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Background Trastuzumab is the cornerstone of adjuvant therapy for HER2-positive breast cancer (BC), but due to de novo or primary resistance, >20% of early-stage patients become resistant. Thus, predicting the mechanisms of trastuzumab resistance would facilitate the planning of specific therapeutic strategies. We performed a case-control study of 26 HER2-positive BC patients who relapsed within 5 years of starting adjuvant treatment and 26 controls, no relapsed, analyzing an extensive gene expression profile. Methods Total RNA was isolated from formalin-fixed paraffin-embedded primary tumors with AllPrep DNA/RNA FFPE Kit (Qiagen) and quantified by Nanodrop, before performing Nanostring gene expression profiling. nCounter Breast Cancer 360 Panel (Nanostring Technologies) was used according to the manufacturer’s instructions to analyze the expression of 770 genes and important signatures for BC (e.g. PAM50). For the statistical analyses, genes were normalized using a ratio of the expression value to the geometric mean of all housekeeping genes on the panel. Housekeeper-normalized data were then log(2) transformed. These data were evaluated by unsupervised selection of genes using consensus clustering with the Partitioning Around Medoids algorithm followed by the association of cluster “representative” genes (i.e. the medoids) with respect to the presence of relapse. The optimal number of clusters was determined by inspecting the consensus matrix cumulative distribution. Given the small sample size, the association between the "representative" genes, demographic and clinical covariates and the risk of relapse was evaluated using a logistic regression model with ELASTIC-NET regularization with α and λ tuned by 5-fold cross-validation. Penalized regression coefficients β are reported. The analyses were performed using R version 4.0.4 and package “ConsensusClusterPlus” and “glmnet”. Results Patient clinical characteristics are reported in Table 1. Relapsed patients showed a higher tumor stage, larger tumor size and greater lymph node involvement than controls (Table 1). Six samples failed the quality control check and so the analyses on gene expression data were performed on 46 subjects who had a distribution of clinical covariates similar to that reported in Table 1. The PAM50 intrinsic subtype, Risk Of Recurrence (ROR) score and ROR category were not associated with relapse status (P = 0.5, 0.5 and 0.1, respectively). The consensus clustering analysis revealed that the optimal number of clusters was 5, and the corresponding medoids were: HDAC6 for cluster 1 (n1=192 genes), WNT4 for cluster 2 (n2=102 genes), BMPR2 for cluster 3 (n3=162 genes), PALB2 for cluster 4 (n4=191 genes), and PARP1 for cluster 5 (n5=84 genes). HDAC6 and BMPR2 showed the highest correlation (r = 0.53, P Table 1.Patient characteristics in relation to Trastuzumab outcome.VariableAllCasesControlsP(n=52)(n=26)(n=26)n(%)n(%)n(%)Age at start adiuvant therapyMean ± sd53.4 ± 11.953.1 ± 12.653.7 ± 11.40.854Menopausal StatusPre-menopause21(40.4)10(38.5)11(42.3)0.777Post-menopause31(59.6)16(61.5)15(57.7)Stage of DiseaseI9(18.8)2(8.0)7(30.4)0.001II20(41.7)7(28.0)13(56.5)III19(39.6)16(64.0)3(13.0)Unknown413Lymph Node StatusNegative18(36.0)4(16.7)14(53.9)0.008Positive32(64.0)20(83.3)12(46.2)Unknown220Tumor SizeT128(53.9)7(26.9)21(80.8) Citation Format: Sara Ravaioli, Roberta Maltoni, Elisabetta Petracci, Michela Palleschi, William Balzi, Francesca Pirini, Maria Maddalena Tumedei, Michele Zanoni, Michela Cortesi, Giovanni Martinelli, Andrea Rocca, Sara Bravaccini. HDAC6 is an unfavorable prognostic factor in HER2-positive breast cancer patients treated with adjuvant trastuzumab [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 P2-13-44.

Simple Summary Circulating tumor cells (CTCs) are rare cells found in the bloodstream of oncologic patients with a central role in the metastatic spread. In this study, we aim at exploring their heterogeneity levels in metastatic breast cancer patients focusing on single cell single nucleotide variant (SNV) and copy number aberration (CNA) analyses. Our results show high levels of heterogeneity, especially concerning SNVs. Further analysis revealed the presence of CNAs associated with breast tumorigenesis, while longitudinal CNA profiling was demonstrated to track clonal selection of CTCs during treatment. Despite this heterogeneity, we found a group of CTCs from different patients sharing common genomic aberrations, such as losses on 15q. Collectively, our findings demonstrate that single-cell molecular analyses could be exploited in future to better address therapeutic strategies. Further investigations to better characterize this mixed population are needed to understand its role in MBC. Circulating tumor cells' (CTCs) heterogeneity contributes to counteract their introduction in clinical practice. Through single-cell sequencing we aim at exploring CTC heterogeneity in metastatic breast cancer (MBC) patients. Single CTCs were isolated using DEPArray NxT. After whole genome amplification, libraries were prepared for copy number aberration (CNA) and single nucleotide variant (SNV) analysis and sequenced using Ion GeneStudio S5 and Illumina MiSeq, respectively. CTCs demonstrate distinctive mutational signatures but retain molecular traces of their common origin. CNA profiling identifies frequent aberrations involving critical genes in pathogenesis: gains of 1q (CCND1) and 11q (WNT3A), loss of 22q (CHEK2). The longitudinal single-CTC analysis allows tracking of clonal selection and the emergence of resistance-associated aberrations, such as gain of a region in 12q (CDK4). A group composed of CTCs from different patients sharing common traits emerges. Further analyses identify losses of 15q and enrichment of terms associated with pseudopodium formation as frequent and exclusive events. CTCs from MBC patients are heterogeneous, especially concerning their mutational status. The single-cell analysis allows the identification of aberrations associated with resistance, and is a candidate tool to better address treatment strategy. The translational significance of the group populated by similar CTCs should be elucidated.

Background Trastuzumab is the cornerstone of adjuvant therapy for HER2-positive breast cancer (BC), but up to 20% of patients relapse. We performed a nested case-control study comparing the gene expression profile of relapsed cases within 5 years from the start of adjuvant trastuzumab and a group of controls not relapsed, in order to understand potential resistance mechanisms and to allow developing alternative strategies.MethodsRNA was isolated from formalin-fixed paraffin-embedded primary tumors with AllPrep DNA/RNA FFPE Kit (Qiagen) and analyzed with nCounter Breast Cancer 360 Panel (Nanostring Technologies), according to manufacturer’s instructions. The analysis of differential expression was performed by DESeq2 R package, on raw counts assuming a negative binomial distribution and using the Benjamini-Hochberg procedure to control the False Discovery Rate (FDR). Pathway enrichment analysis on Differentially Expressed Genes (DEGs) was performed by STRING database v. 11.0. Fisher’s Exact test was used for categorical variables and Wilcoxon-Mann-Whitney test for the continuous ones to analyze the comparison between cases and controls for the clinical and genetic variables. ResultsConsidering a median of > 20 counts per gene as threshold, 653 genes were analyzed. Eight significant DEGs were found between cases and controls, with a FDR < 0,10: AGTR1, Receptor for angiotensin 2 (log2foldchange [log2FC]=-1.83; FDR=0.0017), PGR (log2FC=-2.41, FDR=0.016), ROCK1, Rho-associated protein kinase 1 (log2FC=1.26, FDR=0.042), ITPR1, Inositol 1,4,5-trisphosphate receptor type 1 (log2FC=-0.98, FDR=0.052), MMP3, Stromelysin-1 (log2FC=-0.58, FDR=0.097), MMP9, Matrix Metalloproteinase-9 (log2FC=1.15, FDR=0.097), TIE1, Tyrosine-protein kinase receptor-1 (log2FC= 1.0047, FDR=0.097) and GDF15, Growth Differentiation Factor 15 (log2FC= 1.30, FDR=0.099). After adjustment for multiplicity of the tests, no statistically significant associations were found between gene expression and clinical variables (e.g. tumor size, stage…). The most enriched KEGG pathways were: Estrogen signaling (3 genes out of 133, FDR=0.00088), Vascular smooth muscle contraction (3/119, FDR= 0.00088), cGMP-PKG signaling (3/160, FDR=0.00088), Proteoglycans in cancer (3/195, FDR= 0.00093), Cortisol synthesis and secretion (2/63, FDR=0.0041), Leukocyte transendothelial migration (2/112, FDR= 0.0061), TNF signaling (2/108, FDR=0.0061), IL-17 signaling (2/92, FDR=0.0061) and Calcium signaling (2/179, FDR=0.0078). Conclusions Our preliminary results indicate that altered expression of genes related to inflammation and antigen presentation, adhesion and migration are involved in trastuzumab resistance, highlighting the role of the immune system and of the tumor microenvironment in this particular subtype of BC. For HER2+ BC with extremely poor prognosis, we aim to validate these results, reproducing the genetic landscape of unfavorable prognostic factors, in order to identify new therapeutic targets and gene signatures, able to identify Trastuzumab resistance mechanisms. Table 1. Patient characteristicsGlobalCasesControlsP(42)(18)(24)N %Tumor SizeT1-T24379.631659.2627100.00 Citation Format: Sara Bravaccini, Sara Ravaioli, Roberta Maltoni, Elisabetta Petracci, Michela Palleschi, William Balzi, Francesca Pirini, Giovanni Martinelli, Andrea Rocca. Gene expression profile in HER2-positive breast cancer to predict outcome in patients treated with adjuvant trastuzumab [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 PS10-19.

Background: Immunohistochemistry (IHC) is conventionally used in clinical practice to define Breast Cancer (BC) subtypes, while PAM50 is useful in specific cases. The former is partly subjective and semi-quantitative, the latter is based on gene expression profile. We compared gene expression data obtained by NanoString BC 360™ panel with those obtained by RNAseq on 12 Formalin-Fixed Paraffin-Embedded (FFPE) BC samples. Methods: RNA was isolated from FFPE tumors and the quality was checked before performing NanoString BC 360™ assay and preparing RNAseq library (NEBNext Ultra II RNA Library Prep Kit, Illumina) following the manufacturers’ instructions. Libraries were sequenced on NextSeq500 (Illumina). Reads were aligned using Kallisto and raw read counts were normalized as Transcripts Per Million (TPM). PAM50 and Tumor Inflammation Signature (TIS) were determined on NanoString data. Two-sided t-test on TPM and on normalized NanoString counts was used to compare luminal and Triple Negative (TN) BCs (p Results: Of the 12 BC samples, 3 were luminal A-like, 3 luminal B (HER2+)-like and 6 TN by IHC (Table 1). Pearson’s correlation coefficient between NanoString counts and RNAseq TPM was 0.72. Out of the 734 genes shared by both methods, those differentially expressed between luminal and TN BCs, defined based on IHC, were 135 according to RNAseq TPM, 155 according to NanoString counts and 88 according to both methods. These 88 genes belonged to “cell cycle”, “pathways in cancer”, “HIF-1 signaling”, “progesterone-mediated oocyte maturation”, “prostate cancer” and “basal cell carcinoma” KEGG pathways. When considering the whole transcriptome analyzed by RNAseq, the differential gene expression analysis between luminal and TN/basal BCs was performed according to both IHC and PAM50 classifications. Considering the former, there were 1143 differentially expressed genes out of a total of 20936 (5.5%) and the pathways involved were “fatty acid elongation”, “cell cycle”, “oocyte meiosis”, “p53 signaling” and “alcoholism”. The analysis on the latter classification showed 1608 differentially expressed genes out of 21048 (7.6%), and the enriched pathways were “cell cycle”, “p53 signaling”, “DNA replication”, “alcoholism” and “systemic lupus erythematosus”. Regarding the TIS, no significant differences were found between TN/basal and luminal BCs, comparing both IHC and the PAM50 subtypes. An expected heterogeneity was found among TNBCs, that showed 3 distinct signatures: basal-like immune activated, basal-like immune-suppressed and luminal androgen receptor. Conclusions: This preliminary analysis shows a good concordance between NanoString and RNAseq data. The choice of the method must take into account important factors such as the costs, the number of genes analyzed, the requirement of bioinformatic support, the working time. Further analyses on pathway enrichment will be presented. Table 1. Breast cancer samples characteristics.Sample ID Age Grade T N IHC subtype PAM50 Subtype TIS scoreTNBC signature 0172222aLumALumB7,0NA026031c2aTNBasal7,2BLIA037221cxTNLumA7,4LAR045231c0LumALumA7,5NA053531c1aTNBasal5,5BLIS0675320TNBasal6,2BLIS077931cxLumB HER2+LumB6,7NA0878220LumALumB7,7NA097431a2aTNBasal8,5BLIA106031c1LumB HER2+LumA8,2NA116621b0LumB HER2+LumA6,1NA1241321aTNBasal7,5BLIATIS: Tumor Inflammation Signature; BLIA: Basal-like Immune Activated; BLIS: Basal-like Immune-Suppressed; LAR: Luminal Androgen Receptor. Citation Format: Andrea Rocca, Sara Ravaioli, Eugenio Fonzi, Iros Barozzi, Ylenia Perone, Luca Magnani, Francesca Pirini, Giovanni Martinelli, Sara Bravaccini. Breast cancer subtype classification using NanoString and RNAseq technologies [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 P4-07-06.

In the present work we perform the numerical characterization of the hydrodynamic and the hydroacoustic field of a notional submarine geometry (the BB2 submarine). Starting from a model-scale CFD simulation, the full scale acoustic signature of the submarine is derived. We use large eddy simulation with a wall-layer model and perform laboratory-scale numerical experiments at a value of Reynolds number equal to 1.2 x 106. The conditions are those of a wind-tunnel test. The study is carried out focusing on the submarine in its own basic configuration without propeller to highlight the acoustic properties of the hull by itself and to replicate the silent advancing running (where all the non-essential source of noise are shut down and the speed is minimum to minimize the propeller noise). In this configuration, the wake is the main source of noise. The computational hydroacoustic analysis is based on the Ffowcs-Williams and Hawkings equation, here considered in advective form, which is the most suitable form for wind-tunnel-like problems. The results show that the acoustic spectrum is broadband in the far field and the SPL evaluated in the near field contains the signature of coherent structures associated to the presence of the sail and appendages.

Although gene alterations of the RAS/RAF/MEK/ERK pathway are uncommon in breast cancer, this pathway is frequently activated in breast tumors, implying its role in tumor progression. We describe, after a revision of the literature, the frequency and types of gene alterations affecting this pathway in breast cancer by analyzing some public datasets from cBioPortal. Moreover, we consider their prognostic and predictive impact on treatment response, along with the role of transcriptomic predictors of RAS pathway activation. Our analysis shows that the driver alterations in RAS/RAF/MEK/ERK pathway-related genes are detected in 11% of primary breast cancers. The most frequently mutated genes are NF1 and KRAS, while copy number alterations mainly affect KRAS and BRAF, especially in basal-like tumors. The subgroup of patients carrying these alterations shows a worse prognosis; alterations in NF1 and RAF1 are associated with significantly reduced breast-cancer-specific survival in multivariate analysis. The literature review shows that the pathway is implicated, either by genetic or epigenetic alterations or by signaling network adaptations, in the mechanisms of sensitivity and resistance to a wide range of drugs used in the treatment of breast cancer. A thorough understanding of these alterations is critical for developing combination therapies that can delay or overcome drug resistance.

Resistant tumours are thought to arise from the action of Darwinian selection on genetically heterogenous cancer cell populations. However, simple clonal selection is inadequate to describe the late relapses often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. Here, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare subpopulation of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. We find evidence for sub-clonal expression of a PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers., The development of resistance to endocrine therapy is a significant, clinical problem in breast cancer. Here, the authors identify a rare subpopulation of cells that drive resistance following transcriptional reprogramming.

Background: There are no well-established chemotherapy regimens for metastatic triple negative breast cancer. The combination of a microtubule inhibitor (eribulin) with a nucleoside analog (gemcitabine) may synergistically induce tumor cell death, especially in tumors like triple negative breast cancers (TNBC) characterized by high cell proliferation, aggressive tumor behavior, and chemo-resistance. Materials and Methods:This is an open-label, national multicenter phase II study evaluating the combination of eribulin (0.88 mg/m2) plus gemcitabine (1000 mg/m2) on day 1 and 8, q21 as either first- or second-line treatment of locally advanced or metastatic TNBC.The primary endpoint was the objective response rate (ORR) for evaluable patients (pts). The study was designed according to the Simon's two stage optimal design. We chose the lower activity (p0) of 0.20 and target activity level (p1) of 0.35. A prospective, molecular correlative study has been being carried out on germinal DNA of study population to assess the role of BRCA mutations and single nucleotide polymorphisms (SNPs) in predicting efficacy and toxicity of the combination regimen. Results: From July 2013 to September 2016, 83 evaluable pts (37 in the first stage, 46 in the second one) were enrolled. They received a median number of 6 cycles of treatment (range 1-24). The ORR (CR+PR) was 37.35% (90% CI: 28.47-46.93) and the clinical benefit rate (CR+PR+SD ≥ 24wks) was 48.78% (90% CI: 39.24%-58.39%). The most common grade 3-4 adverse events (> 10% of patients) were neutropenia and liver toxicity. With a median follow-up of 28.8 months, the median progression-free survival (PFS) and overall survival (OS) were 5.1 months (95% CI: 4.2-7.0) and 14.7 months (95% CI: 10.2-20.0), respectively. BRCA1/2 deleterious mutations were observed in 15 (22%) out of 68 genotyped pts. Women with BRCA1/2 mutations were associated with worse ORR, PFS and OS than those with BRCA1/2 wild-type. A panel of SNPs in genes of study drug metabolism pathways was evaluated. Among these, CYP3A4 392A >G and FGD4 2044236G>A SNPs were associated with greater liver toxicity by logistic regression analysis. Furthermore, CDA*2 79A>C, RRM1 2455 A>G, and CYP2C8 416G>A SNPs were associated with poorer overall survival by Cox proportional hazards model. Conclusions:The combination of eribulin and gemcitabine shows promising activity and a moderate toxicity profile in metastatic TNBC. BRCA status and pharmacogenetics tests may help identify pts with high probability of response with negligible toxicity. Citation Format: Musolino A, Cavanna L, Boggiani D, Zamagni C, Frassoldati A, Caldara A, Rocca A, Gori S, Piacentini F, Berardi R, Brandes AA, Foglietta J, Villa F, Pellegrino B, Todeschini R, Tognetto M, Naldi N, Bortesi B, Boni L, Montemurro F, Ardizzoni A. Phase II study of eribulin in combination with gemcitabine for the treatment of patients with locally advanced or metastatic triple negative breast cancer (ERIGE Trial). Clinical and pharmacogenetic results on behalf of the Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC) [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 P1-14-05.

Purpose Androgen receptor (AR) expression occurs in up to 86% of human epidermal growth factor receptor 2-positive (HER2+) breast cancers. In vitro, AR inhibitors enhance antitumor activity of trastuzumab, an anti-HER2 antibody, in trastuzumab-resistant HER2+ cell lines. This open-label, single-arm, phase II study evaluated the efficacy and safety of enzalutamide, an AR-signaling inhibitor, in patients with advanced HER2+ AR+ breast cancer previously treated with trastuzumab. Methods Eligible patients had measurable or non-measurable evaluable disease per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, Eastern Cooperative Oncology Group status ≤ 1, no history of brain metastases, and previously received ≥ 1 anti-HER2 regimen for advanced disease. Patients received 160 mg oral enzalutamide daily and 6 mg/kg intravenous trastuzumab every 21 days until disease progression or unacceptable toxicity. Primary end point was clinical benefit rate at 24 weeks (CBR24); secondary end points included progression-free survival (PFS) and safety. Results Overall, 103 women were enrolled [median age 60 years (range 34–83)]; 62% had received ≥ 3 lines of prior anti-HER2 therapy. CBR24, comprising patients with confirmed partial responses (5%) and durable stable disease at 24 weeks (19%), was 24% in the efficacy evaluable set (n = 89). CBR24 did not seem related to AR-expression levels or hormone receptor status. Median PFS was 3.4 months (95% confidence interval 2.0–3.8). Overall, 97 (94%) patients experienced treatment-emergent adverse events (TEAEs), with fatigue most common (34%). Dyspnea (4%) and malignant neoplasm progression (3%) were the only TEAEs grade ≥ 3 reported in ≥ 3 patients. 22 patients (21%) reported serious TEAEs. Four patients (4%) experienced fatal, non-drug-related TEAEs. Conclusions Enzalutamide plus trastuzumab was well tolerated, and a subset of patients in this heavily pretreated population had durable disease control. Determination of biomarkers is needed to identify patients most likely to benefit from this combination. ClinicalTrials.gov number NCT02091960

Hormone dependent breast cancer (HDBC) is the most commonly diagnosed tumor type in women. Adjuvant endocrine therapies (ET) have been the cornerstone in the clinical management of HDBC patients for over forty years. A vast proportion of HDBC patients incur long periods of clinical dormancy following ET, with tumour awakening appearing at a steady pace for up to 25 years (Pan et al., 2017). Extensive genomic studies have demonstrated that 15-30% of clinical relapses develop recurrent genomic changes which contribute to drug resistance (i.e. ESR1 activating mutations) (Bertucci et al., 2019; Magnani et al., 2017; Razavi et al., 2018). However, even in these cases, there is no conclusive evidence around the pre-existence vs. de novo nature of these events. We previously showed that ETs can trigger and select for dormancy in subpopulations of breast cancer (Hong et al., 2019). In this work we took two novel approaches to investigate the dormancy and awakening roadmap of HDBC cells at unprecedented detail. Firstly, we leveraged a rare cohort of n=5 patients which were treated with primary adjuvant ETs in the absence of surgery (TRACING-HT) to dissect the contribution of genomic aberrations to tumor awakening. Next, we developed a first of its kind evolutionary study in vitro to systematically annotate cancer cells adaptive strategies at single cell level in unperturbed systems during a period of several months (TRADITIOM). Collectively our data suggest that ETs steer HDBC cells into an inherently unstable dormant state. Over time, routes to awakening emerge sporadically and spontaneously in single lineages. Each dormant cell retains an intrinsic awakening probability which we propose is a function of epigenetic decay. Awakening occurs without an external trigger and involves multiple apparent endpoint phenotypes that cannot be fully explained by conventional Darwinian genetic selection processes. Finally, our data show that common genetic hits associated with resistance happen downstream of awakening. Overall, our data have uncovered previously unsuspected roles for stochastic nongenetic events during dormancy with profound clinical implications.

Background The combination of a microtubule inhibitor (eribulin) with a nucleoside analog (gemcitabine) may synergistically induce tumor cell death, particularly in triple negative breast cancer (TNBC) characterized by high cell proliferation, aggressive behavior, and chemo-resistance. Patients and methods This is an open-label, multicenter phase II study evaluating the combination of eribulin (0.88 mg/m2) plus gemcitabine (1000 mg/m2) on days 1 and 8 of a 21-day cycle as either first- or second-line treatment of locally advanced or metastatic TNBC. The primary endpoint was the objective response for evaluable patients. A prospective, molecular correlative study was carried out to assess the role of germinal BRCA pathogenic variants and single nucleotide polymorphisms (SNPs) in predicting efficacy and toxicity of the combination regimen. Results From July 2013 to September 2016, 83 evaluable patients were enrolled. They received a median number of six cycles of treatment. An overall response rate (ORR) of 37.3% (31 patients) was observed, with a complete response rate of 2.4% and a partial response rate of 34.9%; the clinical benefit rate was 48.8%. With a median follow-up of 28.8 months, the median response duration was 6.6 months, the median progression-free survival (PFS) was 5.1 months, and the median overall survival (OS) was 14.5 months. The most common grade 3-4 adverse events were aminotransferase elevation (in 25% of the patients) and neutropenia (in 23.8%). Women with BRCA1/2 pathogenic variants were associated with worse ORR, PFS, and OS than BRCA1/2 wild-type carriers. CYP3A4 and FGD4 SNPs were associated with increased risk of liver toxicity. Three different SNPs in CDA∗2, RRM1, and CYP2C8 genes were significantly associated with poorer OS. Conclusions The combination of eribulin and gemcitabine showed promising activity and a moderate toxicity profile in metastatic TNBC. BRCA status and pharmacogenetics tests may help identify patients with high probability of response with negligible toxicity. EudraCT number 2012-003505-10., Highlights • Eribulin plus gemcitabine showed a remarkable best ORR of 37.3% and a clinical benefit rate of 48.8%. • The most common grade 3/4 toxicities were liver toxicity and neutropenia without febrile neutropenia. • The study regimen partially lost its efficacy in patients harboring BRCA1/2 pathogenic variants. • SNPs in CYP3A4 and FGD4 genes were associated with increased risk of liver toxicity. • Three different SNPs in CDA∗2, RRM1, and CYP2C8 genes were significantly associated with poorer OS.

Purpose: The mTOR complex C1 (mTORC1) inhibitor everolimus in combination with the aromatase inhibitor exemestane is an effective treatment for patients with hormone receptor—positive (HR+), HER2-negative (HER2−), advanced breast cancer (HR+/HER2− aBC). However, everolimus can cause hyperglycemia and hyperinsulinemia, which could reactivate the PI3K/protein kinase B (AKT)/mTORC1 pathway and induce tumor resistance to everolimus. Experimental Design: We conducted a multicenter, retrospective, Italian study to investigate the impact of baseline and on-treatment (i.e., during first 3 months of therapy) blood glucose levels on progression-free survival (PFS) in patients with HR+/HER2− aBC treated with everolimus-exemestane. Results: We evaluated 809 patients with HR+/HER2− aBC treated with everolimus-exemestane as any line of therapy for advanced disease. When evaluated as dichotomous variables, baseline and on-treatment glycemia were not significantly associated with PFS. However, when blood glucose concentration was evaluated as a continuous variable, a multivariable model accounting for clinically relevant patient- and tumor-related variables revealed that both baseline and on-treatment glycemia are associated with PFS, and this association is largely attributable to their interaction. In particular, patients who are normoglycemic at baseline and experience on-treatment diabetes have lower PFS compared with patients who are already hyperglycemic at baseline and experience diabetes during everolimus-exemestane therapy (median PFS, 6.34 vs. 10.32 months; HR, 1.76; 95% confidence interval, 1.15–2.69; P = 0.008). Conclusions: The impact of on-treatment glycemia on the efficacy of everolimus-exemestane therapy in patients with HR+/HER2− aBC depends on baseline glycemia. This study lays the foundations for investigating novel therapeutic approaches to target the glucose/insulin axis in combination with PI3K/AKT/mTORC1 inhibitors in patients with HR+/HER2− aBC.

Background: The aim of this study was to improve activity over single human epidermal growth factor receptor 2 (HER2)-blockade sequential neaodjuvant regimens for HER2-positive breast cancer, by exploiting the concomitant administration of trastuzumab, taxane and anthracycline, while restraining cardiac toxicity with use of liposomal doxorubicin, and by adding metformin, based on preliminary evidence of antitumor activity. Patients and methods: This multi-center, single-arm, two-stage phase II trial, assessed the safety and the activity of a new treatment regimen for HER2-positive, early or locally advanced breast cancer. Patients received six 21-day cycles of non-pegylated liposomal doxorubicin, 50 mg/m2 intravenously (i.v.) on day 1, docetaxel, 30 mg/m2 i.v. on days 2 and 9, trastuzumab, 2 mg/kg/week i.v. on days 2, 9, and 16 (with 4 mg/kg loading dose), in association with metformin 1000 mg orally twice daily. The primary endpoint was the rate of pathological complete response (pCR) in the breast and axilla (ypT0/is ypN0). A subgroup of patients performed a 3-deoxy-3-18F-fluorothymidine positron emission tomography (FLT-PET) at baseline and after one cycle. Results: Among 47 evaluable patients, there were 18 pCR [38.3%, 95% confidence interval (CI) 24.5–53.6%]. A negative estrogen-receptor status, high Ki67, and histological grade 3 were related with pCR, although only grade reached statistical significance. FLT-PET maximum standardized uptake value after one cycle was inversely related to pCR in the breast (odds ratio 0.29, 95% CI 0.06–1.30, p = 0.11). Toxicity included grade 3–4 neutropenia in 70% and febrile neutropenia in 4% of patients, grade 1–2 nausea/vomiting in 60%/38%, and grade 3 in 4%/2%, respectively, grade 1–2 diarrhea in 72%, and grade 3 in 6%. There were two cases of reversible grade 2 left-ventricular ejection-fraction decrease, and one case of sharp troponin-T increase. Conclusions: The concomitant administration of trastuzumab, liposomal doxorubicin, docetaxel, and metformin is safe and shows good activity, but does not appear to improve activity over conventional sequential regimens.

CDK4/6 inhibitors (CDK4/6i) are recommended in patients with estrogen receptor (ER)-positive, HER2-negative advanced breast cancer (ABC). Up to now, no prognostic biomarkers have been identified in this setting. We retrospectively analyzed the expression of progesterone receptor (PR) and Ki67, assessed by immunohistochemistry, in 71 ABC patients treated with CDK4/6i and analyzed the impact of these markers on progression-free survival (PFS). The majority of patients 63/71 (88.7%) received palbociclib, 4 (5.6%) received ribociclib, and 4 (5.6%) received abemaciclib. A higher median value of Ki67 was observed in cases undergoing second-line treatment (p = 0.047), whereas the luminal B subtype was more prevalent (p = 0.005). In the univariate analysis of the first-line setting, luminal A subtype showed a trend towards a correlation with a longer PFS (p = 0.053). A higher continuous Ki67 value led to a significantly shorter PFS. When the interaction between pathological characteristics and line of treatment was considered, luminal B subtype showed a significantly (p = 0.043) worse outcome (Hazard Ratio (HR) 2.84, 1.03&ndash, 7.82 95% Confidence Interval (CI)). PFS in patients undergoing endocrine therapy plus CDK4/6i was inversely correlated with Ki67 expression but not with PR, suggesting that tumor proliferation has a greater impact on cell cycle inhibitors combined with endocrine therapy than PR expression.

Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an "all from one tube" format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2-) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an "all from one tube" format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies.

Background:In the Italian Breast Cancer Intergroup Studies (IBIS) 3 phase III trial, we compared cyclophosphamide, methotrexate, 5-fluorouracil (CMF) alone to sequential epirubicin/CMF regimens in patients with rapidly proliferating early breast cancer (RPEBC). We performed a post hoc analysis in the subgroup of patients with hormone-receptor-positive RPEBC on the prognostic role of progesterone receptor (PgR) status.Methods:RPEBC was defined by thymidine labeling index (TLI) >3% or grade 3 or S-phase >10% or Ki67 >20%. We analyzed 466 patients with hormone-receptor-positive RPEBC receiving sequential epirubicin/CMF regimens followed by tamoxifen, and for whom the status of ER and PgR was available.Results:Considering both cut-off values of 10% and 20%, PgR expression was significantly associated with age, menopausal status, and ER expression; HER2 status was associated with PgR status only at a cutoff value of 20% PgR. Upon univariate analysis, tumor size, nodal status, and PgR were significantly associated with disease-free survival (DFS) and overall survival (OS), while age class and local treatment type were associated only with DFS. Patients with PgR Conclusions:Our results highlight the independent prognostic relevance of PgR expression in patients with hormone-receptor-positive RPEBC treated with adjuvant chemotherapy and endocrine therapy, where the definition of prognostic subgroups is still a major need.

Rationale Patients with, or who develop, metastatic breast cancer have a 5-year relative survival of about 25%. Endocrine therapy clearly improves outcomes in patients with estrogen receptor-positive breast cancer. In the metastatic setting, the primary goal of treatment is to maintain long-term disease control with good quality of life. Rarely, exceptional responders achieve durable disease control, and potential cures cannot be ruled out. Patient concerns We report the case of a 39-year-old woman with primary breast cancer and associated synchronous bone metastases, who experienced a disease response of 12 years with hormonal therapy as maintenance after first line chemotherapy, with a good toxicity profile. Diagnosis The patient was diagnosed with estrogen receptor + human epidermal growth factor receptor 2 (HER2)- metastatic breast cancer with synchronous bone metastases. Interventions This patient was treated with chemotherapy for 6 cycles as a first-line therapy following by endocrine treatment given as a maintenance therapy. Outcomes Our patient experienced a progression-free survival >12 years with an exceptionally good quality of life. Lessons Our anecdotal experience highlights the existence of exceptional responders among patients with hormone receptor-positive metastatic breast cancer, who achieve clinical remission and durable disease control with endocrine therapy. Being able to identify these patients could help in the selection of the best treatment option among the many available.

Circulating tumor cells (CTCs) are a rare population of cells representing a key player in the metastatic cascade. They are recognized as a validated tool for the identification of patients with a higher risk of relapse, including those diagnosed with breast cancer (BC). However, CTCs are characterized by high levels of heterogeneity that also involve copy number alterations (CNAs), structural variations associated with gene dosage changes. In this study, single CTCs were isolated from the peripheral blood of 11 early-stage BC patients at different time points. A label-free enrichment of CTCs was performed using OncoQuick, and single CTCs were isolated using DEPArray. Libraries were prepared from single CTCs and DNA extracted from matched tumor tissues for a whole-genome low-coverage next-generation sequencing (NGS) analysis using the Ion Torrent S5 System. The analysis of the CNA burden highlighted that CTCs had different degrees of aberration based on the time point and subtype. CTCs were found even six months after surgery and shared CNAs with matched tumor tissue. Tumor-associated CNAs that were recurrent in CTCs were patient-specific, and some alterations involved regions associated with BC and survival (i.e., gains at 1q21-23 and 5p15.33). The enrichment analysis emphasized the involvement of aberrations of terms, associated in particular with interferon (IFN) signaling. Collectively, our findings reveal that these aberrations may contribute to understanding the molecular mechanisms involving CTC-related processes and their survival ability in occult niches, supporting the goal of exploiting their application in patients&rsquo, surveillance and follow-up.

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Women with a diagnosis of ductal carcinoma in situ (DCIS) have a high risk of developing a second breast event (SBE). The immune system might play a role in trying to prevent a SBE. Patients diagnosed with DCIS were identified in the population-based cancer registry of Area Vasta Romagna from 1997 to 2010. Median follow-up is 8.5 years. Tumor-infiltrating lymphocytes (TILs) were evaluated both in index DCIS and in SBE. The main endpoint was to assess the association between TILs' levels in index DCIS and risk of a SBE. Out of 496 DCIS patients, 100 SBEs (20.2%) were identified: 55 ipsilateral (11.1%) and 43 contralateral (8.7%). The distribution of TILs was heterogeneous, but significantly associated with grade, necrosis, screen detection and type of surgery. Patients stratified according to TILs percentage (≤5% and >5%) did not show a statistically significant difference in the 5-year cumulative incidence of SBEs: 14.9% (95% CI 11.3–19.1) and 11.0% (95% CI, 6.9–16.2), respectively (p = 0.147). In the subgroup of patients who did not receive radiotherapy, TILs >5% were associated with a reduced risk of SBE (HR 0.34, 95% CI 0.14–0.82, p = 0.016). Although we did not find any significant association between TILs and SBE, further studies evaluating their role according to radiotherapy are warranted.

BACKGROUND: Pathological complete response (pCR) is rare in hormone receptor-positive (HR+) HER2-negative breast cancer (BC) treated with either endocrine therapy (ET) or chemotherapy. Radical resection of locoregional relapse, although potentially curative in some cases, is challenging when the tumor invades critical structures. The oral cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with ET has obtained a significant increase in objective response rates and progression-free survival in patients with advanced BC and is now being evaluated in the neoadjuvant setting. We present a clinical case of a patient with an inoperable locoregional relapse of HR+ HER2-negative BC who experienced pCR after treatment with palbociclib. CASE SUMMARY: We report the clinical case of a 60-year-old patient who presented with an inoperable locoregional relapse of HR+, HER2-negative BC 10 years after the diagnosis of the primary tumor. During a routine follow-up visit, breast magnetic resonance imaging and positron emission tomography/computed tomography revealed a 4-cm lesion in the right subclavicular region, infiltrating the chest wall and extending to the subclavian vessels, but without bone or visceral involvement. Treatment was begun with palbociclib plus letrozole, converting the disease to operability over a period of 6 mo. Surgery was performed and a pCR achieved. Of note, during treatment the patient experienced a very uncommon toxicity characterized by burning tongue and glossodynia associated with dysgeusia, paresthesia, dysesthesia, and xerostomia. A reduction in the dose of palbociclib did not provide relief and treatment with the inhibitor was thus discontinued, resolving the tongue symptoms. Laboratory exams were unremarkable. Given that this was a late relapse, the tumor was classified as endocrine-sensitive, a condition associated with high sensitivity to palbociclib. CONCLUSION: This case highlights the potential of the cyclin-dependent kinase 4/6 inhibitor plus ET combination to achieve pCR in locoregional relapse of BC, enabling surgical resection of a lesion initially considered inoperable.

Fulvestrant is the first selective estrogen receptor (ER) downregulator available in clinical practice. It is a pure antiestrogen with no agonistic effects, leading to degradation of ER alpha, with activity in tamoxifen-resistant breast cancer (BC) models. Pharmacokinetic and pharmacodynamic studies and several postmarketing clinical trials led to the definition of the optimal dose at 500 mg intramuscularly on days 1, 15, and 29 and then every 28 days. Targeting ER alpha, fulvestrant is a cornerstone of treatment in luminal BCs, whose growth is largely driven by the ER pathway. In endocrine therapy-naive patients with hormone receptor-positive, HER2- advanced BC (ABC), fulvestrant yielded significantly longer progression-free survival compared to anastrozole in the Phase III FALCON study. Due to its mechanism of action and pharmacokinetic properties, fulvestrant is an ideal backbone for combination therapies. Preclinical studies have shown synergism with drugs acting on signaling pathways involved in the development of endocrine resistance, among which the cyclin D/cyclin-dependent kinase 4-6/retinoblastoma pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin pathway, contributing to overcoming or delaying endocrine resistance. In the Phase III PALOMA-3 trial, a combination of the cyclin-dependent kinase 4/6 inhibitor palbociclib with fulvestrant significantly improved progression-free survival over fulvestrant alone in women with hormone receptor positive, HER2- ABC progressing during prior endocrine therapy. This led to approval of the combination in this clinical setting. Similar results were obtained with abemaciclib and ribociclib. Combination with pan-PI3K inhibitors, though showing some efficacy, was hampered by the toxicity of these agents, and studies in combinations with more selective inhibitors of the α-catalytic subunit of PI3K are ongoing. Fulvestrant has shown partial activity also in patients with tumors harboring mutations of the ESR1 gene. It is thus a key drug in the treatment of ABC, whose role in combination with new targeted agents is still evolving.

Background Androgen receptor (AR) is widely expressed in breast cancer (BC) but its role in estrogen receptor (ER)-positive tumors is still controversial. The AR/ER ratio has been reported to impact prognosis and response to antiestrogen endocrine therapy (ET). Methods We assessed whether AR in primary tumors and/or matched metastases is a predictor of efficacy of first-line ET in advanced BC. Patients who had received first-line ET (2002–2011) were recruited, while those given concomitant chemotherapy or trastuzumab or pretreated with > 2 lines of chemotherapy were excluded. ER, progesterone receptor (PgR), Ki67 and AR expression were assessed by immunohistochemistry, and HER2 mainly by fluorescent in-situ hybridization. Cut-offs of 1 and 10% immunostained cells were used to categorize AR expression. Results Among 102 evaluable patients, biomarkers were assessed in primary tumors in 70 cases and in metastases in 49, with 17 patients having both determinations. The overall concordance rate between primary tumors and metastases was 64.7% (95% CI 42%-87.4%) for AR status. AR status did not affect TTP significantly, whereas PgR and Ki67 status did. AR/PgR ≥0.96 was associated with a significantly shorter TTP (HR = 1.65, 95% CI 1.05-2.61, p = 0.028). AR status in primary tumors or metastases was not associated with progressive disease (PD) as best response. In contrast, Ki67 ≥ 20% and PgR

Background The presence of human papillomavirus (HPV) DNA in breast cancer (BC) tissues has been widely investigated in recent years. HPV DNA has been detected in ductal lavage fluids and in serum-derived extracellular vesicles of patients with benign or in situ breast lesions. However, there are no data attesting to its presence in liquid biopsies of different BC subtypes or to its impact on prognosis. Methods We analyzed a total of 72 serum samples for the presence of circulating HPV DNA, of which 20 were from luminal A BC (5 relapsed, 15 non relapsed), 17 from luminal B BC (5 relapsed, 12 non relapsed), 15 from triple-negative BC (6 relapsed and 9 non relapsed), 12 from HER2-positive BC (3 relapsed, 9 non relapsed) and 8 from healthy subjects. Circulating DNA was extracted and purified from 500 μl of serum by Qiamp DNA minikit (Qiagen, Milan, Italy) according to the manufacturer's instructions. HPV DNA was assessed by a high-throughput MALDI-TOF mass spectrometry-based method (Mass Array Platform, Agena Bioscience, Hamburg, Germany). The DNA target sequence was amplified by a multiplex PCR with HPV E6 or E7 gene-specific primers. A primer for primer extension annealing to the amplified product was extended at its 3′ terminal base for each HPV type. Results HPV DNA was detected in 5 BC patients but in none of the healthy controls. None of the serum samples analyzed showed HPV DNA types 16 or 18. Four of the 5 BC cases had high-risk HPV DNA (type 39,45,52,59) and one had low-risk HPV DNA (type 73). The 4 patients with high risk HPV DNA had low-grade cervical intraepithelial neoplasia (CIN 1) detected by Pap smear prior to the diagnosis of BC. No relation was found between HPV infection and tumor subtype or prognosis. Our in vitro studies also revealed the active release of HPV DNA into the extracellular vesicle compartment of cervical cancer cells. Table 1.Features of BC patients positive for circulating HPV DNA.Sample IDAge (years)GradingSubtypeDeathRelapseHPV type, riskCervical lesion17471Luminal A-No59, highCIN 139783Luminal B20/03/2015No45, highCIN 145462Luminal B-No39, highCIN 160583TN03/01/2011Yes73, lowHyperplasia63523HER2-positive-Yes52, highCIN 1TN, triple negative; CIN 1:low-grade cervical intraepithelial neoplasia Conclusions Our findings support the feasibility of HPV DNA evaluation by liquid biopsy in BC. They also suggest that circulating HPV DNA in BC patients might be of cervical tissue origin and that the presence of HPV DNA in BC may be a consequence of its spreading from virus-infected tissue such as that of the uterine cervix. Citation Format: Bravaccini S, Ravaioli S, Rocca A, Maltoni R, Cristalli C, Marasco E, De Carolis S, Cricca M, Bonafè M. Human papillomavirus (HPV) DNA detection in breast cancer by liquid biopsy: Something new on the horizon? [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 P2-04-01.

Ductal carcinoma in situ (DCIS) is a heterogeneous disease that has been investigated less extensively than invasive breast cancer. Women with DCIS are mainly treated with conservative surgery almost exclusively followed by radiotherapy. However, as radiation treatment is not always effective, the search for biomarkers capable of identifying DCIS lesions that could progress to invasive cancer is ongoing. Although conventional biomarkers have been thoroughly studied in invasive tumours, little is known about the role played by androgen receptor (AR), widely expressed in DCIS. A series of 42 DCIS patients treated with quadrantectomy and radiotherapy were followed for a period of up to 95 months. Of these, 11 had recurrent DCIS or progressed to invasive cancer. All tumours were analysed for clinical pathological features. Conventional biomarkers and androgen receptor expression were determined by immunohistochemistry. Our results showed that AR was higher in tumours of relapsed patients than non-relapsed patients (P value: 0.0005). Conversely, oestrogen receptor (ER) was higher, albeit not significantly, in non-relapsed patients than in relapsed patients. AR/ER ratio was considerably different in the two subgroups (P value: 0.0033). Area under the curve (AUC) values were 0.85 for AR and 0.80 for the AR/ER ratio. These preliminary results highlight the potentially important role of both AR and the AR/ER ratio as prognostic markers in DCIS.

// Gianluca Tedaldi 1 , Michela Tebaldi 1 , Valentina Zampiga 1 , Rita Danesi 2 , Valentina Arcangeli 3 , Mila Ravegnani 2 , Ilaria Cangini 1 , Francesca Pirini 1 , Elisabetta Petracci 4 , Andrea Rocca 5 , Fabio Falcini 2 , Dino Amadori 5 and Daniele Calistri 1 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 2 Romagna Cancer Registry, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 3 Department of Medical Oncology, Ospedale Infermi, Rimini, Italy 4 Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 5 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy Correspondence to: Daniele Calistri, email: daniele.calistri@irst.emr.it Keywords: hereditary breast and ovarian cancer, multiple-gene panel, next-generation sequencing, bilateral breast cancer, cancer predisposition Received: October 19, 2016 Accepted: March 14, 2017 Published: April 03, 2017 ABSTRACT As new genes predisposing to breast (BC) and ovarian cancer (OC) are constantly emerging, the use of panels of genes analyzed by Next-Generation Sequencing (NGS) is increasing in clinical diagnostics. The identification of a large number of new germline mutations allows for deeper knowledge of cancer predisposition, although raising many questions about patient management. BC and OC patients recruited by our counseling service between 2012-2015 were included in this study. DNA was extracted from peripheral blood and a panel of 94 genes involved in hereditary tumors was analyzed by NGS. Patient clinical features of BC and OC and cancer family history were collected and compared to the patient genetic profile. A total of 255 women were analyzed, 57 of whom had a pathogenic mutation in BRCA1/2 genes, and 17 carried pathogenic mutations in other genes, such as PALB2 , ATM , BRIP1 , RAD51D , MSH6 , PPM1D , RECQL4 , ERCC3 , TSC2 , SLX4 and other Fanconi anemia genes. Patients with a pathogenic mutation in genes other than BRCA1 and BRCA2 showed no significant difference from the BRCA1/2 -mutated carriers with respect to age at diagnosis and clinical features, suggesting that mutations in other genes could pose a high risk of cancer development. These patients had a much higher percentage of bilateral breast cancer (BBC) and a lower rate of OC than BRCA -mutated patients and patients with no pathogenic mutations: as a consequence, the surveillance protocol should be customized to the patient genetic characteristics.

Purpose Combination of anthracyclines with trastuzumab is hampered by cardiotoxicity. Pegylated liposomal doxorubicin and lapatinib could represent a safer alternative to combination therapy. Methods In this phase Ib study with 3 + 3 dose escalation design, patients with HER2-positive advanced breast cancer received pegylated liposomal doxorubicin 30 mg/m2 intravenously on day 1 plus lapatinib 1250 (level 1) or 1500 (level 2) mg/day orally on days 1–21 of each 21-day cycle. The aims were to establish the maximum tolerated dose at first cycle, and the activity and safety of multiple cycles. Results Nine patients out of 11 enrolled were evaluable: 3 at level 1 and 6 at level 2. No dose-limiting toxicities occurred at dose level 1, while 1 (grade 3 diarrhea) occurred at dose level 2, leading to the expansion of this cohort to 6 patients, with no further dose-limiting toxicities. Main grade 1–2 toxicities at first cycle were leucopenia, diarrhea, elevated transaminases, mucositis. Three patients had grade 3 toxicities at subsequent cycles, including colitis, anorexia, stomatitis plus hand-foot syndrome. One partial response, 5 disease stabilizations, and 3 disease progressions were reported. Conclusions Combination of pegylated liposomal doxorubicin and lapatinib is feasible and potentially active in pretreated HER2-positive advanced breast cancer patients. Trial registration NCT02131506 (ClinicalTrials.gov identifier).

// Roberta Maltoni 1 , Valentina Casadio 2, * , Sara Ravaioli 2, * , Flavia Foca 3 , Maria Maddalena Tumedei 2 , Samanta Salvi 2 , Filippo Martignano 2 , Daniele Calistri 2 , Andrea Rocca 1 , Alessio Schirone 1 , Dino Amadori 1 , Sara Bravaccini 2 1 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 2 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 3 Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy * These authors contributed equally to this work Correspondence to: Sara Bravaccini, email: sara.bravaccini@irst.emr.it Keywords: CF-DNA, HER2, PI3KCA, prognosis, breast cancer subtypes Received: August 20, 2016 Accepted: January 23, 2017 Published: February 06, 2017 ABSTRACT As conventional biomarkers for defining breast cancer (BC) subtypes are not always capable of predicting prognosis, search for new biomarkers which can be easily detected by liquid biopsy is ongoing. It has long been known that cell-free DNA (CF-DNA) could be a promising diagnostic and prognostic marker in different tumor types, although its prognostic value in BC is yet to be confirmed. This retrospective study evaluated the prognostic role of CF-DNA quantity and integrity of HER2 , MYC , BCAS1 and PI3KCA , which are frequently altered in BC. We collected 79 serum samples before surgery from women at first diagnosis of BC at Forli Hospital (Italy) from 2002 to 2010. Twenty-one relapsed and 58 non-relapsed patients were matched by subtype and age. Blood samples were also collected from 10 healthy donors. All samples were analyzed by Real Time PCR for CF-DNA quantity and integrity of all oncogenes. Except for MYC , BC patients showed significantly higher median values of CF-DNA quantity (ng) than healthy controls, who had higher integrity and lower apoptotic index. A difference nearing statistical significance was observed for HER2 short CF-DNA ( p = 0.078, AUC value: 0.6305). HER2 short CF-DNA showed an odds ratio of 1.39 for disease recurrence with p = 0.056 (95% CI 0.991-1.973). Our study suggests that CF-DNA detected as liquid biopsy could have great potential in clinical practice once demonstration of its clinical validity and utility has been provided by prospective studies with robust assays.