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We assessed the impact of DNA damage response and repair (DDR) biomarker expressions in 222 node-positive early breast cancer (BC) patients from a previous Phase III GOIM 9902 trial of adjuvant taxanes. At a median follow-up of 64 months, the original study showed no disease-free survival (DFS) or overall survival (OS) differences with the addition of docetaxel (D) to epirubicine-cyclophosphamide (EC). Immunohistochemistry was employed to assess the expression of DDR phosphoproteins (pATM, pATR, pCHK1, γH2AX, pRPA32, and pWEE1) in tumor tissue, and their association with clinical outcomes was evaluated through the Cox elastic net model. Over an extended follow-up of 234 months, we confirmed no significant differences in DFS or OS between patients treated with EC and those receiving D → EC. A DDR risk score, inversely driven by ATM and ATR expression, emerged as an independent prognostic factor for both DFS (HR = 0.41, p < 0.0001) and OS (HR = 0.61, p = 0.046). Further validation in a public adjuvant BC cohort was possible only for ATM, confirming its protective role. Overall, our findings confirm the potential role of the DDR pathway in BC prognostication and in shaping treatment strategies advocating for an integrated approach, combining molecular markers with clinical-pathological factors.

HER2 activating mutations have emerged as oncogenic drivers and therapeutic targets in a variety of human tumors. In breast cancer, these deregulations occur at low frequency, and are mostly detected in HER2-nonamplified, metastatic disease. Preclinical evidence has clarified the role of hotspot mutations in HER2 constitutive activation, defining them as an alternative mechanism to HER2 gene amplification. Furthermore, recent clinical studies have indicated the emergence of newly acquired HER2 deregulations in significant proportions of breast cancer patients who experience disease progression following both endocrine and HER2-targeted therapies. As the involvement of HER2 mutation in therapy resistance may profoundly impact patient outcomes on successive therapies, several clinical trials are currently investigating the efficacy of various HER2-targeted drugs in HER2-mutant breast cancer. In this review, we firstly summarize the structural organization of the HER2 oncogene and its historical impact on breast cancer prognosis and therapeutic advancement. Then, we provide an overview of the frequencies and functional relevance of clinically recurrent HER2 mutations in breast cancer with a special focus on their role in therapeutic resistance. Finally, we provide a collection of the clinical trials that are currently exploring novel therapeutic approaches for this patient subset and discuss the related perspectives and challenges., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Understanding the mechanisms of breast cancer cell communication underlying cell spreading and metastasis formation is fundamental for developing new therapies. ID4 is a proto-oncogene overexpressed in the basal-like subtype of triple-negative breast cancer (TNBC), where it promotes angiogenesis, cancer stem cells, and BRACA1 misfunction. Here, we show that ID4 expression in BC cells correlates with the activation of motility pathways and promotes the production of VEGFA, which stimulates the interaction of VEGFR2 and integrin β3 in a paracrine fashion. This interaction induces the downstream focal adhesion pathway favoring migration, invasion, and stress fiber formation. Furthermore, ID4/ VEGFA/ VEGFR2/ integrin β3 signaling stimulates the nuclear translocation and activation of the Hippo pathway member's YAP and TAZ, two critical executors for cancer initiation and progression. Our study provides new insights into the oncogenic roles of ID4 in tumor cell migration and YAP/TAZ pathway activation, suggesting VEGFA/ VEGFR2/ integrin β3 axis as a potential target for BC treatment., (© 2024. The Author(s).)

Background: Immunotherapy has revolutionized the approach to metastatic triple-negative breast cancers. Atezolizumab was approved for patients with metastatic triple-negative breast cancers whose tumors express PD-L1, determined by SP 142 assay. To assess the availability and practice of SP142 test we administered a survey to all the 15 pathology departments of the Lazio Region during a six-month period., Methods: The survey comprised 12 questions regarding the availability of SP142 in the pathology departments, the percentage of positive tests, the difficulties of pathologists in cases close to cut-off value and the tested samples., Results: The SP142 assay was available in only eight centers. In case of positive result, most centers (5/8, 62.5%) reported values of PD-L1 expression ranging from > 1 to ⩽ 5%, with values close to the cut-off point (⩾ 1% or < 1%) being the greatest challenge.Most of the centers (6/8, 75%) tested material from both their own and other hospitals. In most centers, the evaluations were performed either on primary tumors or metastasis, in particular lymph nodes (5/8, 62.5%), followed by lung (3/8, 37.5%) and liver (1/8, 12.5%) metastasis., Conclusion: Our results raise some important issues concerning the evaluation of PD-L1 in the "real-life" setting, providing strategies for its implementation., Competing Interests: Declaration of conflicting interestsThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Paolo Marchetti (PM) has/had a consultant/advisory role for BMS, Roche- Genentech, MSD, Novartis, Amgen, Merck Serono, Pierre Fabre, Incyte. The other authors declare they have no competing interests.

Pembrolizumab has received approval as a first-line treatment for unresectable/metastatic triple-negative breast cancer (mTNBC) with a PD-L1 combined positive score (CPS) of ≥ 10. However, assessing CPS in mTNBC poses challenges. Firstly, it represents a novel analysis for breast pathologists. Secondly, the heterogeneity of PD-L1 expression in mTNBC further complicates the assessment. Lastly, the lack of standardized assays and staining platforms adds to the complexity. In KEYNOTE trials, PD-L1 expression was evaluated using the IHC 22C3 pharmDx kit as a companion diagnostic test. However, both the 22C3 pharmDx and VENTANA PD-L1 (SP263) assays are validated for CPS assessment. Consequently, assay-platform choice, staining conditions, and scoring methods can significantly impact the testing outcomes. This consensus paper aims to discuss the intricacies of PD-L1 CPS testing in mTNBC and provide practical recommendations for pathologists. Additionally, we present findings from a nationwide Italian survey elucidating the state-of-the-art in PD-L1 CPS testing in mTNBC., Competing Interests: Declaration of Competing Interest Nicola Fusco has received honoraria for consulting, advisory role, speaker bureau, travel, and/or research grants from Merck Sharp & Dohme (MSD), Novartis, AstraZeneca, Roche, Daiichi Sankyo, GlaxoSmithKline (GSK), Gilead, Diaceutics, Adicet Bio, and Sermonix. Mariia Ivanova from Agilent Technologies Denmark ApS. Carmen Criscitiello reports personal fees for consulting, advisory role, and speakers’ bureau from Lilly, Roche, Novartis, MSD, Seagen, Gilead, Daiichi Sankyo, AstraZeneca, and Pfizer. Bruna Cerbelli from MSD and Novartis. Fabio Pagni from MSD, Novartis, Roche, and Amgen. Mauro Mastropasqua from Exact Sciences. Alfredo Santinelli from Roche-Ventana, Novartis, Astrazeneca, Amgen. Giuseppe Curigliano has received honoraria for speaker’s engagement: Roche, Seattle Genetics, Novartis, Lilly, Pfizer, Foundation Medicine, NanoString, Samsung, Celltrion, BMS, MSD; Honoraria for providing consultancy: Roche, Seattle Genetics, NanoString; Honoraria for participating in Advisory Board: Roche, Lilly, Pfizer, Foundation Medicine, Samsung, Celltrion, Mylan; Honoraria for writing engagement: Novartis, BMS; Honoraria for participation in Ellipsis Scientific Affairs Group; Institutional research funding for conducting phase I and II clinical trials: Pfizer, Roche, Novartis, Sanofi, Celgene, Servier, Orion, AstraZeneca, Seattle Genetics, AbbVie, Tesaro, BMS, Merck Serono, Merck Sharp Dome, Janssen-Cilag, Philogen, Bayer, Medivation, Medimmune. Elena Guerini-Rocco from Thermo Fisher Scientific, Novartis, AstraZeneca, Roche, Biocartis, Exact Science, GSK, and Illumina. Paolo Graziano Consulting/Honoraria from Eli-Lilly, Pfizer, Novartis, AstraZeneca, Boehringer-Ingelheim, Roche, MSD, Amgen. Giulia d’Amati from MSD, Roche, Astrazeneca, Daiichi Sankyo. These companies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and/or in the decision to publish the results. All other authors declare no potential conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: This study aimed to develop a clinical-radiomic model based on radiomic features extracted from digital breast tomosynthesis (DBT) images and clinical factors that may help to discriminate between benign and malignant breast lesions., Materials and Methods: A total of 150 patients were included in this study. DBT images acquired in the setting of a screening protocol were used. Lesions were delineated by two expert radiologists. Malignity was always confirmed by histopathological data. The data were randomly divided into training and validation set with an 80:20 ratio. A total of 58 radiomic features were extracted from each lesion using the LIFEx Software. Three different key methods of feature selection were implemented in Python: (1) K best (KB), (2) sequential (S), and (3) Random Forrest (RF). A model was therefore produced for each subset of seven variables using a machine-learning algorithm, which exploits the RF classification based on the Gini index., Results: All three clinical-radiomic models show significant differences (p < 0.05) between malignant and benign tumors. The area under the curve (AUC) values of the models obtained with three different feature selection methods were 0.72 [0.64,0.80], 0.72 [0.64,0.80] and 0.74 [0.66,0.82] for KB, SFS, and RF, respectively., Conclusion: The clinical-radiomic models developed by using radiomic features from DBT images showed a good discriminating power and hence may help radiologists in breast cancer tumor diagnoses already at the first screening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Murtas, Landoni, Ordòñez, Greco, Ferranti, Russo, Perracchio and Vidiri.)

Genomics has greatly increased the understanding of the study of breast cancer (BC) and has shaped the concept of intra-tumor heterogeneity, currently recognized as a propelling force for cancer progression. In this context, knowledge and understanding of metastatic breast cancer (mBC) has somehow lagged behind that of primary breast cancer. This may be explained by the relative scarcity of matched mBC samples, however it is possible that the mutation spectrum obtained from primary BC does not capture the full complexity of the metastatic disease. Here, we provide a few examples supporting this possibility, from public databases. We evoke the need to perform an integrated multi-OMICS characterization of mBC, to obtain a broad understanding of this complex disease, whose evolution cannot be explained solely by genomics. Pertinent to this, we suggest that rather an infrequent use of Patient-Derived -Tumor-Organoids (PDTOs) may be influenced by assuming that the metastatic conditions of PDTOs growth (mPDTOs) should be similar to those of the tissue of origin. We challenge this view by suggesting that the use of "target-organ inspired" growth conditions for mPDTOs, may better fit the emerging knowledge of metastatic disease. Thus, the integrated use of multi-OMICS and of clinically relevant mPDTOs may allow a further understanding of such disease and foster therapeutically relevant advances. We believe that our points may be valid for other solid cancers., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Vascular Endothelial Growth Factor A (VEGFA) is the most commonly expressed angiogenic growth factor in solid tumors and is generated as multiple isoforms through alternative mRNA splicing. Here, we show that lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and ID4 (inhibitor of DNA-binding 4) protein, previously referred to as regulators of linear isoforms of VEGFA, induce back-splicing of VEGFA exon 7, producing circular RNA circ&#95;0076611. Circ&#95;0076611 is detectable in triple-negative breast cancer (TNBC) cells and tissues, in exosomes released from TNBC cells and in the serum of breast cancer patients. Circ&#95;0076611 interacts with a variety of proliferation-related transcripts, included MYC and VEGFA mRNAs, and increases cell proliferation and migration of TNBC cells. Mechanistically, circ&#95;0076611 favors the expression of its target mRNAs by facilitating their interaction with components of the translation initiation machinery. These results add further complexity to the multiple VEGFA isoforms expressed in cancer cells and highlight the relevance of post-transcriptional regulation of VEGFA expression in TNBC cells., (© 2022. The Author(s).)

In metastatic breast cancer (mBC), the change of human epidermal growth factor receptor 2 (HER2) status between primary and metastatic lesions is widely recognized, however clinical implications are unknown. Our study address the question if relevant differences exist between subjects who preserve the HER2 status and those who gain the HER2 positivity when relapsed. Data of patients affected by HER2-positive mBC, treated with pertuzumab and/or trastuzumab-emtansine (T-DM1) in a real-world setting at 45 Italian cancer centers were retrospectively collected and analyzed. From 2003 to 2017, 491 HER2-positive mBC patients were included. Of these, 102 (20.7%) had been initially diagnosed as HER2-negative early BC. Estrogen and/or progesterone receptor were more expressed in patients with HER2-discordance compared to patients with HER2-concordant status (p < 0.0001 and p = 0.006, respectively). HER2-discordant tumors were characterized also by a lower rate of brain metastases (p = 0.01) and a longer disease free interval (p < 0.0001). Median overall survival was longer, although not statistically significant, in the subgroup of patients with HER2-discordant cancer with respect to patients with HER2-concordant status (140 vs 78 months, p = 0.07). Our findings suggest that patients with HER2-positive mBC with discordant HER2 status in early BC may have different clinical, biological and prognostic behavior compared to HER2-concordant patients.

Introduction: p53 and antiapoptotic B-cell leukemia/lymphoma 2 (BLC2) have been proposed as prognostic markers for early breast cancer (BC), although their relationship with conventional parameters and patient prognosis, as well as their distribution within the molecular BC subtypes remains uncertain., Patients and Methods: In this observational study, we analyzed the immunohistochemical expression of p53 and BLC2 in 1099 early BC patients surgically treated between 2000 and 2006 and followed for at least 5 years, also considering their association with pathologic factors and molecular subtypes, as well as their influence on disease-free survival., Results: p53 and BLC2 are distributed differently across molecular subtypes (P < .0001); in particular, p53 positivity and BLC2 negativity seems to be associated with more aggressive conventional tumor phenotypes. Moreover, BLC2 negativity seems to be a significant discriminating factor for disease-free survival (P = .003) according to Kaplan-Meier analysis, while p53 seems to have no discriminating effect. Among patients with discordant p53/BLC2 phenotype, the combination p53 + BLC2 - seems to be associated with the worst outcomes (P = .007) and significantly influenced the clinical course of node-negative patients treated only with hormone therapy (P = .004)., Conclusion: These two biomarkers, in addition to conventional pathologic factors and molecular subtype, could help define the risk and outcome of BC., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A hypercoagulable state may either underlie or frankly accompany cancer disease at its onset or emerge in course of cancer development. Whichever the case, hypercoagulation may severely limit administration of cancer therapies, impose integrative supporting treatments and finally have an impact on prognosis. Within a flourishing research pipeline, a recent study of stage I-IIA breast cancer patients has allowed the development of a prognostic model including biomarkers of coagulation activation, which efficiently stratified prognosis of patients in the study cohort. We are now validating our risk assessment tool in an independent cohort of 108 patients with locally advanced breast cancer with indication to neo-adjuvant therapy followed by breast surgery. Within this study population, we will use our tool for risk assessment and stratification in reference to 1. pathologic complete response rate at definitive surgery, intended as our primary endpoint, and 2. rate of thromboembolic events, intended as our secondary endpoint. Patients' screening and enrollment procedures are currently in place. The trial will be shortly enriched by experimental tasks centered on next-generation sequencing techniques for identifying additional molecular targets of treatments which may integrate current standards of therapy in high-risk patients.

Background: Phospholipase Cγ1 (PLCγ1) is highly expressed in human tumours. Our previous studies reported that both stable and inducible PLCγ1 down-regulation can inhibit formation of breast-cancer-derived experimental lung metastasis. Further, high expression of PLCγ1 and its constitutively activated forms (i.e., PLCγ1-pY1253, PLCγ1-pY783) is associated with worse clinical outcome in terms of incidence of distant metastases, but not of local relapse in T1-T2, N0 breast cancer patients., Methods: In the present retrospective study, we analysed the prognostic role of PLCγ1 in early breast cancer patients stratified according to the St. Gallen criteria and to their menopausal status. PLCγ1-pY1253 and PLCγ1-pY783 protein expression levels were determined by immunohistochemistry on tissue microarrays, and were correlated with patients' clinical data, using univariate and multivariate statistical analyses., Results: In our series, the prognostic value of PLCγ1 overexpression was restricted to Luminal type tumours. From multivariate analyses, pY1253-PLCγ1 High was an independent prognostic factor only in postmenopausal patients with Luminal-B tumours (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.1-5.3; P = 0.034). Conversely, PLCγ1-pY783 High was a remarkably strong risk factor (HR, 20.1; 95% CI, 2.2-178.4; P = 0.003) for pre/perimenopausal patients with Luminal-A tumours., Conclusions: PLCγ1 overexpression is a strong predictive surrogate marker of development of metastases in early Luminal-A and -B breast cancer patients, being able to discriminate patients with high and low risk of metastases. Therefore, targeting the PLCγ1 pathway can be considered of potential benefit for prevention of metastatic disease.

Targeting different members of the Akt pathways is a promising therapeutic chance in solid tumors including breast cancer. The variable expression levels of Akt isoforms with opposite effects on tumor growth and metastasis, however, make it difficult to select the inhibitors to be used for specific breast tumor subtypes. Using in vitro and in vivo models, we demonstrated here that Vav1, ectopically expressed in invasive breast tumors derived cells, downmodulates Akt acting at expression and/or activation levels depending on tumor subtype. The decreased p-Akt1 (Ser473) levels are a common effect of Vav1 upmodulation, suggesting that, in breast tumor-derived cells and independently of their phenotype, Vav1 interferes with signaling pathways ended to specifically recruit Akt1. Only in ER-negative cell lines, the silencing of Vav1 induced the expression but not the activation of Akt2. A retrospective analysis of early invasive breast tumors allowed to establish the prognostic significance of the p-Akt/Vav1 relationship. In particular, low Vav1 levels negatively influence the follow-up of patients with low p-Akt in their primary tumors and subjected to adjuvant chemotherapy. As the use of specific or pan Akt inhibitors may not be sufficient or may even be detrimental, increasing the levels of Vav1 could be a new approach to improve breast cancer outcomes. This might be particularly relevant for tumors with a triple-negative phenotype, for which target-based therapies are not currently available., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Background: This is a retrospective cross sectional study aimed to verify whether Multiplex Ligation-dependent Probe Amplification (MLPA), a quantitative molecular assay, may represent a valuable reflex test in breast cancer with equivocal HER2 expression by immunohistochemistry and HER2 gene signals/nucleus (s/n) ranging between 4.0 and 5.9 by in situ hybridization., Methods: A series of 170 breast carcinomas scored as 2+ for HER2 expression by immunohistochemistry, were selected from our files and analyzed in parallel by silver in situ hybridization and by MLPA. According to ASCO-CAP 2013 guidelines, 54/170 tumors, displaying 4.0-5.9 HER2 gene s/n, were defined as low amplified (ratio ≥ 2) or equivocal (ratio < 2) on the basis of centromere enumeration probe 17 (CEP17) status. An independent set of 108 score 2+ breast cancers represented the external validation set. Concordance between the two techniques was assessed through the use of Cohen's K statistic., Results: A concordance rate of 78.2% (Cohen's K statistic: 0,548 95% CI:[0,419-0,677]) between in situ hybridization and MLPA was found in the whole series of 170 cases and of 55.5% (Cohen's K statistic: -0,043 95% CI:[-0,271-0,184]) in the 54 tumors presenting 4.0-5.9 HER2 gene s/n. By MLPA, we found HER2 amplification or gain in 14% of the 21 BC presenting a disomic status and in 18% of the 33 BC presenting a CEP17 > 2.0. These data were further confirmed in the external validation set. Interestingly, the 54 low amplified/equivocal breast carcinomas presented a frequency of hormonal receptor positivity significantly higher than that observed in the amplified tumors and similar to the non-amplified one (p = 0.016 for estrogen receptor and p = 0.001 for progesterone receptor)., Conclusions: To avoid to offer patients an ineffective therapy, HER2 status should be studied more thoroughly in low amplified and equivocal cases which can have lower response rates and shorter time to progression to trastuzumab. In this context, our data indicate that MLPA may be a reliable, objective supporting test in selecting HER2 positive breast cancer patients.

Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter "tsRNAs") is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called " ts-101 " and " ts-53 ," respectively), ts-46 , and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival., Competing Interests: The authors declare no conflict of interest.

The Hippo kinases MST1/2 and LATS1/2 inhibit the oncoproteins TAZ/YAP and regulate T cell function. Hippo kinases also cooperate with the ATR-Chk1 and ATM-Chk2 pathways, central orchestrators of the DNA damage response (DDR). We hypothesized that MST1/2 and LATS1/2 localization differently impacts the efficacy of neoadjuvant therapy (NAT) in breast cancer, being protective when expressed in the cytoplasm of tumor cells and in tumor-infiltrating lymphocytes, whereas representing molecular determinants of chemoresistance when present in the nucleus as a consequence of their cooperation with the DDR. Diagnostic biopsies from 57 HER2-positive and triple-negative breast cancer patients treated with NAT were immunostained for evaluating the expression of phosphorylated MST1/2 (pMST1/2) and LATS1/2 (pLATS1/2) in tumor-infiltrating lymphocytes (TILs) and in cancer cells. TAZ and Chk1 immunostaining was exploited for investigating subcellular compartment-dependent activity of Hippo kinases. Nuclear pMST1/2 (pMST1/2 nuc ) expression was significantly associated with nuclear expression of Chk1 (p = 0.046), whereas cytoplasmic pMST1/2 (pMST1/2 cyt ) expression was marginally associated with cytoplasmic TAZ staining (p = 0.053). Patients whose tumors expressed pMST1/2 nuc were at increased risk of residual disease after NAT (pCR ypT0/is ypN0: OR 4.91, 95%CI: 1.57-15.30; pCR ypT0 ypN0: OR 3.59, 95%CI 1.14-11.34). Conversely, exclusive cytoplasmic localization of pMST1/2 (pMST1/2 cyt )seemed to be a protective factor (pCR ypT0/is ypN0: OR 0.34, 95%CI: 0.11-1.00; pCR ypT0 ypN0: OR 0.31, 95%CI 0.10-0.93). The subcellular localization-dependent significance of pMST1/2 expression suggests their involvement in different molecular networks with opposite impact on NAT efficacy. Larger studies are warranted to confirm these novel findings.

Since 2007, one-step nucleic acid amplification (OSNA) has been used as a diagnostic system for sentinel lymph node (SLN) examination in patients with breast cancer. This study aimed to define a new clinical cut-off of CK19 mRNA copy number based on the calculation of the risk that an axillary lymph node dissection (ALND) will be positive. We analyzed 1529 SLNs from 1140 patients with the OSNA assay and 318 patients with positive SLNs for micrometastasis (250 copies) and macrometastasis (5000 copies) underwent ALND. Axillary non-SLNs were routinely examined. ROC curves and Youden's index were performed in order to identify a new cut-off value. Logistic regression models were performed in order to compare OSNA categorical variables created on the basis of our and traditional cut-off to better identify patients who really need an axillary dissection. 69% and 31% of OSNA positive patients had a negative and positive ALND, respectively. ROC analysis identified a cut-off of 2150 CK19 mRNA copies with 95% sensitivity and 51% specificity. Positive and negative predictive values of this new cut-off were 47% and 96%, respectively. Logistic regression models indicated that the cut-off of 2150 copies better discriminates patients with node negative or positive in comparison with the conventional OSNA cut-off (p<0.0001). This cut-off identifies false positive and false negative cases and true-positive and true negative cases very efficiently, and therefore better identifies which patients really need an ALND and which patients can avoid one. This is why we suggest that the negative cut-off should be raised from 250 to 2150. Furthermore, we propose that for patients with a copy number that ranges between 2150 and 5000, there should be a multidisciplinary discussion concerning the clinical and bio-morphological features of primary breast cancer before any decision is taken on whether to perform an ALND or not., Competing Interests: The authors have declared that no competing interests exist.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple-negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three-dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6-depleted, triple-negative MDA-MB-231 cells rearranged the actin cytoskeleton and restored epidermal growth factor-mediated invasion. In patients with localized, lymph node-negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse-free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6-fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

To report the results of the DECT trial, a phase II study of locally advanced or operable HER2-positive breast cancer (BC) treated with taxanes and concurrent anthracyclines and trastuzumab. Eligible patients (stage IIA-IIIB HER2-positive BC, 18-75 years, normal organ functions, ECOG ≤1, and left ventricular ejection fraction (LVEF) ≥55%) received four cycles of neoadjuvant docetaxel, 100 mg/m(2) intravenously, plus trastuzumab 6 mg/kg (loading dose 8 mg/kg) every 3 weeks, followed by four 3-weekly cycles of epirubicin 120 mg/m(2) and cyclophosphamide, 600 mg/m(2) , plus trastuzumab. Primary objective was pathologic complete response (pCR) rate, defined as ypT0/is ypN0 at definitive surgery. We enrolled 45 consecutive patients. All but six patients (13.3%) completed chemotherapy and all underwent surgery. pCR was observed in 28 patients (62.2%) overall and in 6 (66.7%) from the inflammatory subgroup. The classification and regression tree analysis showed a 100% pCR rate in patients with BMI ≥25 and with hormone negative disease. The median follow up was 46 months (8-78). Four-year recurrence-free survival was 74.7% (95%CI, 58.2-91.2). Seven patients (15.6%) recurred and one died. Treatment was well tolerated, with limiting toxicity being neutropenia. No clinical cardiotoxicity was observed. Six patients (13.4%) showed a transient LVEF decrease (<10%). In one patient we observed a ≥10% asymptomatic LVEF decrease persisting after surgery. Notwithstanding their limited applicability due to the current guidelines, our findings support the efficacy of the regimen of interest in the neoadjuvant setting along with a fairly acceptable toxicity profile, including cardiotoxicity. Results on BMI may invite further assessment in future studies. J. Cell. Physiol. 231: 2541-2547, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc., (© 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

Eribulin has shown survival advantage and manageable toxicity in heavily pre-treated metastatic breast cancer (mBC). We assessed whether body mass index (BMI) impacts treatment outcomes in 101 patients treated with eribulin at six Italian Oncologic Centers. BMI was addressed as a categorical variable (18.5-24.9 vs. at least 25). Clinical benefit rate (CBR) was assessed overall and in subgroups defined by BMI, line of therapy (LOT), and hormone receptor (HR) status. Analysis of CBR by LOT and HR status were further stratified by BMI. Survival curves were compared using the Kaplan-Meier method and log-rank test. Predictors of survival were tested in Cox models. Patients treated with eribulin as third line showed greater CBR when their BMI was in the lowest category (77.8 vs. 58.1%, P = 0.03). Median progression free survival (PFS) and overall survival (OS) in normal and overweight patients were 4 (95% CI, 3-5) versus 3 (2.1-4) months, P = 0.02 and 13 (11-15) versus 12 (6-18) months, P = 0.96, respectively. Median PFS and OS in estrogen receptor (ER) positive and negative tumours were 4 (3-5) versus 3 (2-4) months, P = 0.005 and 14 (10-18) versus 7 (4-10), P = 0.02, respectively. In multivariate analyses, BMI impacted PFS at a nearly significant extent (P = 0.05), while ER expression significantly affected PFS and OS (P = 0.01 and 0.02, respectively). No relevant findings emerged concerning toxicity. We found evidence of greater efficacy of eribulin in leaner mBC patients, particularly if given as third line and in ER positive tumors. Further studies are warranted to confirm our findings., (© 2015 Wiley Periodicals, Inc.)

Background: Breast cancer is the most common cancer in women worldwide and is the second most common cause of cancer death in women. Electrochemotherapy (ECT) used in early-phase clinical trials for the treatment of primary breast cancer resulted in a not complete tumor necrosis in most cases. The present study was undertaken to analyze the feasibility to use ECT to treat patients with histologically proven unifocal ductal breast cancer. In particular, results of ECT treatment in a clinical case are compared with the ones of a simplified 3D dosimetric model., Methods: This clinical study was conducted with the pulse generator Cliniporator Vitae (IGEA, Carpi, Italy). ECT procedures were performed according to ESOPE standard operating procedures. Five single needle electrodes were used with one positioned in the center of the tumor, and the other four distributed around the nodule. Histological images of the resected tumor are compared with the maps of the electric field obtained with a simplified 3D model in Comsol Multiphysics v 4.3., Results: The results of the clinical case demonstrated a reduced efficacy of the ECT treatment described. The proposed simple numerical model of the breast tumor located in a low conductive tissue suggests that this is due to the reduced electric field induced inside the tumor with such 5 electrodes placement. However, where the electric field is predicted higher than the reversible electroporation threshold (E>400 V/cm), also the histological images confirm the necrosis of the target with a good agreement between the modeled and clinical results., Conclusions: The results suggest the dependence of the effectiveness of the treatment on the careful placement of the electrodes. A detailed planned procedure for the tumor analysis after the treatment is also needed in order to better correlate the single electrode positions and the histological images. Simulation models could be used to identify better electrodes configuration in planning the experimental protocol for ECT treatment of breast tumors.

Nectins are Ca(2+)-independent immunoglobulin-like cell adhesion molecules that compose a family of four members that regulate several cellular activities such as movement, proliferation, survival, differentiation, polarization, and the entry of viruses. Nectin-4 has recently emerged as a metastatis-associated protein in several cancers. Here, we have evaluated the association between the expression of Nectin-4 and the clinical outcome of patients with node-negative, T1/T2 breast cancers.The study group consisted of 197 patients presenting with primary unilateral breast carcinoma (T1/T2), with no evidence of nodal involvement and distant metastases. Nectin-4 protein expression was assessed by immunohistochemistry on tissue microarrays, and the results correlated with the clinical data using Kaplan-Meier curves and multivariate Cox regression analysis. Thirty-four out of 197 tumors (17.3%) exhibited Nectin-4 expression on cell membrane (m-Nectin-4) and 122 out of the 163m-Nectin-4 negative tumors (74.8%) showed high cytoplasmic expression of Nectin-4 (c-Nectin-4(High)). At Kaplan-Meier analysis, m-Nectin-4 positivity was significantly associated with a lower disease-free survival (DFS) and distant relapse-free survival (DRFS) rate in patients with a luminal-A phenotype (P=0.030 and P=0.002, respectively). Multivariate analysis showed that in luminal-A tumors m-Nectin-4 positivity is an independent prognostic factor for DFS (P=0.018) and DRFS (P=0.004), but not for local relapse-free survival (LRFS). On the other hand, c-Nectin-4(High) was significantly associated with higher rates of DFS and LRFS, but not DRFS, in the whole population (P=0.008 and P=0.004, respectively) and in patients with luminal-A tumors (P=0.022 and P=0.018, respectively). In patients with luminal-A tumors, multivariate analysis showed that the prognostic value of c-Nectin-4(Low/Negative) is limited to DFS (P=0.012) and LRFS (P=0.022). We suggest that Nectin-4 represents a prognostic factor and a therapeutic target in luminal-A early stage breast cancer.

Vav1 is one of the signalling proteins normally restricted to hematopoietic cells that results ectopically expressed in solid tumors, including breast cancer. By immunohistochemical analysis on TMAs containing invasive breast tumor from patients without lymph node involvement, we have found that Vav1 is expressed in almost all investigated cancers and shows a peculiar localization inside the nucleus of tumor cells. High amounts of nuclear Vav1 are positively correlated with low incidence of relapse, regardless phenotype and molecular subtype of breast neoplasia. In particular, Kaplan-Meier plots showed an elevated risk of distant metastasis in patients with low Vav1 expression compared with patients with high Vav1 expression in their tumors. Experiments performed with breast tumor-derived cells indicated that Vav1 negatively modulates their invasiveness in vitro and their metastatic efficiency in vivo, possibly by affecting the expression of genes involved in invasion and/or metastasis of breast tumors. Since the high heterogeneity of breast tumors makes difficult to predict the evolution of early breast neoplasias, the evaluation of nuclear Vav1 levels may help in the characterization and management of early breast cancer patients. In particular, Vav1 may serve as a prognostic biomarker and a target for new therapies aimed to prevent breast cancer progression.

Background: An External Quality Assessment (EQA) program was developed to investigate the state of the art of HER2 immunohistochemical determination in breast cancer (BC) in 16 Pathology Departments in the Lazio Region (Italy). This program was implemented through two specific steps to evaluate HER2 staining (step 1) and interpretation (step 2) reproducibility among participants., Methods: The management activities of this EQA program were assigned to the Coordinating Center (CC), the Revising Centers (RCs) and the Participating Centers (PCs). In step 1, 4 BC sections, selected by RCs, were stained by each PC using their own procedures. In step 2, each PC interpreted HER2 score in 10 BC sections stained by the CC. The concordance pattern was evaluated by using the kappa category-specific statistic and/or the weighted kappa statistic with the corresponding 95% Jackknife confidence interval., Results: In step 1, a substantial/almost perfect agreement was reached between the PCs for scores 0 and 3+ whereas a moderate and fair agreement was observed for scores 1+ and 2+, respectively.In step 2, a fully satisfactory agreement was observed for 6 out of the 16 PCs and a quite satisfactory agreement was obtained for the remaining 10 PCs., Conclusions: Our findings highlight that in the whole HER2 evaluation process the two intermediate categories, scores 1+ and 2+, are less reproducible than scores 0 and 3+. These findings are relevant in clinical practice where the choice of treatment is based on HER2 positivity, suggesting the need to share evaluation procedures within laboratories and implement educational programs.

Background: The "proximal-type" epithelioid sarcoma is a very rare kind of mesenchimal tumor characterized by the difficulty in histological diagnosis and the very aggressive biological behavior., Case: We report of a case of a 63 years old woman with a vulvar "proximal-type" epithelioid sarcoma that underwent a radical surgical staging followed by an adjuvant radiotherapy. She is on follow-up care for 14 months and there is no clinical evidence of disease., Conclusion: Even if quite rare the proximal type epithelioid sarcoma should be regarded as a separate entity of particularly aggressive biologic behaviour. Its diagnosis attracts controversies and criticism related to the surgical approach and the choice of an adjuvant therapy., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1508554852942125.

Phospholipase Cγ1 (PLCγ1) is highly expressed in several tumors. We have previously reported that both stable and inducible PLCγ1 down-regulation resulted in an almost complete inhibition of breast cancer-derived experimental lung metastasis formation. The aim of our study is to evaluate the association between the expression of PLCγ1 and of PLCγ1 phosphorylated at Tyr1253 (PLCγ1-pY1253) and at Tyr783 (PLCγ1-pY783) with the clinical outcome of patients with node negative, T1/T2 breast cancers. The study groups consisted of 292 (training set) and 122 (validation set) patients presenting with primary unilateral breast carcinoma (T1-T2), with no evidence of nodal involvement and distant metastases. PLCγ1, PLCγ1-pY1253 and PLCγ1-pY783 protein expression were assessed by immunohistochemistry on tissue microarrays and the results correlated with the clinical data using Kaplan-Meier curves and multivariate Cox regression analysis. Tumor cells while expressing variable proportions of cytoplasmic PLCγ1, express PLCγ1-pY1253 and PLCγ1-pY783 predominantly in the nucleus. High expression of PLCγ1, and of its activated forms, is associated with a worse clinical outcome in terms of incidence of distant metastases, and not of local relapse in T1-T2, N0 breast cancer patients undergone adjuvant chemotherapy. PLCγ1 over-expression appears to be a reliable predictive surrogate marker of development of metastases. Thus, targeting PLCγ1 pathways might represent a potential therapeutic approach for the prevention of metastatic disease in breast cancer., (Copyright © 2012 UICC.)

Human mena (hMENA), a member of the actin cytoskeleton regulators Ena/VASP, is overexpressed in high-risk preneoplastic lesions and in primary breast tumors and has been identified as playing a role in invasiveness and poor prognosis in breast cancers that express HER2. Here we identify a unique isoform, hMENAΔv6, derived from the hMENA alternative splicing program. In an isogenic model of human breast cancer progression, we show that hMENA(11a) is expressed in premalignant cells, whereas hMENAΔv6 expression is restricted to invasive cancer cells. "Reversion" of the malignant phenotype leads to concurrent down-regulation of all hMENA isoforms. In breast cancer cell lines, isoform-specific hMENA overexpression or knockdown revealed that in the absence of hMENA(11a), overexpression of hMENAΔv6 increased cell invasion, whereas overexpression of hMENA(11a) reduced the migratory and invasive ability of these cells. hMENA(11a) splicing was shown to be dependent on the epithelial regulator of splicing 1 (ESRP1), and forced expression of ESRP1 in invasive mesenchymal breast cancer cells caused a phenotypic switch reminiscent of a mesenchymal-to-epithelial transition (MET) characterized by changes in the cytoskeletal architecture, reexpression of hMENA(11a), and a reduction in cell invasion. hMENA-positive primary breast tumors, which are hMENA(11a)-negative, are more frequently E-cadherin low in comparison with tumors expressing hMENA(11a). These data suggest that polarized and growth-arrested cellular architecture correlates with absence of alternative hMENA isoform expression, and that the hMENA splicing program is relevant to malignant progression in invasive disease.