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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.

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.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy (ET) are now a backbone of treatment for hormone receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. CDK4/6i plus ET is more effective than ET alone in this setting; however, the risk of grade 3-4 adverse events also increases. Approved agents in this class have similar efficacies, but important differences due to their structural and pharmacological properties. We review biomarkers and discuss determinants to inform a rational approach to therapy choice when selecting the most appropriate ET and CDK4/6i partners. We also identify subgroups that may benefit from specific ET-CDK4/6i combinations and discuss strategies to overcome resistance. This personalized approach aims to minimize treatment-related toxicities that may affect patient QoL and compliance, and ultimately therapy efficacy.

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

// Martina Bonechi 1, * , Francesca Galardi 1, * , Chiara Biagioni 2 , Francesca De Luca 1 , Mattias Bergqvist 3 , Magnus Neumuller 3 , Cristina Guarducci 1 , Giulia Boccalini 1 , Stefano Gabellini 1 , Ilenia Migliaccio 1 , Angelo Di Leo 1, 4 , Marta Pestrin 1, 4, ** and Luca Malorni 1, 4, ** 1 “Sandro Pitigliani” Translational Research Unit, Hospital of Prato, Prato, Italy 2 Bioinformatics Unit, Hospital of Prato, Prato, Italy 3 Biovica International, Uppsala Science Park, Uppsala, Sweden 4 “Sandro Pitigliani” Medical Oncology Department, Hospital of Prato, Prato, Italy * Co-first authors ** Co-last authors Correspondence to: Luca Malorni, email: luca.malorni@uslcentro.toscana.it Keywords: metastatic breast cancer; endocrine therapy; thymidine kinase-1; liquid biopsy; circulating biomarkers Received: October 15, 2017 Accepted: February 28, 2018 Published: March 27, 2018 ABSTRACT The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum TM assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1 / PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients.

Circulating Tumor Cells (CTCs) represent a “liquid biopsy” of the tumor potentially allowing real-time monitoring of cancer biology and therapies in individual patients. The purpose of the study was to explore the applicability of a protocol for the molecular characterization of single CTCs by Next Generation Sequencing (NGS) in order to investigate cell heterogeneity and provide a tool for a personalized medicine approach. CTCs were enriched and enumerated by CellSearch in blood from four metastatic breast cancer patients and singularly isolated by DEPArray. Upon whole genome amplification 3–5 single CTCs per patient were analyzed by NGS for 50 cancer-related genes. We found 51 sequence variants in 25 genes. We observed inter- and intra-patient heterogeneity in the mutational status of CTCs. The highest number of somatic deleterious mutations was found in the gene TP53, whose mutation is associated with adverse prognosis in breast cancer. The discordance between the mutational status of the primary tumor and CTCs observed in 3 patients suggests that, in advanced stages of cancer, CTC characteristics are more closely linked to the dynamic modifications of the disease status. In one patient the mutational profiles of CTCs before and during treatment shared only few sequence variants. This study supports the applicability of a non-invasive approach based on the liquid biopsy in metastatic breast cancer patients which, in perspective, should allow investigating the clonal evolution of the tumor for the development of new therapeutic strategies in precision medicine.

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.

Until recently, the mainstay of treatment in the majority of hormone receptor (HR)-positive, human epidermal growth factor 2 receptor (HER2)-negative advanced breast cancer (ABC) has consisted of single-agent endocrine therapy (ET). However, as understanding of endocrine resistance has grown, newer targeted agents have come to the fore. Inhibition of cyclin-dependent kinase complexes 4 and 6 (CDK4/6) combined with ET has shown significant activity in HR+ HER2− ABC, with impressive results in terms of progression-free survival (PFS) when compared with ET alone. This review summarizes the seminal findings pertaining to CDK4/6 inhibition in this population, specifically focusing on abemaciclib, contrasted with palbociclib and ribociclib. Potential directions for future studies are discussed, as a way of addressing outstanding issues such as establishing optimal treatment sequencing and agent combinations, appropriate patient selection to derive maximal benefits, predictive biomarkers and the employment of CDK4/6 inhibition beyond the ABC setting.

The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch® and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic “hot spot” regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients.

Circulating Tumor Cells (CTCs) represent a "liquid biopsy of the tumor" which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging. The CellSearch(®) system provides the enumeration of CTCs with prognostic significance in patients with metastatic breast cancer (mBC), but it does not allow their molecular characterization, which might be useful to identify therapeutically relevant targets for individualized treatment. Combining the CellSearch(®) and DEPArray™ technologies allows the recovery of single CTCs as a pure sample for molecular analysis. The purpose of the study was to investigate the heterogeneity of PIK3CA mutational status within single CTCs isolated from individual mBC patients. CTCs were enriched and enumerated by CellSearch(®) in blood samples collected from 39 mBC patients. In 20 out of 39 patients enriched samples with ≥5 CTCs were sorted using DEParray™ to isolate single CTCs or pools of CTCs to be submitted to Whole Genome Amplification (WGA) before sequencing analysis. In 18 out of 20 patients, it was possible to perform PIK3CA sequencing on exons 9 and 20. Twelve subjects were wild type (wt) for the PIK3CA gene. PIK3CA status could also be assessed in pools of CTCs in seven of these patients, with consistent wt status found. Six patients (33%) had a PIK3CA mutation identified. In 2 of the six patients, molecular heterogeneity was detected when mutational analysis was performed on more than one single CTC, including one patient with loss of heterozygosity on both single and pooled CTCs, and one patient with three different PIK3CA variants on single CTCs but PIK3CA wt status on pooled CTC samples. In six out of the 18 cases PIK3CA status was also evaluable on a primary tumor sample. In one of the six cases a discordance in PIK3CA status between the primary (wild-type) and the matched CTC (exon 20 mutation) was observed. This study demonstrates the feasibility of a non-invasive approach based on the liquid biopsy in mBC patients. Moreover, our data suggest the importance of characterizing CTCs at the single cell level in order to investigate the molecular heterogeneity within cells from the same patient.

Circulating tumor cell (CTC) count has prognostic significance in metastatic breast cancer, but the predictive utility of CTCs is uncertain. Molecular studies on CTCs have often been limited by a low number of CTCs isolated from a high background of leukocytes. Improved enrichment techniques are now allowing molecular characterisation of single CTCs, whereby molecular markers on single CTCs may provide a real-time assessment of tumor biomarker status from a blood test or "liquid biopsy", potentially negating the need for a more invasive tissue biopsy. The predictive ability of CTC biomarker analysis has predominantly been assessed in relation to HER2, with variable and inconclusive results. Limited data exist for other biomarkers, such as the estrogen receptor. In addition to the need to define and validate the most accurate and reproducible method for CTC molecular analysis, the clinical relevance of biomarkers, including gain of HER2 on CTC after HER2 negative primary breast cancer, remains uncertain. This review summarises the currently available data relating to biomarker evaluation on CTCs and its role in directing management in metastatic breast cancer, discusses limitations, and outlines measures that may enable future development of this approach.

Background: Palbociclib (PD-0332991) is an orally active, potent and highly selective inhibitor of CDK4/6 that prohibits progression of the cell cycle from G1 into S phase and has shown activity in ER positive breast cancer in vivo and in vitro. Recently, in phase 2 settings, palbocilcib extended PFS in combination with letrozole as first-line hormonal treatment for advanced breast cancer (ABC) (Finn R.S. et al. Cancer Res., Dec 2012; 72: S1-6) and showed single agent activity in patients with ER positive ABC relapsed to several lines of therapy (DeMichele A. et al., ASCO 2013). Given pre-clinical evidence on the relevance of Cyclin D-CDK4/6-E2F signalling in the development of acquired resistance to hormonal therapy, CDK4-6 inhibition might be a strategy to overcome resistance in tumors who have progressed after hormonal therapy for metastatic disease. Study design: This is an investigator initiated, open-label, multicenter, randomized, Phase 2 trial for patients with ER+ HER2 negative ABC who have experienced progression on first or second line hormonal therapy. Other eligibility criteria include: ≤1 previous line of chemotherapy for ABC; measurable disease; and available archival tumor tissue for translational studies. Consenting patients are randomized (1:1) to receive, until progression of disease, unacceptable toxicity or consent withdrawal, either ARM A) palbociclib 125 mg/d orally for 3 weeks followed by 1 week off treatment or ARM B) palbociclib in combination with the hormonal therapy to which the patient has experienced progression in the previous line of treatment (anastrozole 1 mg/d, letrozole 2.5 mg/d, exemestane 25 mg/d, fulvestrant 500mg/4 weeks). Patients are stratified according to: number or prior lines of hormonal therapy (1 vs. 2); disease site (visceral vs. non visceral); duration of prior line of hormonal therapy (>6 vs. ≤6 months); treating center. The primary endpoint is clinical benefit (CB) (i.e. complete response, partial response, stable disease≥ 24 weeks), based on local assessment of response using RECIST 1.1 criteria. Key secondary endpoints are: objective response rate, duration of response, time to progression, progression-free survival, overall survival and assessment of exploratory tissue markers of response. Statistical methods: A two-stage optimal phase 2 design is used to assess the activity of each of the two treatment regimens. Assuming P0≤20% (inactivity), P1≥ 40% (activity) and α = 10%, a sample size of 50 evaluable patients per treatment arm is required for a statistical power equal to 90%. In case of inactivity, enrolment could be stopped in each arm after 25 evaluable patients. This study does not provide adequate power for a two arm comparison. Target accrual: the study is actively recruiting in 3 Italian centers and is expected to involve additional 7 centers in Italy by the end of 2013. The first patient was enrolled in October 2012 and, as per June 2013, a total of 9 patients have been enrolled, 8 remain on treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-6-01.

Background Although dose-dense chemotherapy may reduce breast cancer recurrence and death, phase II studies show that dose-dense docetaxel is poorly tolerated following administration of dose-dense anthracycline-based chemotherapy mainly because of cutaneous toxicity. Material and methods This pilot study was designed to explore feasibility and safety of dose-dense docetaxel after conventional anthracycline-based therapy. Treatment consisted of sequential administration of 4 cycles of 3-weekly epirubicin (90 mg/m 2 ) plus cyclophosphamide (600 mg/m 2 ), followed by 4 cycles of bi-weekly docetaxel with pelfilgrastim on day 2 of each docetaxel cycle. Two docetaxel dose levels were planned: 75 mg/m 2 (D75) and 100 mg/m 2 (D100). Patients could only be assigned to the higher docetaxel dose if no early treatment discontinuations due to toxicity were seen, and a median relative dose intensity of docetaxel >90% among the first 5 evaluable patients was achieved. Results Fifty three patients received 4 cycles of epirubicin/cyclophosphamide (EC). Six patients withdrew from study before commencing docetaxel: four for toxicity, and two who declined further study participation. Eight patients, 2 in the first dose level and 6 in the second dose level, stopped treatment for toxicity after the first cycle of docetaxel and before densification. Therefore these events were not considered early treatment discontinuations. No patients required dose interruption after the second docetaxel administration. Overall 5 patients in the first dose level and 34 patients in the second dose level received 4 cycles of accelerated (dose-dense) docetaxel. No grade 3 or grade 4 toxicities occurred at the first dose level. No grade 4 toxicities occurred at the second dose level, while grade 3 toxicities occurring in >2 patients were myalgia and bone pain (5 and 8 patients respectively, 13% and 20%) and skin-nail toxicity (7 patients, 21%). No dose-reductions or significant treatment delays were required, translating to median relative dose intensity of 100% for docetaxel 75 mg/m 2 , and 99% for 100 mg/m 2 . Conclusions Administration of docetaxel 100 mg/m 2 bi-weekly after conventional EC is feasible in selected early breast cancer patients. Lack of prior exposure to dose-dense anthracycline, as well as the use of stringent criteria implemented in the treatment protocol, might explain the improved safety profile and high treatment compliance observed in this study.

The prognosis of pT1N0M0/stage I breast cancer has generally been considered so favourable that these patients are not routinely offered adjuvant systemic therapy. However, biological heterogeneity within pT1N0M0 dictates diverse outcomes within the subgroup. HER2 gene amplification or protein overexpression is uncommon in pT1N0M0 disease, but, when present, is clearly associated with a higher risk of recurrence. The role of anti-HER2 therapy in these patients is controversial. Few women with node-negative, small tumours were included in the adjuvant trastuzumab trials. There are no robust data on trastuzumab in this patient subset, although subgroup analyses suggest that proportional benefits are independent of T and N. With current guidelines and scheduling, committing to adjuvant trastuzumab involves concurrent chemotherapy, 1 year of treatment and potential cardiotoxicity. A further challenge with anti-HER2 therapy is the potential benefit in patients with demonstrable HER2 positivity within a predominantly HER2-negative tumour. The decision for therapy requires a yes/no answer, but HER2 status derives from a continuum of gene copy number and protein expression. The diagnostic threshold is made more complex by heterogeneity of the HER2 status within a tumour. This review focuses on available data for HER2-positive pT1N0M0 disease and explores the significance of intratumoural HER2 heterogeneity.

Adjuvant systemic therapy targets minimal residual disease. Our current clinical approach in the adjuvant setting is to presume, rather than confirm, the presence of minimal residual disease. Based on assessment of the primary tumor, we estimate an individual’s recurrence risk. Subsequent treatment decisions are based on characteristics of the primary tumor, with the presumption of consistent biology and treatment sensitivity between micrometastases and the primary lesion. An alternative approach is to identify micrometastatic disease. Detection of disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) from peripheral blood collection may offer quantification and biocharacterization of residual disease. This paper will review the prognostic and predictive potential of micrometastatic disease in early breast cancer.

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.

Despite significant improvements in the treatment and outcomes of early-stage breast cancer, the quest continues to find biological and molecular markers that would enable earlier diagnosis or better prediction of treatment efficacy and toxicity. Metabolomics--the latest and one of the most exciting of the 'omic' sciences--has shown early promise as a non-invasive diagnostic aid in ovarian cancer, and may allow the detection of subtle metabolic changes that could have diagnostic, prognostic or predictive value in breast cancer. Routine monitoring of circulating tumour cells (CTCs) has also been advocated as a novel means of detecting breast cancer progression earlier, and identifying alterations in tumour cells that might signal the need for therapy changes. Ongoing studies should help to answer important questions relating to the use of metabolomics and CTC evaluation as new strategies to monitor cancer progression and identify markers of chemotherapy activity and toxicity.

BackgroundAnalysis of circulating tumor cells (CTCs) provides real-time measures of cancer sub-populations with potential for CTC-directed therapeutics. We examined whether lapatinib which binds both HER2 and EGFR could induce depletion of the EGFR-positive pool of CTCs, which may in turn lead to clinical benefits.Patients and methodsPatients with metastatic breast cancer and HER2 non-amplified primary tumors with EGFR-positive CTCs were recruited and lapatinib 1500 mg daily was administered, in a standard two step phase 2 trial.ResultsThere were no responses leading to termination at the first analysis with 16 patients recruited out of 43 screened. In 6 out of 14 (43%) individuals eligible for the efficacy analysis, a decrease in CTCs was observed with most of these having a greater decrease in their EGFR-positive CTC pool.ConclusionsThis is one of the first studies of CTC-directed therapeutics and suggests that lapatinib monotherapy is not having any demonstrable clinical effects by reducing the EGFR-positive pool of CTCs in HER2 non-amplified primary tumors. Our attempt to expand the pool of patients eligible for a targeted therapy was unsuccessful; the role of clonal populations in cancer biology and therapeutic strategies to control them will require extensive evaluation in years to come.Trial registrationClinical trials.gov NCT00820924.

Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; Biocharacterization of circulating tumor cells (CTCs) in the peripheral blood of advanced breast cancer (ABC) patients may represent a real-time tumor biopsy. We assessed HER2 status on CTCs from blood samples of ABC patients. CTCs were separated and stained using the CellSearch System. HER2 status was assessed by immunofluorescence and, when technically feasible, by fluorescence in situ hybridization. Blood samples were obtained from 66 ABC patients. Forty patients had a positive CTC sample (61%) and of these, 15 (37%) had HER2 + CTCs. We found non-concordant results in 32% of cases: 29% (8/28) of HER2-negative primary tumors had HER2-positive CTCs and 42% (5/12) of HER2-positive primary tumors had HER2-negative CTCs ( = 0.278). Our study suggests that a subset of patients with HER2-negative primary tumors develops HER2-positive CTCs during disease progression.

The urokinase plasminogen activator (uPA) system includes uPA and plasminogen activator inhibitor types 1 (PAI-1) and 2 that mainly act by regulating extracellular matrix degradation, and it is involved in tumor progression. The –675 4G/5G polymorphism of the PAI-1 gene regulates PAI-1 activity in serum. We aimed at studying the –675 4G/5G polymorphism of the PAI-1 gene and uPA, PAI-1, and cyclooxygenase-2 (COX-2) immunohistochemical expression in a series of breast cancer cases. Homozygosity for the 4G allele of the PAI-1 gene was associated with node-positive breast cancer (P = .02). We showed a direct correlation between uPA and estrogen receptor expression (P = .03); negative uPA expression was associated with negative hormonal expression, high tumor grade, and high proliferation index (P < .05). A direct correlation was seen between uPA and PAI-1, uPA and COX-2, and PAI-1 and COX-2 expression (P < .05). Interaction between uPA and COX-2 systems in breast cancer deserves further study.

Background: Little is known about the evolution of genetic aberrations during metastatic cancer progression and in response to systemic treatment. Obtaining repeated tissue biopsies is often impractical. On the other hand, it has been shown that circulating tumor cells (CTCs) can be easily followed during disease course and genetic characterization at the single cell level is possible with high reliability [1]. Methods: Individual CTCs of a ER+ and HER2- de novo metastatic breast cancer patient treated with weekly paclitaxel/gemcitabine as first line therapy, were collected at three different time points (before start, after one and two cycles of treatment). Whole peripheral blood was enriched using the CellSearch® system and CTCs were sorted by DEPArray™ device. The whole genome of single CTCs was amplified with Ampli1™ WGA kit and WGA quality was assessed by Ampli1™ QC Kit. Genome wide single cell copy number variation (CNV) profile was evaluated with Agilent SurePrint 180k array comparative genomic hybridization (aCGH). Results: CTCs count at CellSearch was 22, 75 and 15 at three time points respectively. A total of 25 single CTCs were collected and 23 (92%) showed high Genome Integrity Index (GII) as measured by Ampli1™ QC kit (GII = 3 or 4). For each time point multiple CTCs (3, 6 and 3 respectively) were selected for single cell aCGH analysis. The genomic profile was strikingly similar (1q gain, 12p, 13p, 16q and 17p losses) across individual cells of the same blood sample and throughout different time points evaluated. After 3 cycles of therapy a disease progression was documented by CAT-scan. Discussion: The observed high GII, low genetic heterogeneity and stable genome across different time points suggests the presence of an aggressive clone resistant to the treatment and cancer-associated genes analysis for sequence variants by NGS targeted sequencing is ongoing. Further patients with longitudinal follow-up will be enrolled in order to evaluate if the heterogeneity between aCGH profile at a given time point and longitudinal evolution of aCGH profiles can be associated with early treatment response. Results will be presented at the conference. [1] Polzer B, Medoro G, et al, EMBO Mol Med. 2014 Oct 30;6(11):1371-86. Citation Format: Valeria Sero, Francesca De Luca, Anna Doffini, Francesca Galardi, Marta Pestrin, Zbignew T. Czyz, Genny Buson, Giulia Bregola, Chiara Bolognesi, Francesca Fontana, Gianni Medoro, Bernhard Polzer, Angelo Di Leo, Christoph A. Klein, Nicolo Manaresi. Longitudinal genetic characterization of circulating tumor cells in metastatic breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 371. doi:10.1158/1538-7445.AM2015-371

Background The homeobox gene HEX is expressed in several cell types during different phases of animal development. It encodes for a protein localized in both the nucleus and the cytoplasm. During early mouse development, HEX is expressed in the primitive endoderm of blastocyst. Later, HEX is expressed in developing thyroid, liver, lung, as well as in haematopoietic progenitors and endothelial cells. Absence of nuclear expression has been observed during neoplastic transformation of the thyroid follicular cells. Aim of the present study was to evaluate the localization and the function of the protein HEX in normal and tumoral breast tissues and in breast cancer cell lines. Methods HEX expression and nuclear localization were investigated by immunohistochemistry in normal and cancerous breast tissue, as well as in breast cancer cell lines. HEX mRNA levels were evaluated by real-time PCR. Effects of HEX expression on Sodium Iodide Symporter (NIS) gene promoter activity was investigated by HeLa cell transfection. Results In normal breast HEX was detected both in the nucleus and in the cytoplasm. In both ductal and lobular breast carcinomas, a great reduction of nuclear HEX was observed. In several cells from normal breast tissue as well as in MCF-7 and T47D cell line, HEX was observed in the nucleolus. MCF-7 treatment with all-trans retinoic acid enhanced HEX expression and induced a diffuse nuclear localization. Enhanced HEX expression and diffuse nuclear localization were also obtained when MCF-7 cells were treated with inhibitors of histone deacetylases such as sodium butyrate and trichostatin A. With respect to normal non-lactating breast, the amount of nuclear HEX was greatly increased in lactating tissue. Transfection experiments demonstrated that HEX is able to up-regulate the activity of NIS promoter. Conclusion Our data indicate that localization of HEX is regulated in epithelial breast cells. Since modification of localization occurs during lactation and tumorigenesis, we suggest that HEX may play a role in differentiation of the epithelial breast cell.

Tumors have usually been classified by their morphologic appearances. Unfortunately, these current classification schemes have serious drawbacks. They are not able to stratify similar histopathological appearances that will follow significantly different clinical courses or respond differently to chemotherapy. The information that a specific molecular profile correlates with important clinical endpoints should permit physicians to take treatment decisions based on the molecular characteristics of each tumor. Lessons from the metastatic setting have been translated to the adjuvant setting, and several strategies have been evaluated in clinical trials. The expression of estrogen receptors (ER) in breast cancer enables physicians to make treatment decisions related to the use of hormonal manipulations. In this context, the challenge is to define a suitable subgroup of patients who will benefit from the addition of chemotherapy. Otherwise, the lack of ER expression predicts no benefit from hormonotherapy. In this setting chemotherapy plays a central role. The selection of the most appropriate regimen based on HER-2 status remains an uncertain strategy. However, the expression of the oncoprotein HER-2 has been linked to the probability of response to the target-designed monoclonal antibody trastuzumab. The role of trastuzumab in the adjuvant setting is supported by the early results of three large clinical trials presented at the American Society Clinical Oncology meeting in 2005. These trials have shown a striking impact of trastuzumab on the main endpoints such as disease-free survival and overall survival. In this context, the integration of trastuzumab with taxane and anthracycline-based-chemotherapy seems to be the appropriate choice. This review will combine data from breast cancer biology with clinical evidence coming from large phase III trials in the attempt to propose a molecular targeted approach to the adjuvant treatment strategy of early breast cancer patients.

Introduction Presence of circulating tumor cells (CTCs) in peripheral blood of patients with metastatic breast cancer (MBC) represents an independent prognostic factor. Discrepancies in HER2 status between primary breast tumors and CTCs have been reported. Indeed, 5-38% of patients with HER2 negative (HER2-) primary breast cancers show HER2 overexpression in corresponding CTCs at time of metastatic disease. However, the biological explanation of such discrepancies is still matter of debate. Sparse reports indicate that 5-30% of breast cancers show HER2 heterogeneity. We hypothesize that heterogeneity of HER2 expression within HER2- primary tumors might explain the presence of HER2 positive (HER2+) CTCs. Methods We analyzed 14 CTC positive (≥ 2 CTCs/7.5ml blood) MBC patients whose primary tumors were HER2- according to the adjuvant trastuzumab trials criteria. CTCs were evaluated for HER2 expression by immunofluorescence (CellSearch™ System, Veridex LLC) and defined positive if ≥ 50% cells stained for HER2. According to these criteria, seven of the patients had discordant HER2+ CTCs (cases), while seven had concordant HER2- CTCs (controls). For both cases and controls, we evaluated HER2 status by immunohistochemistry on additional tumor-bearing blocks, either from the primary tumor or from synchronous metastatic axillary lymphnodes (average of 3 blocks/patient). HER2 heterogeneity was defined as presence of incomplete or complete membrane staining in Results Five of the seven (71%) discordant cases showed HER2 heterogeneity. Heterogeneity was present either in the additional primary tumor blocks (40%) or in the metastatic axillary lymphnodes (40%) or in both (20%). Intriguingly, none of the concordant cases (controls) showed HER2 heterogeneity. Conclusion Here we show that areas of HER2 expression in HER2- tumors are associated with the presence of HER2+ CTCs at time of metastatic disease, suggesting that HER2+ clones might be selected during cancer progression. Trials investigating whether patients with HER2 heterogeneous tumors might benefit from HER2 targeted therapies are warranted. Disclosure A. Di Leo: Dr. Di Leo received research grants from GSK and honoraria as a speaker at satellite symposia or as a consultant from Roche and GSK. All other authors have declared no conflicts of interest.

Lapatinib is a dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR/ErbB1) and human epidermal growth factor receptor 2 (HER2/ErbB2). EGFR and HER2 overexpression is associated with aggressive breast cancer with a high risk of disease relapse and death. Although lapatinib targets both EGFR and HER2, its effects on HER2 appear to be more critical. The role of lapatinib in the first-line setting remains unclear. A phase II first-line monotherapy lapatinib trial in HER2-therapy-naïve metastatic breast cancer (MBC) patients confirms efficacy in HER2-positive tumors. Retrospective analysis of a phase III, first-line MBC study confirmed incremental benefit from lapatinib and paclitaxel over paclitaxel alone in HER2-positive disease. A prospective phase III study confirms superiority of letrozole and lapatinib over letrozole alone in HER2-positive MBC. Further investigation is required to define the potential first-line role for lapatinib. Particular strengths appear to be its manageable toxicity profile, lack of cross resistance with trastuzumab, activity in central nervous system disease, and synergy in combination with other anticancer therapy. Current limitations are lack of dosing recommendations from early trials, lack of predictive biomarkers beyond HER2 status, and lack of large prospective phase III trials for HER2-positive disease in the first-line setting. The role of lapatinib in HER2-negative disease is unclear.