Purpose
Preliminary reports suggest that extracellular vesicles (EVs) might be a promising biomarker for breast cancer (BC). However, the quantification of plasmatic levels of EVs is a complex task. ...To overcome these limitations, we developed a new, fast, and easy to use assay for the quantification of EVs directly in plasma based on the use of Single-Molecule Array (SiMoA).
Methods
By using SiMoA to identify CD9+/CD63+ EVs, we analyzed plasma samples of 181 subjects (95 BC and 86 healthy controls, HC). A calibration curve, made of a serial dilution of lyophilized standards from human plasma, was used in each run to ensure the obtainment of quantitative results from the assay. In a subgroup of patients, EVs concentrations were estimated in plasma before and after 30 days from cancer surgery. Additional information on the size of EVs were also acquired using a Nanosight system to obtain a clearer understanding of the mechanism underlying the releases of EVs associated with the presence of cancer.
Results
The measured levels of EVs resulted significantly higher in BC patients (median values 1179.1 ng/µl
vs
613.0 ng/µl,
p
< 0.0001). ROC curve was used to define the optimal cut-off level of the test at 1034.5 ng/µl with an AUC of 0.75 95% CI 0.68–0.82. EVs plasmatic concentrations significantly decreased after cancer surgery compared to baseline values (
p
= 0.014). No correlation was found between EVs concentration and clinical features of BC.
Conclusion
SiMoA assay allows plasmatic EVs levels detection directly without any prior processing. EVs levels are significantly higher in BC patients and significantly decreases after cancer surgery.
Immune inflammatory biomarkers are easily obtained and inexpensive blood-based parameters that recently showed prognostic and predictive value in many solid tumors. In this study, we aimed to ...investigate the role of these biomarkers in predicting distant relapse in breast cancer patients treated with neoadjuvant chemotherapy (NACT). All breast cancer patients who referred to our Breast Unit and underwent NACT were retrospectively reviewed. The pre-treatment neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), and pan-immune-inflammation value (PIV) were calculated from complete blood counts. The primary outcome was 5-year distant-metastasis-free survival (DMFS). In receiver operating characteristic analyses, the optimal cutoff values for the NLR, PLR, MLR, and PIV were determined at 2.25, 152.46, 0.25, and 438.68, respectively. High levels of the MLR, but not the NLR, PLR, or PIV, were associated with improved 5-year DMSF in the study population using both univariate (HR 0.52, p = 0.03) and multivariate analyses (HR, 0.44; p = 0.02). Our study showed that the MLR was a significant independent parameter affecting DMFS in breast cancer patients undergoing NACT. Prospective studies are required to confirm this finding and to define reliable cutoff values, thus leading the way for the clinical application of this biomarker.
Abstract
Background
Breast cancer Patient Derived Organoids (PDO) have been demonstrated to be a reliable model to study cancer that promised to replace and reduce the use of animals in pre-clinical ...research. They displayed concordance with the tissue of origin, resuming its heterogenicity and representing a good platform to develop approaches of personalized medicines. Although obtain PDOs from mammary tumour, was a very challenging process, several ongoing studies evaluated them as a platform to study efficacy, sensitivity and specificity of new drugs and exploited them in personalized medicine. Despite tissue organization represented a crucial point to evaluate in a 3-dimensional model, since it could influence drug penetration, morphology of breast cancer PDOs has not been analysed yet. Here, we proposed a complete ultrastructural analysis of breast PDOs obtained from tumour and healthy tissues to evaluate how typical structures observed in mammary gland were resumed in this model.
Methods
81 samples of mammary tissue (healthy or tumour) resulting from surgical resections have been processed to obtain PDO. The resulting PDOs embedded in matrigel drop have been processed for transmission electron microscopy and analysed. A comparison between ones from healthy and ones from cancerous tissue has been performed and PDOs derived from tumour tissue have been stratified according to their histological and molecular subtype.
Result
The morphological analysis performed on 81 PDO revealed an organized structure rich in Golgi, secretion granules and mitochondria, which was typical of cells with a strong secretory activity and active metabolism. The presence of desmosomes, inter and intracellular lumens and of microvilli and interdigitations signified a precise tissue-organization. Each PDO has been classified based on whether or not it possessed (i) peripheral ridges in mitochondria, (ii) intracellular lumens, (iii) intercellular lumens, (iv) micro-vesicles, (v) open desmosomes, (vi) cell debris, (vii) polylobed nuclei, (viii) lysosomes and (ix) secretion granules, in order to identify features coupled with the cancerous state or with a specific histological or molecular subtype.
Conclusion
Here we have demonstrated the suitability of breast cancer PDO as 3-dimensional model of mammary tissue. Besides, some structural features characterizing cancerous PDO have been observed, identifying the presence of distinctive traits.
The host’s immune system plays a crucial role in determining the clinical outcome of many cancers, including breast cancer. Peripheral blood neutrophils and lymphocytes counts may be surrogate ...markers of systemic inflammation and potentially reflect survival outcomes. The aim of the present study is to assess the role of preoperative systemic inflammatory biomarkers to predict local or distant relapse in breast cancer. In particular we investigated ER+ HER2- early breast cancer, considering its challenging risk stratification. A total of 1,763 breast cancer patients treated at tertiary referral Breast Unit were reviewed. Neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR) and lymphocyte-to-monocyte (LMR) ratios were assessed from the preoperative blood counts. Multivariate analyses for 5-years locoregional recurrence-free (LRRFS), distant metastases-free (DMFS) and disease-free survivals (DFS) were performed, taking into account both blood inflammatory biomarkers and clinical-pathological variables. Low NLR and high LMR were independent predictors of longer LRRFS, DMFS and DFS, and low PLR was predictive of better LRRFS and DMFS in the study population. In 999 ER+ HER2- early breast cancers, high PLR was predictive of worse LRRFS (HR 0.42, p=0.009), while high LMR was predictive of improved LRRFS (HR 2.20, p=0.02) and DFS (HR 2.10, p=0.01). NLR was not an independent factor of 5-years survival in this patients’ subset. Inflammatory blood biomarkers and current clinical assessment of the disease were not in agreement in terms of estimate of relapse risk (K-Cohen from -0.03 to 0.02). In conclusion, preoperative lymphocyte ratios, in particular PLR and LMR, showed prognostic relevance in ER+ HER2- early breast cancer. Therefore, they may be used in risk stratification and therapy escalation/de-escalation in patients with this type of tumor.
Type of axillary surgery in breast cancer (BC) patients who convert from cN + to ycN0 after neoadjuvant chemotherapy (NAC) is still debated. The aim of the present study was to develop and validate a ...preoperative predictive nomogram to select those patients with a low risk of residual axillary disease after NAC, in whom axillary surgery could be minimized.
1950 clinically node-positive BC patients from 11 Breast Units, treated by NAC and subsequent surgery, were included from 2005 to 2020. Patients were divided in two groups: those who achieved nodal pCR vs. those with residual nodal disease after NAC. The cohort was divided into training and validation set with a geographic separation criterion. The outcome was to identify independent predictors of axillary pathologic complete response (pCR).
Independent predictive factors associated to nodal pCR were axillary clinical complete response (cCR) after NAC (OR 3.11, p < 0.0001), ER-/HER2+ (OR 3.26, p < 0.0001) or ER+/HER2+ (OR 2.26, p = 0.0002) or ER-/HER2- (OR 1.89, p = 0.009) BC, breast cCR (OR 2.48, p < 0.0001), Ki67 > 14% (OR 0.52, p = 0.0005), and tumor grading G2 (OR 0.35, p = 0.002) or G3 (OR 0.29, p = 0.0003). The nomogram showed a sensitivity of 71% and a specificity of 73% (AUC 0.77, 95%CI 0.75–0.80). After external validation the accuracy of the nomogram was confirmed.
The accuracy makes this freely-available, nomogram-based online tool useful to predict nodal pCR after NAC, translating the concept of tailored axillary surgery also in this setting of patients.
•Axillary surgery after neoadjuvant chemotherapy in breast cancer is still debated.•Several surgical approaches are reported but clear evidences are lacking.•Prediction of axillary status after neoadjuvant chemotherapy is a clinical need.•An nomogram was developed and validated to preoperatively identify ypN0 patients.•This online tool could allow de-escalation of axillary surgery.
List of abbreviations MCs microcalcifications HAp hydroxyapatite Whit whitlockite Mg magnesium DCIS ductal carcinoma in-situ IDC invasive ductal carcinoma XRF X-ray fluorescence WAXS Wide Angle X-ray ...Scattering P phosphorous wMg magnesium mass fraction AUC area under curve a-CaCO3 amorphous calcium carbonate c-CaCO3 crystalline calcium carbonate (calcite) CaC2O4 calcium oxalate FWHM full width at half maximum Dear Editor, Microcalcifications (MCs) are common findings in mammography and can be indicative of different degrees of malignancy, thus requiring multiple stereotaxis vacuum-assisted biopsies under mammography to identify and characterize breast cancer 1, 2. In many cases, however, the presence of MCs is not associated with the presence of a tumor. ...a better understanding of the physical-chemical properties of MCs is needed to assess the relationship between their presence and breast cancer. (A) XRF analysis of MCs from representative samples reporting normal tissue (B1), ductal carcinoma in situ (B5a) and invasive ductal carcinoma (B5b), respectively, reporting the Magnesium mass fraction (wMg) map derived from data taken at 2.5 keV (see Supplementary Figure S2, the intensity maps of P and Mg); values outside the calcifications are plotted as zero; the dashed box defines the same area reported in panels (C) and (E), on contiguous slices. (E) Raman mapping analysis of tissue slices contiguous to those used for XRF and WAXS studies, reporting in false colors the localization of HAp (green), Whit (red), surrounding tissue (light pink) and background (white).
Background:
Systemic inflammatory markers draw great interest as potential blood-based prognostic factors in several oncological settings.
Objectives:
The aim of this study is to evaluate whether ...neutrophil-to-lymphocyte ratio (NLR) and pan-immune-inflammation value (PIV) predict nodal pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) in node-positive (cN+) breast cancer (BC) patients.
Design:
Clinically, cN+ BC patients undergoing NAC followed by breast and axillary surgery were enrolled in a multicentric study from 11 Breast Units.
Methods:
Pretreatment blood counts were collected for the analysis and used to calculate NLR and PIV. Logistic regression analyses were performed to evaluate independent predictors of nodal pCR.
Results:
A total of 1274 cN+ BC patients were included. Nodal pCR was achieved in 586 (46%) patients. At multivariate analysis, low NLR odds ratio (OR) = 0.71; 95% CI, 0.51–0.98; p = 0.04 and low PIV (OR = 0.63; 95% CI, 0.44–0.90; p = 0.01) were independently predictive of increased likelihood of nodal pCR. A sub-analysis on cN1 patients (n = 1075) confirmed the statistical significance of these variables. PIV was significantly associated with axillary pCR in estrogen receptor (ER)−/human epidermal growth factor receptor 2 (HER2)+ (OR = 0.31; 95% CI, 0.12–0.83; p = 0.02) and ER−/HER2− (OR = 0.41; 95% CI, 0.17–0.97; p = 0.04) BC patients.
Conclusion:
This study found that low NLR and PIV levels predict axillary pCR in patients with BC undergoing NAC.
Registration:
Eudract number NCT05798806.
A major concern in the management of Inflammatory Bowel Disease (IBD) is the absence of accurate and specific biomarkers to drive diagnosis and monitor disease status timely and non-invasively. ...Fibroblast activation protein (FAP) represents a hallmark of IBD bowel strictures, being overexpressed in stenotic intestinal myofibroblasts. The present study aimed at evaluating the potential of circulating FAP (cFAP) as an accessible blood biomarker of IBD. Quantitative determination of cFAP was performed by enzyme-linked immunosorbent assay on plasma samples prospectively collected from patients with IBD and control subjects. A discrimination model was established on a training set of 50% patients and validated on independent samples. Results showed that cFAP concentration was reduced in patients with IBD when compared to controls (
p
< 0.0001). Age, sex, smoking, disease location and behavior, disease duration and therapy were not associated with cFAP. The sensitivity and specificity of cFAP in discriminating IBD from controls were 70 and 84%, respectively, based on the optimal cutoff (57.6 ng mL
−1
, AUC = 0.78). Predictions on the test set had 57% sensitivity, 65% specificity, and 61% accuracy. There was no strong correlation between cFAP and routine inflammatory markers in the patients' population. A subgroup analysis was performed on patients with Crohn's disease undergoing surgery and revealed that cFAP correlates with endoscopic mucosal healing. In conclusion, cFAP deserves attention as a promising blood biomarker to triage patients with suspected IBD. Moreover, it might function as a biomarker of post-operative remission in patients with Crohn's disease.
•Development of a fully automated and easily customisable pipeline for microcalcifications analysis requiring only the raw mammograms as input and limited computational resources.•Automatic ...patch-based semantic segmentation using a UNet based network with a custom loss function appositely designed to deal with extremely small lesions.•Classification model for breast microcalcifications malignancy detection trained with a deep transfer-learning approach on a dataset with biopsy-proven labels.•Output classification predictions are analysed with state-of-the-art explainable AI algorithms to visually inspect the contribution of each area of the image to the final classification.
Breast cancer is the world’s most prevalent form of cancer. The survival rates have increased in the last years mainly due to factors such as screening programs for early detection, new insights on the disease mechanisms as well as personalised treatments. Microcalcifications are the only first detectable sign of breast cancer and diagnosis timing is strongly related to the chances of survival. Nevertheless microcalcifications detection and classification as benign or malignant lesions is still a challenging clinical task and their malignancy can only be proven after a biopsy procedure. We propose DeepMiCa, a fully automated and visually explainable deep-learning based pipeline for the analysis of raw mammograms with microcalcifications. Our aim is to propose a reliable decision support system able to guide the diagnosis and help the clinicians to better inspect borderline difficult cases.
DeepMiCa is composed by three main steps: (1) Preprocessing of the raw scans (2) Automatic patch-based Semantic Segmentation using a UNet based network with a custom loss function appositely designed to deal with extremely small lesions (3) Classification of the detected lesions with a deep transfer-learning approach. Finally, state-of-the-art explainable AI methods are used to produce maps for a visual interpretation of the classification results. Each step of DeepMiCa is designed to address the main limitations of the previous proposed works resulting in a novel automated and accurate pipeline easily customisable to meet radiologists’ needs.
The proposed segmentation and classification algorithms achieve an area under the ROC curve of 0.95 and 0.89 respectively. Compared to previously proposed works, this method does not require high performance computational resources and provides a visual explanation of the final classification results.
To conclude, we designed a novel fully automated pipeline for detection and classification of breast microcalcifications. We believe that the proposed system has the potential to provide a second opinion in the diagnosis process giving the clinicians the opportunity to quickly visualise and inspect relevant imaging characteristics. In the clinical practice the proposed decision support system could help reduce the rate of misclassified lesions and consequently the number of unnecessary biopsies.
Extensive intraductal component (EIC) associated to early breast cancer could increase the risk locoregional recurrence, but its impact on distant metastases is still unclear. The aim of the present ...study was to assess the role of EIC on 5-year survival outcomes in patients affected by early breast cancer treated with breast-conserving surgery.
A total of 414 consecutive patients with a minimum follow-up of 60 mo were collected from January 2007 to December 2015. Disease-free survival (DFS), distant metastasis-free survival (DMFS), and locoregional recurrence-free survival at 5 y were assessed considering the presence or absence of EIC and other clinical and pathological features.
Absence of EIC was independently associated with worse 5-year DFS (hazard ratio HR 1.68, P = 0.008) and 5-year DMFS (HR 1.93, P = 0.007), whereas 5-year locoregional recurrence-free survival was not affected (HR 1.50, P = 0.16). Five-year DFS was increased by EIC in T1 patients (P = 0.03) but not in T2 stage. Moreover, EIC was associated to better DFS in G2 (P = 0.03) and G3 patients (P = 0.01) but not in G1 cases.
Our results suggest that EIC is independently correlated with increased 5-year DFS and in particular with 5-year DMFS.
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