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Objective: Neo-adjuvant radiotherapy (NART) for breast cancer has shown promising survival results in retrospective trials. However, there are some obstacles such as a chemotherapy delay, an increased overall treatment time (OTT) and the risk of increasing surgical morbidity. Accelerated radiotherapy (RT) in 5 fractions allows to deliver NART in a very short time span and minimizes the delay of surgery and chemotherapy. This trial investigates this NART schedule for safety, feasibility and OTT. Material and methods: Twenty patients eligible for neo-adjuvant chemotherapy (NACT) and breast conserving surgery, were randomized between NART before NACT or NACT and postoperative RT. In both arms, RT treatment was given in 5 fractions to the whole breast with a simultaneously integrated boost (SIB) on the tumor(bed). Lymph node irradiation was given concomitantly in case of lymph node involvement. OTT was defined as the time from diagnosis to last surgery in the intervention group, while in the control group the time between diagnosis and last RT-fraction was used. In the intervention group NACT-delay was defined as time between diagnosis and start of chemotherapy. Results: 20 patients were included, and 19 patients completed treatment. OTT was significantly shorter in the intervention group (mean 218 days, range 196–253) compared to the control group (mean 237, range 211–268, p = 0.001). The difference in mean duration from diagnosis to the first treatment was a non-significant 4 days longer (31 vs 27 days, p = 0.28), but the start of NACT after diagnosis was delayed by 21 days (48 vs 27 days, p

Purpose: The aim of the study was to compare the difference in survival between invasive ductal (IDC) and lobular carcinoma (ILC)., Methods: Data of patients (n = 1843) with a hormone receptor-positive, HER2-negative, pT1-3 IDC or ILC cancer without distant metastasis, treated at the Ghent University Hospital over the time period 2001-2015, were analyzed., Results: ILC represented 13.9% of the tumors, had a higher percentage of pT3 and pN3 stages than IDC, lymphovascular space invasion (LVSI) was less present and Ki-67 was mostly low. 73.9% of ILCs were grade 2, whereas IDC had more grade 1 and grade 3 tumors. Kaplan-Meier curves and log-rank testing showed a significant worse DFS for ILC with pN ≥ 1 than for their IDC counterpart. In a multivariable Cox regression analysis the histologic tumor type, ductal or lobular, was a determinant of DFS over 120 months (IDC as reference; hazard ratio for ILC 1.77, 95% CI 1.08-2.90) just as the ER Allred score (hazard ratio 0.84, 95% CI 0.78-0.91), LVSI (hazard ratio 1.75, 95% CI 1.12-2.74) and pN3 (hazard ratio 2.29, 95% CI 1.03-5.09). Determinants of OS over ten years were age (hazard ratio 1.05, 95% CI 1.02-1.07), LVSI (hazard ratio 3.62, 95% CI 1.92-6.82) and the ER Allred score (hazard ratio 0.80, 95% CI 0.73-0.89)., Conclusion: The histologic tumor type, ductal or lobular, determines DFS in hormone receptor-positive, HER2-negative, pT1-3 breast cancer besides the ER Allred score, LVSI and pN3., (© 2023. The Author(s).)

Background: The prognostic and predictive role of stromal tumor-infiltrating lymphocytes (sTILs) is undetermined in pleomorphic invasive lobular cancer (pILC). The same applies for the expression of PD-1/PD-L1 in this rare breast cancer subtype. Here, we aimed to investigate the expression of sTILs and analyze the PD-L1 expression levels in pILC., Methods: Archival tissues from sixty-six patients with pILC were collected. The sTIL density was scored as a percentage of tumor area using the following cut-offs: 0%; <5%; 5-9%; and 10-50%. The PD-L1 expression was analyzed using IHC on formalin-fixed, paraffin-embedded tissue sections using SP142 and 22C3 antibodies., Results: A total of 82% of the sixty-six patients were hormone receptor positive and 8% of cases were triple negative (TN), while 10% showed human epidermal growth factor receptor 2 (HER2) amplification. sTILs (≥1%) were present in 64% of the study population. Using the SP142 antibody, 36% of tumors demonstrated a positive PD-L1 score of ≥1%, and using the 22C3 antibody, 28% had a positive PD-L1 score of ≥1. There was no correlation between sTILs or PD-L1 expression and tumor size, tumor grade, nodal status, expression of estrogen receptor (ER), or amplification of HER2. Our data did not show any difference in survival between the three molecular subtypes of pILC with respect to sTILs and PD-L1 expression., Conclusion: This study shows that pILCs show some degree of sTILs and PD-L1 expression; however, this was not associated with a survival improvement. Additional large trials are needed to understand immune infiltration in lobular cancer, especially in the pleomorphic subtype.

Background: One of the current challenges in breast cancer is the appropriate treatment of invasive lobular breast cancer (ILC) and defining the high-risk group within ILC. The biological character of ILC typically translates to a good prognosis, however, several studies have indicated that the long-term prognosis is worse than for patients diagnosed with the more commonly invasive ductal carcinoma. Many genomic tests are now available to determine whether those patients are at high risk (HR) and enable tailored treatment. Unfortunately, most of the studies in which these genomic tests have been evaluated entail retrospective analysis of a prospective trial., Aim: This review focuses on the validation of the available genomic assays based on trials performed in ILC patients, where in some instances, the various subtypes of ILC (classical, pleomorphic, and non-classic type) were taken into account., Results: Using Oncotype DX in retrospective studies, only 1.3%-8% of ILC tumors were categorized as HR tumors. For MammaPrint, 24% of patients were classified as HR, which was associated with poor outcome. In a recent sub-analysis of the MINDACT study comprising 487 ILC patients, 16.2% were high genomic risk. EndoPredict, Prosigna Breast Cancer Prognostic Gene Signature Assay, and the Breast Cancer Index have been validated in patients receiving only endocrine treatment., Conclusion: Although ILC accounts for the second most common breast cancer subtype in women, none of these tests encompass tumor morphology in their algorithms. Prospective studies on ILC with genomic assays are warranted given the various subtypes of and treatment options for this underestimated, but frequently occurring cancer., Relevance for Patients: Genomic assays can be employed in ILC patients to predict the risk of recurrence and identify those patients who might benefit from chemotherapy in addition to their standard treatment regimen., Competing Interests: The authors have no conflicts of interest to declare., (Copyright: © 2022 Author(s).)

Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: In early-stage breast cancer, the cornerstone of treatment is surgery. After breast-conserving surgery, adjuvant radiotherapy has shown to improve locoregional control and overall survival rates. The use of breast radiotherapy in the preoperative (preop) setting is far less common. Nevertheless, it might improve disease-free survival as compared to postoperative radiotherapy. There is also a possibility of downsizing the tumour which might lead to a lower need for mastectomy. There are some obstacles that complicate its introduction into daily practice. It may complicate surgery or lead to an increase in wound complications or delayed wound healing. Another fear of preop radiotherapy is delaying surgery for too long. At Ghent University Hospital, we have experience with a 5-fraction radiotherapy schedule allowing radiotherapy delivery in a very short time span., Methods: Twenty female breast cancer patients with non-metastatic disease receiving preop chemotherapy will be randomized between preop or postoperative radiotherapy. The feasibility of preop radiotherapy will be evaluated based on overall treatment time. All patients will be treated in 5 fractions of 5.7 Gy to the whole breast with a simultaneous integrated boost to the tumour/tumour bed of 5 × 6.2 Gy. In case of lymph node irradiation, the lymph node regions will receive a dose of 27 Gy in 5 fractions of 5.4 Gy. The total duration of therapy will be 10 to 12 days. In the preop group, overall treatment time is defined as the time between diagnosis and the day of last surgery, in the postop group between diagnosis and last irradiation fraction. Toxicity related to surgery, radio-, and chemotherapy will be evaluated on dedicated case-report forms at predefined time points. Tumour response will be evaluated on the pathology report and on MRI at baseline and in the interval between chemotherapy and surgery., Discussion: The primary objective of the trial is to investigate the feasibility of preop radiotherapy. Secondary objectives are to search for biomarkers of response and toxicity and identify the involved cell death mechanisms and the effect of preop breast radiotherapy on the in-situ immune micro-environment., Competing Interests: Competing interestsThis work is funded by Stand up to Cancer (Flemish Cancer Society)., (© The Author(s) 2020.)

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.

Adipose tissue secretes a plethora of adipokines as evidenced by characterization of subcutaneous and visceral adipose tissue secretomes. However, adipose tissue composition and secretion pattern is depot and disease dependent, influencing the adipose tissue secretome. We investigated the secretome of cancer-associated adipose tissue (CAAT) explants from breast cancer patients and explored its role in breast cancer proliferation. CAAT proteins were identified by LC-MS/MS and human protein antibody arrays and stimulated proliferation of three breast cancer cell lines. Kinomics and transcriptomics of MCF-7 breast cancer cells treated with the secretome of CAAT revealed activation of Akt-, ERK- and JNK-pathways and differential expression of activator protein 1 (AP-1) and cAMP responsive element-binding protein (CREB) target genes. The cyclin-dependent kinase (CDK)4/6-inhibitor palbociclib significantly abrogated CAAT-enhanced breast cancer cell proliferation. Our work characterizes the specific breast CAAT protein secretome and reveals its pro-proliferative potency in breast cancer.

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076-1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active.

Objective: We retrospectively compared a protocol using sufentanil and ropivacaine intrathecally with a protocol in which only ropivacaine was administered intrathecally and sufentanil was used epidurally to evaluate whether banning sufentanil from the intrathecal space results in a decreased incidence of adverse fetal heart rate changes., Methods: Some 520 cardiotocographic tracings were examined for changes in fetal heart rate and uterine activity following two different protocols of combined spinal epidural analgesia. Charts were consulted for neonatal and labor outcome., Results: When sufentanil was used epidurally instead of intrathecally, the incidence of adverse changes in fetal heart trace was less, demonstrated by a higher percentage of normal reassuring tracings (74.5% vs. 60.4% when sufentanil was used intrathecally; P=0.007), less tracings showing bradycardia (7.5% vs. 14.1%; P=0.035), and more tracings displaying 3 or more accelerations in fetal heart rate in 45 min (93.5% vs. 83.9%; P=0.003) together with less episodes of tachycardia (3.5% vs. 11.4%; P=0.005). There were no differences in labor and neonatal outcome., Conclusions: Based on fetal heart tracing, it seems favorable to ban sufentanil from the intrathecal compartment.