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

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

Background: Colorectal cancer (CRC) is the fourth most commonly diagnosed and third most deadly cancer worldwide. Surgery is the main treatment option for early disease; however, a relevant proportion of CRC patients relapse. Here, variations among preoperative and postoperative serum metabolomic fingerprint of CRC patients were studied, and possible associations between metabolic variations and cancer relapse were explored. Methods: A total of 41 patients with stage I-III CRC, planned for radical resection, were enrolled. Serum samples, collected preoperatively (t0) and 4–6 weeks after surgery before the start of any treatment (t1), were analyzed via NMR spectroscopy. NMR data were analyzed using multivariate and univariate statistical approaches. Results: Serum metabolomic fingerprints show differential clustering between t0 and t1 (82–85% accuracy). Pyruvate, HDL-related parameters, acetone, and 3-hydroxybutyrate appear to be the major players in this discrimination. Eight out of the 41 CRC patients enrolled developed cancer relapse. Postoperative, relapsed patients show an increase of pyruvate and HDL-related parameters, and a decrease of Apo-A1 Apo-B100 ratio and VLDL-related parameters. Conclusions: Surgery significantly alters the metabolomic fingerprint of CRC patients. Some metabolic changes seem to be associated with the development of cancer relapse. These data, if validated in a larger cohort, open new possibilities for risk stratification in patients with early-stage CRC.

Simple Summary Around 30–40% of patients with early stage colorectal cancer (eCRC) experience relapse after surgery. Current recommendations for adjuvant therapy are based on suboptimal risk-stratification tools. In elderly patients, risk of relapse assessment is particularly important to ultimately avoid unnecessary chemotherapy-related toxicity in this frailer population. Serum metabolomics via NMR spectroscopy may improve risk stratification by identifying patients with residual micrometastases after surgery and thus at higher risk of relapse. We evaluated the serum metabolomic fingerprints of 94 elderly patients with eCRC (65 relapse free and 29 relapsed), and of 75 elderly patients with metastatic disease. Metabolomics efficiently discriminated patients with relapse-free eCRC from those with metastatic disease, correctly predicting relapse in 69% of relapsed eCRC patients. The metabolomic score was strongly and independently associated with prognosis. Our data suggest metabolomics as a valid addition to standard tools to refine risk stratification for eCRC and warrant further investigation. Abstract Adjuvant treatment for patients with early stage colorectal cancer (eCRC) is currently based on suboptimal risk stratification, especially for elderly patients. Metabolomics may improve the identification of patients with residual micrometastases after surgery. In this retrospective study, we hypothesized that metabolomic fingerprinting could improve risk stratification in patients with eCRC. Serum samples obtained after surgery from 94 elderly patients with eCRC (65 relapse free and 29 relapsed, after 5-years median follow up), and from 75 elderly patients with metastatic colorectal cancer (mCRC) obtained before a new line of chemotherapy, were retrospectively analyzed via proton nuclear magnetic resonance spectroscopy. The prognostic role of metabolomics in patients with eCRC was assessed using Kaplan–Meier curves. PCA-CA-kNN could discriminate the metabolomic fingerprint of patients with relapse-free eCRC and mCRC (70.0% accuracy using NOESY spectra). This model was used to classify the samples of patients with relapsed eCRC: 69% of eCRC patients with relapse were predicted as metastatic. The metabolomic classification was strongly associated with prognosis (p-value 0.0005, HR 3.64), independently of tumor stage. In conclusion, metabolomics could be an innovative tool to refine risk stratification in elderly patients with eCRC. Based on these results, a prospective trial aimed at improving risk stratification by metabolomic fingerprinting (LIBIMET) is ongoing.

Introduction: TK1 plays a crucial role in DNA synthesis and is a well-established marker of cell proliferation. We and others have previously described the potential role of TK1 activity (TKa) as predictive biomarker of response to endocrine therapy in HR+/HER2 negative metastatic breast cancer patients. TK1 synthesis is regulated by the E2F pathway, the target pathway of CDK4/6 inhibitors, and TKa has recently been reported as a potential circulating pharmacodynamic marker of CDK4/6 inhibition in breast cancer. However, modulations of TK1 levels and activity during palbociclib treatment and in the development of treatment resistance are unknown. Here, we report how TK1 expression and TKa are modulated in response to palbociclib in a panel of HR+ breast cancer cell lines: both palbociclib-sensitive (PDS) and with acquired resistance to (PDR). Material and methods: We used a panel of 7 PDR HR+ breast cancer models previously developed in our lab via chronic exposure of parental cells (MCF7, T47D, ZR75-1, BT474, MDAMB361 and two MCF7 endocrine resistant derivatives) to escalating doses of palbociclib, from a Starting Treatment Concentration (STC) of 50 nM or 350 nM according to the cell line, up to 1 μM. We analyzed gene expression profiles of PDS cells treated with drug vehicle (DMSO) as a control or palbociclib at STC for 3 days, and PDR cells grown with palbociclib 1 μM. Cell proliferation was assessed by methylene blue assay in MCF7 and BT474 PDS and PDR treated for 3, 6 and 9 days with DMSO, palbociclib STC and 1 μM. In parallel, TKa was measured in cell lysates at 3 days of treatment using the DiviTumTM assay (Biovica, Sweden). Results: Among E2F target genes, gene expression data demonstrated that TK1 was one of the most differentially expressed genes between PDR and PDS treated cells. In PDS cells compared to control, treatment with palbociclib resulted in reduced TK1 expression, with the HER2 positive models (BT474 and MDAMB361) showing the highest reduction. In PDR cells, TK1 expression was higher, but remained slightly inhibited compared to untreated PDS cells. TKa was significantly reduced in PDS cells treated with palbociclib for 3 days compared to vehicle (p Conclusions: TK1 expression and activity are regulated by palbociclib in HR+ breast cancer cell lines, particularly in HER2 positive models. Ongoing studies of TKa in patients treated with palbociclib will assess the role of TKa as a circulating biomarker for predicting and monitoring response to CDK4/6 inhibitors. Citation Format: Bonechi M, Migliaccio I, Benelli M, Romagnoli D, Bergqvist M, Mattsson K, Boccalini G, Capaccioli G, De Luca F, Galardi F, Biagioni C, Risi E, McCartney A, Rossi L, Osborne CK, Schiff R, De Angelis C, Guarducci C, Di Leo A, Malorni L. Effects of palbociclib on thymidine kinase-1 (TK1) in hormone receptor positive (HR+) breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-02.

Background 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.

Precision oncology is an emerging approach in cancer care. It aims at selecting the optimal therapy for the right patient by considering each patient’s unique disease and individual health status. In the last years, it has become evident that breast cancer is an extremely heterogeneous disease, and therefore, patients need to be appropriately stratified to maximize survival and quality of life. Gene-expression tools have already positively assisted clinical decision making by estimating the risk of recurrence and the potential benefit from adjuvant chemotherapy. However, these approaches need refinement to further reduce the proportion of patients potentially exposed to unnecessary chemotherapy. Nuclear magnetic resonance (NMR) metabolomics has demonstrated to be an optimal approach for cancer research and has provided significant results in BC, in particular for prognostic and stratification purposes. In this review, we give an update on the status of NMR-based metabolomic studies for the biochemical characterization and stratification of breast cancer patients using different biospecimens (breast tissue, blood serum/plasma, and urine).

Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.

Background: Chemotherapy added to anti-HER2 agents (H) is the treatment of choice in patients with HER2+ early breast cancer. However, HER2+ tumours are clinically and biologically heterogeneous, and treatment response varies significantly by hormone receptor (HR) status and molecular subtype. Predictive biomarkers are needed in this context. This study assessed whether an RB-1 loss of function gene signature (RBsig) is predictive of response to neoadjuvant chemotherapy in combination with trastuzumab, lapatinib or both, within the NeoALTTO trial. Methods: We collected RNA-sequencing data from pretreatment biopsies derived from the NeoALTTO trial. RBsig expression was computed retrospectively and correlated with pathological complete response (pCR) using receiver-operating characteristic (ROC) curves. The RBsig was dichotomised as High/Low in correspondence to the 25th percentile. Reported p values resulted from Fisher’s exact test. Results: Of 455 NeoALTTO patients, 244 were eligible for this substudy (HR+ n = 129; HR− n = 115). Overall, pCR rate was significantly higher in patients with RBsig High tumours than those with RBsig Low (35% versus 18% respectively; p = 0.01). The area under the ROC curve (AUC) was 0.60 (95% CI 0.52–0.67). A remarkably low pCR rate of 11% was seen in HR+/RBsig Low patients versus 28% in HR+/RBsig High. Conclusions: These results indicate RBsig may add valuable information to HER2 and HR expression, which may in turn inform treatment choices. HR+/HER2+/RBsig Low breast cancers exhibited the poorest pathological response following chemotherapy plus H. Accordingly, in such patients, endocrine therapy in combination with H and, possibly, a CDK4/6 inhibitor, may potentially prove to be a more effective treatment., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

HER2-positive (HER2+) breast cancers show heterogeneous response to chemotherapy, with the ER-positive (ER+) subgroup deriving less benefit. Loss of retinoblastoma tumor suppressor gene (RB1) function has been suggested as a cardinal feature of breast cancers that are more sensitive to chemotherapy and conversely resistant to CDK4/6 inhibitors. We performed a retrospective analysis exploring RBsig, a gene signature of RB loss, as a potential predictive marker of response to neoadjuvant chemotherapy in ER+/HER2+ breast cancer patients. We selected clinical trials of neoadjuvant chemotherapy ± anti-HER2 therapy in HER2+ breast cancer patients with available information on gene expression data, hormone receptor status, and pathological complete response (pCR) rates. RBsig expression was computed in silico and correlated with pCR. Ten studies fulfilled the inclusion criteria and were included in the analysis (514 patients). Overall, of 211 ER+/HER2+ breast cancer patients, 49 achieved pCR (23%). The pCR rate following chemotherapy ± anti-HER2 drugs in patients with RBsig low expression was significantly lower compared to patients with RBsig high expression (16% vs. 30%, respectively; Fisher’s exact test p = 0.015). The area under the ROC curve (AUC) was 0.62 (p = 0.005). In the 303 ER-negative (ER−)/HER2+ patients treated with chemotherapy ± anti-HER2 drugs, the pCR rate was 43%. No correlation was found between RBsig expression and pCR rate in this group. Low expression of RBsig identifies a subset of ER+/HER2+ patients with low pCR rates following neoadjuvant chemotherapy ± anti-HER2 therapy. These patients may potentially be spared chemotherapy in favor of anti-HER2, endocrine therapy, and CDK 4/6 inhibitor combinations.

This abstract was not presented at the symposium.

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

SummaryWe describe a potential role for thymidine kinase-1, a general marker of cellular proliferation, to act as a prognostic biomarker in patients receiving CDK4/6 inhibitors for advanced hormone receptor-positive, HER2-negative breast cancer, with early data suggesting that it may also provide early indication of treatment response.

Introduction: Thymidine kinase is an established marker of cancer cell proliferation and its activity can be measured in blood. We and others have recently shown that baseline and dynamic evaluation of circulating thymidine kinase activity (TKa) during treatment gives prognostic and predictive information in patients with HR+, HER2-negative metastatic BC treated with endocrine therapy alone, as well as in the setting of CDK4/6 inhibition. However, there is limited data regarding the role of TKa as a prognostic biomarker in operable BC. Here we present a retrospective analysis of TKa in serum samples collected in a cohort of premenopausal women with operable BC enrolled in a phase III adjuvant multicenter clinical trial (NCT00201851). Materials and methods: Serum samples were available for 644 (87%) of participants prospectively enrolled in a randomized trial between 2003 and 2009 in South East Asia. All women were premenopausal, had stage II-IIIB HR+ operable BC and uniformly received bilateral surgical oophorectomy concurrent with mastectomy followed by tamoxifen alone for five years. Patients did not receive chemotherapy or targeted therapy pre- or post-operatively. Participants were randomized in the study according to the timing of surgery with respect to the phase of the menstrual cycle. Serum samples were collected preoperatively on the day of surgery. Serum TKa was measured using the ELISA-based DiviTum™ assay (Biovica, Sweden). TKa analysis was performed at a central laboratory, blind to clinical data. Baseline TKa values were correlated with clinico-pathological characteristics and clinical outcome. Clinical outcome was estimated using the Kaplan-Meier method. Results: The majority of patients had both estrogen and progesterone receptor positive tumors (94% and 92% respectively), 65% were HER2 negative (18% positive; 17% unknown). Most had pT2 or pT3 disease (60% and 27% respectively), and more than half were node-positive (pN0 42%, pN1 27%, pN2 19%, pN3 11%, pNx 1%). The overall median TKa value was 65.4 Du/L. At five years, patients with a baseline TKa value below the median had a disease-free survival (DFS) rate of 75% versus 61% in those with a baseline over the median (HR 1.82, 95% CI 1.37-2.4, p Conclusions: This study shows pre-operative TKa measured in serum is a strong prognostic marker in a large cohort of women with HR+ operable BC uniformly treated within a clinical trial. The notable rate of recurrence seen within this cohort of patients derived from non-high income countries may be mainly attributed to the relative degree of disease burden at diagnosis. TKa may be seen as a potential circulating marker of proliferation akin to tumor Ki67, which may provide useful prognostic information to guide adjuvant therapies. Citation Format: Luca Malorni, Ilenia Migliaccio, Chiara Biagioni, Lorenzo Rossi, Irene De Santo, Richard L Love, Amelia McCartney, Mattias Bergqvist, Martina Bonechi, Francesca De Luca, Francesca Galardi, Matteo Benelli, Dario Romagnoli, Emanuela Risi, Laura Biganzoli, Adriano Laudico, Nguyen Van Dinh, Angelo Di Leo. Serum thymidine kinase-1 activity (TKa) as a prognostic marker in premenopausal women with hormone receptor positive (HR+) operable breast cancer (BC) [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 P5-06-11.

The recent arrival of CDK4/6 inhibitor agents, with an approximate doubling of progression-free survival (PFS) associated with their use in hormone receptor-positive, HER2-negative advanced breast cancer (BC), has radically changed the approach to managing this disease. However, resistance to CDK4/6 inhibitors is considered a near-inevitability in most patients. Mechanisms of resistance to these agents are multifactorial, and research in this field is still evolving. Biomarkers with the ability to identify early resistance, or to predict the likelihood of successful treatment using CDK4/6 inhibitors are yet to be identified, and represent an area of unmet clinical need. Here we present selected mechanisms of resistance to CDK4/6 inhibitors, largely focussing on roles of Rb, cyclin E1, and the PIK3CA pathway, with discussion of associated biomarkers which have been investigated and applied in recent pre-clinical and clinical studies. These biological drivers may furthermore influence clinical treatment strategies adopted beyond CDK4/6 resistance.

The landscape of therapeutic options for the treatment of hormone receptor (HR)-positive (HR+) HER2- breast cancer (BC) has been profoundly changed by the introduction of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors into the metastatic setting. Currently all CDK4/6 inhibitors are approved only in the metastatic setting by Food and Drug Administration (FDA) and European Medicine Agency (EMA), whereas their role in the neoadjuvant setting is still at an investigational stage. Exploitation of novel agents such as CDK4/6 inhibitors to improve the efficacy of neoadjuvant endocrine therapy (ET) or to overcome de novo resistance to ET is an area of research under active evaluation. We present a review of the currently available data and ongoing clinical trials that are evaluating the role of CDK4/6 inhibitors in neoadjuvant therapy of HR+ HER2- early BC, and also illustrate translational aspects, such as the potential biomarkers of response to these new therapeutic agents.

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.

Thymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome.The Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression.The median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91-5.89) versus 2.57 months (95% CI: 2.04-3.52) in patients with high sTKa baseline levels (P 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14-4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01-6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker.sTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted.

Background New single-cell isolation technologies are facilitating studies on the transcriptomics of individual cells. Bio-Rad ddSEQ is a droplet-based microfluidic system that, when coupled with downstream Illumina library preparation and sequencing, enables the monitoring of thousands of genes per cell. Sequenced reads show unique features that do not permit the use of freely available tools to perform single cell demultiplexing. Results We present ddSeeker, a tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina experiments. Its application to the Illumina test dataset demonstrates that ddSeeker performs better than Illumina BaseSpace software, enabling a higher recovery of valid reads. We also show its utility in the analysis of an in-house dataset including two read sets characterized by low and high sequencing quality. ddSeeker and its source code are available at https://github.com/cgplab/ddSeeker. Conclusions ddSeeker is a freely available tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina single cell transcriptomic experiments. Electronic supplementary material The online version of this article (10.1186/s12864-018-5249-x) contains supplementary material, which is available to authorized users.

Background: HER2 blockade in combination with chemotherapy (CT) remains the treatment of choice for patients with HER2+ early breast cancer (BC), irrespective of estrogen receptor (ER) status. Patients with ER+/HER2+ early BC may benefit from HER2 blockade in combination with endocrine therapy (ET) and potentially also new targeted agents. Recent data have elucidated cyclin-dependent kinases 4 and 6 (CDK4/6) as key therapeutic targets acting downstream of both ER and HER2 pathways suggesting CDK4/6 inhibitors like palbociclib may be ideal partners for ET in this context. Pre-clinical and clinical data suggest that a gene signature of functional loss of retinoblastoma (RBsig) might predict sensitivity to CT versus CDK4/6 inhibition in ER+/HER2+ early BC. The TOUCH hypothesis is that neoadjuvant therapy with palbociclib + ET + dual HER2 blockade with trastuzumab and pertuzumab may be more active in postmenopausal patients with ER+/HER2+ RBsig LOW early BC while those with RBsig HIGH may require CT. This will be formally tested in the primary objective, exploring the interaction between the RBsig status (HIG or LOW) and treatment activity, assessed by pathological complete response (pCR), of palbociclib+letrozole versus paclitaxel when given with trastuzumab+pertuzumab for ER+/HER2+ primary breast cancer. The study was initially targeting older patients due to the particular appealing of CT de-escalation in this population. Due to the increasing interest for CT de-escalation and the mounting evidence of the potential benefit of such ET combination optimization, TOUCH has been recently amended to extend accrual to postmenopausal women. Trial design: TOUCH is an open-label, multicenter, randomized phase II neoadjuvant trial in postmenopausal patients with ER+/HER2+ primary BC. Eligible patients will be randomized (1:1) to dual HER2 blockade (5 doses trastuzumab + pertuzumab) plus either palbociclib (125 mg/d po; 21 of 28d x 4 cycles) and letrozole (daily x 16 weeks), or paclitaxel (80 mg/m2 iv, d1,8,15 q28 days x 4 cycles), before surgery. RBsig (HIGH vs LOW) will be determined centrally on mandatory pre-treatment biopsies. Patients ≥65 years will receive a baseline geriatric assessment including G8, Instrumental Activity of Daily Living (IADL) and Charlson Comorbidity Index. The primary objective is to explore the interaction between RBsig and treatment activity assessed by pathological complete response (pCR) at time of surgery. Exact logistic regression will test RBsig by treatment interaction (2-sided a=.05) and estimate odds ratios (OR) for treatment effect on pCR according to RBsig status. The sample size provides 86% power, assuming an overall pCR rate of 26%, 50% RBsig LOW, and OR=2.4 vs OR=0.11 for RBsig LOW vs HIGH. Accrual: The trial will recruit 144 patients from approximately 45 Centers in Belgium, France, Italy and Switzerland. The first patient was enrolled in April 2019. Accrual as of mid-June 2020 was 25 patients. The TOUCH trial (IBCSG 55-17) is sponsored and coordinated by IBCSG with financial support from Pfizer and Roche. The trial is conducted in collaboration with Unicancer GERICO and SAKK. NCT03644186 Citation Format: Laura Biganzoli, Etienne Brain, Luca Malorni, Andrea Gombos, Ursula Hasler-Strub, Claudio Zamagni, Camille Chakiba, Heidi Roschitzki-Voser, Meredith M Regan. Phase II randomized trial of neoadjuvant trastuzumab and pertuzumab with either palbociclib plus letrozole or paclitaxel for postmenopausal women with estrogen receptor-positive / HER2-positive breast cancer - The TOUCH trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-28-02.

Background: The CDK4/6 inhibitor palbociclib (P) plus fulvestrant (F) is approved for the treatment of patients (pts) with luminal metastatic breast cancer (MBC) progressed on prior endocrine therapy (ET). Despite clinical activity, a significant proportion of pts in this setting show primary resistance to P+F, with treatment failure within 3-6 months of initiation. To date there is no validated biomarker to identify such pts. Thymidine kinase 1 is a cancer proliferation marker downstream of the CDK4/6 pathway, whose activity can be measured in serum as a readout of tumour proliferation. Circulating thymidine kinase activity (TKa) is a potential prognostic and monitoring marker in pts treated with ET alone or in combination with P for MBC. However, the prognostic value of early changes in TKa during P+F treatment and its role in identifying pts with primary resistance are not yet defined. Here we prospectively investigated the role of serum TKa measured at different timepoints in pts treated with P+F within the PYTHIA trial (IBCSG 53-14/BIG 14-04; NCT02536742), a downstream trial of the AURORA platform (BIG 14-01; NCT02102165). Methods: PYTHIA is a biomarker discovery phase II trial including pts (Aug ‘16 to Jun ’19) with ET-resistant luminal MBC who received P+F at standard schedule and dose with 3-monthly imaging. Serum samples were collected at baseline (D0; n=122), on-treatment at day 11-16 of cycle 1 (D15; n=108), and during the one week off P before initiating cycle 2 (D28: Day 24-37 of Cycle 1; n=108). TKa was measured with DiviTum®, a refined ELISA-based assay. Complete TKa response (CTR) was defined as TKa below the limit of detection (LOD; 20 Du/L) at D15. Cox models evaluated association of log-transformed TKa measurements with progression-free survival (PFS; from initiation of therapy until progression by RECIST criteria or death). Kaplan-Meier method estimated median, 3 and 6 months (95% CI) PFS in groups of patients defined by dichotomizing TKa as “high” or “low” at the median or by CTR. A sample size of 120 provided 80% power to detect a hazard ratio of 2.0 for biomarker with 30-50% prevalence (two-sided α=0.05) after ≥80 events. Results: A total of 122 pts were enrolled. About half had received one prior line of ET for MBC, and 18% had received one prior line of chemotherapy. 48% had visceral metastases and 31% had bone-only disease. TKa at D0 was not associated with clinical characteristics. Median TKa (mTKa) at D0 was 87 Du/L. Overall, 82 pts experienced progression, with a median PFS (mPFS) of 11 months (95% CI: 8.6 - 16). P+F dramatically suppressed mTKa levels at D15, with 90/108 (83%) pts achieving CTR. At D28, TKa showed some rebound in most pts. At each timepoint, higher TKa was significantly and consistently associated with shorter PFS (each p Conclusions: TKa is an independent prognostic biomarker in pts treated with P+F. High baseline TKa and incomplete suppression of TKa during treatment may identify pts with poor prognosis and primary resistance to P+F. TKa may represent a novel biomarker to select pts for alternative treatment modalities. These results warrant further investigation in prospective comparative trials. TimepointBaseline (D0)D151D28TK median value (Du/L) (range)87 ( Citation Format: Luca Malorni, Svitlana Tyekucheva, Florentine S Hilbers, Michail Ignatiadis, Patrick Neven, Marco Colleoni, Stéphanie Henry, Alberto Ballestrero, Andrea Bonetti, Guy Jerusalem, Konstantinos Papadimitriou, Antonio Bernardo, Francois Duhoux, Iain MacPherson, Alastair Thomson, David Mark Davies, Mattias Bergqvist, Matteo Benelli, Amelia McCartney, Heidi De Swert, Barbara Ruepp, Manuela Rabaglio, Rudolf Maibach, Martine Piccart, Meredith M Regan. Serum thymidine kinase activity in patients with luminal metastatic breast cancer treated with palbociclib and fulvestrant within the PYTHIA trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-05.

CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy are the mainstay of treatment for patients with hormone receptor-positive, HER2 negative (HR+/HER2neg) metastatic breast cancer. However, resistance - either de novo or acquired - invariably occurs, leading to treatment failure and cancer progression. Genomic alterations, gene expression data and circulating biomarkers have been correlated to response to treatment, but to date no biomarker has been approved to stratify patients. Treatment strategies after progression on CDK4/6i are yet to be standardized. Current approaches include endocrine therapy alone or in combination with target therapy, or chemotherapy. New agents are in clinical development based on potential mechanisms of acquired resistance. Here we will review recent advancements in biomarkers of response to CDK4/6i, and in post- treatment therapeutic strategies.

Background: The primary objective of this phase IIIb, single arm trial is to study circulating tumor DNA (ctDNA) alterations, their evolution during treatment and association with clinical outcome in patients receiving ribociclib and letrozole as first-line therapy for aBC. Here we report baseline ctDNA mutational status and its correlation with clinical-pathological characteristics and response to treatment on the basis of first imaging evaluation. Methods: From February to December 2018, 287 post-menopausal patients (pts) with hormone receptor positive (HR+) and HER2 negative aBC were enrolled in 47 Italian Centers; 271 pts were suitable for biomarker analysis (Biomarker Analysis Set- BAS). Data on first imaging evaluation were available for 225 pts. Baseline liquid biopsies were analyzed both for Copy Number Variation (CNV) by Oncomine Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific) and for Single Nucleotide Variant (SNV) using 533 amplicon custom AmpliSeq HD panel optimized for the detection of low abundance events in ctDNA. Variants were analyzed as both individual genes and as pre-defined pathways (n=10). Results: At BAS, median age was 66 years (range 47-86). At study entry, 60% (109) and 40% (162) of pts had recurrent and de-novo disease, respectively, with 43% (117) of pts with visceral and 24% (64) with bone-only involvement. At least one CNV alteration or a hotspot mutation was detected in 51% of patients. The most frequently altered genes were PIK3CA (22.14%), TP53 (15.50%), FGFR1 (6.64%), CCND1 (4.80%), CCND2 (4.80%), KMT2C (4.43%), MYC (4.06%), CDK4 (3.69%) AKT1 (3.32%), PTEN (3.32%), ERBB2 (2.58%), CCND3 (2.58%), APC (2.21%) and MAP2K4 (2.21%). More than one alteration was observed in 28% pts (either CNV or hotspots). Mutation in KTM2C or alterations in genes belonging to the “Estrogen Receptor nuclear function” (ERnf) pathway (i.e. KTM2C, ESR1, GATA3 and MYC) were more frequent in patients with recurrent vs. de novo disease (Odds Ratio [OR] =7.69, p=0.0499 for KTM2C; OR=2.70, p=0.0381 for ERnf). MYC copy number gain or alterations in the ERnf genes were more frequent in Estrogen Receptor positive (ER+)/Progesteron Receptor negative (PgR-) compared to ER+/PgR+ tumors (OR= 4.07, p=0.0361 for MYC; OR=3.36, p=0.0073 for ERnf). Copy number gain of FGFRs (FGFR1, 2 and 3) were more frequent in patients with visceral versus bone-only disease (OR=5.26, p=0.0144 for FGFRs) and in pts with more than three metastatic sites (OR=8.05, p=0.0052). At first imaging, the Clinical Benefit Rate (CBR, defined as CR + PR + SD) was 90%, with only 10% of pts showing early disease progression (PD). Thirty pts (11%) had at least one alteration in genes belonging to the CDK4/6 pathway (CCND1, RB1, CDK4 and 6, CDKN2A). These alterations were more frequent in the group of patients with early PD (OR=3.23, p=0.0272). MYC gain, TP53 mutations and alterations in the HER family genes (EGFR, ERBB2, 3 and 4) were also more frequent in pts with early PD compared with pts with CBR (OR 5.16, p=0.0280, OR 3.87, p=0.0058 and OR 4.40, p=0.0426, respectively). Conclusion: At the present analysis, alterations in KTM2C and ERnf seem to characterize recurrent vs de novo aBC while FGFRs are prevalent in patients with more aggressive disease. Interestingly, MYC gain, TP53 mutations, alterations in genes of the HER family and CDK4/6 pathway were more likely detected in patients with early PD suggesting these as potential markers of intrinsic resistance to first-line treatment with ribociclib and letrozole. Further analyses for biomarker dynamics and pharmacogenomics are ongoing. Citation Format: Michelino De Laurentiis, Luca Malorni, Giampaolo Bianchini, Roberta Caputo, Mario Giuliano, Alberto Zambelli, Fabio Puglisi, Lucia Del Mastro, Marco Colleoni, Filippo Montemurro, Giulia Bianchi, Ida Paris, Giacomo Allegrini, Laura Amaducci, Marina Elena Cazzaniga, Michele Orditura, Claudio Zamagni, Vincenzo Adamo, Valentina Guarnieri, Nicola Battelli, Maria Rosaria Valerio, Saverio Cinieri, Sara Bianchetti, Donatella Grasso, Daniela Castelletti, Diletta Valsecchi, Ettore Cotroneo, Grazia Arpino. Bioitalee - Biomarker analysis on liquid biopsies of patients treated with ribociclib and letrozole as first-line therapy for advanced breast cancer (aBC) (NCT03439046) [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 P5-01-07.

Background: The CDK4/6 inhibitors (i) palbociclib (Palbo), ribociclib, and abemaciclib remarkably improved the outcome of patients with metastatic ER+/HER2- breast cancer (BC) and are now under clinical investigation in early BC. Despite high efficacy, de novo and acquired resistance to CDK4/6i is common. Elucidating the molecular basis for sensitivity and resistance to CDK4/6i is crucial to identify predictive biomarkers and novel therapeutic targets to improve patient outcome. Materials and Methods: MCF7, T47D and ZR75-1 parental (P) BC cells and their derivatives made resistant to tamoxifen, estrogen deprivation (EDR), or fulvestrant were used. The P and EDR models of MCF7 and T47D cells were chronically exposed to increasing concentrations of Palbo to generate derivatives with acquired resistance to Palbo (PalboR). The transcriptomic profiles of P, endocrine-resistant (EndoR) and PalboR models were determined by RNA-seq. IC50s were determined by exposing MCF7, T47D, and ZR75-1 P and EndoR lines (n=12) to increasing concentrations of Palbo. Cell growth was assessed by methylene blue staining, and changes in the mRNA and protein levels of key cell cycle molecules were assessed by RT-PCR and Western blot, respectively. Gene expression data from the Cancer Dependency Map (DepMap), baseline tumors from the NeoPalAna (NCT01723774) and neoMONARCH (NCT02441946) neoadjuvant trials, as well as the TCGA and METABRIC datasets were interrogated for correlations of gene signatures and patient outcome (by KMPlot). Results: Palbo treatment resulted in a dose-dependent inhibition of the growth of P and EndoR BC cell lines, with varying degree of sensitivity among the models. GSEA analysis comparing the least sensitive (IC50>350nM) vs. the most sensitive (IC50 Citation Format: Carmine De Angelis, Xiaoyong Fu, Maria Letizia Cataldo, Agostina Nardone, Valerie M. Jansen, Jamunarani Veeraraghavan, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Matteo Benelli, Ilenia Migliaccio, Luca Malorni, Joshua Donaldson, Pier Selenica, David N. Brown, Britta Weigelt, Jorge S. Reis-Filho, Ben H. Park, Sara A. Hurvitz, Dennis J. Slamon, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. High levels of interferon-response gene signatures are associated with de novo and acquired resistance to CDK4/6 inhibitors in ER+ breast cancer [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 GS2-01.

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.

Randomized clinical trials demonstrated that CDK4/6 inhibitors are highly effective in patients with hormone receptor-positive (HR+), HER2-negative (HER2-) metastatic breast cancer in combination with endocrine therapy. The use of CDK4/6 inhibitors in clinics is becoming common for patients with HR+/HER2- metastatic breast cancer and will certainly increase in the near future. However, patients might show de novo or acquired resistance to these drugs. Molecular alterations have been suggested as determinants for de novo resistance to CDK4/6 inhibitors, but have never been validated in a clinical setting. In addition, molecular mechanisms of acquired resistance to palbociclib have been analyzed only in preclinical studies. Here we review the current knowledge on the available preclinical data about the mechanisms of de novo and acquired resistance to CDK4/6 inhibitors in breast cancer, and clinical data about potential biomarkers of response.

Considerable progress has been made in breast cancer treatment in Europe over the past three decades, yet survival rates for metastatic disease remain poor, underlining the need for further advances. While the use of predictive biomarkers for response to systemic therapy could improve drug development efficiency, progress in identifying such markers has been slow. The currently inadequate classification of breast cancer subtypes is a further challenge. Improved understanding of the molecular pathology of the disease has led to the identification of new targets for drug treatment, and evolving classifications should reflect these developments. Further ongoing challenges include difficulties in finding optimal combinations and sequences of systemic therapies, circumventing multidrug resistance and intra-tumor heterogeneity, problems associated with fragmentation in clinical trials and translational research efforts. Adoption of some of the strategies identified in this article may lead to further improvements in outcomes for patients with the disease., SCOPUS: re.j, info:eu-repo/semantics/published

Hormone-receptor-positive breast cancer accounts for the majority of all breast cancers. The evolution of this disease from early stage to the metastatic setting leads to increased heterogeneity and the development of treatment resistance representing a great challenge for management decisions. In this Review, we examine the current evidence that can guide treatment decisions in patients with advanced-stage ER+ breast cancer, discuss how to tackle these therapeutic challenges and provide suggestions for the optimal management of this patient population. Hormone-receptor-positive breast cancer accounts for the majority—up to 80%—of all breast cancers. The evolution of breast cancer from early stage to the metastatic setting leads to increased heterogeneity, the occurrence of new mutations, and the development of treatment resistance representing a great challenge for management decisions. Unfortunately, little data exist to offer guidance in this context, and a reliance on traditional clinical parameters remains when deciding on optimal treatment. In advanced-stage oestrogen receptor-positive (ER+) disease, ongoing issues include the choice between endocrine therapy and chemotherapy, the appropriate sequence of treatment agents, and the incorporation of biological agents, such as everolimus, into the treatment armamentarium. In metastatic disease, repeated biopsies can help to reassess the receptor or genetic mutational status; however, the evidence to support this approach is limited. In this Review, we examine the current evidence that can guide treatment decisions in patients with advanced-stage ER+ breast cancer, discuss how to tackle these therapeutic challenges and provide suggestions for the optimal management of this patient population.

Fulvestrant is a pure estrogen receptor (ER) antagonist devoid of the agonistic properties displayed by tamoxifen in some tissues (1–4). After phase III studies, which demonstrated similar efficacy and an acceptable safety profile for fulvestrant 250mg compared with anastrozole (1,5), fulvestrant 250mg was approved as treatment in postmenopausal women with advanced hormone receptor–positive breast cancer that had progressed or recurred after prior antiestrogen therapy. However, previous preoperative studies showed that short-term exposure to fulvestrant was associated with a dose-dependent reduction in the levels of ER, progesterone receptor, and the cell proliferation–related antigen Ki67 (6,7) for fulvestrant doses up to 250mg. Other phase I and phase III studies also suggested a dose–response effect for fulvestrant (1,5,8). The phase III Comparison of Faslodex in Recurrent or Metastatic Breast Cancer (CONFIRM) trial compared the then-approved dose and dosing schedule of fulvestrant (250mg every 28 days) with a higher-dose regimen (500mg every 28 days plus an additional 500mg on day 14 of the first month only) in postmenopausal women with locally advanced or metastatic ER-positive breast cancer that had recurred or progressed after prior endocrine therapy. The initial results showed that fulvestrant 500mg was associated with a statistically significant increase in progression-free survival (PFS) without increased toxicity, therefore corresponding to a clinically meaningful improvement in benefit vs risk compared with fulvestrant 250mg (9). Based on these data, the 500-mg dose of fulvestrant is now the approved dose in the European Union (approved in March 2010), United States (approved in September 2010), Japan (approved in November 2011), and other countries worldwide. In the CONFIRM study, the assessment of the therapeutic efficacy of both doses of fulvestrant was evaluated by several secondary outcome measures, including overall survival (OS). At the time of the initial analysis, approximately 50% of patients had died. After the reporting of the 50% survival data, which showed a trend in favor of 500mg over 250mg, it was agreed to perform a final survival analysis after 75% of patients had died. Here we report the results of this final OS analysis.

CDK4 and 6 are central regulators of cell cycle entry. The CDK4/6 pathway is frequently deregulated in breast cancer, and strategies to target this pathway have recently been proven to be effective in breast cancer patients. Preclinical and clinical data suggest that CDK4/6 inhibitors might be particularly useful in patients with hormone receptor-positive or HER2-positive tumors, while the role of such inhibitors in triple-negative breast cancer is still controversial.

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.

Mutations in the hotspot ligand-binding domain of the estrogen receptor (ER) gene ESR1 have recently been recognized as mechanisms of endocrine resistance in endocrine receptor-positive metastatic breast cancer (MBC). Accumulating data suggest these mutations develop under the selective pressure of endocrine treatments, and are infrequent in untreated ER-positive breast cancers. In vitro studies show that these mutations confer ligand-independent activity, resistance to estrogen deprivation, and relative resistance to tamoxifen and fulvestrant. Post-hoc retrospective and prospective analyses of ESR1 mutations in patients with MBC have consistently found that these mutations are markers of poor prognosis and predict resistance to aromatase inhibitors (AIs). These results warrant further investigation and prospective validation in dedicated studies. Moreover, studies are ongoing to clarify the activity of novel drugs in the context of metastatic endocrine resistant luminal breast cancer harboring ESR1 mutations. In this review, we summarize the pre-clinical and clinical findings defining the characteristics of ESR1 mutant breast cancer, and highlight the potential clinical developments in this field.

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

Background Adjuvant treatment decisions for pts with eCRC are currently based on suboptimal risk stratification factors, especially for elderly pts. Metabolomics measures multiple cancer-related metabolites with potential to identify new biomarkers in this setting. We have shown that serum metabolomics can discriminate pts with eCRC from pts with metastatic CRC (mCRC). We hypothesized that a metabolomic score derived from pts with mCRC may identify pts with eCRC with an increased risk of relapse. This hypothesis was tested in a cohort of elderly pts with eCRC. Methods Serum samples from 103 pts aged ≥70 were collected from four clinical trials with 5 years follow up. These samples were derived from 55 pts with eCRC, and 48 with mCRC. Samples were retrospectively analyzed via proton nuclear magnetic resonance (1H NMR), to assess their metabolomic fingerprints. A Random Forest (RF) classification model was built using a training set of 30 eCRC pts free from relapse at 5 years and all mCRC pts (N = 48). This model was then applied to a validation set constituted by the remaining eCRC pts (10 relapse-free and 15 relapsed). A risk-of-recurrence score was built on the basis of the likelihood of the sample being misclassified as metastatic. Results In the eCRC group, 44% (n = 24) received adjuvant chemotherapy, and 27% (n = 15) experienced relapse. In the training set, the RF model discriminated eCRC from mCRC with an accuracy of 74.4%. The RF risk of recurrence score correlated with relapse, with an AUC of 0.754 in ROC analysis. In the training set, by maximizing specificity and sensitivity, a threshold for the RF score was set at 0.55. In the validation set, using this threshold, an AUC of 0.727 in ROC analysis, and a prediction accuracy of 76% (73.3% sensitivity, 80% specificity) were obtained in predicting relapse. Conclusions Serum metabolomics performed on post-operative samples of elderly pts with eCRC identifies pts with higher risk of relapse with good accuracy. This may represent a tool to refine risk stratification in this population, to maximize the benefit from adjuvant chemotherapy. Based on these results, a prospective trial is ongoing (LIquid BIopsy and METabolomics in CRC - LIBIMET). Legal entity responsible for the study The authors. Funding Fondazione Sandro Pitigliani per la Lotto Contro i Tumori - ONLUS. Disclosure S. Di Donato: Advisory / Consultancy, Travel / Accommodation / Expenses: Amgen; Advisory / Consultancy, Travel / Accommodation / Expenses: Lilly; Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: Servier; Travel / Accommodation / Expenses: Celgene; Advisory / Consultancy, Travel / Accommodation / Expenses: Sanofi; Advisory / Consultancy, Travel / Accommodation / Expenses: Bayer. E. Mori: Travel / Accommodation / Expenses: Bayer. L. Malorni: Honoraria (self): AstraZeneca; Advisory / Consultancy: Novartis; Travel / Accommodation / Expenses: Janssen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Pfizer; Travel / Accommodation / Expenses: Roche. D. Becheri: Travel / Accommodation / Expenses: Daiichi-Sankyo; Travel / Accommodation / Expenses: Bristol-Myers Squibb. A. Di Leo: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Bayer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Celgene; Advisory / Consultancy, Travel / Accommodation / Expenses: Daiichi-Sankyo; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Eisai; Advisory / Consultancy: Genentech; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Genomic Health; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pierre Fabre; Advisory / Consultancy, Travel / Accommodation / Expenses: Puma Biotechnology; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche. L. Biganzoli: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Astrazeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Celgene; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Eisai; Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Genomic Health; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Ipsen; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pierre Fabre; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche. All other authors have declared no conflicts of interest.

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.

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.

// Luca Malorni 1,2 , Silvano Piazza 3,4 , Yari Ciani 3 , Cristina Guarducci 1 , Martina Bonechi 1 , Chiara Biagioni 1,2 , Christopher D. Hart 2 , Roberto Verardo 3 , Angelo Di Leo 1,2 and Ilenia Migliaccio 1 1 “Sandro Pitigliani” Translational Research Unit, Hospital of Prato-AUSL Toscana Centro, Istituto Toscano Tumori, Prato, Italy 2 “Sandro Pitigliani” Medical Oncology Department, Hospital of Prato-AUSL Toscana Centro, Istituto Toscano Tumori, Prato, Italy 3 Laboratorio Nazionale CIB (LNCIB), Area Science Park, and Functional Genomics & Bioinformatics Units, Trieste, Italy 4 Bioinformatics Core Facility, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy Correspondence to: Ilenia Migliaccio, email: // Luca Malorni, email: // Keywords : breast cancer, CDK4/6 inhibitors, retinoblastoma loss of functions Received : July 14, 2016 Accepted : August 03, 2016 Published : September 13, 2016 Abstract Palbociclib is a CDK4/6 inhibitor that received FDA approval for treatment of hormone receptor positive (HR+) HER2 negative (HER2neg) advanced breast cancer. To better personalize patients treatment it is critical to identify subgroups that would mostly benefit from it. We hypothesize that complex alterations of the Retinoblastoma (Rb) pathway might be implicated in resistance to CDK4/6 inhibitors and aim to investigate whether signatures of Rb loss-of-function would identify breast cancer cell lines resistant to palbociclib. We established a gene expression signature of Rb loss-of-function (RBsig) by identifying genes correlated with E2F1 and E2F2 expression in breast cancers within The Cancer Genome Atlas. We assessed the RBsig prognostic role in the METABRIC and in a comprehensive breast cancer meta-dataset. Finally, we analyzed whether RBsig would discriminate palbociclib-sensitive and -resistant breast cancer cells in a large RNA sequencing-based dataset. The RBsig was associated with RB1 genetic status in all tumors ( p

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

Background. New imaging tests such as computed tomography [CT], [18F]fluorodeoxyglucose-positron emission tomography (FDG-PET) scanning and magnetic resonance imaging (MRI) are not recommended for staging or follow up of asymptomatic patients with EBC according to current guidelines. However, frequently these tests are requested even in the absence of a clinical indication. Variations in practice patterns have significant cost implications and no clear impact on cancer outcomes. Aim. In the present study we analyzed how the availability of novel and more expensive imaging technique has changed staging and follow-up modalities in EBC patients and if these changes affect costs. Methods. Two thousand and five patients with EBC diagnosed between January 2005 and December 2008 were identified using clinical computerized medical records from 639 general practitioners assisting 915,689 inhabitants of the Campania, a southern region of Italy. For each year EBC incidence, the mean number of diagnostic tests per patient (N/Pt) done during the first year after the initial diagnosis and the mean costs per each year were evaluated and compared by Anova test. Results. We identified 576, 489, 474 and 497 newly diagnosed cases of EBC in 2005, 2006, 2007 and 2008 respectively. Table 1 Table 1 describes changes in the mean number of different imaging tests requested per patient in these years. Overall, there was a significant increase of the mean number of imaging tests done per patient from 2005 to 2008 (P Conclusions. TC scan, PET scan and MRI employment for patient with EBC in daily clinical practice has been steadily growing over the past four years. However, there are no data to support their role in routine breast cancer staging or surveillance in asymptomatic patients. More studies are needed to characterize which subset of patients deserve more intensive staging and follow up procedures as costs related to these imaging test prescriptions are relevant. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-09-06.

12031Background: TK1 plays a critical role in DNA synthesis and cell proliferation. The DiviTum assay measures serum TK1 activity (sTKa), reflecting cancer cell proliferation. Recent studies sugges...