Nuovi approcci proteomici per l'identificazione di potenziali marcatori di neoplasie pancreatiche

Lo sviluppo di approcci rapidi ed automatizzati come la tecnologia multidimensionale di identificazione proteica (MudPIT) sta rendendo la proteomica uno strumento sempre pi\uf9 efficiente per l\u2019analisi delle proteine in miscele complesse, permettendo l\u2019identificazione di nuovi marcatori biologici che sono di importanza critica per una migliore comprensione della biologia dei tumori e per rendere la sua rilevazione pi\uf9 precoce e meno invasiva. Lo scopo del presente studio era quello di identificare nuove proteine rilasciate dalle cellule di adenocarcinoma duttale del pancreas, usando piccole quantit\ue0 di campione ed un sistema automatizzato; le evidenze sperimentali cos\uec ottenute sarebbero state utilizzate per verificare in vitro ed in vivo, con metodiche pi\uf9 tradizionali quali western blot, RT-PCR ed immunoistochimica, la presenza delle proteine selezionate come possibili marcatori. \uc8 stata dunque applicata la tecnologia MudPIT, che incorpora la cromatografia capillare bidimensionale e la spettrometria di massa in tandem, per l\u2019analisi di piccole quantit\ue0 di surnatanti privi di siero derivanti dalla coltura di cellule Suit-2 non trattate oppure attivate con esteri del forbolo e ionoforo. I potenziali marcatori prescelti sono stati valutati in altre linee cellulari di cancro del pancreas, adenocarcinomi primitivi e xenotrapiantati in topi nu/nu. L\u2019analisi MudPIT effettuata su campioni di 10 \u3bcl di surnatanti ha permesso l\u2019identificazione complessiva di 46 proteine tra cellule attivate e non trattate. Di queste proteine, 21 sono classificate come secrete sui database pubblicamente disponibili e 10 non erano state precedentemente associate al carcinoma duttale del pancreas. Questo gruppo comprende le proteine CSPG2/versican, Mac25/angiomodulina, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, ciclofilina B, K2 microglobulina, ed ICA69. Le evidenze sperimentali che queste proteine siano rilasciate dalle cellule tumorali in vivo sono state ottenute, per CSPG2/versican e Mac25/angiomodulina, mediante immunoistochimica. L\u2019analisi \ue8 stata eseguita tanto su tumori primitivi quanto su di un modello, consistente in cellule della linea Suit-2 incluse in una matrice amorfa (Matrigel\uae) e trapiantate per una settimana in topi atimici. Si \ue8 inoltre dimostrato, mediante il confronto tra cellule non trattate ed attivate con esteri del forbolo, che l\u2019analisi MudPIT pu\uf2 fornire dati semiquantitativi correlati con la quantit\ue0 relativa di proteina presente nel campione analizzato; anche quest\u2019osservazione \ue8 stata convalidata mediante misurazione del diverso livello di espressione di tre proteine rispettivamente inibite (Mac25/angiomodulina), non modificate (CSPG2/versican) ed indotte (MMP-1). Si \ue8 poi indagata, su una casistica di 100 pazienti con varie patologie pancreatiche, l\u2019espressione di forme solubili di uPAR (suPAR), il cui ligando uPA era tra le proteine maggiormente indotte in seguito all\u2019attivazione delle cellule in vitro. L\u2019analisi \ue8 stata fatta utilizzando una metodica immunoenzimatica (saggio ELISA) su sieri ed urine dei casi disponibili presso la biobanca della clinica chirurgica dell\u2019Universit\ue0 di Verona. E\u2019 stato riscontrato un significativo incremento dei valori di suPAR nei sieri di pazienti affetti da adenocarcinoma duttale del pancreas rispetto alle altre patologie infiammatorie o neoplastiche del pancreas; i dati delle urine, pur se meno netti, indicano comunque una tendenza simile ed incoraggiano ad un incremento del numero di campioni sui quali effettuare ulteriori analisi. Confrontato con altre metodiche, dunque, MudPIT \ue8 stato in grado di fornire in modo rapido e riproducibile dati su di una serie di molecole rilasciate da cellule neoplastiche, che hanno quindi caratteristiche di sicuro interesse quali potenziali marcatori di malattia potenzialmente rilevabili nei liquidi biologici. Gli sviluppi futuri di tale approccio comprendono, oltre all\u2019ampliamento dell\u2019analisi MudPIT su un numero maggiore di linee cellulari, lo sviluppo di nuovi reagenti per l\u2019identificazione quelle molecole per le quali essi non sono attualmente disponibili.Non disponibil

To metabolomics and beyond: a technological portfolio to investigate cancer metabolism

Tumour cells have exquisite flexibility in reprogramming their metabolism in order to support tumour initiation, progression, metastasis and resistance to therapies. These reprogrammed activities include a complete rewiring of the bioenergetic, biosynthetic and redox status to sustain the increased energetic demand of the cells. Over the last decades, the cancer metabolism field has seen an explosion of new biochemical technologies giving more tools than ever before to navigate this complexity. Within a cell or a tissue, the metabolites constitute the direct signature of the molecular phenotype and thus their profiling has concrete clinical applications in oncology. Metabolomics and fluxomics, are key technological approaches that mainly revolutionized the field enabling researchers to have both a qualitative and mechanistic model of the biochemical activities in cancer. Furthermore, the upgrade from bulk to single-cell analysis technologies provided unprecedented opportunity to investigate cancer biology at cellular resolution allowing an in depth quantitative analysis of complex and heterogenous diseases. More recently, the advent of functional genomic screening allowed the identification of molecular pathways, cellular processes, biomarkers and novel therapeutic targets that in concert with other technologies allow patient stratification and identification of new treatment regimens. This review is intended to be a guide for researchers to cancer metabolism, highlighting current and emerging technologies, emphasizing advantages, disadvantages and applications with the potential of leading the development of innovative anti-cancer therapies

Protein Tyrosine Phosphatase Gamma (PTPγ) is a Novel Leukocyte Marker Highly Expressed by CD34+ Precursors

Protein Tyrosine Phosphatase gamma (PTPγ) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPγ is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPγ mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPγ specific antibody that recognizes the protein by flow cytometry. PTPγ expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPγ was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34+ haematopoietic precursors express high PTPγ level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPγ as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation

Elevated urinary levels of urokinase-type plasminogen activator receptor (uPAR) in pancreatic ductal adenocarcinoma identify a clinically high-risk group

BACKGROUND: The urokinase plasminogen activator receptor is highly expressed and its gene is amplified in about 50% of pancreatic ductal adenocarcinomas; this last feature is associated with worse prognosis. It is unknown whether the level of its soluble form (suPAR) in urine may be a diagnostic-prognostic marker in these patients. METHODS: The urinary level of suPAR was measured in 146 patients, 94 pancreatic ductal adenocarcinoma and 52 chronic pancreatitis. Urine from 104 healthy subjects with similar age and gender distribution served as controls. suPAR levels were normalized with creatinine levels (suPAR/creatinine, ng/mg) to remove urine dilution effect. RESULTS: Urinary suPAR/creatinine values of pancreatic ductal adenocarcinoma patients were significantly higher (median 9.8; 25(th)-75(th )percentiles 5.3-20.7) than those of either healthy donors (median 0; 0-0.5) or chronic pancreatitis patients (median 2.7; 0.9-4.7). The distribution of values among cancer patients was widespread and asymmetric, 53% subjects having values beyond the 95(th )percentile of healthy donors. The values of suPAR/creatinine did not correlate with tumour stage, Ca19-9 or CEA levels. Higher values correlated with poor prognosis among non-resected patients at univariate analysis; multivariate Cox regression identified high urinary suPAR/creatinine as an independent predictor of poor survival among all cancer patients (odds ratio 2.10, p = 0.0023), together with tumour stage (stage III odds ratio 2.65, p = 0.0017; stage IV odds ratio 4.61, p < 0.0001) and female gender (odds ratio 1.85, p = 0.01). CONCLUSIONS: A high urinary suPAR/creatinine ratio represents a useful marker for the identification of a subset of patients with poorer outcome

A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

Background: Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. Methods: TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results: Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions: The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions

Ultra-Mutation in IDH Wild-Type Glioblastomas of Patients Younger than 55 Years is Associated with Defective Mismatch Repair, Microsatellite Instability, and Giant Cell Enrichment

Glioblastomas (GBMs) are classified into isocitrate dehydrogenase (IDH) mutants and IDH wild-types (IDH-wt). This study aimed at identifying the mutational assets of IDH-wt GBMs in patients aged 18-54 years for which limited data are available

Genetic alterations analysis in prognostic stratified groups identified TP53 and ARID1A as poor clinical performance markers in intrahepatic cholangiocarcinoma

The incidence and mortality rates of intrahepatic cholangiocarcinoma have been rising worldwide. Few patients present an early-stage disease that is amenable to curative surgery and after resection, high recurrence rates persist. To identify new independent marker related to aggressive behaviour, two prognostic groups of patient were selected and divided according to prognostic performance. All patients alive at 36 months were included in good prognostic performers, while all patients died due to disease within 36 months in poor prognostic performers. Using high-coverage target sequencing we analysed principal genetic alterations in two groups and compared results to clinical data. In the 33 cases included in poor prognosis group, TP53 was most mutated gene (p\u2009=\u20090.011) and exclusively present in these cases. Similarly, ARID1A was exclusive of this group (p\u2009=\u20090.024). TP53 and ARID1A are mutually exclusive in this study. Statistical analysis showed mutations in TP53 and ARID1A genes and amplification of MET gene as independent predictors of poor prognosis (TP53, p\u2009=\u20090.0031, ARID1A, p\u2009=\u20090.0007, MET, p\u2009=\u20090.0003 in Cox analysis). LOH in PTEN was also identified as marker of disease recurrence (p\u2009=\u20090.04) in univariate analysis. This work improves our understanding of aggressiveness related to this tumour type and has identified novel prognostic markers of clinical outcome

ERG alterations and mTOR pathway activation in primary prostate carcinomas developing castration-resistance

Abstract Introduction One of the most common sites of distant metastasization of prostate cancer is bone, but to date reliable biomarkers able to predict the risk and timing of bone metastasization are still lacking. Patients and methods Surgically resected paraffin embedded samples from 12 primary prostate cancers that developed metachronous bone metastasis at different time points were studied (six cases within 2 years, six cases after 5 years from surgery). A targeted next-generation DNA and RNA sequencing able to assess simultaneously mutations, copy number alterations and fusion events of multiple genes was used. Immunohistochemistry was used to assess mTOR pathway activation. Results Rearrangements of ETS family genes, molecular alterations in PTEN and TP53 genes were detected in 10, 6 and 5 cancers, respectively. Nine samples showed TMPRSS2-ERG fusions, which were associated with increased ERG expression at immunohistochemistry. mTOR pathway activation was documented in 6 patients, with a clear trend of prevalence in late-metastatic patients (p = 0.08). Conclusions A simultaneous next-generation targeted DNA and RNA sequencing is applicable on routine formalin-fixed paraffin-embedded tissues to assess the multigene molecular asset of individual prostate cancers. This approach, coupled with immunohistochemistry for ERG and mTOR pathway proteins, may help to better characterize prostate cancer molecular features with a potential impact on clinical decisions

IDH-wild type glioblastomas featuring at least 30% giant cells are characterized by frequent RB1 and NF1 alterations and hypermutation

: Giant cell glioblastoma (GC-GBM) is a rare variant of IDH-wt GBM histologically characterized by the presence of numerous multinucleated giant cells and molecularly considered a hybrid between IDH-wt and IDH-mutant GBM. The lack of an objective definition, specifying the percentage of giant cells required for this diagnosis, may account for the absence of a definite molecular profile of this variant. This study aimed to clarify the molecular landscape of GC-GBM, exploring the mutations and copy number variations of 458 cancer-related genes, tumor mutational burden (TMB), and microsatellite instability (MSI) in 39 GBMs dichotomized into having 30-49% (15 cases) or\u2009 65\u200950% (24 cases) GCs. The type and prevalence of the genetic alterations in this series was not associated with the GCs content (&lt;\u200950% or 65\u200950%). Most cases (82% and 51.2%) had impairment in TP53/MDM2 and PTEN/PI3K pathways, but a high proportion also featured TERT promoter mutations (61.5%) and RB1 (25.6%) or NF1 (25.6%) alterations. EGFR amplification was detected in 18% cases in association with a shorter overall survival (P\u2009=\u20090.004). Sixteen (41%) cases had a TMB\u2009&gt;\u200910 mut/Mb, including two (5%) that harbored MSI and one with a POLE mutation. The frequency of RB1 and NF1 alterations and TMB counts were significantly higher compared to 567 IDH wild type (P\u2009&lt;\u20090.0001; P\u2009=\u20090.0003; P\u2009&lt;\u20090.0001) and 26 IDH-mutant (P\u2009&lt;\u20090.0001; P\u2009=\u20090.0227; P\u2009&lt;\u20090.0001) GBMs in the TCGA PanCancer Atlas cohort. These findings demonstrate that the molecular landscape of GBMs with at least 30% giant cells is dominated by the impairment of TP53/MDM2 and PTEN/PI3K pathways, and additionally characterized by frequent RB1 alterations and hypermutation and by EGFR amplification in more aggressive cases. The high frequency of hypermutated cases suggests that GC-GBMs might be candidates for immune check-point inhibitors clinical trials