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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.

PDF file - 34K, Supplementary Table S1. ROR cutoff values for risk groups defined by nodal status.

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.

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.

PURPOSE The Onco type DX Recurrence Score (RS), Prosigna Prediction Analysis of Microarray 50 (PAM50) Risk of Recurrence (ROR), EndoPredict (EP), and Breast Cancer Index (BCI) are used clinically for estimating risk of distant recurrence for patients receiving endocrine therapy. Discordances in estimates occur between them. We aimed to identify the molecular features that drive the tests and lead to these differences. PATIENTS AND METHODS Analyses for RS, ROR, EP, and BCI were conducted by the manufacturers in the TransATAC sample collection that consisted of the tamoxifen or anastrozole arms of the ATAC trial. Estrogen receptor–positive/human epidermal growth factor receptor 2 (HER2)–negative cases without chemotherapy treatment were included in which all four tests were available (n = 785). Clinicopathologic features included in some tests were excluded from the comparisons. Estrogen, proliferation, invasion, and HER2 module scores from RS were used to characterize the respective molecular features. Spearman correlation and analysis of variance tests were applied. RESULTS There were moderate to strong correlations among the four molecular scores (ρ = 0.63-0.74) except for RS versus ROR (ρ = 0.32) and RS versus BCI (ρ = 0.35). RS had strong negative correlation with its estrogen module (ρ = −0.79) and moderate positive correlation with its proliferation module (ρ = 0.36). RS’s proliferation module explained 72.5% of ROR’s variance, while the estrogen module explained only 0.6%. Most of EP’s and BCI’s variation was accounted for by the proliferation module (50.0% and 54.3%, respectively) and much less by the estrogen module (20.2% and 2.7%, respectively). CONCLUSION In contrast to common understanding, RSs are determined more strongly by estrogen-related features and only weakly by proliferation markers. However, the EP, BCI, and particularly ROR scores are determined largely by proliferative features. These relationships help to explain the differences in the prognostic performance of the tests.

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.

Background: Oncotype DX Recurrence Score (RS), Prosigna PAM50 ROR score, the EndoPredict score (EP, EPclin) and the Breast Cancer Index (BCI) all provide substantial molecular prognostic information and are used in clinical practice for guiding the treatment for large numbers of patients with primary ER+/HER2- breast cancer. Despite this, there is incomplete information on the concordance between these scores and very little understanding of the molecular features that drive the individual scores. Here we report from a unique database (i) the correlation between all 4 scores when measured by the respective manufacturers in the TransATAC set of samples and (ii) the degree to which each of the scores is driven by proliferation, oestrogen signalling, HER2 signalling and invasive properties insomuch as these are reflected by each component module of the RS test (PM, EM, HM and IM, respectively). Methods: The TransATAC tumour sample set was collected from patients in the tamoxifen or anastrozole arms from the ATAC clinical trial of 5 years of endocrine therapy in postmenopausal patients with primary hormone receptor positive breast cancer. Cases were included if HER2- and all 4 scores were available. None of the patients received adjuvant chemotherapy. EP and ROR both include some clinical features in their final score but components were excluded for the purposes of this study. The RS module scores were provided by the manufacturer. The genes that contribute to the module scores are as follows: PM: Ki67, STK15, Survivin, CCNB1, MYBL2; EM: ER, PGR, BCL2, SCUBE2; HM: GRB7, HER2; IM: MMP11, CTSL2). Spearman rank correlation was used to study the association between molecular components. Results: Data was available for all four molecular scores for 785 TransATAC patients. Correlations were moderate to strong for RS vs EP (r=0.63), ROR vs EP (r=0.68), ROR vs BCI (r=0.74) and EP vs BCI (r=0.67) but were weak for RS vs ROR (r=0.32) and RS vs BCI (r=0.35). The correlation between each score with the RS module scores are shown in the Table. Molecular scoreRS moduleRSROREPBCIPM0.360.860.710.74EM-0.790.00-0.38-0.10HM-0.04-0.14-0.15-0.18IM0.260.380.340.32 The RS had a strong negative correlation with its EM (r= -0.79) and a weak positive correlation with its PM (r= 0.36) although the latter was stronger when the PM was thresholded; 78% of the cases had a PM score below the threshold of 6.5 and were assigned a value of 6.5 in the RS with no correlation between RS and PM in these samples (r=-0.03). ROR showed a very strong correlation with the PM (r=0.86), but none with the EM (r: 0.00). EP and BCI both showed strong correlation with the PM (r= 0.71 and 0.74, respectively) and with low or near absent correlation with EM (r= -0.38 and -0.10, respectively). There was little correlation between the HM with each of the scores while the IM correlation was similar for each score varying from 0.26 to 0.38. Of the two module components of BCI, MGI was strongly associated with the PM (r= 0.84) and not at all with the EM while H/I moderately correlated with PM (r=0.51) and weakly correlated with EM (r= -0.31). Conclusion: EP scores are moderately correlated with each of the other 3 scores while RS scores are dissimilar to those from the ROR and BCI. In contrast to common thinking the RS is driven only weakly by proliferative features and more by those related to ER/PR pathways. The EP, BCI and particularly ROR are dominated by proliferative features. These relationships provide and explanation for the differences that we and others have reported in the prognostic performance of the tests for both early and late recurrence. Citation Format: Richard Buus, Ivana Sestak, Mitch Dowsett, Ralf Kronenwett, Sean Ferree, Catherine Schnabel, Frederick L Baehner, Jack Cuzick. Correlation between and molecular drivers of oncotype DX, prosigna, EndoPredict and breast cancer index: A TransATAC study [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 P3-07-05.

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.

Background: Therapeutic recommendations for adjuvant treatment of hormone-receptor positive breast cancer patients depend on the individual recurrence risk. A number of genomic assays introduced to achieve this goal, but it's still questioned if they actually offer superior risk assessment compared to traditional risk evaluation by experienced clinicians. This study was designed to compare the prognostic accuracy of PAM-50 to clinical judgment. Methods: Based on the real data of a large adjuvant trial cohort (ABCSG-8, postmenopausal HR positive breast cancer patients), we created online-questionnaires including demographic, histological, and local-therapy details, with and without results of PAM50 ROR score. Out of 14 international breast cancer experts asked for individual patient's risk evaluation (low, intermediate, high) and therapy recommendations, 9 completed the questionnaire. Patient data were described by Kaplan-Meier estimates of distant disease free survival (DDFS) stratified by risk group. Cox regression models were compared using the Akaike Information Criterion (AIC). Results: 10 years DDFS and hazard ratios for distant recurrences stratified by risk-group as estimated giving in Table 1: 10 years DDFS and hazard ratios for distant recurrences stratified by risk-group as estimated Low riskIntermediate riskHigh risk n (%)10y DDFS,%(95%CI)n (%)10y DDFS,%(95%CI)n (%)10y DDFS, %(95%CI) HR(95% CI) HR(95% CI) HR(95% CI)Clinical only: AIC 817.6269 (43)93.0(89.8-96.2)289 (46)89.7(85.9-93.5)73 (11)76.6(66.1-87.1) 0.68(0.39,1.20) 1 2.57(1.41,4.65)PAM50 ROR: AIC 804.8241 (34)96.5(93.1-99.1)210 (33)89.2(84.7-93.7)207 (33)82.5(76.9-88.2) 0.27(0.11,0.62) 1 1.66(0.99,2.78)Combined: AIC 813.4232 (37)95.7(93.0-98.5)282 (45)87.8(83.7-92.0)117 (18)81.7(74.2-89.2) 0.42(0.22,0.84) 1 1.90(1.11,3.24) Adding genomic information to clinical risk factors leads to escalation of therapeutic recommendations (i.e. additional chemotherapy, extended adjuvant endocrine) in 20% of patients, and de-escalation in 13% of patients. Conclusions: Clinical judgment accurately identified the patients at high risk of relapse, but was clearly inferior to multi-genomic testing using the PAM-50 ROR score in differentiating low from intermediate risk. Particularly when avoiding unnecessary escalated therapy is the strategic goal, the addition of PAM-50 testing to clinical judgment offers improved accuracy in predicting low vs. intermediate risk of breast cancer recurrence. Citation Format: Devyatko Y, Filipits M, Greil R, Balic M, Bago-Horvath Z, Singer C, Fitzal F, Steger G, Gray B, Ferree S, Fesl C, Soelkner L, von Minckwitz G, Gnant M. The impact of clinical risk assessment versus PAM-50 ROR score on prognosis and therapeutic decision making in patients with hormone-receptor positive early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-17-05.

The OncoAnalyses for RS, ROR, EP, and BCI were conducted by the manufacturers in the TransATAC sample collection that consisted of the tamoxifen or anastrozole arms of the ATAC trial. Estrogen receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative cases without chemotherapy treatment were included in which all four tests were available (n = 785). Clinicopathologic features included in some tests were excluded from the comparisons. Estrogen, proliferation, invasion, and HER2 module scores from RS were used to characterize the respective molecular features. Spearman correlation and analysis of variance tests were applied.There were moderate to strong correlations among the four molecular scores (ρ = 0.63-0.74) except for RS versus ROR (ρ = 0.32) and RS versus BCI (ρ = 0.35). RS had strong negative correlation with its estrogen module (ρ = -0.79) and moderate positive correlation with its proliferation module (ρ = 0.36). RS's proliferation module explained 72.5% of ROR's variance, while the estrogen module explained only 0.6%. Most of EP's and BCI's variation was accounted for by the proliferation module (50.0% and 54.3%, respectively) and much less by the estrogen module (20.2% and 2.7%, respectively).In contrast to common understanding, RSs are determined more strongly by estrogen-related features and only weakly by proliferation markers. However, the EP, BCI, and particularly ROR scores are determined largely by proliferative features. These relationships help to explain the differences in the prognostic performance of the tests.

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.

Biomarkers based on transcriptional profiling can be useful in the measurement of complex and/or dynamic physiological states where other profiling strategies such as genomic or proteomic characterization are not able to adequately measure the biology. One particular advantage of transcriptional biomarkers is the ease with which they can be measured in the clinical setting using robust platforms such as the NanoString nCounter system. The nCounter platform enables digital quantitation of multiplexed RNA from small amounts of blood, formalin-fixed, paraffin-embedded tumors, or other such biological samples that are readily available from patients, and the chapter uses it as the primary example for diagnostic assay development. However, development of diagnostic assays based on RNA biomarkers on any platform requires careful consideration of all aspects of the final clinical assay a priori, as well as design and execution of the development program in a way that will maximize likelihood of future success. This chapter introduces transcriptional biomarkers and provides an overview of the design and development process that will lead to a locked diagnostic assay that is ready for validation of clinical utility.

Biomarkers based on transcriptional profiling can be useful in the measurement of complex and/or dynamic physiological states where other profiling strategies such as genomic or proteomic characterization are not able to adequately measure the biology. One particular advantage of transcriptional biomarkers is the ease with which they can be measured in the clinical setting using robust platforms such as the NanoString nCounter system. The nCounter platform enables digital quantitation of multiplexed RNA from small amounts of blood, formalin-fixed, paraffin-embedded tumors, or other such biological samples that are readily available from patients, and the chapter uses it as the primary example for diagnostic assay development. However, development of diagnostic assays based on RNA biomarkers on any platform requires careful consideration of all aspects of the final clinical assay a priori, as well as design and execution of the development program in a way that will maximize likelihood of future success. This chapter introduces transcriptional biomarkers and provides an overview of the design and development process that will lead to a locked diagnostic assay that is ready for validation of clinical utility.

Clinical benefit from checkpoint inhibitors has been associated in a tumor-agnostic manner with two main tumor traits. The first is tumor antigenicity, which is typically measured by tumor mutation burden, microsatellite instability (MSI), or Mismatch Repair Deficiency using gene sequence platforms and/or immunohistochemistry. The second is the presence of a pre-existing adaptive immune response, typically measured by immunohistochemistry (e.g. single analyte PD-L1 expression) and/or gene expression signatures (e.g. tumor "inflamed" phenotype). These two traits have been shown to provide independent predictive information. Here we investigated the potential of using gene expression to predict tumor MSI, thus enabling the measurement of both tumor antigenicity and the level of tumor inflammation in a single assay, possibly reducing sample requirement, turn-around time, and overall cost.Using The Cancer Genome Atlas RNA-seq datasets with the greatest MSI-H incidence, i.e. those from colon (n = 208), stomach (n = 269), and endometrial (n = 241) cancers, we trained an algorithm to predict tumor MSI from under-expression of the mismatch repair genes MLH1, PMS2, MSH2, and MSH6 and from 10 additional genes with strong pan-cancer associations with tumor hypermutation. The algorithms were validated on the NanoString nCounter™ platform in independent cohorts of colorectal (n = 52), endometrial (n = 11), and neuroendocrine (n = 4) tumors pre-characterized using the MMR immunohistochemistry assay.In the validation cohorts, the algorithm showed high prediction accuracy of tumor MSI status, with sensitivity of at least 88% attained at thresholds chosen to achieve 100% specificity. Furthermore, MSI status was compared to the Tumor Inflammation Signature (TIS), an analytically validated diagnostic assay which measures a suppressed adaptive immune response in the tumor and enriches for response to immune checkpoint blockade. TIS score was largely independent of MSI status, suggesting that measuring both parameters may identify more patients that would respond to immune checkpoint blockade than either assay alone.Development of a gene expression signature of MSI status raises the possibility of a combined diagnostic assay on a single platform which measures both tumor antigenicity and presence of a suppressed adaptive immune response. Such an assay would have significant advantages over multi-platform assays for both ease of use and turnaround time and could lead to a diagnostic test with improved clinical performance.

Clinical variables and several gene signature profiles have been investigated for the prediction of (distant) recurrence in several trials. These molecular markers are significantly correlated with overall and late distant recurrences. Here, we retrospectively explore whether age and body mass index (BMI) affect the prediction of these molecular scores for distant recurrence in postmenopausal women with hormone receptor-positive breast cancer in the transATAC trial. 940 postmenopausal women for whom the Clinical Treatment Score (CTS), immunohistochemical markers (IHC4), Oncotype Recurrence Score (RS), and the Prosigna Risk of Recurrence Score (ROR) were available were included in this retrospective analysis. Conventional BMI groups were used (N = 865), and age was split into equal tertiles (N = 940). Cox proportional hazard models were used to determine the effect of a molecular score for the prediction of distant recurrence according to BMI and age groups. In both the univariate and bivariate analyses, the effect size of the IHC4 and RS was strongest in women aged 59.8 years or younger. Trends tests for age were significant for the IHC4 and RS, but not for the CTS and ROR, for which most prognostic information was added in women aged 60 years or older. The CTS and ROR scores added significant prognostic information in all three BMI groups. In both the univariate and bivariate analyses, the IHC4 provided the most prognostic information in women with a BMI lower than 25 kg/m(2), whereas the RS did not add prognostic information for distant recurrence in women with a BMI of 30 kg/m(2) or above. Molecular scores are increasingly used in women with breast cancer to assess recurrence risk. We have shown that the effect size of the molecular scores is significantly different across age groups, but not across BMI groups. The results from this retrospective analysis may be incorporated in the identification of women who may benefit most from the use of these molecular scores, but our findings need further evaluation before these scores can be used in clinical decision making.

Importance: 20-25% of patients with early stage HER2-positive breast cancer develop tumor relapse after adjuvant trastuzumab. Identification of such patients is a key goal for clinical management decisions. Objective: To assess molecular heterogeneity among early stage HER2-positive patients using the Prosigna™ algorithm, to define intrinsic subtypes, and to determine the clinical significance of such heterogeneity. Design: The NanoString® platform was used to measure the abundance of the PAM50 subtype signature transcripts. Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna™ algorithm to define intrinsic subtype and risk scores. Subtypes were evaluated for recurrence-free survival following chemotherapy with or without trastuzumab. Setting: Samples were obtained from a multi-center randomized phase III trial of chemotherapy versus chemotherapy plus trastuzumab. Participants: All tumors were centrally evaluated for HER2 positivity, defined as IHC 3+ and/or FISH >2.0; 1392 patients were evaluated for molecular subtype. Intervention(s): Patients received adjuvant chemotherapy (doxorubicin plus cyclophosphamide followed by paclitaxel) (n=484) or chemotherapy plus trastuzumab (n=908). Main Outcome Measure(s): The primary outcome was recurrence-free survival as a function of subtype and treatment. Results: Patients with HER2-positive tumors with HER2-enriched features comprised about 70% of the sample cohort, and these individuals received significant benefit from adjuvant trastuzumab (HR=0.68, 95%CI: 0.52, 0.89, p=0.005), as did the relatively fewer patients (291/1392) with Luminal-type tumors (HR=0.52, 95%CI: 0.32, 0.85, p=0.01). The sample cohort contained a small number of patients with tumors having Basal-like features (97/1392), and the data suggest that these individuals may have received less benefit from trastuzumab, beyond that received from chemotherapy alone (HR=1.06, 95%CI:0.53,2.13, p=0.87). Conclusions: The majority of HER2-positive tumors are classified as HER2-enriched or Luminal using the Prosigna algorithm, and patients with such tumors benefit from adjuvant trastuzumab. About 10% of HER2-positive tumors exhibit Basal-like genomic features, and such tumors appear to recur at fairly similar frequency irrespective of treatment with chemotherapy or chemotherapy plus trastuzumab. Patients with HER2-positive/Basal-like tumors may represent a cohort that should be considered for enrollment in trials to evaluate emerging novel HER2-targeted agents, other targeted therapies, or combinations of both approaches. Support provided in part by CA129949 and CA15083. Citation Format: Perez EA, Ballman KV, Mashadi-Hossein A, Tenner KS, Kachergus JM, Norton N, Necela BM, Carr JM, Ferree S, Perou CM, Cheang MCU, Thompson EA. Intrinsic subtype and therapeutic response among early stage HER2-positive breast tumors from the North Central cancer treatment group (Alliance) N9831 trial. [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 P3-07-04.

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.

Background: The PAM50-based (Prosigna) risk of recurrence (ROR) score and intrinsic subtypes are prognostic for women with high-risk breast cancer. We investigate the predictive ability of Prosigna regarding the effectiveness of cyclophosphamide-based adjuvant chemotherapy in premenopausal patients with high-risk breast cancer. Methods: Prosigna assays were performed on the NanoString platform in tumors from participants in Danish Breast Cancer Group (DBCG) 77B, a four-arm trial that randomized premenopausal women with high-risk early breast cancer to no systemic treatment, levamisole, oral cyclophosphamide (C) or cyclophosphamide, methotrexate and fluorouracil (CMF). Results: In total, this retrospective analysis included 460 women (40% of the 1146 randomized patients). The continuous Prosigna ROR score was prognostic in the no systemic treatment group (unadjusted P

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.

This secondary analysis of a randomized clinical trial evaluates the prognostic value of 6 multigene signatures in women with early estrogen receptor–positive breast cancer who have received 5 years of endocrine therapy., Key Points Question What is the comparative performance of prognostic multigene signatures for estimation and risk stratification of overall and late distant recurrence in estrogen receptor–positive ERBB2-negative breast cancer? Findings In this biomarker analysis of data from a randomized clinical trial, a combination of multigene expression tests with clinical information was associated with improved prognostic value for distant recurrences and risk stratification specifically in women with node-positive disease. Differences in the prognostic value for late distant recurrence were observed. Meaning The combination of clinical and molecular information may enhance the prognostic value for distant recurrence and risk stratification in estrogen receptor–positive, ERBB2-negative breast cancer, particularly for women with node-positive disease., Importance Multiple molecular signatures are available for managing estrogen receptor (ER)–positive breast cancer but with little direct comparative information to guide the patient’s choice. Objective To conduct a within-patient comparison of the prognostic value of 6 multigene signatures in women with early ER-positive breast cancer who received endocrine therapy for 5 years. Design, Setting, and Participants This retrospective biomarker analysis included 774 postmenopausal women with ER-positive ERBB2 (formerly HER2)–negative breast cancer. This analysis was performed as a preplanned secondary study of data from the Anastrozole or Tamoxifen Alone or Combined randomized clinical trial comparing 5-year treatment with anastrozole vs tamoxifen with 10-year follow-up data. The signatures included the Oncotype Dx recurrence score, PAM50-based Prosigna risk of recurrence (ROR), Breast Cancer Index (BCI), EndoPredict (EPclin), Clinical Treatment Score, and 4-marker immunohistochemical score. Data were collected from January 2009, through April 2015. Main Outcomes and Measures The primary objective was to compare the prognostic value of these signatures in addition to the Clinical Treatment Score (nodal status, tumor size, grade, age, and endocrine treatment) for distant recurrence for 0 to 10 years and 5 to 10 years after diagnosis. Likelihood ratio (LR) statistics were used with the χ2 test and C indexes to assess the prognostic value of each signature. Results In this study of 774 postmenopausal women with ER-positive, ERBB2-negative disease (mean [SD] age, 64.1 [8.1] years), 591 (mean [SD] age, 63.4 [7.9] years) had node-negative disease. The signatures providing the most prognostic information were the ROR (hazard ratio [HR], 2.56; 95% CI, 1.96-3.35), followed by the BCI (HR, 2.46; 95% CI, 1.88-3.23) and EPclin (HR, 2.14; 95% CI, 1.71-2.68). Each provided significantly more information than the Clinical Treatment Score (HR, 1.99; 95% CI, 1.58-2.50), the recurrence score (HR, 1.69; 95% CI, 1.40-2.03), and the 4-marker immunohistochemical score (HR, 1.95; 95% CI, 1.55-2.45). Substantially less information was provided by all 6 molecular tests for the 183 patients with 1 to 3 positive nodes, but the BCI (ΔLR χ2 = 9.2) and EPclin (ΔLR χ2 = 7.4) provided more additional prognostic information than the other signatures. Conclusions and Relevance For women with node-negative disease, the ROR, BCI, and EPclin were significantly more prognostic for overall and late distant recurrence. For women with 1 to 3 positive nodes, limited independent information was available from any test. These data might help oncologists and patients to choose the most appropriate test when considering chemotherapy use and/or extended endocrine therapy. Trial Registration isrctn.com Identifier: ISRCTN18233230

1533Background: Gene expression profiling assays are not commonly used for therapy selection for breast cancer patients with germline BRCA mutations but a complete molecular characterization could ...

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.

Purpose We have previously shown that the PAM50-based risk of recurrence (ROR) score is significantly correlated with distant recurrence in both the translational research cohort within the Arimidex, Tamoxifen Alone or in Combination (ATAC) trial (TransATAC) and Austrian Breast and Colorectal Cancer Study Group 8 (ABCSG 8) randomized trials. Here, we focus on the ROR score for predicting distant recurrence after 5 years of follow-up in a combined analysis of these two randomized trials. Methods Long-term follow-up data and tissue samples were obtained from 2,137 postmenopausal women with hormone receptor–positive early-stage breast cancer from the ABCSG 8 and TransATAC trials. We used Cox proportional hazard regression models to determine the prognostic value of ROR for distant recurrence beyond 5 years in the combined data set. Results A total of 2,137 women who did not have a recurrence 5 years after diagnosis were included in the combined analyses. The Clinical Treatment Score (CTS) was the strongest prognostic factor 5 years after diagnosis (univariable: likelihood ratio [LR] χ2 = 94.12, bivariable: LR χ2 = 61.43). The ROR score was significantly prognostic by itself in years 5 to 10. In the node-negative/human epidermal growth factor receptor 2–negative subgroup, more prognostic value for late distant recurrence was added by the ROR score compared with the CTS. Conclusion The ROR score added clinically meaningful prognostic information to the CTS in all patients and all subgroups in the late follow-up period. These results suggest that the ROR score may be helpful for separating patients into risk groups who could be spared or potentially benefit from extended hormonal therapy beyond 5 years of treatment.

Improved understanding of risk of recurrence (ROR) is needed to reduce cases of recurrence and more effectively treat breast cancer patients. The purpose of this study was to examine how a gene-expression profile (GEP), identified by Prosigna, influences physician adjuvant treatment selection for early breast cancer (EBC) and the effects of this influence on optimizing adjuvant treatment recommendations in clinical practice. A prospective, observational, multicenter study was carried out in 15 hospitals across Spain. Participating medical oncologists completed pre-assessment, post-assessment, and follow-up questionnaires recording their treatment recommendations and confidence in these recommendations, before and after knowing the patient’s ROR. Patients completed questionnaires on decision-making, anxiety, and health status. Between June 2013 and January 2014, 217 patients enrolled and a final 200 were included in the study. Patients were postmenopausal, estrogen receptor positive, human epidermal growth hormone factor negative, and node negative with either stage 1 or stage 2 tumors. After receiving the GEP results, treatment recommendations were changed for 40 patients (20%). The confidence of medical oncologists in their treatment recommendations increased in 41.6% and decreased in 6.5% of total cases. Patients reported lower anxiety after physicians made treatment recommendations based on the GEP results (p Though this study does not include evaluation of the impact of GEP on long-term outcomes, it was found that GEP results influenced the treatment decisions of medical oncologists and their confidence in adjuvant therapy selection. Patients’ anxiety about the selected adjuvant therapy decreased with use of the GEP.

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.

Adjuvant chemotherapy and endocrine therapy for early breast cancer have had a considerable impact on outcomes (1), but a substantial number of women, especially those with estrogen receptor (ER)–positive tumors, remain at risk for late recurrences. The annual rate is in excess of 2% for at least 15 years, even after 5 years of tamoxifen therapy (2), and currently it is not possible to identify a group of such women who can be considered as cured (3,4). This remains true for at least 10 years for women treated for 5 years with an aromatase inhibitor (5). Most of the studies of prevention of late relapse have been performed in women receiving tamoxifen as initial adjuvant endocrine therapy for early ER-positive breast cancer. The MA17 trial clearly showed that extended adjuvant therapy with letrozole after 5 years of tamoxifen prolongs disease-free survival and overall survival, regardless of the patient’s nodal status involvement (6). Brewster et al. (7) found that ER positivity, nodal involvement, and grade were all associated with increased risk of late recurrence. It is of importance to determine to what extent the newer immunohistochemical and molecular scores can help in predicting late recurrence. Recent publications from the transATAC (Anastrozole, Tamozifen, Alone or in Combination) cohort have demonstrated that the Oncotype DX recurrence score (RS) (8), the immunohistochemical (IHC4) score (9) and the PAM50-based risk of recurrence (ROR) score (10) all provide additional information beyond that available for clinical variables, summarized in the clinical treatment score (CTS), about the risk of distant recurrence in postmenopausal women with hormone receptor–positive breast cancer treated with anastrozole or tamoxifen, but it is unknown how much of this effect extends beyond 5 years. Here, we investigate the relationship between clinical variables, immunohistochemical markers, and these scores for the prediction of distant recurrence separately in years 0 to 5 and years 5 to 10 after diagnosis for postmenopausal women with early hormone receptor–positive breast cancer.

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-χ2 = 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.

Background Genomic data from human epidermal growth factor receptor 2-positive (HER2+) tumors were analyzed to assess the association between intrinsic subtype and clinical outcome in a large, well-annotated patient cohort. Methods Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna algorithm on the NanoString platform to define intrinsic subtype, risk of recurrence scores, and risk categories for 1392 HER2+ tumors. Subtypes were evaluated for recurrence-free survival (RFS) using Kaplan-Meier and Cox model analysis following adjuvant chemotherapy (n = 484) or chemotherapy plus trastuzumab (n = 908). All statistical tests were two-sided. Results Patients with HER2+ tumors from N9831 were primarily scored as HER2-enriched (72.1%). These individuals received statistically significant benefit from trastuzumab (hazard ratio [HR] = 0.68, 95% confidence interval [CI] = 0.52 to 0.89, P = .005), as did the patients (291 of 1392) with luminal-type tumors (HR = 0.52, 95% CI = 0.32 to 0.85, P = .01). Patients with basal-like tumors (97 of 1392) did not have statistically significantly better RFS when treated with trastuzumab and chemotherapy compared with chemotherapy alone (HR = 1.06, 95% CI = 0.53 to 2.13, P = .87). Conclusions The majority of clinically defined HER2-positive tumors were classified as HER2-enriched or luminal using the Prosigna algorithm. Intrinsic subtype alone cannot replace conventional histopathological evaluation of HER2 status because many tumors that are classified as luminal A or luminal B will benefit from adjuvant trastuzumab if that subtype is accompanied by HER2 overexpression. However, among tumors that overexpress HER2, we speculate that assessment of intrinsic subtype may influence treatment, particularly with respect to evaluating alternative therapeutic approaches for that subset of HER2-positive tumors of the basal-like subtype.

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.

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...

203 Background: The development and analytical performance of the Tumor Inflammation Signature (TIS) assay has been described previously. The TIS is an investigational use RNA expression assay on the nCounter Dx Analysis System, which is being evaluated as a patient enrichment biomarker for treatment with pembrolizumab single agent across multiple solid tumor types. Here we describe the analytical validation of the RNA input range and analytical precision starting from RNA isolated from formalin fixed paraffin embedded (FFPE) tissue blocks. Methods: Analytical validation of TIS assay performance across an RNA input range was performed using samples from 11 tumor types. The analytical precision between sites, instruments, reagent lots, and users was measured, using RNA samples isolated from FFPE tissue blocks. Results: The assay was validated across the specified RNA input range with ≥ 94% concordance at the minimum specified RNA input (50ng). The total standard deviation of the TIS score was < 0.04 units across three sites with ≥98% concordance between sites. The 6 users across the three sites did not significantly contribute to the assay variability. There was 100% concordance in biomarker high/low categorization between multiple reagent lots and multiple instruments. Conclusions: The analytical performance of the NanoString TIS assay has been validated to give consistent results across the RNA input range and between site, instrument, assay user, and reagent kit lot. The assay is well suited for decentralized clinical testing and is currently under investigation as a biomarker to enrich for response to anti-PD1 therapy across multiple tumor types.

Background The four intrinsic subtypes of breast cancer, defined by differential expression of 50 genes (PAM50), have been shown to be predictive of risk of recurrence and benefit of hormonal therapy and chemotherapy. Here we describe the development of Prosigna™, a PAM50-based subtype classifier and risk model on the NanoString nCounter Dx Analysis System intended for decentralized testing in clinical laboratories. Methods 514 formalin-fixed, paraffin-embedded (FFPE) breast cancer patient samples were used to train prototypical centroids for each of the intrinsic subtypes of breast cancer on the NanoString platform. Hierarchical cluster analysis of gene expression data was used to identify the prototypical centroids defined in previous PAM50 algorithm training exercises. 304 FFPE patient samples from a well annotated clinical cohort in the absence of adjuvant systemic therapy were then used to train a subtype-based risk model (i.e. Prosigna ROR score). 232 samples from a tamoxifen-treated patient cohort were used to verify the prognostic accuracy of the algorithm prior to initiating clinical validation studies. Results The gene expression profiles of each of the four Prosigna subtype centroids were consistent with those previously published using the PCR-based PAM50 method. Similar to previously published classifiers, tumor samples classified as Luminal A by Prosigna had the best prognosis compared to samples classified as one of the three higher-risk tumor subtypes. The Prosigna Risk of Recurrence (ROR) score model was verified to be significantly associated with prognosis as a continuous variable and to add significant information over both commonly available IHC markers and Adjuvant! Online. Conclusions The results from the training and verification data sets show that the FDA-cleared and CE marked Prosigna test provides an accurate estimate of the risk of distant recurrence in hormone receptor positive breast cancer and is also capable of identifying a tumor's intrinsic subtype that is consistent with the previously published PCR-based PAM50 assay. Subsequent analytical and clinical validation studies confirm the clinical accuracy and technical precision of the Prosigna PAM50 assay in a decentralized setting. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0129-6) contains supplementary material, which is available to authorized users.

Theories of DNA electrophoretic separations generally treat the DNA as a free draining polymer moving in an electric field at a rate that depends on the effective charge density of the molecule. Separations can occur in sieving media ranging from ultradilute polymer solutions to tightly cross-linked gels. It has recently been shown that DNA is not free-draining when both electric and nonelectric forces simultaneously act on the molecule, as occurs when DNA collides with a polymer during electrophoretic separations. Here we show that a semidilute polymer solution screens the hydrodynamic interaction that results from the application of these forces. Fluorescently labeled DNA tethered at one end in a semidilute solution of hydroxyl-ethyl cellulose stretch more in an electric field than they stretch in free solution, and approach free-draining behavior. The steady stretching behavior is predicted without adjustable parameters by a theory developed by Stigter using a hydrodynamic screening length found from effective medium theory. Data on the relaxation of stretched molecules after the electric field is removed agree with the Rouse model prediction, which neglects hydrodynamic interactions. The slowest relaxation time constant, tau(R), scales with chain length as tau(R) approximately L(1.9+/-0.17) when analyzed by the data collapse method, and as tau(R) approximately L(2.17+/-0.17) when analyzed by multiexponential fit.

During electrophoretic separations of DNA in a sieving medium, DNA molecules stretch from a compact coil into elongated conformations when encountering an obstacle and relax back to a coil upon release from the obstacle. These stretching dynamics are thought to play an important role in the separation mechanism. In this article we describe a silicon microfabricated device to measure the stretching of tethered DNA in electric fields. Upon application of an electric field, electro-osmosis generates bulk fluid flow in the device, and a protocol for eliminating this flow by attaching a polymer brush to all silicon oxide surfaces is shown to be effective. Data on the steady stretching of DNA in constant electric fields is presented. The data corroborate the approximate theory of hydrodynamic equivalence, indicating that DNA is not free-draining in the presence of both electric and nonelectric forces. Finally, these data provide the first quantitative test of a Stigter and Bustamante's detailed theory of electrophoretic stretching of DNA without adjustable parameters. The agreement between theory and experiment is good.

8 Background: Pembrolizumab is a humanized anti-PD1 antibody that is approved for use in advanced melanoma, recurrent or metastatic head and neck squamous cell carcinoma, and metastatic non-small-cell lung cancer. It has also shown clinical activity in a number of other tumor types in clinical trials, but there is need for a precise and accurate test that can identify patients most likely to benefit from therapy. We have previously described the development and analytical performance of a NanoString RNA expression clinical trial assay, referred to here as the Tumor Inflammation Signature (TIS) assay, which is being evaluated as a patient enrichment biomarker in multiple solid tumor types for treatment with pembrolizumab. Here we describe additional performance data and analytical verification of reproducibility from tissue and RNA input range in multiple tumor types. Methods: Linearity and specificity were assessed with single targets or pools of in vitro transcribed RNAs with sequences matching the 18 biomarker and 10 normalization gene probe targets. The verification of the previously reported analytical precision from RNA and reproducibility from tissue was performed using independent samples from 11 tumor types. The biological variability of the signature within a patient sample was evaluated by testing multiple core punches from formalin fixed paraffin embedded (FFPE) tissue blocks. Results: The TIS assay’s measurement of the 28 genes was linear across a wide dynamic range ( ≥ 4 logs) and was highly specific with < 1% cross reactivity between probes. The assay was verified across the specified RNA input range with > 90% concordance at the low end (50ng) of the RNA input range. The total standard deviation of the anti-PD1 Predictor Score from tissue was verified as < 5% of the signature score range and > 90% concordance in biomarker high/low categorization within the biological replicates. Conclusions: The analytical performance of the NanoString TIS assay was verified to be robust. The assay is well suited for decentralized clinical testing and is currently under investigation to identify responders to anti-PD1 therapy in multiple tumor types in several clinical studies.

Background: A number of prognostic signatures have been developed for the prediction of breast cancer recurrence in the past decade. We have developed two signatures (Clinical Treatment Score (CTS), four immunohistochemical markers (IHC4)) and validated four prognostic signatures (Oncotype Dx Recurrence Score (RS), PAM50-based Prosigna (ROR), Breast Cancer Index (BCI), and EndoPredict (EPclin)) in the TransATAC cohort. Here, we compare the prognostic performance of these six signatures for distant recurrence (DR) in years 0-10, and specifically in years 5-10 after treatment cessation. Methods: 1231 postmenopausal women with hormone receptor positive and HER2-negative breast cancer had at least one test performed. Of these, 818 women had data on all six signatures available. IHC4, RS and BCI (linear) are molecular only signatures whereas CTS, ROR and EPclin include clinicopathological factors. The primary endpoint was DR and the primary objective was to compare the prognostic value of the six signatures in terms of DR for years 0-10, 0-5, and 5-10. Secondary objectives included the comparison of the prognostic performance for node-negative and node-positive patients separately and the additional prognostic performance of each signature to the others. Likelihood ratio statistics (LR-χ2) were used to assess the prognostic information of each signature alone or in combination with other signatures. Results: Median follow-up for this analysis was 9.94 years (IQR 8.01-10.09) and a total of 126 DR were recorded. 818 women with HER2-negative disease for whom data of all six signatures were available were included in this analysis. For all patients, CTS and EPclin were the most prognostic signatures in years 0-10 (CTS: LR-χ2=124.9; EPclin: LR-χ2=116.2) and years 5-10 (CTS: LR-χ2=59.6; EPclin: LR-χ2=56.8) in the univariate analysis. The other four signatures performed similarly well in years 0-5, but of those only BCI and ROR provided substantial prognostic information in years 5-10 (BCI: LR-χ2=25.3; ROR: LR-χ2=43.8). In multivariate analyses comparing the added information of the molecular signatures over CTS, IHC4 and BCI provided the most information (IHC4: ΔLR-χ2=19.0; BCI: ΔLR-χ2=19.8). In node-negative patients (72.3%), the ROR showed the most prognostic value in years 0-10 (LR-χ2=48.6) and years 5-10 (LR-χ2=31.3) whereas the RS was least prognostic in this patient group. For patients with node-positive disease (27.7%), the CTS and EPclin were the most prognostic and the other four signatures provided much less prognostic information for this patient population (data not shown). Conclusion: Overall, the CTS and EPclin were the most prognostic signatures for DR and also added significant prognostic value to the other scores in women with HER2-negative disease, primarily due to the incorporation of nodal status in these signatures. For women with node-negative disease, the ROR, BCI, and EPclin signatures provided most prognostic value whereas for those with positive nodes CTS and EPclin were most prognostic. Our analyses showed that the inclusion of clinic-pathological factors into gene signatures is highly important for deriving an accurate prognostic assessment, particularly in node-positive patients. Citation Format: Sestak I, Buus R, Cuzick J, Dubsky P, Kronenwett R, Ferree S, Sgroi D, Schnabel C, Baehner R, Mallon E, Dowsett M. Comprehensive comparison of prognostic signatures for breast cancer in TransATAC [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 S6-05.

The recent increase in research and applications of oligonucleotides in new drug design has stimulated a need to determine the three-dimensional structures of oligonucleotides and their complexes with various ligands. Three-dimensional structure determination relies on X-ray diffraction data from high-quality crystals. However, only a very limited number of RNA molecules have been crystallized, and a broad record of experience in oligonucleotide crystallization is lacking. Osmotic dewatering is a crystallization method in which the oligonucleotide and its precipitant are concentrated at a controlled rate by the removal of water through a reverse-osmosis membrane using a specified concentration gradient. Therefore, rates of nucleation and crystal growth can be controlled. In this study, a transport model was developed to predict the water removal rates at various concentration gradients. Experimental dewatering rate data agreed very well with theoretical calculations. Model predictions were applied to different osmotic dewatering crystallization devices to crystalize a model RNA, “U-U dodecamer”. High-quality dodecamer crystals were successfully grown and yielded a maximum X-ray diffraction resolution of 2.46 A and usable resolution of 3.0 A. The results of this research will facilitate the applications of osmotic dewatering to crystallographic studies and industrial purification of biopharmaceuticals.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin's lymphoma with two distinct molecular subtypes defined by Cell-of-Origin (COO): germinal center B-cell (GCB) and activated B-cell (ABC). COO is being evaluated as a predictive biomarker in multiple DLBCL clinical trials hence the need for an accurate and precise companion diagnostics (CDx). NanoString LST is an investigational multiplexed digital gene expression assay performed on the nCounter® Dx Analysis System that identifies COO from FFPE tissue with a turnaround time of 2-3 days (Wallden B et al, JCO 2015; Storhoff J et al, Blood 2015). The test is based on the Lymph2Cx gene expression assay which profiles 15 classifier genes and 5 housekeeping genes to compute a linear predictor score (LPS) and determine the COO subtype (Scott D et al, Blood 2014). To establish a CDx, both analytical and clinical validations are required using the locked algorithm and assay procedure. Clinical validity of the LST is currently being evaluated in a global Phase 3 study in which the assay is used to prospectively select patients with ABC-type DLBCL to receive in a randomized fashion R2-CHOP or R-CHOP (Nowakowski G et al, JCO 2016). The analytical validation studies described herein were designed to evaluate the analytical performance of the test across multiple laboratory sites, operators, instruments, reagent lots, and RNA input levels. Design: Reproducibility was measured across 3 sites and 6 operators by testing replicate tissue sections from 43 FFPE DLBCL tumor blocks following independent pathology review at each site. The testing included both core needle and surgical biopsy specimens. The standard deviation (SD) of the LPS output was analyzed as a continuous variable using a linear mixed model to estimate assay variance, and the site-to-site concordance was evaluated. Analytical precision was measured across 3 sites and 6 operators by testing 5 pooled DLBCL tumor RNA samples representing each COO subtype including samples near the subtype thresholds. The sensitivity of the assay was evaluated by testing 4 RNA levels within the recommended input range (62.5 ng, 125 ng, 500 ng, 1000 ng) and 2 input levels outside of this range (50 ng and 1250 ng) using 14 DLBCL tumor RNA samples and 2 reagent lots. The percentage of test samples passing all QC metrics was determined at each input level, and the subtype concordance was evaluated compared to the nominal test input level (500 ng). Interference of human genomic DNA was assessed by omitting DNase from the assay procedure. The impact of including non-tumor tissue was assessed by omitting the tissue macrodissection step from the assay. Results: In the multi-site reproducibility study, the average site-to-site LST subtype concordance with independent pathology review was >97% with no ABC-to-GCB discordances (or vice versa). The overall range of LPS spanned approximately 5000 units. The total SD was Conclusions: The investigational NanoString LST has been analytically validated for identifying COO subtypes across multiple testing laboratories. The test provides a highly precise, sensitive and rapid method for measuring COO on FFPE DLBCL tumor specimens, including both excisional and core needle biopsies. Disclosures Chen: NanoString Technologies, Inc.: Employment, Other: Stock option. Dennis:NanoString Technologies, Inc.: Employment, Other: Stock option. Danaher:NanoString Technologies, Inc.: Employment, Other: Stock option. Wallden:NanoString Technologies, Inc.: Employment. Hood:NanoString Technologies, Inc.: Employment, Other: Stock option. Ren:NanoString Technologies, Inc.: Employment, Other: Stock option. Liu:NanoString Technologies, Inc.: Employment, Other: Stock option. Dowidar:NanoString Technologies, Inc.: Employment, Other: Stock option. Sullivan:NanoString Technologies, Inc.: Employment, Other: Stock option. Haffner:NanoString Technologies, Inc.: Employment, Other: Stock option. Cesano:NanoString Technologies, Inc.: Employment, Other: Stock option. Ferree:NanoString Technologies, Inc.: Employment, Other: Stock option, Patents & Royalties: NanoString Technologies, Inc.. Storhoff:NanoString Technologies, Inc.: Employment, Other: Stock option.

3034Background: Pembrolizumab is a humanized anti-PD1 antibody that is FDA approved for use in patients with advanced melanoma and in selected patients with metastatic non-small-cell lung cancer. I...

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.