Search

Background: In the era of targeted therapies, chemotherapy (CT) is still a valuable treatment option for patients (pts) with HER2 negative metastatic breast cancer (MBC). The identification of predictive and prognostic markers might improve treatment response and survival. Here we investigated the role of tumor subtypes, circulating tumor cells (CTCs), and mutations in genes or pathways of interest, in predicting response to CT and prognosis of HER2 negative MBC pts within AARES trial. Methods: AARES is an open label multicentric randomised phase 2 trial comparing a DNA-damaging (arm A: cisplatin 25 mg/m2/day, day 1-3 + cyclophosphamide 600 mg/m2 day 1) versus (vs) a non-DNA-damaging (arm B: capecitabine 1000 mg/m2 bid/day, day 1-14 + vinorelbine 60 mg/m2/day, day 1,8) CT regimen in pts with HER2 negative MBC. Archival tumor tissue samples and blood samples were collected at baseline. The Cell SearchTM system was used for CTCs isolation and enumeration and a cut-off of 5 CTC/7,5 ml was used. Tumor subtypes were based on locally assessed hormone receptors and HER2 status. The luminal-like subtype (Lum) was defined by ER or PR positivity, while the triple negative subtype (TN) by ER, PR, and HER2 negativity. Tissue samples were used for DNA extraction and next generation sequencing analysis using a target enrichment panel of 170 genes (TruSight 170, Illumina). Primary endpoint was objective response rate (ORR) assessed for the two treatment arms. Progression free survival (PFS) and overall survival (OS) were evaluated as secondary endpoints, and estimated with the Kaplan-Meier method. ORR, PFS and OS were correlated with CTC counts, tumor subtypes, and mutational status of genes/pathway of interest (PIK3CA, TP53, BRCA1, BRCA2, PI3K/AKT pathway (PI3K/AKT)). Results: AARES enrolled 102 pts from 2011 to 2016 across 9 Italian centers. Of these, 77 pts were evaluable for ORR. Median follow up was 32 months. Overall, median age was 57. The majority of pts had Lum tumors (73%), while 27% had TN. 86% of pts had visceral metastases, and 52% had 3 or more metastatic sites. 48% of pts received the study treatment as 1st line, 32% as 2nd line, and 19% as 3rd line. Out of the 77 pts with ORR data, 41 (53%) were CTC- and 36 (47%) CTC+. A larger proportion of pts with Lum tumors were CTC+ (54% n=30), while TN tumors were mainly CTC- (71% n=15). 49 pts had adequate tumor tissue for sequencing analysis. TP53 mutations (mut) were found in 33% of pts, BRCA1/2 mut in 14%, PIK3CA mut in 35%, and PI3K/AKT mut in 47%. ORR was 24% and 37% in arm A vs arm B, respectively (p= 0.2), and no difference in ORR was observed by tumor subtypes (Lum vs TN), and CTCs count (+ vs -). PFS and OS were assessable on the whole population (n=102) and did not significantly differ by treatment arms. Median OS (mOS) was higher in Lum pts compared to TN (24 months (mo) vs 15.4 mo, p=0.048) while median PFS (mPFS) did not differ according to tumor subtypes (p=0.28). Overall, CTCs count was not significantly associated with mPFS or OS however in pts with TN tumors and CTC-, mPFS and mOS were significantly longer (mPFS: 4.44 mo vs 2.56 mo in CTC- and CTC+ subgroups, respectively, p=0.00087 and mOS: 22.07 mo vs 3.57 mo in CTC- and CTC+ subgroups, respectively, p< 0.0001). On the other hand, no differences were observed in Lum pts categorized according to CTC status (p=0.83 for mPFS, p=0.54 for mOS). Finally, pts with PI3K/AKT mut had a significantly worse PFS compared to wild type in the overall population (p=0.0091) as well as in Lum (p=0.034) and TN pts (p=0.0052). Conclusions: CTCs and tumor subtypes were not predictive of response to CT regimens in HER2 negative MBC pts. A number of CTC >5 in TN MBC, and mutations in the PI3K/AKT pathway, identified a subgroup of pts with worse prognosis, potentially candidates for alternative treatments. Further studies are needed to confirm these results in a larger population. Citation Format: Emanuela Risi, Marta Pestrin, Chiara Biagioni, Dario Romagnoli, Ilenia Migliaccio, Francesca Galardi, Francesca De Luca, Matteo Benelli, Erica Moretti, Giuseppina Sanna, Luca Livraghi, Silvia Cappadona, Roberta Di Marsico, Domenico Amoroso, Angelo Martignetti, Angela S. Ribecco, Elena Rota Caremoli, Luigi Coltelli, Fabio Puglisi, Luca Malorni, Laura Biganzoli. Circulating tumor cells, immunohistochemical subtypes, and genes mutation as prognostic markers in HER2 negative metastatic breast cancer patients candidates to chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-45.

Time to progression with E2 and Tam +/- Dox in xenografts of MCF7 Tet-off Vector control clone 3.

Genes up-regulated in Tam-R and putatively associated with EGF-induced ER binding sites (genes containing an AP-1 motif are shown in bold)

The transcription factor AP-1 is downstream of growth factor (GF) receptors (GFRs) and stress-related kinases, both of which are implicated in breast cancer endocrine resistance. Previously, we have suggested that acquired endocrine resistance is associated with increased activity of AP-1 in an in vivo model. In this report, we provide direct evidence for the role of AP-1 in endocrine resistance. First, significant overlap was found between genes modulated in tamoxifen resistance and a gene signature associated with GF-induced estrogen receptor (ER) cistrome. Interestingly, these overlapping genes were enriched for key signaling components of GFRs and stress-related kinases and had AP-1 motifs in their promoters/enhancers. Second, to determine a more definitive role of AP-1 in endocrine resistance, AP-1 was inhibited using an inducible dominant-negative (DN) cJun expressed in MCF7 breast cancer cells in vitro and in vivo. AP-1 blockade enhanced the antiproliferative effect of endocrine treatments in vitro, accelerated xenograft tumor response to tamoxifen and estrogen deprivation in vivo, promoted complete regression of tumors, and delayed the onset of tamoxifen resistance. Induction of DN-cJun after the development of tamoxifen resistance resulted in dramatic tumor shrinkage, accompanied by reduced proliferation and increased apoptosis. These data suggest that AP-1 is a key determinant of endocrine resistance by mediating a global shift in the ER transcriptional program.Implications: AP-1 represents a viable therapeutic target to overcome endocrine resistance. Mol Cancer Res; 14(5); 470–81. ©2016 AACR.

Supplementary Table 1: Genes up-regulated in Tam-R and putatively associated with EGF-induced ER binding sites.

Time to response, time to complete response, and rate of complete response to endocrine treatment +/- Dox in xenografts of MCF7 Tet-off Vector Control clone 3.

Genes down-regulated in Tam-R and putatively associated with EGF-induced ER binding sites (genes containing an AP-1 motif are shown in bold)

Full gel

Legend for supplementay figures

Supplemental Figure Legends

QC analysis

Baseline characteristics of studied c-TREnd biomarker population - including all patients with at least one sample available for analysis at any timepoint(s), compared to the overall original TREnd cohort. Fisher exact test was used to calculate p-values. *Denotes patients included in intention-to-treat population of TREnd, despite breach of eligibility criteria (discovered post-enrolment).

Supplementary Figure S1. ER, PR, and HER2 mRNA and protein levels across endocrine-sensitive (parental) and endocrine-resistant BC cell lines. Supplementary Figure S2. Reduced sensitivity to CDK4/6 inhibition is associated with IFN-signaling in ER+ and endocrine-resistant derivative BC cell lines. Supplementary Figure S3. The cyclin D-CDK4/6-Rb axis is altered in ER+ cell lines with acquired resistance to palbociclib. Supplementary Figure S4. Effect of acute IFN-signaling activation on sensitivity to palbociclib in ER+ BC cell lines. Supplementary Figure S5. Proteomic profiles of MCF7 P, MCF7 EDR, and their PalboR derivatives. Supplementary Figure S6. Relationship between IFN-signaling and Rb status in ER+/HER2- BC. Supplementary Figure S7. DNMT1 mRNA levels in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S8. Mutations of DNA damage response genes are not associated with IFN-signaling in ER+ BC. Supplementary Figure S9. Levels of ER and ER signalling in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S10. HALLMARK gene sets significantly up-regulated in ER+/HER2- BC patients stratified by the status (+/-) of RBsig and IRPS. Supplementary Figure S11. Impact of selected IRPS genes on relapse-free survival of ER+ BC patients.

Cox proportional hazards analyses

PFS and OS of PAM50 and OncotypeDx risk categories

Supplementary Table S1. Details of antibodies used in this study. Supplementary Table S2. Q-PCR primers. Supplementary Table S3. HALLMARK gene-set enrichment analysis (GSEA) of baseline tumor samples from the NeoPalANA trial. Supplementary Table S4. Palbociclib IC50 values in MCF7 P, MCF7 EDR, T47D P-LM, and T47D EDR palbociclib-resistant (PalboR)-LM cell lines. Supplementary Table S5. Top-20 Gene Ontology (GO) terms significantly enriched in PalboR cell lines. Supplementary Table S6. HALLMARK gene sets significantly up-regulated in cell lines with acquired resistance to palbociclib. Supplementary Table S7. RPPA of MCF7 P, MCF7 EDR, and their PalboR derivatives.

Supplemental Materials and Methods and Materials present a complete description of methods, reagents, and statistics required to reproduce the experiments in the paper.

Purpose:Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC.Experimental Design:Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor–positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the “To Reverse ENDocrine Resistance” (TREnd) trial (n = 46).Results:Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients (n = 8) showing an increase—correlating with a worse outcome than those with decreased/stable TKa (n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05).Conclusions:TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib.

Purpose: Fulvestrant is an estrogen receptor (ER) antagonist and an approved treatment for metastatic estrogen receptor–positive (ER+) breast cancer. With the exception of ER levels, there are no established predictive biomarkers of response to single-agent fulvestrant. We attempted to identify a gene signature of response to fulvestrant in advanced breast cancer.Experimental Design: Primary tumor samples from 134 patients enrolled in the phase III CONFIRM study of patients with metastatic ER+ breast cancer comparing treatment with either 250 mg or 500 mg fulvestrant were collected for genome-wide transcriptomic analysis. Gene expression profiling was performed using Affymetrix microarrays. An exploratory analysis was performed to identify biologic pathways and new signatures associated with response to fulvestrant.Results: Pathway analysis demonstrated that increased EGF pathway and FOXA1 transcriptional signaling is associated with decreased response to fulvestrant. Using a multivariate Cox model, we identified a novel set of 37 genes with an expression that is independently associated with progression-free survival (PFS). TFAP2C, a known regulator of ER activity, was ranked second in this gene set, and high expression was associated with a decreased response to fulvestrant. The negative predictive value of TFAP2C expression at the protein level was confirmed by IHC.Conclusions: We identified biologic pathways and a novel gene signature in primary ER+ breast cancers that predicts for response to treatment in the CONFIRM study. These results suggest potential new therapeutic targets and warrant further validation as predictive biomarkers of fulvestrant treatment in metastatic breast cancer. Clin Cancer Res; 22(23); 5755–64. ©2016 AACR.

Purpose:Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes.Experimental Design:Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes.Results:Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis.Conclusions:Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Correlation between IHC and transcript level by microarray

PFS and RORP classifier

Supplementary Methods and Materials from Development of Resistance to Targeted Therapies Transforms the Clinically Associated Molecular Profile Subtype of Breast Tumor Xenografts

The effectiveness of therapies targeting specific pathways in breast cancer, such as the estrogen receptor or HER2, is limited because many tumors manifest resistance, either de novo or acquired, during the course of treatment. To investigate molecular mechanisms of resistance, we used two xenograft models of estrogen receptor–positive (ER+) breast cancer, one with and one without HER2 overexpression (MCF7/HER2-18 and MCF7 wt, respectively). Mice with established tumors were assigned to the following treatment groups: estrogen supplementation (E2), estrogen deprivation (ED), ED plus tamoxifen (Tam), all with or without the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (G). Another group received ED plus the antiestrogen fulvestrant (MCF7 wt only). Tumors with acquired or de novo resistance to these endocrine therapies were profiled for gene expression and compared with tumors in the E2 control group. One class of genes underexpressed in endocrine-resistant tumors (relative to E2-treated tumors) were estrogen inducible in vitro and associated with ER+ human breast cancers (luminal subtype). Another class of genes overexpressed in tumors with acquired resistance in both models represented transcriptional targets of HER2 signaling and was associated with ER−/HER2+ human cancers (ERBB2+ subtype). A third class of genes overexpressed in MCF7/HER2-18 tumors exhibiting de novo resistance to tamoxifen was associated with ER+ human cancers but not with estrogen-regulated genes. Thus, in response to various endocrine therapy regimens, these xenograft breast tumors shut down classic estrogen signaling and activate alternative pathways such as HER2 that contribute to treatment resistance. Over time, the molecular phenotype of breast cancer can change. [Cancer Res 2008;68(18):7493–501]

INTRODUCTION: Somatic mutations of the PIK3CA gene, encoding for the class IA PI3K p110α subunit, are the most common activating mutations in breast cancer (BC), occurring in 30-50% of early and in 28% of metastatic ER+/HER2- disease. We have previously demonstrated that PIK3CA mutations co-occurring with copy number (CN) gain (PIK3CA-mut/gain) are associated with worse outcome in patients with early hormone receptor positive and HER2 negative (HR+/HER2-) BC, including those receiving endocrine therapy (ET). Here we aim to evaluate the prognostic role of PIK3CA-mut/gain in patients with metastatic HR+/HER2- BC treated with chemotherapy, ET alone or in combination with targeted therapies (CDK4/6 inhibitors or everolimus) included in a publicly available dataset of BC. PATIENTS AND METHODS: We accessed genomic, clinical, treatment and outcome data from 1365 patients with HR+/HER2- BC included in the MSK-2018 dataset. Patients treated for distant metastases were selected. PIK3CA status, considering both protein-affecting mutations and CN alterations, was derived from pre-treatment biopsies data. Tumors were classified as: PIK3CA wt and CN neutral (-wt/neut), PIK3CA wt with CN gain (-wt/gain), PIK3CA mutant and CN neutral (-mut/neut), and PIK3CA mutant with CN gain (-mut/gain). Tumors with PIK3CA loss were excluded from the analyses. For each patient and treatment type, only progression free survival (PFS) from the first available line of treatment was analyzed, thus excluding data concerning possible retreatments with the same drug in subsequent lines. Multivariate Cox proportional hazard models were used to assess the association between PIK3CA status and PFS including treatment line and the four categories of PIK3CA as co-variates. RESULTS: The proportion of PIK3CA-mut/gain was 15.4% in the chemotherapy group (n=454), 17.3% in the ET alone group (n= 352), 13.4% in the ET plus CDK4/6 inhibitors group (n= 268) and 10.5% in the ET plus everolimus group (n= 143). As expected, in all groups treatment line was significantly and independently associated with PFS. In the ET alone group, patients whose tumors had gain in PIK3CA CN, with or without co-occurring mutations, were at significantly higher risk of progression compared with PIK3CA-wt/neut (HR 2.2 [1.53-3.2] p Citation Format: Ilenia Migliaccio, Marta Paoli, Emanuela Risi, Chiara Biagioni, Laura Biganzoli, Matteo Benelli, Luca Malorni. PIK3CA mutations co-occurring with copy number gain identify patients with adverse outcome and potentially different treatment sensitivity among hormone receptor positive and HER2 negative metastatic breast cancer [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 P5-13-13.

Background: ctDNA analysis is emerging as an attractive non-invasive approach to characterize tumor biology, describe its evolution over time, and predict treatment benefit. Here, we assessed the prognostic and predictive role of baseline and dynamic ctDNA analysis in HR+/HER2- aBC patients (pts) treated with R+L. Methods: 287 postmenopausal pts were enrolled in the BioItaLEE trial (NCT03439046). Liquid biopsies were collected at baseline (D0; n=263), day 15 of cycle 1 (D15; n=238), day 1 of cycle 2 (C2D1; n=241) and at first imaging (FI, at approximately 12 weeks; n=206). ctDNA analysis was carried out using a 533-amplicon Custom AmpliSeq HD Panel, with amplicons covering the coding exons of 39 BC-related genes (limit of detection: 0.1%). Target mutations were defined as single-nucleotide variant (SNV) or Insertion/Deletion detected at D0. When multiple target mutations were detected, the one with the highest variant allele frequency (VAF) was considered. The association between pre-treatment and on-treatment ctDNA dynamics with progression-free survival (PFS) was assessed using Multivariate Cox models. VAF clearance was defined as 100% decrease in a target mutation. Results: Median follow-up was 26.9 months and median PFS was 23.39 (20.8-NE) months. At baseline, target mutations were detected in 113 pts (43.0%), whereas 150 pts were wild-type (wt). Mean (SD) pre-treatment VAF at D0 was 11.3% (14.4). The absence of a target mutation at D0 was associated with good prognosis (HR: 0.41, 95% CI: 0.27–0.61; p Citation Format: Giampaolo Bianchini, Luca Malorni, Grazia Arpino, Alberto Zambelli, Fabio Puglisi, Lucia Del Mastro, Marco Colleoni, Filippo Montemurro, Giulia Bianchi, Ida Paris, Giacomo Allegrini, Marina Elena Cazzaniga, Michele Orditura, Claudio Zamagni, Stefano Tamberi, Daniela Castelletti, Matteo Benelli, Maurizio Callari, Angela Santoro, Michelino De Laurentiis. Circulating tumor DNA (ctDNA) dynamics in patients with hormone receptor positive (HR+)/HER2 negative (HER2-) advanced breast cancer (aBC) treated in first line with ribociclib (R) and letrozole (L) in the BioItaLEE trial [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 GS3-07.

DNA-methylation alterations are common in cancer and display unique characteristics that make them ideal markers for tumor quantification and classification. Here we present MIMESIS, a computational framework exploiting minimal DNA-methylation signatures composed by a few dozen informative DNA-methylation sites to quantify and classify tumor signals in tissue and cell-free DNA samples. Extensive analyses of multiple independent and heterogenous datasets including >7200 samples demonstrate the capability of MIMESIS to provide precise estimations of tumor content and to enable accurate classification of tumor type and molecular subtype. To assess our framework for clinical applications, we designed a MIMESIS-informed assay incorporating the minimal signatures for breast cancer. Using both artificial samples and clinical serial cell-free DNA samples from patients with metastatic breast cancer, we show that our approach provides accurate estimations of tumor content, sensitive detection of tumor signal and the ability to capture clinically relevant molecular subtype in patients’ circulation. This study provides evidence that our extremely parsimonious approach can be used to develop cost-effective and highly scalable DNA-methylation assays that could support and facilitate the implementation of precision oncology in clinical practice.

Introduction: Breast cancer (BC) is a heterogeneous disease characterized by different molecular "intrinsic" subtypes (PAM50) with distinct prognostic and therapeutic implications. DNA methylation (DNAm) is one of the most studied epigenetic mechanisms involved in BC tumorigenesis and progression. Recently, many studies have demonstrated the capability of DNAm-based assays in detecting and monitoring the disease in liquid biopsy samples from patients with BC, thus confirming promising role of DNAm-based biomarkers in the context of precision oncology. Here we present a novel computational method that exploits a minimal set of informative, clonal DNAm sites to estimate tumor content and molecular subtype in BC tissue samples with potential application for liquid biopsy analyses. Methods: DNAm data of TCGA-BRCA comprising 737 tumor samples, including 135 Basal-like (Basal), 46 HER2-enriched (HER2e) and 556 Luminal A/B (LumA/B) samples, and 96 normal tissue samples were downloaded from GDC Data Portal. To estimate tumor purity, we selected DNAm sites based on the following characteristics: (1) AUC>0.8 or AUC350 samples, high accuracy was obtained for LumA/B and Basal samples while HER2e samples showed lower F1-score due to a drop of recall, though obtaining precision >80% in both datasets. Based on these results, a DNAm-based targeted-seq assay has been designed and will be applied to a series of plasma samples from patients with metastatic BC collected within our center. First results of the assay will be presented during the meeting. Citation Format: Dario Romagnoli, Francesca Galardi, Francesca De Luca, Chiara Biagioni, Erica Moretti, Laura Biganzoli, Ilenia Migliaccio, Luca Malorni, Matteo Benelli. A minimal DNA-methylation signature to estimate tumor content and molecular subtype in breast cancer tissue samples with potential application to liquid biopsy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-05-02.

We previously showed that metabolomics predicts relapse in early breast cancer (eBC) patients, unselected by age. This study aims to identify a "metabolic signature" that differentiates eBC from advanced breast cancer (aBC) patients, and to investigate its potential prognostic role in an elderly population.Serum samples from elderly breast cancer (BC) patients enrolled in 3 onco-geriatric trials, were retrospectively analyzed via proton nuclear magnetic resonance (1H NMR) spectroscopy. Three nuclear magnetic resonance (NMR) spectra were acquired for each serum sample: NOESY1D, CPMG, Diffusion-edited. Random Forest (RF) models to predict BC relapse were built on NMR spectra, and resulting RF risk scores were evaluated by Kaplan-Meier curves.Serum samples from 140 eBC patients and 27 aBC were retrieved. In the eBC cohort, median age was 76 years; 77% of patients had luminal, 10% HER2-positive and 13% triple negative (TN) BC. Forty-two percent of patients had tumors2 cm, 43% had positive axillary nodes. Using NOESY1D spectra, the RF classifier discriminated free-from-recurrence eBC from aBC with sensitivity, specificity and accuracy of 81%, 67% and 70% respectively. We tested the NOESY1D spectra of each eBC patient on the RF models already calculated. We found that patients classified as "high risk" had higher risk of disease recurrence (hazard ratio (HR) 3.42, 95% confidence interval (CI) 1.58-7.37) than patients at low-risk.This analysis suggests that a "metabolic signature", identified employing NMR fingerprinting, is able to predict the risk of disease recurrence in elderly patients with eBC independently from standard clinicopathological features.

Background: Thymidine kinase-1 is a cell proliferation marker downstream of the CDK4/6 pathway, whose activity can be measured in serum to reflect tumor proliferation. The CDK4/6 inhibitor palbociclib (P) is approved for the treatment of patients (pts) with hormone receptor positive metastatic breast cancer (MBC) in first or second line endocrine-based treatment settings. Approximately 10-15% of pts exhibit de novo resistance to P, with circulating levels of thymidine kinase activity (TKa) previously shown as a potential marker of early treatment resistance. Therapeutic strategies to address primary resistance to P are currently lacking. Little is known of the clinical efficacy of alternative dosing schedules of P, and its effect on TKa. Here we report serum TKa measured at different timepoints from samples collected within the MA38 (NCT02630693) study. Methods: MA38 is an open label randomised Phase 2 trial comparing two different schedules of P plus second-line ET in pts with ER-positive, HER2-negative MBC. Pts were assigned to receive physician’s choice ET plus either standard P dosing (125mg daily for 21 days on a 28-day cycle), or 100mg daily continuously. Serum samples were collected at baseline (BL; n=135), at 12 weeks (W12; n=122) and 24 weeks (W24; n=95). TKa was measured with DiviTum®, a refined ELISA-based assay (lower limit of detection [LLOD] = 100 DuA). Kaplan-Meier method estimated BL, W12 and W24 (95% CI) median PFS (mPFS; from randomization until progression by RECIST criteria or death) and overall survival (OS; from randomization until death from any cause) in groups of patients defined by dichotomizing TKa as “high” or “low” at the median. Results: MA38 enrolled 180 pts from December 2015 and February 2017 across Canada. Median follow up was 19 months. Overall, the median age was 60, and 90% of pts were post-menopausal. All pts had estrogen receptor-positive disease, and 64% had visceral metastases. On study, 56% received fulvestrant with P, 34% aromatase inhibitor and 10% tamoxifen. TKa was successfully measured in 100% of samples. Median TKa (mTKa) at BL was 234 DuA (IQR 138.5 - 438). BL TKa was not associated with clinical or pathological characteristics. TKa was prognostic at BL with mPFS of 5.5 months (mo) in pts with high TKa vs 16.3 mo with low TKa (HR=2.43; 95% CI, 1.6-3.7; p< 0.001). Similar results were obtained employing other previously reported cut off values. At multivariate analysis, BL TKa was independent from other prognostic factors including age, ECOG status and presence of visceral metastases (adjusted HR= 2.34; 95%CI 1.5- 3.6; p < 0.001). In terms of OS, BL TKa was an independent prognostic factor (adjusted HR=2.0; 95% CI, 1.1-3.7; p=0.02). At 12 mo, OS rate was 68% in pts with high BL TKa vs 92% in low TKa. Both for PFS and OS, no interaction between BL TKa and study arm was observed. At W12 mTKa was 129.5 DuA (IQR 100 - 219.8) and below LLOD (IQR 100 - 180) at W24. At these timepoints, landmark analyses showed no significant difference in PFS according to TKa. However, at W12 high TKa was significantly associated with worse OS (HR 2.0; 95%CI 1.0- 4.0; p=0.03), with a similar trend at W24 (HR 2.5; 95%CI 0.9-6.4; p=0.06). Conclusions: Baseline TKa is a reliable prognostic marker of both PFS and OS in pts treated with P and ET, further substantiating previous data. Monitoring TKa during treatment may provide important clinical information. A significant relationship between TKa and assigned treatment arm was not observed, suggesting TKa is not influenced by P treatment dose or intensity. These data confirm the role of baseline TKa as a new marker for patient stratification, and supports further investigation for the assessment of the clinical utility of TKa as a monitoring biomarker in the advanced setting. Citation Format: Amelia McCartney, Chiara Biagioni, Bingshu Chen, Lois Shepherd, Karen Gelmon, Anil A. Joy, Wendy Parulekar, Mattias Bergqvist, Ilenia Migliaccio, Angela Leo, Matteo Benelli, Emanuela Risi, Erica Moretti, Luca Livraghi, Laura Biganzoli, Luca Malorni. Serum thymidine kinase activity as a prognostic marker in women with metastatic breast cancer treated with two different schedules of palbociclib plus second-line endocrine therapy within the CCTG MA38 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 P5-02-27.

Background: We have previously identified two potentially predictive signatures of palbociclib resistance: the RBsig, composed of E2F1/E2F2 dependent genes, which is correlated with genetic loss of RB1, and the ratio between the gene expression levels of CCNE1 to RB1 (CCNE1/RB1). Both signatures have been previously tested in vitro and in neoadjuvant studies with palbociclib. The present analysis aims to explore the prognostic and predictive role of RBsig and CCNE1/RB1 in the pivotal phase III randomized trials PALOMA-2 and PALOMA-3. Materials and methods: Gene expression data from the PALOMA-2 and PALOMA-3 datasets were generated using the HTG EdgeSeq Oncology BM Panel, as previously described. Of the 87 genes composing RBsig, 46 were available within the EdgeSeq dataset and were used for the analyses; CCNE1 and RB1 were both available. RBsig was calculated as the mean of the Z-score scaled gene expression (log) of the 46 genes; CCNE1/RB1 was computed as the log ratio between the mRNA expression of CCNE1 and RB1. High and low values of RBsig and CCNE1/RB1 were defined based on the third quartile (Q3) as cutoff or as continuous variables. The prognostic/predictive effect of the signatures in terms of PFS was tested using Cox proportional hazard models and the Wald test. Results: The 46-genes RBsig versus the original signature showed excellent correlation in the METABRIC dataset (R=0.99), confirming its reliability as a surrogate of the original RBsig using EdgeSeq data. In both PALOMA-2 and PALOMA-3, RBsig high was significantly associated with a worse outcome compared to RBsig low in the palbociclib arm but not in the control arm [PALOMA-2: HR 1.4 (95% CI 1.0, 2.0) p=0.029 for palbociclib arm; HR 1.1 (95% CI 0.7, 1.6) p=0.71 for control arm. PALOMA-3: HR 1.7 (95% CI 1.1, 2.6) p=0.01 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p= 0.49 for control arm]. However, in both studies RBsig was not predictive of palbociclib resistance both when considered as a continuous variable and when dichotomized at Q3. Similarly to RBsig, in PALOMA- 3 patients with CCNE1/RB1 high tumors treated in the palbociclib arm showed a significantly worse outcome compared to those with CCNE1/RB1 low but this effect was not observed in those treated in the control arm [HR 1.6 (95% CI 1.1- 2.5) p= 0.03 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p=0.5 for control arm]. In addition, CCNE1/RB1 as a continuous variable was predictive of palbociclib benefit in PALOMA-3 (interaction p= 0.047). These effects were not observed in PALOMA-2. Conclusions: RBsig is a prognostic biomarker in patients treated with palbociclib, suggesting it may help in patients’ risk stratification. CCNE1/RB1 is predictive of palbociclib benefit in PALOMA-3, but not in PALOMA-2 probably due to the different patient populations and characteristics. Further studies of these biomarkers in patients treated with CDK4/6 inhibitors in the metastatic as well in the adjuvant setting are warranted. Citation Format: Luca Malorni, Matteo Benelli, Yuan Liu, Shibing Deng, Zhe Zhang, Cristina Guarducci, Angela Leo, Agostina Nardone, Francesca Galardi, Emanuela Risi, Erica Moretti, Dario Romagnoli, Chiara Biagioni, Marta Paoli, Laura Biganzoli, Ilenia Migliaccio. Prognostic and predictive role of RBsig and CCNE1/RB1 gene-expression signatures in patients with advanced breast cancer treated with palbociclib in combination with endocrine therapy in the PALOMA-2 and 3 trials [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 P5-02-17.

Purpose: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. Experimental Design: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. Results: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. Conclusions: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

HER2+ early breast cancer is a heterogeneous disease, comprising all the intrinsic breast cancer subtypes. The only biomarker available nowadays for anti-HER2 treatment selection is HER2 status itself, but estrogen receptor (ER) status is emerging as a robust predictive marker within HER2+ disease. In this Perspective, we discuss the biological and clinical differences between patients with HER2+/ER-positive (ER+) disease versus those with HER2+/ER-negative (ER-neg) tumors, namely, short-term and long-term (>5 years after diagnosis) prognosis, response to neoadjuvant treatment and benefit from adjuvant anti-HER2–targeted therapies. We also address other possible biomarkers to be used for patient selection in future clinical trials, such as gene signatures, PAM50 subtypes, tumor-infiltrating lymphocytes, PIK3CA mutations, and changes in Ki67 score during treatment and discuss their limitations. Finally, we suggest new clinical trial designs that can have an impact on clinical practice, aiming to test treatment deescalation separately for patients with HER2+/ER+ and HER2+/ER-neg tumors. We also propose an integrated classification of HER2+ disease, comprising DNA, RNA, protein expression, and microenvironment characteristics, in order to identify those tumors that are truly “HER2-addicted” and may benefit the most from anti-HER2 treatment.