Background The aim of this study was to investigate whether the PAM-50-based 46-gene assay carries prognostic value for risk of local recurrence of breast cancer. Methods The Austrian Breast and Colorectal Cancer Study Group (ABCSG) 8 RCT compared 5 years of tamoxifen with tamoxifen for 2 years followed by anastrozole for 3 years in postmenopausal women with endocrine receptor-positive breast cancer. This study included patients from the trial who had breast-conserving surgery for whom tumour blocks were available for PAM-50 analysis. Results Tumour blocks from 1204 patients who had breast-conserving surgery were available for the PAM-50 analysis, and 1034 of these received radiotherapy. After a median follow-up of 10.8 years, 23 local events had been observed, corresponding to an overall local recurrence risk of 2.2 per cent. Univariable competing-risk analysis demonstrated that patients at low risk according to PAM-50 analysis (risk-of-recurrence (ROR) score less than 57) had a significantly lower incidence of local recurrence than those in the high-risk group at 5 years (0.1 (95 per cent c.i. 0 to 0.7) versus 2.2 (0.9 to 4.6) per cent respectively; subhazard ratio (SHR) 17.18, 95 per cent c.i. 2.06 to 142.88; P = 0.009) and 10 years (0.9 (0.4 to 2.0) versus 3.8 (1.9 to 6.6) per cent; SHR 4.76, 1.72 to 13.17; P = 0.003). Multivariable analyses that included ROR score, age, tumour size, nodal status, type of surgery, tumor grade, and trial-specific endocrine therapy confirmed that ROR score was an independent prognostic factor for risk of local recurrence. Analysis of the women randomized to radiotherapy or control after breast conservation showed that PAM-50 was not predictive of radiotherapy effect. Conclusion PAM-50 can be used as a prognostic tool for local recurrence risk in postmenopausal women with hormone receptor-positive breast cancer treated with endocrine therapy. The test was not predictive for the benefit of radiotherapy.

Background: The Tumor Inflammation Signature (TIS) is an investigational use RNA expression assay on the NanoString nCounter Dx Analysis System, which provides a measure of tumor inflammation across multiple solid tumor types. TIS measures immune genes in tumors from multiple origins, and it is possible that inclusion of tissue-specific interferents, such as non-tumor lymphoid aggregates (NTLA) could influence TIS performance. Here we describe the validation of the reproducibility of the TIS assay starting from FFPE tissues and robustness of the TIS across 8 potential tissue interferents. Methods: TIS includes both review of an H&E tumor slide by a Pathologist and sample processing of unstained slides by an assay user. Analytical validation of the reproducibility of the TIS assay from 3 different Pathologists and 3 different assay users was performed using at least 10 patient specimens for 11 different tumor types (>110 independent tumors tested) from excisional and core biopsies. The robustness of the TIS assay to potential tissue interferents (genomic DNA, adjacent non-tumor tissue, NTLA, mucin, hemorrhagic, necrotic, and fibrotic tissue) was also assessed. Results: The assay was validated to be reproducible with ≥ 95% concordance in assay results between independent pathologists. The total standard deviation of the TIS score was less than 2% of the score range from tissue including different pathology review and users. The interference studies demonstrated that the presence of mucin, necrotic, hemorrhagic and fibrotic tissue did not influence TIS results. However, if not properly removed, contamination with large concentrations of genomic DNA, non-tumor tissue, and NTLA can reduce biomarker concordance by increasing the TIS score. Conclusions: The analytical performance of the NanoString TIS assay has been validated to be reproducible between users and pathologists when potential interferents are removed as instructed in the assay procedures. TIS is well suited for decentralized clinical testing and use as a potential biomarker to enrich for patients based on their inflamed phenotype across multiple tumors. Legal entity responsible for the study: NanoString Technologies, Inc. Funding: NanoString Technologies, Inc. Disclosure: B. Wallden, S. Church, S. Zimmerman, S. Popa, A. Sullivan, C. Ngouenet, E. Harris, N. Dowidar, A. Bergdahl, S. Ferree: Employee, Stockholder: NanoString Technologies, Inc. I. Pekker, P. Danaher: Employee, Stockholder at the time of the study: NanoString Technologies, Inc. C. Schaper: Paid consultant: NanoString Technologies, Inc.

Abstract 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: 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. u003e2cm), grade (I, II, III, or unknown), age (≤65 vs. u003e65yr), 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, Kiboll 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.

Abstract 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<0.0001; Luminal B Odds ratio=0.059 per unit increase, p<0.0001). Conversely, higher HER2E and Basal-like correlations were associated with increased probability of response (HER2E Odds ratio=11.2 per unit increase, p<0.0001; Basal-like Odds ratio=9.0 per unit increase, p<0.0001). Increased proliferation-score (p-score) was also associated with increased probability of response to NAC (Odds ratio=3.43 per unit increase, p<0.0001). Conclusions: In a representative cohort of breast cancer patients, both the magnitude of the subtype correlation to the prototypical centroids as well as p-score, as determined by the Prosigna Assay, were strong predictors of response to NAC. This data underlines the importance of molecular testing for optimal systemic therapy indications in EBC. 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.

Abstract 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.

Abstract 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: In the adjuvant treatment of hormone receptor-positive (HR+) breast cancer, variables like tumour size, grade and nodal status have great impact on therapy decisions. As most node-positive patients with HR+ breast cancer currently receive adjuvant chemotherapy improved methods for characterization of individuals' metastasis risk are needed to reduce overtreatment.Patients and methods: Tissue specimens from node-positive patients of the ABCSG-8 and ATAC trials who received adjuvant tamoxifen and/or anastrozole were included in this study. Analysing RNA from paraffin blocks using the PAM50 test, the primary objective was to evaluate the prognostic information of the risk of recurrence (ROR) score added to combined clinical standard variables in patients with one positive node (1N+) and in patients with two or three positive nodes (2-3N+), using log-likelihood ratio tests.Results: At a median follow-up of 9.6 years, distant metastases occurred in 97 (18%) of 543 node-positive patients. In a multivariate analysis, the PAM50-derived ROR score provided reliable prognostic information in addition to and beyond established clinical factors for 1N+ (P < 0.0001) and 2-3N+ patients (P = 0.0002). Ten-year distant recurrence risk was significantly increased in the high-risk compared with the low-risk group derived from ROR score for 1N+ [25.5%, 95% confidence interval (CI) 17.5% to 36.1% versus 6.6%, 95% CI 3.3% to 12.8%] and compared with the combined low/intermediate risk group for 2-3N+ patients (33.7%, 95% CI 25.5% to 43.8% versus 12.5%, 95% CI 6.6% to 22.8%). Additionally, the luminal A intrinsic subtype (IS) exhibited significantly lower risk of distant recurrence compared with the luminal B subtype in 1N+ and 2-3N+ patients.Conclusion: PAM50 ROR score and IS can identify node-positive patient subgroups with limited risk of metastasis after endocrine therapy, for whom adjuvant chemotherapy can be spared. The PAM50 test is a valuable tool in determining treatment of node-positive early-stage breast cancer patients.

546 Background: The Prosigna (PAM50) risk of recurrence (ROR) score has been validated in two randomized clinical trials to predict 10yr DR in EBC patients treated with ET alone. Here we examine the value of PAM50 for predicting risk of DR in a comprehensive nationwide cohort from Denmark consisting of all postmenopausal women diagnosed with HR+ EBC allocated to 5yr of ET alone. Methods: Using the population based DBCG database FFPE primary tumor blocks and follow-up data were collected from all patients diagnosed from 2000-2003 (N = 2749) who by nationwide guidelines were allocated to 5yr of ET alone. PAM50 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, Intermediate, or High risk based upon pre-specified ROR cutoffs varied by number of positive nodes. Results: Blocks from 2749 patients were identified and data from 2722 samples (1256 N0, 1466 N1) were included in the analysis (99%). Median follow-up was 9.25yr. High risk patients (n = 1200) had a DR risk of 20.8% [95%CI: 18.3-23.4] at 10 years, compared to 4.3% [2.9-6.2] for Low risk patients (n = 733). These figures were consistent across nodal status. Adding ROR to a Fine and Gray's proportional sub-hazards model containing clinical and pathological variables significantly improved the model (likelihood ratio: p < 0.0001; HR for a 20-point change in ROR = 1.7 [1.5-1.9]. Luminal B tumors (N = 977, DR risk = 18.0% [15.4-20.9]) and Her2-enriched tumors (N = 203, DR risk = 27.7% [21.5-34.3]) had a significantly worse outcome than Luminal A (N = 1515, DR risk = 7.7% [6.2-9.3]), both p < 0.0001. Conclusions: To our knowledge, this is the first genomic study of breast cancer on a comprehensive nationwide population. Prosigna (PAM50) improved the prediction of outcome over and above standard clinical and pathological variables in this DBCG cohort devoid of physician selection bias. PAM50 can reliably identify patients in a real world setting who may be spared overtreatment with chemotherapy.

BackgroundPAM50 is a 50-gene test that is designed to identify intrinsic breast cancer subtypes and generate a Risk of Recurrence (ROR) score. It has been developed to be carried out in qualified routine hospital pathology laboratories.Patients and MethodsOne thousand four hundred seventy-eight postmenopausal women with estrogen receptor (ER)+ early breast cancer (EBC) treated with tamoxifen or tamoxifen followed by anastrozole from the prospective randomized ABCSG-8 trial were entered into this study. Patients did not receive adjuvant chemotherapy. RNA was extracted from paraffin blocks and analyzed using the PAM50 test. Both intrinsic subtype (luminal A/B, HER2-enriched, basal-like) and ROR score were calculated. The primary analysis was designed to test whether the continuous ROR score adds prognostic value in predicting distant recurrence (DR) over and above standard clinical variables.ResultsIn all tested subgroups, ROR score significantly adds prognostic information to the clinical predictor (P < 0.0001). PAM50 assigns an intrinsic subtype to all cases, and the luminal A cohort had a significantly lower ROR at 10 years compared with Luminal B (P < 0.0001). Significant and clinically relevant discrimination between low- and high-risk groups occurred also within all tested subgroups.Conclusion(s)The results of the primary analysis, in combination with recently published results from the ATAC trial, constitute Level 1 evidence for clinical validity of the PAM50 test for predicting the risk of DR in postmenopausal women with ER+ EBC. A 10-year metastasis risk of <3.5% in the ROR low category makes it unlikely that additional chemotherapy would improve this outcome—this finding could help to avoid unwarranted overtreatment.Clinical trial numberABCSG 8: NCT00291759.

Abstract Background: Extended adjuvant endocrine therapy beyond 5 years is known to be of benefit to some oestrogen receptor (ER+) patients. Given the significant burden extended endocrine therapy presents, individual prediction of late recurrence risk would be highly valuable. We have previously shown that the PAM50 based Risk of Recurrence (ROR) score is significantly correlated with late recurrence in both the transATAC and ABCSG8 cohorts. Here, we assess the value of the ROR score for predicting distant recurrence beyond 5 years in a combined analysis of these two cohorts. Methods: Long-term follow-up data and tissue samples were obtained from 2,485 postmenopausal women with hormone receptor positive (HR+) early breast cancer from the ABCSG-8 and transATAC trials. We used univariate and multivariate models to determine the prognostic value of ROR (with tumour size) for distant recurrence in years 5-10 in the combined dataset. Changes in likelihood ratio tests (DLR-χ2) are presented. Results: A total of 2137 women who did not have a recurrence in years 0-5 were included in the combined analyses of late recurrence risk. In the univariate and multivariate analyses, CTS was the strongest prognostic score in the late follow-up period (ΔLR-χ2 = 95.17, ΔLR-χ2 = 61.65, respectively) in all patients. The ROR score also added significant prognostic information in years 5-10 in the univariate and multivariate analyses, but somewhat less than the CTS (ΔLR-χ2 = 66.09, ΔLR-χ2 = 32.57, respectively) (Table 1). The risk of distant recurrence at 10 years for the low risk group was 5.7% (95% CI 4.5% to 7.2%), for intermediate risk 14.6% (12.0% to 17.6%), and for high risk 29.3% (25.5% to 33.6%). Patients with a luminal A subtype had a 70% lower risk of late distant recurrence than those with a luminal B subtype (HR = 0.30 (0.21-0.43), P<0.0001). Conclusions: The results of this combined analyses showed that the ROR score added prognostic information to CTS in all patients and in all patient subgroups in the late follow-up period. The risk of distant recurrence in years 5-10 was statistically significantly different for the low, intermediate risk and high risk groups. These results suggest that the ROR score may be used to separate patients into risk groups who could be spared or could benefit from extended hormonal therapy beyond 5 years of treatment. Hazard Ratio (HR) and changes in likelihood ratio test (DLR-χ2) for univariate and multivariate analyses according to all groups. Univariate Multivariate* HR (95% CI)ΔLR-χ2 (P-value)HR (95% CI)ΔLR-χ2 (P-value)All patients (N = 2137) CTS2.05 (1.80-2.34)95.17 (<0.0001)1.88 (1.63-2.17)61.65 (<0.0001)ROR2.88 (2.22-3.72)66.09 (<0.0001)2.14 (1.65-2.79)32.57 (<0.0001)Node-negative (N = 1580) CTS2.10 (1.57-2.81)22.17 (<0.0001)1.65 (1.19-2.30)8.28 (0.004)ROR2.67 (1.86-3.85)28.50 (<0.0001)2.13 (1.44-3.15)14.61 (0.0001)Node-positive (N = 557) CTS1.94 (1.58-2.37)37.48 (<0.0001)1.82 (1.47-2.25)26.90 (<0.0001)ROR2.72 (1.89-3.92)29.72 (<0.0001)2.28 (1.57-3.30)19.14 (<0.0001)HER2-negative (N = 1974) CTS2.02 (1.76-2.32)79.67 (<0.0001)1.84 (1.58-2.15)51.09 (<0.0001)ROR3.02 (2.29-4.00)62.06 (<0.0001)2.31 (1.73-3.07)33.48 (<0.0001)*Addition of either score to each other Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-04.

Introduction: Despite great improvements in overall outcomes of endocrine-responsive breast cancer, the main challenge in improving long-term cure of these patients remains their inherent risk for late relapse. Particularly in patients with initially favorable molecular characteristics (e.g. luminal A), the annual relapse risk persists well beyond 5 years follow-up. Extending adjuvant therapy may be an option to reduce risk of late metastasis. It would be of great value to differentiate patients at high vs. low risk of late relapse for clinical decision making and patient counseling. The goal of this work is to evaluate the ability of the PAM50 signature to predict late metastasis in a large cohort of endocrine responsive breast cancer. Methods/patients: NanoString has developed a PAM50-based breast cancer gene signature assay that is used to categorize a breast tumor specimen into intrinsic breast cancer subtypes and to provide a risk-of-recurrence (ROR) score. Using the nCounter DX Analysis System, this assay directly measures the mRNA expression levels of 58 different genes in a single hybridization. NanoString's assay has been optimized to be performed with FFPE tissue samples in local hospital pathology laboratories. This ROR score has been clinically validated in several studies, and has been suggested to predict late recurrences in a transATAC study. Out of 3,714 patients of ABCSG-8, 1,671 patients could be re-consented to this study, with 1,620 FFPE blocks available. Out of those, 1,478 (91.2%) passed the PAM50 analysis. Results: PAM50 ROR provided significant additional prognostic information to clinical factors with respect to late distant-relapse-free survival (DRFS) (Chi-Square 15.3, p < 0.0001) and late RFS (Chi-Square 11.4, p = 0.0007) in multivariate models, with probabilities for DRFS after year 5 of 98.7% for low ROR patients vs. 91.5% for high ROR patients. This was true both for node-positive and node negative disease. Discussion: PAM50 ROR can successfully be used to differentiate patients with respect to their risk for late metastasis, in addition to established clinicopathological risk factors. This ability to predict late metastasis may be used in the future to identify patients with endocrine-responsive breast cancer who need or alternatively who can be spared extended adjuvant therapy. Disclosure: S. Ferree: employee of nano string J.W. Cowens: employee of nano string All other authors have declared no conflicts of interest.

Abstract Aim: To assess the performance of the ROR score in predicting DR for postmenopausal patients with ER+ EBC treated with tamoxifen or tamoxifen followed by anastrozole when the PAM50 test is performed in a routine hospital pathology laboratory. Background: The multi-analyte gene-expression tests (Oncotype DX, MammaPrint) that are currently used in clinical practice to assess prognosis after endocrine therapy in primary ER+ breast cancer are performed in a central referral laboratory. PAM50 is a 50-gene test in development that is designed to be performed in local routine hospital pathology laboratories and has been optimized to separate intrinsic disease subtypes which are used to generate a ROR score. This ROR score has been clinically validated and demonstrated to contain more prognostic information than Oncotype Dx RS in the TransATAC patient population (Dowsett, SABCS, 2011). Methods: 1,251 women from the prospective randomized ABCSG-8 trial were entered into this study: the entry criteria were the availability of their FPET block and informed consent. Three unstained 10 micron sections and 1 H&E slide for each patient were sent to an independent academic pathology laboratory at BCCA where tissue review, manual micro-dissection and RNA extraction were performed. PAM50 analysis was then conducted on 250 ng of the extracted RNA using the NanoString nCounter® technology: both intrinsic subtype and ROR score were calculated. The median follow up of the patients entered into this study is 10 years. 912 are node negative and 339 are node positive. The baseline characteristics of the patients entered showed them to be a representative sample of the overall ABCSG-8 trial population. The primary analysis is designed to test whether the continuous ROR score adds prognostic value over and above the standard clinical variables, using a likelihood ratio test. Results: Results are currently being derived and will be presented in full at SABCS. Discussion: This large clinical trial serves both as a clinical validation study of the PAM 50 test and as a demonstration that a complex multi-analyte gene-expression test can be performed in a routine hospital pathology laboratory while meeting the same quality metrics as a central referral laboratory. The results of the primary analysis, when combined with the results already reported from the Trans ATAC population, yield Level 1 evidence for clinical validity of the PAM50 test for predicting the risk of DR in postmenopausal women with ER+ EBC and form the basis of the design of the clinical utility studies of the PAM 50 test. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-02.

Abstract Purpose: To assess the prognostic value of the PAM50 risk-of-recurrence (ROR) score on late distant recurrence (beyond 5 years after diagnosis and treatment) in a large cohort of postmenopausal, endocrine-responsive breast cancer patients. Experimental Design: The PAM50 assay was performed on formalin-fixed paraffin-embedded whole-tumor sections of patients who had been enrolled in the Austrian Breast and Colorectal Cancer Study Group Trial 8 (ABCSG-8). RNA expression levels of the PAM50 genes were determined centrally using the nCounter Dx Analysis System. Late distant recurrence-free survival (DRFS) was analyzed using Cox models adjusted for clinical and pathologic parameters. Results: PAM50 analysis was successfully performed in 1,246 ABCSG-8 patients. PAM50 ROR score and ROR-based risk groups provided significant additional prognostic information with respect to late DRFS compared with a combined score of clinical factors alone (ROR score: ΔLRχ2 15.32, P < 0.001; ROR-based risk groups: ΔLRχ2 14.83, P < 0.001). Between years 5 and 15, we observed an absolute risk of distant recurrence of 2.4% in the low ROR-based risk group, as compared with 17.5% in the high ROR-based risk group. The DRFS differences according to the PAM50 ROR score were observed for both node-positive and node-negative disease. Conclusion: PAM50 ROR score and ROR-based risk groups can differentiate patients with breast cancer with respect to their risk for late distant recurrence beyond what can be achieved with established clinicopathologic risk factors. Clin Cancer Res; 20(5); 1298–305. ©2014 AACR.

Abstract Aim: To assess the performance of the risk of recurrence (ROR) score in predicting RFS and DRFS for postmenopausal patients with primary ER+ breast cancer treated with anastrozole or tamoxifen and compare this with the performance of Oncotype Dx and IHC4 for both N0 and N+ patients. Background: OncotypeDx Recurrence Score (RS) is used to assess RR after endocrine therapy in primary ER+ breast cancer and is valid for both tamoxifen and anastrozole (Dowsett ref). IHC4 is a 4-panel set of IHC markers (ER, PgR, HER2, Ki67) that was shown to provide as much prognostic accuracy as RS in the translational arm of the ATAC trial (TransATAC) of anastrozole versus tamoxifen alone or combined and subsequently independently validated (Cuzick et al, JCO, 2011, in press). PAM50 is 50-gene test that has been optimised to separate intrinsic disease subtypes and is used to generate a ROR score. Good correlation has been shown between the ROR and RS but no large scale comparisons of their ability to predict clinical outcome have been conducted. Methods: PAM50 analysis was conducted using NanoString technology on 125 ng RNA extracted from all 1026 samples from the TransATAC study that had at least 500ng RNA available. Nodal status was known on 986: N+, 271; N-neg 715. ROR scores were calculated and their relationship with RFS and DRFS assessed in both the N-neg and N+ categories. The prognostic value of ROR when added to standard clinical variables was assessed using the likelihood ratio chi-square. Finally, the relative accuracy of prognosis in this patient population using ROR, RS and IHC4 was evaluated by comparing the C-index for each test. Results: Results are currently being derived and will be presented in full. Discussion: This is the first large-scale clinical comparison of 2 prominent multi-gene predictors for use in FFPE material. The results will be instrumental in defining the preferred method for use in assessing residual risk of recurrence in patients treated with endocrine therapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S4-5.

PURPOSE:Risk of distant recurrence (DR) among women with estrogen receptor (ER) -positive early breast cancer is the major determinant of recommendations for or against chemotherapy. It is frequently estimated using the Oncotype DX recurrence score (RS). The PAM50 risk of recurrence (ROR) score provides an alternative approach, which also identifies intrinsic subtypes. PATIENTS AND METHODS:mRNA from 1,017 patients with ER-positive primary breast cancer treated with anastrozole or tamoxifen in the ATAC trial was assessed for ROR using the NanoString nCounter. Likelihood ratio (LR) tests and concordance indices (c indices) were used to assess the prognostic information provided beyond that of a clinical treatment score (CTS) by RS, ROR, or IHC4, an index of DR risk derived from immunohistochemical assessment of ER, progesterone receptor, human epidermal growth factor receptor 2 (HER2), and Ki67. RESULTS:ROR added significant prognostic information beyond CTS in all patients (Δ LR-χ(2) = 33.9; P < .001) and in all four subgroups: node negative, node positive, HER2 negative, and HER2 negative/node negative; more information was added by ROR than by RS. C indices in the HER2-negative/node-negative subgroup were 0.73, 0.76, and 0.78 for CTS, CTS plus RS, and CTS plus ROR, respectively. More patients were scored as high risk and fewer as intermediate risk by ROR than by RS. Relatively similar prognostic information was added by ROR and IHC4 in all patients but more by ROR in the HER2-negative/node-negative group. CONCLUSION:ROR provides more prognostic information in endocrine-treated patients with ER-positive, node-negative disease than RS, with better differentiation of intermediate- and higher-risk groups.