Search

BACKGROUND We proposed that a test for sensitivity to the adjuvant endocrine therapy component of treatment for patients with stage II-III breast cancer (SET2,3) should measure transcription related to estrogen and progesterone receptors (SETER/PR index) adjusted for a baseline prognostic index (BPI) combining clinical tumor and nodal stage with molecular subtype by RNA4 (ESR1, PGR, ERBB2, and AURKA). PATIENTS AND METHODS Patients with clinically high-risk, hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative (HR+/HER2-) breast cancer received neoadjuvant taxane-anthracycline chemotherapy, surgery with measurement of residual cancer burden (RCB), and then adjuvant endocrine therapy. SET2,3 was measured from pre-treatment tumor biopsies, evaluated first in an MD Anderson Cancer Center (MDACC) cohort (n = 307, 11 years' follow-up, U133A microarrays), cut point was determined, and then independent, blinded evaluation was carried out in the I-SPY2 trial (n = 268, high-risk MammaPrint result, 3.8 years' follow-up, Agilent-44K microarrays, NCI Clinical Trials ID: NCT01042379). Primary outcome measure was distant relapse-free survival. Multivariate Cox regression models tested prognostic independence of SET2,3 relative to RCB and other molecular prognostic signatures, and whether other prognostic signatures could substitute for SETER/PR or RNA4 components of SET2,3. RESULTS SET2,3 added independent prognostic information to RCB in the MDACC cohort: SET2,3 [hazard ratio (HR) 0.23, P = 0.004] and RCB (HR 1.77, P < 0.001); and the I-SPY2 trial: SET2,3 (HR 0.27, P = 0.031) and RCB (HR 1.68, P = 0.008). SET2,3 provided similar prognostic information irrespective of whether RCB-II or RCB-III after chemotherapy, and in both luminal subtypes. Conversely, RCB was most strongly prognostic in cancers with low SET2,3 status (MDACC P < 0.001, I-SPY2 P < 0.001). Other molecular signatures were not independently prognostic; they could effectively substitute for RNA4 subtype within the BPI component of SET2,3, but they could not effectively substitute for SETER/PR index. CONCLUSIONS SET2,3 added independent prognostic information to chemotherapy response (RCB) and baseline prognostic score or subtype. Approximately 40% of patients with clinically high-risk HR+/HER2- disease had high SET2,3 and could be considered for clinical trials of neoadjuvant endocrine-based treatment.

Background Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SETER/PR and PI3Kges), and measures expression of selected activating point mutations and key breast cancer genes. Methods Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin’s concordance correlation coefficient (CCC) to measure concordance, including precision (Rho) and accuracy (Bias), and nonparametric tests (Wilcoxon, Levene’s) to compare the modified (NEW) SET4 assay to the original (OLD) SET4 assay and to whole transcriptome RNAseq using RNA from matched fresh frozen (FF) and FFPE samples from 12 primary breast cancers. Results The modified (NEW) SET4 assay measured single transcripts (pER/PR (p=0.002) more reproducibly in technical replicates from FFPE samples. The modified SET4 assay was more precise for measuring single transcripts (Rho 0.966 vs 0.888, pER/PR (Rho 0.985 vs 0.968) or PI3Kges (Rho 0.985 vs 0.946) in FFPE, compared to FF samples. It was also more precise than wtRNAseq of FFPE for measuring transcripts (Rho 0.986 vs 0.934, pER/PR (Rho 0.993 vs 0.915, p=0.004), but not PI3Kges (Rho 0.988 vs 0.945, p=0.051). Accuracy (Bias) was comparable between protocols. Two samples carried a PIK3CA mutation, and measurements of transcribed mutant allele fraction was similar in FF and FFPE samples and appeared more precise with the modified SET4 assay. Amplification efficiency (reads per UMI) was consistent in FF and FFPE samples, and close to the theoretically expected value, when the library size exceeded 400,000 aligned reads. Conclusions Modifications to the targeted RNAseq protocol for SET4 assay significantly increased the precision of UMI-based and reads-based measurements of individual transcripts, multi-gene signatures, and mutant transcript fraction, particularly with FFPE samples.

Background We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II–III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance. Methods We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin’s concordance correlation coefficient (CCC) was used to measure concordance between measurements. Results SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1–9% coefficient of variation over 20 weekly tests, linear range 3.0–11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts). Conclusions Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.

Background: The SET2,3 index combines an accurate measure of transcription related to both estrogen and progesterone receptors (SETER/PR index) with a baseline prognostic index (BPI) derived from c-T Stage, c-N status and molecular subtype by RNA4 (ESR1, PGR, ERBB2, and AURKA). SET2,3 index was translated from a microarray-based signature to a customized hybridization assay that yields highly reproducible results from routine pathology tissue blocks. Methods: The SET2,3 index was calculated from microarray data prepared from baseline biopsies of HR+/HER2- cancers prior to neoadjuvant taxane-anthracycline chemotherapy followed by adjuvant endocrine therapy in: 1) a test set of 307 samples from the MDACC cohort with 8 years median follow-up (Affymetrix U133A microarrays, Symmans et al JAMA 2011), and 2) an independent blinded validation set of 268 high-risk Mammaprint (MP) samples from the I-SPY2 clinical trial with 3.8 years median follow-up (Agilent 44K microarrays, Agendia). The cut-point for high versus low SET2,3 was defined in the MDACC cohort and then tested in the I-SPY2 cohort. Association between the SET2,3 index and distant relapse-free survival (DRFS) was evaluated in each cohort using multivariate Cox regression models. One model adjusted for residual cancer burden (RCB) after neoadjuvant chemotherapy and its interaction with the SET2,3 index, also including treatment arm (experimental vs. control) in the I-SPY 2 trial. Another model adjusted for other prognostic gene expression signatures (GES) for recurrence score (RS), MP, or endopredict (EP) in the MDACC cohort, and actual MP in the I-SPY2 trial. A third model tested the ability of prognostic GES to substitute for RNA4 subtype (part of BPI) or SETER/PR index as components of the SET2,3 index. Results: Predicted sensitivity to endocrine therapy (SET2,3) and response to neoadjuvant chemotherapy (RCB) were independently prognostic, with nonsignificant (NS) interaction term, in multivariate analyses of the MDACC study: SET2,3 HR 0.26 (p=0.016); RCB HR 2.04 (p Conclusions: SET2,3 index of endocrine transcriptional activity in a pre-treatment core biopsy added independent significant prognostic information to any baseline prognostic score and response to neoadjuvant chemotherapy. Approximately 40% of clinical or genomic (MP) high-risk HR+/HER2- cancers had high SET2,3 index, and their response to neoadjuvant chemotherapy (RCB class) was not prognostic. This suggests that SET2,3 index might be used to select appropriate candidates for endocrine-based neoadjuvant treatments in clinical trials. Citation Format: Lili Du, Christina Yau, Lamorna Brown-Swigart, Rebekah Gould, Gillian L Hirst, I-SPY2 Consortium, Marieke van der Noordaa, Isabelle Bedrosian, Rachel M Layman, Vicente Valero, Laura van't Veer, Laura Esserman, W. Fraser Symmans. Prognostic contribution of predicted sensitivity to endocrine therapy (SET) prior to neoadjuvant chemotherapy for stage II-III hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-04.

Background: Previous studies have independently validated the prognostic relevance of residual cancer burden (RCB) after neoadjuvant chemotherapy. We used results from several independent cohorts in a pooled patient-level analysis to evaluate the relationship of RCB with long-term prognosis across different phenotypic subtypes of breast cancer, to assess generalisability in a broad range of practice settings. Methods: In this pooled analysis, 12 institutes and trials in Europe and the USA were identified by personal communications with site investigators. We obtained participant-level RCB results, and data on clinical and pathological stage, tumour subtype and grade, and treatment and follow-up in November, 2019, from patients (aged ≥18 years) with primary stage I–III breast cancer treated with neoadjuvant chemotherapy followed by surgery. We assessed the association between the continuous RCB score and the primary study outcome, event-free survival, using mixed-effects Cox models with the incorporation of random RCB and cohort effects to account for between-study heterogeneity, and stratification to account for differences in baseline hazard across cancer subtypes defined by hormone receptor status and HER2 status. The association was further evaluated within each breast cancer subtype in multivariable analyses incorporating random RCB and cohort effects and adjustments for age and pretreatment clinical T category, nodal status, and tumour grade. Kaplan-Meier estimates of event-free survival at 3, 5, and 10 years were computed for each RCB class within each subtype. Findings: We analysed participant-level data from 5161 patients treated with neoadjuvant chemotherapy between Sept 12, 1994, and Feb 11, 2019. Median age was 49 years (IQR 20–80). 1164 event-free survival events occurred during follow-up (median follow-up 56 months [IQR 0–186]). RCB score was prognostic within each breast cancer subtype, with higher RCB score significantly associated with worse event-free survival. The univariable hazard ratio (HR) associated with one unit increase in RCB ranged from 1·55 (95% CI 1·41–1·71) for hormone receptor-positive, HER2-negative patients to 2·16 (1·79–2·61) for the hormone receptor-negative, HER2-positive group (with or without HER2-targeted therapy; p

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SETER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts (all had high SETER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: The clinical management of patients with metastatic HR-positive breast cancer is often uncertain due to decreased sensitivity to anti-estrogen therapy over time. Recently, we developed a targeted RNAseq based 18-transcript SET ER/PR assay of endocrine sensitivity from biopsies of metastatic cancer. In this work we assess the effect of pre-analytical factors, specifically RNA extraction methods for FFPE tissue samples, on the reliability of the targeted RNAseq assay. Methods: FFPE blocks and matched fresh frozen (FF) sections from 12 tumors were collected at MD Anderson Cancer Center. RNA from FFPE slides was extracted in duplicate using three kits (Norgen, Qiagen, Roche), and RNAseq libraries from all samples were prepared using Kapa Total RNAseq kit. Targeted RNA libraries were prepared using droplet-based PCR (RainDance), and also by transcriptome-wide RNAseq for comparison. Reads were mapped to genomic sequence using STAR and expression was quantified using RSEM. Expression data were normalized based on expression of 10 reference genes. The effect of FFPE RNA extraction kit on the reliability of the SET index was assessed using linear mixed effects model (LME) analysis, and agreement with FF was assessed using the concordance correlation coefficient (CCC). Results: Analysis of the whole transcriptome RNAseq data confirmed minimal 3'-end transcript bias from FFPE samples, irrespective of transcript size or FFPE kit. All 18 genes included in the SET index had high overall concordance between FFPE and FF (median CCC percentile=98.8, range 57.2-99.9 for Norgen; similar for the other two kits) and relatively consistent bias across genes, as estimated by the random effects of the LME model. Furthermore, compared to random 18-gene indices, concordance in the SET index values between FF and FFPE was higher than 99.8% of the random samples, verifying the analytical reliability of the selected genes. For the targeted RNAseq assay, RNA from FFPE extracted with the Norgen kit showed the highest concordance compared to FF (CCC=0.956, 95%CI 0.871-0.985). In general, the analytical variation of SET from FFPE samples was greater than that from FF (1.71-2.71 fold greater), with the lowest variation associated with the Norgen kit. The SET index values from targeted RNAseq for both FF and FFPE samples were consistently lower compared to transcriptome-wide RNAseq but were highly correlated, with the Norgen kit having the highest correlation between targeted and transcriptome-wide RNAseq (rho=0.915). Conclusions: All three FFPE RNA extraction kits have excellent analytical performance compared to FF samples. The Norgen kit may be marginally better yielding higher concordance with FF and lower analytical variation between replicates. All genes in the SET ER/PR showed very good analytical performance in comparison to random indices and individual genes. Targeted gene RNA sequencing appears very promising as a platform for clinical deployment of quantitative assays, showing only a small (fixable) bias compared to RNAseq. Citation Format: Marczyk M, Fu C, Lau R, Du L, Trevarton AJ, Sinn BV, Gould RE, Symmans WF, Hatzis C. Pre-analytical effects of FFPE extraction methods on targeted and whole transcriptome sequencing assays for endocrine sensitivity in metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-20.

For primary triple-negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NAC), higher pretreatment tumor-infiltrating lymphocytes (TILs) correlates with increased pathologic complete response (pCR) rates, and improved survival. We evaluated the added prognostic value of residual disease (RD) TILs to residual cancer burden (RCB) in predicting survival post-NAC.We combined four TNBC NAC patient cohorts who did not achieve pCR. RD TILs were investigated for associations with recurrence-free survival (RFS), and overall survival (OS) using Cox models with stromal TILs as a continuous variable (per 10% increment). The likelihood ratio test was used to evaluate added prognostic value of RD TILs.A total of 375 RD TNBC samples were evaluable for TILs and RCB. The median age was 50 years, with 62% receiving anthracycline/taxane chemotherapy. The RCB class after NAC was 11%, 50%, and 39% for I, II, and III, respectively. The median RD TIL level was 20% (IQR 10-40). There was a positive correlation between RD TIL levels and CD8+ T-cell density (ρ = 0.41). TIL levels were significantly lower with increasing post-NAC tumor (P = 0.005), nodal stage (P = 0.032), but did not differ by RCB class (P = 0.84). Higher RD TILs were significantly associated with improved RFS (HR: 0.86; 95% CI 0.79-0.92; P 0.001), and improved OS (HR: 0.87; 95% CI 0.80-0.94; P 0.001), and remained significant predictors in multivariate analysis (RFS P = 0.032; OS P = 0.038 for OS). RD TILs added significant prognostic value to multivariate models including RCB class (P 0.001 for RFS; P = 0.021 for OS). The positive prognostic effect of RD TILs significantly differed by RCB class for RFS (PInt=0.003) and OS (PInt=0.008) with a greater magnitude of positive effect observed for RCB class II than class III.TIL levels in TNBC RD are significantly associated with improved RFS and OS and add further prognostic information to RCB class, particularly in RCB class II.

Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Rationale: A robust index for gene expression related to activity of estrogen (ESR1) and progesterone (PGR) receptors could predict sensitivity to endocrine therapy in metastatic breast cancer. Methods: Transcripts correlated with ESR1 and PGR expression in 389 hormone receptor-positive breast cancer samples (Affymetrix U133A microarrays) were ranked for reliability according to their pre-analytical (intratumoral heterogeneity, biopsy type) and analytical reproducibility. Eighteen target and ten reference genes were selected and summarized as the SETER/PR index. The SETER/PR index was evaluated in a different set of 140 biopsies from distant metastases of hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer, and in additional pre-analytical and analytical sample cohorts. Thereafter, SETER/PR was translated to a customized format for application to formalin-fixed and paraffin-embedded (FFPE) sections. Results: Higher SETER/PR in a metastasis was associated with longer progression-free survival (PFS, 9 vs. 2 months) and overall survival (OS, 50 vs. 19 months) following endocrine therapy in the cohort with metastatic breast cancer (MBC) and relapsed disease (n=79), so a cut point was defined in that cohort. SETER/PR was also significantly associated with PFS after adjusting for PR status of the metastasis, presence of visceral metastases, number of previous relapse events, and clinical history of previous sensitivity to endocrine therapy (HR 0.485, 95%CI 0.265 – 0.889, p = 0.019). Technically, SETER/PR was highly reproducible under different pre-analytical and analytical conditions, including host organ contamination. The translated SETER/PR assay used a single 10 µm FFPE tissue section, did not require RNA purification, and represented the microarray results from matched fresh samples with excellent agreement (correlation = 0.980, n = 31). Conclusion: The SETER/PR index is a new biomarker to predict PFS and OS for patients with HR+/HER2- MBC who receive endocrine therapy. The assay is applicable to FFPE tissue sections from small biopsies of metastases. Additional independent (blinded) validation studies will be necessary to confirm these results.Rationale: A robust index for gene expression related to activity of estrogen (ESR1) and progesterone (PGR) receptors could predict sensitivity to endocrine therapy in metastatic breast cancer. Methods: Transcripts correlated with ESR1 and PGR expression in 389 hormone receptor-positive breast cancer samples (Affymetrix U133A microarrays) were ranked for reliability according to their pre-analytical (intratumoral heterogeneity, biopsy type) and analytical reproducibility. Eighteen target and ten reference genes were selected and summarized as the SETER/PR index. The SETER/PR index was evaluated in a different set of 140 biopsies from distant metastases of hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer, and in additional pre-analytical and analytical sample cohorts. Thereafter, SETER/PR was translated to a customized format for application to formalin-fixed and paraffin-embedded (FFPE) sections. Results: Higher SETER/PR in a metastasis was associated with longer progression-free survival (PFS, 9 vs. 2 months) and overall survival (OS, 50 vs. 19 months) following endocrine therapy in the cohort with metastatic breast cancer (MBC) and relapsed disease (n=79), so a cut point was defined in that cohort. SETER/PR was also significantly associated with PFS after adjusting for PR status of the metastasis, presence of visceral metastases, number of previous relapse events, and clinical history of previous sensitivity to endocrine therapy (HR 0.485, 95%CI 0.265 – 0.889, p = 0.019). Technically, SETER/PR was highly reproducible under different pre-analytical and analytical conditions, including host organ contamination. The translated SETER/PR assay used a single 10 µm FFPE tissue section, did not require RNA purification, and represented the microarray results from matched fresh samples with excellent agreement (correlation = 0.980, n = 31). Conclusion: The SETER/PR index is a new biomarker to predict PFS and OS for patients with HR+/HER2- MBC who receive endocrine therapy. The assay is applicable to FFPE tissue sections from small biopsies of metastases. Additional independent (blinded) validation studies will be necessary to confirm these results. Citation Format: Sinn BV, Tsai T-H, Lau R, Fu C, Gould R, Murthy R, King TA, Hatzis C, Kwiatkowski DN, Valero V, Symmans WF. SETER/PR - A robust 18-gene predictor of sensitivity to endocrine therapy in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-23.

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SETER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts (all had high SETER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript.

Background: Achieving a pathologic complete response (pCR) has been shown on the patient level to predict excellent long-term event-free survival outcomes. Residual cancer burden (RCB) quantifies the extent of residual disease for patients who did not achieve pCR. We have previously observed in the I-SPY 2 TRIAL that while metastatic events outside the central nervous system (CNS) were dramatically reduced in the setting of pCR, the incidence of CNS metastasis remained similar across RCB classes, raising the possibility that these CNS events may be independent of response in the breast. In this study, we evaluate the type and sites of recurrences by RCB in a large pooled dataset, which allows for analysis within subtype, to validate these findings. Methods: 5161 patients pooled across 12 institutions/trials with available RCB and event-free survival (EFS) data were included in this analysis. EFS was calculated as the interval between treatment initiation, and locoregional recurrence, distant recurrence or death from any cause; patients without event are censored at time of last follow-up. The median follow-up is 4.6 years. We summarized the EFS event type, further sub-dividing the distant recurrence events (DR) by their site of relapse (CNS-only, CNS and other sites, Non-CNS). We used a competing risk (Fine-Gray) model to assess which of these site-specific relapses differ between RCB classes and estimated the cumulative incidence of CNS-only and non-CNS events at 5 years. Analyses were performed across the entire study population and within HR/HER2 defined subtypes. Results: Among the 5161 subjects, there were 1164 EFS events, including 92 (7.9%) local recurrences (without distant recurrence and/or death) and 1072 distant recurrence-free survival (DRFS) events. Among the DRFS events, 158 patients died without a distant recurrence. 914 experienced distant recurrences, including 90 (9.8%) with CNS-only, 145 (15.9%) with CNS and other sites, 664 (72.6%) with non-CNS distant recurrence; 15 (1.6%) patients had missing recurrence site information. Table 1 summarizes the cumulative incidence of CNS-only and non-CNS recurrence at 5 years and the proportion of CNS-only recurrences among DR events by RCB class overall and within each HR/HER2 subtypes. The incidence of CNS-only recurrences was low and similar across RCB classes. In contrast, the incidence of non-CNS recurrences increases with increasing RCB. As a result, CNS-only recurrences are proportionally higher within the RCB-0 and RCB-I than in the RCB-II and RCB-III groups, largely because of the low DR event rate and relative low frequency of non-CNS recurrence events within the RCB-0 and RCB-I classes. Overall, 27% of the recurrences in the setting of pCR (RCB-0) are due to CNS-only recurrences.Conclusions: Consistent with previous studies, our large pooled analysis confirmed that CNS-only recurrences are uncommon but appear similar across RCB groups, independent of response, suggesting that the CNS is a treatment sanctuary site. In contrast, non-CNS recurrence rates increase as RCB increases. These findings suggest that inclusion of CNS-only recurrences as an outcome event may impact the association between neoadjuvant therapy response and long-term outcomes in the context of current therapies. Novel therapies that cross the blood brain barrier will be needed to impact CNS recurrence rates. Table 1: Cumulative Incidence of CNS Only and non-CNS Distant Recurrences at 5 years and proportion of CNS-only events among DR eventsRCB Class0IIIIIIpOverall (5161)N16766622017806Cum. Inc. CNS Only2%2%2%1%0.627Cum. Inc. Non-CNS3%6%16%27% Citation Format: Sonal Shad, Marieke van der Noordaa, Marie Osdoit, Diane de Croze, Anne-Sophie Hamy, Marick Lae, Fabien Reyal, Miguel Martin, María Del Monte-Millán, Sara López-Tarruella, I-SPY 2 TRIAL Consortium, Judy C Boughey, Matthew P Goetz, Tanya Hoskin, Rebekah Gould, Vicente Valero, Gabe Sonke, Tessa G Steenbruggen, Maartje van Seijen, Jelle Wesseling, John Bartlett, Stephen Edge, Mi-Ok Kim, Jean Abraham, Carlos Caldas, Helena Earl, Elena Provenzano, Stephen-John Sammut, David Cameron, Ashley Graham, Peter Hall, Lorna Mackintosh, Fan Fang, Andrew K Godwin, Kelsey Schwensen, Priyanka Sharma, Angela DeMichele, Janet Dunn, Louise Hiller, Larry Hayward, Jeremy Thomas, Kimberly Cole, Lajos Pusztai, Laura Van't Veer, Fraser Symmans, Laura Esserman, Christina Yau. Site of recurrence after neoadjuvant therapy: A multi-center pooled analysis [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 PD13-02.

Background: Recent studies have demonstrated independent validation of the prognostic relevance of residual cancer burden (RCB) after neoadjuvant chemotherapy. However, a pooled subject-level analysis of multiple cohorts is needed to determine estimates of long-term prognosis for each class of RCB in each phenotypic subtype of breast cancer (BC) to better inform on patient outcomes. Also, a pooled subject-level analysis allows more detailed analyses of generalizability of the prognostic meaning of RCB assessments in a broader experience of practice settings. Method: Subject-level RCB results, with relevant clinical and pathologic stage, tumor subtype and grade, demographic, treatment and follow-up data from 11 institutes/trials are being collected for combined analysis. The association between the continuous RCB index and event-free survival (EFS), and distant recurrence free survival (DRFS) were assessed using mixed effect Cox models with the incorporation of random RCB coefficients to account for between-study heterogeneity. We will also allow for differences in baseline hazard across biological BC subtypes and, if needed, across studies as well. In addition to this stratified mixed effect model, a multivariate analysis adjusting for age, T-category, nodal status and grade was performed within each subtype. In addition, mixed effect Cox models will be employed to evaluate association between RCB index with EFS and DRFS within each HR/HER2 subtype. Kaplan Meier estimates of EFS and DRFS at 5 and 10 years were computed for each RCB class within subtype. Results: We analyzed subject-level data from 9 institutes/trials representing 4077 patients currently available from an anticipated final total of 4,800 patients (to be presented at the meeting). There were 950 EFS and 876 DRFS events during follow up (median 65 months, IQR: 70 months). RCB index (continuous) was independently prognostic within each subtype: HR+/HER2- (EFS HR (per unit increase in RCB index) =1.64, 95%CI 1.48-1.82; DRFS HR=1.68, 1.51-1.87), HR+/HER2+ (EFS HR=1.80, 1.57-2.05; DRFS HR=1.93, 1.67-2.24), HR-/HER2+ (EFS HR=2.15, 1.76-2.62; DRFS HR=2.10, 1.77-2.50), and HR-/HER2- (EFS HR=2.05, 1.89-2.22; DRFS HR=2.16, 1.90-2.46); and remained prognostic in multivariate models adjusting for age, grade, and clinical T and N stage at diagnosis. Table 1 contains the response rate and estimated EFS at 5 years and 10 years for each RCB class within each HR/HER2 phenotype (DRFS results were similar). Conclusions: Long-term prognosis after pCR was similarly excellent in all phenotypic subtypes. RCB index and classification was independently and strongly prognostic in all subtypes, and generalizable to multiple practice settings. Prognostic differences by RCB class occurred within 5 years in HR- BC, but extended to 10 years in HR+ BC. RCB-I had slightly worse EFS than pCR in HR- BC and HR+/HER2+ BC (after 5 years), but the same EFS as pCR in HR+/HER2- BC. Complete analysis of all subjects, including neoadjuvant treatments, will be presented at the meeting. PhenotypeOutcomepCRRCB-IRCB-IIRCB-IIIHR+/HER2-Frequency (%)11%10%52%27%(N=1467)5 yr EFS (95% CI)91% (86-96)93% (89-98)82% (79-85)70% (65-75)10 yr EFS (95% CI)84% (75-93)88% (82-95)71% (67-75)52% (46-58)HR+/HER2+Frequency (%)38%18%35%9%(N=762)5 yr EFS (95% CI)94% (91-97)93% (88-98)78% (73-84)49% (37-65)10 yr EFS (95% CI)91% (86-96)79% (70-90)65% (59-73)42% (29-60)HR-/HER2+Frequency (%)66%11%18%5%(N=550)5 yr EFS (95% CI)93% (90-96)88% (79-97)60% (50-71)45% (30-69)10 yr EFS (95% CI)90% (86-94)84% (74-95)56% (46-68)45% (30-69)HR-/HER2-Frequency (%)41%13%33%13%(N=1293)5 yr EFS (95% CI)92% (90-94)85% (79-91)68% (63-72)28% (21-36)10 yr EFS (95% CI)87% (82-91)80% (72-88)63% (58-68)24% (18-33) Citation Format: Christina Yau, Marieke van der Noordaa, Jane Wei, Marie Osdoit, Fabien Reyal, Anne-Sophie Hamy, Marick Lae, Miguel Martin, Maria del Monte, I-SPY 2 TRIAL Consortium, Judy C Boughey, Rebekah Gould, Jelle Wesseling, Tessa Steenbruggen, Maartje van Seijen, Gabe Sonke, Stephen Edge, Stephen-John Sammut, Elena Provenzano, Jean Abraham, Peter Hall, Ashley Graham, Lorna Mackintosh, David Cameron, Alice Wang, Priyanka Sharma, Kimberly Cole, Lajos Pusztai, Mi-Ok Kim, Laura van ‘t Veer, Laura Esserman, W. Fraser Symmans. Residual cancer burden after neoadjuvant therapy and long-term survival outcomes in breast cancer: A multi-center pooled analysis [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 GS5-01.

Purpose To determine the long-term prognosis in each phenotypic subset of breast cancer related to residual cancer burden (RCB) after neoadjuvant chemotherapy alone, or with concurrent human epidermal growth factor receptor 2 (HER2)–targeted treatment. Methods We conducted a pathologic review to measure the continuous RCB index (wherein pathologic complete response has RCB = 0; residual disease is categorized into three predefined classes of RCB index [RCB-I, RCB-II, and RCB-III]), and yp-stage of residual disease. Patients were prospectively observed for survival. Three patient cohorts received paclitaxel (T) followed by fluorouracil, doxorubicin, and cyclophosphamide (T/FAC): original development cohort (T/FAC-1), validation cohort (T/FAC-2), and independent validation cohort (T/FAC-3). Another validation cohort received FAC chemotherapy only, and a fifth cohort received concurrent trastuzumab (H) with sequential paclitaxel and fluorouracil, epirubicin, and cyclophosphamide (FEC; H+T/FEC). Phenotypic subsets were defined by hormone receptor (HR) and HER2 status at diagnosis, classified as HR-positive/HER2-negative, HER2-positive (HR-negative/HER2-positive or HR-positive/HER2-positive), or triple receptor–negative. Relapse-free survival estimates were determined from Kaplan-Meier analysis and compared using the log-rank test. Results Five cohorts (T/FAC-1 [n = 219], T/FAC-2 [n = 262], T/FAC-3 [n = 342], FAC [n = 132], and H+T/FEC [n = 203]) had median event-free follow-up of 13.5, 9.1, 6.8, 16.4, and 7.1 years, respectively. Continuous RCB index was prognostic within each phenotypic subset, independent of other clinical-pathologic variables. RCB classes stratified prognostic risk overall, within each phenotypic subset, and within yp-stage categories. Estimates of 10-year relapse-free survival rates in the four RCB classes (pathologic complete response, RCB-I, RCB-II, and RCB-III) were 86%, 81%, 55%, and 23% for triple receptor–negative; 83%, 97%, 74%, and 52% for HR-positive/HER2-negative in the combined T/FAC cohorts; and 95%, 77%, 47%, and 21% in the H+T/FEC cohort. Conclusion RCB was prognostic for long-term survival after neoadjuvant chemotherapy in all three phenotypic subsets of breast cancer. Our institutional findings should be externally validated.

The residual cancer burden index was developed as a method to quantify residual disease ranging from pathological complete response to extensive residual disease. The aim of this study was to evaluate the inter-Pathologist reproducibility in the residual cancer burden index score and category, and in their long-term prognostic utility. Pathology slides and pathology reports of 100 cases from patients treated in a randomized neoadjuvant trial were reviewed independently by five pathologists. The size of tumor bed, average percent overall tumor cellularity, average percent of the in situ cancer within the tumor bed, size of largest axillary metastasis, and number of involved nodes were assessed separately by each pathologist and residual cancer burden categories were assigned to each case following calculation of the numerical residual cancer burden index score. Inter-Pathologist agreement in the assessment of the continuous residual cancer burden score and its components and agreement in the residual cancer burden category assignments were analyzed. The overall concordance correlation coefficient for the agreement in residual cancer burden score among pathologists was 0.931 (95% confidence interval (CI) 0.908-0.949). Overall accuracy of the residual cancer burden score determination was 0.989. The kappa coefficient for overall agreement in the residual cancer burden category assignments was 0.583 (95% CI 0.539-0.626). The metastatic component of the residual cancer burden index showed stronger concordance between pathologists (overall concordance correlation coefficient=0.980; 95% CI 0.954-0.992), than the primary component (overall concordance correlation coefficient=0.795; 95% CI 0.716-0.853). At a median follow-up of 12 years residual cancer burden determined by each of the pathologists had the same prognostic accuracy for distant recurrence-free and survival (overall concordance correlation coefficient=0.995; 95% CI 0.989-0.998). Residual cancer burden assessment is highly reproducible, with reproducible long-term prognostic significance.

Background: We developed SET4 as a targeted, droplet-based, next-generation RNA sequencing assay to measure both the SETER/PR index of gene expression and the percent of estrogen receptor (ER) transcripts with point mutation in the ligand-binding domain (LBD) in metastatic biopsies of Stage IV breast cancer. The SETER/PR index (31 genes) is a cumulative measure of gene expression for transcripts associated with ER and progesterone receptor (PR), excluding those with a direct role in proliferation. High SETER/PR could indicate increased sensitivity to endocrine therapy, whereas LBD mutations indicate resistance but might also induce high SETER/PR. Methods: Targeted needle biopsies from a metastatic site were prospectively obtained from 82 patients with HR+/HER2- breast cancer at time of any progression event, and preserved in RNAlater. Samples were prepared for targeted sequencing on a MiSeq by combining purified total RNA with SET4 primers and RT-PCR master mix into single molecule-formatted picodroplets using a RainDrop Source instrument, followed by thermal cycling and sample indexing. Calculated SETER/PR index and percent ER transcripts with LBD mutations were evaluated as continuous variables and compared to progression-free and overall survival using Cox regression analysis with log-rank test, if the next treatment after biopsy included endocrine therapy. Results: The average read depth for the LBD of the ER transcript was 33,475X (range: 1230-180,889X), confidently detecting mutation at 1% frequency. LBD mutations were identified in 17% (14/82) of metastases (range of mutated transcripts 1%-98%). LBD mutations (>10% of transcripts) were only observed in metastases with higher SETER/PR (above the median). In patients who next received endocrine therapy (n=58), higher SETER/PR predicted longer progression-free (PFS) (HR=0.37, p=0.0004, Δ median PFS 9 months) and overall survival (OS) (HR=0.49, p=0.03). The predictions were more pronounced in patients without LBD mutation (PFS HR=0.32, p=0.0001, Δ median PFS 13 months; OS HR=0.42, p=0.01). Currently, there are insufficient cases with LBD mutation for reliable survival analysis. Conclusion: The SET4 assay measured the percent of ER transcripts with activating LBD mutation (≥1% prevalence) and also downstream ER/PR-related transcription. High SETER/PR predicted longer PFS and OS with endocrine therapy. While activating LBD mutations may be associated with endocrine resistance of Stage IV cancer, they were associated with high SETER/PR index. Consequently, metastatic cancers with high SETER/PR index and no LBD mutation in ER transcripts were particularly sensitive to endocrine therapy. This single assay unraveled a possible interaction between genotype, phenotype, and treatment outcome; and is currently being evaluated in a larger cohort of patients. Citation Format: Rosanna Lau, Lily Fu, Michael Samuels, Rashmi K. Murthy, Bruno Sinn, Jane Yu, Rebekah Gould, Jennifer Litton, Alda Tam, Stacy Moulder, Daniel Booser, Debu Tripathy, Vicente Valero, Fraser Symmans. A targeted RNA-seq assay to measure activating ER mutations and ER/PR-associated gene expression predicts sensitivity to endocrine therapy for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1796. doi:10.1158/1538-7445.AM2017-1796

Background: In 2007 we described residual cancer burden (RCB) as a measure of pathologic response after neoadjuvant chemotherapy and demonstrated that RCB was strongly prognostic for 5-year survival outcomes. Herein, we update the long-term outcome of this original cohort, validate our results in an independent patient group, and report outcome according to hormone receptor (HR) and HER2 receptor status. Methods: Our original RCB development cohort received paclitaxel followed by fluorouracil, doxorubicin and cyclophosphamide (T/FAC) and the original validation cohort received FAC alone as neoadjuvant chemotherapy for stage II or III breast cancer (JCO 2007;25:4414-22). We subsequently evaluated RCB in 2008 in another independent validation cohort of patients treated with neoadjuvant T/FAC. These patient cohorts were followed up until 2013. The T/FAC cohorts were combined to test RCB within phenotypic subsets of HER2+, HR+/HER2-, and triple receptor-negative breast cancer (TNBC). Pre-defined RCB classes of pathologic complete response (pCR), minimal (RCB-I), moderate (RCB-II) and extensive (RCB-III) residual disease were compared with respect to relapse-free survival (RFS) using the likelihood ratio test and estimates of 10-year RFS (with 95% confidence interval) were determined from Kaplan-Meier analysis. We excluded patients with HER2-positive (HER2+) cancer who received neoadjuvant or adjuvant trastuzumab. Results: Median follow up of event-free survivors was 142 months for the original T/FAC development cohort (N = 220), 91 months for the T/FAC validation cohort (N = 277), and 198 months for the FAC (N = 169) cohort. The estimates of 10-year RFS by RCB class for each cohort, respectively, were 86%, 81% and 95% for pCR; 87%, 81% and 85% for RCB-I; 76%, 62%, and 62% for RCB-II; and 38%, 36% and 38% for RCB-III. Table 1 presents the frequency and the estimates of 10-year RFS for each RCB class within each phenotypic subset of breast cancer (HER2+, TNBC, and HR+/HER2-) in the combined T/FAC cohorts (N = 497). Frequency and estimated 10-year RFS of RCB classes in breast cancer subsets after T/FAC chemotherapy HER2+TNBCHR+/HER2-RCB ClassFreqRFS (95% CI)FreqRFS (95% CI)FreqRFS (95% CI)pCR36%83% (66 to 92)35%82% (63 to 92)10%83% (59 to 93)RCB-I16%69% (41 to 86)15%82% (53 to 94)13%96% (74 to 99)RCB-II30%50% (31 to 66)33%54% (38 to 68)60%76% (68 to 83)RCB-III18%32% (13 to 52)17%18% (6 to 36)17%45% (27 to 61)RFS, relapse-free survival; Freq, frequency; CI, confidence interval. RCB was prognostic in all cohorts and subgroups (p Conclusions: RCB after neoadjuvant chemotherapy was strongly prognostic in all phenotypic subsets of breast cancer patients over a decade of follow up, even for HR+/HER2- cancers. In HER2-negative breast cancers, RCB-I had similar prognosis to pCR, whereas RCB-III for HR+/HER2- and RCB-II/III for TNBC defined high-risk patient populations following neoadjuvant chemotherapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-02.

Background: Individuals with excellent pathologic response (complete response or minimal residual cancer burden, pCR+RCB-I) to neoadjuvant chemotherapy have prolonged survival, and several chemotherapy regimens have resulted in improved pathologic response rates. However, how to estimate expected improvements in disease free survival (DFS) in a clinical trial based on improvement in pathologic response remains uncertain. The purpose of this study was to develop a statistical tool to estimate improvements in DFS based in improvements in pCR/RCB-I in triple negative (TNBC) and HR+/HER2- breast cancer subtypes. Methods: 387 clinical stage II (73%) and III (23%) breast cancers who received neoadjuvant T/FAC chemotherapy at MD Anderson Cancer Center were included in this analysis (N = 127 TNBC, N = 260 HR+/HER2-). Patients received adjuvant endocrine therapy if HR+. To evaluate the association between pCR rate and survival we used within-subtype stratified bootstrap analysis with biased resampling from the two response groups (pCR and RCB-I) to generate 500 bootstrapped populations with a range of different response rates. Survival was based on the Kaplan-Meier estimator with its variance obtained from the bootstrap standard error. Power was estimated as the probability of no overlap in the 95% log confidence intervals of survival in 500 bootstrap replicates of populations of different sizes and response rates. Results: Excellent pathologic response (pCR or RCB-I), was observed in 50% and 23% in the TNBC and ER+/HER2- cohorts respectively. The median follow-up was 7.6 years (range 0.1 to 13.4 years); 48 and 59 relapses occurred in the TNBC and HR+ subtypes, respectively. Bootstrap analysis showed a linear dependence of the 5-year DFS on the pCR/RCB-I rate, with a slope of 0.472 for TNBC suggesting a 4.7% improvement in DFS for every 10% increase in response rate. A more modest slope of 0.129 was observed for ER+/HER2- cancers. The sample size of a randomized 2-arm study required to show with 80% power a statistically significant improvement in 5-year DFS that corresponds to 75% response rate compared to baseline response rate of 50% and 23% in TNBC and ER+/HER2- would be 1144 and 1688 cases respectively. We are providing an open-access web-based calculator to estimate improvements in DFS and for sample size calculations for randomized clinical trials with combined endpoints of pCR/RCB-I and survival. Conclusions: We observed a linear increase in DFS with increase in pathologic response rate in the evaluated cohort. The slope depends on breast cancer subtype - it is greater in TNBC than in ER+ cancers - and expected to be influenced by the stage distribution in the study population. These results could help provide a basis for powering studies with combined response and survival endpoints. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-37.