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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.

Supplemental Figure Legends

QC analysis

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

Cox proportional hazards analyses

PFS and OS of PAM50 and OncotypeDx risk categories

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

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

Correlation between IHC and transcript level by microarray

PFS and RORP classifier

INTRODUCTION: Somatic mutations of the PIK3CA gene, encoding for the class IA PI3K p110α subunit, are the most common activating mutations in breast cancer (BC), occurring in 30-50% of early and in 28% of metastatic ER+/HER2- disease. We have previously demonstrated that PIK3CA mutations co-occurring with copy number (CN) gain (PIK3CA-mut/gain) are associated with worse outcome in patients with early hormone receptor positive and HER2 negative (HR+/HER2-) BC, including those receiving endocrine therapy (ET). Here we aim to evaluate the prognostic role of PIK3CA-mut/gain in patients with metastatic HR+/HER2- BC treated with chemotherapy, ET alone or in combination with targeted therapies (CDK4/6 inhibitors or everolimus) included in a publicly available dataset of BC. PATIENTS AND METHODS: We accessed genomic, clinical, treatment and outcome data from 1365 patients with HR+/HER2- BC included in the MSK-2018 dataset. Patients treated for distant metastases were selected. PIK3CA status, considering both protein-affecting mutations and CN alterations, was derived from pre-treatment biopsies data. Tumors were classified as: PIK3CA wt and CN neutral (-wt/neut), PIK3CA wt with CN gain (-wt/gain), PIK3CA mutant and CN neutral (-mut/neut), and PIK3CA mutant with CN gain (-mut/gain). Tumors with PIK3CA loss were excluded from the analyses. For each patient and treatment type, only progression free survival (PFS) from the first available line of treatment was analyzed, thus excluding data concerning possible retreatments with the same drug in subsequent lines. Multivariate Cox proportional hazard models were used to assess the association between PIK3CA status and PFS including treatment line and the four categories of PIK3CA as co-variates. RESULTS: The proportion of PIK3CA-mut/gain was 15.4% in the chemotherapy group (n=454), 17.3% in the ET alone group (n= 352), 13.4% in the ET plus CDK4/6 inhibitors group (n= 268) and 10.5% in the ET plus everolimus group (n= 143). As expected, in all groups treatment line was significantly and independently associated with PFS. In the ET alone group, patients whose tumors had gain in PIK3CA CN, with or without co-occurring mutations, were at significantly higher risk of progression compared with PIK3CA-wt/neut (HR 2.2 [1.53-3.2] p Citation Format: Ilenia Migliaccio, Marta Paoli, Emanuela Risi, Chiara Biagioni, Laura Biganzoli, Matteo Benelli, Luca Malorni. PIK3CA mutations co-occurring with copy number gain identify patients with adverse outcome and potentially different treatment sensitivity among hormone receptor positive and HER2 negative metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-13.

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

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

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

Background: Thymidine kinase-1 is a cell proliferation marker downstream of the CDK4/6 pathway, whose activity can be measured in serum to reflect tumor proliferation. The CDK4/6 inhibitor palbociclib (P) is approved for the treatment of patients (pts) with hormone receptor positive metastatic breast cancer (MBC) in first or second line endocrine-based treatment settings. Approximately 10-15% of pts exhibit de novo resistance to P, with circulating levels of thymidine kinase activity (TKa) previously shown as a potential marker of early treatment resistance. Therapeutic strategies to address primary resistance to P are currently lacking. Little is known of the clinical efficacy of alternative dosing schedules of P, and its effect on TKa. Here we report serum TKa measured at different timepoints from samples collected within the MA38 (NCT02630693) study. Methods: MA38 is an open label randomised Phase 2 trial comparing two different schedules of P plus second-line ET in pts with ER-positive, HER2-negative MBC. Pts were assigned to receive physician’s choice ET plus either standard P dosing (125mg daily for 21 days on a 28-day cycle), or 100mg daily continuously. Serum samples were collected at baseline (BL; n=135), at 12 weeks (W12; n=122) and 24 weeks (W24; n=95). TKa was measured with DiviTum®, a refined ELISA-based assay (lower limit of detection [LLOD] = 100 DuA). Kaplan-Meier method estimated BL, W12 and W24 (95% CI) median PFS (mPFS; from randomization until progression by RECIST criteria or death) and overall survival (OS; from randomization until death from any cause) in groups of patients defined by dichotomizing TKa as “high” or “low” at the median. Results: MA38 enrolled 180 pts from December 2015 and February 2017 across Canada. Median follow up was 19 months. Overall, the median age was 60, and 90% of pts were post-menopausal. All pts had estrogen receptor-positive disease, and 64% had visceral metastases. On study, 56% received fulvestrant with P, 34% aromatase inhibitor and 10% tamoxifen. TKa was successfully measured in 100% of samples. Median TKa (mTKa) at BL was 234 DuA (IQR 138.5 - 438). BL TKa was not associated with clinical or pathological characteristics. TKa was prognostic at BL with mPFS of 5.5 months (mo) in pts with high TKa vs 16.3 mo with low TKa (HR=2.43; 95% CI, 1.6-3.7; p< 0.001). Similar results were obtained employing other previously reported cut off values. At multivariate analysis, BL TKa was independent from other prognostic factors including age, ECOG status and presence of visceral metastases (adjusted HR= 2.34; 95%CI 1.5- 3.6; p < 0.001). In terms of OS, BL TKa was an independent prognostic factor (adjusted HR=2.0; 95% CI, 1.1-3.7; p=0.02). At 12 mo, OS rate was 68% in pts with high BL TKa vs 92% in low TKa. Both for PFS and OS, no interaction between BL TKa and study arm was observed. At W12 mTKa was 129.5 DuA (IQR 100 - 219.8) and below LLOD (IQR 100 - 180) at W24. At these timepoints, landmark analyses showed no significant difference in PFS according to TKa. However, at W12 high TKa was significantly associated with worse OS (HR 2.0; 95%CI 1.0- 4.0; p=0.03), with a similar trend at W24 (HR 2.5; 95%CI 0.9-6.4; p=0.06). Conclusions: Baseline TKa is a reliable prognostic marker of both PFS and OS in pts treated with P and ET, further substantiating previous data. Monitoring TKa during treatment may provide important clinical information. A significant relationship between TKa and assigned treatment arm was not observed, suggesting TKa is not influenced by P treatment dose or intensity. These data confirm the role of baseline TKa as a new marker for patient stratification, and supports further investigation for the assessment of the clinical utility of TKa as a monitoring biomarker in the advanced setting. Citation Format: Amelia McCartney, Chiara Biagioni, Bingshu Chen, Lois Shepherd, Karen Gelmon, Anil A. Joy, Wendy Parulekar, Mattias Bergqvist, Ilenia Migliaccio, Angela Leo, Matteo Benelli, Emanuela Risi, Erica Moretti, Luca Livraghi, Laura Biganzoli, Luca Malorni. Serum thymidine kinase activity as a prognostic marker in women with metastatic breast cancer treated with two different schedules of palbociclib plus second-line endocrine therapy within the CCTG MA38 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-27.

Background: We have previously identified two potentially predictive signatures of palbociclib resistance: the RBsig, composed of E2F1/E2F2 dependent genes, which is correlated with genetic loss of RB1, and the ratio between the gene expression levels of CCNE1 to RB1 (CCNE1/RB1). Both signatures have been previously tested in vitro and in neoadjuvant studies with palbociclib. The present analysis aims to explore the prognostic and predictive role of RBsig and CCNE1/RB1 in the pivotal phase III randomized trials PALOMA-2 and PALOMA-3. Materials and methods: Gene expression data from the PALOMA-2 and PALOMA-3 datasets were generated using the HTG EdgeSeq Oncology BM Panel, as previously described. Of the 87 genes composing RBsig, 46 were available within the EdgeSeq dataset and were used for the analyses; CCNE1 and RB1 were both available. RBsig was calculated as the mean of the Z-score scaled gene expression (log) of the 46 genes; CCNE1/RB1 was computed as the log ratio between the mRNA expression of CCNE1 and RB1. High and low values of RBsig and CCNE1/RB1 were defined based on the third quartile (Q3) as cutoff or as continuous variables. The prognostic/predictive effect of the signatures in terms of PFS was tested using Cox proportional hazard models and the Wald test. Results: The 46-genes RBsig versus the original signature showed excellent correlation in the METABRIC dataset (R=0.99), confirming its reliability as a surrogate of the original RBsig using EdgeSeq data. In both PALOMA-2 and PALOMA-3, RBsig high was significantly associated with a worse outcome compared to RBsig low in the palbociclib arm but not in the control arm [PALOMA-2: HR 1.4 (95% CI 1.0, 2.0) p=0.029 for palbociclib arm; HR 1.1 (95% CI 0.7, 1.6) p=0.71 for control arm. PALOMA-3: HR 1.7 (95% CI 1.1, 2.6) p=0.01 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p= 0.49 for control arm]. However, in both studies RBsig was not predictive of palbociclib resistance both when considered as a continuous variable and when dichotomized at Q3. Similarly to RBsig, in PALOMA- 3 patients with CCNE1/RB1 high tumors treated in the palbociclib arm showed a significantly worse outcome compared to those with CCNE1/RB1 low but this effect was not observed in those treated in the control arm [HR 1.6 (95% CI 1.1- 2.5) p= 0.03 for palbociclib arm; HR 1.2 (95% CI 0.7, 1.9) p=0.5 for control arm]. In addition, CCNE1/RB1 as a continuous variable was predictive of palbociclib benefit in PALOMA-3 (interaction p= 0.047). These effects were not observed in PALOMA-2. Conclusions: RBsig is a prognostic biomarker in patients treated with palbociclib, suggesting it may help in patients’ risk stratification. CCNE1/RB1 is predictive of palbociclib benefit in PALOMA-3, but not in PALOMA-2 probably due to the different patient populations and characteristics. Further studies of these biomarkers in patients treated with CDK4/6 inhibitors in the metastatic as well in the adjuvant setting are warranted. Citation Format: Luca Malorni, Matteo Benelli, Yuan Liu, Shibing Deng, Zhe Zhang, Cristina Guarducci, Angela Leo, Agostina Nardone, Francesca Galardi, Emanuela Risi, Erica Moretti, Dario Romagnoli, Chiara Biagioni, Marta Paoli, Laura Biganzoli, Ilenia Migliaccio. Prognostic and predictive role of RBsig and CCNE1/RB1 gene-expression signatures in patients with advanced breast cancer treated with palbociclib in combination with endocrine therapy in the PALOMA-2 and 3 trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-17.

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.

Differentially DNA methylated regions (DMRs) inform on the role of epigenetic changes in cancer. We present Rocker-meth, a new computational method exploiting a heterogeneous hidden Markov model to detect DMRs across multiple experimental platforms. Through an extensive comparative study, we first demonstrate Rocker-meth excellent performance on synthetic data. Its application to more than 6,000 methylation profiles across 14 tumor types provides a comprehensive catalog of tumor type-specific and shared DMRs, and agnostically identifies cancer-related partially methylated domains (PMD). In depth integrative analysis including orthogonal omics shows the enhanced ability of Rocker-meth in recapitulating known associations, further uncovering the pan-cancer relationship between DNA hypermethylation and transcription factor deregulation depending on the baseline chromatin state. Finally, we demonstrate the utility of the catalog for the study of colorectal cancer single-cell DNA-methylation data., Matteo Benelli et al. present Rocker-meth, a new Hidden Markov Model (HMM)-based method, to robustly identify differentially methylated regions (DMRs). They use Rocker-meth to analyse more than 6000 methylation profiles across 14 cancer types, providing a catalog of tumor-specific and shared DMRs.

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.

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

Background: 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 is a piece about the global importance of archives and archivists. From the Global Seed Vault in Svalbard, near the North Pole, that helped fight hunger in the war in Syria to the archives of Australia that have assisted thousands of victims of the “Stolen Generations” reclaim their identity, this piece shows how archivists help tackle global issues in today’s interconnected society. Recalling the heroic acts of the custodian of the legendary manuscripts of Timbuktu, as the city was besieged by rebels and extremists, the authors challenge archivists to explore how far they would go to protect archives and preserve them for humanity. Finally, partnerships and cooperation both within and outside the archival world are discussed, emphasising that the “possibilities are endless” when archivists bring the archives “out of the box and into the world”.

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.

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.

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.

Introduction Gene fusions (GFs) are genomic alterations with oncogenic potential. This class of alterations is highly actionable with targeted therapies showing high rates of durable responses in agnostic settings such as tumors with NTRK fusions. Recurrent GFs are rare in metastatic breast cancer (mBC) and ESR1 fusions drive endocrine resistance. AURORA is a molecular screening program for patients with mBC early in the course of their disease, collecting primary tumors, metastatic samples, liquid samples and longitudinal clinical follow-up data. Experimental procedures Patients are enrolled at the diagnosis of metastatic disease or after 1 line of therapy. Targeted gene sequencing of 411 BC genes is performed on the primary tumor, a metastatic sample, whole blood for germline variants and, for a subset of these genes, on tumor DNA from a baseline plasma sample. RNA-seq is performed on primary and metastatic tumor tissue samples. GFs were identified by rnafusion, a pipeline implementing 5 widely-used tools. Only GFs detected by at least 3 tools or 2 tools and 1 GF database match were considered for downstream analysis. Selected GFs were validated in-silico by FusionInspector. GFs detected in independent human normal tissue datasets were filtered out. Findings were correlated to available genomics, transcriptomics and clinical data. Results GFs data were generated from 316 paired primary/metastatic samples from 158 patients with curated clinical and genomic data: 97 ER+/HER2- (61.4%), 37 triple-negative (23.4%) and 24 HER2+ (15.2%). A total of 538 fusions were called in the primary samples (mean = 3.4 per sample) and 707 in the metastatic samples (mean = 4.5) with a validation rate of 72%. The gene fusion burden in metastatic samples was higher than in the primary (p Conclusions We report on the characterization of GFs in a large cohort of patients with mBC. Through the analysis of matched primary and metastatic tumor samples from 158 patients, we delineated the landscape of acquired gene fusions in BC. We observed a significant increase of gene fusion burden in metastatic compared to corresponding primary samples, involving key BC genes. Fusions involving actionable genes were associated with shorter PFS. Additional integrative analyses combining detected GFs and available genomics, gene expression and/or patients' treatments and outcomes are ongoing and will be presented during the meeting. Citation Format: Matteo Benelli, Chiara Biagioni, Danai Fimereli, Florentine S. Hilbers, Claudia De Angelis, Ana Vivancos, David Venet, Andrea Vingiani, Alexandre Irrthum, Veerle Van Dooren, Peter Willem Vuylsteke, Sonia Servitja, Jorge Reis-Filho, Giuseppe Curigliano, Mafalda Oliveira, Martine Piccart, Philippe Aftimos. Characterization of gene fusions in paired primary and metastatic samples of breast cancer in the AURORA molecular screening program [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2488.

Introduction: CDK4/6 inhibitors, such as palbociclib, target the cyclin-dependent kinases 4 and 6, essential for cell cycle regulation. Preclinical studies have suggested potential determinants of palbociclib sensitivity and clinical data have recently indicated loss-of function mutations in RB1 and increased expression of CCNE1 as potential biomarkers of resistance in a fraction of breast cancer (BC) patients. In line with these studies, using a panel of resistant luminal BC cell lines made resistant to palbociclib we recently demonstrated a common transcriptional signature of resistance to this agent, including the over-expression of CCNE1 and under-expression of RB1. Based on these data, we developed a minimal signature of de-novo resistance to palbociclib (ratio between CCNE1 and RB1, CCNE1/RB1) and demonstrated its prognostic role in both preclinical and clinical datasets. Gene expression signatures, such as CCNE1/RB1, are limited to the analysis of tissue samples because they are not easily detectable in liquid biopsies. Based on the key role DNA-methylation (DNAm) plays in regulating gene expression in human cells, we hypothesized that DNAm could be used as surrogate of our gene expression signature, with the further advantage of being easily adapted to non-invasive screenings. Exploiting this concept, here we report on a DNAm-based signature mirroring CCNE1/RB1 and its potential role in predicting resistance to palbociclib in BC patients. Materials and methods: Seven estrogen receptor positive breast cancer cell lines with acquired in vitro resistance to palbociclib were used. DNAm profiles of untreated parental cells and of their palbociclib-resistant counterparts were generated through Illumina Infinium MethylationEPIC BeadChip. For each cell line, candidate DNAm sites associated with resistance to palbociclib were selected by differential DNAm analysis in resistant versus sensitive counterparts. To identify DNAm sites/regions distinguishing CCNE1/RB1 high vs low patients, RNA-seq data from ER+/HER2- patients in the TCGA-BRCA dataset were considered and patients were classified as CCNE1/RB1 high (≥90% quantile) or low (≤10% quantile). Matched DNAm data were exploited to perform differential DNAm analysis between CCNE1/RB1 high and low samples by Rocker-meth, a tool recently developed by our group to perform differential DNAm analysis. The pharmacogenomic cancerrxgene dataset was used to test the association between the CCNE1/RB1 DNAm signature and the measure of effectiveness of palbociclib across multiple BC cell lines. Results: From the differential methylation analysis of CCNE1/RB1 high versus low ER+/HER2- BC patients from the TCGA-BRCA dataset we identified the set of sites and regions that compose our DNAm-based signature. In-silico functional analysis of differentially methylated sites and regions showed significant enrichment of regulatory-related terms. Both hyper- and hypo- DNAm sites in resistant versus untreated sensitive cell lines were significantly enriched in the set of differentially methylated sites in CCNE1/RB1 high versus low TCGA samples. In the cancerrxgene dataset, the CCNE1/RB1 DNAm signature correlated with higher IC50 in breast cancer cell lines. Conclusions: This study shows that DNAm is a potential predictor of resistance to palbociclib in ER+/HER2- patients. The DNAm-based signature mirroring CCNE1/RB1 developed using TCGA-BRCA dataset was validated in our preclinical BC cell lines and associated with effectiveness of palbociclib in an independent BC cell line dataset. Ongoing studies on the analysis of DNAm in liquid biopsies of metastatic luminal BC patients will evaluate the reliability of this DNAm signature in predicting resistance to palbociclib. Citation Format: Matteo Benelli, Martina Bonechi, Dario Romagnoli, Chiara Biagioni, Ilenia Migliaccio, Francesca De Luca, Francesca Galardi, Angelo Di Leo, Luca Malorni. A DNA-methylation signature to predict resistance to the CDK4/6 inhibitor palbociclib [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 P4-04-07.

CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb. CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation., Biomarkers: Prognostic indicator for resistance to cell cycle–targeted drug The expression levels of two genes involved in controlling the cell cycle offers a promising predictive indicator of whether women with hormone receptor–positive breast cancer are likely to develop resistance to the drug palbociclib. A team led by Luca Malorni and Ilenia Migliaccio from the Hospital of Prato, Italy, analyzed gene and protein profiles in a panel of breast cancer cell lines with differential sensitivity to palbociclib. The researchers found that, compared to palbociclib-sensitive cell lines, those that were drug-resistant tended to have elevated expression of one cell-cycle–related gene, called CCNE1, and reduced expression of another, called Rb. The ratio of CCNE1:Rb expression proved to be an accurate prognostic factor among patients enrolled in a clinical trial of palbociclib, performing better than either CCNE1 or Rb levels on their own.

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.

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.

CDK4/6 inhibitors, such as palbociclib, target the cyclin-dependent kinases 4 and 6, essential for cell cycle regulation. CDK4/6 inhibitors are established treatment options in patients with endocrine receptor positive, HER2 negative metastatic breast cancer (BC). We recently conducted a transcriptomic study of seven palbociclib resistant luminal BC cell lines, demonstrating a common transcriptional signature of resistance to palbociclib, including the over-expression of CCNE1 and under-expression of RB1. Genomic analysis based on ddPCR and whole exome sequencing (WES) demonstrated CCNE1 over-expression due to gene amplification in only two cell lines, and RB1 under-expression due to gene deletion in 3 out of 7 cell lines. This prompts the hypothesis that more complex molecular mechanisms of CCNE1 over-expression / RB1 under-expression may exist. To improve the characterization of the molecular mechanisms underlying the resistance to palbociclib, we performed single-cell transcriptomic profiling (single-cell RNA-seq, scRNA-seq) of seven BC cell line resistant to palbociclib and their sensitive counterparts. Cells were isolated using the Bio-Rad ddSEQ single cell isolator and sequenced in order to obtain about 100,000 reads per cell. A total of 10,557 cells with at least 2,000 genes read per cell were selected for downstream analysis, corresponding to 5,116 palbociclib sensitive cells and 5,441 palbociclib resistant cells. Considering the 5,000 most variable genes, dimensionality reduction analysis based on t-distributed stochastic neighbor embedding (tSNE) clearly segregated cells depending on their cell type, as expected based on previously generated bulk RNA-seq data. Using previously identified top cell-type specific differentially expressed genes between resistant and sensitive cell lines (absolute log2(fold-change) > 0.5), dimensionality reduction analysis correctly classified 98.2% of cells based on their sensitivity/resistant status, demonstrating the quality of our scRNA-seq data. Interestingly, we observed a small fraction ( This study represents, to our knowledge, the first attempt to generate a single-cell transcriptomic profiling of a large panel of luminal BC cell lines resistant to palbociclib. Results from this study might lead to the identification of new biomarkers of de-novo and acquired resistance to palbociclib, which would assist to better personalize treatment of patients with endocrine receptor positive BC. Citation Format: Matteo Benelli, Giulia Boccalini, Dario Romagnoli, Martina Bonechi, Chiara Biagioni, Rachel Schiff, Carmine De Angelis, Angelo Di Leo, Ilenia Migliaccio, Luca Malorni. Single-cell transcriptomic characterization of luminal breast cancer cell lines with acquired resistance to the CDK4/6 inhibitor palbociclib [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 3012.