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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.

Background: This study provides insights into the treatment use and outcomes of metastatic non-small cell lung cancer (NSCLC) patients in a real-world setting prior to and after the availability of immuno-oncology (IO) regimens in the first line (1L). Methods: Metastatic NSCLC patients, who initiated systemic 1L anticancer treatment from 2014 to 2020, were identified from health records. Patients were grouped into Pre-1L IO and Post-1L IO, according to the availability of pembrolizumab 1L monotherapy at the date of initiating 1L systemic anticancer treatment. Patient characteristics, treatment patterns and outcomes were assessed by the cohort. Overall survival (OS) and real-world progression-free survival (rwPFS) were calculated using the Kaplan-Meier method. Results: The most common 1L treatment was platinum-based chemotherapy regimens in both groups (≥46%), followed by single-agent chemotherapy (27.0%) in Pre-1L IO and pembrolizumab (26.0%) in Post-1L IO. Median OS was 6.2 (95% CI 5.5–7.4) in Pre- and 8.9 months (95% CI 7.5–10.6) in Post-1L IO, while rwPFS was 3.7 (95% CI 3.3–4.2) and 4.7 months (95% CI 3.9–5.7), respectively. Conclusions: Even if a small proportion of patients received a 1L IO, the data showed an improved survival outcomes in the Post-1L IO group.

Breast cancer (BC) is the most common diagnosed cancer and the second leading cause of cancer-associated death in women, with the triple negative (TNBC) subtype being characterized by the poorest prognosis. New therapeutic targets are urgently needed to overcome the high metastatic potential, aggressiveness and poor survival of these tumors. Trop2 transmembrane glycoprotein, acting as an intracellular calcium signal transducer, recently emerged as a new potential target in epithelial cancers, in particular in breast cancer.We summarize the key features of Trop2 structure and function, describing the therapeutic strategies targeting this protein in cancer. Particular attention is paid to antibody-drug conjugates (ADCs), actually representing the most successful strategy.ADCs targeting Trop2 recently received an accelerated FDA approval for the therapy of metastatic TNBC. The prospects for these novel ADCs in BC subtypes other than TNBC are discussed, taking into account the main pitfalls relative to Trop2 structure and function.

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.

The risk of relapse for early breast cancer (BC) patients persists even after decades and to date, no specific and sensitive effective circulating biomarker for recurrence prediction has been identified yet. The international guidelines do not recommend the assessment of the serum tumor markers CEA and CA15-3 in the follow-up of asymptomatic early BC patients. In our institute, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori”, as part of the E.Pic.A study, which was designed to assess the economic appropriateness of integrated care pathways in early BC, the use of CEA and CA15-3 as circulating tumor biomarkers in early BC patients was evaluated in 1502 patients one year after surgery, from 2015 to 2018, with an overall expense of EUR 51,764. A total of EUR 47,780 (92%) was used for execution of circulating tumor markers in early BC patients with stage 0, I and II tumors, neglecting the current guidelines and considered inappropriate by our professional board. We found that no patients with stage I BC experienced relapse in the 365 days after surgery, and in any case examination of the circulating markers CEA and CA15-3 was considered crucial for diagnosis of relapse. Our findings suggest that this inadequacy is a low-value area, supporting the reallocation of economic resources for interventions of a higher value for patients.

open 14 no Breast cancer (BC) is the most commonly diagnosed malignant tumor in women worldwide, and the leading cause of cancer death in the female population. The percentage of patients experiencing poor prognosis along with the risk of developing metastasis remains high, also affecting the resistance to current main therapies. Cancer progression and metastatic development are no longer due entirely to their intrinsic characteristics, but also regulated by signals derived from cells of the tumor microenvironment. Extracellular vesicles (EVs) packed with DNA, RNA, and proteins, are the most attractive targets for both diagnostic and therapeutic applications, and represent a decisive challenge as liquid biopsy-based markers. Here we performed a study based on a multiplexed phenotyping flow cytometric approach to characterize BC-derived EVs from BC patients and cell lines, through the detection of multiple antigens. Our data reveal the expression of EVs-related biomarkers derived from BC patient plasma and cell line supernatants, suggesting that EVs could be exploited for characterizing and monitoring disease progression. open Bandini E.; Rossi T.; Scarpi E.; Gallerani G.; Vannini I.; Salvi S.; Azzali I.; Melloni M.; Salucci S.; Battistelli M.; Serra P.; Maltoni R.; Cho W.C.; Fabbri F. Bandini E.; Rossi T.; Scarpi E.; Gallerani G.; Vannini I.; Salvi S.; Azzali I.; Melloni M.; Salucci S.; Battistelli M.; Serra P.; Maltoni R.; Cho W.C.; Fabbri F.

Trop-2 is an ideal candidate for targeted therapeutics because it is a transmembrane protein with an extracellular domain overexpressed in a wide variety of tumors, and is upregulated in normal cells. Consequently, several Trop-2-targeted drugs have recently been developed for clinical use, such as anti-Trop-2 antibodies. Sacituzumab govitecan, a Trop-2-directed antibody and topoisomerase inhibitor drug conjugate, was recently approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of metastatic triple-negative breast cancer and metastatic urothelial cancer. In Italy, this treatment cannot be used in clinical practice because it has not yet been approved by the Agenzia Italiana del Farmaco (AIFA, Rome, Italy). In Italy, this is not a new problem, in fact, when a new compound is approved by the U.S. and Europe, there is often a delay in its approval for use. The adoption of universal guidelines and the standardization of Trop-2 evaluation is urgently needed.

Breast cancer (BC) is a disease characterized by high degrees of heterogeneity at morphologic, genomic, and genetic levels, even within the same tumor mass or among patients. As a consequence, different subpopulations coexist and less represented clones may have a selective advantage, significantly influencing the outcome of BC patients. Circulating tumor cells (CTCs) represent a rare population of cells with a crucial role in metastatic cascade, and in recent years have represented a fascinating alternative to overcome the heterogeneity issue as a “liquid biopsy”. However, besides the raw enumeration of these cells in advanced epithelial tumors, there are no CTC-based assays applied in the clinical practice to improve personalized medicine. In this review, we report the latest findings in the field of CTCs for intra-tumoral heterogeneity unmasking in BC, supporting the need to deepen their analysis to investigate their role in metastatic process and include the molecular characterization in the clinical practice. In the future, CTCs will be helpful in monitoring patients during treatment, as well as to better address therapeutic strategies.

Background COVID-19 severity is uneven between genders. We hypothesized a role of hormonal therapies in the severity of COVID-19 in breast cancer (BC) patients via the modulation of SARS-CoV-2 susceptibility genes. Patients and Methods We mined the Emilia Romagna region (Italy) registries to compare the rates of hospitalization and mortality for COVID-19 in 2020 amongst 24628 BC patients. Next, we analyzed the modulation of ACE2, TMPRSS2 and NRP1 gene expression and the susceptibility to SARS-CoV-2 infection by tamoxifen, fulvestrant and 17β-estradiol on human ER+ MCF-7 cells in vitro.Results The hospitalization rate observed for 4784 tamoxifen treated BC patients was the lowest (OR, 0.41; 95% CI, 0.18-0.94; p=0.04) among hormonal therapies and no fatalities occurred. A standard mortality rate reduction has been observed also for patients treated with aromatase inhibitors (SMR: 0.73; 95% CI, 0.45-0.90). In vitro experiments showed that fulvestrant, but not tamoxifen, increases ACE2, TMPRSS2 and NRP1 gene expression and susceptibility to SARS-CoV-2 infection and that 17β-estradiol reduces significantly TMPRSS2 and NRP1 expression.Conclusions Tamoxifen treated BC patients showed a reduced rate of hospitalization and strikingly no fatalities for COVID-19. In vitro experiments confirmed a protective role of tamoxifen while an increased susceptibility to SARS-CoV-2 infection of ER+ cells treated with fulvestrant was observed.

Circulating tumor cells (CTCs) are a rare population of cells found in the bloodstream and represent key players in the metastatic cascade. Their analysis has proved to provide further core information concerning the tumor. Herein, we aim at investigating CTCs isolated from a 32-year-old patient diagnosed with triple negative spindle-shaped metaplastic breast cancer (MpBC), a rare tumor poorly responsive to therapies and with a dismal prognosis. The molecular analysis performed on the primary tumor failed to underline effective actionable targets to address the therapeutic strategy. Besides the presence of round-shaped CTCs, cells with a spindle shape were present as well, and through molecular analysis, we confirmed their malignant nature. This aspect was coherent with the primary tumor histology, proving that CTCs are released regardless of their morphology. Copy number aberration (CNA) profiling and variant analysis using next-generation sequencing (NGS) showed that these cells did not harbor the alterations exhibited by the primary tumor (PIK3CA G1049A mutation, MYC copy number gain). However, despite the great heterogeneity observed, the amplification of regions involved in metastasis emerged (8q24.22–8q24.23). Our findings support the investigation of CTCs to identify alterations that could have a role in the metastatic process. To the best of our knowledge, this is the first examination of CTCs in an MpBC patient.

Background: Due to its high incidence, evaluating performance of care delivered to breast cancer patients is a crucial issue. The multidisciplinary panel E.Pic.A. (Economic Appropriateness of an Integrated Care Pathway) defined a set of key performance indicators (KPIs) to evaluate economic waste in breast cancer healthcare interventions. Methods: The E.Pic.A. panel identified the principal KPIs that are crucial within the breast cancer care pathway to evaluate the performance of care. KPIs were defined taking into account their reliability, validity, usability and feasibility of measurement through the linkage between multiple routine healthcare data sources. Results: Seven KPIs were identified: three on instrumental diagnostics, two on surgery and two on treatment. The three KPIs regarding instrumental diagnostics are aimed at assessing the inappropriateness of diagnostic tests performed before and after the index surgery. The two KPIs regarding surgery measure the inappropriateness of possible repeated interventions considering the time elapsed from the index surgery. The two KPIs regarding oncologic therapy measure the inappropriateness about the administration time of adjuvant therapy and radiotherapy considering the time elapsed from the index surgery. Conclusion: E.Pic.A methodology could help to evaluate economic waste in healthcare interventions with the objective of redirecting resources to interventions with greater value. (HTA & Market Access)

The immune system is a dynamic feature of each individual and a footprint of our unique internal and external exposures. Indeed, the type and level of exposure to physical and biological agents shape the development and behavior of this complex and diffuse system. Many pathological conditions depend on how our immune system responds or does not respond to a pathogen or a disease or on how the regulation of immunity is altered by the disease itself. T-cells are important players in adaptive immunity and, together with B-cells, define specificity and monitor the internal and external signals that our organism perceives through its specific receptors, TCRs and BCRs, respectively. Today, high-throughput sequencing (HTS) applied to the TCR repertoire has opened a window of opportunity to disclose T-cell repertoire development and behavior down to the clonal level. Although TCR repertoire sequencing is easily accessible today, it is important to deeply understand the available technologies for choosing the best fit for the specific experimental needs and questions. Here, we provide an updated overview of TCR repertoire sequencing strategies, providers and applications to infectious diseases and cancer to guide researchers’ choice through the multitude of available options. The possibility of extending the TCR repertoire to HLA characterization will be of pivotal importance in the near future to understand how specific HLA genes shape T-cell responses in different pathological contexts and will add a level of comprehension that was unthinkable just a few years ago.

Introduction Multiple myeloma (MM) is a hematological malignancy in which patients present with bone marrow infiltration of clonal terminally-differentiated plasma cells. Monoclonal protein in the serum and/or urine is frequently detected. Over the past decade, important progress has been made in the comprehension of disease biology and treatment personalization. Much work has been put into the development of chimeric antigen receptor (CAR) gene-modified T-cell therapy thought to be a promising therapeutic option for pluritreated patients with refractory MM. Evidence acquisition We performed an analysis of clinical trials registered at the international repository clinicaltrials.gov using "CAR" OR "CAR T" AND "multiple myeloma" as search terms to understand what were the antigens targeted by CAR T strategies and what was the trade-off of their exploitation. The search retrieved a list of 103 trials that was manually filtered to eliminate follow-up and observational or not-pertinent trials. Evidence synthesis Most studies employed anti-BCMA targeting either alone (62/94; 66%), or in combination with a second target (12/94; 13%). The second target most studied was SLAMF7 (CD319) explored by 4/94 (4%) clinical trials. Other antigens investigated and described here include: CD44v6, CD38, CD138, MUC1, CD56, CD19, Igk light chain, Lewis Y, CD229 and GPRC5D. Conclusions Targeting an appropriate antigen(s) is the key to both safety and efficacy of CAR T approaches in MM as there is dearth of tumor-specific antigens. Most antigens tested are merely enriched on MM cells. Working with tumor-enriched antigens requires careful assessment of the balance between harm (toxicity) and benefit (disease eradication) to the patient. This review provides an up-to-date overview of the avenues that are being explored.

Breast cancer (BC) is the most diagnosed carcinoma and the leading cause of cancer death in female over 100 countries. Thanks to the advance in therapeutic strategies, patients' survival has improved. However, the lack of response to treatments and drug resistance are still a main concern, demanding for new therapeutic approaches, in particular for the advanced stages of the disease. Androgen receptor (AR) is gaining increasing interest as a fourth targetable receptor in BC, however, its regulation in BC cells is still poorly understood. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. Here, we identified miR-9-5p as an inhibitor of AR expression, we validated the inverse correlation between miR-9-5p and AR in primary BC samples and we further identified a feedback loop in which androgen agonists of AR up-regulate miR-9-5p. We also provided evidence that miR-9-5p elicits anti-proliferative effects in BC cell lines regardless of their estrogen receptor status. Finally, we showed that miR-9-5p can revert AR-downstream signaling even in presence of AR-agonists, highlighting the role of this miR in the hormonal response of BC. In conclusion, this study supports the role of miR-9-5p as an anti-proliferative miR in BC and as a central modulator of AR-signaling response to circulating androgens in BC.

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: This study estimated the economic and organisational impact of using subcutaneous injection (SC) formulations of two monoclonal antibodies (rituximab and trastuzumab) in patients with n...

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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Highlights • The choice of adjuvant therapy in elderly patients should consider several factors. • There is a lack of universal and unique guidelines to define elderly patients. • Biological age is the result of genetics, lifestyle, nutrition and comorbidities. • AT choice in elderly patients must consider chronological and biological age. • The geriatric assessment and the research results should be evaluated in AT choice., Ma and colleagues reported in their study on 12,004 elderly patients published on Breast J. 2020, that adjuvant chemotherapy was not associated with overall survival. Given the toxicities associated with systemic treatments, caution recommendation or omission of chemotherapy may be considered in elderly patient selection especially when comorbidities are present. We agree with authors final conclusions but we want to highlight that to define the adjuvant therapy in BC elderly patients several factors need to be taken into account. One of the main issues is the lack of universal and unique guidelines to define elderly patients. In clinical practice it can be very difficult to estimate the benefit/risk ratio in elderly patients because chemotherapy-induced toxicity is worse than in younger individuals. For these reasons, beyond comorbidities, the choice of adjuvant therapy for elderly patients must also be based both on chronological and biological age. Moreover, the multidisciplinary team for the elderly patient evaluation should include both the geriatrician and the molecular biologist.

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.