High-Resolution Raman Imaging of >300 Patient-Derived Cells from Nine Different Leukemia Subtypes: A Global Clustering Approach.

Leukemia comprises a diverse group of bone marrow tumors marked by cell proliferation. Current diagnosis involves identifying leukemia subtypes through visual assessment of blood and bone marrow smears, a subjective and time-consuming method. Our study introduces the characterization of different leukemia subtypes using a global clustering approach of Raman hyperspectral maps of cells. We analyzed bone marrow samples from 19 patients, each presenting one of nine distinct leukemia subtypes, by conducting high spatial resolution Raman imaging on 319 cells, generating over 1.3 million spectra in total. An automated preprocessing pipeline followed by a single-step global clustering approach performed over the entire data set identified relevant cellular components (cytoplasm, nucleus, carotenoids, myeloperoxidase (MPO), and hemoglobin (HB)) enabling the unsupervised creation of high-quality pseudostained images at the single-cell level. Furthermore, this approach provided a semiquantitative analysis of cellular component distribution, and multivariate analysis of clustering results revealed the potential of Raman imaging in leukemia research, highlighting both advantages and challenges associated with global clustering.

Preliminary Study of Axillary Lymphatic Drainage in Cutaneous Melanoma Patients: A Cross-Sectional Study.

Background: The axilla is a region of fundamental importance for the implications during oncological surgery, and there are many classifications of axillary lymph node subdivision: on the basis of studies on women with breast cancer, we used Clough's and Li's classification. However, currently we do not have a gold-standard classification regarding axillary lymphatic drainage in melanoma patients. Purpose: Our aim was to evaluate how these classifications could be adapted to sentinel lymph node evaluation in skin-melanoma patients and to look for a possible correlation between the most recent classifications of axillary lymph node location and Oeslner's classification, one of the most common anatomical classifications still widespread today. Methods: We analyzed data from 21 patients who underwent sentinel lymph node biopsy between January 2021 and January 2022. Results: Our study demonstrates that, to an extent, there is a possible difference in the use of the various classifications, hinting at possible limits of each. The data we obtained underline how cutaneous melanoma presents extremely heterogenous lymphatic drainage at the level of the axillary cavity. However, the limited data in our possession do not allow us to obtain, at the moment, results that are statistically significant, although we are continuing to enroll patients and collect data. Conclusions: Results of this study support the evidence that the common classifications used for breast cancer do not seem to be exhaustive. Therefore, a specific axillary lymph node classification is necessary in skin melanoma patients.

miRNAs and Alzheimer's Disease: Exploring the Role of Inflammation and Vitamin E in an Old-Age Population.

Alzheimer's disease (AD) is the most frequent cause of dementia worldwide and represents one of the leading factors for severe disability in older persons. Although its etiology is not fully known yet, AD may develop due to multiple factors, including inflammation and oxidative stress, conditions where microRNAs (miRNAs) seem to play a pivotal role as a molecular switch. All these aspects may be modulated by nutritional factors. Among them, vitamin E has been widely studied in AD, given the plausibility of its various biological functions in influencing neurodegeneration. From a cohort of old-aged people, we measured eight vitamin E forms (tocopherols and tocotrienols), thirty cytokines/chemokines, and thirteen exosome-extracted miRNAs in plasma of subjects suffering from subjects affected by AD and age-matched healthy controls (HC). The sample population included 80 subjects (40 AD and 40 HC) with a mean age of 77.6 ± 3.8 years, mostly women (45; 56.2%). Of the vitamin E forms, only α-tocopherol differed between groups, with significantly lower levels in AD. Regarding the examined inflammatory molecules, G-CSF, GM-CSF, INF-α2, IL-3, and IL-8 were significantly higher and IL-17 lower in AD than HC. Among all miRNAs examined, AD showed downregulation of miR-9, miR-21, miR29-b, miR-122, and miR-132 compared to controls. MiR-122 positively and significantly correlated with some inflammatory molecules (GM-CSF, INF-α2, IL-1α, IL-8, and MIP-1ß) as well as with α-tocopherol even after correction for age and gender. A final binary logistic regression analysis showed that α-tocopherol serum levels were associated with a higher AD probability and partially mediated by miR-122. Our results suggest an interplay between α-tocopherol, inflammatory molecules, and microRNAs in AD, where miR-122 may be a good candidate as modulating factor.

CSA Antisense Targeting Enhances Anticancer Drug Sensitivity in Breast Cancer Cells, including the Triple-Negative Subtype.

Breast cancer (BC) is the most common cancer with the highest frequency of death among women. BC is highly heterogenic at the genetic, biological, and clinical level. Despite the significant improvements in diagnosis and treatments of BC, the high rate of cancer recurrence and resistance to treatment remains a major challenge in clinical practice. This issue is particularly relevant in Triple-Negative Breast Cancer (TNBC) subtype, for which chemotherapy remains the main standard therapeutic approach. Here, we observed that BC cells, belonging to different subtypes, including the TNBC, display an increased expression of Cockayne Syndrome group A (CSA) protein, which is involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division and that recently was found to confer cell robustness when it is up-regulated. We demonstrated that CSA ablation by AntiSense Oligonucleotides (ASOs) drastically impairs tumorigenicity of BC cells by hampering their survival and proliferative capabilities without damaging normal cells. Moreover, suppression of CSA dramatically sensitizes BC cells to platinum and taxane derivatives, which are commonly used for BC first-line therapy, even at very low doses not harmful to normal cells. Finally, CSA ablation restores drug sensitivity in oxaliplatin-resistant cells. Based on these results, we conclude that CSA might be a very attractive target for the development of more effective anticancer therapies.

Analysis of the Different Lymphatic Drainage Patterns during Sentinel Lymph Node Biopsy for Skin Melanoma.

In the last two decades, studies of lymphoscintigraphy imaging in lymphatic mapping reported an extreme heterogeneity of skin lymphatic drainage of some skin area, in contrast with the previous scientific literature. The aim of this study was to investigate the presence of any correlations between the topographical location of cutaneous melanoma and the topographical location of sentinel lymph nodes. Data from 165 patients undergoing sentinel lymph node biopsy between January 2013 and May 2021 were analyzed, demonstrating that melanomas in the Lumbar region presented a significant more heterogeneous drainage by site than those in the Scapular region (p < 0.01) and that melanomas in the Subscapular region were significantly more heterogeneous by laterality (unilateral vs. bilateral) than those in the Scapular region (p < 0.05). Results of this study supported the evidence of multiple lymphatic drainage as regards the sentinel node biopsy performed in skin melanoma located on the dorsal subscapular region and lumbar region. For this reason, the association of preoperative lymphoscintigraphy with another imaging evaluation is needed in these critical cutaneous areas. Recent technical developments enabling fluorescence lymphography together with indocyanine green have significantly improved the visualization of lymphatic drainage patterns at a microscopic level. In the preoperative phase, any doubt can be resolved by associating the SPET-CT scan to lymphoscintigraphy, while during the intraoperative phase, an additional evaluation with indocyanine green can be performed in doubtful cases. The aim of the duplex lymphatic mapping (pre and/or intraoperative) is an accurate search of sentinel nodes, in order to reduce the rate of false negatives.

Epigenetic Mechanisms of Inflammasome Regulation.

The innate immune system represents the host's first-line defense against pathogens, dead cells or environmental factors. One of the most important inflammatory pathways is represented by the activation of the NOD-like receptor (NLR) protein family. Some NLRs induce the assembly of large caspase-1-activating complexes called inflammasomes. Different types of inflammasomes have been identified that can respond to distinct bacterial, viral or fungal infections; sterile cell damage or other stressors, such as metabolic imbalances. Epigenetic regulation has been recently suggested to provide a complementary mechanism to control inflammasome activity. This regulation can be exerted through at least three main mechanisms, including CpG DNA methylation, histones post-translational modifications and noncoding RNA expression. The repression or promotion of expression of different inflammasomes (NLRP1, NLRP2, NLRP3, NLRP4, NLRP6, NLRP7, NLRP12 and AIM2) through epigenetic mechanisms determines the development of pathologies with variable severity. For example, our team recently explored the role of microRNAs (miRNAs) targeting and modulating the components of the inflammasome as potential biomarkers in bladder cancer and during therapy. This suggests that the epigenetic control of inflammasome-related genes could represent a potential target for further investigations of molecular mechanisms regulating inflammatory pathways.

Relationship between cellular and exosomal miRNAs targeting NOD-like receptors in bladder cancer: preliminary results.

BACKGROUND: Exosomes are membrane vesicles secreted by both cancerous and normal cells which play important roles during intercellular communications and oncogenic transformation. Many reports highlight the importance of exosomal microRNAs (miRNAs) as pro-tumorigenic mediators during carcinogenesis, since they regulate all these pathways at the post-transcriptional level. Since bladder cancer cells have a high immunogenic potential, from one hand, and inflammation process is intimately connected to carcinogenesis, from the other, the interest in analyzing inflammasome-related exo-miRNAs is apparent. The modulation of miRNAs targeting NOD-like receptor mRNAs in patients harboring bladder cancer has been assessed in our previous studies. METHODS: In the present report, we characterized the previously selected miRNAs in the soluble fraction of the same bladder cancer patient cohort, stratified according to the risk of recurrence and progression. Exosome precipitation and isolation were performed; the expression levels of exosomal miRNAs were compared with their cellular counterparts. RESULTS: An up-regulation of exosomal miR-141-3p and miR-19a-3p with respect to urine sediment was reported. Linear regression analysis showed a significant negative correlation for the same miRNAs. Moreover, exosomal miRNAs increased in low risk compared to high risk patients, which was opposite to that observed for urine sediment. CONCLUSIONS: Our work demonstrated the inverse correlation between exosomal and cellular miRNAs in patients with bladder cancer. The fact that these miRNAs were higher in exosomal than cellular fraction allowed us to hypothesize their active compartmentalization during cancer progression, suggesting their potential role as cancer messengers.

Renal Artery Embolization Before Radical Nephrectomy for Complex Renal Tumour: Which are the True Advantages?

INTRODUCTION: Renal artery embolization is performed before radical nephrectomy (RN) for renal mass in order to induce preoperative infarction and to facilitate surgical intervention through decrease of intraoperative bleeding. Moreover, in metastatic renal cancer it seems to stimulate tumour-specific antibodies, even if no established benefits in clinical response or survival have been reported. The role of preoperative renal artery embolization (PRAE) in management of renal masses has been often debated and its real benefits are still unclear. Nevertheless, in huge and complex renal masses, which are often characterized by a high and anarchic blood supply and rapid local invasion, radical nephrectomy can be challenging even for skilled surgeons. The aim of this prospective randomized study was to evaluate the effectiveness and safety of PRAE in complex masses by comparing perioperative outcomes of RN with and without PRAE. MATERIALS AND METHODS: From December 2015 to May 2018 we enrolled prospectively 64 patients who underwent RN for localized (T2a-b) or locally advanced (T3 and T4) or advanced (N+, M+) renal cancers. Patients were divided in two groups. The first group included 30 patients who underwent PRAE; in the second group we enrolled 34 patients who did not undergo RN without PRAE. Perioperative outcomes in terms of operative time, blood loss, transfusion rate and length of hospitalization were evaluated. Statistical analysis was performed using GraphPad Prism 6.0 software. RESULTS: Median blood loss was 250 ml (50-500) and 400 ml (50-1000) in the first and second group, respectively, with a statistically significant difference (p=0.0066). Median surgical time was 200 min (90-390) and 240 min (130-390) in PRAE and No-PRAE group (p=0.06), respectively. No major complications occurred after embolization. Overall complication rate in Group 1 and 2 was 46.7% (14/30) and 50% (17/34), respectively (p=0.34). No major complications occurred in both groups. The mean follow up was 21,5 months. CONCLUSIONS: Our results prove PRAE to be a safe procedure with low complications rate. To our experience, PRAE seems to be a useful tool in surgical management of a large mass and advanced disease.

Detection and scavenging of hydroxyl radical via D-phenylalanine hydroxylation in human fluids.

Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .OH attack, using a detection system based on Fenton reaction-mediated D-phenylalanine (D-Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D-Phe on .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N'-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl-L-cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 ± 0.12 times, p < 0.01). Furthermore, HD treatment determined a significant reduction of serum OHSC concentration (54.63 ± 2.82%, p < 0.001). Our results provide evidence that Fenton reaction-mediated D-Phe hydroxylation is a suitable method for routine and non-invasive evaluation of .OH detection and its scavenging in human biological fluids.

Characterization of inflammasome-related genes in urine sediments of patients receiving intravesical BCG therapy.

BACKGROUND: Nowadays, the intravesical Bacillus Calmette-Guérin (BCG) instillation is the method of choice for the postsurgical treatment of high-grade nonmuscle-invasive bladder cancer , to reduce both recurrence rate and risk of progression. BCG is hypothesized to correct the immune system disequilibrium occurring during carcinogenesis, through an immunostimulation with detrimental effects for tumoral cells. Inflammation plays a crucial role in tumor progression. The deregulation of inflammasomes upon carcinogenesis underlines its importance both in physiologic and pathologic human conditions. Nucleotide oligomerization domain-like receptors (NLRs) are key components of this molecular platform and the increase in expression of some members of nucleotide oligomerization domain-like receptors family (NLRP3, NLRP4, NLRP9, and NLR family apoptosis inhibitory protein [NAIP]) in urothelial carcinoma was already demonstrated in our previous work. The first aim of the present work was to estimate whether these inflammasome-related genes show alterations during BCG instillations. The expression levels of NLRP3, NLRP4, NLRP9, and NAIP were assessed in the urine sediments from patients, which underwent surgery for superficial high-grade bladder cancer and further subjected to serial BCG instillations. The eventual association between NLR expression and recurrence was also evaluated. The expression of CK20 mRNA as confirmed marker of bladder cancer was also assayed. METHODS: Urine were sampled from patients harboring high-grade superficial bladder cancer and treated postsurgically with weekly BCG instillations for 6 weeks (induction cycle, I). Urine sediments were processed and resulting RNA was reverse transcribed and used for amplification by real-time PCR. RESULTS: After surgery, CK20 levels decreased significantly whereas NLRP4 and NLRP9 genes showed an increase. NLRP3 and NAIP remained substantially unmodified. CK20 mRNA decreased at the end of the induction cycle. NLRP3 did not show relevant modifications. The expression levels of NLRP4 and NLRP9 decreased significantly after 2 BCG administrations and remained substantially downregulated during the whole induction cycle. CK20 was higher in recurrence cases before BCG administration compared to the recurrence-free group, while no significant difference after BCG therapy was recorded. NLRP4 and NLRP9 were higher in patients with recurrence before BCG administration. CONCLUSIONS: The study underlines the importance of NLRP4 and NLRP9 in urothelial carcinoma and if these preliminary data will be confirmed in larger cohort studies, the assessment of NLRP4 and NLRP9 expression levels could help to predict the BCG failure, playing a relevant role in decision making for early radical surgery.

Expression of urinary miRNAs targeting NLRs inflammasomes in bladder cancer.

AIM OF THE STUDY: Inflammasome, a large complex of NOD-like receptors (NLRs), drives tumor growth and progression. The present study aimed at exploring the alteration in expression of urinary inflammasome-related microRNAs (miRNAs) in bladder cancer (BC). Our previous report demonstrated the up-regulation of NLRs genes (NLRP3, NLRP4, NLRP9 and NAIP) in urine sediments of patients harboring BC. The expression levels of miRNAs targeting these NLRs (miR-146a-5p, miR-106a-5p, miR-17-5p, miR-223-3p, miR-141-3p, miR-19a-3p, miR-145-5p, miR-185-5p) were assayed in the same patient cohort. MATERIALS AND METHODS: Forty-six subjects affected by BC, 28 healthy controls (CTR0) and 31 subjects with histologically confirmed bladder inflammation (CTR1) were recruited. Total RNA was extracted from urine sediment and resulting cDNA was used for amplification by real-time polymerase chain reaction. MiRNA expression levels were evaluated and compared among selected groups. Patients were further stratified according to tumor stage, grade and risk of recurrence and progression. Moreover, non-muscle invasive low-grade and high-grade (HG) BC patients were compared. RESULTS: MiR 141-3p and miR-19a-3p expression decreased in CTR1 with respect to both BC and CTR0. In contrast, miR-146a-5p was up-regulated in BC compared with CTR0. MiR106a-5p, miR17-5p and miR19a-5p were significantly up-regulated in HG, high-risk (HR) and non-muscle invasive HG BC patients, while miR-185-5p was significantly higher in muscle invasive tumors, according to T stage stratification. CONCLUSION: The increased expression of miRNAs targeting NLRs in HG and HR BC patients is in accordance with the decrease in NLR mRNAs observed in our previous report. These data corroborate the direct role of NLR genes and respective regulatory miRNAs in BC making these inflammasome-related molecules a reliable non-invasive tool for BC diagnosis.

Different levels of serum microRNAs in prostate cancer and benign prostatic hyperplasia: evaluation of potential diagnostic and prognostic role.

INTRODUCTION: Diagnosis of prostate cancer (PCa) is based on prostate biopsy that is performed when prostate specific antigen (PSA) is persistently altered over time and/or abnormal digital rectal examination is found. Serum PSA levels increase in both PCa and benign prostatic hyperplasia, leading to an increased number of unnecessary biopsies. There is an urgent need to unravel PCa-specific molecular signatures. PATIENTS AND METHODS: This study aimed at characterizing a panel of circulating micro RNAs (miRNAs) that could distinguish PCa from benign prostatic hyperplasia in a population of age-matched patients with increased PSA levels. Both miRNAs targeting genes involved in PCa onset and miRNAs whose role in PCa has been highlighted in other studies were included. For this purpose, let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-24-3p, miR-23b-3p, miR-27-b-3p, miR-106a-5p, miR-20b-5p, miR-18b-5p, miR-19b-2-5p, miR-363-3p, miR-497, miR-195, miR-25-3p, miR-30c-5p, miR-622, miR-874-3p, miR-346 and miR-940 were assayed through real-time PCR in 64 patients with PCa and compared with 60 patients with benign prostatic hyperplasia. The ability of miRNAs to predict the stage of disease was also analyzed. RESULTS: Let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-18b-5p and miR-25-3p were able to discriminate patients with PCa from those harboring benign prostatic hyperplasia, both presenting altered PSA levels (>3 ng/mL). MiR-25-3p and miR-18b-5p showed the highest sensitivity and specificity to predict PCa, respectively. The combination of these two miRNAs improved the overall sensitivity. A correlation between pathological Gleason score and miRNA expression levels was reported; miR-363-3p, miR-26a-5p, miR-26b-5p, miR-106a-5p, miR-18b-5p, miR-25-3p and let-7i decreased in expression concomitantly with an increase in malignancy. CONCLUSION: This study confirms serum miRNAs to be reliable candidates for the development of minimally invasive biomarkers for the diagnosis and prognosis of PCa, particularly in those cases where PSA acts as a flawed marker.

New Trends in Acute Management of Colonic Diverticular Bleeding: A Systematic Review.

Colonic diverticular disease is the most common cause of lower gastrointestinal bleeding. In the past, this condition was usually managed with urgent colectomy. Recently, the development of endoscopy and interventional radiology has led to a change in the management of colonic diverticular bleeding.The aim of this systematic review is to define the best treatment for colonic diverticular bleeding.A systematic bibliographic research was performed on the online databases for studies (randomized controlled trials [RCTs], observational trials, case series, and case reports) published between 2005 and 2014, concerning patients admitted with a diagnosis of diverticular bleeding according to the PRISMA methodology.The outcomes of interest were: diagnosis of diverticulosis as source of bleeding; incidence of self-limiting diverticular bleeding; management of non self-limiting bleeding (endoscopy, angiography, surgery); and recurrent diverticular bleeding.Fourteen studies were retrieved for analysis. No RCTs were found. Eleven non-randomized clinical controlled trials (NRCCTs) were included in this systematic review. In all studies, the definitive diagnosis of diverticular bleeding was always made by urgent colonoscopy. The colonic diverticular bleeding stopped spontaneously in over 80% of the patients, but a re-bleeding was not rare. Recently, interventional endoscopy and angiography became the first-line approach, thus relegating emergency colectomy to patients presenting with hemodynamic instability or as a second-line treatment after failure or complications of hemostasis with less invasive treatments.Colonoscopy is effective to diagnose diverticular bleeding. Nowadays, interventional endoscopy and angiographic treatment have gained a leading role and colectomy should only be entertained in case of failure of the former.

Expression of inflammasome-related genes in bladder cancer and their association with cytokeratin 20 messenger RNA.

BACKGROUND: Inflammation plays a crucial role in different stages of cancer development and has long been associated with various types of cancer. Strong associations between dysregulated inflammasome activity and human heritable and acquired inflammatory diseases highlight the importance of this pathway in the immune response. The inflammasome is a large complex of NOD-like receptors called NLRs and drives growth and progression of different tumors. The aim of the present study was the characterization of some NLR genes, NLRP3, NLRP4, NLRP9, and NAIP, in urine sediment of patients with bladder cancer. Cytokeratin 20 and survivin were used as confirmed markers of bladder cancer. BASIC PROCEDURES: For this study, 3 groups of subjects were considered: patients harboring bladder cancer, subjects affected by bladder inflammation (CTR1), and healthy subjects (CTR0). Total RNA was extracted from urine sediments and resulting complementary DNA was used for amplification by real-time polymerase chain reaction. Results were stratified according to tumor stage, grade, and risk of progression and recurrence. MAIN FINDINGS: The expression of cytokeratin 20 was always significantly higher in patients with bladder cancer when compared with that in both the tumor-free groups. NLRP3, NLRP4, NLRP9, and NAIP were overexpressed in patients with BCa when compared with that in CTR0. Stratification according to tumor stage, grade, and risk of recurrence and progression showed NLRP up-regulations in patients with early-stage cancer. NAIP was overexpressed in high-risk patients in comparison to CTR0 and in high-grade patients compared with CTR0 and CTR1. PRINCIPAL CONCLUSIONS: These data are relevant to demonstrate the role of inflammasome in urothelial carcinoma, making NLR genes in urine sediment potential candidates for bladder cancer diagnosis.

Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications.

We aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confirmed benign prostatic hyperplasia (BPH). The stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the effect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutoff of acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments.