PAM50 Risk Of Recurrence Score Research Articles

Predicting invasive disease-free survival in ER-positive, HER2-negative early breast cancer using the PAM50 risk-of-recurrence score: a retrospective analysis using single-center long-term follow-up data of postmenopausal Japanese patients

BackgroundThe prognostic value of the risk-of-recurrence (ROR) score calculated using PAM50 has been validated using clinical trials and patient cohorts. This study aimed to investigate the prognostic value of the PAM50 ROR score in Japanese patients with early breast cancer using long-term follow-up data.MethodsWe enrolled postmenopausal patients with ER-positive, HER2-negative, stage I–II breast cancer who had undergone surgery at the Kyoto University Hospital between 2008 and 2014. The intrinsic subtype and ROR score were calculated using PAM50. The primary endpoint was invasive disease-free survival (IDFS).ResultsWe enrolled 146 patients, of whom 47 (32%) patients had node-positive disease, and 36 (25%) had received neoadjuvant or adjuvant chemotherapy. The proportions of intrinsic subtypes for luminal A, luminal B, HER2-enriched, and basal-like subtypes were 67%, 27%, 3%, and 2%, respectively. The median follow-up duration was 8.4 (range 6.3–10.0) years, and 21 IDFS events were observed. Based on the ROR score, 37%, 33%, and 30% of the patients were classified as low, intermediate, and high risks, respectively. Patients in the high-risk group had a significantly worse 8-year IDFS rate than those in the low-to-intermediate-risk groups (75.1% vs. 91.6%, p = 0.04). The same trend was observed in patients with and without neoadjuvant or adjuvant chemotherapy.ConclusionsUsing long-term follow-up data, this study showed that the ROR score can predict the prognosis of ER-positive, HER2-negative early breast cancer in Japanese postmenopausal patients. Further investigations are required to confirm the prognostic value of the ROR score in Asian populations.

Association of early menarche with breast tumor molecular features and recurrence

BackgroundEarly menarche is an established risk factor for breast cancer but its molecular contribution to tumor biology and prognosis remains unclear.MethodsWe profiled transcriptome-wide gene expression in breast tumors (N = 846) and tumor-adjacent normal tissues (N = 666) from women in the Nurses’ Health Studies (NHS) to investigate whether early menarche (age < 12) is associated with tumor molecular and prognostic features in women with breast cancer. Multivariable linear regression and pathway analyses using competitive gene set enrichment analysis were conducted in both tumor and adjacent-normal tissue and externally validated in TCGA (N = 116). Subgroup analyses stratified on ER-status based on the tumor were also performed. PAM50 signatures were used for tumor molecular subtyping and to generate proliferation and risk of recurrence scores. We created a gene expression score using LASSO regression to capture early menarche based on 28 genes from FDR-significant pathways in breast tumor tissue in NHS and tested its association with 10-year disease-free survival in both NHS (N = 836) and METABRIC (N = 952).ResultsEarly menarche was significantly associated with 369 individual genes in adjacent-normal tissues implicated in extracellular matrix, cell adhesion, and invasion (FDR ≤ 0.1). Early menarche was associated with upregulation of cancer hallmark pathways (18 significant pathways in tumor, 23 in tumor-adjacent normal, FDR ≤ 0.1) related to proliferation (e.g. Myc, PI3K/AKT/mTOR, cell cycle), oxidative stress (e.g. oxidative phosphorylation, unfolded protein response), and inflammation (e.g. pro-inflammatory cytokines IFNα\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\alpha$$\\end{document} and IFNγ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\gamma$$\\end{document}). Replication in TCGA confirmed these trends. Early menarche was associated with significantly higher PAM50 proliferation scores (β = 0.082 [0.02–0.14]), odds of aggressive molecular tumor subtypes (basal-like, OR = 1.84 [1.18–2.85] and HER2-enriched, OR = 2.32 [1.46–3.69]), and PAM50 risk of recurrence score (β = 4.81 [1.71–7.92]). Our NHS-derived early menarche gene expression signature was significantly associated with worse 10-year disease-free survival in METABRIC (N = 952, HR = 1.58 [1.10–2.25]).ConclusionsEarly menarche is associated with more aggressive molecular tumor characteristics and its gene expression signature within tumors is associated with worse 10-year disease-free survival among women with breast cancer. As the age of onset of menarche continues to decline, understanding its relationship to breast tumor characteristics and prognosis may lead to novel secondary prevention strategies.

Abstract P2-23-16: Clinico-Pathological and Molecular Characterization of HER2-Enriched Breast Tumors Independently of HER2 Status

Abstract INTRODUCTION: Breast cancer (BC) is a highly prevalent and heterogeneous disease, entailing different so-called intrinsic subtypes (IS) according to gene expression, namely Luminal A, Luminal B, HER2-Enriched (HER2E) and Basal-like, as well as a normal breast-like group. The HER2E is still a poorly understood entity, which needs further biologic characterization to improve therapeutic management. MATERIAL AND METHODS: Patients (pts) treated at Hospital Clinic (Barcelona, Spain) over 18 years with a diagnosis of metastatic BC, with available matched primary and metastatic tumor samples for gene expression analyses were retrospectively recruited. Using the nCounter® Breast Cancer 360 panel, we studied the expression of 776 genes pertaining to different tumor and immune pathway-related signatures, with a focus on HER2E vs. non-HER2E tumors. Significant changes were considered at a false discovery rate (FDR) &amp;lt; 5%. Main clinicopathological features were also compared. IS changes from primary to metastatic disease were assessed. RESULTS: Ninety-one pts with paired tumor samples were included. Briefly, primary tumors were 67.0% hormone receptor-positive (HR+)/HER2-negative (HER2-), 14.8% were HER2-positive (HER2+) and 18.2% were triple-negative (TNBC). IS distribution in primary tumors was the following: 25 Luminal A (28%), 22 Luminal B (24%), 24 HER2E (26%), 12 Basal-like (13%), and 8 Normal-like (9%). In contrast, IS distribution in metastatic tumors was the following: 13 Luminal A (14%), 22 Luminal B (24%), 34 HER2-E (37%), 16 Basal-like (18%), and 6 Normal-like (7%). The HER2E disease proved to be a relatively stable subtype, with 16 (66.7%) tumors not changing IS in the transition to metastatic disease (p=0.078). Particularly, within HR+/HER2-, HER2+ and TNBC, 8/12 (66.7%), 8/10 (80.0%) and 1/2 (50.0%) tumors remained HER2E. Conversely, an overall significant switch from Luminal to non-Luminal tumors at the metastatic progression was observed (p=0.031), with 14/19 (73.7%) new non-Luminal BC being HER2E. When considering all primary and metastatic tumors, HER2E were observed to be less frequently estrogen receptor (ER) positive than non-HER2E tumors (55.8% vs. 78.7%; p=0.002), with lower mean progesterone receptor (PR) levels (15.3% vs. 25.8%; p=0.027). Compared to the other subtypes (as a group), the PAM50 risk of recurrence score (ROR-P) was higher for HER2E tumors (59.1 vs. 41.7; p &amp;lt; 0.001), which were also more frequently HER2+ (36.5% vs. 5.8%) and less likely HR+/HER2- (50.0% vs. 73.6%) and TNBC (13.5% vs. 20.7%), compared to non-HER2E (p &amp;lt; 0.001). No significant differences in grade, TILs, Ki67 and histotype were observed. Overall, 140/776 genes were significantly downregulated in HER2E vs. non-HER2E tumors, including genes related to cell adhesion, migration, DNA damage repair, apoptosis, estrogen signalling pathway, senescence. Conversely, 178/776 genes were significantly upregulated, including the tyrosine-kinase receptor FGFR4, genes involved in nuclear activity, DNA transcription regulation, MAP-kinase signaling pathways, proliferation, cytokine response, epithelial-to-mesenchymal transition (EMT), cell cycle progression, and immune-related genes such as CD274 (PD-L1 gene). DISCUSSION: A switch towards more aggressive IS from primary to advanced disease was observed, with an increase in HER2E prevalence. HER2E tumors, which tend to maintain their IS at the metastatic disease, showed upregulation of genes related to proliferation, survival and EMT, coherently with their well-known worse prognosis. FGFR4 and CD274 upregulation, along with downregulation of genes involved in DNA damage repair suggest these tumors might benefit from targeted therapeutic approaches. Genomic higher risk was not convoyed by consistent clinicopathological features, except for lower PR levels and HER2 overexpression at IHC. Further characterization according to primary/metastatic and HR status is ongoing. Intrinsic subtype distribution across primary and metastatic breast tumors Citation Format: Francisco Javier Muñoz-Carrillo, Laia Paré, Benedetta Conte, Adela Rodríguez, Patricia Galván, Esther Sanfeliu, Blanca González-Farré, Claudette Falato, Isabel Garcia-Fructuoso, Barbara Adamo, Nuria Chic, Olga Martínez-Sáez, Tomás Pascual, Reinaldo Moreno, Montserrat Muñoz, Fara Brasó-Maristany, Aleix Prat, Francesco Schettini, Maria Vidal. Clinico-Pathological and Molecular Characterization of HER2-Enriched Breast Tumors Independently of HER2 Status [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-16.

Sample Preparation Approach Influences PAM50 Risk of Recurrence Score in Early Breast Cancer.

Simple SummaryThe PAM50 risk of recurrence (ROR) score is predictive of the risk of distant recurrence and the benefits of adjuvant therapy in early breast cancer. The Prosigna assay utilizes RNA from tumor cells selected via macrodissection of formalin-fixed, paraffin-embedded (FFPE) tissue sections to measure the activity of the PAM50 genes. An alternative and widely used extraction method is RNA purification from fresh-frozen (FF) bulk tissue without enriching the tumor cellularity. However, the impact of the RNA preparation approach on ROR scores and subsequent treatment selection has not been systematically evaluated. We compared the different approaches and found high correlation between risk of recurrence scores estimated from macrodissected FFPE tissue and scores estimated from bulk FF tumor tissue. However, important discrepancies were revealed for luminal tumors, which may have consequences for treatment recommendations for these patients.The PAM50 gene expression subtypes and the associated risk of recurrence (ROR) score are used to predict the risk of recurrence and the benefits of adjuvant therapy in early-stage breast cancer. The Prosigna assay includes the PAM50 subtypes along with their clinicopathological features, and is approved for treatment recommendations for adjuvant hormonal therapy and chemotherapy in hormone-receptor-positive early breast cancer. The Prosigna test utilizes RNA extracted from macrodissected tumor cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, RNA extracted from fresh-frozen (FF) bulk tissue without macrodissection is widely used for research purposes, and yields high-quality RNA for downstream analyses. To investigate the impact of the sample preparation approach on ROR scores, we analyzed 94 breast carcinomas included in an observational study that had available gene expression data from macrodissected FFPE tissue and FF bulk tumor tissue, along with the clinically approved Prosigna scores for the node-negative, hormone-receptor-positive, HER2-negative cases (n = 54). ROR scores were calculated in R; the resulting two sets of scores from FFPE and FF samples were compared, and treatment recommendations were evaluated. Overall, ROR scores calculated based on the macrodissected FFPE tissue were consistent with the Prosigna scores. However, analyses from bulk tissue yielded a higher proportion of cases classified as normal-like; these were samples with relatively low tumor cellularity, leading to lower ROR scores. When comparing ROR scores (low, intermediate, and high), discordant cases between the two preparation approaches were revealed among the luminal tumors; the recommended treatment would have changed in a minority of cases.

Abstract C045: Outcomes of hormone-receptor positive, HER2-negative breast cancers by race and tumor biologic features

Abstract Background: Black women have higher breast cancer mortality than white women, particularly within the hormone receptor positive, human epidermal growth factor receptor 2 negative (HR+/HER2-) clinical subtype. Interactions between tumor biology and treatment factors are complex and racial disparities in early events (such as 5-year recurrence risk) are not well characterized. Methods: Using data from the Carolina Breast Cancer Study (Phase 3, 2008-2013), a large population-based study enrolling 50% black and 50% white women from 44 counties in North Carolina, we estimated associations between race and recurrence among non-metastatic HR+/HER2- tumors, overall and within subgroups defined by PAM50 Risk of Recurrence score (ROR-PT, calculated from subtype, proliferation level, and tumor size), PAM50 intrinsic subtype, and tumor grade. We also compared treatment patterns by race among HR+/HER2- patients with high-risk disease. Relative frequency differences (RFD), interpretable as the percentage difference between index and referent groups, were estimated using multivariable linear regression. Recurrence risk was estimated using survival curves standardized for age and stage and inverse probability-weighted Cox models. Results: Among 1,775 eligible women, black women had higher recurrence risk relative to white women (crude hazard ratio: 1.8, 95% confidence interval [CI]: 1.3, 2.5), which remained elevated after standardizing for age and clinical covariates (hazard ratio: 1.4, CI: 1.0, 1.9). In stratified analyses, racial disparities persisted among women with high ROR-PT score (5-year standardized recurrence risk 18.9% in black vs. 12.5% in white women) and in high grade patients (5-year standardized recurrence risk 16.6% in black vs. 12.0% in white women). Black women with high grade tumors were significantly less likely to initiate endocrine therapy (RFD: -8.4%, CI: -15.9, -1.0) and experienced treatment delay more often than white women (RFD: +10.4%, CI: 1.8, 19.0). Conclusions: Racial disparities in HR+/HER2- breast cancer recurrence persist within high-risk subgroups. Efforts to identify treatment inequities and other causes of variation in cancer treatment are critical to reducing outcome disparities. Note: This abstract was not presented at the conference. Citation Format: H. Shelton Earp, Andrew F Olshan, Lisa A Carey, Charles M Perou, Melissa A Troester, Halei C. Benefield, Katherine E Reeder-Hayes, Hazel B Nichols, Benjamin C Calhoun, Michael I Love, Xuezheng Sun, Erin L Kirk, Joseph Geradts, Katherine A Hoadley, Stephen R Cole. Outcomes of hormone-receptor positive, HER2-negative breast cancers by race and tumor biologic features [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C045.

Abstract P2-18-06: Value of PAM50 risk of recurrence score in patients with HR positive HER2 negative early breast cancer and intermediate risk: A single cancer center institution prospective observational study

Abstract Background: Currently, clinico-pathological characteristics (CPC) such as tumor size, lymph node status, tumor grade, proliferation, lympho-vascular invasion and percentage of cells expressing estrogen (ER) and progesterone receptors (PR) are usually considered in order to guide decisions on adjuvant systemic therapy for women with early-stage breast cancer (EBC). The indication of adjuvant chemotherapy (ACT) or not is relatively easy in women with ER+/HER2- EBC with either poor or very good CPC. However, this decision is much more difficult in those with intermediate CPC (essentially defined by grade II tumor with low PR expression and/or intermediate proliferation). The PAM50-based Prosigna risk of recurrence (ROR) is one of the multigene assays that have been evaluated and recommended for guiding treatment decision. We report here our experience on the impact of ROR in treatment decision-making in women with intermediate CPC. Patients and methods: Between March 2016 and May 2019, ROR was performed in 200 consecutive women with intermediate CPC ER+/HER2- EBC. The clinical decision (ACT or not) was prospectively recorded before and after ROR assessment and finally after the consultation with each patient. Data were summarized by frequency and percentage for categorical variables and by median and range for continuous variables. Associations between variables will be assessed using Chi-square or Fisher’s exact test for qualitative variables and the Mann-Whitney for continuous variables. All reported p-values were two-sided. For all statistical tests, differences were considered significant at the 5% level. Statistical analysis was performed using STATA v13 software. Results: Median age at diagnostic was 57 years [30-79] and 119 (59.5%) were post-menopausal. Median tumor size was 18mm [8-70mm]. Most tumors were PR+ (n=153, 76.5%), no special type (NST) carcinomas (n=172, 86%), Elston and Ellis grade II (n=183, 91.5%), with Ki67 value between 10 and 20 % (n=117, 62.6%). Lympho vascular invasion was observed in 38 tumors (19%), 124 patients were pN0 (62%), 20 pNmi (10%) and 55 pN1a (27.5%). Using the immunohistochemical (IHC)-based definition proposed by Prat et al. (JCO 2013), 90 tumors were luminal A (45%) and 110 luminal B (55%). There was 59.5% of concordance between IHC subtype classification and PAM50. ROR distribution was as follows: ROR-low (n=33; 16.5%); ROR-intermediate (n=75; 37.5%); and ROR-high (n=92; 46%). The clinical decision of modalities of adjuvant medical therapy switched after ROR assessment for 74 patients (37%): -26 patients from ACT plus endocrine therapy (ET) to ET only (13%); 9 and 17 have respectively a ROR-low or -intermediate and 23 a luminal A tumor according to PAM50, -48 patients from ET only to ACT plus ET (24%); 7 and 41 have respectively a ROR-intermediate or high and 44 a luminal B tumor. Importantly, 8 of them don’t received ACT at the issue of the consultation with the medical oncologist. Conclusion: In our experience, the use of ROR leads us more often to escalate than de-escalate adjuvant therapy, proving that the definition of the group with intermediate prognosis remains uncertain and variable according medical teams. Citation Format: Vincent Nicolaï, Julia Gilhodes, Jean Louis Lacaze, Mony Ung, Eleonora De Maio, Raphaelle Duprez-Paumier, Charlotte Vaysse, Thomas Filleron, Eva Jouve, Gabrielles Selmes, Camille Franchet, Carole Massabeau, Cyprian Chira, Francoise Izar, Henri Roché, Anne Pradines, Florence Dalenc. Value of PAM50 risk of recurrence score in patients with HR positive HER2 negative early breast cancer and intermediate risk: A single cancer center institution prospective observational 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 P2-18-06.

On-treatment biomarkers can improve prediction of response to neoadjuvant chemotherapy in breast cancer

BackgroundNeoadjuvant chemotherapy is increasingly given preoperatively to shrink breast tumours prior to surgery. This approach also provides the opportunity to study the molecular changes associated with treatment and evaluate whether on-treatment sequential samples can improve response and outcome predictions over diagnostic or excision samples alone.MethodsThis study included a total of 97 samples from a cohort of 50 women (aged 29–76, with 46% ER+ and 20% HER2+ tumours) with primary operable breast cancer who had been treated with neoadjuvant chemotherapy. Biopsies were taken at diagnosis, at 2 weeks on-treatment, mid-chemotherapy, and at resection. Fresh frozen samples were sequenced with Ion AmpliSeq Transcriptome yielding expression values for 12,635 genes. Differential expression analysis was performed across 16 patients with a complete pathological response (pCR) and 34 non-pCR patients, and over treatment time to identify significantly differentially expressed genes, pathways, and markers indicative of response status. Prediction accuracy was compared with estimations of established gene signatures, for this dataset and validated using data from the I-SPY 1 Trial.ResultsAlthough changes upon treatment are largely similar between the two cohorts, very few genes were found to be consistently different between responders and non-responders, making the prediction of response difficult. AAGAB was identified as a novel potential on-treatment biomarker for pathological complete response, with an accuracy of 100% in the NEO training dataset and 78% accuracy in the I-SPY 1 testing dataset. AAGAB levels on-treatment were also significantly predictive of outcome (p = 0.048, p = 0.0036) in both cohorts. This single gene on-treatment biomarker had greater predictive accuracy than established prognostic tests, Mammaprint and PAM50 risk of recurrence score, although interestingly, both of these latter tests performed better in the on-treatment rather than the accepted pre-treatment setting.ConclusionChanges in gene expression measured in sequential samples from breast cancer patients receiving neoadjuvant chemotherapy resulted in the identification of a potentially novel on-treatment biomarker and suggest that established prognostic tests may have greater prediction accuracy on than before treatment. These results support the potential use and further evaluation of on-treatment testing in breast cancer to improve the accuracy of tumour response prediction.

Novel 18-gene signature for predicting relapse in ER-positive, HER2-negative breast cancer

BackgroundSeveral prognostic signatures for early oestrogen receptor-positive (ER+) breast cancer have been established with a 10-year follow-up. We tested the hypothesis that signatures optimised for 0–5-year and 5–10-year follow-up separately are more prognostic than a single signature optimised for 10 years.MethodsGenes previously identified as prognostic or associated with endocrine resistance were tested in publicly available microarray data set using Cox regression of 747 ER+/HER2− samples from post-menopausal patients treated with 5 years of endocrine therapy. RNA expression of the selected genes was assayed in primary ER+/HER2− tumours from 948 post-menopausal patients treated with 5 years of anastrozole or tamoxifen in the TransATAC cohort. Prognostic signatures for 0–10, 0–5 and 5–10 years were derived using a penalised Cox regression (elastic net). Signature comparison was performed with likelihood ratio statistics. Validation was done by a case-control (POLAR) study in 422 samples derived from a cohort of 1449.ResultsNinety-three genes were selected by the modelling of microarray data; 63 of these were significantly prognostic in TransATAC, most similarly across each time period. Contrary to our hypothesis, the derived early and late signatures were not significantly more prognostic than the 18-gene 10-year signature. The 18-gene 10-year signature was internally validated in the TransATAC validation set, showing prognostic information similar to that of Oncotype DX Recurrence Score, PAM50 risk of recurrence score, Breast Cancer Index and IHC4 (score based on four IHC markers), as well as in the external POLAR case-control set.ConclusionsThe derived 10-year signature predicts risk of metastasis in patients with ER+/HER2− breast cancer similar to commercial signatures. The hypothesis that early and late prognostic signatures are significantly more informative than a single signature was rejected.

Abstract P2-13-05: Racial differences in recurrence of hormone receptor-Positive breast cancers in the Carolina breast cancer study

Abstract Racial disparities in breast cancer outcome are most pronounced among women with hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR+/HER2-) disease. Several factors may contribute to this disparity, including biologic heterogeneity within the clinical HR+/HER2- subtype, and differences in access to treatment. In this study, we leverage the unique resources of the Carolina Breast Cancer Study Phase III (CBCS-III), a large prospective cohort study including tumor bio-specimens and detailed clinical annotation from black and white women with breast cancer, to examine potential contributors to racial disparities in recurrence of HR+/HER2- breast cancer. The CBCS-III cohort includes 2,998 women recruited between 2008 and 2013 by rapid case ascertainment from 44 counties in North Carolina, stratified by age (&amp;gt;50 versus &amp;lt;50) and race (black vs white). For this analysis, we included 1,629 women with stage I-III, HR+/HER2- clinical phenotype. We performed Cox proportional hazards regression to assess the effect of race on recurrence-free survival (RFS). Model 1 was minimally adjusted for age. Model 2 additionally adjusted for clinical factors likely to vary by race and to affect recurrence risk, including stage, grade, and the receipt of adjuvant chemotherapy. Finally, for patients who had specimens available (n=935), models were fit adjusting for clinical variables as well as PAM50 Risk of Recurrence score with both proliferation index and tumor size (ROR-PT) (Model 3) or molecular subtype dichotomized as Luminal A versus other (Model 4). With a median follow-up time of 5.5 years, 8.6% (118/1371) of whites and 14% (196/1403) among blacks had recurrence. After adjustment for age, black patients were at significantly higher risk for recurrence (HR=2.01, 95% CI 1.40-2.88), and this disparity was only partially attenuated by adjusting for baseline clinical factors (HR 1.62, 95% CI 1.11-2.36). Additional adjustment for molecular features by either ROR-PT score or molecular subtype further attenuated, but did not eliminate, the disparity in recurrence risk (HR=1.51, 95% CI 0.81,2.81 using ROR-PT and HR=1.44, 95% CI 0.77-2.69 using subtype category). Results are summarized in Table 1. Models of Recurrence Free Survival by Race (white = ref) HR95% CIModel 12.01(1.40, 2.88)Model 21.62(1.11, 2.36)Model 31.51(0.81, 2.81)Model 41.44(0.77, 2.69)Adjusted for: (1) age; (2)age, AJCC stage, grade, chemo; (3)ROR-PT, assay technique factor, and variables in Model 2; (4)PAM 50 subtype, assay technique factor, and variables in Model 2. In this recent population-based cohort, black women with HR+/HER2 negative disease remain twice as likely as their white counterparts to experience a recurrence within 5 years. Clinical features at disease presentation do not fully explain these outcome differences, while adjustment for biologic heterogeneity by either ROR-PT score or molecular subtype attenuates the observed disparity more than clinical features alone. There are likely residual factors not measured in this analysis, such as endocrine therapy under-use or other biologic differences, which may be targetable determinants of survival disparities. These hypotheses are being actively explored in CBCS-III. Citation Format: Reeder-Hayes KE, Sun X, Olshan A, Carey LA, Troester MA. Racial differences in recurrence of hormone receptor-Positive breast cancers in the Carolina breast cancer study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-13-05.

PAM50 Risk of Recurrence Score Predicts 10-Year Distant Recurrence in a Comprehensive Danish Cohort of Postmenopausal Women Allocated to 5 Years of Endocrine Therapy for Hormone Receptor-Positive Early Breast Cancer.

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.

The ability of PAM50 risk of recurrence score to predict 10-year distant recurrence in hormone receptor-positive postmenopausal women with special histological subtypes

Introduction: 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.Methods: 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 size >20 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.Results: 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).Conclusion: 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 size >20 mm or 1 to 3 positive lymph nodes) and ER-positive, HER2-normal early breast cancer.

Abstract P2-05-16: Establishment of molecular profiling for individual treatment decisions in early breast cancer – Clinical impact of PAM50 and PAM50 risk of recurrence score after more than 16 years follow up

Abstract Background Molecular profiling has recently been included in recommendations for decisions on adjuvant treatment in breast cancer (BrCa). However, the use of molecular profiling has not yet been widely established in all countries. Additional studies may give important information about the clinical relevance of the tests. Aims The study aims to discover the long term prognostic impact of PAM50 and PAM50 ROR score on survival for early BrCa pts according to treatment, with comparison to the routine clinical and histopathological parameters. Patients and methods Unselected early BrCa pts (n=651) from the Oslo Micromet project (n=920) having available FFPE primary tumor tissue were included in the current study. The pts were enrolled from 1995-1998. Follow up status is available for distant disease (median FU 7 years) and BrCa death (16.0-19.7 years after study inclusion). Clinical and histopathological parameters have been collected from the hospital records. FFPE tissue sections were macrodissected, RNA isolated from the dissected tumor tissue, followed by analysis of the PAM50 gene list on the Nanostring Platform. The samples were run in research mode and the raw data was sent to Nanostring (Seattle) for determination of the PAM50 subtype and ROR score. Results Of the 651 included pts, 323 did not receive any adjuvant systemic treatment (pT1pN0 patients), 161 tamoxifen only, the rest chemotherapy+/-tamoxifen. Twelve preoperatively treated pts were excluded from the analyses. Of the 639 remaining pts, PAM50 molecular profiling defined 52.3% as LumA, 26.8% LumB, 10.6% HER2enriched and 10.3% Basal. Multivariate analysis showed that the PAM50 intrinsic subtypes yielded prognostic information in addition to the established clinicopathological variables (pT, Grade, pN, age HR/HER2 subgroups, systemic treatment)(BCSS: HazardR vs LumA: 2.7 (95% CI 1.7-4.1) for LumB, 3.5 (1.8-6.8) for HER2enriched, 1.8 (0.8-4.2) for Basal). For the HR+HER2- pts, the risk classification by ROR score was an independent prognostic factor (BCSS: HazardR vs low risk: 3.1 (1.2-8.1) for intermediate, 6.6 (2.5-17.1) for high risk). In univariate analysis, the PAM50 intrinsic subtype classification separated clinical outcome both for all pts, for no adjuvant treated pts (both p&amp;lt;0.001, log rank), for the HR+HER2- (p&amp;lt;0.001), HR+HER2+ (p=0.061) and HR-HER2- (p=0.015) subgroups. Among the pT1-2pN0 HR+HER2- pts with no adjuvant treatment (n=222), risk classification by ROR score categorized 52.7% of the pts as low risk with excellent prognosis (BrCa death 4.2%), 29.7% as intermediate risk (BrCa death 16.7%) and 17.6% as high risk (BrCa death 35.9%)(p&amp;lt;001, log rank). For the pT1-2pN0-1 HR+HER2- pts who received adjuvant tamoxifen only (n=102), a low and similar risk of BrCa death was observed among the low and intermediate ROR risk groups. The high risk group had poor prognosis (BrCa death 32.7%)(p&amp;lt;0.001). Similar results were obtained for patients classified as LumA. Conclusions PAM50 subtype classification and ROR score improves classification of BrCa pts into prognostic groups, allowing more precise identification of future recurrence risk and improved basis for adjuvant treatment decisions. Citation Format: Naume B, Borgen E, Falk RS, Ohnstad HO, Lien TG, Aaserud M, Sveli MAT, Kyte JA, Kristensen V, Geitvik G, Schlichting E, Wist E, Sørlie T, Russnes H. Establishment of molecular profiling for individual treatment decisions in early breast cancer – Clinical impact of PAM50 and PAM50 risk of recurrence score after more than 16 years follow up [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 P2-05-16.

Identifying clinically relevant prognostic subgroups of postmenopausal women with node-positive hormone receptor-positive early-stage breast cancer treated with endocrine therapy: a combined analysis of ABCSG-8 and ATAC using the PAM50 risk of recurrence score and intrinsic subtype.

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. 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. 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. 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.

Prediction of late distant recurrence after 5 years of endocrine treatment: a combined analysis of patients from the Austrian breast and colorectal cancer study group 8 and arimidex, tamoxifen alone or in combination randomized trials using the PAM50 risk of recurrence score.

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. 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. 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] χ(2) = 94.12, bivariable: LR χ(2) = 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. 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.

The PAM50 Risk-of-Recurrence Score Predicts Risk for Late Distant Recurrence after Endocrine Therapy in Postmenopausal Women with Endocrine-Responsive Early Breast Cancer

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. 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. 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: ΔLRχ(2) 15.32, P < 0.001; ROR-based risk groups: ΔLRχ(2) 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. 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.

Predicting distant recurrence in receptor-positive breast cancer patients with limited clinicopathological risk: using the PAM50 Risk of Recurrence score in 1478 postmenopausal patients of the ABCSG-8 trial treated with adjuvant endocrine therapy alone

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

Comparison of PAM50 Risk of Recurrence Score With Oncotype DX and IHC4 for Predicting Risk of Distant Recurrence After Endocrine Therapy

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. 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. ROR added significant prognostic information beyond CTS in all patients (Δ LR-χ(2) = 33.9; P < .001) and in all four subgroups: node negative, node positive, HER2 negative, and HER2 negative/node negative; more information was added by ROR than by RS. C indices in the HER2-negative/node-negative subgroup were 0.73, 0.76, and 0.78 for CTS, CTS plus RS, and CTS plus ROR, respectively. More patients were scored as high risk and fewer as intermediate risk by ROR than by RS. Relatively similar prognostic information was added by ROR and IHC4 in all patients but more by ROR in the HER2-negative/node-negative group. 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.

Identifying clinically relevant prognostic subgroups in node-positive postmenopausal HR+ early breast cancer patients treated with endocrine therapy: A combined analysis of 2,485 patients from ABCSG-8 and ATAC using the PAM50 risk of recurrence (ROR) score and intrinsic subtype.

506 Background: Most postmenopausal women with node positive HR+ EBC receive adjuvant chemotherapy. We hypothesized that a molecular-based characterization of residual risk after endocrine therapy using the ROR score and IS may identify node-positive patient subgroups with limited long-term recurrence risk after endocrine therapy better than clinical-pathological risk assessment by clinical treatment score (CTS) alone. Methods: Long-term follow-up and tissue samples were obtained from 2,485 postmenopausal HR+ patients from the ABCSG-8 (N=1,478) and transATAC (N=1,007) trials. The PAM50 test was conducted on RNA extracted from paraffin blocks using the NanoString nCounter Analysis system. The ability of ROR, IS and ROR-defined risk groups (ROR-RG) to add prognostic information to CTS was assessed by the likelihood ratio test in a prospectively defined analysis plan. Results: Patients in the combined data set were grouped by the number of positive nodes into 1 (N1), 2 (N2), or 2 or 3 (N2-3),Baseline hazards for these subgroups were similar in the two trials. ROR score, IS and ROR-RG added statistically significant prognostic information (10-year distant recurrence risk) beyond CTS in all groups. In patients with one positive node, the absolute 10-year risk of distant recurrence was 6.6% [95% CI: 3.3%-12.8%] in the PAM-50-low risk group (40% of patients) and 8.4 % [5.3%-13.3%] in the Luminal A subgroup (69% of patients). Conclusions: The results of this combined analysis demonstrate that a significant proportion of N1 EBC patients have very limited long term recurrence risk and suggest the same for some N2 patients. The PAM50 ROR score, IS and ROR-RG reliably provide additional prognostic information beyond CTS and may be useful in deciding which women with node-positive HR+ EBC can be spared adjuvant chemotherapy. [Table: see text]