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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.

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).

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.

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.

The addition of supplemental food sources for natural enemies is a growing component of conservation and augmentative biological control. Supplemental foods can be used to retain natural enemies when prey are scarce and to promote survival, fecundity, longevity, and development of natural enemy populations, especially generalist natural enemies. Amblydromella caudiglans (Schuster) (Acari: Phytoseiidae) is one of the most abundant predatory mites found in commercial apple orchards in Washington, USA, and contributes to spider mite control. However, because its widespread presence in commercial apple orchards was only recently discovered, how supplementary food sources affect its performance is unknown. In laboratory studies, we evaluated the performance (fecundity, retention, prey consumption) of the generalist phytoseiid A. caudiglans on commercially available supplemental food sources, including factitious prey (Ephestia eggs and Artemia brine shrimp cysts), and pollens of apple, pear, and cattail. We determined that A. caudiglans will not consume Ephestia eggs and performs best on cattail and pear pollens. Combinations of food sources did not enhance the performance of this predator compared to the best performing single-sources. The presence of alternative food sources did not decrease A. caudiglans predation of twospotted spider mite nymphs, except for Artemia brine shrimp cysts, which had a substantial handling time. These results lay the groundwork for identifying a way to promote and retain this natural enemy in tree fruit cropping systems through the use of food resource applications or floral plantings.

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.

Background Circulating tumor cells (CTCs) are prognostic in patients with advanced breast cancer (ABC). However, no data exist about their use in patients treated with palbociclib. We analyzed the prognostic role of CTC counts in patients enrolled in the cTREnd study, a pre-planned translational sub-study of TREnd (NCT02549430), that randomized patients with ABC to palbociclib alone or palbociclib plus the endocrine therapy received in the prior line of treatment. Moreover, we evaluated RB1 gene expression on CTCs and explored its prognostic role within the cTREnd subpopulation. Methods Forty-six patients with ER-positive, HER2-negative ABC were analyzed. Blood samples were collected before starting palbociclib treatment (timepoint T0), after the first cycle of treatment (timepoint T1), and at disease progression (timepoint T2). CTCs were isolated and counted by CellSearch® System using the CellSearch™Epithelial Cell kit. Progression-free survival (PFS), clinical benefit (CB) during study treatment, and time to treatment failure (TTF) after study treatment were correlated with CTC counts. Samples with ≥ 5 CTCs were sorted by DEPArray system® (DA). RB1 and GAPDH gene expression levels were measured by ddPCR. Results All 46 patients were suitable for CTCs analysis. CTC count at T0 did not show significant prognostic value in terms of PFS and CB. Patients with at least one detectable CTC at T1 (n = 26) had a worse PFS than those with 0 CTCs (n = 16) (p = 0.02). At T1, patients with an increase of at least three CTCs showed reduced PFS compared to those with no increase (mPFS = 3 versus 9 months, (p = 0.004). Finally, patients with ≥ 5 CTCs at T2 (n = 6/23) who received chemotherapy as post-study treatment had a shorter TTF (p = 0.02). Gene expression data for RB1 were obtained from 19 patients. CTCs showed heterogeneous RB1 expression. Patients with detectable expression of RB1 at any timepoint showed better, but not statistically significant, outcomes than those with undetectable levels. Conclusions CTC count seems to be a promising modality in monitoring palbociclib response. Moreover, CTC count at the time of progression could predict clinical outcome post-palbociclib. RB1 expression analysis on CTCs is feasible and may provide additional prognostic information. Results should be interpreted with caution given the small studied sample size.

Background Metronomic chemotherapy can induce disease control in patients with metastatic breast cancer (MBC) and has better safety profiles than conventional chemotherapy. Evidence suggests that cytotoxics can be anti-angiogenic in pre-clinical models and may have synergistic effects when combined with anti-vascular endothelial growth factor therapies. Patients and Methods Patients pretreated with ≥ 1 prior line of therapy for MBC received oral cyclophosphamide 50 mg daily in combination with oral vinorelbine at escalating doses of 20 mg (V20), 30 mg (V30), and 40 mg (V40) 3 times per week, and intravenous bevacizumab 15 mg/kg every 3 weeks. Patients with human epidermal growth factor receptor 2-positive disease were given the same regimen plus standard trastuzumab. Doses were escalated when 3 patients completed 3 treatment cycles of V20 and V30, without experiencing dose-limiting toxicities. The recommended dose was then tested in a further 6 patients. Circulating tumour cells and circulating endothelial cells (CEC) were measured in 30 mL of whole blood samples at baseline, after cycle 1, and at the disease progression. Results Fifteen patients were recruited from June 2013 to October 2015. The median age was 61 years (range, 29-72 years); 80% had estrogen receptor-positive and 33% had human epidermal growth factor receptor 2-positive disease. At least 67% had visceral metastases, and 80% had received ≥ 2 lines of prior treatment. No dose-limiting toxicities were observed at the 3 dose-levels, making V40 the recommended dose. Overall 8 (53%) patients developed grade 2 adverse events (arthralgia, n = 3 [20%]; asthenia, n = 2 [13%]; diarrhea, n = 2 [13%]; leukopenia, n = 2 [13%]). Bevacizumab was associated with grade 3 hypertension (n = 3 [20%]). Stable disease as best response was observed in 11 (73.3%) patients. The clinical benefit rate was 66.6% (10/15 patients). The median time to progression was 6.9 months. At baseline, CECs were more commonly detectable than circulating tumor cells; however, no statistical correlation was found between CEC kinetics and response. Conclusion A metronomic vinorelbine dose of 40 mg combined with cyclophosphamide and bevacizumab is a promising treatment regimen in pretreated patients with MBC.

Background. Partial breast irradiation (PBI) is a reasonable alternative for a whole breast irradiation (WBI) in selected early stage breast cancer patients. The 5-year analysis of the APBI IMRT Florence phase 3 trial showed no significant difference in terms of ipsilateral tumor recurrence (IBTR) and survival rates between the two arms. We present the 10-year follow up data. Methods. From March 2005 to June 2013, women aged more than 40 years affected by early BC, with a maximum pathological tumor size of 25 mm, were randomly assigned in a 1:1 ratio to receive either WBI using three-dimensional conformal radiotherapy (3DCRT) or accelerated PBI (APBI) using intensity-modulated radiotherapy (IMRT) technique. Patients in the APBI arm received a total dose of 30 Gy to the tumor bed in five daily fractions. The WBI arm received 50 Gy in 25 fractions, followed by a boost on the tumor bed of 10 Gy in five fractions. The primary end-point, IBTR rate is now assessed at 10 years in the ITT population, as well as secondary endpoints: overall survival (OS), breast cancer specific survival (BCSS), distant metastasis free survival (DMFS), contralateral breast cancer (CBC), and locoregional recurrences (LRR). This trial is registered with ClinicalTrials.gov, number NCT02104895. Results. Of the 520 patients, 260 were enrolled in the APBI-arm and 260 in the WBI-arm; median age was 62.8 years (63.6 in the APBI and 61.6 in the WBI arm, p=0.20). Both treatment-arms achieved a median 10-year follow-up (10.1 in APBI-arm and 10.4 in WBI-arm, p=0.39) and were comparable regarding age, tumor size, grade, tumor type, and adjuvant endocrine treatment. No significant difference in terms of IBTR rates between treatment arms was shown (log rank test p=0.58). In the group who received APBI, the 5-year IBTR was 1.96% (5 events; 95% CI: 0.3;3.7) and the 10-year IBTR was 3.74% (9 events; 95% CI: 1.5;6.3). In the group who received WBI, the 5-year IBTR rate was 1.2% (3 events; 95% CI: 0;2.5) and the 10-year IBTR was 2.5% (6 events; 95% CI: 0.7;4.5). HR for APBI patients compared with WBI patients was 1.33 (p=0.58; 95% CI: 0.49;3.56). There were no significant differences between treatment arms regarding survival outcomes. OS (log rank test p=0.33): APBI 95.4% (95% CI: 93.6;97.2) versus WBI 94.3% (95% CI: 92.3;96.3); HR for APBI patients 0.66 (p=0.33; 95% CI: 0.29;1.53). BCSS (log rank test p=0.55): APBI 98% (95% CI: 96.8;99.2) versus WBI 97.5% (95% CI: 96.2;98.8); HR for APBI patients 0.68 (P=0.55; 95% CI: 0.19;2.42). DMFS (log rank test p=0.45): APBI 97.4% (95% CI: 96.0;98.8) versus WBI 96.1% (95% CI: 94.4;97.8); HR for APBI patients 0.67 (p=0.45; 95% CI: 0.24;1.89). Cumulative incidence estimates of 10-year LRR were 3.9% (95% CI: 2.24;5.56) for APBI versus 3.0% (95% CI: 1.53;4.47) for WBI (log rank test p=0.59). CBC observed after APBI were 5 (1.9%) as compared to 9 (3.5%) after WBI (p=0.42). Conclusion. IBTR rate after 10 years in patients with early breast cancer who were treated with APBI using IMRT technique in 5 fractions is rare and not significantly different from patients treated with WBI. OS, BCSS, DMFS, and LRR control are also comparable. Thus, APBI should be considered a reasonable alternative for a WBI in early breast cancer patients. Citation Format: Icro Meattini, Calogero Saieva, Sara Lucidi, Monica lo Russo, Vieri Scotti, Isacco Desideri, Livia Marrazzo, Gabriele Simontacchi, Monica Mangoni, Carlotta Becherini, Luca Visani, Lisa Paoletti, Erica Moretti, Luca Triggiani, Marco Bernini, Lorenzo Orzalesi, Luis Sanchez, Jacopo Nori, Stefania Pallotta, Simonetta Bianchi, Lorenzo Livi. Accelerated partial breast or whole breast irradiation after breast conservation surgery for patients with early breast cancer: 10-year follow up results of the APBI IMRT Florence randomized phase 3 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS4-06.

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

Patients with luminal early breast cancer are at risk of relapse, even after five years of adjuvant endocrine therapy. To date, no biomarkers have been clinically validated to identify those patients at risk of late recurrence, who might benefit from extended adjuvant endocrine therapy. In recent decades, multiple clinical trials have tested the role of extending adjuvant endocrine therapies in patients with luminal disease. However, the data currently at our disposal are conflicting. This article reviews all the major trials concerning extended adjuvant endocrine regimens, and formulates some general conclusions and hypotheses of future study.

Background The combination of platinum with 5-fluorouracil has scarcely been studied in metastatic breast cancer. As this combination does not lead to significant hepatic metabolism, in some clinical situations it may prove useful, especially in cases with liver dysfunction and an urgent clinical need for rapid tumor shrinkage. A retrospective study was conducted to evaluate the efficacy and safety of the combination of cisplatin and 5-fluorouracil in patients with metastatic breast cancer with significant alterations of biochemistry. Patients and methods A total of 109 patients with metastatic breast cancer and liver dysfunction were treated; time-to-progression, overall survival and trends in liver function were evaluated. Results The median time-to-progression was 3.4 months, and median overall survival was 7.8 months. About 50% of patients obtained a complete, partial or stable biochemical response and 24 patients were subsequently able to receive additional therapies. Conclusion Our results show that this therapeutic doublet represents a clinically effective, safe and well-tolerated treatment option for patients with metastatic breast cancer and liver dysfunction.

In the early years of the twentieth century, the great structural, social and cultural changes in American society included a growing number of immigrants arriving from the poorest regions of Europe. For the first time, the issues of immigration, assimilation and social integration became the most important problems facing American society. In the optimistic climate of the so-called progressive era, social reformers thought that these problems could be solved by the science of pedagogy, as applied to the educational needs of foreign immigrants. This essay centres on the pedagogical efforts of Italian-American educator Angelo Patri, who attempted to integrate Italian-American children into the fabric of American society through education. It starts by assessing Patri’s early writings, such as A Schoolmaster of the Great City, and his private and professional papers. In doing so, his work is situated in the debate on progressive education alongside pedagogue Maria Montessori, demonstrating his central role ...

Background: The CDK4/6 inhibitor palbociclib (P) is approved for the treatment of luminal metastatic breast cancer (MBC) in combination with endocrine therapy (ET). It leads to reduced phosphorylation of the Rb protein resulting in a decrease in E2F activity and eventual cell cycle arrest. Thymidine kinase 1 is a well-known cancer proliferation marker downstream of the E2F pathway, whose activity can be measured in plasma samples as readout of tumor proliferation. Circulating thymidine kinase activity (TKa) is a prognostic marker in MBC patients (pts) treated with ET, both when measured at baseline and during treatment. TKa has been previously shown to decrease in pts treated with neoadjuvant P+ET for 15 days, which was attributed as pharmacodynamic change on P treatment. The predictive value of TKa changes during treatment with P, as well as the dynamics of TKa changes on P-containing treatments are not yet comprehensively defined. Here we investigate the role of plasma TKa measured at different timepoints in a cohort from the TREnd study (NCT02549430). Methods: TREnd was a randomized phase II trial that allocated 115 pts with moderately pre-treated luminal MBC to receive single-agent P or P plus the same ET agent that was received in the prior line of therapy. Plasma samples were collected at baseline (T0; n=45), at day 1 of cycle 2 (T1; n=45) and at disease progression (T2; n= 36) from 46 consenting pts. TKa was measured with DiviTum®, a refined ELISA-based assay. Patients were dichotomized as high/low at T0 based on an optimal cut-off (260 Du/L) determined by maximally selected rank statistics, and on the median value at T2 (250 Du/L). Dynamic changes between T0 and T1 were deemed meaningful if >10% of T1 or T0, whichever was greatest. Clinical outcome was estimated using the Kaplan-Meier method. Results: Median TKa (mTKa) at T0 was 73 Du/L (range 20-4302). As expected, P-containing treatment reduced mTKa levels at T1 (37 Du/L, range 20-4504). Conversely, at disease progression, TKa increased compared to T0 (mTKa at T2, 250 Du/L, range 20-3653). Median time to progression (mTTP) in pts with low TKa at T0 (n= 33) was 8.5 months, compared to 5.6 months in pts with high TKa (n= 12). Interestingly, pts with an increase in TKa at T1 (n=9) had a mTTP of only 3.1 months compared to pts with stable/reduced TKa (N=35), who showed a mTTP of 9 months. Considering the potential significance of TKa measured at disease progression, pts with high levels at T2 (n=18) had a worse outcome on subsequent post-study treatment (both chemotherapy and ET) compared to those with lower levels (n=18) (mTTP at T2, 2.9 vs 8.9 months, respectively). Conclusions: These data suggest for the first time that TKa may be a useful prognostic biomarker for non-invasive monitoring of MBC in the context of treatment with P. These results warrant further investigation in larger sample sets. Citation Format: Luca Malorni, Chiara Biagioni, Francesca De Luca, Martina Bonechi, Giuseppe Curigliano, Alessandro M. Minisini, Erica Moretti, Mattias Bergqvist, Karin Mattsson, Emanuela Risi, Ilenia Migliaccio, Stefania Vitale, Stefano Gabellini, Amelia McCartney, Irene De Santo, Francesca Galardi, Giulia Boccalini, Matteo Benelli, Lorenzo Rossi, Laura Biganzoli, Angelo Di Leo. Plasma thymidine kinase activity in patients with luminal metastatic breast cancer treated with Palbociclib within the phase II TREnd trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4416.

At the turn of the twentieth century, many Italian intellectuals opposed women’s participation in the public sphere, maintaining that women could not engage in politics due to their exclusive love for their biological children. Contemporary feminists countered this notion by promoting the idea of social motherhood. Sibilla Aleramo and Maria Montessori, better known for their work in feminist literature and early childhood education, respectively, made important contributions to this debate by implementing, theorizing, and popularizing the notion of social motherhood. This essay traces the strategies the two intellectuals used to demonstrate how women could act as political subjects via a socialization of the maternal functions. In her novel Una donna, Aleramo offered a fictional portrait of the social mother. Influenced by it and by the feminist debate on motherhood, Montessori conceptualized the notion of social motherhood as both a socialization of maternal duties and the expansion of women’s ma...

Aim: Anthracyclines are commonly used in breast cancer, although they lack validated predictive biomarkers. We explored the interaction between TOP2A protein by quantitative immunofluorescence (QIF) and anthracycline sensitivity. Patients & methods: Patients with estrogen receptor-negative breast cancer received neoadjuvant epirubicin. Pretreatment biopsies were analyzed using AQUA®. Total, cytoplasmic (C) and nuclear (N) TOP2A protein concentrations were expressed as QIF scores and compared with pathologic complete response (pCR), TOP2A by immunohistochemistry, TOP2A mRNA, TOP2A and HER2 gene status, and Ki-67 level. Results: In total, 76 cases were assessable. C, N, and total scores did not correlate with pCR, or other markers. The N:C ratio differed significantly by HER2 status. No pCRs occurred in patients in the lowest N:C quartile. Conclusion: Although no relevant correlation between TOP2A QIF scores and pCR was found, N:C ratio may have a negative predictive role, and may merit further exploration in a multifactorial predictive model that includes tumor and host factors.

Triple negative breast cancers, which are defined by lack of expression of estrogen, progesterone, or HER2 receptors, represent approximately 15% of all breast cancers, although they account for a much higher proportional of breast cancer mortality. This is due both to their innate aggressive biological characteristics, but also to lack of effective therapies. Conventional chemotherapy is currently the only treatment option, thus there is a critical need to find new and effective targeted therapies in this disease. While investigation of agents such as poly (ADP-ribose) polymerase (PARP) inhibitors and EGFR inhibitors continues, results from recent clinical trials indicate that these therapies are not as active in sporadic triple negative breast cancers as initially hoped. It is important therefore to consider other emerging therapeutic agents. Mutation in p53 is found in the vast majority of triple negative breast cancers, and as such is a target of particular interest. Within this review, several agents with potential activity against aberrant p53 signaling have been considered, as a novel approach to finding an effective targeted therapy for this aggressive breast cancer subtype.

In Quota zero, Pietro Saitta traces a genealogy of the marginality of the Italian South, specifically the city of Messina. The author looks at the intersection between the ‘Southern question’ and t...

BACKGROUND Trastuzumab is part of the standard treatment for HER-2 positive breast cancer patients, but not all patients respond to trastuzumab. Altered expression levels for microRNAs in cancer cells have been correlated with prognosis and response to chemotherapy. We hypothesized that altered expression levels for miRNAs in plasma are associated with sensitivity to trastuzumab in patients with HER-2 positive breast cancer.

The past decade has seen extensive research into potential markers of responsiveness to anthracycline therapy in breast cancer patients; however, such markers have remained elusive. The status of both human epidermal growth factor receptor-2 (HER2) and topoisomerase II alpha (TOP2A) genes has been investigated in this context, but neither can be considered a clinically reliable predictor of response to anthracyclines. Expression of the TOP2A protein is affected by a number of factors not always related to gene copy number, and these might provide future potential markers. Recent studies have suggested that stroma-related signatures could predict anthracycline resistance, and increased expression of tissue inhibitor of metalloproteinases 1 (TIMP1) has also been implicated in resistance. Deficiency of the BRCA1 and BRCA2 genes may render cells more sensitive to topoisomerase inhibitors such as anthracyclines by disrupting repair of damaged DNA. Chromosome 17 polysomy has also been associated with increased responsiveness to anthracyclines. Given the complexity of topoisomerases and DNA repair pathways, it may be that a multifactorial approach, rather than reliance on a single biomarker, is needed to identify anthracycline-sensitive patients.

Fulvestrant is a pure estrogen antagonist that binds, blocks and downgrades the estrogen receptor (ER). Its unique mechanism of action is its antitumor activity after progression on prior endocrine therapy. Fulvestrant has shown activity in ER-dependent cells that are ligand independent. Fulvestrant has been approved at 250 mg/month for postmenopausal women with hormone-sensitive advanced breast cancer after progression or recurrence on antiestrogen therapy. The fulvestrant 500 mg regimen has just received approval by the EMA and the US FDA, supported by dose-dependent ER downregulation and the recent results of the clinical trial CONFIRM. Fulvestrant in combination with systemic lowering of estrogen has shown no improvement over fulvestrant alone. Combination therapy with inhibitors of growth factor signaling may have greater efficacy and is under exploration. To enhance the benefit of fulvestrant and improve outcomes for individuals with ER-positive breast cancer, greater understanding of resistance mechanisms is required. A key issue is identification of patients with ER-positive disease who retain sensitivity to antiestrogen therapy after progression on tamoxifen and/or aromatase inhibitors.

With increased use of aromatase inhibitors (AIs) in the adjuvant and first-line metastatic settings for postmenopausal women with estrogen receptor (ER)-positive breast cancer, systemic relapse or progressive metastatic disease on AIs is an increasingly encountered clinical scenario. Overcoming resistance is a priority. Patients with AI-resistant disease represent a heterogeneous population, with diverse mechanisms of resistance dictating varied sensitivity to subsequent treatment. Cells with persistent dependence on ER signaling may be inhibited by fulvestrant, in which case dose-dependent ER downregulation and activity favor high-dose, loading schedule fulvestrant. In direct contrast, cells with long-term estrogen deprivation with adaptive estrogen hypersensitivity may be inhibited by exposure to estrogen. Cell survival by alternate growth signaling pathways may be inhibited by targeted agents. Currently missing, however, are predictive biomarkers to identify underlying resistance mechanisms and guide effective post-AI therapy for individual patients.

More than a decade of research into identification of individual predictive biomarkers for anthracyclines has been disappointing. Despite extensive attempts to identify clinically robust predictive biomarkers, the issue of the specific biological subgroup of early breast cancer patients to derive anthracycline benefit remains elusive. In particularly, the use of HER2 and the anthracycline drug target, topoisomerase II alpha (topo IIa), as sole markers has yielded inconsistent results. Regulation of topo IIa, DNA repair pathways and host features complicate the anthracycline story. See Fig. 1. We may need to abandon the quest for a sole predictive marker and re-search anthracycline activity with multifactorial predictive tools. Anthracyclines are DNA-damaging agents; however, they cannot bind directly to DNA. They bind and inhibit the topo IIa enzyme. Topoisomerase enzymes relieve the torsional stresses created by separation of the supercoiled DNA double-helix during transcription and replication. Disruption of topo IIa leads to double-strand DNA breaks, with subsequent cell death if the cell is unable to repair the damaged DNA. Theoretically, anthracyclines would be particularly active in the setting of overexpression of the topo IIa protein, coupled with impaired DNA repair. However, prediction of anthracycline benefit by topo IIa is inconsistently reported. Indeed, prediction of anthracycline activity has been reported in a conflicting mix of reports for several markers, including HER2, topo IIa and duplication of the chromosome 17 centromere. Regarding HER2, the Canadian MA.5 trial showed a significant predictive role for positive HER2 status [1]. In contrast, a substudy of the UK BR9601 trial showed improved outcomes from anthracyclines in the setting of normal HER1-3 status [2]. Many other studies showed a trend in favour of HER2, but were individually underpowered to robustly test treatment interactions [3–7]. A meta-analysis based on abstracted data from eight previously published studies concluded that anthracycline benefit was confined to HER2-positive disease [8]. However, diversity in patients, treatment and biological subtypes in the meta-analyses limit robust conclusions. A clear mechanistic link between HER2 and anthracycline activity remains elusive. Results also conflict for topo IIa, with support for [4, 5, 9–13] and against a predictive role [2, 14–16]. The Canadian MA.5 trial reported significant predictive value of pooled TOP2A aberrations, but not of TOP2A deletion or amplification independently [10]. Combined analysis of the UK NEAT/BR9601 trials showed no predictive role for TOP2A [14]. A recently presented meta-analysis assessed TOP2A in four phase III adjuvant studies comparing anthracyclines with CMF [17]. The interim analysis showed a clinically modest and statistically borderline predictive value for TOP2A amplification. The only prospective study to address this question, the TOP trial, was a single arm, single-agent neoadjuvant epirubicin analysis [9]. Whilst not yet formally published or peer reviewed, this trial is important as it is also the only trial to assess This is an invited commentary to article doi:10.1007/s10549-010-0931-y.

Purpose of review The benefit from anthracycline-based vs. nonanthracycline-based adjuvant therapy is not experienced by all breast cancer patients. Identification of the individuals to derive this benefit may be guided by predictive biomarkers. This review focuses on the search for biomarkers, particularly focusing on the potential roles for HER-2 and/or topoisomerase IIalpha. Recent findings Clarification of differential sensitivity to anthracyclines is complicated due to disease heterogeneity, complexity of underlying biological pathways, biomarker detection methods and features of study design. Meta-analyses suggest anthracycline benefit is restricted to patients with HER-2 amplified disease. However, diversity within HER-2 positive and HER-2 negative subgroups limits the use of HER-2 status as an independent marker. Certainly, subgroups within HER-2 negative disease have demonstrable incremental benefit from anthracycline-based therapy. Regarding topoisomerase IIalpha, the best method of detection and predictive role remain unclear. Summary Although progress has been made in defining breast cancer subgroups and identifying patients with general chemosensitivity, we do not yet have reliable predictive markers for anthracyclines. With current evidence, neither HER-2 status nor topoisomerase IIalpha status can be considered clinically valuable in guiding prescription of anthracyclines. Disease heterogeneity may dictate prediction by tumour profiles, rather than any single marker. These profiles may incorporate a panel of markers, including not only tumour features, such as HER-2 and topoisomerase IIalpha, but also host-determined features, such as stroma and stroma-anthracycline interaction. A new generation of well powered clinical trials that attempt to incorporate breast cancer heterogeneity may bridge the gap between available results and individual patient care.

Taxanes are very effective agents in several types of cancer. However, their activity is counterbalanced by side effects that could represent a limitation of their use in older cancer patients. This review aims at evaluating whether or not there are data supporting a tailored use of standard taxanes i.e. docetaxel and paclitaxel in elderly patients with the aim to increase their therapeutic index. In addition, recent data on the role of nanoparticle albumine-bound paclitaxel in breast cancer are discussed in this paper.

MicroRNAs (miRNAs) comprise a class of small endogenous non-coding RNA molecules that play critical roles in modulating numerous biological pathways by regulating gene expression. High throughput and expression profile studies have shown that miRNAs are commonly dysregulated in human cancer. The study of the mechanisms of action of miRNAs has uncovered an entirely new repertoire of potential biomarkers and generegulated pathways. MiRNAs may function as oncogenes or tumour suppressor genes and are characterised by unique properties such as stability, tissue specificity, ease of detection and manipulation. Additionally, miRNAs are differentially expressed in the blood of cancer patients versus healthy donors, providing a rationale for the detection of miRNAs as diagnostic and prognostic circulating biomarkers. Recent studies have shown that specific expression patterns of miRNAs could be used as potential predictors of response to treatments in clinical trials. Finally, manipulation of miRNA expression in cancer could be used for future molecular therapies.

1002 Background: P is approved for treatment of HR+/HER2− mBC combined with ET. There is paucity of clinical data of single-agent P in ET resistant pts. Pre-clinical data suggest P may partially reverse endocrine resistance, though this is yet to be tested in pts. Methods: This Phase II, open-label, multicenter study enrolled post-menopausal pts with HR+ HER2− mBC who progressed on 1 or 2 prior ETs. Pts were randomized to P (125 mg/d 3 w on/1 w off) alone or to continue their current ET (aromatase inhibitor or fulvestrant) in combination with P (same schedule as P arm). The primary endpoint was clinical benefit rate (CBR) [complete response (CR), partial response (PR) and stable disease (SD) for > 6 months (mo)]. Secondary endpoints were adverse events (AE) and additional measures of efficacy. A two-stage optimal design assessed treatment activity in each arm assuming activity as CB≥40% (α and β = 10%). Exploratory comparisons were planned for safety and efficacy endpoints. Results: 115 pts were enrolled (ITT population) 58 in the P arm and 57 in the P+ET arm. In both arms, 67% of pts had the study treatment as second line ET, 33% as third line, and about 1/3 of pts also received 1 prior chemotherapy for mBC. CBR was similar in both arms: 54% (95% CI 42 - 67%) with P+ET, and 60% (95% CI 48 -73%) with P alone. Median duration of CB was longer with P+ET (11.5 mo; 95% CI 8.6 – 17.8) than with P (6 mo; 95% CI 3.9 - 9.9) (HR 0.31, 95% CI 0.1 - 0.7, p-value 0.001, exploratory). Objective response rate (ORR; CR, PR) was 11% (95% CI 3 - 19%) and 7% (95% CI 0.4 -13%) with P+ET and P, respectively. PFS was 10.8 mo (95% CI 5.6 - 12.7) with P+ET and 6.5 mo (95% CI 5.4 - 8.5) with P alone (HR 0.69, 95% CI 0.4 - 1.1, p-value 0.13, exploratory). AEs were in line with previous data. Conclusions: Single agent P has clinical activity in ET pre-treated HR+/HER2– mBC pts. The observed increase in PFS and duration of CB with P+ET may suggest that P could reverse resistance to the prior line of ET. Translational studies are ongoing to explore potential biomarkers in this setting. Clinical trial information: NCT02549430.

In the past, treatment decisions regarding adjuvant chemotherapy in early breast cancer (EBC) were made solely based on clinicopathologic factors. However, with increased awareness of the importance of underlying tumor biology, we are now able to use genomic analyses to determine molecular breast cancer subtype and thus identify patients with tumors that are chemotherapy resistant and unlikely to benefit from the addition of chemotherapy. Although genomics has allowed some patients to avoid chemotherapy—specifically those with luminal A–like breast cancer—these assays do not indicate which regimen is most appropriate. For this, consideration must be given to the combination of underlying tumor biology, tumor stage, and patient characteristics, such as age and tolerability of side effects.

Summary Chemotherapy is the only evidence based adjuvant systemic treatment option in triple negative breast cancer (TNBC). Despite emerging results for targeted biological therapies for this subpopulation, lack of robust results does not currently support their use beyond the confines of a clinical trial. Conventional systemic chemotherapy remains the standard of care and is curative in a minority of patients. There is no defined standard chemotherapy and there is currently no robust, prospective, randomized data to advise different use of specific chemotherapy agents in TNBC as compared to non-TNBC. Data suggest high sensitivity to chemotherapy, however it is yet to be determined whether this increased sensitivity is agent/regimen specific or whether it reflects general chemosensitivity. This review will focus on systemic chemotherapy in early TNBC, particularly anthracyclines and platinums, and potential predictive tools to guide chemotherapy use.

Triple-negative breast tumours are defined as oestrogen receptor (ER)-negative, progesterone receptor (PgR)-negative and human epidermal growth factor receptor 2 (HER2)-negative. Beyond this common negative triplet, however, triple-negative breast cancers (TNBC) are not an homogeneous subgroup. There is substantial intra-subgroup diversity in tumour biology, prognosis and treatment sensitivity. Triple-negative breast cancer accounts for 15−20% of all breast cancer cases; however, it accounts for a disproportionate number of breast cancer relapses and deaths. Studies in early breast cancer have shown patients with TNBC to have a worse outcome compared with patients with non-TNBC [1]. Interestingly, neo-adjuvant studies highlight a subgroup of TNBC patients, approximately 20%, with highly chemosensitive disease and post-chemotherapy outcomes as favourable as their non-TNBC counterparts [2]. A key issue with TNBC is the accurate diagnosis of histological subtype and ER, PgR and HER2 status. ER, PgR and HER2 measurement has been associated with concerns regarding reliability and reproducibility, with remarkable reported rates of discordance. Furthermore, prior arbitrary thresholds for defining positivity for ER and PgR status, including 1%, 10% and 20%, have varied between laboratories and clinical trials. Clarity is offered by recent St Gallen and ASCO-CAP guidelines that define endocrine responsiveness as 1%. TNBC includes several morphological subtypes. Most are high-grade ductal carcinomas (not otherwise specified) but the triple-negative phenotype also occurs in medullary, apocrine and squamous cell carcinomas. Some subtypes, such as medullary carcinoma, are associated with a better prognosis and may not require aggressive chemotherapy. In contrast to endocrine-sensitive and HER2-positive breast cancers, which are defined by the presence of specific targets for matched targeted therapy, there is currently no targeted therapy for TNBC. There are promising new targeted biological therapies, such as PARP inhibitors; however, chemotherapy remains the mainstay of systemic therapy for TNBC. The biological diversity displayed within TNBC does not support such an empirical approach; however, a lack of robust predictive biomarkers prevents a more individualised approach to the choice of adjuvant therapy. Predictive markers could guide chemotherapy use by identification of the subset of TNBC patients with chemosensitive disease, or more specifically to predict sensitivity or resistance to individual chemotherapeutic agents. However, currently there are no clinical tools to recommend use, or omission, of specific chemotherapeutics. Conventional thirdgeneration combination chemotherapy is the standard treatment. Rates of pathological complete response to conventional neoadjuvant anthracycline/taxane-based chemotherapy are reported to be within the range of 13−45% [2]. Optimal adjuvant systemic therapy for TNBC is difficult to define because of a lack of robust prospective data in triple-negative restricted trial populations. Most clinical data derive from retrospective exploratory subgroup analyses from biologically unselected trial populations, and small underpowered studies. Most data reveal a highly chemosensitive TNBC subset. Lack of randomised phase III trials with a standard therapy control arm make it difficult to determine whether this increased sensitivity is agent-specific or whether it reflects general sensitivity to chemotherapy (Fig. 1). Triple-negative breast cancers are associated with a high proliferative rate, which may render them particularly sensitive to chemotherapy. Furthermore, a subset of TNBCs displays phenotypic overlap with BRCA1-mutation-related breast cancers. BRCA1 is critical in homologous recombination, a cellular process of double-strand DNA damage repair, and in the cell cycle arrest necessary for DNA repair.

Human epidermal growth factor receptor-2 (HER-2) and topoisomerase IIalpha (topo IIalpha) co-inhabit chromosome 17. In the search for predictive biomarkers to refine clinical prescription of cytotoxic agents, both HER-2 and topo IIalpha are under exploration for their potential role in identifying individuals with early breast cancer who may benefit from anthracycline therapy. Whilst recent meta-analyses support a predictive role for HER-2 amplification, it remains unclear whether HER-2 is the critical biomarker or whether it is a surrogate marker for topo IIalpha alteration, a known drug target of anthracyclines. The major limitation in considering HER-2 as a single marker is heterogeneity within the subgroups of HER-2 positive and HER-2 negative disease. For topo IIalpha, current data is inconclusive. Issues plaguing this field are technical variability in marker definition, complex regulation pathway of topo IIalpha and lack of prospective, adequately powered studies. With current evidence, neither HER-2 nor topo IIalpha gene status can be considered clinically valuable markers for anthracycline benefit. This paper will focus on issues relating to reliable detection and predictive analyses of HER-2 and topo IIalpha, and highlight potential developments in improving individualized approach to anthracycline use in early breast cancer patients.

In this issue of Journal of Clinical Oncology, Bartlett et al report the results of a retrospective study evaluating human epidermal growth factor receptor (HER) family membrane receptors and topoisomerase II(TOP2A) as markers predicting sensitivity to anthracycline-based therapy in early breast cancer patients. In the original phase III clinical trial, patients were randomly allocated to one of two study arms, either an all intravenous cyclophosphamide, methotrexate, and fluorouracil (CMF) regimen administered every 3 weeks or a sequential regimen consisting of full-dose, singleagent epirubicin followed by the same type of CMF regimen. Treatment duration was 24 weeks in both study arms. Archival primary tumor samples were collected and made available for a centralized biomarker analysis in 322 of 374 patients who participated in the clinical trial. Protein expression was evaluated by immunohistochemistry. HER2 and TOP2A gene aberrations were investigated by fluorescent in situ hybridization. The study has, in our opinion, two interesting features. It investigates, for the first time to our knowledge, the interaction between the HER family membrane receptors and anthracycline activity in the context of a randomized trial for early-stage breast cancer patients. Furthermore, this is the first study (of five) suggesting that TOP2A gene amplification is not a helpful marker in predicting sensitivity to anthracyclines. Interestingly enough, the authors report that, in contradiction with the a priori study hypothesis, patients with normal HER1, HER2, and HER3 levels derive the largest benefit from the anthracyclinebased therapy. Conversely and surprisingly, no superiority for anthracyclines over CMF is seen in the cohort of patients who have tumors with overexpression of at least one of the three HER family membrane receptors. On the basis of previously reported literature, the cohort of patients with HER1 to HER3 normal tumors is expected to be characterized by a high level of expression of hormone receptors (estrogen and progesterone receptors) and low proliferation rates. Why should an anthracycline-based therapy be more active than CMF in this specific subgroup of patients with endocrine-sensitive tumors? A possible explanation is that, in this cohort of patients, the level of ovarian suppression achieved with a full-dose, anthracycline-based regimen is expected to be more pronounced than with all intravenous CMF administered every 3 weeks. Supporting this explanation is the fact that almost 55% of the study population had a preor perimenopausal status. The present article and the clinical trial report do not provide details on the percentage of preand perimenopausal women with endocrine-sensitive tumors who actually received adjuvant tamoxifen for 5 years sequentially after adjuvant chemotherapy. The clinical trial report specifies that adjuvant hormonal treatment was not restricted and that 46% of those patients who were candidates for adjuvant tamoxifen were to receive this treatment concomitantly with chemotherapy. Accordingly, we speculate that a relevant proportion of the HER1 to HER3 normal population was preor perimenopausal, had endocrine-sensitive tumors, and did not receive adjuvant tamoxifen for 5 years sequentially after chemotherapy. Under these conditions, a full-dose, anthracycline-based therapy might produce a clinical benefit over a nonintensive CMF regimen that is independent of a cytotoxic mechanism and that is mediated by endocrine effects (ie, interference with ovarian function). Arguing against this hypothesis is the fact that rates of chemotherapy-related amenorrhea reported in the clinical trial report are 73% and 74% for the anthracycline-based and CMF regimens, respectively. It is meaningful to emphasize that rates of chemotherapy-related amenorrhea can vary over time and that differences in amenorrhea rates between two treatment groups can emerge and become clinically relevant several months after the end of adjuvant chemotherapy. Unfortunately, the limited study sample size (146 patients with HER1 to HER3 normal tumors) makes any reliable conclusion impossible in subgroup analysis according to hormone receptors and menopausal status. Hence, the conclusion that the suggested superiority of anthracyclines over CMF in the cohort of HER1 to HER3 normal tumors is mediated by endocrine effects has to remain hypothetical. Differently from the HER1 to HER3 normal cohort, the group of tumors carrying overexpression of at least one of the three markers is biologically heterogeneous, potentially represented by HER2-positive, triple-negative, and moderately hormone-sensitive tumors. Accordingly, it is difficult to formulate any hypothesis to explain the observed results in this specific cohort (ie, lack of superiority of anthracyclines over CMF). It is relevant to emphasize that, in the cohort of HER1to HER3-positive tumors, the hazard ratio for the comparison of anthracyclines with CMF has a wide 95% CI. This makes the results unstable and, at the same time, highlights two different problems encountered in the vast majority of studies looking at molecular predictive markers—lack of adequate statistical power and biologic heterogeneity of the study population. As alluded to earlier, this is the first time that a retrospective analysis from a randomized trial suggests that TOP2A gene amplification JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 26 NUMBER 31 NOVEMBER 1 2008