Methods: Using the population based clinical DBCG database FFPE primary tumor blocks, treatment, and follow-up data were collected from all patients diagnosed from 2000-2003 with HR+, postmenopausal EBC (N0-N1) who by nationwide guidelines were allocated to 5yr of ET alone, N=2,722 (1256 N0, 1466 N1). PAM50 intrinsic subtype classification (Luminal A, Luminal B, HER2-enriched, Basal-like) was conducted using the NanoString nCounter®Analysis System. Univariate and multivariate analyses tested the ability of PAM50 to predict DR. Patients were categorized as Low or High Risk based upon pre-specified ROR cutoff value of 40. HER2 positive tumors by immunohistochemistry were excluded from analysis. Results: Median follow-up was 9.25 years. Risk of 10yr DR by ROR and PAM50 (Luminal A and Luminal B) is shown in the table by ILBC as compared to invasive ductal breast cancer (IDBC) type. Cumulative incidence for 10 yr distant recurrence (DR)Histological subtypeRisk Group % [95% CI](N row%)Intrinsic Subtype % [95% CI](N row%)Histological subtype% [95% CI](N) LowHighLuminal ALuminal BAllDuctal3.8 [2.5-5.6] (738 35%)16.6 [14.4-18.8] (1388 65%)6.6 [5.1-8.4] (1174 59%)18.1 [15.2-21.3] (832 41%)12.1% [10.6-13.7] (2126)Lobular9.7 [5.5-15.4] (181 53%)23.9 [16.8-31.6] (159 47%)12.7 [8.6-17.8] (256 77%)24.1 [14.5-35.1] (77 23%)16.4 [12.2-21.1] (340) In this specific cohort the ILBC subgroup had a worse 10yr DR of 16.4% [12.2-21.1] as compared to IDBC of 12.1% [10.6-13.7] (Gray´s test, p = 0.046). A significant difference regarding the distribution of ILBC into both High and Low Risk Group and intrinsic subtypes as compared to IDBC was identified (p < 0.0001). Molecular intrinsic subtype analysis for ILBC by Prosigna (PAM50) showed DR 12.7 [8.6-17.8] for Luminal A vs 24.1 [14.5-35.1] for Luminal B. The difference between ILBC subtypes was not significant (p = 0.09). Adding ROR to a Fine and Gray's proportional sub-hazards model containing clinical and pathological variables significantly improved the model for ILBC, (likelihood ratio: p = 0.0001); HR for a 20-point change in ROR = 1.84 [1.37-2.48] notable with DR 9.7 [5.5-15.4] for the Low Risk Group. Conclusions: Following 5-yr of endocrine treatment alone patients with ILBC in this large population-based study had an inferior DR as compared to patients with IDBC and the apparent difference between ILBC and IDBC must be interpreted with caution. Citation Format: Laenkholm A-V, Jensen M-B, Buckingham W, Schaper C, Knoop A, Eriksen JO, Rasmussen BB, Ferree S, Haffner T, Kiboel T, Ejlertsen B. Prediction of 10yr distant recurrence (DR) using the Prosigna® (PAM50) assay in histological subgroups of a Danish breast cancer group (DBCG) cohort of postmenopausal Danish women with hormone receptor-positive (HR+) early breast cancer (EBC) allocated to 5yr of endocrine therapy (ET) alone [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-10.

Background: Several multigene assays are available for managing ER-positive, HER2-negative early stage breast cancer but with little direct comparative information within this patient population. Existing evidence suggests that current multigene assays provide broadly equivalent risk information for the population of women with ER-positive, HER2-negative breast cancers. However, for the individual patient tests may provide differing risk categorization (Bartlett et al, JNCI 2016). We have previously demonstrated the prognostic value of different commercially available tests including Prosigna (ROR) and Oncotype Dx (RS) (Dowsett et al, JCO 2013; Sestak et al, JAMA Onc 2018). Here, we investigate risk classification and 10-year DR rates in patients that had discordant risk categorization between ROR and RS. Methods: A total of 663 postmenopausal women with ER-positive, HER2-negative, node-negative early stage breast cancer from the TransATAC study for whom both signatures were available were included in this analysis. The primary endpoint was distant recurrence (DR). Predefined ROR cut-off points for low/intermediate and intermediate/high of 40/60 were used. Comparisons were made using two sets of RS cut-off points 1) TailoRX cut-off points of 10/25 and 2) original cut-off points of 18/31. Kaplan-Meier curves were used to estimate the mean risk of DR after 10 years of follow-up in predefined risk groups. Results: Using original RS cut-off points, 25 patients (3.8%) had a high-low discordance compared to ROR, with a total of 295 (44.5%) discordant cases. Using the TailoRx cut-off points, 40 patients (6.0%) had a high-low discordance compared to ROR, with a total of 396 (59.7%) discordant cases. Applying original RS cut-off points, patients categorized into ROR low but into intermediate or high by RS (86/365 (23.6%)) had a 10-year DR rate of only 6.3% (2.7-14.6) (Table). In contrast, patients categorized into RS low but into intermediate or high by ROR (144/423 (48.6%)) had a 10-year DR rate of 13.4% (8.3-21.2). Using the TailoRx cut-off points, patients categorized into ROR low but into intermediate or high by RS (261/365 (71.5%)) had a 10-year DR rate of 2.9% (1.4-5.9), while patients with an RS low but intermediate or high by ROR (62/166 (37.3%)) had a 10-year DR rate of 18.2% (10.2-31.2) (Table). Conclusion: Discordance in risk categorization at the individual patient-level between commercially available multigene assays for early stage breast cancer is not uncommon. Our results show that node-negative patients that were classified by ROR as low risk, regardless of RS risk stratification, had an excellent 10-year DR risk with endocrine therapy alone. In contrast, patients that were classified as RS low risk by either cut-off points but were classified as intermediate or high risk by ROR had a significantly worse 10-year outcome. Our results indicate that ROR better categorized women with node negative disease into respective risk groups. Risk stratification and 10-year DR risk (%) according to ROR and RS cut-off points.ROR cut-offsLow (N=365)Intermediate/High(N=298)Original RS cut-offsNumber10-year risk (%)Number10-year risk (%)Low (N=423)2791.5%14413.4%Intermediate/High (N=240)866.3%15424.8%TailoRx cut-offsLow (N=166)1042.1%6218.2%Intermediate/High (N=497)2612.9%23619.5% Citation Format: Ivana Sestak, Sean Ferree, Itay Shemesh, Wesley Buckingham, Jack Cuzick, Mitchell Dowsett. Discordant classification and outcomes between Prosigna and Oncotype Dx Recurrence Score for ER-positive, HER2-negative, node-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-05.

Purpose The PAM50-based Prosigna risk of recurrence (ROR) score has been validated in randomized clinical trials to predict 10-year distant recurrence (DR). The value of Prosigna for predicting DR was examined in a comprehensive nationwide Danish cohort consisting of postmenopausal women with hormone receptor–positive early breast cancer treated with 5 years of endocrine therapy alone. Patients and Methods Using the population-based Danish Breast Cancer Cooperative Group database, follow-up data were collected on all patients diagnosed from 2000 through 2003 who, by nationwide guidelines, were treated with endocrine therapy for 5 years. Primary tumor blocks from 2,740 patients were tested with Prosigna and, after determination of human epidermal growth factor receptor 2 (HER2) status, data from 2,558 hormone receptor–positive/HER2-negative samples were analyzed, including 1,395 node-positive patients. Fine and Gray models were applied to determine the prognostic value of ROR for DR. Results Median follow-up for recurrence was 9.2 years. Twenty-six percent of the node-positive patients were classified as low ROR (n = 359) with a DR risk of 3.5% (95% confidence interval [CI], 1.9% to 6.1%) versus a DR risk of 22.1% (95% CI, 18.6% to 25.8%) at 10 years for patients classified as high ROR (n = 648). Node-negative patients classified as low and high ROR had a risk of DR of 5.0% (95% CI, 2.9% to 8.0%) and 17.8% (95% CI, 14.0% to 22.0%), respectively. Luminal B tumors (n = 947; DR risk, 18.4% [95% CI: 15.7% to 21.3%]) had a significantly worse outcome than luminal A tumors (n = 1,474,;DR risk, 7.6% [95% CI: 6.1% to 9.2%]; P < .001). Conclusion Prosigna ROR score improved the prediction of outcome in this nationwide Danish population. In a real-world setting, Prosigna can reliably identify node-negative patients and a significant proportion of patients with one to three positive nodes who can be spared treatment with adjuvant chemotherapy.

Background: Prosigna's ROR score was demonstrated as a strong predictor of response to NAC in a representative cohort of EBC patients including HR+/HER2- N0-N1 patients.1 Given that the ROR score is partially derived from the correlation of the tumor's expression profile to that of the four prototypical intrinsic subtypes, we determined the relative strength of the association between each subtype correlation and the likelihood of response to NAC. Methods: We analyzed 294 FFPE breast cancer samples from pts treated with NAC (anthracyclines and taxanes) in a multi-center Spanish cohort. The Prosigna Assay was performed on the NanoString nCounter® Dx Analysis System at HU Virgen de la Victoria de Málaga/CIMES-UMA. Pathologic complete response (pCR) was used as the primary endpoint for this study and was determined using the Miller and Payne scoring criteria. Results: Mean patient age in this population was 50 (±11yr). Apart from targeted therapy, all patients received a standard neoadjuvant treatment regimen consisting of 8-12 cycles of anthracyclines and taxanes. 58% of patients were HR+/HER2- while 24% were classified as HER2+ and 18% were TNBC patients. Of the 311 pts samples previously tested, subtype correlation data was available for 294. Overall subtype concordance between IHC and Prosigna was 72% (K=0.66). The overall pCR rate in this population was 24.9%. Prosigna subtype breakdown in the full study population was 60 Luminal A, 118 Luminal B, 69 HER2-enriched and 47 Basal-like with response rates of 7.2%, 7.2%, 46.2% and 57.4%, respectively. We found that in all study populations, subtype correlation was a strong predictor of response to NAC. Tumors with expression profiles that correlated well with the Luminal prototypical centroids were found to be largely unresponsive to NAC (Luminal A Odds ratio=0.074 per unit increase, p 1Rodriguez B, Lavado Fernandez A, Ribelles N, et al. Prosigna (PAM50) to predict response to neoadjuvant chemotherapy (NAC) in HR+/HER2- early breast cancer (EBC) patients. J Clin Oncol 33, 2015 (suppl; abstr 11049). Citation Format: Chica Parrado R, Jiménez-Rodríguez B, Sánchez Rovira P, Álvarez M, Vicioso L, Fernandez AI, de Luque V, José Lozano M, Villar E, Zarcos I, Ramírez C, González-Hermoso C, Jeiranian A, Dowidar N, Schaper C, Buckingham W, Ferree S, Ribelles N, Rodrigo I, Prat AP, Alba E. Prosigna® subtype correlation is a strong predictor of response to neoadjuvant chemotherapy (NAC) in early breast cancer (EBC) study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-15.

Background: HR+EBC patients are routinely treated with both adjuvant radiation therapy (RT) and ET. RT is considered an important tool for achieving local control of disease. A limited number of biomarkers have been demonstrated to predict LR. In a previously performed retrospective analysis of a randomized trial, Prosigna (PAM50) risk of recurrence (ROR) score identified low risk patients with a local recurrence rate of 1.6% at 9.5yr median follow-up. In this study, we seek to validate the ability of ROR to predict LR in a comprehensive nationwide cohort from Denmark. Methods: Using the population based DBCG database primary FFPE tumor blocks and follow-up data were collected from all postmenopausal Danish women diagnosed from 2000-2003 with HR+EBC (N=2,722). Prosigna (PAM50) on the NanoString nCounter® Dx Analysis System assigned each patient an ROR score and associated risk group based on pre-specified cutoffs. Patients are also assigned an intrinsic subtype (Luminal A, Luminal B, Her2-Enriched, Basal-Like) based on gene expression. Univariate and multivariate analyses were performed to assess the ability of Prosigna (PAM50) to predict LR. Results: 48 local recurrences were observed with median follow-up of 9.25 yr. Continuous ROR was significantly associated with LR in univariate and multivariate models including node status (0, 1, 2, or 3 positive nodes), tumor size (≤2 vs.>2cm), grade (I, II, III, or unknown), age (≤65 vs.>65yr), and local treatment (mastectomy (MX), lumpectomy+RT, or MX+RT) (p=0.036 and p=0.049, respectively). Clinicopathologic variables were not significant in the multivariate model alone or in combination (p=0.85 for full model excluding ROR). Utilization of a pre-specified LR cutoff, hazard ratio (HR) and [95%CI, p-value] for high risk vs. low risk patients in a univariate analysis was 1.96 [1.11-3.46, p=0.0205] and 2.04 [1.08-3.83, p=0.0275] in the multivariate analysis. 10-year cumulative incidence of LR for low risk patients was 1.7% [1.1%-2.6%]. Similarly, 10-year cumulative incidence of LR for Luminal A patients was 1.7% [1.1%-2.6%]. 10-year cumulative incidence for high risk patients was 2.3% [1.3%-3.2%]. Conclusions: In a large population-based study of n=2,722 patients, Prosigna (PAM50) predicted LR over standard variables. These data validate a pre-specified cutoff separating patients at high and low risk of LR. Additional studies of Prosigna (PAM50) in RT-untreated populations are ongoing. Citation Format: Ole Eriksen J, Jensen M-B, Laenkholm A-V, Kibøll T, Bruun Rasmussen B, Knoop AS, Ferree S, Haffner T, Buckingham W, Schaper C, Ejlertsen B. Validation of prediction of local recurrence (LR) by Prosigna® (PAM50) in a Danish breast cancer cooperative group (DBCG) cohort of hormone receptor positive (HR+), postmenopausal early breast cancer (EBC) patients allocated to 5yr of endocrine therapy (ET). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-10.

Background: The role of the HER2-enriched (HER2E) subtype determined by the Prosigna Assay in the neoadjuvant setting has remained largely uncharacterized. In this study, we examine whether Prosigna can identify a subgroup of HER2+ patients for whom combination neoadjuvant therapy that includes trastuzumab (Herceptin) is associated with a greater likelihood of pathological complete response (pCR). Methods: In this single-arm retrospective analysis, 75 patients determined to be HER2+ by IHC were treated with a neoadjuvant regimen (NAC) consisting of 8-12 cycles of anthracyclines and taxanes as well as Herceptin. The Prosigna Assay was performed on the NanoString nCounter® Dx Analysis System at HU Virgende la Victoria de Málaga/CIMES-UMA. pCR was used as the endpoint for this study and was determined using the Miller & Payne scoring criteria. Results: Mean patient age for this study population was 49 (±11.1yr) and all patients were determined to be HER2+ by IHC. The overall pCR rate in this patient population was 46.2%. Of the 75 patient samples analyzed for this study, 59 (78.6%) were HER2E, 4 (5.3%) were Luminal A and 12 (16.1%) were Luminal B, as identified by the Prosigna Assay. Of the 16 tumors classified as Luminal (A or B) by Prosigna within this HER2+ population, only 2 (12.5%) responders were observed. Categorical analysis revealed that Prosigna subtype predicted response to a NAC regimen combined with Herceptin (Odds ratio [Her2E vs. non-Her2E]=6.4, p=0.023). Further analysis of the Her2E subtype revealed that tumors with profile expression that correlated well with the prototypical Her2E centroid were significantly more likely to respond to combination NAC and Herceptin (Odds ratio [Unit increase of 1 in Her2E correlation]=88.2, p=0.004). Conclusions: The results of this study indicate that HER2+ patients with greater correlations to the HER2E subtype have an increased likelihood of response to combination neoadjuvant regimens that included HER2-targeted therapy. Citation Format: Santonja Á, Ribelles N, Jiménez-Rodríguez B, Sánchez Rovira P, Álvarez M, Vicioso L, Isabel Fernandez A, de Luque V, Fernández de Sousa C, Villar E, Zarcos I, Ramírez C, González-Hermoso C, Jeiranian A, Dowidar N, Schaper C, Buckingham W, Ferree S, Jiménez A, Prat A, Alba E. Prosigna® intrinsic subtyping predicts response to neoadjuvant combination therapy in study that includes herceptin within HER2+ (IHC) patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-14.

Purpose: Most hormone receptor (HR)+/HER2− breast cancer patients respond unfavorably to neoadjuvant chemotherapy (NAC); however, genomic tests may identify those patients who are likely to benefit. Using the Prosigna assay, we first evaluated the technical performance of core needle biopsy (CNB) tissues. We then determined whether Prosigna risk of relapse (ROR) score and intrinsic subtype predicted response to NAC in HR+/HER2− patients using CNB samples. Experimental Design: Using the NanoString's nCounter Dx analysis system and a development tissue sample set, we established tissue requirements and assay output variance. We then evaluated the concordance in subtype and correlation in ROR between CNBs and corresponding surgical resection specimens (SRS) in a second independent sample set. Finally, we analyzed 180 independent CNB samples from HR+/HER2− patients who were treated with NAC and correlated ROR and intrinsic subtype with pathologic response. Results: Intra- and interbiopsy variabilities were 2.2 and 6.8 ROR units, respectively. Subtype concordance within multiple CNBs was high for the 4- and 3-subtype classifications (k = 0.885 and 0.889, respectively). Correlation in Prosigna ROR score observed between paired CNBs and SRS was high (r ≥ 0.90), and subtype concordance was also high for the 4- and 3-subtype classifications (kappa = 0.81 and 0.91, respectively). Prosigna results obtained from the HR+/HER2− patient samples showed that both ROR (P = 0.047) and intrinsic subtype (OR LumA vs. non-LumA = 0.341, P = 0.037) were significant predictors of response to NAC. Conclusions: Prosigna ROR and intrinsic subtype are readily obtained from CNB samples in normal practice and reliably predict response to NAC in HR+/HER2− patients. Clin Cancer Res; 22(3); 560–6. ©2015 AACR.

e24243Background: The heterogeneity of breast cancer is driven by many factors, including processes driven by tumor, stroma, and immune cell populations. Using PAM50 and Tumor Inflammation Signatur...

Background: Enzalutamide is an orally administered androgen receptor (AR) inhibitor approved by the FDA for use in men with metastatic castrate-resistant prostate cancer. A recent phase II study of enzalutamide in patients with advanced, AR positive, TNBC (NCT01889238) demonstrated significant improvements in both PFS and OS for patients whose tumors exhibited a gene expression (Gx) profile enriched in AR signaling and luminal biology. A PAM50-based signature was developed from the phase 2 study which used next generation RNA sequencing (NGS) to identify patients likely to respond to enzalutamide. We transitioned the test to the NanoString (NS) nCounter® Analysis System using Prosigna reagents to support clinical validation in a phase 3 trial. Here we describe the development and analytical performance of the NanoString Androgen Gene Expression Profiling Assay-1 (NS-AR-01). Methods: The NS-AR-01 algorithm coefficients were calibrated from the Predict AR algorithm by testing FFPE tumor tissue from patients who were pre-screened but not enrolled in the phase II study with both platforms (NGS and NS). Three unique algorithms were developed and subsequently challenged with an independent sample set with NGS data to provide an unbiased evaluation of the concordance of the platforms. A pre-specified clinical accuracy verification study was performed through prediction of NS-AR-01 scores from the NGS Gx data from the patients included in the phase 2 study efficacy analysis. The final NS-AR-01 algorithm was selected based on performance in the clinical accuracy verification. The final NS-AR-01 algorithm was evaluated in the 118 patients included in the ITT analysis, as well as those treated with 0-1 lines of prior therapy. The analytical performance of the assay was characterized by testing precision from RNA, reproducibility from FFPE tissue, sensitivity to RNA input amounts, and the impact of common interferents. Results: All three algorithm translations met the pre-specified clinical accuracy verification acceptance criteria. The final NS-AR-01 algorithm generated a hazard ratio most similar to that observed from the NGS algorithm. The total standard deviation when testing multiple FFPE sections from the same block was < 1.5% of the score range with an empirical concordance rate of 100% for biomarker status. The range of RNA input specified for Prosigna was successfully verified for NS-AR-01 (125ng–500ng total RNA). The assay was demonstrated to be robust to common interferents including non-tumor tissue. Conclusions: Based on these results, NS-AR-01 is an accurate, precise, and robust assay for the identification of advanced TNBC patients who may respond to treatment with enzalutamide. The assay is well suited to clinical applications, and its ability to identify responders to enzalutamide will be evaluated in future investigational studies.Background: Enzalutamide is an orally administered androgen receptor (AR) inhibitor approved by the FDA for use in men with metastatic castrate-resistant prostate cancer. A recent phase II study of enzalutamide in patients with advanced, AR positive, TNBC (NCT01889238) demonstrated significant improvements in both PFS and OS for patients whose tumors exhibited a gene expression (Gx) profile enriched in AR signaling and luminal biology. A PAM50-based signature was developed from the phase 2 study which used next generation RNA sequencing (NGS) to identify patients likely to respond to enzalutamide. We transitioned the test to the NanoString (NS) nCounter® Analysis System using Prosigna reagents to support clinical validation in a phase 3 trial. Here we describe the development and analytical performance of the NanoString Androgen Gene Expression Profiling Assay-1 (NS-AR-01). Methods: The NS-AR-01 algorithm coefficients were calibrated from the Predict AR algorithm by testing FFPE tumor tissue from patients who were pre-screened but not enrolled in the phase II study with both platforms (NGS and NS). Three unique algorithms were developed and subsequently challenged with an independent sample set with NGS data to provide an unbiased evaluation of the concordance of the platforms. A pre-specified clinical accuracy verification study was performed through prediction of NS-AR-01 scores from the NGS Gx data from the patients included in the phase 2 study efficacy analysis. The final NS-AR-01 algorithm was selected based on performance in the clinical accuracy verification. The final NS-AR-01 algorithm was evaluated in the 118 patients included in the ITT analysis, as well as those treated with 0-1 lines of prior therapy. The analytical performance of the assay was characterized by testing precision from RNA, reproducibility from FFPE tissue, sensitivity to RNA input amounts, and the impact of common interferents. Results: All three algorithm translations met the pre-specified clinical accuracy verification acceptance criteria. The final NS-AR-01 algorithm generated a hazard ratio most similar to that observed from the NGS algorithm. The total standard deviation when testing multiple FFPE sections from the same block was < 1.5% of the score range with an empirical concordance rate of 100% for biomarker status. The range of RNA input specified for Prosigna was successfully verified for NS-AR-01 (125ng–500ng total RNA). The assay was demonstrated to be robust to common interferents including non-tumor tissue. Conclusions: Based on these results, NS-AR-01 is an accurate, precise, and robust assay for the identification of advanced TNBC patients who may respond to treatment with enzalutamide. The assay is well suited to clinical applications, and its ability to identify responders to enzalutamide will be evaluated in future investigational studies. Citation Format: Danaher P, Skewis L, Mashadi-Hossein A, Ram N, Gowen-MacDonald J, Harris E, Ferree S, Buckingham W. Development of a Prosigna® (PAM50)-based classifier for the selection of advanced triple negative breast cancer (TNBC) patients for treatment with enzalutamide [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-01.

Staphylococcus aureus (SA) is one of the most important human pathogens, causing both nosocomial and community-acquired infections (1)(2). The occurrence of methicillin-resistant SA (MRSA) has increased steadily worldwide and now accounts for a substantial portion of all staphylococcal infections in US hospitals (3). To develop preventive measures, a rapid screening method, along with accurate and timely identification of MRSA, is essential. The existing techniques for doing so are either time-intensive (culturing of bacteria on selective media), relatively insensitive (use of latex agglutination), or expensive and easily susceptible to operator error (such as PCR). We describe a method designed for clinical laboratories using oligonucleotides conjugated to gold nanoparticles. We avoid radioactivity, fluorescence, or target amplification (such as PCR), and use a simple and rapid hybridization-based approach in a microarray format, with ClearRead™ technology to detect specific genomic sequences (4). The ClearRead procedure involves a two-step process: the first involves the hybridization of target to oligonucleotides conjugated to gold nanoparticles as well as oligonucleotides attached to a solid matrix; the second step involves the catalytic deposition of silver on the gold nanoparticle, providing a sixfold amplification of signal (Fig. 1A⇓ ) (4)(5). The differentiation at isothermal hybridization temperatures is a result of the sharp melting transitions characteristic of nanoparticle probes (5). Previous methods based on this property (6)(7) have required the use of PCR and have not directly assayed for genomic DNA. Our assay, on the other hand, requires minimal amounts of genomic DNA (∼500 ng, or ∼108 DNA molecules) and has been used to reliably identify MRSA from liquid cultures, based on the detection of the mecA and tuf genes. Resistance to methicillin is mediated by the presence of penicillin-binding protein 2a, encoded by the mecA gene (8)(9). We are …

Recent advances in molecular profiling of breast cancer have given clinicians the tools required to make better treatment decisions for patients. Building an accurate representation of the biology of a particular tumor is key for: patient selection, therapeutic monitoring, and rational combination therapy design. The NanoString PanCancer Pathways, PanCancer Immune Profiling and PanCancer Progression Panels enable researchers to quickly analyze the expression of up to 770 genes (per panel) and construct a comprehensive view of the biology of a particular tumor. The PanCancer Pathways Panel groups genes into 13 canonical driver pathways and provides both an expression value for each gene based on digital counts of transcripts and a Pathway Score that describes the relative dysregulation of each pathway. The Immune Profiling Panel measures the expression level of target genes that are specific to immune cell types and immune cell functions. Differential expression of each gene, relative abundance of immune cell types and abundance of tumor-specific antigens can be analyzed with the Immune Profiling panel. The Progression Panel analyzes the expression level of genes within four major biological processes that are associated with tumor growth and invasiveness. Together, these panels allow holistic characterization of the biologically meaningful attributes of a tumor. In this proof-of-concept study, we analyzed 59 FFPE primary breast tumor samples along with 10 normal breast tissues using the PanCancer panels as well as the Prosigna Gene Signature Assay. We grouped the tumor samples by intrinsic subtype and explored pathway dysregulation using the PanCancer Pathways Panel, the immune landscape using the Immune Profiling Panel and the metastatic potential of the tumor using the Progression panel. For data analysis purposes, we used NanoString's PanCancer Advanced Analysis software. In each panel's data we compared the Prosigna subtypes at the single gene and the pathway level. We measured differential expression of various genes across subtypes as well as overall changes in pathway activation and suppression. Using the Immune Profiling Panel, we further compared relative abundance of the various immune cells across subtype. The distribution of intrinsic subtype, as determined by the Prosigna Assay, in the 59 breast tumors was as follows: 16 (27.1%) Luminal A; 20 (33.9%) Luminal B; 13 (22.0%) Her2 Enriched; and 10 (17.0%) BasalLike. PanCancer Pathways analysis of these tumor samples along with 10 normal breast tissues revealed that dysregulation of certain canonical pathways characterizes each intrinsic subtype. In BasalLike tumors, we found that genes involved in the TGF-b pathway are significantly downregulated relative to Luminal A tumors and normal breast tissues. Further analysis with the Immune Profiling Panel revealed that the relative abundance of Mast cells is reduced while that of type 2 Th (Th2) cells is increased in Basal Like tumors relative to Luminal A and normal breast tissue. These results suggest that pathways associated with angiogenesis are downregulated in Basal Like breast cancer and favor the recruitment of immune cells associated with hypoxic conditions. These results confirm findings from multiple previous studies. In this study, we show that the NanoString PanCancer Pathways, Immune Profiling and Progression panels reveal associations between intrinsic breast cancer subtype and specific pathway dysregulation as well as related changes in the immune landscape of the tumor. We demonstrate that a comprehensive view of the biology of a tumor can be readily obtained with the NanoString platform and the PanCancer Panels. Citation Format: Lucas Dennis, Patrick Danaher, Maribeth Eagan, Andrew White, Nathan Elliot, Namratha Ram, Gayathri Balasundaram, Arthur Jeiranian, Seely Kaufmann, Rich Boykin, Lindy Irving, Wesley Buckingham, Sean Ferree, Christina Bailey, Joseph Beechem. Building a comprehensive view of tumor biology in breast cancer by combining NanoString's Prosigna assay with the Pancancer Pathways, Immune Profiling, and Progression Panels. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A49.

The molecular probes and associated instrumentation necessary to perform genetic analyses are typically expensive, complex, and prone to error. While techniques such as real-time polymerase chain reaction (PCR) and gene expression analysis have provided a wealth of information previously unattainable, their utility in clinical diagnostics has yet to be realized due to the aforementioned limitations. Nanosphere Inc. has developed a gold nanoparticle-based platform for sequence specific DNA detection that is well-suited for clinical diagnostics due to its cost-effectiveness, simplicity, and accuracy. Thirteen nanometer gold nanoparticle probes, stabilized by a shell of oligonucleotides using proprietary attachment chemistries, enable highly sensitive and specific detection of bacterial genomic DNA sequences without synthetic amplification techniques on a glass array. After silver staining, light scattered by the nanoparticle probes is collected with robust, cost-effective instrumentation. It is the unique features of Nanosphere's detection methodology that provide the necessary steps forward to allow for genetic analyses to become part of routine clinical diagnostics.

569 Background: Pathological complete response following NAC is associated with improved survival. CESP is a novel algorithm derived from the GEICAM 2006-03 clinical trial which is based on the ass...

11049 Background: Prosigna has been clinically validated in 2 large randomized studies to predict the risk of distant and late recurrence in HR+/HER2- patients treated with endocrine therapy.NAC ef...

Background: The PROSIGNA® (PAM50) gene assay has been validated on formalin-fixed paraffin embedded (FFPE) surgical resection specimens (SRS) to identify the intrinsic subtypes of breast cancer and to estimate the 10-year risk of recurrence (ROR) in post-menopausal patients treated with adjuvant endocrine therapy. However, demonstration of the ability to perform PAM50 assay in diagnostic core biopsy specimens (CBS) before primary surgery and/or systemic therapy could be clinically useful. The objectives of this study were to 1) evaluate the feasibility of performing the PAM50 assay in CBS and 2) compare the PAM50 results from paired CBS and SRS. Methods and materials: Baseline tissue surface area, cellularity and RNA yield (obtained after ∼10 FFPE 10μm sections) were determined in CBS from 30 newly diagnosed breast cancer patients. The tissue volume requirements determined from these samples reflected the lower 95% confidence limits of a minimum RNA concentration of >20ng/µL. The RNA yield and assay pass rate of the established tissue volume requirements were then tested in 30 independent CBSs. Intrinsic subtype concordance, and ROR score variability, were determined from 1) multiple extractions of the same CBS (10 independent cases for a total of 84 extractions) and 2) multiple CBS of the same tumor (30 independent cases for a total of 79 CBS). To test the PAM50 assay concordance between paired CBS and SRS, the following PAM50 data were evaluated in an independent and retrospective set of 33 paired samples: 4-class subtype classification (Luminal A, Luminal B, HER2-enriched and Basal-like), 3-class subtype classification (Luminal A/B, HER2-enriched and Basal-like), ROR score (0-100), proliferation score and the correlation to each subtype centroid. Correlation and concordance between CBS and SRS were estimated using Pearson coefficients and multi-rater kappa values, respectively. Results: Baseline median surface area, cellularity and RNA yield concentration were 10.2 mm2, 45% and 155.3 ng/µl, respectively. Correlation of surface area and cellularity with RNA yield concentration was low (Person coefficient 12 mm2 = 2 10-micron slides, 6-12 mm2 = 4 slides; 95%. Conclusions: The PAM50 assay in CBS is feasible and measurements are comparable with surgical resections, which suggest that PAM50 can be performed on diagnostic core biopsy tissues. Citation Format: Aleix Prat, Patricia Galván, Wesley Buckingham, Maria Vidal, Sherley Díaz, Paolo Nuciforo, Sean Ferree, Barbara Adamo, Santiago Ramon y Cajal, Vicente Peg. Feasibility of the PROSIGNA® multigene test in core biopsies and comparison to corresponding surgical breast cancer sections [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-06.

The development of a nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic applications is described. The technology utilizes gold nanoparticles derivatized with thiol modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized oligonucleotide capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. Amplification with silver allows for detection and quantitation by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, silver amplified gold nanoparticle probes provide for a approximately 1000-fold increase in sensitivity. Furthermore, direct detection of non-amplified genomic DNA from infectious agents is afforded through increased specificity and even identification of single nucleotide polymorphisms (SNP) in human genomic DNA appears feasible.