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Background The OlympiAD trial evaluated the PARP inhibitor (PARPi) olaparib versus a non-platinum standard chemotherapy in HER2-negative metastatic breast cancer (MBC) patients with a germline BRCA1/2 (gBRCA) mutation. Olaparib resulted in improved progression-free survival (PFS) and doubled the response rate vs chemotherapy. Nevertheless, the response rate to PARPi is 60% in the gBRCA1/2 population with MBC (current approval), suggesting a limited positive predictive value of gBRCA1/2 status. Moreover, patients with other relevant Homologous Recombination Repair defects (HRD) such as PALB2 or RAD51C/D mutation carriers, or HRD epigenetic silencing, are not captured with a gBRCA analysis. We have previoulsy shown that the functional HRD biomarker RAD51, tested in FFPE tumor samples using an optimized immunofluorescence-based assay, is associated with platinum response in early TNBC and PARPi response in preclinical BC models. We hypothesize that the RAD51 test would help to expand the clinical benefit of PARPi by predicting response to olaparib in MBC with germline/somatic BRCA1/2, PALB2 or RAD51C/D mutation and beyond. Study design RADIOLA is an open-label, single-arm, multicentre phase II study evaluating treatment with olaparib in male or female ≥18 years patients with HER2-negative MBC with ≤ two prior chemotherapy lines in two cohorts: cohort 1 (N=41) with known germline/somatic BRCA1/2, PALB2 or RAD51C/D mutation; cohort 2 (N=25) with functional HRD, namely RAD51-low score (≤10%), in wild-type/unknown mutation status at study entry. All patients will receive olaparib 300mg po BID until progression or unacceptable toxicity. Primary objective will assess, in terms of overall response rate (ORR), the capacity of the RAD51 score to predict olaparib efficacy in cohort 1. Secondary objectives include PFS, clinical benefit rate, duration of response, safety in both cohorts and ORR in cohort 2. Recruitment Recruitment (11 sites) started in March 2022. As of July 2022, 7 patients have been enrolled in Spain. Funding This study is financially supported by AstraZeneca. Citation Format: Judith Balmaña, Tomás Pascual, Alba Llop-Guevara, Pablo Tolosa, Isabel Blancas, Maria-Eva Perez-Lopez, Barbara Adamo, Iris Teruel, Jose Ponce, Marta Gonzalez, Gemma Viñas, Laura Lema, Francisco Javier Salvador, Mª Teresa Martinez, Alejandra Espinosa, Aleix Prat, Violeta Serra. SOLTI-1910: Predicting olaparib sensitivity in patients with unresectable locally advanced/metastatic HER2-negative breast cancer with BRCA1/2, PALB2, RAD51C/D mutations or HRD by the RAD51 test: RADIOLA TRIAL [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 OT3-11-02.

Background: Palbociclib might be less effective than ribociclib in HR+/HER2- and HER2-enriched BC. This hypothesis is currently being tested in the HARMONIA prospective phase III clinical trial (NCT05207709). Here, we evaluated the downstream biological effects of both drugs using cell lines and patient tumor samples. Methods: Three HR+ BC cell lines (i.e., MCF7, T47D and BT474) were treated at 2 different dose levels of palbociclib or ribociclib (i.e., 100 nM and 500 nM) +/- fulvestrant (1 nM) for 72 hours (h). PAM50 gene signatures were determined on the nCounter, as well as phosphorylation of RB (p-RB) by Western Blot and senescence-associated β-galactosidase activity by FACS. In vitro experiments were performed in triplicates. PAM50 gene signatures were obtained from 49 paired baseline versus week-2 samples and 49 paired baseline versus surgery samples of the CORALLEEN phase II study (Prat, Lancet Oncol. 2020), which treated 49 women with PAM50 Luminal B HR+/HER2- early BC with neoadjuvant ribociclib (600 mg daily) plus endocrine therapy (ET). PAM50 signature scores were also evaluated in publicly available data from 23 paired baseline versus week-2 samples and 16 paired baseline versus surgery samples of the NEOPALANA phase II trial (Ma, Clin Cancer Res. 2017) which treated 50 patients with HR+/HER2- early BC with palbociclib (125 mg daily) plus anastrozole. Changes in PAM50 signatures upon CDK4/6i were determined by paired t-tests and significant analysis of microarray (SAM). Results: Across all cell lines, both palbociclib and ribociclib statistically significantly reduced p-RB at 72h with both doses (i.e., 100 and 500 nM) compared to non-treated cells (p< 0.001). Senescence was also observed at 72h with both doses. Both drugs +/- ET significantly increased the Luminal A signature and decreased Luminal B and proliferation signatures with both doses. However, the HER2-enriched signature was only significantly reduced when both CDK4/6 inhibitors were given at 500 nM. In tumor samples from the CORALLEEN and NEOPALANA phase II studies, a similar change in PAM50 biology was observed with both drugs, namely an increase in Luminal A signature and a decrease in Luminal B and proliferation signatures after 2 weeks of treatment and at surgery. At 2 weeks of treatment, the HER2-enriched signature was significantly decreased in both studies with ribociclib (p< 0.001) and palbociclib (p< 0.001). However, the decrease in the HER2-enriched signature was only observed in surgical samples of patients treated with ribociclib (p< 0.001), but not palbociclib (p=0.194). A difference in sample size could explain this result. Nevertheless, in CORALLEEN, the median number of days between the last dose of ribociclib and surgery was 13.1 days (range: 1-78). In NEOPALANA, the median number of days between the last dose of palbociclib and surgery was 29 days (range: 8-49), except for 8 patients who received additional 10-12 days of palbociclib immediately before surgery (Ma, Clin Cancer Res. 2017). In patients who underwent surgery at 8 days or before, the HER2-enriched signature was significantly decreased for both ribociclib (p< 0.001) and palbociclib (p=0.013). Interestingly, in patients that underwent surgery after >8 days from the last dose, a significant reduction of the HER2-enriched signature was only observed with ribociclib (p< 0.001), but not with palbociclib (p=0.500). Conclusions: Both palbociclib and ribociclib have similar effects on PAM50 biology when given at the same dose. However, in clinical practice, palbociclib is given at a lower dose than ribociclib, and although HER2-enriched signature is significantly decreased in tumors after 2 weeks of CDK4/6i+ET, this effect is only maintained at later time points with ribociclib, indicating a dose-dependent efficacy of CDK4/6i in this biologically aggressive subtype. Citation Format: Natàlia Lorman-Carbó, Olga Martínez-Sáez, Aranzazu Fernandez-Martinez, Patricia Galván, Nuria Chic, Barbara Adamo, Maria Vidal, Montserrat Muñoz, Charles M. Perou, Joaquín Gavilá, Tomás Pascual, Aleix Prat, Fara Brasó-Maristany. Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) [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 P1-13-16.

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

Immune-checkpoint inhibitors (ICI) have revolutionized the therapeutic landscape of cancer. However, optimal patient selection is still an unmet need. One-hundred-forty-six patients with metastatic cancer candidates to ICI at the Hospital Clinic of Barcelona Clinical Trials Unit were prospectively recruited in this observational study. Blood samples were collected at different timepoints, baseline LIPI score calculated and pre-ICI archived tissues retrieved to evaluate PD-L1, tumor-infiltrating lymphocytes (TILs) and PD1 mRNA levels. Tumor assessments were centrally reviewed by RECIST 1.1 criteria. Associations with overall response rates (ORR), durable clinical benefit (DCB), progression-free survival (PFS) and overall survival (OS) were performed with univariable/multivariable logistic and Cox regressions, where appropriate. At a median follow-up of 26.9 months, median PFS and OS were 2.7 and 12.9 months. Response rates were 17.8% with duration of response (DOR) of 4.4 months. LIPI score was independently associated with PFS (p = 0.025) and OS (p p = 0.005). Time-to-best response (TTBR) and ORR (p p p = 0.028) and DCB (univariate p = 0.043). PD1 mRNA levels were strikingly associated to complete responses (p = 0.021). To resume, in our prospective observational pan-cancer study, baseline LIPI score, immunotherapy-naïve status, cancer type and RT before starting ICI were the most relevant clinical factors independently correlated with immunotherapy outcomes. Longer TTBR seemed to associate with better survival, while PD1 mRNA and PD-L1 protein levels might be tumor-agnostic predictive factors of response to ICI and should be furtherly explored.

In advanced HER2-positive (HER2+) breast cancer, the new antibody-drug conjugate trastuzumab deruxtecan is more effective compared with trastuzumab emtansine (T-DM1). However, trastuzumab deruxtecan can have considerable toxicities, and the right treatment sequence is unknown. Biomarkers to guide the use of anti-HER2 therapies beyond HER2 status are needed. Here, we evaluated if preestablished levels of ERBB2 mRNA expression according to the HER2DX standardized assay are associated with response and survival following T-DM1. In ERBB2 low, medium, and high groups, the overall response rate was 0%, 29%, and 56%, respectively (P

This file contains additional details on the microarray analysis and normalization.

Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA–wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation. Mol Cancer Ther; 14(4); 920–30. ©2015 AACR.

This file contains the compiled Supplemental Figures S1-S9. Figure S1 is a flowchart describing the process of normalizing the microarray data for the SUM149 model for the different scanners used. Figure S2 depicts the scanner differences in the microarray data for the SUM149 model before and after normalization. Figure S3 is a flowchart depicting the analytical process used to analyze and compare the SUM149 and MDA-MB-436 models microarray results, and Figure S4 shows the difference in scale between models before and after the scaling process for visual comparison of the SUM149 and MDA-MB-436 models. Figure S5 demonstrates the lack of survival benefit of Carboplatin +/- ABT88 in the two BRCA-wt models, MDA-MB-468 and MDA-MB-231BR. Figure S6 contains representative images of IHC cC3 staining for the BRCA-wt model, MDA-MB-468. Figure S7 shows the unsupervised clustering of the BRCA-mut SUM149 and MDA-MB-436 models with treatment. Figure S8 contains the supervised clustering based on carboplatin treatment in these two models. Figure S9 demonstrates the expression levels of the 38 commonly regulated genes in both models with carboplatin treatment.

This file contains the differentially expressed genes and enriched pathways lists from the gene expression microarray analyses in Supplemental Table S1, with sample information in S1a. Lists are for the following comparisons: SUM149 Control vs. ABT888 (S1b), MDA-MB-436 Control vs. ABT888 (S1c), SUM149 Control vs. Carboplatin (S1d), MDA-MB-436 Control vs. Carboplatin (S1e), SUM149 Control vs Carboplatin+ABT888 (S1f), MDA-MB-436 Control vs Carboplatin+ABT888 (S1g), SUM149 Carboplatin vs Carboplatin+ABT888 (S1h), MDA-MB-436 Carboplatin vs Carboplatin+ABT888 (S1i). DAVID pathway enrichment analysis results are shown for the significantly up-regulated (S1j) and down-regulated (S1k) genes with Carboplatin treatment for both models.

This file contains the figure legends for Supplemental Figures S1-S9.

Survival outcomes based on the PAM50MET score in the BOLERO-2 trial. (A) Progression-free survival using a predefined cutoff in patients treated with placebo. (B) Progression-free survival using a predefined cutoff in patients treated with everolimus. (C) Overall survival using a predefined cutoff in patients treated with placebo. (D) Overall survival using a predefined cutoff in patients treated with everolimus. Interaction tests between PAM50 and treatment arm for PFS and OS were statistically non-significant (all p-values >0.05). PAM50MET, final model.

Purpose: Most hormone receptor (HR)+/HER2− breast cancer patients respond unfavorably to neoadjuvant chemotherapy (NAC); however, genomic tests may identify those patients who are likely to benefit. Using the Prosigna assay, we first evaluated the technical performance of core needle biopsy (CNB) tissues. We then determined whether Prosigna risk of relapse (ROR) score and intrinsic subtype predicted response to NAC in HR+/HER2− patients using CNB samples.Experimental Design: Using the NanoString's nCounter Dx analysis system and a development tissue sample set, we established tissue requirements and assay output variance. We then evaluated the concordance in subtype and correlation in ROR between CNBs and corresponding surgical resection specimens (SRS) in a second independent sample set. Finally, we analyzed 180 independent CNB samples from HR+/HER2− patients who were treated with NAC and correlated ROR and intrinsic subtype with pathologic response.Results: Intra- and interbiopsy variabilities were 2.2 and 6.8 ROR units, respectively. Subtype concordance within multiple CNBs was high for the 4- and 3-subtype classifications (k = 0.885 and 0.889, respectively). Correlation in Prosigna ROR score observed between paired CNBs and SRS was high (r ≥ 0.90), and subtype concordance was also high for the 4- and 3-subtype classifications (kappa = 0.81 and 0.91, respectively). Prosigna results obtained from the HR+/HER2− patient samples showed that both ROR (P = 0.047) and intrinsic subtype (OR LumA vs. non-LumA = 0.341, P = 0.037) were significant predictors of response to NAC.Conclusions: Prosigna ROR and intrinsic subtype are readily obtained from CNB samples in normal practice and reliably predict response to NAC in HR+/HER2− patients. Clin Cancer Res; 22(3); 560–6. ©2015 AACR.

PDF file - 797K, Figure S1. Barrier phenotype of human brain microvascular endothelial cells and astrocytes in monocultures and co-cultures. Figure S2. alpha/beta-crystallin does not affect cell viability of TNBC cells in standard monolayer culture. Figure S3. alpha/beta-crystallin overexpression increases liver metastases in an orthotopic model of TNBC. Figure S4. Silencing alpha/beta-crystallin inhibits liver metastases in an orthotopic model of TNBC. Figure S5. alpha/beta-crystallin does not affect proliferation or apoptosis in primary mammary tumors and brain metastatic lesions determined at autopsy. Table S1. Patient characteristics Table S2. alpha/beta-crystallin expression in paired tumors. Table S3. Association of alpha/beta-crystallin expression with breast cancer subtype. Table S4. Incidence of metastases in mice in orthotopic models of TNBC .

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Supplementary Data

Biological changes that occur during metastatic progression of breast cancer are still incompletely characterized. In this study, we compared intrinsic molecular subtypes and gene expression in 123 paired primary and metastatic tissues from breast cancer patients. Intrinsic subtype was identified using a PAM50 classifier and χ2 tests determined the differences in variable distribution. The rate of subtype conversion was 0% in basal-like tumors, 23.1% in HER2-enriched (HER2-E) tumors, 30.0% in luminal B tumors, and 55.3% in luminal A tumors. In 40.2% of cases, luminal A tumors converted to luminal B tumors, whereas in 14.3% of cases luminal A and B tumors converted to HER2-E tumors. We identified 47 genes that were expressed differentially in metastatic versus primary disease. Metastatic tumors were enriched for proliferation-related and migration-related genes and diminished for luminal-related genes. Expression of proliferation-related genes were better at predicting overall survival in metastatic disease (OSmet) when analyzed in metastatic tissue rather than primary tissue. In contrast, a basal-like gene expression signature was better at predicting OSmet in primary disease compared with metastatic tissue. We observed correlations between time to tumor relapse and the magnitude of changes of proliferation, luminal B, or HER2-E signatures in metastatic versus primary disease. Although the intrinsic subtype was largely maintained during metastatic progression, luminal/HER2-negative tumors acquired a luminal B or HER2-E profile during metastatic progression, likely reflecting tumor evolution or acquisition of estrogen independence. Overall, our analysis revealed the value of stratifying gene expression by both cancer subtype and tissue type, providing clinicians more refined tools to evaluate prognosis and treatment. Cancer Res; 77(9); 2213–21. ©2017 AACR.

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Supplemental Methods, Tables and Figures Tables Table S1. CES association with chemotherapy sensitivity (measured as pCR) in the MDACC-based dataset. Table S2. CES association with chemotherapy sensitivity (measured as Residual Cancer Burden [RCB]) in the MDACC-based dataset. Model A. Table S3. CES association with chemotherapy sensitivity (measured as Residual Cancer Burden [RCB]) in the MDACC-based dataset. Model B. Table S4. Univariate association of CES and various signatures with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S5. Association of CES and PAM50 Proliferation Signature with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S6. Association of CES and CHEMOPRED Signature with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S7. Association of CES and the proliferation component of the Genomic Health Index (GHI; OncotypeDX Recurrence Score) with chemotherapy sensitivity in HR+/HER2- negative disease from the MDACC-based dataset. Table S8. Association of CES and Genomic Grade Index (GGI) Signature with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S9. Association of CES and SET index Signature with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S10. Association of CES and RCBPRED Signature with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S11. Association of CES and DLDA30 Signature with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S12. Association of CES and ROR-P Signature with chemotherapy sensitivity in HR+/HER2-negative disease from the MDACC-based dataset. Table S13. Association of CES and Ki67 by IHC with chemotherapy sensitivity in HR+/HER2-negative disease of the Malaga cohort. 7 Table S14. Association of CES and PAM50 ROR with chemotherapy sensitivity in HR+/HER2-negative disease of the Malaga cohort. Table S15. CES association with endocrine sensitivity in the Marsden dataset (n=103). Table S16. CES association with endocrine sensitivity in the Marsden dataset within HER2-negative disease (n=89). Figures Fig. S1. Association of CES with Miller-Payne following chemotherapy in HR+/HER2- negative disease from the Malaga-based cohort. Fig. S2. CES association with endocrine sensitivity in the Edinburgh dataset (n=120). (A) Tumor volume changes of each patient and response classification. (B) Association of CES and other variables with response (defined as at least 70% reduction by 90 days) in the overall population. (C) Association of CES and other variables with response within HER2-negative disease. Fig. S3. Association of CES with pCR in the combined dataset from the MDACC- and Malaga-based cohorts.

Clinical-pathological and genomic variable (n = 21) used to calculate the PAM50MET model. PAM50MET, final model.

Purpose:Predicting prognosis in HR+/HER2− metastatic breast cancer (MBC) might be clinically useful; however, no validated prognostic biomarkers exist in this setting to date.Patients and Methods:In phase III, EGF30008 trial, 484 patients with HER2− MBC who received letrozole and placebo or lapatinib were selected. PAM50 data, ECOG performance status, visceral disease, number of metastasis, biopsy type, and age were evaluated. A progression-free survival (PFS) Cox model was evaluated. The final model (PAM50MET) with a prespecified cutoff was validated in patients (n = 261) with HR+/HER2− advanced breast cancer (aBC) from BOLERO-2 (phase III trial that evaluated exemestane and placebo or everolimus).Results:In EGF30008, prognostic models with PAM50 plus clinical variables yielded higher C-index values versus models with only PAM50 or clinical variables. The PAM50MET model combined 21 variables: 2 PAM50 subtypes, basal signature, 14 genes, and 4 clinical variables. In EGF30008, the optimized cutoff was associated with PFS [HR = 0.37; 95% confidence interval (CI), 0.29–0.47; P < 0.0001] and overall survival (OS; HR = 0.37; 95% CI, 0.27–0.51; P < 0.0001). The median (months; 95% CI) PFS and OS were 22.24 (19.0–24.9) and not reached in PAM50MET-low versus 9.13 (8.15–11.0) and 33.0 (28.0–40.0) in PAM50MET-high groups, respectively. In BOLERO-2, the PAM50MET-low was associated with better PFS (HR = 0.72; 95% CI, 0.53–0.96; P = 0.028) and OS (HR = 0.51; 95% CI, 0.35–0.69; P < 0.0001). The median (months) (95% CI) PFS and OS were 6.93 (5.57–11.0) and 36.9 (33.4–NA) in PAM50MET-low versus 5.23 (4.2–6.8) and 23.5 (20.2–28.3) in PAM50MET-high groups, respectively.Conclusions:PAM50MET is prognostic in HR+/HER2− MBC, and further evaluation might help identify candidates for endocrine therapy only or novel therapies.

Purpose:We do not yet have validated biomarkers to predict response and outcome within hormone receptor–positive/HER2-positive (HR+/HER2+) breast cancer. The PAM50-based chemo-endocrine score (CES) predicts chemo-endocrine sensitivity in hormone receptor–positive/HER2-negative (HR+/HER2−) breast cancer. Here, we evaluate the relationship of CES with response and survival in HR+/HER2+ breast cancer.Experimental Design:Intrinsic subtype and clinicopathologic data were obtained from seven studies in which patients were treated with HER2-targeted therapy either with endocrine therapy (ET) or with chemotherapy (CTX). CES was evaluated as a continuous variable and categorically from low to high scores [CES-C (chemo-sensitive), CES-U (uncertain), and CES-E (endocrine-sensitive)]. We first analyzed each dataset individually, and then all combined. Multivariable analyses were used to test CES association with pathologic complete response (pCR) and disease-free survival (DFS).Results:A total of 457 patients were included (112 with ET and 345 with CTX). In the combined cohort, CES-C, CES-U, and CES-E were identified in 60%, 23%, and 17% of the patients, respectively. High CES (i.e., CES-E) was associated with a lower probability of achieving pCR independently of clinical characteristics, therapy, intrinsic subtype, and study (adjusted OR = 0.42; P = 0.016). A total of 295 patients were analyzed for DFS with a median follow-up of 66 months. High CES was also associated with better DFS (adjusted HR, 0.174; P = 0.003) independently of pCR, clinical characteristics and intrinsic subtype. In patients with residual disease, the adjusted DFS HR of CES was 0.160 (P = 0.012).Conclusions:In HER2+/HR+ breast cancer, CES is useful for predicting chemo-endocrine sensitivity and provides additional prognostication beyond intrinsic subtype and clinicopathologic characteristics.

Purpose:To assess palbociclib in combination with trastuzumab with or without endocrine therapy in patients with HER2-positive advanced breast cancer.Patients and Methods:PATRICIA is a prospective, open-label, multicenter phase II trial. Patients had received 2–4 prior lines of anti-HER2–based regimens. Treatment consisted of palbociclib 200 mg daily for 2 weeks and 1 week off plus trastuzumab. The study was based on a Simon two-stage design comprising three cohorts: estrogen receptor (ER)-negative (cohort A), ER-positive (cohort B1), and ER-positive with letrozole (cohort B2). ER-positive patients were randomized to cohorts B1 or B2. Primary endpoint was progression-free survival rate at 6 months (PFS6). Secondary objectives included safety and evaluation of the PAM50 intrinsic subtypes.Results:Seventy-one patients were recruited (n = 15 in cohort A and 28 in each cohort B). The PFS6 rate in cohorts A, B1, and B2 was 33.3% (5/15), 42.8% (12/28), and 46.4% (13/28), respectively. Regarding safety, grade 1–2 and 3–4 toxicities occurred in 97.7% and 84.4% of patients, respectively. The most common grade 3–4 toxicities were neutropenia (66.4%) and thrombocytopenia (11.3%). Regarding PAM50, 59 (83.1%) tumors were profiled. Luminal disease defined by PAM50 was found independently associated with longer PFS compared with non-luminal disease (10.6 vs. 4.2 months median PFS; adjusted hazard ratio = 0.40; P = 0.003).Conclusions:Palbociclib in combination with trastuzumab is safe and exhibits promising survival outcomes in trastuzumab pretreated ER-positive/HER2-positive advanced breast cancer with a PAM50 Luminal A or B subtype. The enrollment was stopped prematurely, and a new randomized cohort was opened in this population.

Purpose: Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a high incidence of brain metastases and poor clinical outcomes. The molecular chaperone αB-crystallin is predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor phenotype in preclinical models. We investigated the potential role of αB-crystallin in brain metastasis in TNBCs.Experimental Design: αB-crystallin expression in primary breast carcinomas and brain metastases was analyzed by immunohistochemistry among patients with breast cancer with brain metastases. αB-crystallin was overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain microvascular endothelial cells (HBMEC) or extracellular matrix proteins, transendothelial migration, and transmigration across a HBMEC/astrocyte coculture blood–brain barrier (BBB) model were examined. In addition, the effects of overexpressing or silencing αB-crystallin on brain metastasis in vivo were investigated using orthotopic TNBC models.Results: In a cohort of women with breast cancer brain metastasis, αB-crystallin expression in primary breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even among patients with TNBC. Stable overexpression of αB-crystallin in TNBC cells enhanced adhesion to HBMECs, transendothelial migration, and BBB transmigration in vitro, whereas silencing αB-crystallin inhibited these events. αB-crystallin promoted adhesion of TNBC cells to HBMECs, at least in part, through an α3β1 integrin–dependent mechanism. αB-crystallin overexpression promoted brain metastasis, whereas silencing αB-crystallin inhibited brain metastasis in orthotopic TNBC models.Conclusion: αB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential biomarker and drug target for this aggressive disease. Clin Cancer Res; 20(1); 56–67. ©2013 AACR.

Supplementary Figure S1 and Supplementary Tables S1-S2. Supplementary Figure S1. Diagram of tissue block usage for CNB Development Study and Chemo-prediction Validation Study. Supplemental Table S1. Top-10 and bottom-10 Prosigna gene-correlations between paired CNBs and SRS. Supplemental Table S2. Distribution of the Prosigna subtypes within the three main pathology-based groups in 39 independent metastatic samples.

Supplementary Data

Purpose: Hormone receptor–positive (HR+) breast cancer is clinically and biologically heterogeneous, and subgroups with different prognostic and treatment sensitivities need to be identified.Experimental Design: Research-based PAM50 subtyping and expression of additional genes was performed on 63 patients with HR+/HER2− disease randomly assigned to neoadjuvant multiagent chemotherapy versus endocrine therapy in a phase II trial. The biology associated with treatment response was used to derive a PAM50-based chemoendocrine score (CES). CES's predictive ability was evaluated in 4 independent neoadjuvant data sets (n = 675) and 4 adjuvant data sets (n = 1,505). The association of CES, intrinsic biology, and PAM50 risk of relapse (ROR) was explored across 6,007 tumors.Results: Most genes associated with endocrine sensitivity were also found associated with chemotherapy resistance. In the chemotherapy test/validation data sets, CES was independently associated with pathologic complete response (pCR), even after adjusting for intrinsic subtype. pCR rates of the CES endocrine–sensitive (CES-E), uncertain (CES-U), and chemotherapy-sensitive (CES-C) groups in both data sets combined were 25%, 11%, and 2%, respectively. In the endocrine test/validation data sets, CES was independently associated with response. Compared with ROR, >90% of ROR-low and ROR-high tumors were identified as CES-E and CES-C, respectively; however, each CES group represented >25% of ROR-intermediate disease. In terms of survival outcome, CES-C was associated with poor relapse-free survival in patients with ROR-intermediate disease treated with either adjuvant endocrine therapy only or no adjuvant systemic therapy, but not in patients treated with (neo)adjuvant chemotherapy.Conclusions: CES is a genomic signature capable of estimating chemoendocrine sensitivity in HR+ breast cancer beyond intrinsic subtype and risk of relapse. Clin Cancer Res; 23(12); 3035–44. ©2016 AACR.

Background: HoR+ breast cancer (BC) is an heterogeneous disease, comprising 4 molecular subtypes (i.e.Luminal A [LumA], Luminal B [LumB]), HER2-Enriched [HER2E] and Basal-like [BL]). In HoR+ MBC, non-LumA subtypes appear to be associated with poorer survival outcome compared to LumA. Here, we present a meta-analysis to validate the prognostic value of IS inHoR+ MBC. Methods: We performed a systematic review and trial-level meta-analysis of all available prospective phase II/III trials in HoR+ MBC where the prognostic role of the IS was assessed in terms of progression-free survival (PFS) or time-to-progression (TTP), if the former was not available. Hazard ratios (HR) and 95% confidence intervals (CI) had to be available or computable. Subgroup analyses according to treatment, menopausal and HER2 status were also performed. The random-effect model of Der Simonian and Laird was applied. Heterogeneity was assessed through Cochran’s Q and I2. Funnel plot with Egger’s and Begg’s test for publication bias, multiple sensitivity analyses and risk of bias analysis were also conducted. Revman 5.4 and R 3.6.1 for MacOS X were used for statistical analyses. Tests were two-sided and the threshold for significance was set at p Table 1.Included studies characteristicsStudy ReferenceTrial NameBreast CancerSubgroupMenopausal StatusTrial TypeTreatmentNon-Luminal ALuminal APrat A. et al. The Oncologist 2019BOLERO2HR+/HER2-negPostmenopausalRandomizedEverolimus+exemestane vs exemestane139122Jørgensen C.L.T. et al. Acta Oncologica 2014DBCGMainly HR+/HER2-negMixedRandomizedDocetaxel+gemcitabine vs docetaxel18684Prat A. et al. JAMA Oncol 2016EGF30008HR+/HER2+ and negPostmenopausalRandomizedLetrozole+lapatinib vs letrozole424377Prat A. et al. J Clin Oncol 2021MONALEESA 2HR+/HER2-negPostmenopausalRandomizedRibociclib+letrozole vs letrozole--Prat A. et al. J Clin Oncol 2021MONALEESA 3HR+/HER2-negPostmenopausalRandomizedRibociclib+fulvestrant vs fulvestrant618542Prat A. et al. J Clin Oncol 2021MONALEESA 7HR+/HER2-negPremenopausalRandomizedRibociclib+AI/TAM vs AI/TAM--Ciruelos E. et al. Clin Can Res 2020PATRICIAHR+/HER2+PostmenopausalNon-RandomizedPalbociclib+/-letrozole+trastuzumab3410 Citation Format: Francesco Schettini, Olga Martínez-Sáez, Nuria Chic, Fara Brasó-Maristany, Patricia Galván, Debora Martínez, Laia Paré, Maria Vidal, Barbara Adamo, Montserrat Muñoz, Tomás Pascual, Eva Ciruelos, Charles M Perou, Lisa A Carey, Aleix Prat. Prognostic value of intrinsic subtypes (IS) in hormone receptor-positive (HoR+) metastatic breast cancer (MBC): A systematic review and meta-analysis of prospective trials [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 P4-07-08.

BACKGROUND: In early-stage triple negative breast cancer (TNBC), the addition of carboplatin (CBDCA) to neoadjuvant chemotherapy (CT) increases pathologic complete response (pCR) and relapsed-free survival. However, it is unclear whether CBDCA improves overall survival (OS). In addition, the prognostic and/or predictive role of pathogenic germline variants (PGV) in BRCA1/2 genes and other cancer risk in this setting is not fully understood. Here, we assessed the efficacy of (neo)adjuvant CBDCA and the prognostic and predictive role of a panel of 14 genes in patients (pts) with eTNBC. METHODS: This is a retrospective study on 117 pts diagnosed with early-stage TNBC between 2000-2021 at Hospital Clinic of Barcelona. Eighty-one pts (69%) were candidates for PGV testing. Overall, 14 genes (PGV) (BRCA1, BRCA2, PALB2, BRIP1, CHEK2, TP53, ATM, RAD51C, RAD51D, BARD1, MLH1, MSH2, MSH6 and PMS2) were assessed using the TruSight hereditary cancer panel (Illumina MySeq platform) according to local guidelines. Univariable and multivariable logistic regression and Cox regression analyses were performed to identify clinical and molecular predictors of pCR and relapse-free survival (RFS), respectively. Chi-squared or Fisher’s exact tests were used to assess characteristics’ distribution as appropriate. RESULTS: Of 117 pts, 83 (71%) received CT in the neoadjuvant setting and 28 (24%) in the adjuvant setting. CBDCA was added to standard CT in 68 pts (82%) in the neoadjuvant cohort and 7 pts (6%) in the adjuvant cohort. Among pts with germline testing, 32/81 (39%) harbored PGV. BRCA1 was the most frequently mutated gene (18/32, 56%), followed by BRCA2 (5/32, 16%), PALB2 (4/32, 13%), BRIP1 (2/32, 6%), CHEK2, TP53 and PMS2 (3/32, 6%). Percentages of pts receiving CBDCA were similar between patients with and without PGV (14/16,87% and wild type (53/67,79%)(p=0.120). In the neoadjuvant cohort (n=83), CBDCA was the only variable significantly associated with pCR at both univariate (pCR rates of 58.5% with CBCDA and 14.3% without CBCDA; odds ratio [OR]=8.2 [95% CIs 2.0-55.7], p=0.008) and remained statistically significant after adjusting for PGV, tumor size and nodal status (OR=6.9 [95% CIs 1.4-53.0], p=0.028). pCR rates according to PGV are reported in Table 1. In terms of RFS, addition of CBDCA to neoadjuvant therapy (hazard ratio [HR]=0.2 [0.1-0.45], p< 0.001), PGV (HR=0.2 [0.05-0.9], p=0.048), pCR (HR=0.2 [0.1-0.6], p=0.004) and nodal status (HR=5.1 [1.7-15.2], p< 0.003) were significantly associated with RFS in univariate analyses. In a multivariable model, CBDCA remained an independent predictor of improved RFS along with pCR. When the neoadjuvant and adjuvant cohort were pooled together (n=117), platinum-based CT remained significantly associated with better RFS (HR=0.2 [0.14-0.86], p=0.021) regardless of time of administration (i.e., neoadjuvant or adjuvant). With a median follow-up of 5 years, CBCDA use was not found associated with OS (HR=0.7 [0.3-1.8], p= 0.560). CONCLUSIONS: The addition of CBDCA to standard CT was significantly associated with pCR and RFS but not OS, consistent with the phase III data. The benefit in terms of RFS was independent of the presence and the type of pathogenic germline alterations. Table 1. pCR rates according to PGV Citation Format: Adela Rodríguez Hernández, Benedetta Conte, Laia Fernández, Fara Brasó-Maristany, Belén Pastor, Miriam Potrony, Lorena Moreno, Elia Grau, Joan Antón Puig-Butillé, Aurora Sánchez, Blanca González-Farré, Esther Sanfeliu, Olga Martínez-Sáez, Claudette Falato, Maria Vidal, Nuria Chic, Tomás Pascual, Francesco Schettini, Montserrat Muñoz, Francesc Balaguer, Aleix Prat, Barbara Adamo. Efficacy of platinum-based chemotherapy and germline mutational status in early-stage triple-negative breast cancer: a unicenter retrospective analysis with long-term follow-up [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-09.

Purpose:To assess palbociclib in combination with trastuzumab with or without endocrine therapy in patients with HER2-positive advanced breast cancer.Patients and Methods:PATRICIA is a prospective, open-label, multicenter phase II trial. Patients had received 2–4 prior lines of anti-HER2–based regimens. Treatment consisted of palbociclib 200 mg daily for 2 weeks and 1 week off plus trastuzumab. The study was based on a Simon two-stage design comprising three cohorts: estrogen receptor (ER)-negative (cohort A), ER-positive (cohort B1), and ER-positive with letrozole (cohort B2). ER-positive patients were randomized to cohorts B1 or B2. Primary endpoint was progression-free survival rate at 6 months (PFS6). Secondary objectives included safety and evaluation of the PAM50 intrinsic subtypes.Results:Seventy-one patients were recruited (n = 15 in cohort A and 28 in each cohort B). The PFS6 rate in cohorts A, B1, and B2 was 33.3% (5/15), 42.8% (12/28), and 46.4% (13/28), respectively. Regarding safety, grade 1–2 and 3–4 toxicities occurred in 97.7% and 84.4% of patients, respectively. The most common grade 3–4 toxicities were neutropenia (66.4%) and thrombocytopenia (11.3%). Regarding PAM50, 59 (83.1%) tumors were profiled. Luminal disease defined by PAM50 was found independently associated with longer PFS compared with non-luminal disease (10.6 vs. 4.2 months median PFS; adjusted hazard ratio = 0.40; P = 0.003).Conclusions:Palbociclib in combination with trastuzumab is safe and exhibits promising survival outcomes in trastuzumab pretreated ER-positive/HER2-positive advanced breast cancer with a PAM50 Luminal A or B subtype. The enrollment was stopped prematurely, and a new randomized cohort was opened in this population.

BACKGROUND. The prognostic and predictive value of the two nonluminal (human epidermal growth factor receptor 2 [HER2]‐enriched and basal‐like) subtypes within advanced hormone receptor‐positive (HR+) breast cancer is currently unknown. MATERIALS AND METHODS. This study retrospectively analyzed 261 tumors (80.7% primary; 19.3% metastatic) from the BOLERO‐2 study; BOLERO‐2 randomized 724 patients with advanced HR+/HER2‐negative breast cancer to everolimus plus exemestane or placebo plus exemestane. Tumors were classified using a PAM50 subtype predictor. Multivariable Cox regression analyses tested the independent prognostic significance of PAM50, and associations between PAM50 subtypes and treatment upon progression‐free survival (PFS) were evaluated. RESULTS. Subtype distribution was 46.7% luminal A (n = 122), 21.5% HER2‐enriched (n = 56), 15.7% luminal B (n = 41), 14.2% normal‐like (n = 37), and 1.9% basal‐like (n = 5); HER2‐enriched subtypes were more common in metastatic versus primary tumors (32.0% vs. 18.7%; p = .038). Median PFS differences between luminal and nonluminal (6.7 vs. 5.2 months; adjusted hazard ratio, 0.66; 95% confidence interval [CI], 0.47–0.94; p = .020) and HER2‐enriched and non‐HER2‐enriched subtypes (5.2 vs. 6.2 months; adjusted hazard ratio, 1.53; 95% CI, 1.07–2.19; p = .019) were significant. Everolimus plus exemestane significantly improved median PFS versus placebo plus exemestane among patients with HER2‐enriched tumors (5.8 vs. 4.1 months; adjusted hazard ratio, 0.49; 95% CI, 0.26–0.90; p = .034); however, the association between HER2‐enriched tumors and everolimus benefit was nonsignificant (p = .433). CONCLUSION. The HER2‐enriched subtype was identified in a substantial proportion of advanced HR+/HER2‐negative breast tumors, and was a consistent biomarker of poor prognosis. Tailored therapies are therefore needed for HER2‐enriched tumors in the advanced HR+/HER2‐negative breast cancer setting. IMPLICATIONS FOR PRACTICE. Using 261 tumor samples from the BOLERO‐2 phase III clinical trial, this study shows that a substantial proportion (20%–30%) of hormone receptor‐positive (HR+)/human epidermal growth factor receptor 2 (HER2)‐negative advanced breast cancers do not have a luminal A or B gene expression profile. This group of patients with nonluminal disease has a poor survival outcome regardless of the addition of everolimus to exemestane. This is the second study that confirms the prognostic value of this biomarker. Overall, these findings indicate a necessity to design novel clinical trials targeting nonluminal disease within HR+/HER2‐negative breast cancer.

Purpose:We do not yet have validated biomarkers to predict response and outcome within hormone receptor–positive/HER2-positive (HR+/HER2+) breast cancer. The PAM50-based chemo-endocrine score (CES) predicts chemo-endocrine sensitivity in hormone receptor–positive/HER2-negative (HR+/HER2−) breast cancer. Here, we evaluate the relationship of CES with response and survival in HR+/HER2+ breast cancer.Experimental Design:Intrinsic subtype and clinicopathologic data were obtained from seven studies in which patients were treated with HER2-targeted therapy either with endocrine therapy (ET) or with chemotherapy (CTX). CES was evaluated as a continuous variable and categorically from low to high scores [CES-C (chemo-sensitive), CES-U (uncertain), and CES-E (endocrine-sensitive)]. We first analyzed each dataset individually, and then all combined. Multivariable analyses were used to test CES association with pathologic complete response (pCR) and disease-free survival (DFS).Results:A total of 457 patients were included (112 with ET and 345 with CTX). In the combined cohort, CES-C, CES-U, and CES-E were identified in 60%, 23%, and 17% of the patients, respectively. High CES (i.e., CES-E) was associated with a lower probability of achieving pCR independently of clinical characteristics, therapy, intrinsic subtype, and study (adjusted OR = 0.42; P = 0.016). A total of 295 patients were analyzed for DFS with a median follow-up of 66 months. High CES was also associated with better DFS (adjusted HR, 0.174; P = 0.003) independently of pCR, clinical characteristics and intrinsic subtype. In patients with residual disease, the adjusted DFS HR of CES was 0.160 (P = 0.012).Conclusions:In HER2+/HR+ breast cancer, CES is useful for predicting chemo-endocrine sensitivity and provides additional prognostication beyond intrinsic subtype and clinicopathologic characteristics.