IDH1 mutation is detectable in plasma cell-free DNA and is associated with survival outcome in glioma patients.

BACKGROUND: Circulating cell-free DNA (cfDNA, liquid biopsy) is a powerful tool to detect molecular alterations. However, depending on tumor characteristics, biology and anatomic localization, cfDNA detection and analysis may be challenging. Gliomas are enclosed into an anatomic sanctuary, which obstacles the release of cfDNA into the peripheral blood. Therefore, the advantages of using liquid biopsy for brain tumors is still to be confirmed. The present study evaluates the ability of liquid biopsy to detect IDH1 mutations and its correlation with survival and clinical characteristics of glioma patients. METHODS: Blood samples obtained from glioma patients were collected after surgery prior to the adjuvant therapy. cfDNA was extracted from plasma and IDH1 p.R132H mutation analysis was performed on a digital droplet PCR. χ2-test and Cohen k were used to assess the correlation between plasma and tissue IDH1 status, while Kaplan Meier curve and Cox regression analysis were applied to survival analysis. Statistical calculations were performed by MedCalc and GraphPad Prism software. RESULTS: A total of 67 samples were collected. A concordance between IDH1 status in tissue and in plasma was found (p = 0.0024), and the presence of the IDH1 mutation both in tissue (138.8 months vs 24.4, p < 0.0001) and cfDNA (116.3 months vs 35.8, p = 0.016) was associated with longer median OS. A significant association between IDH1 mutation both in tissue and cfDNA, age, tumor grade and OS was demonstrated by univariate Cox regression analysis. No statistically significant association between IDH1 mutation and tumor grade was found (p = 0.10). CONCLUSIONS: The present study demonstrates that liquid biopsy may be used in brain tumors to detect IDH1 mutation which represents an important prognostic biomarker in patients with different types of gliomas, being associated to OS.

Endothelial Progenitor Cells: An Appraisal of Relevant Data from Bench to Bedside.

The mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow is well known to be present in several clinical settings, including acute coronary syndrome, heart failure, diabetes and peripheral vascular disease. The aim of this review was to explore the current literature focusing on the great opportunity that EPCs can have in terms of regenerative medicine.

Androgen receptor expression inversely correlates with histological grade and N stage in ER+/PgRlow male breast cancer.

PURPOSE: Androgen Receptor (AR) positivity is often displayed in breast cancer and especially in Male Breast Cancer (MBC), where it appears to be a heterogeneous feature, with its expression ranging between 38 and 81% of cases. Given the fact that circulating androgens represent the most important sex hormones in males and that breast carcinogenesis is characteristically subjected to hormonal mechanisms, our purpose was to investigate the clinicopathological significance of AR in MBC assessing if its expression could be associated with parameters of tumor aggressiveness. METHODS: Clinical and pathological data were retrospectively reviewed for male patients with a diagnosis of invasive breast cancer. AR status was detected by immunohistochemistry on formalin-fixed, paraffin-embedded tumoral tissue sections. Correlations between AR expression and histopathological features were assessed using univariate and multiple comparisons where appropriate, assuming P values < 0.05 as statistically significant. RESULTS: The study included 44 consecutive male patients. AR expression ranged between 10 and 98% and the majority of cases presented a moderate to high expression of this receptor. Adopting a 20% PgR cut-off, statistical analyses highlighted a different behavior of AR: in ER+/PgRhigh group, it positively correlated with the other steroid receptors pointing out the importance of hormonal cross-talk: in ER+/PgRlow group, AR status inversely correlated with histological grade and lymph node status. CONCLUSION: Hormonal factors reveal to play a crucial role in MBC carcinogenesis and progression. Intriguingly, in ER+/PgRlow tumors AR expression significantly correlates with lymph node status, hinting at a favorable biological role of AR in this tumor subgroup.

From Tuscany to Vienna: Paolo Mascagni's anatomic models in the Josephinum.

Paolo Mascagni (Pisa 1755-Siena 1815) was one of the major members of the cultural and scientific community in Tuscany in the XIX century. After initial interest in geological investigations, he successfully turned his attention to medical sciences. In 1801, Mascagni was appointed as Professor of Anatomy at the University of Pisa, with additional charge of Lecturer at the Hospital of Santa Maria Nuova in Florence. Within his teaching activity he also designed several anatomic drawings which were employed for wax models of human organs. Such models, which are still visible in the La Specola museum in Florence, were so appreciated for their accuracy, that the curators of the Viennese Josephinum requested copies of them for teaching purposes. The current paper explores the relationship between Mascagni and the Josephinum, and aims to point out the importance of the Italian contribution to the education of the students of the Viennese Medical University.

Overexpression of TK1 and CDK9 in plasma-derived exosomes is associated with clinical resistance to CDK4/6 inhibitors in metastatic breast cancer patients.

PURPOSE: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) improve progression-free survival (PFS) in patients with hormone receptor-positive (HR+) advanced breast cancer. However, a better knowledge of predictive biomarkers of response and resistance to CDK4/6i is needed. Therefore, the present article addresses the role of the mRNA expression of thymidine kinase 1 (TK1), CDK4, 6 and 9 in plasma-derived exosomes and their relevance in the pharmacologic activity of CDK4/6i. METHODS: Blood samples of 40 HR+/HER2- advanced breast cancer patients were collected before (T0) the administration of palbociclib plus hormonal therapy and after 3 months (T1). RNA was isolated from exosomes and analysed for the expression of TK1, CDK 4, 6 and 9 by digital droplet PCR (ddPCR). RESULTS: A higher value of TK1 copies/ml at baseline (T0) was significantly associated with the number of previous lines of chemotherapy (p = 0.009). In patients with PD, a significant increase was observed in the number of copies/ml of TK1 (p = 0.01) and CDK9 (p = 0.03) comparing T1 vs. T0 values. No significant correlations between response to treatment and clinical parameters were found at univariate analysis. High baseline CDK4 expression was significantly correlated with longer PFS in patients treated with fulvestrant + palbociclib (low versus high: 6.45 months vs. not reached, p = 0.01). CONCLUSIONS: The present study demonstrates that, in plasma-derived exosomes, high baseline CDK4 mRNA levels are associated with response to palbociclib plus hormonal therapy, while the increase in TK1 and CDK9 mRNA copies/ml is associated with clinical resistance.

Dabrafenib and Trametinib prolong coagulation through the inhibition of tissue factor in BRAFv600e mutated melanoma cells in vitro.

BACKGROUND: Neoplastic cells promote a hypercoagulable state by the expression of cell surface proteins, such as tissue factor. In BRAFv600 mutated melanoma patients upon BRAF inhibitors, a hypercoagulable state correlates with prognosis, while a down-regulation of the hemostatic parameters is observed in patients responders as compared to non responders. The present study was intended to better clarify the strict relationship between coagulation mediators and target therapy in melanoma. METHODS: The expression of tissue factor was investigated after the treatment with the BRAF inhibitor Dabrafenib and the MEK inhibitor Trametinib in the BRAFv600e mutated melanoma cell lines A-375 and SK-MEL-28, together with its ability to activate the coagulation cascade. RESULTS: Dabrafenib and Trametinib caused the down-regulation of TF in both cell lines A-375 and SK-MEL-28. For the cell line A-375 the effect was evident both at RNA and procoagulant activity; for the cell line SK-MEL-28 only at RNA level without any variation of the protein. Interestingly, when in contact with plasma deficient of factor VII, both cell lines were not able to activate the coagulation cascade. CONCLUSIONS: The present study provides the first in vitro observation that tissue factor expressed in melanoma cells may contribute to the modulation of the coagulation state of patients in the treatment with BRAF inhibitors.

Gastric cancer prevention targeted on risk assessment: Gastritis OLGA staging.

Gastric cancer (GC) ranks among the most lethal epithelial malignancies, and its striking mortality rate prompts a global prevention strategy. Helicobacter pylori (H. pylori) gastritis is the main GC promoter, and the 2014 Global Kyoto conference recognized H. pylori gastritis as a (treatable) infectious disease. It is therefore plausible that any large-scale intervention for H. pylori eradication would result in cleansing the world of the fifth cause of cancer-related death. Atrophic gastritis is the cancerization field in which GCs (both intestinal and diffuse histotypes) mainly develop. Discontinuing the inflammatory cascade triggered by H. pylori is tantamount to preventing GC. For patients (still infected or eradicated) who have already developed gastric atrophy, the severity/topography of the atrophic changes correlates with their cancer risk. Gastritis OLGA (Operative Link for Gastritis Assessment) staging consistently ranks the atrophy-associated cancer risk, providing a solid clinical/biological rationale for establishing patient-specific surveillance programs. By combining primary and secondary prevention strategies, gastric cancer is a preventable disease.

Association of Glutathione S-Transferase P-1 (GSTP-1) rs1695 polymorphism with overall survival in glioblastoma patients treated with combined radio-chemotherapy.

Glioblastoma (GBM) is the most frequent malignant primary brain tumor in adults and, despite recent advances, the prognosis for this cancer remains dismal. The aims of this study were to test the influence of XRCC1 rs25487, XRCC3 rs861539, XRCC3 rs1799794, RAD51 rs1801320 and GSTP-1 rs1695 single nucleotide polymorphisms on progression free survival (PFS) and overall survival (OS) in GBM patients treated with radiotherapy (RT) and temozolomide (TMZ). Fifty GBM patients treated with upfront radio-chemotherapy (RT 60 Gy/30 sessions; TMZ 75 mg/m2 during RT and 200 mg/m2 days 1 â†’ 5 every 28 days) were enrolled. Survival curves were calculated using the Kaplan-Meier method, and the log-rank test was used to evaluate differences between curves. A trend to a statistically significant association with PFS in univariate and multivariate COX regression analysis was found with GSTP-1 rs1695 polymorphism (p = 0.087 and p = 0.097 on univariate and multivariate analyses, respectively). Conversely, the same GSTP-1 rs1695 SNP revealed a statistically significant association with OS (p = 0.007 and p = 0.042 on univariate and multivariate analysis, respectively). Our pharmacogenetic prospective study suggests that GSTP-1 rs1695 genotypes can be associated with different OS in GBM patients treated with RT and TMZ.

Histological study of atraumatic periprosthetic fractures: does atypical periprosthetic fracture exist?

PURPOSE: Is it possible a correlation between some periprosthetic femoral fractures and atypical fractures? CASE: We present a case of a 77-year-old woman with atypical periprosthetic femoral fracture. The patient had a history of long-term bisphosphonate use. We performed an open reduction, a synthesis of the fracture and a histological exam. The patient stopped the bisphosphonate (BF) therapy. Three months later, before starting the teriparatide treatment, the patient had a re-fracture so we did a second osteosynthesis and began a teriparatide therapy. After six months, the radiography showed a bone healing at the fracture site. RESULT: The histological examination confirmed the diagnosis of atypical femoral fracture. CONCLUSION: At first, the fracture showed a delayed union which led to a new surgery, as often happens in BF-related atypical fractures. Appropriate treatment (BF suspension and teriparatide beginning) permitted fracture healing. The atypical characteristic of the fracture was confirmed by histological exam.Some periprosthetic femoral fractures in patients treated with BF, especially in long time therapies, should be suspected as atypical fractures and a specific medical treatment should be performed, as well as a correct surgical treatment.

Micro-RNA-21 (biomarker) and global longitudinal strain (functional marker) in detection of myocardial fibrotic burden in severe aortic valve stenosis: a pilot study.

AIMS: Myocardial fibrosis (MF) is a deleterious consequence of aortic valve stenosis (AVS). Global longitudinal strain (GLS) is a novel left ventricular (LV) functional parameter potentially useful to non-invasively estimate MF. MicroRNAs (miRNAs) are non-coding small ribonucleic acids (RNA) modulating genes function, mainly through RNA degradation. miRNA-21 is a biomarker associated with MF in pressure overload. The aim of the present study was to find an integrated algorithm for detection of MF using a combined approach with both bio- and functional markers. METHODS: Thirty-six patients (75.2 ± 8 y.o.; 63 % Female) with severe AVS and preserved LV ejection fraction (EF), candidate to surgical aortic valve replacement (sAVR) were enrolled. Clinical, bio-humoral evaluation (including plasmatic miRNA-21 collected using specific tubes, PAXgene, for stabilization of peripheral RNA) and a complete echocardiographic study, including GLS and septal strain, were performed before sAVR. Twenty-eight of those patients underwent sAVR and, in 23 of them, an inter-ventricular septum biopsy was performed. Tissues were fixed in formalin and embedded in paraffin. Sections were stained with Hematoxylin and Eosin for histological evaluation and with histochemical Masson trichrome for collagen fibers. The different components were calculated and expressed as micrometers(2). To evaluate tissue miRNA components, sections 2-µm thick were cut using a microtome blade for each slide. Regression analysis was performed to test association between dependent variable and various predictors included in the model. RESULTS: Despite a preserved EF (66 ± 11 %), patients presented altered myocardial deformation parameters (GLS -14,02 ± 3.8 %; septal longitudinal strain, SSL -9.63 ± 2.9 %; septal longitudinal strain rate, SL-Sr -0.58 ± 0.17 1/s; Septal Longitudinal early-diastolic strain rate, SL-SrE 0.62 ± 0.32 1/s). The extent of MF showed an inverse association with both GLS and septal longitudinal deformation indices (GLS: R(2) = 0.30; p = 0.02; SSL: R(2) = 0.36; p = 0.01; SL-Sr: R(2) = 0.39; p < 0.001; SL-SrE: R(2) = 0.35; p = 0.001). miRNA-21 was mainly expressed in fibrous tissue (p < 0.0001). A significant association between MF and plasmatic miRNA-21, alone and weighted for measures of structural (LVMi R(2) = 0.50; p = 0.0005) and functional (SSL R(2) = 0.35; p = 0.006) remodeling, was found. CONCLUSIONS: In AVS, MF is associated with alterations of regional and global strain. Plasmatic miRNA-21 is directly related to MF and associated with LV structural and functional impairment.

Accessibilome of human glioblastoma: collagen-VI-alpha-1 is a new target and a marker of poor outcome.

Functional targeted therapy has unfortunately failed to improve the outcome of glioblastoma patients. Success stories evidenced by the use of antibody-drug conjugates in other tumor types are encouraging, but targets specific to glioblastoma and accessible through the bloodstream remain scarce. In the current work, we have identified and characterized novel and accessible proteins using an innovative proteomic approach on six human glioblastomas; the corresponding data have been deposited in the PRIDE database identifier PXD001398. Among several clusters of uniquely expressed proteins, we highlight collagen-VI-alpha-1 (COL6A1) as a highly expressed tumor biomarker with low levels in most normal tissues. Immunohistochemical analysis of glioma samples from 61 patients demonstrated that COL6A1 is a significant and consistent feature of high-grade glioma. Deposits of COL6A1 were evidenced in the perivascular regions of the tumor-associated vasculature and in glioma cells found in pseudopalisade structures. Retrospective analysis of public gene-expression data sets from over 300 glioma patients demonstrated a significant correlation of poor patient outcome and high COL6A1 expression. In a proof-of-concept study, we use chicken chorioallantoic membrane in vivo model to show that COL6A1 is a reachable target for IV-injected antibodies. The present data warrant further development of human COL6A1 antibodies for assessing the quantitative biodistribution in the preclinical tumor models.

Glioblastoma and Internal Carotid Artery Calcium Score: A Possible Novel Prognostic Partnership?

Purpose: Clinical evidence suggests an association between comorbidities and outcome in patients with glioblastoma (GBM). We hypothesised that the internal carotid artery (ICA) calcium score could represent a promising prognostic biomarker in a competing risk analysis in patients diagnosed with GBM. Methods: We validated the use of the ICA calcium score as a surrogate marker of the coronary calcium score in 32 patients with lung cancer. Subsequently, we assessed the impact of the ICA calcium score on overall survival in GBM patients treated with radio-chemotherapy. Results: We analysed 50 GBM patients. At the univariate analysis, methyl-guanine-methyltransferase gene (MGMT) promoter methylation (p = 0.048), gross total tumour resection (p = 0.017), and calcium score (p = 0.011) were significant prognostic predictors in patients with GBM. These three variables also maintained statistical significance in the multivariate analysis. Conclusions: the ICA calcium score could be a promising prognostic biomarker in GBM patients.

Cost analysis of archives in the pathology laboratories: from safety to management.

CONTEXT: Despite the reluctance to invest and the challenging estimation of necessary supporting costs, optimising the archives seems to be one of the hottest topics in the future management of the pathology laboratories. Historically, archives were only partially designed to securely store and organise tissue specimens, and tracking systems were often flawed, posing significant risks to patients' health and legal ramifications for pathologists. OBJECTIVE: The current review explores the available data from the literature on archives' management in pathology, including comprehensive business plans, structure setup, outfit, inventories, ongoing conservation and functional charges. DATA SOURCES: Electronic searches in PubMed-MEDLINE and Embase were made to extract pertinent articles from the literature. Works about the archiving process and storage were included and analysed to extract information. Prepublication servers were ignored. Italian Institutional Regional databases for public competitive bidding processes were queried too. CONCLUSIONS: A new emergent feeling in the pathology laboratory is growing for archives management; the digital pathology era is a great opportunity to apply innovation to tracking systems and samples preservation. The main aim is a critical evaluation of the return of investment in developing automatic and tracked archiving processes for improving not only quality, efficacy and efficiency of the labs but also patients' healthcare.

Cervical Adenocarcinoma: A Still Under-investigated Malignancy.

BACKGROUND/AIM: The aim of the study was to correlate the expression of mismatch repairs proteins (MMR), programmed-death-ligand1 (PDL-1), and estro-progestinic receptors (ER/PgR) in tissue samples from a series of cervical adenocarcinoma (ADC) patients with their clinicopathological features. MATERIALS AND METHODS: Thirty-nine ADC specimens were retrospectively retrieved from the Division of Pathology of the University Hospital of Pisa from 2015 to 2021. Histological subtype, grade (G), Silva pattern, presence of lymph vascular space invasion (LVI), and perineural invasion (PNI) were annotated. On representative samples, immunostaining for ER/PgR, MLH1, PMS2, MSH2, MSH6, and PDL-1(sp142) was performed. RESULTS: Thirty-five ADCs were HPV-associated usual type (24 invasive and 11 in situ), 2 were clear cell type, one was a minimal deviation adenocarcinoma (MDA), and one was an invasive stratified mucin-producing carcinoma (iSMC). ADC associated with LVI were mostly G2-3, whereas those associated also with PNI were G3 with Silva pattern C. No difference in the expression of ER/PgR was observed with a dichotomic age stratification (51 years) of patients. Only 6 ADCs were MMR-deficient, all of them were of the usual type (4 invasive and 2 in situ). The heterodimer MLH-1/PMS2 was the one most frequently altered (5/6), whereas only one case had MSH6 loss. None of ADCs express PDL-1, except iSMC which showed PDL-1 expression >1% in neoplastic cells. CONCLUSION: Both invasive and in situ usual type ADCs indicate MMR deficiency, highlighting how this could be an early event in tumorigenesis. None of the cases, except for iSMC, express PDL-1.

Positive p16 Immunostaining Is an Independent Prognostic Variable for Disease-free Survival and Overall Survival in Patients With Squamous Cell Carcinoma of the Vulva Treated With Radical Surgery and Inguinofemoral Lymphadenectomy: An Italian Single Center Retrospective Study.

BACKGROUND/AIM: The expression of the cyclin-dependent kinase inhibitor p16 correlates with the presence of human papillomavirus. The purpose of this investigation was to assess the prognostic relevance of p16 expression in patients with vulvar squamous cell carcinoma (VSCC) treated with radical surgery followed by adjuvant (chemo) radiation in selected cases. PATIENTS AND METHODS: Seventy-eight patients were analyzed retrospectively. RESULTS: Positive p16 immunostaining was detected in 19 (24.4%) patients. Five-year disease-free survival (DFS) and 5-year overall survival (OS) were better in p16-positive compared to p16-negative patients (83.9% versus 37.3% p=0.002 and 91.7% versus 57.6%, p=0.003, respectively). p16 expression retained prognostic relevance at multivariate analysis for both DFS and OS. CONCLUSION: p16 expression was detected in 24.4% of patients with VSCC and was found to be an independent prognostic variable for both DFS and OS.

The Prognostic Impact of Gender, Therapeutic Strategies, Molecular Background, and Tumor-Infiltrating Lymphocytes in Glioblastoma: A Still Unsolved Jigsaw.

Despite the adoption of novel therapeutical approaches, the outcomes for glioblastoma (GBM) patients remain poor. In the present study, we investigated the prognostic impact of several clinico-pathological and molecular features as well as the role of the cellular immune response in a series of 59 GBM. CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) were digitally assessed on tissue microarray cores and their prognostic role was investigated. Moreover, the impact of other clinico-pathological features was evaluated. The number of CD4+ and CD8+ is higher in GBM tissue compared to normal brain tissue (p < 0.0001 and p = 0.0005 respectively). A positive correlation between CD4+ and CD8+ in GBM is present (rs = 0.417-p = 0.001). CD4+ TILs are inversely related to overall survival (OS) (HR = 1.79, 95% CI 1.1-3.1, p = 0.035). The presence of low CD4+ TILs combined with low CD8+ TILs is an independent predictor of longer OS (HR 0.38, 95% CI 0.18-0.79, p = 0.014). Female sex is independently related to longer OS (HR 0.42, 95% CI 0.22-0.77, p = 0.006). Adjuvant treatment, methylguanine methyltransferase (MGMT) promoter methylation, and age remain important prognostic factors but are influenced by other features. Adaptive cell-mediated immunity can affect the outcomes of GBM patients. Further studies are needed to elucidate the commitment of the CD4+ cells and the effects of different TILs subpopulations in GBM.

Value of Artificial Intelligence in Evaluating Lymph Node Metastases.

One of the most relevant prognostic factors in cancer staging is the presence of lymph node (LN) metastasis. Evaluating lymph nodes for the presence of metastatic cancerous cells can be a lengthy, monotonous, and error-prone process. Owing to digital pathology, artificial intelligence (AI) applied to whole slide images (WSIs) of lymph nodes can be exploited for the automatic detection of metastatic tissue. The aim of this study was to review the literature regarding the implementation of AI as a tool for the detection of metastases in LNs in WSIs. A systematic literature search was conducted in PubMed and Embase databases. Studies involving the application of AI techniques to automatically analyze LN status were included. Of 4584 retrieved articles, 23 were included. Relevant articles were labeled into three categories based upon the accuracy of AI in evaluating LNs. Published data overall indicate that the application of AI in detecting LN metastases is promising and can be proficiently employed in daily pathology practice.

Sparc-like protein 1 is a new marker of human glioma progression.

High-grade gliomas (glioblastomas) are the most common and deadly brain tumors in adults, currently with no satisfactory treatment available. Apart from de novo glioblastoma, it is currently accepted that these malignancies mainly progress from lower grade glial tumors. However, the molecular entities governing the progression of gliomas are poorly understood. Extracellular and membrane proteins are key biomolecules found at the cell-to-cell communication interface and hence are a promising proteome subpopulation that could help understand the development of glioma. Accordingly, the current study aims at identifying new protein markers of human glioma progression. For this purpose, we used glial tumors generated orthotopically with T98G and U373 human glioma cells in nude mice. This setup allowed also to discriminate the protein origin, namely, human (tumor) or mouse (host). Extracellular and membrane proteins were selectively purified using biotinylation followed by streptavidin affinity chromatography. Isolated proteins were digested and then identified and quantified employing 2D-nano-HPLC-MS/MS analysis. A total of 23 and 27 up-regulated extracellular and membrane proteins were identified in the T98G and U373 models, respectively. Approximately two-thirds of these were predominantly produced by the tumor, whereas the remaining proteins appeared to be mainly overexpressed by the host tissue. Following extensive validation, we have focused our attention on sparc-like protein 1. This protein was further investigated using immunohistochemistry in a large collection of human glioma samples of different grades. The results showed that sparc-like protein 1 expression correlates with glioma grade, suggesting the possible role for this protein in the progression of this malignancy.