Molecular Grade Index Research Articles

Immunohistochemical Expression of Caspase1 and Epidermal Growth Factor Receptor in Invasive Breast Carcinoma and Their Biological and Prognostic Associations.

Despite advances in breast carcinoma therapies, drug resistance mechanisms as anti-apoptosis and anti-pyroptosis limit the application of these therapies. This work assesses the immunohistochemical (IHC) expression of Caspase1 and EGFR in breast carcinoma and analyzes their clinicopathological associations as prognostic markers and potential therapeutic targets. Caspase1/EGFR expression patterns are utilized to specify breast carcinoma patients who may benefit from these therapies. After reviewing the hematoxylin and eosin-stained slides and the routine breast carcinoma IHC stains (estrogen receptors, progesterone receptors, HER2/NEU, Ki-67) by two pathologists and preparation of tissue microarray blocks, anti-Caspase-1 and EGFR IHC staining was performed using Horseradish Peroxidase (HRP) technique. Intensity and percentage-based scoring was applied dividing the 153 included breast carcinomas into Caspase1-negative and positive expression groups; and EGFR low and overexpression groups. Groups were statistically analyzed in relation to age, tumor size, histological and molecular subtype, grade, nodal status, metastasis/recurrence, TNM stage and Ki-67 proliferation index. Kaplan-Meier's analysis was used to compare disease-free survival (DFS) and overall survival (OS). Combined patterns based on Caspase1 and EGFR expression status were created to stratify patients into prognostic groups. Caspase1 was positive in 54.2% of breast carcinomas and its positivity was significantly associated with smaller tumor size, absence of metastasis/recurrence, luminal A and B molecular subtypes and longer OS (p<0.05). EGFR overexpression was detected in 32.7% of carcinomas and was significantly associated with larger tumor size, TNBLBC and a shorter OS (p<0.05). Caspase1-negative/EGFR-overexpression pattern comprised 14.4% of carcinomas and had the worst prognostic associations including larger tumor size, metastasis/recurrence, TNBLBC subtype and shortest OS (p=0.002, 0.002, 0.004 and ≤0.001 respectively). Conclusions: Combined Caspase1/EGFR IHC expression may provide a tool for selection of patients who benefit from combined EGFR-inhibitors with miR-155-5p down-regulators or photodynamic therapy via induction of apoptosis/pyroptosis in EGFR-overexpression carcinomas through enhanced Caspase1 signaling.

Abstract PO2-01-08: Differential HOXB13 gene expression and promoter methylation analysis in breast cancer

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature comprising two functional biomarker panels: the Molecular Grade Index (MGI) and BCI (H/I), which is a ratio of the HOXB13 and IL17BR gene expression. Several studies have shown that BCI (H/I) is a predictive biomarker for extended adjuvant endocrine therapy benefit in hormone receptor-positive (HR+) early-stage breast cancer. Molecular mechanisms underlying endocrine responsiveness remain to be fully explored. The objective of this analysis is to evaluate the correlation between gene expression and methylation in breast cancer patients displaying different HOXB13 mRNA expression levels from The Cancer Genome Atlas (TCGA) project. Methods: Data from methylation microarrays and mRNA sequencing were examined in combination with clinical metadata from 1,095 TCGA breast cancer patients. After removing outliers, HR+ samples were ordered based on their HOXB13 expression levels and the top 10% of samples (HOXB13-high; n=95) and bottom 10% (HOXB13-low; n=95) were selected for further analysis. Differentially expressed genes (DEGs) were determined by comparing all protein-coding genes between the HOXB13-high and HOXB13-low sets, based on an absolute log-fold change above 2 and a Benjamini-Hochberg adjusted p-value threshold of 0.01. Among the subset of samples with matching methylation data (n=60 for the HOXB13-high group and n=48 for the HOXB13-low group), hypo-methylated and hyper-methylated differentially methylated probes (DMPs) were identified based on at least 10% change of β (i.e., average promoter methylation) and a Benjamini-Hochberg adjusted p-value threshold of 0.01. Motif and transcription factor (TF) enrichment analyses were identified at a minimum incidence of 10 and a lower boundary of 1.5 for the 95% confidence interval of the odds ratio. Results: Our analysis identified a total of 613 DEGs between the HOXB13-high and HOXB13-low groups. Gene ontology analysis of DEGs revealed a statistically significant enrichment of genes associated with PTK6 regulated cell cycle, oncogene-induced cellular senescence, and regulation of T cell activation. Analysis of protein-protein interactions of differentially expressed genes revealed differences in key biological processes including changes in cell cycle regulation, p53 pathway, and IL-17 signaling. A total of 5,295 hyper-methylated and 580 hypo-methylated DMPs were discovered when comparing HOXB13-high and HOXB13-low samples. Changes in global methylation patterns enabled classification of breast cancer samples with high or low HOXB13 expression with an accuracy of 0.90. Reconstruction of gene regulatory networks from DNA methylation and transcriptome profiles elucidated significant pairs of DMPs and differentially expressed genes. Specifically, a total of 197 differentially expressed genes were attributed to hyper-methylated DMPs, including genes involved in response to estrogen, activation of HOX genes during differentiation, and drug-mediated inhibition of CDK4/6 activity. Additionally, 8 differentially expressed genes were attributed to hypo-methylated DMPs and were associated with proteasomal protein catabolic processes. Lastly, motif analysis of transcriptional factors for the hyper-methylated DMPs revealed enrichment of binding motifs attributed to FOXA1, ESR1, XBR1, and FOXP1. Conclusion: A comprehensive and comparative analysis of mRNA expression levels and methylation patterns between TCGA breast cancer samples with high and low expression of HOXB13 revealed key signaling pathways and biological processes that may provide insights on the molecular mechanism of HOXB13 in the regulation of response to endocrine therapy in HR+ breast cancer. Citation Format: Natalia Siuliukina, Yi Zhang, Kai Treuner, Mark Pegram. Differential HOXB13 gene expression and promoter methylation analysis in breast cancer [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 PO2-01-08.

Abstract P2-03-05: HOXB13/IL17RB-low breast cancers are predicted to respond to PIK3CA inhibitors independent of PIK3CA mutational status

Abstract Background: The Breast Cancer Index is a gene expression-based signature comprising two functional biomarker panels, the Molecular Grade Index (MGI) and the two-gene ratio, HOXB13/IL17BR (H/I). MGI measures tumor proliferation, while H/I measures estrogen signaling. Integration of MGI and H/I provides a single score that quantifies the risk distant recurrence, while H/I alone predicts benefit from extended endocrine therapy (EET). In multiple EET trials, we have shown that hormone receptor-positive (HR+) breast cancer (BC) patients with H/I-high biomarker expression benefit from EET, while those with H/I-low expression do not benefit from such therapy. Previous protein-protein interaction (PPI) dysregulation (dysreg) analysis of H/I-low BC tumors with drug-response-associated dysreg in BC cell lines revealed a significant correlation with response to two PIK3CA inhibitors, suggesting that PIK3CA pathway may represent an exploitable therapeutic vulnerability for H/I-low BC patients. However, given that PIK3CA is frequently mutated in ER+ tumors, the PPI dysreg analysis may merely reflect enrichment of PIK3CA genomic alterations within the H/I-Low BCs. Thus, to determine if a genetic abnormality within the H/I-low BCs accounts for the correlation with PIK3CA inhibitor drug response in BC cell lines, we performed targeted DNA sequencing of H/I-high and H/I-low BC tumors. Methods: DNA was extracted from 44 H/I-high and 30-H/I-low BC samples using a Qiaqen DNeasy Blood &amp; Tissue Kit. Samples were then prepared using the Archer VariantPlex kits according to a modified protocol and sequenced in batches on the Illumina NextSeq platform. To determine if a genetic abnormality within H/I-low BCs accounts for the PPI dysreg correlation with PIK3CA drug response, the magnitude of the sample’s drug associated PPI dysregs was compared to its mutational status. Geneset enrichment (GSEA) functional profiles between BC tissue PPI dysregs and BC cell line drug response-associated PPI dysregs, identified two PIK3CA inhibitors as potential therapeutic drugs for H/I-low tumors. For each PIK3CA drug, the set of leading-edge genes associated with the genesets important to the observed correlation were isolated. Z-score transformed PPI dysreg count data across the BC samples was used to calculate the average dysreg scores for each sample based on the drug-associated leading-edge genes. The samples were sorted based on scores associated with each drug and GSEA was run on each ranked list looking for an association between the dysreg magnitude and PIK3CA mutation status. Results: Targeted DNA sequencing identified copy number variants for in H/I-high and low tumors, the most common of which are: ERBB2 (3/44 H/I-high; 1/30 H/I-low; p: 0.642), FGF19 (3/44 H/I-high; 1/30; p: 0.642), CCND1 (3/44 H/I-high; 1/30 H/I-low; p: 0.642). Sequencing identified a similar distribution of single nucleotide variants (SNVs) between the H/I-high and H/I-low groups. The most common SNVs identified in order of prevalence are PIK3CA (18/44, H/I-high; 6/30, H/I-low; p: 0.078), TP53 (12/44 H/I-high; 6/30 H/I-low; p: 0.585), CDH1 (4/44 H/I-high; 5/30 H/I-low; p: 0.471), BRCA2 (4/44 H/I-high; 2/30 H/I-low; p: 1). No significant difference in mutational prevalence was identified between the H/I-high and H/I-low BCs. No-significant associations between the PPI dysreg magnitude of the drug-associated leading-edge genes and PIK3CA mutational status was observed (AZD6482 FDR=0.736 and A66 FDR=0.95). Conclusion: No significant genetic alteration, including PIK3CA mutational status, was identified between H/I-high and H/I-low groups. Thus, protein-protein interaction (PPI) dysregulation analysis identifies H/I-low BC tumors as those that are predicted to response to PIK3CA inhibitors independent of PIK3CA mutational status. Citation Format: Baris Boyraz, Grace Kirkpatrick, Stefan T. Kaluziak, Robert Morris, Johannes Kreuzer, Wilhelm Haas, Anthony John Iafrate, Dennis Sgroi. HOXB13/IL17RB-low breast cancers are predicted to respond to PIK3CA inhibitors independent of PIK3CA mutational 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 P2-03-05.

Abstract P2-03-13: Breast Cancer Index (BCI) identifies fewer patients with high risk of late recurrence and high likelihood of benefit from extended endocrine therapy with invasive lobular compared to invasive ductal carcinoma

Abstract Background: Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the first and second most common histologic subtypes of breast cancer. Both IDC and ILC present distinguishing clinicopathologic features that contribute to differences in response to treatment and long-term prognosis. BCI is a validated gene expression assay that provides the risk of overall (0-10y) and late (5-10y) distant recurrence (DR) and predicts the likelihood of benefit from extended endocrine therapy (EET). In this analysis, BCI results between groups of HR+ ILC and IDC breast cancer patients were compared. Methods: The BCI Clinical Database for Correlative Studies is an IRB-approved de-identified database containing &amp;gt;50 clinicopathologic and molecular variables from cases submitted for BCI testing in clinical practice (N=19,126). Molecular variables include BCI Prognostic score, HOXB13/IL17BR ratio (H/I), and molecular grade index (MGI). Clinicopathologic variables were abstracted from pathology reports when available. Chi-squared tests and Kruskal-Wallis tests were used to compare categorical and numeric factors, respectively, between IDC and ILC subgroups. Kaplan-Meier survival analysis and Cox proportional hazards regression were used to analyze BCI Predictive performance in the lobular patients from IDEAL study. Results: The current study included 3814 patients submitted for BCI testing during years 4-7 post-diagnosis with available histologic subtype data (80.5% IDC; 13.2% ILC; 3.0% mixed; 3.3% other). Among those with either ductal (n=3072) or lobular (n=504) carcinomas (71% node-negative and 29% node-positive), patients with ILC were older compared to IDC (&amp;gt;70 y: 17% vs 12%). Clinically, ILC was generally less aggressive than IDC (Grade 3: 7% vs 21%; lymphovascular invasion: 9% vs 20%; HER2+: 2% vs 13%; Ki67 % positive stained cells: 29% vs 46%; p&amp;lt; 0.001 for all comparisons), with the exception that ILC had larger tumors than IDC (T2/T3: 46% vs 24%) due to its unique histology. This was consistent with BCI Prognostic results showing fewer ILC patients at high risk for late DR compared to IDC (43% vs 55%, p&amp;lt; 0.001). BCI (H/I) showed a similar trend with fewer patients with High Likelihood to benefit from EET (39% vs 43%) although the difference was not statistically significant (p=0.169). The combination of BCI prognostic and predictive results revealed that more ILC patients were classified as Low Risk/Low Likelihood of benefit (43% vs 38%) and fewer were called High Risk/High Likelihood of benefit (25% vs 35%) (p&amp;lt; 0.001) compared to IDC. The IDEAL BCI translational study included 142 ILC patients with 9.3 years of median follow-up. Similar to the BCI Clinical Database results, ILC was associated with less aggressive disease than IDC (Grade 3: 18% vs 41%; HER2+: 11% vs 24%). 39% and 61% of ILC patients were classified as BCI (H/I)-High and -Low, respectively. Given the small sample size, BCI (H/I)-High showed a non-significant absolute benefit of 11.9% (HR=0.44, 95% CI 0.09-2.14; p=0.298) and BCI (H/I)-Low showed no benefit (HR=2.63, 95% CI 0.70-9.93; p=0.138). An analysis of the 3-way interaction among treatment, biomarker and histology showed a non-significant p-value of 0.28, suggesting BCI (H/I) provides similar predictive information in ILC as in the overall population. Conclusion: BCI identified a smaller proportion of patients with ILC at High Risk of late DR and High Likelihood of benefit from EET compared to IDC. Data from the IDEAL translational study showed that while fewer patients with ILC were identified as BCI (H/I)-High, they still derived similar absolute benefit compared to the overall cohort, while those classified as BCI (H/I)-Low derived no benefit from EET. Citation Format: Otto Metzger, Neeta Parimi, Natalia Siuliukina, Yi Zhang, Kai Treuner, Gerrit-Jan Liefers. Breast Cancer Index (BCI) identifies fewer patients with high risk of late recurrence and high likelihood of benefit from extended endocrine therapy with invasive lobular compared to invasive ductal carcinoma [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 P2-03-13.

Abstract P3-11-10: Estrogen receptor reprogramming by HOXB13 and GATA3 in ER-positive breast cancer cells

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature comprising two functional biomarker panels. The Molecular Grade Index (MGI) is composed of five genes that measure tumor proliferation. BCI (H/I) is a ratio of the HOXB13 and IL17BR genes and measures estrogen signaling. Integration of MGI and BCI (H/I) provides a single prognostic score that quantifies the risk of both late (5-10 years) and overall (0-10 years) distant recurrence. BCI (H/I) has been validated to predict benefit from extended endocrine therapy across multiple adjuvant endocrine treatment backgrounds in several prospective-retrospective studies. Hormone receptor responses are dependent on genome-wide hormone receptor binding patterns. In hormone-dependent cancers, nuclear receptor binding patterns are frequently reprogrammed by other transcription factors. The homeobox transcription factor HOXB13 has previously been shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis, where it colocalizes with FOXA1 at reprogrammed AR binding sites. Similarly, the transcription factor GATA3 was found to mediate enhancer accessibility at regulatory regions involved in estrogen receptor (ER)-mediated transcription. We have shown previously that HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. In this study, we have further characterized the role of HOXB13 in reprograming the ER cistrome and evaluated potential interactions with GATA3. Methods: HOXB13 was expressed in MCF-7 and T47D cells by electroporating HOXB13 mRNA or eGFP mRNA as control. Cells were harvested after 18 hours and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1, GATA3 and histone H3K27ac. Chromatin accessibility was assessed by ATAC-seq. After aligning reads with Bowtie2, peak calling, data integration and motif enrichment analysis was performed using HOMER v4.10. Results: ChIP-seq analysis of T47D cells expressing HOXB13 confirmed previous results in MCF-7 cells, where binding of HOXB13 to a significant number of genomic binding sites induced changes in binding of ER, FOXA1 and GATA3 compared to control cells. Integrative analysis of ATAC-seq and ChIP-seq data revealed that HOXB13-induced reprogramming results in open chromatin that frequently exhibits increased acetylation of histone H3K27, a hallmark of transcriptional activation. While both increased chromatin accessibility and H3K27 acetylation were associated with HOX and AP-1 motif enrichment of the underlying DNA sequences, newly open chromatin was specifically co-enriched for motifs for pioneering factors such as FOXA1 and GRHL1. Conclusion: This study lends further support to a model of HOXB13-mediated reprogramming of the ER cistrome in breast cancer. Motif analysis of HOXB13-induced chromatin opening suggests interactions with other pioneer transcription factors including FOXA1 and GRHL1, while HOXB13-induced transcriptional activation is associated with motifs for activating factors such as AP-1. These results will be expanded to inducible cell lines to further characterize the effects of HOXB13 expression on ER binding and function. Citation Format: Kai Treuner, Camila De Arruda Saldanha, Sven Heinz. Estrogen receptor reprogramming by HOXB13 and GATA3 in ER-positive breast cancer cells [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 P3-11-10.

Abstract P2-03-18: Clinical use of Breast Cancer Index for prediction of late breast cancer recurrence and prediction of benefit in extended endocrine therapy: A single institution experience

Abstract Background: The decision to extend adjuvant endocrine therapy beyond five years is often individualized. Breast Cancer Index (BCI) (Biotheranostics, Inc.) is a validated multigene-expression tissue-based analytic tool used in early hormone-positive breast cancer to predict the response to extended endocrine therapy, based on the HOXB13/IL17BR ratio (H/I ratio), and prognosis 5-10 years after diagnosis, based on the molecular grade index (MGI) and H/I ratio. It is presumed that if BCI shows a high likelihood of benefit from endocrine therapy (BCI-high), the treating clinician would recommend extended adjuvant endocrine therapy and if the primary prediction shows a low likelihood of benefit (BCI-low), the clinician would recommend discontinuation of therapy; however, this may not always be practiced. The Clinical Treatment Score post-5 years (CTS5) is a validated clinicopathologic tool that provides a calculated risk assessment of late distant recurrence (LDR). Clinicians may use the calculated CTS5 score in deciding whether to extend adjuvant endocrine therapy and forego additional prognostic testing, such as BCI. The aim of the study is to understand how clinicians integrate BCI results into medical decision making and to determine the correlation with CTS5 scores in making the decision to offer extended endocrine therapy. Methods: This is a single institution retrospective study. All patients within Henry Ford Health Systewho had BCI testing ordered, between April 2016 and January 2022, were included. Recommendations regarding extended endocrine therapy were collected. CTS5 scores were calculated based on patient’s age, tumor size, tumor grade, and number of involved lymph nodes. If available, the 21-gene recurrence scores (Oncotype-DX) and Ki-67 scores were collected for additional comparison. Results: A total of 165 female patients were included in this study. The average age at diagnosis was 58.5 ± 10.4 years old; 116 (70%) were Caucasian, 35 (21%) were African American; 132 patients (80%) were post-menopausal. The decision regarding extending endocrine therapy was concordant with BCI predictive results in 93.3% of patients; endocrine therapy was continued in 87% of patients in the BCI-high group and discontinued in 95% of patients in the BCI-low group (p&amp;lt; 0.001). In comparing the categorical results of BCI predictive scores (low vs. high) and the CTS5 results (low vs. intermediate vs. high) univariate analysis did not detect a statistically significant relationship. In comparing BCI prognostic LDR risk percentage with the CTS5 LDR (5–10 year) risk percentage, a statistically significant, but weak, positive correlation was observed (Pearson correlation coefficient of 0.38487, p-value= &amp;lt; 0.0001). The odds of a BCI-high primary prediction were 1.031 (95% CI: 1.005, 1.057) times higher for every-one percentage increase in Ki-67 (p-value=0.0172), 1.052 (95% CI: 1.003, 1.057) times higher for every one-unit increase in Oncotype-DX score (p-value=0.0353) and 1.232 (95% CI: 124, 1.351) times higher for every-one percentage increase in BCI prognostic LDR risk assessment (p-value &amp;lt; 0.0001). No statistically significant association was found between the BCI predictive score and age, race, stage, lymph node involvement, and HER2 receptor positivity. Conclusions: This single-institution study revealed that clinicians make decisions regarding extended endocrine therapy that are usually concordant with BCI predictive results. CTS5 results weakly correlate with BCI prognostic results but may not provide conclusive information to support a clinical decision. For patients with early hormone-receptor positive breast cancer the BCI tissue-based analysis may influence a decision regarding extending endocrine therapy. Citation Format: Rebecca Chacko, Manasi M. Godbole, Kylie Springer, Haythem Ali, Vrushali Dabak. Clinical use of Breast Cancer Index for prediction of late breast cancer recurrence and prediction of benefit in extended endocrine therapy: A single institution experience [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 P2-03-18.

The Breast Cancer Index Registry Study: Initial analysis of the first 1,000 patients.

e12513 Background: The Breast Cancer Index (BCI) is a gene expression-based biomarker that reports two results: BCI Predictive [BCI (H/I)], which predicts benefit from extended endocrine therapy (EET), and BCI Prognostic, which combines the Molecular Grade Index (MGI) with BCI (H/I) to provide individualized risk of both late (5-10 years) and overall (0-10 years) distant recurrence. The NCCN clinical practice guideline recommends use of BCI for prediction of benefit from EET in early stage, hormone receptor positive (HR+) breast cancer patients. Methods: The BCI Registry study was designed to prospectively evaluate the long-term clinical outcome, medication adherence and quality of life in HR+ early-stage breast cancer patients receiving BCI testing as part of routine clinical care. The BCI registry will enroll approximately 3,000 patients after completion of 4-7 years of primary adjuvant endocrine therapy. Patients will be followed for disease recurrence for at least 10 years from diagnosis. Patients extending endocrine therapy will be assessed annually for medication adherence. To evaluate the impact of BCI results on EET decision making, both the physician and patient will complete Decision Impact Questionnaires. Clinicopathological data are collected within the ClinCapture electronic data capture system. The BCI Registry Study is registered on ClinicalTrials.gov under NCT04875351. Results: The BCI Registry Study completed enrollment of the first 1,000 patients in January 2022. Clinical data were available for 942 patients (76.8% T1; 51.3% grade II; 75.4% N0). BCI test results were reported for 842 patients. 315 patients (37.4%) were classified as BCI (H/I)-High and 527 patients (62.6%) as BCI (H/I)-Low. Further, in node-negative (N0) patients, BCI classified 314 N0 patients (47.9%) with high risk for late distant recurrence and 342 patients (52.1%) with low risk. In node-positive (N1) patients, BCI classified 138 patients (74.2%) as high risk for late distant recurrence and 48 patients (25.8%) as low risk. The associated clinicopathological factors are shown in the table. Conclusions: The analysis of the first 1,000 BCI Registry patients confirms the expected distribution of clinicopathological factors. BCI by H/I status and prognostic risk groups are consistent with previous clinical validation studies. Clinical trial information: NCT04875351. [Table: see text]

Abstract PD9-09: Breast cancer index and assessment of tumor proliferation by molecular grade index (MGI) within distinct HOXB13/IL17BR (H/I) subsets

Abstract Background: Previous studies have demonstrated that proliferation- and ER-associated gene expression shows a time-dependent interaction with risk of recurrence. Breast Cancer Index (BCI) is a gene expression-based test that integrates biomarker panels of both proliferation (Molecular Grade Index, MGI), and endocrine response (HOXB13/IL17BR, H/I). BCI has been validated as a significant and independent prognostic factor both for risk of overall (0-10y) and late (&amp;gt;5y) distant recurrence and is predictive of extended endocrine benefit in patients with early-stage, hormone receptor-positive (HR+) breast cancer. In this study, correlative analyses were performed to evaluate molecular measures of proliferation and ER signaling by MGI and H/I that underly recurrence risk across clinical categories in patients receiving BCI testing as part of routine care. Methods: Analysis was performed using the BCI Clinical Database for Correlative Studies, an IRB-approved de-identified database which contains &amp;gt;50 data elements including clinicopathologic and molecular variables from 19,126 clinical cases submitted for BCI testing. Descriptive analyses assessed the distribution of MGI by H/I categories based on time of testing or BCI intervention point (time of diagnosis &amp;lt;1y for early recurrence vs 4-7y post diagnosis for late recurrence), lymph node status (N0 vs. N+), age (&amp;lt;50y vs. ≥50y), tumor size (T1-T3), and grade (1-3) to understand the molecular features that may be associated with recurrence in HR+ disease. Results: Analysis included 18,853 patients (88% ≥50y, 73% N0, 70% pT1, 51% grade 2). Comparative analysis of patients tested with BCI at &amp;lt;1y vs. those recurrence-free and tested at 4-7y post diagnosis showed a similar proportion of patients in the H/I categories (H/I-High: 44%1y, 43% 4-7y, P=0.510). An increased proportion of MGI-High was observed in 4-7y (61%) vs. 1y (53%) (P&amp;lt;0.001). The distribution of MGI categories within H/I-Low and High subsets was also similar based nodal status (N0 vs. N+), age (&amp;lt;50y vs. ≥50y), and tumor size. However, H/I status was inversely proportional to tumor grade (P&amp;lt;0.001): the proportion of H/I-Low decreased with increasing grade (Grade 1: 70%, 2: 59%, 3: 36%), whereas the proportion of H/I-High patients increased (Grade 1: 30%, 2: 41%, 3: 64%). In the H/I-Low subset, as grade increased, the proportion of MGI-Low patients decreased (1: 45%, 2: 23%, 3: 5%), while MGI-High remained relatively consistent across grade (1: 25%, 2: 36%, 3: 31%). In H/I-High patients, MGI-High increased as grade increased (1: 11%, 2: 26%, 3: 59%). Conclusion: BCI functional characterization using MGI for tumor proliferation and H/I for estrogen signaling in this large-scale analysis showed that over half (59%,1y vs. 67%, 4-7y) of endocrine responsive tumors (H/I-High) were also highly proliferative (MGI-High) irrespective of time of testing. Results showed that tumor grade correlated with MGI and H/I status, indicating that a subset of high-grade tumors were highly proliferative as well as endocrine responsive. These findings suggest that proliferation and endocrine signaling are combinatorial molecular drivers of recurrences in HR+ breast cancer. Table 1.Biomarker categorization by H/I and MGI status by time of testing, nodal status, age, tumor size, and grade.H/I-LowH/I-HighPatientsNMGI-Low (%)MGI-High (%)MGI-Low (%)MGI-High (%)Time of testing&amp;lt;1y1278282718264-7y1305725321429Nodal StatusN0950227311428N+344222311631Age&amp;lt;50y475824361129≥50y1409526301529Tumor SizeT1464930311524T2182619321435T319927311527Grade1224245251911239812336152631530531559 Citation Format: Reshma Mahtani, Yuan Yuan, Kari B Wisinski, Jay Morris, Max Salganik, Yi Zhang, Catherine A Schnabel, Vk Gadi. Breast cancer index and assessment of tumor proliferation by molecular grade index (MGI) within distinct HOXB13/IL17BR (H/I) subsets [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-09.

Abstract P3-08-03: HOXB13 is coamplified with HER2 in a subset of breast cancers

Abstract Background: The Breast Cancer Index (BCI) is an algorithmic gene expression-based signature comprised of two functional biomarker panels, the Molecular Grade Index (MGI) and the two-gene ratio, HOXB13/IL17BR. The HOXB13/IL17BR ratio has been shown to predict endocrine response across several different treatment scenarios, and the homeobox factor, HOXB13, is the primary determinant of endocrine benefit and response. Previously, we found that HOXB13 gene expression was positively correlated with HER2 in estrogen receptor-positive (ER+) but not ER-negative (ER-) tumors. To validate these findings, we examined HOXB13 gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. In addition, considering that the HOXB13 gene locus resides 9 megabases from the HER2 locus at chromosome 17q21-22, we examined whether HOXB13 was coamplified with HER2 in HER2-positive breast cancers. Methods: Messenger RNA (mRNA) sequencing (mRNAseq) data from the TCGA breast cancer dataset were used to correlate HOXB13 gene expression with immunohistochemical (IHC)-based HER2 expression in ER+ (ER+PR+, ER+PR-) and ER- (ER-PR+ and ER-PR-) breast cancers. Thresholded copy number data from GISTIC output and mRNAseq data from the TCGA datasets were used to correlate HER2 and HOXB13 gene amplification (at least 4 inferred copies) with HOXB13 gene expression in IHC-based ER+ (n= 562) and ER- (n=163) breast cancer subsets. Formalin-fixed paraffin embedded (FFPE) samples from 80 consecutive HER2-amplified invasive breast carcinomas (64 ER+, 16 ER-) diagnosed between 2010 and 2013 were retrospectively collected from the MGH clinical archives and processed for fluorescence in situ hybridization (FISH) for HER2 (spectrum orange) HOXB13 (spectrum green) and CEP17 (spectrum blue). A ratio of &amp;gt; 2.0 HER2 and HOXB13 to CEP17 signals in at least 60 interphase tumor cell nuclei was considered as amplification of HER2 and HOXB13.Results: In the TCGA breast tumors, HOXB13 gene expression was significantly higher in HER2 positive (by IHC status) tumors in both ER+ (p= 3.7 x10-5) and ER- (p= 4.7 x10-4) tumors compared to HER2-negative samples. In HER2+ TCGA breast tumors, genomic analysis revealed co-amplification of HOXB13 with HER2 in 27% of all tumors, and 25.3% and 31.7% in the ER+ and ER- subsets, respectively. Amplification of the HOXB13 locus alone was observed in 2.4% of HER2-non amplified ER+ and ER- TCGA tumors. In the TCGA cohort, median HOXB13 gene expression was not significantly different when comparing HER2-amplified and HER2/HOXB13 coamplified ER+ (p= 0.33) and ER- (p=0.052) tumors, HER2-amplified and HER2-nonamplified ER+ (p=0.42) and ER- (p=0.67) tumors, and HER2/HOXB13 coamplified and HER2-nonamplified ER+ (p=0.50) and ER- (p=0.11) tumors. Lastly, in the consecutive case series of 80 HER2-amplified breast cancers from MGH, HOXB13 was coamplified in 23% of all tumors, and 23.4% and 18.75% in the ER+ and ER- subsets, respectively. Conclusion: HOXB13 was coamplified with HER2 in a substantial subset of TCGA and MGH primary breast cancers irrespective of ER status. Citation Format: Dennis Sgroi, Maristela Onozato, Irene Mitsiades, Ryan McMullin, Daniel Hicks, Angela Volorio, Esther Rheinbay. HOXB13 is coamplified with HER2 in a subset of breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-08-03.

Abstract OT1-10-01: The Breast Cancer Index registry study: A prospective multi-center observational study to evaluate patient outcome, clinical impact, and medication adherence in HR+ breast cancer patients considering treatment with extended endocrine therapy

Abstract Background: Hormone receptor-positive (HR+) is the most common molecular subtype of breast cancer accounting for approximately 2 out of every 3 patients. Adjuvant anti-estrogen therapy is the cornerstone of systemic therapy, underscoring the critical role of endocrine treatment which affects the majority of women with breast cancer. Furthering efforts to better understand endocrine response and resistance and implementing genomic tools to help individualize patient selection for endocrine therapy are of ongoing importance to improve outcomes. Breast Cancer Index (BCI) is a gene expression signature composed of the Molecular Grade Index (MGI) and HOXB13/IL17BR (H/I) that predicts the likelihood of extended endocrine benefit and quantifies the risk of both late (5-10 years) and overall (0-10 years) distant recurrence. BCI has recently been incorporated into the NCCN clinical practice guideline as a biomarker for prediction of benefit from extended endocrine therapy in patients with early-stage, HR+ breast cancer. The objective of the BCI Registry Study is to collect real-world evidence at a national level aimed at advancing patient care and optimizing duration of endocrine treatment and will be conducted in two phases. First, long-term patient outcomes and the impact of BCI on decision-making based on physician and patient reported outcomes as well as medication adherence to extended endocrine therapy will be assessed. Secondly, individual patient data on over 40 clinical variables will be collected and partnered with molecular profiling and genomic data for each patient to enable correlative analysis and novel biomarker research of endocrine sensitivity and resistance. Study Description: The BCI Registry Study is a prospective, multi-center, observational data registry and biospecimen repository for stage I-III HR+ breast cancer patients. The main objective of the Registry Study is prospective characterization of BCI predictive and prognostic performance. The primary endpoint is time to distant recurrence (TTDR), defined as the interval between the time of BCI testing and the first metastasis at distant organs. Secondary endpoints include time to any recurrences and medication adherence score over time. Eligibility criteria includes confirmed diagnosis of HR+ early-stage invasive breast carcinoma (ductal, lobular, or mixed ductal/lobular), either HER2 negative or positive and either node-negative or node-positive tumors with 1-3 positive lymph nodes (N1). Patients must have completed 4 to 7 years of primary adjuvant endocrine therapy and have pre-treatment tumor tissue available from a previous breast surgery or biopsy. Both physicians and patients will complete a pre- and post-test Decision Impact Questionnaire to assess the impact of BCI results on extended endocrine therapy decision-making. Patients who elect to complete 10 years of endocrine therapy will answer a Medication Adherence Questionnaire during annual follow-up visits. Follow-up of patients will end 10 years from initial breast cancer diagnosis. The Registry plans to enroll approximately 3,000 patients across approximately 40 study sites in the United States. Enrollment for the study began in Q2 2021, and 324 patients have been enrolled as of July 2021. For more information, please visit the ClinicalTrials.gov registration: NCT04875351. Citation Format: Joyce A O'Shaughnessy, Jenny R Fox, Carlos A Encarnación, Brandon O'Neal, Kai Treuner, Tara Sanft, Rachel C Jankowitz, Mark D Pegram, Catherine A Schnabel, Sami G Diab. The Breast Cancer Index registry study: A prospective multi-center observational study to evaluate patient outcome, clinical impact, and medication adherence in HR+ breast cancer patients considering treatment with extended endocrine therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-10-01.

Abstract PS17-32: Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature that consists of two functional biomarker panels, HOXB13/IL17BR (H/I) and Molecular Grade Index (MGI), that interrogate important proliferation and estrogen signaling pathways in breast cancer. The BCI prognostic score reports individualized risk of overall and late distant recurrence and is based on the algorithmic combination of the H/I ratio and MGI, while the predictive component, BCI (H/I), reports a categorical prediction of high versus low likelihood of benefit from extended endocrine therapy.The homeobox transcription factor HOXB13 has previously been shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis, where it colocalizes with FOXA1 at reprogrammed AR binding sites. The aim of the current study was to characterize the potential role of HOXB13 in estrogen receptor (ER) reprogramming by analyzing changes in the global ER binding pattern induced by transient overexpression of HOXB13 in breast cancer cells. In addition, gene ontology (GO) analysis was performed to delineate the functional role of HOXB13 in modulating estrogen signaling and response to endocrine therapy.Methods: HOXB13 was overexpressed in MCF-7 cells by electroporating HOXB13 mRNA, or eGFP mRNA as control. Cells were harvested at different time points and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1 and H3K27ac. After aligning reads with Bowtie2, peak calling and data integration were performed using HOMER v4.10. Gene ontology (GO) analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT v4.0.4) (http://great.stanford.edu/) to identify annotations enriched among genes near ER genomic binding sites.Results: ChIP-seq analysis revealed substantial binding of HOXB13 to a large number of genomic binding sites compared to the eGFP control. HOXB13 overexpression in ER+ breast cancer cells significantly increased ER binding with a majority of ER binding sites being co-bound by FOXA1 and HOXB13. GO analysis of both proximal and distal genomic regions showed significant enrichment of genes associated with mammary gland development, such as “mammary gland epithelium development”, “mammary gland development”, and “mammary gland epithelial cell differentiation”.Conclusion: Findings from this analysis show that transient HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. Genomic regions newly bound by ER are enriched for genes involved in mammary gland and epithelial differentiation suggesting that HOXB13 activates ER transcriptional programs that link oncogenic and developmental pathways. These results will be compared to ongoing experiments using an inducible HOXB13 expression system to assess the effects of HOXB13 expression on ER binding and function in response to estradiol and tamoxifen. Citation Format: Kai Treuner, Dominic Schenone, Christopher Benner, Catherine A Schnabel, Sven Heinz. Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-32.

Abstract PD2-11: Breast cancer index is a molecular signature of endocrine responsiveness that determines extended endocrine benefit independent of prognostic risk

Abstract Background: For patients with early stage, hormone receptor positive (HR+) breast cancer, extension of endocrine therapy beyond 5 years reduces the ongoing risk of late distant recurrence and new primary breast cancer but is associated with potentially serious toxicities and side effects that can impair quality of life. While risk stratification by clinicopathological factors and prognostic biomarkers is recommended to guide patient selection, predictive biomarkers that are directly informative of therapeutic response are critical for clinical utility. Breast Cancer Index (BCI) is a gene expression-based signature that reports: 1) BCI Prognostic (BCI Score), which combines the Molecular Grade Index (MGI) with the HOXB13/IL17BR (H/I) ratio to provide individualized risk of late (post-5y) distant recurrence, and 2) BCI Predictive (BCI H/I), which predicts benefit from extended endocrine therapy. In this study, BCI and endocrine benefit from 5 vs 2.5y of extended letrozole were examined by comparing the deterministic effects of prognostic risk vs endocrine response in patients treated in the IDEAL trial.Methods: 908 patients with available tumor tissue were included in this pre-specified and blinded analysis, with recurrence-free interval (RFI) as the primary endpoint. Kaplan-Meier and Cox proportional hazards regression were performed using pre-defined assay cut-points. Clinically high prognostic risk was defined as pN+ and ≥pT2; low prognostic risk was defined as pN0 or pN1 and pT1 or G1. Genomic risk was defined by BCI prognostic categories (High vs Low risk). Results: Patients classified with endocrine responsive disease by BCI (H/I)-High status showed a statistically significant benefit from 5 vs 2.5y of letrozole irrespective of clinical risk, whereas those classified as BCI (H/I)-Low did not show benefit in either clinical risk category. Both clinically High and Low risk patients derived a similar magnitude of benefit when stratified as BCI (H/I)-High (12.5% and 15.1%, respectively). Evaluation of BCI (H/I) predictive performance in the context of BCI prognostic risk categories showed similar results. In particular, patients with High Risk/High Likelihood to benefit showed significantly improved outcomes with extended therapy (HR 0.42 [0.20-0.89]; absolute benefit 10.7%; P=0.020), whereas those with High Risk but Low Likelihood to benefit did not show response to extended endocrine therapy (HR 1.05 [0.58-1.88]; absolute benefit -0.7%; P=0.880), despite considerable risk of late distant recurrence (~20%). Conclusion: Results from this study highlight distinct tumor biology underlying recurrence risk vs endocrine responsiveness in HR+ early stage breast cancer. BCI (H/I) as a molecular signature to identify patients with endocrine responsive disease is a key determinant of benefit and improved outcome with extended endocrine therapy. Prediction of endocrine benefit with BCI (H/I) is a more actionable approach vs risk assessment to individualize discontinuation of or continued treatment with prolonged durations of endocrine therapy. Prognostic RiskBCI Predictive NHR (95% CI)P valueClinical RiskHigh Risk: N+/ T2+ (N=353)High BCI(H/I)1620.32 (0.10-0.98)0.035Low BCI(H/I)1911.13 (0.55-2.31)0.742Low Risk: N0 or N1 &amp; T1 or grade 1 (N=404)High BCI(H/I)1910.15 (0.03-0.66)0.004Low BCI(H/I)2130.75 (0.33-1.70)0.484Genomic RiskHigh BCI Score(N=621)High BCI(H/I)3410.42 (0.20-0.89)0.020Low BCI(H/I)2801.05 (0.58-1.88)0.880Low BCI Score(N=227)High BCI(H/I)600.35 (0.04-3.38)0.344Low BCI(H/I)1670.64 (0.22-1.92)0.425 Citation Format: Gerrit-Jan Liefers, Iris Noordhoek, Kai Treuner, Hein Putter, Yi Zhang, Jenna Wong, Elma Meershoek-Klein Kranenbarg, Marjolijn Duijm-de Carpentier, Cornelis JH Van De Velde, Catherine A Schnabel. Breast cancer index is a molecular signature of endocrine responsiveness that determines extended endocrine benefit independent of prognostic risk [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD2-11.

Abstract P6-04-17: Role of HOXB13 in modulating estrogen signaling in breast cancer cells

Abstract Background: The ratio of HOXB13 (H) and IL17BR (I) gene expression levels in hormone receptor-positive (HR+) primary early stage breast tumors define a predictive index (H/I) that, combined with the Molecular Grade Index (MGI), constitute the Breast Cancer Index (BCI). The BCI score (MGI+H/I prognostic model) provides individualized risk of overall (0-10 years) and specifically late (5-10 years) distant recurrence, and BCI (H/I) predicts the benefit of extended endocrine therapy (EET). Hormone receptor responses are dependent on genome-wide hormone receptor binding patterns. In hormone-dependent cancers, nuclear receptor binding patterns are frequently reprogrammed by other transcription factors. For example, in breast cancer cells, GATA3 has been shown to reprogram estrogen receptor (ER) binding and to alter gene expression. Similarly, HOXB13 was previously shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis. To assess whether HOXB13 expression is associated with regulation of ER binding, global ER binding data in highly HOXB13-expressing, tamoxifen-resistant breast cancer cells to the ER binding pattern in HOXB13-low, tamoxifen-sensitive breast cancer cells were compared. The study objective was to understand whether HOXB13 contributes to ER reprogramming in early-stage breast cancer. Methods: Global ER, HOXB13 and FOXA1 DNA binding patterns and the enhancer activation mark histone H3 lysine 27 acetylation (H3K27ac) were characterized in BT474 cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq). To compare ER binding in highly HOXB13-expressing, tamoxifen-resistant BT474 cells to published ER binding data in HOXB13-low, tamoxifen-sensitive MCF-7 breast cancer cells, we downloaded published ER, FOXA1, GATA3 and H3K27ac ChIP-seq data from MCF-7 cells. After aligning reads with Bowtie2, peak calling and data integration was performed using HOMERv4.10. Results: Analysis of highly HOXB13-expressing BT474 cells identified a large number of HOXB13 binding sites, which overlapped with approximately half of the ER binding sites in these cells. Many of these binding sites were co-bound by FoxA1, a general pioneer factor that has been shown to also colocalize with HoxB13 at reprogrammed AR binding sites in human prostate tumors. Interestingly, most of these locations are distinct from the genomic binding sites of ER in HOXB13-low MCF-7 cells, where ER frequently binds to MCF-7-specific sites in conjunction with GATA3. Conclusion: Our preliminary evidence suggests that HOXB13 influences the estrogen receptor binding pattern in human breast cancer cells and that ER binding sites differ between HOXB13 high- and low-expressing cell lines, which may underlie their differential responses to endocrine therapy. These results will be compared to ongoing experiments assessing the effects of conditional expression or loss of HOXB13 on ER binding and function in HOXB13-negative and HOXB13-positive cells, respectively. Citation Format: Kai Treuner, Michael Hayes, Christopher Benner, Catherine Schnabel, Sven Heinz. Role of HOXB13 in modulating estrogen signaling in breast cancer cells [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 P6-04-17.

Abstract P2-08-42: Use of breast cancer index to analyze tumor proliferation and endocrine responsiveness in genomic intermediate risk patients

Abstract Background: Breast Cancer Index (BCI) is a genomic assay that stratifies patients (pts) for cumulative 10-year and late (post–5-year) risk of distant recurrence and predicts the likelihood of extended endocrine therapy (EET) benefit based on the algorithmic analysis of gene expression from two functional gene cassettes: 1) The Molecular Grade Index (MGI), which contains 5 proliferation genes and 2) The HoxB13/IL17BR (H/I) ratio, an endocrine response biomarker. BCI more precisely resolves genomic intermediate risk patients into low- and high-risk groups (Sestak, Clin Cancer Res 2016) and adds significant prognostic data beyond clinical features (ie, clinical treatment score; Sestak, JAMA Oncol 2017) by interrogating different aspects of tumor biology such as proliferation and endocrine response. The objective of this study was to measure tumor proliferation and endocrine responsiveness using MGI and H/I respectively and evaluate their correlation with age in genomic intermediate risk pts. Methods: This study utilized a subset of cases from the BCI Clinical Database for Correlative Studies, an IRB-approved de-identified database that contains clinicopathologic and molecular variables from clinical cases submitted for BCI testing. Genomic intermediate risk cases were defined as LN-, HER2- (or HER2 status unknown) with 21-gene recurrence scores (RS) of 11 to 25 using cut-points from the TAILORx study. Quantitative scores for MGI and H/I were derived by algorithmic analysis of BCI gene expression. Age groups (&amp;lt;50y and ≥51y) were determined using the date of diagnosis. MGI and H/I were evaluated in two different genomic intermediate risk groups: RS 11-15 and RS 16-25. Pearson correlation coefficients were used to determine the correlation between MGI, BCI, H/I, and genomic intermediate risk scores for patients &amp;lt;50y and ≥51y. Results: Of the 441 pts with RS and BCI results, 303 (69%) with genomic intermediate risk were analyzed. The median MGI score in pts ≥51y was higher in the RS 16-25 group compared to RS 11-15, but there was no difference in MGI score between the two genomic intermediate RS risk groups for pts &amp;lt;50y. In contrast to MGI, median H/I was higher in the RS 16-25 group irrespective of age, with 37% of pts &amp;lt;50y and 41% of pts ≥51y having tumors predicted as more likely to benefit from EET using validated cut-points for H/I. There was no significant correlation between tumor proliferation (MGI: r=0.166) or endocrine responsiveness (H/I: r=0.244) with genomic intermediate-risk RS 11-25 group. Conclusion: These data, which show variations in tumor proliferation and endocrine signaling based on age and genomic intermediate risk group, highlight the importance of measuring different features of tumor biology for risk classification and prediction of therapy response. The absence of correlations between tumor proliferation and genomic intermediate risk and age, and between estrogen signaling and genomic intermediate risk and age, suggests that assays such as BCI that combine distinct aspects of tumor biology for prognosticating risk of recurrence and prediction of benefit from endocrine therapy provide additional value for individualizing the management of patients with early-stage ER+ breast cancer. Citation Format: Yan F, Master AK, Israel MA, Liu J, Zhang Y, Schnabel CA, Gadi VK. Use of breast cancer index to analyze tumor proliferation and endocrine responsiveness in genomic intermediate risk patients [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 P2-08-42.

Residual risk assessment with the Breast Cancer Index (BCI) for prediction of late distant recurrence (DR) in patients from the TransATAC study.

529Background: The Breast Cancer Index (BCI) is a gene-expression based signature comprised of two complementary functional domains: the molecular grade index (MGI) for tumor proliferation, and the...

Abstract P6-09-03: Gene expression patterns in younger versus older HR+ breast cancer patients: An age-related analysis of HoxB13/IL17BR (H/I), proliferation status, and quantitative hormone receptor expression

Abstract Background: HR+ breast cancer in younger vs older patients may have distinct biological features. The Breast Cancer Index (BCI) is a gene expression-based assay that includes two component biomarkers: the HoxB13/IL17BR (H/I ratio), an endocrine response biomarker; and the molecular grade index (MGI), a set of proliferation-related genes. The objective of this study was to assess age-related associations with endocrine sensitivity (H/I), proliferative status (MGI), and quantitative expression of ER and PR. Methods: Data were extracted from the BCI Clinical Database for Correlative Studies, an IRB-approved de-identified database containing clinicopathologic and molecular variables from clinical cases submitted for BCI testing. Molecular results from H/I, MGI, and quantitative (qPCR–based) ER and PR were analyzed across age groups. Chi-squared tests and ANOVA were used to compare the results between age groups (&amp;lt;40y, 40-49y, 50-59y, 60-69y, and ≥70y). Results: Analyses included 19,126 patients (median age at diagnosis 58.6y; 4.5% &amp;lt;40y, 20.6% 40-49y, 28.9% 50-59y, 32.5% 60-69y, and 13.6% ≥70y). Proliferation status (MGI) was significantly higher in patients &amp;lt;40y and 40-49y compared to older groups (P&amp;lt;.0001).H/I analysis indicated a similar distribution of high versus low endocrine responsiveness across all groups (P=.94), except the 40-49y group, in which fewer patients had high H/I (44.2% in &amp;lt;40y, 39.4% in 40-49y, 43.7% in 50-59y, 43.7% in 60-69y, and 43.1% in ≥70y; P=.0001). Median qER increased with age (P&amp;lt;0.0001), while qPR was similar across all age groups except for the 40-49y group (P=.57), in which expression was higher (P&amp;lt;.0001). Conclusion: Results from &amp;gt;19,000 patients with early-stage HR+ breast cancer and BCI testing showed a broad distribution in all variables. Tumors from the youngest patients (&amp;lt;40y) had the highest expression of proliferative genes and the lowest quantitative ER expression. However, endocrine response, according to the H/I biomarker, does not appear to be strongly linked to age. Citation Format: Melisko M, Chien AJ, Poage GM, Salganik M, Schnabel CA, Ruddy KJ, Blackwell K. Gene expression patterns in younger versus older HR+ breast cancer patients: An age-related analysis of HoxB13/IL17BR (H/I), proliferation status, and quantitative hormone receptor expression [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 P6-09-03.

Abstract P1-06-09: Correlation of breast cancer index (BCI) results to lymphovascular invasion in early stage HR+ breast cancer

Abstract Background: Positive lymphovascular invasion (LVI) is a negative prognostic factor for women with early-stage ER+ breast cancer. LVI, along with other clinicopathologic factors such as larger tumor size, higher grade and positive nodal status, increase a patient's risk of late (post-5y) recurrence. Breast Cancer Index (BCI) is a validated gene expression-based assay for patients with early-stage HR+ breast cancer that reports an individualized risk of late distant recurrence based on a combination of the HOXB13/I17BR ratio and the molecular grade index (MGI). The correlation of LVI and individualized risk stratification by genomic analysis is not well characterized. Therefore, this study evaluated risk stratification by BCI based on the presence or absence of LVI. Methods: A population of 2,613 patients with known LVI status were identified in the Breast Cancer Index Clinical Database for Correlative Studies, an IRB-approved de-identified database which contains clinicopathologic and molecular variables of more than 19,000 clinical cases submitted for BCI testing. LVI was recorded as either positive or negative based on pathology report review. BCI results based on LVI status from LN- (n=2035) and LN+ patients (n=578) were evaluated separately. Chi-squared tests were used to compare BCI results between LVI groups. Results: In analyses of 2,613 patients with LVI data available (median age 59.1 y; range 28-89y; 74% ≥50y), 18.3% of patients showed evidence of LVI (LVI-pos). In comparison to the LVI-neg tumors submitted for BCI testing, the LVI-pos tumors had a higher proportion of grade 3 tumors (33% vs 16%, p&amp;lt;0.0001), more LVI-pos tumors were 2.0 cm or greater (45% vs 23%, p&amp;lt;0.0001), a higher percentage LVI-pos patients had node-positive disease (51% vs 16%, p&amp;lt;0.0001), and a higher proportion of LVI-pos tumors showed high Ki67 (Ki67 ≥14%; 64% vs 51%, p=0.004). A correlation between LVI positivity and high BCI prognostic risk was observed, with a higher proportion of LVI-pos patients classified as high risk of late distant recurrence in both the LN- (68% vs 49%, p&amp;lt;0.0001) and LN+ subsets (84% vs 70%, p&amp;lt;0.0001) compared to LVI-neg patients. LVI-pos patients had a higher median molecular proliferative status (MGI) compared to LVI-neg patients regardless of nodal status (p&amp;lt;0.0001 for both). In contrast to the categorical LVI prognostic factor, the wide distribution of BCI individual risk scores provides additional resolution that identifies a substantial subset of LVI positive tumors (32%) that that have a low risk of late recurrence by genomic analysis. Conclusion: While BCI Prognostic stratification correlated with LVI status, BCI identified a subset of patients with LVI positivity as having a low risk of late distant recurrence that otherwise would have an unfavorable prognosis based on LVI and/or LN positivity. These findings help to characterize differential patient stratification based on an individualized assessment of tumor biology versus LVI for patients considering EET. Citation Format: Vulchi M, Sagalnik M, Schnabel CA, Abraham J. Correlation of breast cancer index (BCI) results to lymphovascular invasion in early stage HR+ 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 P1-06-09.

Abstract P1-06-10: Correlative analysis of breast cancer index (BCI) restratification of 21-gene recurrence score (RS) in patients with hormone receptor-positive (HR+), node-negative breast cancer

Abstract Background: In a cross-stratification analysis performed within the TransATAC cohort, Breast Cancer Index (BCI) and 21-gene Recurrence Score (RS) had a concordance of 58.2% (Sestak et al., Clin Cancer Res, 2016). Restratification by BCI of the low and intermediate RS risk groups led to subgroups with significantly different rates (P &amp;lt; 0.001 and P = 0.003, respectively); in contrast, restratified subgroups created by RS of BCI risk groups did not differ significantly. The objective of this study was to analyze the concordance of BCI and RS test results in HR+, node-negative (LN-) patients tested in the real-world setting and to investigate molecular, clinical, and pathologic correlates within discrepant cases. Methods: This study utilized a subset of cases from the BCI Clinical Database for Correlative Studies, an IRB-approved de-identified database which contains clinicopathologic and molecular variables from clinical cases submitted for BCI testing. Clinicopathologic variables, abstracted from pathology reports, were available for a subset of cases. This analysis evaluated cases from LN- patients with available RS data. Concordance was evaluated between BCI Prognostic risk groups (Low, High) and RS risk groups (Low, Intermediate, High based on TAILOR Rx cutpoints [0-10, 11-25, and 26+]). Fisher's Exact tests were used to compare molecular (HoxB13/IL17BR [H/I] endocrine response biomarker and Molecular Grade Index [MGI] proliferation marker) and clinicopathologic (age, grade, size, HER2, Ki67) data in discrepant risk groups. Results: There were 456 LN- patients included. Median age was 58.0y (range 27.2-84.0y; 73.9% ≥50y); 33.1%/50.1%/16.8% were grade 1/2/3; and 24.0%/59.5%/15.3% were T1ab/T1c/T2. BCI classified 47.8% (n=218) of patients as Low Risk vs 52.2% (n=238) as High Risk. RS classified 17.1% (n=78), 67.1% (n=306), and 15.8% (n=72) of patients as Low, Intermediate, and High Risk, respectively. BCI restratified RS-Low patients as high risk in 17.1% of cases, restratified RS-Intermediate as Low Risk in 48.4% and High Risk in 51.6%, and restratified RS-High as Low risk in 20.8% of cases. In RS-Low patients, only H/I (P=0.0004) and MGI (P=0.047) were significantly correlated with restratification to BCI-High Risk. In RS-Intermediate patients, H/I (P&amp;lt;0.0001), MGI (P&amp;lt;0.0001), grade (P&amp;lt;0.0001), and Ki67 &amp;gt;20% (P=0.0003) were significantly correlated with restratification by BCI to High or Low Risk. In RS-High patients, H/I (P=0.0008), MGI (P&amp;lt;0.0001), grade (P=0.016) were significantly correlated with restratification to Low Risk. Conclusion: BCI restratified a substantial proportion of patients in each RS risk group. Based on previous studies demonstrating that BCI has improved prognostic ability for assessment of risk of late distant recurrence (Sgroi et al., Lancet Oncol, 2013), these results highlight the clinical utility of BCI within all RS risk groups. The estrogen signaling pathway biomarker H/I and proliferative biomarkers (MGI, grade, Ki67) were associated with restratification by BCI, while age, HER2 status, and tumor size were not. Citation Format: Yan F, Master AK, Israel MA, Liu J, Schnabel CA, Hurvitz S, Gadi VK. Correlative analysis of breast cancer index (BCI) restratification of 21-gene recurrence score (RS) in patients with hormone receptor-positive (HR+), node-negative 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 P1-06-10.

Abstract P1-07-18: Clinicopathologic and molecular correlates of breast cancer index (BCI) results in patients with HR+, LN- breast cancer that are high risk of late distant recurrence (DR) / low likelihood of benefit from extended endocrine therapy (EET)

Abstract Background: BCI is a gene expression assay for patients with early stage HR+ breast cancer that provides 2 results: BCI Predictive, based on the HoxB13/IL17BR (H/I) ratio, reports a prediction of high vs low likelihood of benefit from EET; BCI Prognostic, based on the algorithmic combination of H/I and a set of proliferation-based genes, reports the risk of late distant recurrence (DR). Clinical actionability is distinct based on the 4 possible combinations of prognostic and predictive results. To better characterize patients classified by BCI as having a high risk of late DR but a low likelihood of benefit from EET, we assessed clinicopathologic and molecular correlates in this subset. Methods: The BCI Clinical Database for Correlative Studies is a de-identified database containing &amp;gt;50 clinicopathologic and molecular variables from cases submitted for BCI in clinical practice (N=19,126). Clinicopathologic variables abstracted from pathology reports were available for a subset of these cases. Molecular proliferation status (molecular grade index [MGI]) and clinicopathologic parameters were examined in the 4 possible BCI result categories of BCI Prognostic (High vs Low risk) and BCI Predictive (High vs Low H/I). Chi-squared tests and ANOVA were used to compare BCI results within subsets. Results: Analyses included 3843 LN- pts with clinicopathologic data: Median age was 59.1y (range 26-89y; 74% ≥50y); 30.9%, 51.7%, and 17.4% were Grade 1, 2, and 3, respectively; 27.8%, 48.9%, 21.7%, and 1.6% were T1a/b, T1c, T2, and T3, respectively. BCI categorized 41.4% of pts as having Low risk/Low likelihood of benefit, 31.3% with High risk/High benefit, 18.0% with High risk/Low benefit, and 9.3% with Low risk/High benefit. Patients with High Risk/Low Benefit had increased median proliferation scores (MGI), and a greater proportion of pts with grade 2/3 tumors and high Ki67 scores compared to pts with Low Risk/Low Benefit (P&amp;lt;.0001 for all). In contrast, there were only modest differences in clinicopathologic parameters between patients with High Risk/ Low Benefit and those with High Risk/High Benefit. Conclusion: In characterizing the molecular and clinical correlates in BCI cases with a high risk of late DR but low likelihood of benefit from EET, we found that higher proliferative status was associated with classification of high risk of DR. Future studies might investigate whether patients with this molecular pattern might benefit from combinatorial therapy (e.g., CDK 4/6 inhibitors) with EET. This study highlights the importance of predictive biomarkers for individualized EET therapy recommendation. Citation Format: Royce M, Poage G, Israel MA, Schnabel CA, Holmes FA. Clinicopathologic and molecular correlates of breast cancer index (BCI) results in patients with HR+, LN- breast cancer that are high risk of late distant recurrence (DR) / low likelihood of benefit from extended endocrine therapy (EET) [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 P1-07-18.

Likelihood of late relapse assessed by the Breast Cancer Index (BCI) in LN-invasive lobular compared to invasive ductal carcinoma.

e12025 Background: Women with HR+ breast cancer remain at risk for late distant recurrences (DR) despite optimal adjuvant therapy. Invasive lobular carcinoma (ILC) is the 2nd most common subtype of invasive breast cancer. When compared to invasive ductal carcinoma (IDC), ILC has distinguishing clinical and pathologic characteristics that may result in different response to therapy and long term prognosis. BCI is a validated gene expression-based assay for pts with early stage HR+ breast cancer that provides risk of late (5-10 y) DR and predicts likelihood of benefit of extended endocrine therapy (EET). Here, we compared BCI assay results in pts with HR+, LN- ILC vs IDC. Methods: The BCI Clinical Database for Correlative Studies is an IRB-approved de-identified database that contains &gt;50 clinicopathologic and molecular variables from cases submitted for BCI in clinical practice (N=14,463). Molecular variables include BCI Prognostic score, HoxB13/IL17BR ratio (H/I), and molecular grade index (MGI). Clinicopathologic variables were abstracted from pathology reports (available for a subset of cases). LN- pts with available pathologic data were analyzed. Chi-squared tests were used to compare BCI results between IDC and ILC subgroups. Results: Analyses included 2554 LN- pts with available histologic subtype data (80.7% IDC; 13.7% ILC; 2.6% mixed; 2.9% other). Median age was 59.3y (range 27-89y; 74% ≥ 50y). BCI Prognostic had a broad distribution of individual risk assessment in both IDC and ILC. However, BCI Prognostic had a lower median BCI scores (P&lt;0.0001) in ILC, and classified a smaller proportion of pts with ILC as high risk for late DR compared to IDC (36.5% vs 53.1%; P&lt;0.0001). IDC had a higher median molecular proliferative status (MGI) compared to ILC (P&lt;0.0001). BCI Predictive (H/I) identified a slightly decreased proportion of pts with ILC benefiting from extended endocrine therapy compared to IDC (38.2% vs 41.1%). There was no difference in quantitative ER or PR (by PCR) between groups. Conclusions: BCI identified a smaller proportion of pts with ILC at high risk of late DR compared to IDC. Further studies evaluating outcomes are warranted to further validate BCI in pts with ILC.