Multigene Expression Assay Research Articles

Abstract PO2-19-07: PROOFS – Pre-/perimenopausal patients with HR+/HER2- EBC with intermediate to high clinical and low genomic risk, treated by endocrine treatment (ET) plus ovarian function suppression (OFS) or chemotherapy followed by ET

Abstract Background During the past years, clinical routine has shifted away from relying solely on clinicopathologic factors toward increasing use of multigene expression assays in guiding treatment decisions regarding adjuvant chemotherapy (CT) for hormone receptor-positive (HR+), HER2-negative (HER2-) early breast cancer (EBC) with 0 to 3 positive lymph nodes. In case of a low genomic risk, there is strong consensus in favor of endocrine treatment (ET) alone for postmenopausal patients, regardless of clinical risk (supported by data from planB, ADAPT, RxPonder, TailorX, MINDACT). However, there is still uncertainty regarding optimal therapy for pre- and perimenopausal patients with a similar clinical/genomic risk profile. Since the observed benefit of CT in this patient group may be partly attributable to CT-induced ovarian function suppression (OFS), some of these patients could be spared CT by adding OFS to endocrine therapy. The West German Study Group (WSG) initiated the PROOFS-registry to create a real-world database on how to optimally treat these patients. Eligibility Criteria PROOFS (NCT05792150) aims at the long-term follow up of pre- or perimenopausal patients with HR+/HER2- EBC with an intermediate to high clinical risk of recurrence and low genomic risk measured by MammaPrint(R). Additional endocrine response assessment (Ki67) after a short preoperative endocrine therapy is encouraged. Up to 25% of the study population may have node-positive disease. Participants undergo standard-of-care treatment at physician´s discretion and according to clinical routine, which may include CT neoadjuvant or adjuvant + ET, ET + OFS in premenopausal, or ET +/- OFS in perimenopausal women. Specific Aims PROOFS aims to give insights in the real-world use of OFS and to confirm an excellent outcome in pre-/perimenopausal women with HR+/HER2- early BC with an intermediate to high clinical risk of recurrence and low genomic risk treated by ET +/- OFS alone (without CT). In addition, quality of life is captured. Statistical Methods The primary objective is to demonstrate the 5-year distant recurrence-free interval (dRFI) in patients treated by ET +/- OFS alone. The null hypothesis H(0) is: 5-year dRFI < 92%. Secondary endpoints for all patients are 5- and 10-year dRFI, distant disease-free survival (dDFS), overall survival (OS), breast cancer-free interval (BCFI), quality of life (QLQ BR23 and QLQ-C30) and therapy adherence. Cox regression models, Kaplan-Meier method and log-rank tests will be applied for survival analysis. Linear mixed models will be utilized to quantitatively describe the course of quality of life-scores as well as therapy adherence, and to conduct group comparisons. Present and Target Accrual The study is open for recruitment of 1500 patients (approximately 20% receiving CT and 80% receiving ET +/- OFS) at up to 100 centers in Germany (June 2023: 24 open for recruitment). Funding AGENDIA N.V., Amsterdam Contact Information rachel.wuerstlein@med.uni-muenchen.de Citation Format: Rachel Wuerstlein, Lotta Ada Fischer, Nadia Harbeck, Monika Graeser, Ulrike Nitz, Matthias Christgen, Sherko Kuemmel, Christine zu Eulenburg, Matthias Warm, Oleg Gluz. PROOFS – Pre-/perimenopausal patients with HR+/HER2- EBC with intermediate to high clinical and low genomic risk, treated by endocrine treatment (ET) plus ovarian function suppression (OFS) or chemotherapy followed by ET [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-19-07.

Association between 21-gene-assay and detection of disseminated tumor cells in patients with early breast cancer: results from the IRMA trial

PurposeDisseminated tumor cells (DTCs) in the bone marrow (BM) are known to be of prognostic value for patients with early breast cancer (EBC). In addition to histopathological features, multigene expression assays, such as the commercially available 21-gene Breast Recurrence Score® assay, have been validated for evaluating prognosis and making decisions concerning adjuvant treatment in EBC. In a previous retrospective study from our group, the 21-gene assay was shown to be associated with DTC-detection. A secondary endpoint of the prospective IRMA trial was to evaluate the association between Recurrence Score® (RS) result and tumor cell dissemination in patients with EBC.MethodsDTC-status and RS result were assessed in patients with ER-positive/HER2-negative EBC with 0–3 pathologic lymph nodes who underwent primary surgical treatment at the Department for Women’s Health of Tuebingen University, Germany.ResultsPatients with a high RS result (≥ 26) were more frequently DTC-positive (22.6%) than patients with a low RS result (8.6%, p = 0.034). The odds for DTC-positivity increased with rising RS values (p = 0.047).ConclusionWe therefore confirm that a high genomic risk is associated with tumor cell dissemination into the BM. Further trials are needed to investigate whether therapeutic decisions could be further individualized by combining DTC-status and prognostic gene signature testing.

Prognostic and predictive impact of gene expression in node-positive early breast cancer patients receiving dose-dense versus standard-dose adjuvant chemotherapy.

The utility of multigene expression assays in advanced (≥ 4 positive lymph nodes) early breast cancer (EBC) is limited. We conducted exploratory transcriptomic analysis of 758 genes (Breast Cancer 360 panel, nCounter® platform; NanoString) in primary tumor samples collected during a phase 3 trial comparing adjuvant taxane-containing dose-dense chemotherapy (ddCTX) versus standard-dosed chemotherapy (stCTX) in resected EBC with ≥ 4 positive lymph nodes. Prognostic and predictive associations with disease-free survival (DFS) and overall survival (OS) were evaluated by Cox regression with false discovery rate (FDR) adjustment. Data were available from tumor samples of 141/226 patients (median follow-up: 14 years). Several genes/signatures, including immune markers, showed prognostic relevance in unadjusted analyses. Of these, two remained significant after multiplicity adjustment: a positive effect on DFS of programmed cell death 1 ligand-2 (PD-L2) in the ddCTX arm (univariate HR: 0.53, FDR-adjusted P = 0.036) and a negative effect on OS of HER2-enriched (HER2-E) signature in the stCTX arm (univariate HR: 5.40, FDR-adjusted P = 0.036). Predictive analyses showed greater DFS benefit of ddCTX in tumors with high antigen processing machinery (APM) expression (multivariate interaction P = 0.024). Multigene expression assays have a prognostic and predictive potential in advanced EBC, and further investigation is warranted in order to identify candidates for de-escalated treatment. In addition, intrinsic subtype and immune gene expression have predictive potential.

Abstract P6-01-27: Utilization of the Oncotype Dx Assay for Young Patients with Early Stage, Hormone-Receptor Positive, HER2-Negative Breast Cancer in an Integrated Health System

Abstract Background: Oncotype Dx (ODX) is one of the most widely used prognostic multigene expression assays for early-stage, hormone receptor-positive (HR+), HER2-negative (HER2-) breast cancer. Previous studies demonstrated reduction in chemotherapy in patients with a low ODX Recurrence Score (RS). Over recent years, utilization of this tool has increased in young breast cancer (YBC) patients (age≤40 years), a population often underrepresented in randomized clinical trials. The purpose of this retrospective study was to assess the utilization of this assay over time in an integrated health system and whether ODX RS results altered clinical practice. Methods: YBC patients with T1-T2N0 HR+HER2- breast cancer were retrospectively evaluated. Descriptive analysis examined the association between tumor characteristics, year of diagnosis, ODX testing, treatment, and recurrence outcomes. ODX risk categories were defined as: low risk 0-15; intermediate risk 16-25; high risk > 25. Recurrence was determined by the date of confirmation on pathology or imaging. Bivariate analysis compared characteristics between groups using Fisher exact tests for categorical variables and t-tests and nonparametric tests for continuous variables. Results: From 2008-2018, 1,436 Stage I-III YBC patients were diagnosed with invasive breast cancer, and 455 met eligibility criteria for ODX testing. Median follow-time (interquartile range, IQR) was 4.9 (2.8, 7.9) years for the 255 women who were tested and 5.7 (3.5, 8.7) years for the 200 women who were not tested (p< 0.05). Prior to 2018, 52.1% of patients were tested; after 2017, ODX testing rate increased to 88%. Overall, there were 255 patients who underwent ODX testing (55.9%). The median age (IQR) of patients who had an ODX test was 38.0 (35.0, 39.0). Of 225 tested patients, 42.0% (n=107) were White, 6.3% (n=16) Black, 33.7% (n=86) Asian/Pacific Islander, 15.7% (n=40) Hispanic, and 2.3% (n=6) identified as Other. There was no overall difference in testing based on ethnicity (p=0.42). More patients with grade 1 versus grade 3 disease were tested, 61.5% vs 45.2% (p=0.02 from overall Fisher exact test). Adjuvant chemotherapy was administered to 61.0% (122/200) patients who were not tested, whereas 38.4% (98/255) of those tested received chemotherapy (p< 0.001). In tested patients, 6% of low-risk (RS 0-15), 37% of intermediate risk (RS 16-25), and 92% of high risk (RS >25) patient received adjuvant chemotherapy. Among patients with T2 lesions, a higher proportion not tested (90.8% [59/65]) received adjuvant chemotherapy compared with those not tested (57.1% [40/70]). There were no differences in recurrence based on ODX testing, 11.0% (22/200) not tested vs. 9.4% (24/255) tested (p=0.26). Conclusions: Utilization of ODX testing increased after 2017. A significantly lower proportion of women who underwent ODX testing received chemotherapy, compared with women not tested for ODX. A higher percent of women with T2 cancer received chemotherapy if testing was not completed, which may reflect a greater fear of recurrence in younger patients. Further investigation is needed to better understand this potential risk of overtreatment in the YBC population. Citation Format: Ashley Aller, Jeanne A. Darbinian, Raymond Liu, Gillian Kuehner, Alison Savitz, Patience Odele, Laurel Habel, Brooke Vuong. Utilization of the Oncotype Dx Assay for Young Patients with Early Stage, Hormone-Receptor Positive, HER2-Negative Breast Cancer in an Integrated Health System [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 P6-01-27.

Recurrence Score® Result Impacts Treatment Decisions in Hormone Receptor-Positive, HER2-Negative Patients with Early Breast Cancer in a Real-World Setting-Results of the IRMA Trial.

Patients with hormone receptor-positive (HR+), HER2-negative (HER2-) early breast cancer (eBC) with a high risk of relapse often undergo adjuvant chemotherapy. However, only a few patients will gain benefit from chemotherapy. Since classical tumor characteristics (grade, tumor size, lymph node involvement, and Ki67) are of limited value to predict chemotherapy efficacy, multigene expression assays such as the Oncotype DX® test were developed to reduce over- and undertreatment. The IRMA trial analyzed the impact of Recurrence Score® (RS) assessment on adjuvant treatment recommendations. The RS result was assessed in patients with HR+/HER2- unilateral eBC with 0-3 pathologic lymph nodes who underwent primary surgical treatment at the Department for Women's Health of Tuebingen University, Germany. Therapy recommendations without knowledge of the RS result were compared to therapy recommendations with awareness of the RS result. In total, 245 patients underwent RS assessment. Without knowledge of the RS result, 92/245 patients (37.6%) would have been advised to receive chemotherapy. After RS assessment, 56/245 patients (22.9%) were advised to undergo chemotherapy. Chemotherapy was waived in 47/92 patients (51.1%) that were initially recommended to receive it. Chemotherapy was added in 11/153 patients (7.2%) that were recommended to not receive it initially. Using the RS result to guide adjuvant treatment decisions in HR+/HER2- breast cancer led to a substantial reduction of chemotherapy. In view of the results achieved in prospective studies, the RS result is among other risk-factors suitable for the individualization of adjuvant systemic therapy.

52P RNA sequencing-based single sample predictors of molecular subtype and risk of recurrence for clinical assessment of early-stage breast cancer

Multigene expression assays for molecular subtypes and biomarkers can aid clinical management of early invasive breast cancer. Based on RNA-sequencing we aimed to develop single-sample predictor (SSP) models for conventional clinical markers, molecular intrinsic subtype and risk of recurrence (ROR).

Photoperiod in recirculation aquaculture systems and timing of seawater transfer affect seawater growth performance of Atlantic salmon (Salmo salar)

AbstractProduction of Atlantic salmon smolts in recirculation aquaculture systems (RAS) is growing, and novel production protocols using continuous light in RAS are being implemented in the industry. In the present study, Atlantic Salmon parr were exposed to either a traditional protocol (short‐day winter signal [12:12 L:D] for 6 weeks) or to continuous light. Both photoperiods were applied in freshwater (FW) and brackish water RAS. Salmon from all treatments were transferred to seawater pens at 200 and 600 g and grown until slaughter size. A control group was smoltified with a 6‐week short‐day winter signal and kept in FW until sea transfer at 100 g. Continuous light gave a higher growth rate in RAS but reduced feed intake and growth and increased feed conversion ratio during the first 8 weeks in seawater. However, at slaughter, fish exposed to continuous light was bigger than fish given a winter signal because of the higher growth rate in RAS. Slaughter weight was lowest in fish transferred to sea at 600 g, despite having the highest day‐degree sum during their life span. The best performing group was the control group transferred at 100 g. All treatments handled transfer to seawater and survival and maturation were not affected by the treatments in RAS. The immune status was examined with a multigene expression assay on BioMark HD platform from parr stage to 5–7 months after seawater transfer. Overall, there was no significant effect of photoperiod or salinity on the expression of the selected immune genes. In sum, the results from this study indicate that using continuous light in RAS may have negative effects on performance shortly after transfer in fish transferred to sea at 200 g, whereas at 600 g, all treatments had reduced growth after transfer irrespective of treatment in RAS.

Oncotype DX Recurrence Score in premenopausal women.

In the past 20 years, clinicians have shifted away from relying solely on clinicopathologic indicators toward increasing use of multigene expression assays in guiding treatment decisions regarding adjuvant chemotherapy for early-stage hormone receptor (HR)-positive, HER2-negative breast cancer. Oncotype DX Recurrence Score (RS) is one of the most widely used multigene assays when considering indications for adjuvant chemotherapy, and guidelines have recently incorporated its use in women with early HR-positive HER2-negative breast cancer and up to three positive lymph nodes. While multiple retrospective and prospective clinical studies have demonstrated that most women with a low- to mid-range RS (0–25) can safely forgo chemotherapy, premenopausal women remain an important subgroup for which recommendations based on RS are ill-defined. The majority of patients included in clinical trials and retrospective analyses validating the use of RS have been postmenopausal women. In the subgroup of premenopausal women with HR-positive HER2-negative breast cancer, studies indicate that traditional clinicopathologic methods for assessing risk continue to be powerful tools when combined with RS to predict benefit from chemotherapy. This suggests that there is an element of uncaptured risk inherent to the premenopausal state that evades characterization by RS alone. This review describes the evidence that has supported the recommendation of RS in clinical guidelines, specifically focusing on data for its current use in premenopausal women. We review available data regarding the impact of the menstrual cycle on hormonally regulated gene expression, which may drive variations in the RS. Further research on the reliability and interpretation of the RS in the premenopausal subgroup is necessary and represents a gap in knowledge of how the RS should be applied in premenopausal women.

The clinical utility of gene expression assays in breast cancer patients with 0-3 involved lymph nodes.

Multigene expression assays are prognostic for recurrence in hormone-receptor positive 2 (HER-2) negative breast cancer, and, in some cases, predictive of benefit from chemotherapy or extended endocrine therapy. The results of these assays may be used to guide treatment recommendations for early HER-2 negative breast cancer. We review the results of trials establishing the clinical utility of several commercially available gene expression assays.

Multigene Expression Assay for Assessment of the Immune Status of Atlantic Salmon.

We report the development of a multigene gene expression assay on the BioMark HD platform for the evaluation of immune competence (ImCom) in farmed Atlantic salmon. The first version of the assay included 92 genes selected on the basis of transcriptome analyses in 54 trials that challenged the immune system; annotations were taken into account to represent the key pathways of innate and adaptive immunity. ImCom was tested on samples collected from seven independent projects. Fish were reared from the start feeding to eight months in the sea at eight units in different parts of Norway. Several tissues were analyzed. Linear discriminant analysis (LDA) showed that no more than 10 genes were required to separate groups, and a set of 46 immune genes was sufficient for any task. The second version of the assay was tested in the gills of two groups of high-performing healthy smolts and in groups with intermediate and high mortality rates (IM and HM, respectively). A set of 645 gill samples from clinically healthy Atlantic salmon was used as a reference. The IM group showed general suppression of immunity. All HM group salmon were above the threshold by the squared deviation from the reference. This group showed marked upregulation of genes involved in acute stress and inflammation: mmp-9, mmp-13, hsp70, il-1b, lect2, and cathelicidin. Further work will clarify the boundaries of the norm and explore various cases of impaired immunity.

Machine learning for RNA sequencing-based intrinsic subtyping of breast cancer

Stratification of breast cancer (BC) into molecular subtypes by multigene expression assays is of demonstrated clinical utility. In principle, global RNA-sequencing (RNA-seq) should enable reconstructing existing transcriptional classifications of BC samples. Yet, it is not clear whether adaptation to RNA-seq of classifiers originally developed using PCR or microarrays, or reconstruction through machine learning (ML) is preferable. Hence, we focused on robustness and portability of PAM50, a nearest-centroid classifier developed on microarray data to identify five BC “intrinsic subtypes”. We found that standard PAM50 is profoundly affected by the composition of the sample cohort used for reference construction, and we propose a strategy, named AWCA, to mitigate this issue, improving classification robustness, with over 90% of concordance, and prognostic ability; we also show that AWCA-based PAM50 can even be applied as single-sample method. Furthermore, we explored five supervised learners to build robust, single-sample intrinsic subtype callers via RNA-seq. From our ML-based survey, regularized multiclass logistic regression (mLR) displayed the best performance, further increased by ad-hoc gene selection on the global transcriptome. On external test sets, mLR classifications reached 90% concordance with PAM50-based calls, without need of reference sample; mLR proven robustness and prognostic ability make it an equally valuable single-sample method to strengthen BC subtyping.

High FOXA1 protein expression might predict late recurrence in patients with estrogen-positive and HER2-negative breast cancer.

Multi-gene expression assays have been developed with the aim of predicting late recurrence in patients with estrogen receptor (ER)-positive breast cancer. However, establishment of alternative markers based on immunohistochemistry is also important for achieving practical use. Based on our previous study, forkhead box A1 (FOXA1) protein was tested as a potentially useful predictive marker for late recurrence. 117 patients with ER-positive HER2-negative invasive breast cancer who developed distant metastasis following curative surgery were retrospectively investigated. We also evaluated responsiveness to endocrine therapy according to FOXA1 expression. Furthermore, publicly available mRNA microarray data were analyzed to examine patterns of metastasis according to FOXA1 mRNA expression, employing the Kaplan-Meier plotter. High expression of FOXA1 was an independent factor predicting long disease-free survival (DFS), along with small tumor size (p = 0.010 and 0.016, respectively). Discrimination of DFS was improved by combining these two factors, i.e., patients with FOXA1-high small tumors had the longest DFS while those with FOXA1-low large tumors had the shortest DFS. Moreover, we revealed that risk of distant metastasis started to increase after the completion of adjuvant endocrine therapy in patients with FOXA1-high tumors. Among patients who developed distant metastasis, those with FOXA1-high tumors had significantly longer DFS. We believe our data to raise the possibility of FOXA1 being a useful predictive marker for late recurrence and to provide new insights into the biology of FOXA1-high breast cancers.

Adjuvant endocrine treatment for estrogen receptor (ER)-positive/HER2-negative breast cancer.

Approximately 70% of women with breast cancer worldwide receive a diagnosis of ER-positive/ HER2-negative disease. Recurrence-free survival of the node-negative population of these women is approximately 85%, and most are treated with endocrine chemotherapy. Endocrine treatment for ERpositive/HER2-negative early breast cancer is a major treatment strategy. Breast cancer clinicians have gained much experience with this issue, but new data regarding adjuvant endocrine therapy have recently been accumulated. In the integrated analyses of the Suppression of Ovarian Function Trial (SOFT) and Tamoxifen and Exemestane Trial (TEXT) trials, the addition of ovarian function suppression (OFS) to tamoxifen showed a significant improvement in both 8-year disease-free survival [hazard ratio (HR) 0.76, 95% confidence interval (CI): 0.62-0.93] and overall survival (HR 0.67, 95% CI: 0.48-0.92) compared with tamoxifen alone for premenopausal patients with breast cancer, with the assumption of 5 years as an appropriate duration for OFS. The ATLAS trial showed that, compared with 5 years of tamoxifen, 10 years of tamoxifen provided further benefit to women with HR-positive early breast cancer in recurrence [5-9 years: rate ratio (RR) 0.90, 95% CI: 0.79-1.07; ≥10 years: RR 0.75, 95% CI: 0.62-0.90; and all years: log-rank P=0.002] and breast cancer mortality (5-9 years: RR 0.97, 95% CI: 0.79-1.18; ≥10 years: RR 0.71, 95% CI: 0.58-0.88; and all years: log-rank P=0.01), particularly after reaching 10 years, which is considered a "carry over" benefit. In this review, adjuvant endocrine treatment for ER-positive/HER2-negative early breast cancer is discussed. Particular areas of focus are premenopausal patients, especially regarding the addition of OFS, postmenopausal patients, the duration of adjuvant endocrine therapy, the treatment implications of multigene expression assays, and adjuvant endocrine therapy for ductal carcinoma in situ. Some of this evidence supports the clinical management of breast cancer patients as well as clinical studies in the future.

Abstract P6-16-01: Refined local recurrence risk estimates based on a multigene expression assay combined with clinicopathological features significantly impacts radiotherapy recommendation in patients with low/intermediate risk DCIS treated with breast-conserving surgery

Abstract Background: Guidelines recommend that breast radiation (RT) can be omitted for patients with a low risk of local recurrence (LR) after breast-conserving surgery (BCS) for DCIS. The inability to identify women at low risk of LR (<10%) at 10 years after BCS has hampered efforts to de-escalate therapy for DCIS. The Oncotype DX Breast DCIS Score® (DS), a 12-gene expression assay, predicts LR risk after BCS. The revised DS report adds information from the DS with clinicopathological features (CPF) (age, tumor size, year) (BCRT 2018;169;358-690) to provide refined estimates of LR risk and can better identify patients with low 10-year LR risk (<10%) after BCS. We evaluated the impact of refined LR risk estimates on its ability to change radiation oncologists’ recommendations for RT in women with low/moderate risk DCIS treated with BCS. Methods: Prospective cohort study of women with low/moderate risk pure DCIS treated with BCS. Cases with age <45 yrs, margins <1mm, tumor >2.5cm, multifocality, or prior breast cancer were excluded. Baseline CPFs, the DS and risk of LR were collected. Pre-assay, the radiation oncologist provided an estimate of 10-year LR risk without RT and a preliminary recommendation for RT. Post-assay, final recommendations were recorded. The primary outcome was change in treatment recommendation by the radiation oncologist. Target sample size was 220 to provide data on 200 evaluable patients with adequate precision. Results: 217 patients were evaluable: mean age, 63 years and mean tumor size, 1.1 cm. Nuclear grade was low in 26 (12%), intermediate in 116 (53%), and high in 75 (35%) of patients. Mean DS = 32; 140 (64%) low (<39), 32 (15%) intermediate (39-54), 45 (21%) high risk DS (≥5). The assay lead to a change in treatment recommendation in 35.2% (76/216) (95% CI: 29.1-41.8) of cases. The proportion of cases recommended to receive RT decreased from 79% (N=171) pre-assay to 50% post-assay (p<.001). This was due to a significant increase in the proportion of cases (46%) with a predicted low risk of LR (<10%) post-assay and recommendations to omit RT for those with low LR risk. Pre-assay, physician estimates of 10-yr LR risks after BCS based on CPFs alone were <10% in 13 (6%) cases, 10-15% in 76 (35%) cases and >15% in 128 (59%) cases. Post-assay, estimated 10-yr LR risk after BCS was <10% in 101 (46%) patients (83% recommended no RT), 10-15% in 67 (31%) (RT recommended in 66%) and >15% in 49 (22%) (RT recommended in 98%). Conclusion: The use of the DCIS Score combined with CPFs identifies more women with an estimated low (<10%) 10-yr LR risk after BCS leading to a significant change in treatment recommendations with a decrease in recommendations for RT following BCS compared to CPFs alone. Table 1.Post-assay 10-yr LR risk<10%10-15%>15%(N=101)(N= 67)(N=49)Pre-assay estimated 10-yr LR risk• <10% (N=13)922• 10-15% (N=76)50188• >15% (N=128)424739RT recommended• Yes17 (17%)44 (66%)48 (98%)• No83 (83%)23 (34%)1 (2%) Citation Format: Eileen Rakovitch, Anne Koch, Laval Grimard, Hany Soliman, Christiaan Stevens, Francisco Perera, Iwa Kong, Senti Senthelal, Margaret Anthes, Ericka Wiebe, Jeffrey Cao, Mira Goldberg, Sally Smith, Luciana Spadafora, Sameer Parpia, Timothy Whelan. Refined local recurrence risk estimates based on a multigene expression assay combined with clinicopathological features significantly impacts radiotherapy recommendation in patients with low/intermediate risk DCIS treated with breast-conserving surgery [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 P6-16-01.

Abstract P3-08-09: The 21-gene recurrence score and chemotherapy use in triple negative breast cancer (TNBC) and HER2 positive breast cancer: A National Cancer Database study

Abstract Background The 21-gene (Oncotype DX) Recurrence Score (RS) is a multi-gene expression assay that is both prognostic and predictive of adjuvant chemotherapy (AdjCT) benefit in estrogen receptor (ER) positive/HER2 negative breast cancer (BC). Use of the RS in TNBC and HER2+ BC is not recommended by national guidelines. Using the National Cancer Database (NCDB), we sought to evaluate whether oncologists use the RS in these subtypes. Additionally, we assessed the prognostic effects of the RS in node negative patients (pts) who did or did not receive adjuvant chemotherapy. Methods Pts with TN and HER2+ BC diagnosed from 2010-2015 in the NCDB were analyzed. Pts with neoadjuvant therapy or stage IV were excluded. Cases with RS testing were classified as low (RS 0-17), intermediate (RS 18-30) or high (RS 31+). Analysis was performed using multivariable logistic regression; overall survival (OS) was analyzed using the Kaplan-Meier method with log-rank tests. Results 142,330 pts were evaluable: 64,830 TNBC and 77,500 HER2+ (21,768 ER-/HER2+ and 55,732 ER+/HER2+). In these subtypes, RS was performed in 5,369 (3.8%) pts as follows: 1,479 (2.3%) TNBC, 185 (0.8%) ER-/HER2+, and 3,705 (6.6%) ER+/HER2+. Given the small ER-/HER2+ cohort, we focused on TNBC and ER+/HER2+. Within these subtypes, factors associated with RS testing included lower grade, smaller pathologic tumor size, pathologic N0 status, white race, age 60-69, and ILC or IMC histology (each p<0.005). Of the TNBC pts tested, the RS distribution was: low 16.3%, intermediate 13.1% and high 70.6%, while for ER+/HER2+ it was low 30.5%, intermediate 30.7% and high 38.8% (p<0.001). AdjCT was less frequently recommended in pts with low RS; however, this varied by tumor subtype with AdjCT recommended for 66.8% of TNBC with low RS and 37.9% of ER+/HER2+ with low RS. For intermediate RS, rates of AdjCT recommendation were 74.3% and 73.7%, and for high RS were 94.0% and 93.2% for TNBC and ER+/HER2+ respectively. In a multivariable analysis among pts with low RS, factors associated with AdjCT recommendation included younger age, larger pathologic tumor size, node positive disease, and higher grade. In AdjCT untreated TNBC, 5 yr OS did not differ for low RS (96.5%) vs intermediate RS (95.2%, p=0.82). In contrast OS was significantly worse for high RS (76.7%) than the other two groups, each p≤0.04. In AdjCT untreated ER+/HER2+, 5 yr OS was significantly better for low RS (96.7%) vs intermediate RS (92.5%, p=0.03) and vs high RS (92.1%, p=0.003), with no difference between intermediate RS vs high RS (p=0.32). Evaluating pts who did receive AdjCT, there was no significant difference in 5 yr OS for either subtype according to RS (p=0.46 and p=0.83). Interestingly, in patients with low RS, 5 yr OS was similar with or without AdjCT in both TNBC (89.5% vs 96.5%, p=0.25) and ER+/HER2+ (97.6% vs 96.7%, p=0.47). Conclusions RS testing is being conducted in a small fraction of pts with TN and HER2+ BC with lower clinical risk features. The observation that RS is prognostic for survival in AdjCT untreated patients is hypothesis generating, and suggests that further evaluation of the RS and other multigene assays in ER negative and HER2+ BC is warranted. Citation Format: Boughey JC, Hoskin TL, Cocco D, Day CN, Leon-Ferre R, Habermann EB, Goetz MP. The 21-gene recurrence score and chemotherapy use in triple negative breast cancer (TNBC) and HER2 positive breast cancer: A National Cancer Database study [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 P3-08-09.

Abstract P4-12-05: Cost-effectiveness analysis of second-generation multi-gene expression prognostic assays compared with the standard 21-gene recurrence score assay to guide adjuvant therapy decisions in women with early stage breast cancer

Abstract Second-generation multi-gene expression assays can generate comprehensive molecular risk scores which may inform adjuvant chemotherapy decisions in women with early breast cancer. The 12-gene EPclin score assay (EndoPredict®) and 50-gene PAM50-risk of recurrence (ROR) score assay (Prosigna®) appeared to have better prognostic value when compared to the current north American standard 21-gene recurrence score (RS) assay (Oncotype DX®). We sought to investigate the cost-effectiveness of using EPclin and ROR score assays versus RS assay in women with axillary lymph node-negative (LN−), hormone receptor–positive (HR+), and human epidermal growth factor receptor 2–negative (HER2−) early-stage operable breast cancer (ESBC) from the perspective of the Canadian public healthcare system. We developed a Markov model to project the lifetime clinical and economic consequences of operable LN- HR+ HER2− ESBC. We assumed that women within each risk category by RS assay (low, intermediate and high) would be reclassified to binary risk categories (low and high) by EPclin score assay and to three risk categories (low, intermediate and high) by ROR score assay. The decision model was parameterized using 10-year follow up data from retrospective analyses of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial, cost data from the London Regional Cancer Program (Ontario, Canada) and secondary sources. Costs are presented in 2017 Canadian dollars. Future costs and benefits were discounted at 5%. EPclin and ROR score-based strategies led to an increase of 0.04 and 0.02 quality adjusted life years (QALY)/person and a decrease in cost of $917 and $600/person respectively, resulting in both strategies being cost-saving compared to RS-based strategy. Incorporating the EPclin and ROR score assays in place of the current standard RS-assay for operable LN- HR+ HER2− ESBC patients in Canada would result in total gains of 469 and 250 QALYs/year and total savings of $11.5 and $7.5 million/year, respectively. EPclin compared to ROR score-based strategy led to an increase of 0.02 QALY/person and a decrease in cost of $317/person, resulting in EPclin score-based strategy being dominant. Our results were most sensitive to the proportion of women classified by EPclin and ROR score assays to different risk categories and who received adjuvant chemotherapy. A value-of-information analysis revealed that the total expected value of perfect information about the EPclin and ROR score assays' clinical impact was $95 and $55 million/year, respectively. Our results indicate that the EPclin and ROR score assays are both clinically and economically attractive for patients with operable LN- HR+ HER2− ESBC in the Canadian healthcare setting. Both assays should be considered for adoption in place of the current standard RS-assay for this patient population. The EPclin compared to the ROR score assay appears to be clinically more promising and provides greater value for money in the Canadian healthcare system. Field evaluations of the EPclin and ROR score assays in real-world Canadian clinical practice are associated with a large societal benefit and warranted. Citation Format: Hannouf MB, Zaric GS, Brackstone M. Cost-effectiveness analysis of second-generation multi-gene expression prognostic assays compared with the standard 21-gene recurrence score assay to guide adjuvant therapy decisions in women with early stage breast cancer [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 P4-12-05.

Abstract P1-09-17: EndoPredict multigene test in the prediction of response and outcome after neoadjuvant hormonal treatment with letrozole adn palbociclib (OOTR-N007)

Abstract Background: The EndoPredict [EP] is a multi-gene expression assay designed to predict the replapse-free survival of patients with estrogen receptor [ER] positive, HER2 negative breast cancer treated with adjuvant endocrine therapy alone. Relative expression levels of the eight prognostic genes [AZGP1, BIRC5, DHCR7, IL6ST, MGP, RBBP8, STC2, UBE2C] and 3 reference genes were analyzed by real-time PCR system. EP low-risk patients have a better RFS when treated with endocrine therapy. One study indicated that EP high-risk patients are more sensitive to anathracycline-based neoadjuvant chemotherapy than low-risk patient. Palbociclib is an orally active potent and highly selective reversible inhibitor of CDK4/6. N007 study aims to evaluate the efficacy of the pre-operative use of letrozole plus palbociclib for ER positive, HER2 negative post-menopausal breast cancer. Whether EP could predict for response and outcome to such treatment will be evaluated. Method: Twenty patients were treated. EP assays were intended to be tested for all patients. RNA were extracted from paired FFPE tumour blocks [core biopsies and corresponding surgical sections]. EP score were calculated for the first 7 paired samples. Also the EP Clin Score which integrate both mRNA of prognostic genes expression and their pathological parameters, including tumor size and nodal status, were also evaluated. The change in EP score were compared with that of Ki67 and also with the pathologic tumor response. Results: The results of the first 7 patients were presented. The overall pCR rate rate was 14.3%[1/7]. 85.7% [6/7] samples were classified as low risk group. Paired T-test showed EP score is significantly lower [P=0.0014] after neoadjuvant hormonal treatment [NHT]. The change of EP score before and after treatment corresponds to the change in Ki67. Both have a similar prediction of response. EP scores either remained high or showed minimal change for patients with stable disease [SD]. The subsequent EP Clin score was high for one of such patients, indicating high clinical risk. The corresponding Ki67 did not show any change and was low after treatment. ResultsSubjectEP (before)EP (after)Ki67 (before)Ki67 (after)Change in EPChange in Ki67EP CLin0014.93.75%1%-1.2.4%2.70028.24.750%3%-3.5.47%2.3003107.113%13%-2.90%4.30045.6-10%--5.6-100%-0057.75.39%1%-2.4-8%2.80063.72.220%8%-1.5-12%2.60074.43.710%5%-0.7-5%3.4 Conclusion: pCR has been used as an endpoint for neoadjuvant chemotherapy trial. Our data suggest that the EP score can be reliably determined in core biopsy specimens and can be applied as endpoint on NHT trials. Changes in EP score correlated with that of Ki67 and also with response. However, changes in EP score may have a better prediction of response than Ki67. EP Clin score, which integrates clinical parameters, may predict subsequent risk and clinical outcome after treatment. The data of all the 20 patients will be presented at the meeting. Citation Format: Chow LWC, Toi M. EndoPredict multigene test in the prediction of response and outcome after neoadjuvant hormonal treatment with letrozole adn palbociclib (OOTR-N007) [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 P1-09-17.

Multigene Expression Assay and Benefit of Radiotherapy After Breast Conservation in Ductal Carcinoma in Situ.

Most women with ductal carcinoma in situ (DCIS) will receive breast-conserving surgery (BCS) and radiation (RT). RT can be omitted for women at low risk of local recurrence (LR). The Oncotype DX DCIS score (DS) predicts LR risk after BCS alone. This study assesses the impact of RT and DS on LR risk. Population-based cohort analysis of individuals with DCIS treated by BCS ± RT from 1994-2003. Treatment and outcomes were determined by linkage and chart review. We used a propensity score-adjusted multivariable model to examine associations between DS and LR and evaluate the impact of RT. All statistical tests were two-sided. The cohort includes 571 individuals treated by BCS alone, 689 cases treated with BCS + RT. Median follow-up was 9.4 years. On multivariable analysis, factors associated with LR include RT, age at diagnosis, tumor size, and multifocality. Adjusting for these factors, the DS risk group was statistically significantly associated with LR risk (hazard ratio high/intermediate = 1.75, 95% confidence interval = 1.28 to 2.41, P < .001). Women with a low-risk DS treated by BCS alone had an LR risk of 10.6% at 10 years and a small benefit from RT, while those with a high DS had a higher risk of LR (25.4%) after BCS alone and greater benefit from RT. A subgroup of patients with favorable clinicopathological features had a high-risk DS; these patients had a higher than expected risk of LR after BCS alone and a greater benefit with RT. The DS molecular assay improves risk stratification and estimates of RT benefit in individuals with DCIS treated with breast-conserving therapy.

Estimating the OncotypeDX score: validation of an inexpensive estimation tool.

OncotypeDX, a multi-gene expression assay, has been incorporated into clinical practice as a prognostic and predictive tool. However, its use in resource-constrained international healthcare systems is limited. Here we develop and validate a simplified model using clinicopathologic criteria to predict OncotypeDX score. Patients with estrogen receptor (ER) and/or progesterone receptor (PR)-positive and HER2-negative invasive ductal carcinoma for whom the OncotypeDX test was successfully performed between 09/2008 and 12/2011 were retrospectively identified. Tumor size, nuclear and histologic grade, lymphovascular invasion, and ER and PR status were extracted from pathology reports. Data were split into a training dataset comprising women tested 09/2008-04/2011, and a validation dataset comprising women tested 04/2011-12/2011. Using the training dataset, linear regression analysis was used to identify factors associated with OncotypeDX score, and to create a simplified risk score and identify risk cutoffs. Estrogen and progesterone receptors, tumor size, nuclear and histologic grades, and lymphovascular involvement were independently associated with OncotypeDX. The full model explained 39% of the variation in the test data, and the simplified risk score and cutoffs assigned 57% of patients in the test data to the correct risk category (OncotypeDX score<18, 18-30, >30). 41% of patients were predicted to have OncotypeDX score<18, of these 83, 16, and 2% had true scores of<18, 18-30, and>30, respectively. Awaiting an inexpensive test that is prognostic and predictive, our simplified tool allows clinicians to identify a fairly large group of patients (41%) with very low chance of having high-risk disease (2%).

145O - Prognostic role for derived neutrophil-to-lymphocyte ratio in early breast cancer

Inflammation is a hallmark of cancer. Elevated markers of host inflammation have been associated with worse outcomes in several solid tumors. Here, we explore the prognostic role of the derived neutrophil-to-lymphocyte ratio (dNLR) in subgroups of women with early breast cancer. This was a retrospective analysis of women with early breast cancer included in the GEICAM/9906 trial, a randomized phase III study of adjuvant FEC compared to FEC-paclitaxel (FEC-P) in breast cancer patients with axillary involvement. dNLR was calculated as the ratio of neutrophils divided by the difference between total leukocytes and neutrophils measured in peripheral blood before the start of chemotherapy. The primary objective was disease free survival (DFS) and overall survival (OS) was a secondary endpoint. Univariable Cox regression was used to explore the prognostic and predictive value of dNLR (explored as median cutoff and grouped in quartiles). Subgroups by PAM50 subtype and hormonal receptor expression were subsequently analyzed. The current analysis comprised 1243 patients from 65 Spanish sites with a median follow-up of 10 years. Median age was 50 years (range 23-76), 66% of patients had available PAM50 subtype determination, of which 22% of the tumors were Luminal A, 21% Luminal B, 14% Her2-enriched, 6% Basal-like, and 3% Normal-like. ER+/PgR+ and ER-/PgR- subtypes comprised 47% and 13% of evaluable tumors, respectively. Median dNLR was 1.35 (IQR 1.08 – 1.71). For Her2-enriched patients by PAM50, a dNLR > median was significantly associated with worse DFS, p = 0.03 (HR: 1.63; 95%CI: 1.04 – 2.54). For non-luminal patients by PAM50, a dNLR > median and a high dNLR explored by quartiles were associated with worse DFS (p = 0.02 and p = 0.03, respectively). For patients with ER-/PgR- tumors, a high dNLR grouped in quartiles was associated with worse DFS and OS (p < 0.001 and p = 0.007, respectively). dNLR is associated with worse DFS in women with Her2-enriched and non-luminal intrinsic subtypes defined by PAM50 multigene expression assay. High dNLR is also associated with worse DFS and OS in women with ER-/PgR- tumors (IHC). Further studies are warranted to confirm these findings.