CMA mediates resistance in breast cancer models.

BACKGROUND: Breast cancer (BC) is the most common malignancy in women and the second leading cause of cancer-related death; chemoresistance is still a clinical challenge mainly because of the different molecular features of this kind of tumour. Doxorubicin (Doxo) is widely used despite its adverse effects and the common onset of resistance. Chaperone-Mediated Autophagy (CMA) has been identified as an important mechanism through which chemotherapeutics can exert their cytotoxic effects and, in this context, LAMP-2A, the key player of CMA, can be a useful biomarker. METHODS: A cohort of patients and breast cancer cells have been screened for Doxo effect and CMA activation by analysing the LAMP-2A level. Molecular silencing has been used to clarify CMA role in BC responsiveness to treatments. Low Doxo doses were combined with other drugs (TMZ or PX-478, a HIF-1α inhibitor) to evaluate their cytotoxic ability and their role in modulating CMA. RESULTS: In this paper, we showed that CMA is an important mechanism mediating the responsiveness of breast cancer cell to different treatments (Doxo and TMZ, as suggested by triple negative cells that are TMZ-resistant and fails to activate CMA). The LAMP-2A expression level was specific for different cell lines and patient-derived tumour subtypes, and was also useful in discriminating patients for their survival rates. Moreover, molecular silencing or pharmacological blockage of HIF-1α activity reverted BC resistance to TMZ. The combination of low-dose Doxo with TMZ or PX-478 showed that the drug associations have synergistic behaviours. CONCLUSION: Here, we demonstrated that CMA activity exerts a fundamental role in the responsiveness to different treatments, and LAMP-2A can be proposed as a reliable prognostic biomarker in breast cancer. In this context, HIF-1α, a potential target of CMA, can also be assessed as a valuable therapeutic target in BC in view of identifying new, more efficient and less toxic therapeutic drug combinations. Moreover, the possibility to combine Doxo with other drugs acting on different but coherent molecular targets could help overcome resistance and open the way to a decrease in the dose of the single drugs.

Establishment and Morphological Characterization of Patient-Derived Organoids from Breast Cancer.

BACKGROUND: Patient-derived organoids (PDO) technology represents an emerging tool for the study of tumor biology and drug responsiveness, thus being useful to design personalized medicine approaches. Despite several studies and clinical trials are ongoing using PDO from colorectal and pancreatic cancer, only few research papers have been published exploiting PDO from breast cancer. Here, we have developed a new protocol to establish PDO from surgical and biopsy samples. Furthermore, we have set up also the methodologies adopted for culture and morphological evaluations. RESULTS: Surgical and core biopsy specimens collected from 33 patients with diagnosis of breast cancer have been processed using the protocols here described obtaining PDO from cancerous and healthy mammary tissue (when available) in a quick and easy way with good yields. The more critical aspects influencing the yield were the characteristic of the tissue of origin (healthy vs tumor tissue) and the amount of material obtained after enzymatic digestion process. Success rate from healthy samples was about 20,83%, while this percentage was higher in samples from cancer tissue (i.e. 87,5%). Also the morphological characterization of breast cancer PDO by brightfield and transmission electron microscopy has been reported. CONCLUSIONS: Despite obtaining some organoids from a surgical or biopsy specimen is not a difficult procedure, the establishment of a stable organoid line able to grow and replicate, suitable for long-term biobank storage, is not so obvious. A novel, simple and quick procedure to obtain PDO from surgical and biopsy samples is here proposed to achieve high success rate .

Deep-Manager: a versatile tool for optimal feature selection in live-cell imaging analysis.

One of the major problems in bioimaging, often highly underestimated, is whether features extracted for a discrimination or regression task will remain valid for a broader set of similar experiments or in the presence of unpredictable perturbations during the image acquisition process. Such an issue is even more important when it is addressed in the context of deep learning features due to the lack of a priori known relationship between the black-box descriptors (deep features) and the phenotypic properties of the biological entities under study. In this regard, the widespread use of descriptors, such as those coming from pre-trained Convolutional Neural Networks (CNNs), is hindered by the fact that they are devoid of apparent physical meaning and strongly subjected to unspecific biases, i.e., features that do not depend on the cell phenotypes, but rather on acquisition artifacts, such as brightness or texture changes, focus shifts, autofluorescence or photobleaching. The proposed Deep-Manager software platform offers the possibility to efficiently select those features having lower sensitivity to unspecific disturbances and, at the same time, a high discriminating power. Deep-Manager can be used in the context of both handcrafted and deep features. The unprecedented performances of the method are proven using five different case studies, ranging from selecting handcrafted green fluorescence protein intensity features in chemotherapy-related breast cancer cell death investigation to addressing problems related to the context of Deep Transfer Learning. Deep-Manager, freely available at https://github.com/BEEuniroma2/Deep-Manager , is suitable for use in many fields of bioimaging and is conceived to be constantly upgraded with novel image acquisition perturbations and modalities.

A three-gene signature marks the time to locoregional recurrence in luminal-like breast cancer.

BACKGROUND: Gene expression profiling (GEP)-based prognostic signatures are being rapidly integrated into clinical decision making for systemic management of breast cancer patients. However, GEP remains relatively underdeveloped for locoregional risk assessment. Yet, locoregional recurrence (LRR), especially early after surgery, is associated with poor survival. PATIENTS AND METHODS: GEP was carried out on two independent luminal-like breast cancer cohorts of patients developing early (≤5 years after surgery) or late (>5 years) LRR and used, by a training and testing approach, to build a gene signature able to intercept women at risk of developing early LRR. The GEP data of two in silico datasets and of a third independent cohort were used to explore its prognostic value. RESULTS: Analysis of the first two cohorts led to the identification of three genes, CSTB, CCDC91 and ITGB1, whose expression, derived by principal component analysis, generated a three-gene signature significantly associated with early LRR in both cohorts (P value <0.001 and 0.005, respectively), overcoming the discriminatory capability of age, hormone receptor status and therapy. Remarkably, the integration of the signature with these clinical variables led to an area under the curve of 0.878 [95% confidence interval (CI) 0.810-0.945]. In in silico datasets we found that the three-gene signature retained its association, showing higher values in the early relapsed patients. Moreover, in the third additional cohort, the signature significantly associated with relapse-free survival (hazard ratio 1.56, 95% CI 1.04-2.35). CONCLUSIONS: Our three-gene signature represents a new exploitable tool to aid treatment choice in patients with luminal-like breast cancer at risk of developing early recurrence.

Accelerating the experimental responses on cell behaviors: a long-term prediction of cell trajectories using Social Generative Adversarial Network.

The incremented uptake provided by time-lapse microscopy in Organ-on-a-Chip (OoC) devices allowed increased attention to the dynamics of the co-cultured systems. However, the amount of information stored in long-time experiments may constitute a serious bottleneck of the experimental pipeline. Forward long-term prediction of cell trajectories may reduce the spatial-temporal burden of video sequences storage. Cell trajectory prediction becomes crucial especially to increase the trustworthiness in software tools designed to conduct a massive analysis of cell behavior under chemical stimuli. To address this task, we transpose here the exploitation of the presence of "social forces" from the human to the cellular level for motion prediction at microscale by adapting the potential of Social Generative Adversarial Network predictors to cell motility. To demonstrate the effectiveness of the approach, we consider here two case studies: one related to PC-3 prostate cancer cells cultured in 2D Petri dishes under control and treated conditions and one related to an OoC experiment of tumor-immune interaction in fibrosarcoma cells. The goodness of the proposed strategy has been verified by successfully comparing the distributions of common descriptors (kinematic descriptors and mean interaction time for the two scenarios respectively) from the trajectories obtained by video analysis and the predicted counterparts.

Discovering the hidden messages within cell trajectories using a deep learning approach for in vitro evaluation of cancer drug treatments.

We describe a novel method to achieve a universal, massive, and fully automated analysis of cell motility behaviours, starting from time-lapse microscopy images. The approach was inspired by the recent successes in application of machine learning for style recognition in paintings and artistic style transfer. The originality of the method relies i) on the generation of atlas from the collection of single-cell trajectories in order to visually encode the multiple descriptors of cell motility, and ii) on the application of pre-trained Deep Learning Convolutional Neural Network architecture in order to extract relevant features to be used for classification tasks from this visual atlas. Validation tests were conducted on two different cell motility scenarios: 1) a 3D biomimetic gels of immune cells, co-cultured with breast cancer cells in organ-on-chip devices, upon treatment with an immunotherapy drug; 2) Petri dishes of clustered prostate cancer cells, upon treatment with a chemotherapy drug. For each scenario, single-cell trajectories are very accurately classified according to the presence or not of the drugs. This original approach demonstrates the existence of universal features in cell motility (a so called "motility style") which are identified by the DL approach in the rationale of discovering the unknown message in cell trajectories.

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model.

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

Aesthetic results following breast cancer surgery: A prospective study on 6515 cases from ten Italian Senonetwork breast centers.

Breast cancer treatment has deeply changed in the last decades, since clinical and oncological cure cannot be achieved without patient's satisfaction in term of aesthetic outcomes. Several methods have been proposed to objectively assess these results. However, Italian breast centers have not yet agreed on measurable, reproducible and validated aesthetic outcome indicators to monitor their performance. METHODS: The study was designed and conducted by Senonetwork, a not-for-profit association of Italian breast centers. Ten breast centers were selected based on specific eligibility criteria. This multicentre observational prospective study recruited 6515 patients with diagnosis of in situ or invasive breast cancer who underwent breast surgery in the years 2013-2016. Thirteen indicators of aesthetic results and of related quality of care were analyzed. Data collection and analysis were conducted using a common study database. RESULTS: On average, seven out of ten centers were able to collect data on the proposed indicators with a proportion of missing values < 25%. By expert consensus based on study results, some seven indicators have been defined as "mandatory" while the remaining six have been defined as "recommended" because they require further refinement before they can be proposed for monitoring aesthetic outcomes or because there are doubts on the feasibility of data collection. The minimum standard is reached for 5 of 13 indicators. This finding and the wide range between centers reveal that there is ample room for improvement. CONCLUSIONS: From the present study useful measurable aesthetic parameters have emerged, leading to the definition of target objectives that breast centers can use for benchmarking and improvement of quality of care.

Does conjugation strategy matter? Cetuximab-conjugated gold nanocages for targeting triple-negative breast cancer cells.

The efficient targeting of cancer cells depends on the success of obtaining the active targeting of overexpressed receptors. A very accurate design of nanoconjugates should be done via the selection of the conjugation strategy to achieve effective targeted nanoconjugates. Here, we present a detailed study of cetuximab-conjugated nonspherical gold nanocages for the active targeting of triple-negative breast cancer cells, including MDA-MB-231 and MDA-MB-468. A few different general strategies were selected for monoclonal antibody conjugation to the nanoparticle surface. By varying the bioconjugation conditions, including antibody orientation or the presence of a polymeric spacer or recombinant protein biolinker, we demonstrate the importance of a rational design of nanoconjugates. A quantitative study of gold content via ICP-AES allowed us to compare the effectiveness of cellular uptake as a function of the conjugation strategy and confirmed the active nature of nanoparticle internalization in cancer cells via epidermal growth factor receptor recognition, corroborating the importance of the rational design of nanomaterials for nanomedicine.

Different effects of vertical banded gastroplasty and Roux-en-Y gastric bypass on meal inhibition of ghrelin secretion in morbidly obese patients.

A decrease in ghrelin plasma levels in morbidly obese patients subjected to bariatric surgery has been considered to help increase body weight loss. Contradictory results have been described after Roux-en-Y gastric bypass (RYGBP), and no study to date has compared RYGBP and vertical banded gastroplasty (VBG), the two main operations performed in the United States. We investigated the effects of RYGBP (10 patients) and VBG (12 patients) on basal and postmeal ghrelin plasma levels in 22 morbidly obese patients (20 F and 2 M), mean age 42.1 +/- 3.7 years, mean weight 115 +/- 3.9 kg, mean body mass index (BMI) 43.5 +/- 1.7. Before surgery and after a 20% reduction in BMI, ghrelin concentrations (pg/mL; radioimmunoassay [RIA], DRG Diagnostics, Germany) were measured in all patients 45 min before and for 3 h after a standard liquid meal (Osmolite RTH solution, 500 mL, 504 kcal). The results were expressed as mean +/- SD. Differences between times and groups were evaluated by Student's t-test and one-way analysis of variance (ANOVA). We found that basal ghrelin plasma levels were reduced after RYGBP (to 73.1 +/- 6 pg/mL, p < .05) but increased after VBG (to 172 +/- 26 pg/mL, p < .0009). After a standard liquid meal, ghrelin plasma levels decreased significantly over 1 h in VBG patients, whereas they remained unchanged in RYGBP patients. Since these results were obtained under the same metabolic and anthropometric conditions, we conclude that RYGBP acts through permanent inhibition of ghrelin secretion, whereas VBG merely restores the mechanisms of ghrelin regulation by nutrients.

A rare case of Brooke-Spiegler syndrome: integrated surgical treatment of multiple giant eccrine spiradenomas of the head and neck in a young girl.

INTRODUCTION: The authors present a "four-step" integrated surgical protocol to treat a rare case of multiple giant eccrine spiradenoma (ES) of the head and neck in a young patient. PRESENTATION OF CASE: An 18-year-old female patient presented with multiple swellings in the head and neck regions. The patient had a severe psychological trauma with a negative impact on her social life. Physical examination revealed multiple papulo-nodular swellings measuring between 5 cm × 8 cm and up to 10 cm × 20 cm in size with cerebriform aspect and soft consistency. Major lesions were located in the scalp, frontal area, neck, occipitotemporal, and retroauricular regions. Tissue biopsy found a benign composite adnexal neoplasm consisted in ES, trichoepithelioma, and cylindroma, a typical feature of Brooke-Spiegler Syndrome. A staged excision was planned, and available reconstructive options were considered. Scalp reconstruction included tissue expansions, advancement flaps, skin grafts, and dermal regeneration template (Integra®). All treatments were successful, and no recurrence was observed. The patient returned to a normal social life, and a radical excision with satisfying aesthetic results was achieved. DISCUSSION: Although adnexal tumors are benign in most of the cases, these lesions are prone to arise in the craniofacial region, thereby causing aesthetic discomfort associated with pain, hemorrhage, and infection to the patient every day. Furthermore, there is a potential risk of malignant transformation. These concerns demonstrate the need to establish a surgical protocol for the treatment of adnexal tumors. CONCLUSIONS: Our integrated surgical approach showed excellent aesthetic and functional results with benefits to the patient's life and complete oncological excision.

Cerebellar tumour presenting with pathological laughter and gelastic syncope.

There is no report of patients in whom pathological laughter, a rare condition characterized by uncontrollable episodes of laughter usually triggered by unrelated stimuli, was ever closely associated with a loss of consciousness overtly linked with the onset of such uncontrollable laughter, also referred to as a gelastic syncope. A 53-year-old man presented with a 4-month history of syncope following intense and uncoordinated laughter. Physical and neurological examination was normal and the patient had no other typical cerebellar signs. We found a mass in the cerebellar vermis abutting the floor of the fourth ventricle, which upon histological examination after surgery proved to be an ependymoma. We emphasize that pathological laughter and gelastic syncope could represent unique and sole features of a cerebellar disorder.

H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

Evaluation of systolic blood pressure control in elderly patients with isolated systolic hypertension.

The aim of this retrospective study was the evaluation of systolic blood pressure (SBP) control in elderly patients (pts) with isolated systolic hypertension (ISH). We assessed SBP control (i.e. average of 2 clinic BP measurements < 140 mmHg) in 152 pts (44 M, 108 F, 75 +/- 6 years) with ISH (149/84 +/- 17/6 mmHg), treated for at least 3 months by general practitioners or specialists with treatments of their choice. Most antihypertensive drugs were used at starting doses in monotherapy or combination treatment, as usual in clinical practice. ECG was abnormal in 82/152 pts (54.0%). All pts were divided in 2 groups according to SBP control. The 2 groups were compared by chi-square test for categorical variables and by Mann-Whitney test for quantitative variables. A p value < 0.05 was considered statistically significant. The global SBP control rate was 41.4% (63/152 pts). BP was higher in pts with poor SBP control, as expected, but the 2 groups were similar for sex distribution, age, prevalence of other cardiovascular risk factors and type of care (general practitioner or specialist). Pts with poor SBP control had a higher prevalence of abnormal ECG tracings (p = 0.003), a lower prevalence of combination regimes (p = 0.007) and prescriptions of dihydropyridine calcium antagonists or thiazide diuretics (p = 0.006). Global SBP control rate in our retrospective study in pts with ISH was unsatisfactory. Use of dihydropyridines or thiazides, drugs of choice in the management of ISH according to ESH/ESC and JNCVII guidelines, as single drugs or in combination regimes, can improve BP control and prevent cardiac damage.

Vertical banded gastroplasty modifies plasma ghrelin secretion in obese patients.

BACKGROUND: Restrictive bariatric surgery causes weight loss through substantial decline of appetite with satiety after meals. Reduction of plasma ghrelin levels after Roux-en-Y gastric bypass and laparoscopic adjustable gastric banding could contribute to these effects, although contradictory results have been reported. The only restrictive operation still not yet investigated is vertical banded gastroplasty (VBG). We studied the effects of VBG on basal plasma ghrelin levels and meal-mediated inhibition. METHODS: 12 morbidly obese patients, 11 female and 1 male, were studied before and after VBG, when the BMI fell by 20%. The control group consisted of 6 lean volunteers. Active ghrelin was determined by RIA after overnight fasting and after the administration of a liquid meal. RESULTS: Obese patients preoperatively had significantly lower basal plasma ghrelin levels than lean volunteers, and the meal did not inhibit ghrelin secretion. After VBG and 20% BMI loss, basal plasma ghrelin levels increased and the reduction caused by a meal recovered. CONCLUSIONS: Weight loss caused by VBG is associated with higher plasma ghrelin levels in obese patients. The operation restores the normal adaptation of the A- cells of the stomach to a meal.

Noninvasive assessment of coronary artery stenoses by multidetector-row spiral computed tomography: comparison with conventional angiography.

BACKGROUND: Coronary artery disease (CAD) is the most common cause of hospitalization and mortality in many industrialized countries. We analysed the diagnostic accuracy of multi-detector row spiral computed tomography (MDCT) in determining mid- to high-grade coronary artery stenoses (> 50%). METHODS: Sixty-nine patients with suspected CAD were referred to MDCT coronary angiography. Patients with a heart rate above 60 bpm received 20-40 mg propranol before the scan. The left main (LM), the left anterior descending artery (LAD), the first diagonal branch (D1), the right coronary artery (RCA) and the proximal tract of the circumflex artery (LCX) were independently evaluated by two blinded observers and screened for > 50% stenoses. The mean values of MDCT coronary narrowings assessed by two observers were compared to quantitative coronary angiography. RESULTS: MDCT correctly detected 95 of 123 coronary lesions (sensitivity 77.2%) and absence of stenoses was correctly identified in 388 of 426 segments (specificity 91%). The sensitivity for the LM, LAD, RCA and the proximal tract of LCX was 100%, 86.5%, 69.8% and 80% respectively. Classification of patients as having 1-vessel, 2-vessels, 3-vessels or left main disease was accurate in 75.4% (46/61) of patients. CONCLUSIONS: MDCT technology, combined with heart rate control, allows reliable noninvasive detection of hemodynamically significant CAD.

Estimating temporal independence of radio-telemetry data on animal activity

Radio-telemetry is an excellent tool for gathering data on the biology of animals and their interactions with the environment they inhabit. Many methods have been developed for analyses of spatial information, on home range size and utilization density. Activity patterns are often described using radio-tracking data, but no generally accepted method is currently available specifically for determining the temporal independence of this type of data for statistical inference. Activity rhythms have generally been analysed by ecologists with the assumption that data are temporally independent, or by subjectively fixing an independence interval, based on attributes of their ranging behaviour. Although some good approximations of activity patterns can be obtained in these ways, we underline the need for a functionally correct method of estimating independence interval. Here we use semi-variograms to estimate the minimum interval required for the readings to be sequentially independent. This geostatistical tool is applied to the analysis of data on activity of Chilean foxes (Pseudalopex culpaeus) and Chacoan peccaries (Catagonus wagneri). Data were collected in the field by radio-tracking over 24-hr periods, with readings on activity state taken every 15 min. The spatial dimension in which the theory of geostatistics lies has been transferred into the time dimension, so that the correlation interval is expressed in time units (min). Time of independence as estimated by the variogram was 110 min for foxes, while data on peccaries indicated that they have long periods of activity, more suitable for time-series analysis. Copyright 1999 Academic Press.

Diffuse leptomeningeal gliomatosis with osteoblastic metastases and no evidence of intraaxial lesions.

An autopsy-proved case of cerebral and medullary leptomeningeal gliomatosis and diffuse osteoblastic metastases without evidence of intraaxial tumor is described. MR findings included diffuse thickening of the cerebral and medullary leptomeninges on T1-weighted, proton density-weighted, and T2-weighted images and abnormal enhancement of the sulci and cisterns of the cerebrum, brain stem, cerebellum, and medulla on postcontrast T1-weighted images. MR also showed several areas of replacement of the normal bone marrow of the skull. No intraaxial lesion was seen.

Metastatic melanoma of the gallbladder.

A 38-year-old man arrived at our clinic with symptoms and investigation results (U.S. scan and MR) suggestive of acute calculous cholecystitis. He gave a past history of excision of a stage I melanoma of the shoulder. Metastatic disease was suspected following measurement of CA 19.9 levels and the CT scan. The patient underwent laparotomy and cholecystectomy; pathological examination confirmed the presence of a malignant melanoma metastatic lesion of the gallbladder.