Tumor-Infiltrating Lymphocytes and PD-L1 Expression in Pleomorphic Lobular Breast Carcinoma.

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.

Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility.

BACKGROUND: Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. RESULTS: Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine. CONCLUSIONS: In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.

Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics.

The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT; six preservation and five non-preservation) and three blood processing intervals (BPI; 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies.

GRACE-trial: a randomised active-controlled trial for vulvovaginal atrophy in patients with breast cancer on endocrine therapy - study protocol.

INTRODUCTION: Breast cancer is the most common cancer type in women worldwide. Due to hormone receptor positivity in the majority of the breast cancer tumours is endocrine therapy a crucial part in the treatment landscape of breast cancer. Endocrine therapy consists of the use of selective oestrogen-receptor modulators or aromatase inhibitors. These medicines generate a hypoestrogenic environment by reducing circulating oestrogen or by altering the effect of oestrogen on tissue cells by receptor blockade. As a common side effect, vulvovaginal atrophy occurs in the majority of patients with breast cancer using endocrine therapy. Vulvovaginal atrophy has a significant impact on physical and psychological well-being due to negative influence on quality-of-life, self-esteem and sexuality. As a consequence, adherence to endocrine therapy for the standard duration of 5-10 years is challenging, resulting in higher rates of therapy interruption, leading to poorer prognosis with shorter distant disease-free survival. The standard treatment for vulvovaginal atrophy in postmenopausal women is based on the use of local hormonal treatment. However, when a patient has a history of breast cancer, delay of treatment and undertreatment are ubiquitous. METHODS AND ANALYSIS: In this first ever prospective randomised trial patients with breast cancer on endocrine therapy with vulvovaginal atrophy will be treated with the available local treatment modalities with a 1:1:1:1 randomisation: oestrogen, dehydroepiandrosterone, moisturisers and a co-treatment of oestrogen and probiotics. Patient-reported outcomes measurements will be implemented to investigate the efficacy of the implemented treatments. Safety of the treatments will be evaluated by assessing systemic sex hormones concentrations. ETHICS AND DISSEMINATION: This study was approved by the Ethical Committee of Ghent University Hospital and by the Federal Agency for Medicines and Health Products. Results will be published in peer-reviewed journals and released in international conferences. TRIAL REGISTRATION NUMBER: 2021-001921-31.

Safety of pre- or postoperative accelerated radiotherapy in 5 fractions: A randomized pilot trial.

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 < 0.001). NART did not result in additional surgery complications. CONCLUSION: This pilot trial is the first to report on accelerated NART in 5 fractions with SIB. NART before NACT resulted in a shorter OTT with good safety results.

Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions.

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.

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine.

Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

The generation and use of recombinant extracellular vesicles as biological reference material.

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.

Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.

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.

The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue.

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.