Non-invasive detection and quantification of circulating tumor DNA by DNAm signatures is emerging as powerful tool for monitoring disease status in pts with metastatic cancer. We recently developed MIMESIS, a computational workflow to design tumor agnostic DNAm signatures of few dozen sites for TC and molecular subtype estimation in liquid biopsy. Here we applied a MIMESIS informed assay to a retrospective cohort of mBC pts (MIMESIS-BC). 72 mBC pts (42 ER+/HER2-, 2 ER+/HER2 N/A, 8 ER+/HER2+, 6 ER-/HER2+, 14 ER-/HER2-, up to 6 previous lines of treatment) and 11 healthy individuals (ctrl) were included. Plasma from mBC pts was collected before starting treatment (T0, n=58) and after the first cycle (T1, n=52) (n=38 pts T0 and T1). MIMESIS-BC for TC, PAM50 molecular subtypes and ER status (n=139 sites) was applied to circulating free DNA as described in the MIMESIS study. Samples were defined as TC+ when TC>0. TC values as continuous variable and TC classes (+/-) were associated with OS and PFS. Concordance of subtype and ER status by MIMESIS-BC with IHC-based from primary tumor was estimated by chi-square and percent agreement. TC was not detected in ctrl. A significantly higher median TC was observed at T0 compared to T1 (5.6% (range 0-73%) vs 0% (range 0-47%); p=0.02). 39 TC+ samples at T0 showed 61% agreement for molecular subtype (p=0.002) and 86% for ER status (p<0.001). In the entire population, PFS and OS were significantly associated with TC values both at T0 and T1 (max p=0.002). TC+ pts had significantly worse OS compared to TC- at T0 (HR=2.1 CI=1.1-3.9 p=0.02) and T1 (HR=2.0 CI=1.1-3.6 p=0.03) and worse PFS at T1 (HR=2.9 CI=1.5-5.4 p<0.001). In ER+ pts, TC+ at T0 and T1 associated with worse PFS (HR=2.6 CI=1.2-5.3 p=0.008, and HR=3.8 CI=1.7-8.2 p<0.001) and OS (HR=3.5 CI=1.6-7.6 p=0.001, HR=3.0 CI=1.4-6.1 p=0.002), while in ER- pts TC classes showed no association with outcome. In a small and heterogenous retrospective cohort of mBC pts, MIMESIS-BC showed promising capabilities for prognostic stratification and subtype estimation from liquid biopsy. Validation in an extended prospective cohort is ongoing.

Metabolomics studies metabolites in biological samples. Cancer can impair metabolism, so the pattern of altered metabolites could reproduce a "cancer signature". This analysis aimed at identifying a "metabolic signature" that could differentiate elderly eBC patients (pts) from elderly advanced breast cancer (aBC) pts, as well as at investigating its prognostic role in terms of disease recurrence (DR). Serum samples from elderly BC pts enrolled in 3 onco-geriatric trials coordinated by the Medical Oncology Division of Prato, were retrospectively analyzed via proton nuclear magnetic resonance (NMR) spectroscopy. Three NMR spectra were acquired for each serum sample (NOESY1D, CPMG and Diffusion-edited). Random Forest (RF) models were calculated. The ability of metabolomics to predict BC relapses was assessed using Kaplan–Meier curves with calculation of the hazard ratio (HR) and p-value by Log-Rank test. Serum samples from 140 pts with eBC and 27 with aBC were collected between 2008 and 2018. In the aBC cohort, median age was 79 years (95% CI, 70-88); 25.9% of pts were hormone receptor positive HER2 negative (HR+HER2-), 29.6% HER2 positive (HER2+), 11.1% triple negative (TN); data missing in 33.3% of cases. In the eBC cohort, median age was 76 years (95% CI, 70-91); 30.7% of pts were HR+HER2-, 48.6% HER2+, 11.4% TN; data missing in 3.6% of cases. In this cohort, 39.3% of pts had tumors larger than 2 cm, and 41.4% had positive axillary lymph nodes. Using NOESY1D spectra, the RF classifier discriminated free from recurrance eBC from aBC with a sensitivity, specificity and accuracy of 81.5%, 66.7% and 70% respectively, performing better than the other spectra. Therefore, we tested the NOESY1D spectra of each eBC pt on the RF models already calculated. If the RF model classified the sample as relapsed, it was considered at "high risk" of DR. Our analysis showed that pts classified as "high risk" had a higher risk of DR (HR 3.39, 95% CI 1.59-7.24, p=0.00084). This analysis suggests that a "metabolic signature", identified employing NMR spectral profiling, is able to predict the risk of DR in elderly pts with eBC. Further studies are needed to confirm these data.

Cyclin-dependent kinase 4 and 6 inhibitors like palbociclib (P) are a mainstay of treatment for ER+/HER2- ABC; however intrinsic or acquired resistance is a major clinical issue. We performed a mutational analysis on ctDNA samples from patients (pts) included in the c-TREnd study, the translational cohort of the TREnd trial (NCT02549430) which randomized pts to receive P alone or in combination with the endocrine treatment (ET) to which they had progressed in the previous line of ET. Forty-six pts were enrolled in c-TREnd. Plasma was collected before treatment (T0), after the first cycle of therapy (T1) and at the time of progression (T2). Hybridization and capture were performed using the TruSight Tumor 170 Kit (Illumina). Single nucleotide variants (SNVs) were detected and annotated using LoFreq and Oncotator, and further refined by ABEMUS. Tumor Mutational burden (TMB) was the sum of silent and non-silent mutations. Tumor fraction (TF) was based on the dispersion of Copy Number Alteration (CNA) genomic profiles. Progression free survival (PFS) was estimated using the Kaplan–Meier method and compared with the log-rank test. Thirty-two pts (87 samples), 14 from the P arm and 18 from the P+ET arm, were included in the final analysis. The most frequently mutated genes at T0 were ESR1 (23%), PIK3CA (17%), AR, FGFR2 and TP53 (10%). At T0, mutations in ESR1, but not in PIK3CA, were significantly prognostic (median PFS – mPFS 3.7 mo vs 11 mo in ESR1 mut vs WT, p=0.015). A significantly worse mPFS was observed when a broader analysis of PI3K pathway (adding AKT1, PTEN, TSC1/2 and BRAF) was performed (mPFS 5.2 m in mut vs 10.8 m in WT, p=0.04). Mutations in AR tended to confer a better, although not statistically significant, mPFS (14.2 mo vs 5.5 in WT, p=0.29). At T2 we observed the emergence of 9 new mutations in 7 genes (ESR1, AKT1, ARID1A, BRIP1, CCNE1, MTOR and TP53). TMB and TF at T0 or TF change between T1 and T0 were not associated with PFS. Mutations in ESR1 and in PI3K pathway genes were associated with worse prognosis in pts treated with P, while TMB and TF were not. Larger studies are needed to validate these observations.

Resistance to CDK4/6i is inevitable. CTC count is prognostic in ABC, but its role in pts treated with CDK4/6i is not well defined. Genetic loss of RB1 is a known yet infrequent marker of CDK4/6i resistance. We assessed the prognostic role of CTC count and gene-expression (GE) levels of RB1 in CTCs in pts receiving P. The TREnd trial (NCT02549430) randomized pts with endocrine resistant ABC to either P alone or P plus the endocrine therapy received in the prior line of treatment. In TREnd, blood samples were prospectively collected in CellSave® tubes before starting P (T0), after the first cycle (T1) and at disease progression (T2). CTCs were isolated and counted by CellSearch® System (CS) using CellSearchTM Epithelial Cell kit. Samples with ≥5CTCs were sorted by DEPArray system® (DA). RNA extraction and retro-transcription for GE experiments were performed by Cell Lysis Two-Step RT-qPCR. RB1 and GAPDH GE levels were measured by ddPCR, with a multiplex assay with a sensitivity of 30-10 pg of cDNA, set up on three different cell lines sensitive and resistant to P. 46 pts were suitable for CTC analysis. CTC count at T0 did not show significant prognostic value in terms of progression free survival (PFS). However, pts with at least 1 detectable CTC at T1 (n=26) had a worse PFS than those with 0 CTCs (n=16) (p=0.02). Similar results were observed with a cut-off of 5 CTCs (p=0.04). At T1, 7 out of 39 pts had an increase of at least 3 CTCs which proved prognostic (p=0.01). Pts with ≥5CTCs at T2 (n=6/23) who received chemotherapy as post-study treatment had a shorter time to treatment failure (p=0.02). DA sorting was conducted on 20/46 pts and GE data for RB1 were obtained from 19 pts. CTCs showed heterogeneous RB1 expression. Pts with detectable expression of RB1 in at least one time-point had better, but not significant, outcomes than those with undetectable levels. Persistence or an increase in CTCs after one cycle of P may identify pts with worse outcome. High CTC counts at disease progression on P may indicate poor post-treatment prognosis. Measuring RB1 GE levels on CTCs by ddPCR is feasible, but its clinical significance is yet unclear.

Abstract Introduction: TK1 plays a crucial role in DNA synthesis and is a well-established marker of cell proliferation. We and others have previously described the potential role of TK1 activity (TKa) as predictive biomarker of response to endocrine therapy in HR+/HER2 negative metastatic breast cancer patients. TK1 synthesis is regulated by the E2F pathway, the target pathway of CDK4/6 inhibitors, and TKa has recently been reported as a potential circulating pharmacodynamic marker of CDK4/6 inhibition in breast cancer. However, modulations of TK1 levels and activity during palbociclib treatment and in the development of treatment resistance are unknown. Here, we report how TK1 expression and TKa are modulated in response to palbociclib in a panel of HR+ breast cancer cell lines: both palbociclib-sensitive (PDS) and with acquired resistance to (PDR). Material and methods: We used a panel of 7 PDR HR+ breast cancer models previously developed in our lab via chronic exposure of parental cells (MCF7, T47D, ZR75-1, BT474, MDAMB361 and two MCF7 endocrine resistant derivatives) to escalating doses of palbociclib, from a Starting Treatment Concentration (STC) of 50 nM or 350 nM according to the cell line, up to 1 μM. We analyzed gene expression profiles of PDS cells treated with drug vehicle (DMSO) as a control or palbociclib at STC for 3 days, and PDR cells grown with palbociclib 1 μM. Cell proliferation was assessed by methylene blue assay in MCF7 and BT474 PDS and PDR treated for 3, 6 and 9 days with DMSO, palbociclib STC and 1 μM. In parallel, TKa was measured in cell lysates at 3 days of treatment using the DiviTumTM assay (Biovica, Sweden). Results: Among E2F target genes, gene expression data demonstrated that TK1 was one of the most differentially expressed genes between PDR and PDS treated cells. In PDS cells compared to control, treatment with palbociclib resulted in reduced TK1 expression, with the HER2 positive models (BT474 and MDAMB361) showing the highest reduction. In PDR cells, TK1 expression was higher, but remained slightly inhibited compared to untreated PDS cells. TKa was significantly reduced in PDS cells treated with palbociclib for 3 days compared to vehicle (p<0.05). TKa response to palbociclib was more dramatic in BT474 cells as compared to MCF7. As expected, palbociclib inhibited cell proliferation in PDS models, with a significant reduction observed only after 6 days of treatment, suggesting that TKa may be an early marker of growth inhibition in response to palbociclib. No significant alterations in TKa were observed in PDR cells, at any dose of palbociclib. Similarly, proliferation rate was not affected by palbociclib in PDR cells. Conclusions: TK1 expression and activity are regulated by palbociclib in HR+ breast cancer cell lines, particularly in HER2 positive models. Ongoing studies of TKa in patients treated with palbociclib will assess the role of TKa as a circulating biomarker for predicting and monitoring response to CDK4/6 inhibitors. Citation Format: Bonechi M, Migliaccio I, Benelli M, Romagnoli D, Bergqvist M, Mattsson K, Boccalini G, Capaccioli G, De Luca F, Galardi F, Biagioni C, Risi E, McCartney A, Rossi L, Osborne CK, Schiff R, De Angelis C, Guarducci C, Di Leo A, Malorni L. Effects of palbociclib on thymidine kinase-1 (TK1) in hormone receptor positive (HR+) breast cancer cell lines [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 P6-09-02.

Background HER2 blockade in combination with chemotherapy (CT) remains the treatment of choice for patients with early HER2+ BC, irrespective of ER status. Patients with HER2+ early BC co-expressing ER may benefit from HER2 blockade in combination with endocrine therapy (ET) and new targeted agents. Elderly patients benefit from HER2 blockade as much as younger ones but they have higher risks of adverse events, mostly induced by the CT partner, making de-escalation of CT appealing. Recent data have elucidated cyclin-dependent kinases 4 and 6 (CDK4/6) as key therapeutic targets functioning downstream of both ER and HER2 pathways suggesting CDK4/6 inhibitors like Pal may be ideal partners for ET in this context. Pre-clinical and clinical data suggest that a gene signature of functional loss of Retinoblastoma (RBsig) might predict sensitivity to CT vs CDK4/6i in ER+/HER2+ early BC. The TOUCH hypothesis is that neoadjuvant therapy with Pal + ET + dual HER2 blockade may be more active in patients with ER+/HER2+ RBsig LOW early BC while those with RBsig HIGH may require CT. Trial design TOUCH is an open-label, multicenter, randomized phase 2 neoadjuvant trial. 144 patients ≥65 years with ER+/HER2+ primary BC will be randomized (1:1) to dual HER2 blockade (5 doses T+P) + either Pal (125 mg/d po; 21 of 28d x 4 cycles) + L (daily x 16 weeks), or Pac (80 mg/m2 iv, d1,8,15 q28 days x 4 cycles), before surgery. RBsig (HIGH vs LOW) will be determined centrally on mandatory pre-treatment biopsies. Baseline geriatric assessment includes G8, Instrumental Activity of Daily Living, Charlson comorbidity index. The primary objective is to explore the interaction between RBsig and treatment activity assessed by pathological complete response (pCR). Exact logistic regression will test RBsig by treatment interaction (2-sided a=.05) and estimate odds ratios (OR) for pCR. The sample size provides 86% power, assuming an overall pCR rate of 26%, and OR = 2.4 vs OR = 0.11 for RBsig LOW vs HIGH. Accrual began April 2019. Clinical trial identification NCT03644186. Legal entity responsible for the study International Breast Cancer Study Group. Funding Pfizer, Roche. Disclosure L. Biganzoli: Advisory / Consultancy, consultant and in an advisory role: AstraZeneca; Advisory / Consultancy, consultant and in an advisory role: Celgene; Advisory / Consultancy, consultant and in an advisory role: Eisai; Advisory / Consultancy, consultant and in an advisory role: Genomic Health; Advisory / Consultancy, consultant and in an advisory role: Ipsen; Advisory / Consultancy, consultant and in an advisory role: Lilly; Advisory / Consultancy, consultant and in an advisory role: Novartis; Advisory / Consultancy, consultant and in an advisory role: Pfizer; Advisory / Consultancy, consultant and in an advisory role: Pierre Fabre; Advisory / Consultancy, consultant and in an advisory role: Roche; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Genomic Health; Research grant / Funding (institution): Novartis. E. Brain: Honoraria (self), receipt of honoraria or consultation fees: Roche; Honoraria (self), receipt of honoraria or consultation fees: Pfizer; Honoraria (self), receipt of honoraria or consultation fees: AstraZeneca; Honoraria (self), receipt of honoraria or consultation fees: BMS; Honoraria (self), receipt of honoraria or consultation fees: Celgene; Honoraria (self), receipt of honoraria or consultation fees: Clinigen; Honoraria (self), receipt of honoraria or consultation fees: Hospira; Honoraria (self), receipt of honoraria or consultation fees: Janssen; Honoraria (self), receipt of honoraria or consultation fees: Mylan; Honoraria (self), receipt of honoraria or consultation fees: OBI Pharma; Honoraria (self), receipt of honoraria or consultation fees: Puma; Honoraria (self), receipt of honoraria or consultation fees: Samsung. L. Malorni: Research grant / Funding (self), research funding and consulting fees: Pfizer. All other authors have declared no conflicts of interest.

Background The activity of palbociclib as a single agent in advanced breast cancer has not been extensively studied, with the only available clinical data limited to heavily pretreated patients. Preclinical data suggests palbociclib may partially reverse endocrine resistance, though this hypothesis has not been evaluated in previous clinical studies. This phase II, open-label, multicenter study examined the activity of palbociclib monotherapy, as well as palbociclib given in combination with the same endocrine therapy (ET) that was received prior to disease progression, in postmenopausal women with moderately pretreated, estrogen receptor-positive, HER2 negative advanced breast cancer. Patients and methods Eligible women with advanced disease which had progressed on one or two prior ETs were randomized 1 : 1 to receive either palbociclib alone, or palbociclib in combination with the ET as previously received. Primary end point was clinical benefit rate (CBR); secondary end points included progression-free survival (PFS). Results Between October 2012 and July 2016, a total of 115 patients were randomized. The CBR was 54% [95% confidence interval (CI): 41.5-63.7] for combination therapy, and 60% (95% CI: 47.8-72.9) for monotherapy. Median PFS was 10.8 months (95% CI: 5.6-12.7) for combination therapy, and 6.5 months (95% CI: 5.4-8.5) for monotherapy [hazard ratio (HR) 0.69; 95% CI: 0.4-1.1, exploratory P-value = 0.12]. Exploratory analyses revealed the PFS advantage for combination therapy was seen in the subgroup of patients who received prior ET for >6 months (HR 0.53; 95% CI: 0.3-0.9, exploratory P-value = 0.02), but not in those who received prior ET for ≤6 months. Conclusion Palbociclib has clinical activity as a single agent in women with moderately pretreated, oestrogen receptor-positive, HER2-negative advanced breast cancer. Palbociclib may have potential to reverse endocrine resistance in patients with a history of previous durable response to ET. Clinical trial information NCT02549430.

Trials of adjuvant therapy in breast cancer patients have been conducted for over 40 years. These trials have usually been guided by the results of advanced breast cancer trials testing new agents [1.Anampa J. Makower D. Sparano JA. Progress in adjuvant chemotherapy for breast cancer: an overview.BMC Med. 2015; 13: 195Crossref PubMed Scopus (196) Google Scholar]. Cytotoxics, such as anthracyclines and taxanes, have become a standard of care in the adjuvant therapy setting after many trials confirmed their clinical activity in advanced disease [2.Peto R. Davies C. Early Breast Cancer Trialists' Collaborative Group (EBCTCG) et al.Comparisons between different polychemotherapy regimens for early breast cancer: meta-analyses of long-term outcome among 100,000 women in 123 randomised trials.Lancet. 2012; 379: 432-444Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar]. The same considerations apply to endocrine therapies such as tamoxifen and aromatase inhibitors [3.Dowsett M. Forbes J.F. Early Breast Cancer Trialists' Collaborative Group (EBCTCG) et al.Aromatase inhibitors versus tamoxifen in early breast cancer: patient-level meta-analysis of the randomised trials.Lancet. 2015; 386: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar], or targeted agents such as trastuzumab [4.Loibl S. Gianni L. HER2-positive breast cancer.Lancet. 2016; Google Scholar]. Bevacizumab has been largely investigated in ER+/ER negative and HER-2 negative advanced breast cancer patients in the context of Phase III trials either in the first- or second-line setting. Results from almost all of these trials have shown that the combination of bevacizumab and chemotherapy (in most of the cases a single-agent taxane) is superior to chemotherapy alone in terms of progression-free survival (primary study end-point). No convincing overall survival improvements have been shown in patients receiving bevacizumab [5.Miller K. Wang M. Gralow J. et al.Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer.N Engl J Med. 2007; 357: 2666-2676Crossref PubMed Scopus (2712) Google Scholar, 6.Miles D.W. Chan A. Dirix L.Y. et al.Phase III study of bevacizumab plus docetaxel compared with placebo plus docetaxel for the first-line treatment of human epidermal growth factor receptor 2-negative metastatic breast cancer.J Clin Oncol. 2010; 28: 3239-3247Crossref PubMed Scopus (791) Google Scholar, 7.Robert N.J. Dieras V. Glaspy J. et al.RIBBON-1: randomized, double-blind, placebo-controlled, phase III trial of chemotherapy with or without bevacizumab for first-line treatment of human epidermal growth factor receptor 2-negative locally recurrent or metastatic breast cancer.J Clin Oncol. 2011; 29: 1252-1260Crossref PubMed Scopus (802) Google Scholar, 8.Brufsky A.M. Hurvitz S. Perez E. et al.RIBBON-2: a randomized, double-blind, placebo-controlled, phase III trial evaluating the efficacy and safety of bevacizumab in combination with chemotherapy for second-line treatment of human epidermal growth factor receptor 2-negative metastatic breast cancer.J Clin Oncol. 2011; 29: 4286-4293Crossref PubMed Scopus (336) Google Scholar]. Survival improvements in advanced breast cancer trials have rarely been observed with chemotherapy or endocrine therapy tested in the first- or second-line setting. However, it would be difficult to argue that anti-cancer agents such as tamoxifen, aromatase inhibitors, anthracyclines, and taxanes are not active agents in the treatment of breast cancer. It makes sense to consider that an overall survival improvement is certainly the ultimate goal of any new anti-cancer treatment, but it may not be a sensitive enough end-point in metastatic breast cancer trials for several reasons, such as inadequate statistical power, a cross-over design, or interference on survival from subsequent lines of therapy delivered after progression on first- or second-line trial treatments [9.Sargent D.J. Hayes DF. Assessing the measure of a new drug: is survival the only thing that matters?.J Clin Oncol. 2008; 26: 1922-1923Crossref PubMed Scopus (61) Google Scholar]. In the current issue ofAnnals of Oncology, Bell et al. present the final efficacy analysis results of the BEATRICE trial, a phase III trial comparing in the adjuvant treatment of centrally confirmed triple-negative breast cancer (TNBC) patients a ‘standard of care’ adjuvant chemotherapy regimen to the same regimen combined with bevacizumab and followed by single-agent bevacizumab for a total of 1 year of treatment [10.Bell R. Brown J. Parmer M. et al.Final efficacy and updated safety results of the randomized phase III BEATRICE trial evaluating adjuvant bevacizumab-containing therapy in triple-negative early breast cancer.Ann Oncol. 2017; 28: 754-760Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]. The results of this trial, reported in 2013 at 30 months [11.Cameron D. Brown J. Dent R. et al.Adjuvant bevacizumab-containing therapy in triple-negative breast cancer (BEATRICE): primary results of a randomised, phase 3 trial.Lancet Oncol. 2013; 14: 933-942Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar] and now at 56 months median follow-up, do not show any convincing evidence concerning the value of bevacizumab in the adjuvant setting and the authors correctly conclude that incorporating bevacizumab into an adjuvant chemotherapy regimen for TNBC patients is not recommended in standard treatment practice. Other phase III trials testing bevacizumab in early breast cancer, including ER + or HER-2+ patients, have provided disappointing [12.von Minckwitz G. Eidtmann H. Rezai M. et al.Neoadjuvant chemotherapy and bevacizumab for HER2-negative breast cancer.N Engl J Med. 2012; 366: 299-309Crossref PubMed Scopus (435) Google Scholar, 13.Miller K. O’Neill A.M. Dang C.T. et al.Bevacizumab (Bv) in the adjuvant treatment of HER2-negative breast cancer: final results from Eastern Cooperative Oncology Group E5103.J Clin Oncol. 2014; 32Google Scholar, 14.Slamon D.J. Swain S.M. Buyse M. et al.Primary results from BETH, a phase 3 controlled study of adjuvant chemotherapy and trastuzumab ± bevacizumab in patients with HER2-positive, node-positive or high risk node-negative breast cancer.Cancer Res. 2013; 73: S01-S03Crossref Google Scholar] or controversial [15.Bear H.D. Tang G. Rastogi P. et al.Neoadjuvant plus adjuvant bevacizumab in early breast cancer (NSABP B-40 [NRG Oncology]): secondary outcomes of a phase 3, randomised controlled trial.Lancet Oncol. 2015; 16: 1037-1048Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar] results. It is our opinion that the lack of an overall survival improvement reported in the advanced breast cancer trials with bevacizumab cannot be the only and major determinant of the negative results observed in BEATRICE. As previously mentioned, many other trials testing endocrine or chemotherapy agents in the advanced setting have not led to convincing survival benefits; however, the same agents have successfully shown relevant activity in adjuvant therapy trials [2.Peto R. Davies C. Early Breast Cancer Trialists' Collaborative Group (EBCTCG) et al.Comparisons between different polychemotherapy regimens for early breast cancer: meta-analyses of long-term outcome among 100,000 women in 123 randomised trials.Lancet. 2012; 379: 432-444Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar,3.Dowsett M. Forbes J.F. Early Breast Cancer Trialists' Collaborative Group (EBCTCG) et al.Aromatase inhibitors versus tamoxifen in early breast cancer: patient-level meta-analysis of the randomised trials.Lancet. 2015; 386: 1341-1352Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar]. What other factors might play a role in the observed lack of benefit from bevacizumab in the context of the BEATRICE trial? In the last few years, we have realized that TNBC is heterogeneous in terms of biology and clinical behaviour. There is evidence that several TNBC sub-types can be identified by gene-expression profile and/or single biomarker analysis. More interestingly, it has been observed that the different TNBC sub-types do not differ only in terms of biology but also in terms of clinical behaviour, and in terms of sensitivity to different anti-cancer agents [16.Lehmann B.D. Bauer J.A. Chen X. et al.Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies.J Clin Invest. 2011; 121: 2750-2767Crossref PubMed Scopus (3461) Google Scholar]. At the time the study was designed, these notions on TNBC biological and clinical heterogeneity were not entirely clear and the study represented a first and important attempt to tailor adjuvant therapy treatment according to the breast cancer subtypes previously identified with the ‘first generation’ gene expression profile studies [17.Perou C.M. Sørlie T. Eisen M.B. et al.Molecular portraits of human breast tumours.Nature. 2000; 406: 747-752Crossref PubMed Scopus (11695) Google Scholar]. Now, a trial for TNBC early breast cancer patients would probably take into account the intrinsic TNBC heterogeneity as well as the potential interactions between TNBC and the host immune response. In addition, reviewing the pre-clinical evidence which in 2007 motivated the design of BEATRICE [18.Foekens J.A. Peters H.A. Grebenchtchikov N. et al.High tumor levels of vascular endothelial growth factor predict poor response to systemic therapy in advanced breast cancer.Cancer Res. 2001; 61: 5407-5414PubMed Google Scholar,19.Holmgren L. O’Reilly M.S. Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression.Nat Med. 1995; 1: 149-153Crossref PubMed Scopus (1681) Google Scholar], one could argue that nowadays the same data may appear as preliminary and not informative enough to activate a large adjuvant therapy trial, as the authors point out in the discussion section of their manuscript. Furthermore, subgroup analyses of advanced or neo-adjuvant breast cancer trials testing bevacizumab, presented after that the study was activated, reported controversial results in terms of the interaction between bevacizumab activity and TNBC [5.Miller K. Wang M. Gralow J. et al.Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer.N Engl J Med. 2007; 357: 2666-2676Crossref PubMed Scopus (2712) Google Scholar,12.von Minckwitz G. Eidtmann H. Rezai M. et al.Neoadjuvant chemotherapy and bevacizumab for HER2-negative breast cancer.N Engl J Med. 2012; 366: 299-309Crossref PubMed Scopus (435) Google Scholar,20.Bear H.D. Tang G. Rastogi P. et al.Bevacizumab added to neoadjuvant chemotherapy for breast cancer.N Engl J Med. 2012; 366: 310-320Crossref PubMed Scopus (400) Google Scholar,21.Brufsky A. Valero V. Tiangco B. et al.Second-line bevacizumab-containing therapy in patients with triple-negative breast cancer: subgroup analysis of the RIBBON-2 trial.Breast Cancer Res Treat. 2012; 133: 1067-1075Crossref PubMed Scopus (96) Google Scholar]. Last but not least, bevacizumab in the BEATRICE trial, given pragmatically for 1 year, was combined with various chemotherapy regimens which differed in terms of agents, doses, schedules and duration. It has emerged that weekly paclitaxel is likely to be the most active cytotoxic agent to be combined with bevacizumab [22.Shaked Y. Henke E. Roodhart J.M. et al.Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents.Cancer Cell. 2008; 14: 263-273Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar]. Of note, no taxanes were administered in the adjuvant setting in approximately one-third of patients from BEATRICE and in the remaining two-thirds weekly paclitaxel was not the only taxane regimen [10.Bell R. Brown J. Parmer M. et al.Final efficacy and updated safety results of the randomized phase III BEATRICE trial evaluating adjuvant bevacizumab-containing therapy in triple-negative early breast cancer.Ann Oncol. 2017; 28: 754-760Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,11.Cameron D. Brown J. Dent R. et al.Adjuvant bevacizumab-containing therapy in triple-negative breast cancer (BEATRICE): primary results of a randomised, phase 3 trial.Lancet Oncol. 2013; 14: 933-942Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar]. One factor strongly supporting the activation of such a trial in TNBC was the need to improve prognosis in this particular cohort of breast cancer patients. In this regard, a relevant question could be: ‘is an unmet need a strong enough justification to activate a large adjuvant therapy trial in absence of fully supportive pre-clinical and clinical data?’ BEATRICE can potentially teach us that strong pre-clinical data and convincing clinical interactions between the tested agent and TNBC may be needed before embarking on large adjuvant therapy trials. A reasonable compromise has to be reached between the need to rapidly identify new active agents for adjuvant therapy and the risk of negative trial results due to an insufficient amount of data supporting the experimental approach. This is, in our opinion, one of the most inspiring messages deriving from BEATRICE. As a last note, we believe that the authors of this trial have to be congratulated, not only because in the context of a large adjuvant therapy trial—fully addressed to TNBC patients—they have shown that bevacizumab, as delivered in this trial, is not a valuable treatment option, but also because they have shown that TNBC is not necessarily associated with a poor clinical outcome. In BEATRICE, the 5-year invasive disease-free survival rate was 77% for patients in the control arm (37% of the trial population presented with node-positive disease). This is important information for both patients and clinicians and it may have an impact not only for the design of future trials in TNBC but also for daily practice, i.e. the intensity of chemotherapy regimens (number of cycles, dose-density, choice of cytotoxics) can be modulated according to patient wishes/expectations and to traditional prognostic factors such as tumor size and number of positive nodes [23.Turner N. Biganzoli L. Malorni L. et al.Adjuvant chemotherapy: which patient? What regimen? American Society of Clinical Oncology educational book/ASCO.Am Soc Clin Oncol Meeting. 2013; 4: 3-8Google Scholar]. Furthermore, considering that in BEATRICE biological samples have been systematically collected and banked, this trial represents a unique opportunity to run translational studies aiming to identify potential predictive bio-markers for bevacizumab activity and to achieve new insights into the biology and clinical behaviour of TNBC. None declared.

Background: HER2+ breast cancers (BC) are clinically and biologically heterogeneous, with approximately half being ER+. Compared to other BC subtypes, HER2+ ER+ tumors display among the lowest rates of pathological complete response (pCR) following neoadjuvant chemotherapy (NACT) +/− anti-HER2 agents (anti-HER2). Yet in spite of this, HER2+/ER+ patients (pts) are typically treated with NACT plus anti-HER2, with the subsequent related toxicity. Currently there is a lack of predictive biomarkers that identify which subgroups of pts will not respond to such therapy. Inactivation of the Retinoblastoma (Rb) signalling pathway is a frequent event in BC. Previously developed gene-signatures of Rb loss-of-function have shown strong prognostic value and prediction of response to NACT. However, none has been extensively studied in the context of HER2+/ER+ BC. We have recently developed a gene-signature of RB-1 loss-of-function (RBsig) that is prognostic in luminal A-like and luminal B-like BC. Here we report the results of a retrospective in-silico study aimed to determine whether low expression of the RBsig in HER2+/ER+ BC correlates with a low pCR rate following NACT +/− anti-HER2. Methods: We performed a PubMed search for clinical trials of NACT +/− anti-HER2 (trastuzumab, lapatinib, or both) in HER2+ BC pts, and selected studies which had available gene expression data, hormone receptors status and pCR information. In-silico analyses of correlation between RBsig expression and pCR were performed using receiver-operating characteristic (ROC) curves and Fisher exact test to assess the prediction performance of the signature score. The threshold RBsig score was set at the 50th percentile of the score distribution. Results: Out of 16 identified studies, 10 fulfilled the inclusion criteria and were included in the analysis (514 pts). Overall, of the 211 HER2+/ER+ BC pts, 49 achieved pCR (23%); the pCR rate following NACT +/− anti-HER2 of pts with RBsig low expression was significantly lower compared to pts with RBsig high expression (16% vs 30%, respectively; Fisher exact test p=0.0098).The area under the ROC curve (AUC) was 0.62 (95% confidence interval (CI) 0.54-0.7, p=0.005). Results were similar for pts receiving NACT alone (94 pts; pCR rate 13% vs 28% in RBsig low vs RBsig high, respectively; Fisher exact test p=0,06; AUC 0.62, 95% CI 0.5-0.74, p=0.043) or combined with anti-HER2 (117 pts; pCR rate 18% vs 33% in RBsig low vs RBsig high, respectively; Fisher exact test p=0,049; AUC 0.61, 95% CI 0.5-0.72, p=0.041). In 303 HER2+/ ER− pts treated with NACT +/− anti-HER2, the pCR rate was 42%. No correlation was found between RBsig expression score and pCR rate in this group (pCR rate 42% vs 43% in RBsig low vs RBsig high, respectively; Fisher exact test p=0.53; AUC 0.5, 95% CI 0.43-0.56, p=0.973). Conclusions: RBsig identifies a subset of HER2+/ER+ pts with a low pCR rate following NACT +/− anti-HER2. We hypothesize that this signature has the potential to identify pts for whom chemotherapy could be avoided in favour of combinations of endocrine therapy and target therapies. Further refinement and validation in an independent dataset is warranted. Citation Format: Risi E, Grilli A, Migliaccio I, Biagioni C, Guarducci C, Bonechi M, Hart CD, Biganzoli L, Bicciato S, Di Leo A, Malorni L. A RB-1 loss-of-function gene-signature (RBsig) predicts resistance to neoadjuvant chemotherapy in HER2+/ER+ breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-13.