Proposal of a classification of cannulation damage in vascular access grafts based on clinical, ultrasound, and microscopic observations.

INTRODUCTION: Arteriovenous grafts (AVGs) serve as an alternative to native arteriovenous fistulas (AVFs) in the context of hemodialysis patient life planning. AVGs are more susceptible to developing outflow stenosis (due to intimal hyperplasia), thrombosis, and infections. However, an often overlooked contributor to AVG failure is cannulation damage. The objective of this paper is to assess the impact of cannulations on AVGs. We aim to establish a classification of AVG damage by comparing clinical data and ultrasound images with microscopic morphological findings obtained from explanted grafts. MATERIALS AND METHODS: This study is conducted at a single center. We included all patients who underwent AVG creation between 2011 and 2019. Comprehensive data on clinical history, follow-up, and complications were collected and reviewed. Duplex ultrasound (DUS) characteristics were documented, and all grafts explanted during the analysis period underwent optical microscopy evaluation. Finally, clinical data, along with DUS and microscopic findings, were integrated to derive a damage classification. RESULTS: During the study period, 247 patients underwent 334 early cannulation AVGs. The median follow-up duration was 714 days (IQR 392, 1195). One hundred eleven (33%) grafts were explanted. Clinical data and DUS findings were utilized to formulate a four-grade classification system indicating increasing damage. CONCLUSION: Cannulation damage alone does not solely account for AVG failure. It results from a biological host-mediated process that promotes the growth of intimal hyperplasia at the cannulation sites. This process is not clinically significant within the initial 2 years after AVG creation.

Immunological characterization of a long-lasting response in a patient with metastatic triple-negative breast cancer treated with PD-1 and LAG-3 blockade.

In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.

Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Accuracy of Human Papillomavirus (HPV) Testing on Urine and Vaginal Self-Samples Compared to Clinician-Collected Cervical Sample in Women Referred to Colposcopy.

In the context of cervical cancer prevention, where human papillomavirus (HPV) infection is pivotal, HPV testing is replacing Pap Smear in primary screening. This transition offers an opportunity for integrating self-sampling to enhance coverage. We evaluated the accuracy of HPV testing using self-collected urine and vaginal samples, comparing them to physician-collected cervical swabs. From a cohort of 245 women with abnormal cytology, we collected self-sampled vaginal, urine, and clinician-administered cervical specimens. Employing Anyplex™II HPV28 assay, outcomes revealed HPV positivity rates of 75.1% (cervical), 78.4% (vaginal), and 77.1% (urine). Significant, hr-HPV detection concordance was observed between self-taken cervical samples and clinical counterparts (k = 0.898 for vaginal; k = 0.715 for urine). This study extends beyond accuracy, highlighting self-collected sample efficacy in detecting high-grade cervical lesions. The insight underscores self-sampling's role in bolstering participation and aligns with WHO's goal to eliminate cervical cancer by 2030.

Hematopoietic Stem Cell (HSC)-Independent Progenitors Are Susceptible to Mll-Af9-Induced Leukemic Transformation.

Infant acute myeloid leukemia (AML) is a heterogeneous disease, genetically distinct from its adult counterpart. Chromosomal translocations involving the KMT2A gene (MLL) are especially common in affected infants of less than 1 year of age, and are associated with a dismal prognosis. While these rearrangements are likely to arise in utero, the cell of origin has not been conclusively identified. This knowledge could lead to a better understanding of the biology of the disease and support the identification of new therapeutic vulnerabilities. Over the last few years, important progress in understanding the dynamics of fetal hematopoiesis has been made. Several reports have highlighted how hematopoietic stem cells (HSC) provide little contribution to fetal hematopoiesis, which is instead largely sustained by HSC-independent progenitors. Here, we used conditional Cre-Lox transgenic mouse models to engineer the Mll-Af9 translocation in defined subsets of embryonic hematopoietic progenitors. We show that embryonic hematopoiesis is generally permissive for Mll-Af9-induced leukemic transformation. Surprisingly, the selective introduction of Mll-Af9 in HSC-independent progenitors generated a transplantable myeloid leukemia, whereas it did not when introduced in embryonic HSC-derived cells. Ex vivo engineering of the Mll-Af9 rearrangement in HSC-independent progenitors using a CRISPR/Cas9-based approach resulted in the activation of an aberrant myeloid-biased self-renewal program. Overall, our results demonstrate that HSC-independent hematopoietic progenitors represent a permissive environment for Mll-Af9-induced leukemic transformation, and can likely act as cells of origin of infant AML.

Monkeypox: A Histopathological and Transmission Electron Microscopy Study.

The global outbreak of human monkeypox virus (hMPXV1) in 2022 highlighted the usefulness of dermatological manifestations for its diagnosis. Infection by the human monkeypox virus thus necessitated inclusion in the diagnostic repertoire of dermatopathology. To assess the histopathological and microscopical findings of cutaneous lesions related to hMPXV infection, we analyzed skin biopsies from patients with positive MPXV DNA polymerase chain reaction presenting with a typical course of hMPXV1 infection. The most prominent histopathological findings were ascribable to a pustular stage in which epidermal necrosis with areas of non-viable keratinocytes and a "shadow cell" appearance were evident; in some cases, the deep portion of the hair follicle and the acrosyringial epithelium were affected. The main cytopathic modifications included ballooning keratinocytes, followed by Guarnieri bodies and a ground glass appearance of the keratinocytes' nuclei, together with a dense mixed inflammatory cell infiltrate with prominent neutrophil exocytosis. Transmission electron microscopy analysis demonstrated viral particle aggregates in the cytoplasm of keratinocytes, without any involvement of the nucleus. Interestingly, we also found the presence of viral particles in infected mesenchymal cells, although to a lesser extent than in epithelial cells. Through this study, we contributed to expanding the histological and microscopic knowledge of the human mpox virus, a key step to understanding current and potential future trends of the disease, as well as of other Orthopoxvirus infections.

Follicular Lymphoma Microenvironment Traits Associated with Event-Free Survival.

The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease "virtually" incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution (intrafollicular, IF and extrafollicular, EF) of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte (CD8, CD4, PD1) and macrophage (CD68, CD163, MA4A4A)-associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan-Meier estimates were linked with shorter EFS (event-free survival), with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts.

Myelomonocytic cells in giant cell arteritis activate trained immunity programs sustaining inflammation and cytokine production.

OBJECTIVE: Trained immunity (TI) is a de facto memory program of innate immune cells, characterized by immunometabolic and epigenetic changes sustaining enhanced production of cytokines. TI evolved as a protective mechanism against infections; however, inappropriate activation can cause detrimental inflammation and might be implicated in the pathogenesis of chronic inflammatory diseases. In this study, we investigated the role of TI in the pathogenesis of giant cell arteritis (GCA), a large-vessel vasculitis characterized by aberrant macrophage activation and excess cytokine production. METHODS: Monocytes from GCA patients and from age- and sex-matched healthy donors were subjected to polyfunctional studies, including cytokine production assays at baseline and following stimulation, intracellular metabolomics, chromatin immunoprecipitation-qPCR, and combined ATAC/RNA sequencing. Immunometabolic activation (i.e. glycolysis) was assessed in inflamed vessels of GCA patients with FDG-PET and immunohistochemistry (IHC), and the role of this pathway in sustaining cytokine production was confirmed with selective pharmacologic inhibition in GCA monocytes. RESULTS: GCA monocytes exhibited hallmark molecular features of TI. Specifically, these included enhanced IL-6 production upon stimulation, typical immunometabolic changes (e.g. increased glycolysis and glutaminolysis) and epigenetic changes promoting enhanced transcription of genes governing pro-inflammatory activation. Immunometabolic changes of TI (i.e. glycolysis) were a feature of myelomonocytic cells in GCA lesions and were required for enhanced cytokine production. CONCLUSIONS: Myelomonocytic cells in GCA activate TI programs sustaining enhanced inflammatory activation with excess cytokine production.

Evaluation of Human Papilloma Virus (HPV) Genotyping and Viral Load Determination as Diagnostic Biomarkers of Cervical Cancer Risk.

HPV testing in cervical cancer screening programs offers the possibility of introducing molecular standardized biomarkers for the triage of HPV-positive women. This study aimed to evaluate the role of HPV genotyping and viral load as possible diagnostic biomarkers of high-grade cervical lesions (CIN2+) by performing a preliminary evaluation of a new HPV test. Cervical specimens were obtained from 200 women referred for a colposcopy. Samples were tested using both Anyplex™ II HR-HPV as well as OncoPredict HPV® Screening (SCR) and quantitative typing (QT). Using a cycle threshold cutoff (Ct) of 36.8 for the SCR assay and 1.27 log10 (viral copies/104 cells) for the QT assay, relative clinical sensitivity for CIN2+ and relative clinical specificity for CIN2- as compared to Anyplex™ II HR-HPV were, respectively, 0.92 and 1.00 for SCR and 1.35 and 1.24 for QT. The distribution of high-risk HPV (HR-HPV) genotypes (p = 0.009) as well as the viral copy numbers (CIN2-: 3.7 log10 (viral copies/104 human cells); CIN2+: 4.3 log10 (viral copies/104 human cells); p = 0.047) were found to differ in women with high- and low-grade cervical lesions, suggesting a possible role of HPV genotyping and normalized viral load as potential biomarkers to identify women at increased risk of cervical lesions.

Dual Functionalized Liposomes for Selective Delivery of Poorly Soluble Drugs to Inflamed Brain Regions.

Dual functionalized liposomes were developed to cross the blood−brain barrier (BBB) and to release their cargo in a pathological matrix metalloproteinase (MMP)-rich microenvironment. Liposomes were surface-functionalized with a modified peptide deriving from the receptor-binding domain of apolipoprotein E (mApoE), known to promote cargo delivery to the brain across the BBB in vitro and in vivo; and with an MMP-sensitive moiety for an MMP-triggered drug release. Different MMP-sensitive peptides were functionalized at both ends with hydrophobic stearate tails to yield MMP-sensitive lipopeptides (MSLPs), which were assembled into mApoE liposomes. The resulting bi-functional liposomes (i) displayed a < 180 nm diameter with a negative ζ-potential; (ii) were able to cross an in vitro BBB model with an endothelial permeability of 3 ± 1 × 10−5 cm/min; (iii) when exposed to functional MMP2 or 9, efficiently released an encapsulated fluorescein dye; (iv) showed high biocompatibility when tested in neuronal cultures; and (v) when loaded with glibenclamide, a drug candidate with poor aqueous solubility, reduced the release of proinflammatory cytokines from activated microglial cells.

A case of medical liability involving an unexpected systemic amyloidosis.

The authors present a case of fatal amyloid cardiomyopathy, which was diagnosed only upon autopsy. A 57-year-old man was admitted to the hospital for scheduled percutaneous cardiac procedure of transcatheter radiofrequency ablation due to persistent atrial fibrillation and atrial flutter. Ventricular fibrillation was recorded in the monitor 2 h after the surgical procedure. Therefore, he was defibrillated and intubated, but he died for nosocomial pneumonia 26 days after being admitted. A judicial autopsy was ordered by the prosecutor due to an alleged medical malpractice. The autopsy confirmed the cause of death being pneumonia, but also revealed an occult restrictive cardiomyopathy with a thick and firm myocardium. Viscera samples were then collected for microscopic examination. Histopathologic analysis showed diffuse amyloid deposits in the myocardium, especially in the perivascular and subendocardial spaces. Amyloid deposits were also detected in all the other organs, except for the brain. Furthermore, immunohistochemistry for light chains was performed on the heart tissue sample, resulting to be positive. In the case presented herein, autopsy and histopathologic examination were crucial to diagnose an occult systemic amyloidosis (AL-type). In fact, it has been observed that the rarity of systematic amyloidosis and its unusual clinical onset were at first mistakenly perceived as a medical malpractice due to a technical error within the catheter ablation for atrial fibrillation. As a consequence, upon discussing the clinical and medicolegal implications concerning the case, the focus was placed on the undiagnosed systemic amyloidosis and on the causality between surgical procedure and the patient's death.

Finding the seed of recurrence: Hepatocellular carcinoma circulating tumor cells and their potential to drive the surgical treatment.

The treatment for hepatocellular carcinoma (HCC) relies on liver resection, which is, however, burdened by a high rate of recurrence after surgery, up to 60% at 5 years. No pre-operative tools are currently available to assess the recurrence risk tailored to every single patient. Recently liquid biopsy has shown interesting results in diagnosis, prognosis and treatment allocation strategies in other types of cancers, since its ability to identify circulating tumor cells (CTCs) derived from the primary tumor. Those cells were advocated to be responsible for the majority of cases of recurrence and cancer-related deaths for HCC. In fact, after being modified by the epithelial-mesenchymal transition, CTCs circulate as "seeds" in peripheral blood, then reach the target organ as dormant cells which could be subsequently "awakened" and activated, and then initiate metastasis. Their presence may justify the disagreement registered in terms of efficacy of anatomic vs non-anatomic resections, particularly in the case of microvascular invasion, which has been recently pointed as a histological sign of the spread of those cells. Thus, their presence, also in the early stages, may justify the recurrence event also in the contest of liver transplant. Understanding the mechanism behind the tumor progression may allow improving the treatment selection according to the biological patient-based characteristics. Moreover, it may drive the development of novel biological tailored tests which could address a specific patient to neoadjuvant or adjuvant strategies, and in perspective, it could also become a new method to allocate organs for transplantation, according to the risk of relapse after liver transplant. The present paper will describe the most recent evidence on the role of CTCs in determining the relapse of HCC, highlighting their potential clinical implication as novel tumor behavior biomarkers able to influence the surgical choice.

Gα15 in early onset of pancreatic ductal adenocarcinoma.

The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded Gα15 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5' GNA15 promoter region was associated with ectopic expression of Gα15 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of Gα15 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic Gα15 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11.

HPV 16 and 18 contribute to development of anal dysplasia in HIV infection irrespective of gender and sexual orientation.

OBJECTIVES: The aim of the present study was too investigate prevalence and persistence of human papilloma virus (HPV) and cytological abnormalities (CAs) in the anal swabs of people living with HIV (PLWH): men who have sex with men (MSM), men who have sex with women (MSW) and women (W). METHODS: Between March 2010 and January 2019, an anal swab for cytological and HPV genotyping tests was offered to all PLWH attending our clinic. Logistic regression analysis was conducted to identify predictors of infection. RESULTS: In all, 354 PLWH were screened: 174 MSM, 90 MSW and 61 W. Prevalence of at least one high-risk (HR) HPV was higher in MSM (91%) and W (85%) than in MSW (77%) (P < 0.05). Cytological abnormalities were found in 21.1% of the entire population. At multivariable regression analysis a lower risk for HPV infection was found for W than for MSM [odds ratio = 0.24 (95% confidence interval: 0.115-0.513)] and for MSW than for MSM [0.37 (0.180-0.773)] and there was a significantly higher risk of CAs in PLWH with HPV 16 and 18 [3.3 (1.04-10.49)]. A total of 175 PLWH (103 MSM, 33 MSW and 26 W) had at least one follow-up visit (T1) after a median (interquartile range) follow-up of 3.6 (2.1-5.7) years. The acquisition rate of HR-HPV was high, with 66.7% of PLWH negative for HR-HPV at T0 who became positive at T1 (P < 0.001). The prevalence of CAs was stable (20.6%). A significant association between CAs at T1 and persistence of HPV-16 and/or 18 was found (P < 0.05). CONCLUSIONS: HPV 16 and 18 are associated with the presence and development of CAs irrespective of sexual orientation.

Comparison of tumor microenvironment in primary and paired metastatic ER+/HER2- breast cancers: results of a pilot study.

BACKGROUND: Tumor microenvironment (TME) is a dynamic setting and changes in TILs and their subpopulations are potential candidates to influence the metastatic process. Aim of this pilot study is to describe the changes occurring between primary breast cancers and their paired metastases in terms of TILs composition. To assess if these changes influence the process of metastasis development, we used a control group of patients. METHODS: We retrospectively identified 18 Luminal patients, for whom primary and metastatic tissue were available (cases) and 18 paired-matched patients (controls), not relapsed after at least 9 years of follow-up, and we quantified TILs and their composition (i.e. T CD8+ and CD4+/FOXP3+). The presence of TILs was defined as ≥10%. RESULTS: Our results showed that the microenvironment composition of relapsed patients was poor of TILs (median = 5%, I-III quartiles = 0.6-5%), CD8+ (2.5%, 0-5%) and CD4+/FOXP3 + (0%, 0-0.6%) in the primary tumor. Comparable results were observed in their related metastases (TILs 3.8%, 0.6-5%; CD8+ 0%, 0-1.3%; CD4+/FOXP3+ 0%,0-1.9%). On the contrary, the microenvironment in the control group was richer of TILs (5%, 5-17.5%) in comparison to cases, both in primary tumor (p = 0.035) and related metastases (p = 0.018). Although CD8+ in controls were similar to cases at primary tumor (p = 0.6498), but not at metastasis (p = 0.0223), they expressed only one part on the TILs subpopulations (p = 0.0060), while TILs in the cases at primary tumor were almost completely CD8+ (p = 0.5034). CONCLUSIONS: These findings suggest that the lack of activation of immune system in the primary tumor might influence the multifactor process of cancer progression.

Reduced Levels of ABCA1 Transporter Are Responsible for the Cholesterol Efflux Impairment in ß-Amyloid-Induced Reactive Astrocytes: Potential Rescue from Biomimetic HDLs.

The cerebral synthesis of cholesterol is mainly handled by astrocytes, which are also responsible for apoproteins' synthesis and lipoproteins' assembly required for the cholesterol transport in the brain parenchyma. In Alzheimer disease (AD), these processes are impaired, likely because of the astrogliosis, a process characterized by morphological and functional changes in astrocytes. Several ATP-binding cassette transporters expressed by brain cells are involved in the formation of nascent discoidal lipoproteins, but the effect of beta-amyloid (Aß) assemblies on this process is not fully understood. In this study, we investigated how of Aß1-42-induced astrogliosis affects the metabolism of cholesterol in vitro. We detected an impairment in the cholesterol efflux of reactive astrocytes attributable to reduced levels of ABCA1 transporters that could explain the decreased lipoproteins' levels detected in AD patients. To approach this issue, we designed biomimetic HDLs and evaluated their performance as cholesterol acceptors. The results demonstrated the ability of apoA-I nanodiscs to cross the blood-brain barrier in vitro and to promote the cholesterol efflux from astrocytes, making them suitable as a potential supportive treatment for AD to compensate the depletion of cerebral HDLs.

miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators.

BACKGROUND: Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance. METHODS: The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy. RESULTS: miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor. CONCLUSIONS: Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Video Abstract.

Detection of TP53 Clonal Variants in Papanicolaou Test Samples Collected up to 6 Years Prior to High-Grade Serous Epithelial Ovarian Cancer Diagnosis.

Importance: The low 5-year survival rate of women with high-grade serous epithelial ovarian cancer (HGS-EOC) is related to its late diagnosis; thus, improvement in diagnosis constitutes a crucial step to increase the curability of this disease. Objective: To determine whether the presence of the clonal pathogenic TP53 variant detected in matched primary tumor biopsies can be identified in DNA purified from Papanicolaou test samples collected from women with HGS-EOC years before the diagnosis. Design, Setting, and Participants: This cohort study was conducted among a single-center cohort of women with histologically confirmed diagnosis of HGS-EOC recruited at San Gerardo Hospital, Monza, Italy, from October 15, 2015, to January 4, 2019. Serial dilutions of DNA derived from tumor samples and DNA extracted from healthy women's Papanicolaou test samples were analyzed to define the sensitivity and specificity of droplet digital polymerase chain reaction assays designed to detect the TP53 variants identified in tumors. All available brush-based Papanicolaou test slides performed up to 6 years before diagnosis were investigated at the Mario Negri Institute, Milano, Italy. Data were analyzed from October 2018 to December 2019. Main Outcomes and Measures: The presence of tumor pathogenic TP53 variants was assessed by the droplet digital polymerase chain reaction approach in DNA purified from Papanicolaou test samples obtained from the same patients before diagnosis during cervical cancer screenings. Results: Among 17 included patients (median [interquartile range] age at diagnosis, 60 [53-69] years), Papanicolaou tests withdrawn before diagnosis presented tumor-matched TP53 variants in 11 patients (64%). In 2 patients for whom longitudinal Papanicolaou tests were available, including 1 patient with Papanicolaou tests from 25 and 49 months before diagnosis and 1 patient with Papanicolaou tests from 27 and 68 months before diagnosis, the TP53 clonal variant was detected at all time points. Conclusions and Relevance: These findings suggest that noninvasive early molecular diagnosis of HGS-EOC is potentially achievable through detection of TP53 clonal variants in the DNA purified from Papanicolaou tests performed during cervical cancer screening.

Two ultrastaging protocols for the detection of lymph node metastases in early-stage cervical and endometrial cancers.

OBJECTIVE: To date, there is no universal consensus on which is the optimal ultrastaging protocol for sentinel lymph node (SLN) evaluation in gynecologic malignancies. To estimate the impact of different ultrastaging methods of SLNs on the detection of patients with nodal metastases in early-stage cervical and endometrial cancers and to describe the incidence of low-volume metastases between two ultrastaging protocols. METHODS: We retrospectively compared two ultrastaging protocols (ultrastaging-A vs ultrastaging-B) in patients with clinical stage I endometrial cancer or FIGO stage IA-IB1 cervical cancer who underwent primary surgery including SLN biopsy from October 2010 to December 2017 in our institution. The histologic subtypes and grades of the tumors were evaluated according to WHO criteria. Only SLNs underwent ultrastaging, while other lymph nodes were sectioned and examined by routine hematoxylin and eosin (H&E). RESULTS: Overall 224 patients were reviewed (159 endometrial cancer and 65 cervical cancer). Lymph node involvement was noted in 15% of patients with endometrial cancer (24/159): 24% of patients (9/38) with the ultrastaging protocol A and 12% (15/121) with the ultrastaging protocol B (p=0.08); while for cervical cancer, SLN metastasis was detected in 14% of patients (9/65): 22% (4/18) in ultrastaging-A and 11% (5/47) in ultrastaging-B (p=0.20). Overall, macrometastasis and low-volume metastases were 50% and 50% for endometrial cancer and 78% and 22% for cervical cancer. Median size of nodal metastasis was 2 (range 0.9-8.5) mm for the ultrastaging-A and 1.2 (range 0.4-2.6) mm for the ultrastaging-B protocol in endometrial cancer (p=0.25); 4 (range 2.5-9.8) mm for ultrastaging-A and 4.4 (range 0.3-7.8) mm for ultrastaging-B protocol in cervical cancer (p=0.64). CONCLUSION: In endometrial or cervical cancer patients, the incidence of SLN metastasis was not different between the two different types of ultrastaging protocol.