Analisi Mutazionale dei geni KRas e BRAF in carcinomi sincroni del colon

1. RIASSUNTO Introduzione Nei paesi occidentali il tumore del colon-retto (CRC, Colon Rectal Cancer) rappresenta la terza causa di morte dovuta a neoplasie maligne. Ad oggi gli studi condotti su CRC hanno riportato che una piccola percentuale di essi, che varia dall’ 1-7%, è rappresentata da CRC sincroni, definiti come 2 o più carcinomi primitivi riscontrati nello stesso individuo al momento della prima diagnosi. Sebbene negli ultimi anni si è assistito ad un aumento dell’incidenza dei tumori del colon-retto, è stata comunque riscontrata una concomitante diminuzione della mortalità ad esso dovuta, attribuibile soprattutto ad una adeguata prevenzione, ad una diagnosi precoce e all’utilizzo di terapie farmacologiche mirate, il cui sviluppo è stato possibile grazie ad una conoscenza più approfondita della biologia di tali tumori. Infatti per il trattamento dei CRC, negli ultimi anni sono state messe a punto terapie farmacologiche mirate basate sulla somministrazione di anticorpi monoclonali (mAb) rivolti verso il recettore per il fattore di crescita epiteliale (EGFR), il quale controlla la cascata di trasduzione del segnale Ras-RAF-MAP-Kasi, mediante l'attivazione di diversi effettori molecolari, tra cui KRas e BRAF, ed è risultato essere iper-espresso in una elevata percentuale di CRC in stadio avanzato. Tuttavia, il trattamento con mAb anti-EGFR è risultato essere efficace soltanto nel 10-20% dei casi. A questo riguardo, studi recenti hanno dimostrato che la resistenza a tale trattamento può essere associata alla presenza di mutazioni a carico dei geni KRas e BRAF, per i quali sono state riportate frequenze di mutazione del 30-40% e del 5-15% rispettivamente. Tali mutazioni determinano l'attivazione costitutiva delle proteine da essi codificate, con conseguente induzione della cascata Ras-RAF-MAP-Kasi, indipendentemente dalla stimolazione del recettore EGFR. Alla luce dei risultati sopra riportati, la determinazione dello stato mutazionale di tali geni ha assunto un ruolo essenziale nell'identificazione dei pazienti affetti da carcinoma del colon-retto che possono essere trattati con gli mAb anti-EGFR. Obiettivo L'obiettivo del mio studio è quello di analizzare la distribuzione delle mutazioni a carico dei geni KRas e BRAF in pazienti affetti da tumore sincrono del colon, per valutare l’eterogeneità di tali tumori e stabilire, inoltre, se per questi pazienti sia sufficiente eseguire l’analisi mutazionale su uno solo dei tumori o su tutti quelli presenti al momento della diagnosi. Materiali e Metodi L’analisi mutazionale dei codoni 12 e 13 dell'esone 2 del gene KRas e dell'esone 15 del gene BRAF è stata eseguita su 31 pazienti affetti da CRC sincroni, selezionati da una casistica consecutiva di 500 casi (6%), operati presso la chirurgia dell’Azienda-Ospedaliera-Universitaria Pisana e diagnosticati presso l’Anatomia Patologica III dello stesso presidio ospedaliero, nel periodo compreso tra il 2006- 2009. Il DNA da analizzare è stato estratto da tessuto tumorale fissato in formalina ed incluso in paraffina (FFPE), mediante macrodissezione manuale. Successivamente, gli esoni di interesse sono stati amplificati mediante PCR, e l'analisi mutazionale è stata eseguita utilizzando le tecniche di SSCP e di sequenziamento genomico diretto. Risultati L’analisi mutazionale dei codoni 12 e 13 dell'esone 2 del gene KRas e dell'esone 15 del gene BRAF è stata eseguita su una casistica di 31 pazienti affetti da CRC sincroni, per un totale di 70 tumori. Dal punto di vista biologico, lo stato mutazionale del gene KRas è risultato essere omogeneo in 14 pazienti, risultati tutti wild-type, (45%,14/31), ed eterogenei in 17 pazienti (55%, 17/31). Di questi ultimi, in 14 pazienti è stata riscontrata la presenza contemporanea di tumori wild-type e tumori mutati, e in 3 pazienti i tumori presenti avevano mutazioni diverse. Lo stato mutazionale del gene BRAF, invece, è risultato essere omogeneo in 29 pazienti (94%, 29/31), dei quali 27 hanno riportato tutti i tumori wild-type e 2 hanno riportato tutti i tumori mutati, ed eterogeneo in 2 pazienti (6%, 2/31), caratterizzati dalla presenza contemporanea di tumori wild-type e tumori mutati. Conclusioni Dai risultati ottenuti nello studio da me condotto è possibile osservare che un’elevata percentuale di CRC sincroni (55%, 17/31) ha mostrato uno stato mutazionale eterogeneo per i geni KRas e BRAF. Dunque, in questi pazienti, per definire lo stato mutazionale è necessario condurre l’analisi mutazionale di tali geni su tutti i tumori presenti al momento della diagnosi

Inverse association of circulating SIRT1 and adiposity. A study on underweight, normal weight, and obese patients

Context: Sirtuins (SIRTs) are NAD+-dependent deacetylases, cellular sensors to detect energy availability, and modulate metabolic processes. SIRT1, the most studied family member, influences a number of tissues including adipose tissue. Expression and activity of SIRT1 reduce with weight gain and increase in conditions of starvation. Objective: To focus on SIRT1 plasma concentrations in different conditions of adiposity and to correlate SIRT1 with fat content and distribution, energy homeostasis and inflammation in under-weight, normal-weight, and obese individuals. Materials and Methods: 21 patients with anorexia nervosa, 26 normal-weight and 75 patients with obesity were evaluated. Body fat composition by dual-energy X-ray absorptiometry, ultrasound liver adiposity, echocardiographic epicardial fat thickness (EFT), inflammatory (ESR, CRP, and fibrinogen), and metabolic (FPG, insulin, LDL- and HDL-cholesterol, triglycerides) parameters, calculated basal metabolic rate (BMR) and plasma SIRT1 (ELISA) were measured. Results: SIRT1 was significantly higher in anorexic patients compared to normal-weight and obese patients (3.27 ± 2.98, 2.27 ± 1.13, and 1.36 ± 1.31 ng/ml, respectively). Linear regression models for each predictor variable adjusted for age and sex showed that SIRT1 concentration was inversely and significantly correlated with EFT, fat mass %, liver fat content, BMR, weight, BMI, WC, LDL-cholesterol, insulin, ESR. Stepwise multiple regression analysis revealed that age and EFT were the best independent correlates of SIRT1 (β = -0.026 ± 0.011, p = 0.025, and β = -0.516 ± 0.083, p < 0.001, respectively). Conclusions: Plasma SIRT1 shows a continuous pattern that inversely follows the whole spectrum of adiposity. SIRT1 significantly associates with EFT, a strong index of visceral fat phenotype, better than other indexes of adiposity studied here

Maturation signatures of conventional dendritic cell subtypes in COVID‐19 suggest direct viral sensing

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19, which negatively affects T-cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T-cell responses limit disease severity. Understanding how cDCs cope with SARS-CoV-2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures, with the upregulation of interferon-stimulated genes and interleukin (IL)-6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti-apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS-CoV-2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T-cell activation

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells

Whether human IL-10-producing regulatory T&nbsp;cells (“Tr1”) represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4 + T-cell subsets, including conventional cytotoxic CD4 + T&nbsp;cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4 + T&nbsp;cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes + GzmK + T&nbsp;cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4 + Eomes + T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes + Tr1-like cells are effector cells of a unique GzmK-expressing CD4 + T-cell subset

Rationale and design of an independent randomised controlled trial evaluating the effectiveness of aripiprazole or haloperidol in combination with clozapine for treatment-resistant schizophrenia

<p>Abstract</p> <p>Background</p> <p>One third to two thirds of people with schizophrenia have persistent psychotic symptoms despite clozapine treatment. Under real-world circumstances, the need to provide effective therapeutic interventions to patients who do not have an optimal response to clozapine has been cited as the most common reason for simultaneously prescribing a second antipsychotic drug in combination treatment strategies. In a clinical area where the pressing need of providing therapeutic answers has progressively increased the occurrence of antipsychotic polypharmacy, despite the lack of robust evidence of its efficacy, we sought to implement a pre-planned protocol where two alternative therapeutic answers are systematically provided and evaluated within the context of a pragmatic, multicentre, independent randomised study.</p> <p>Methods/Design</p> <p>The principal clinical question to be answered by the present project is the relative efficacy and tolerability of combination treatment with clozapine plus aripiprazole compared with combination treatment with clozapine plus haloperidol in patients with an incomplete response to treatment with clozapine over an appropriate period of time. This project is a prospective, multicentre, randomized, parallel-group, superiority trial that follow patients over a period of 12 months. Withdrawal from allocated treatment within 3 months is the primary outcome.</p> <p>Discussion</p> <p>The implementation of the protocol presented here shows that it is possible to create a network of community psychiatric services that accept the idea of using their everyday clinical practice to produce randomised knowledge. The employed pragmatic attitude allowed to randomly allocate more than 100 individuals, which means that this study is the largest antipsychotic combination trial conducted so far in Western countries. We expect that the current project, by generating evidence on whether it is clinically useful to combine clozapine with aripiprazole rather than with haloperidol, provides physicians with a solid evidence base to be directly applied in the routine care of patients with schizophrenia.</p> <p>Trial Registration</p> <p><b>Clincaltrials.gov Identifier</b>: NCT00395915</p

Early Deep Anterior Lamellar Keratoplasty (DALK) for Acanthamoeba Keratitis Poorly Responsive to Medical Treatment

Purpose: To evaluate the success (eradication of infection) or failure (recurrence of infection in the cornea or sclera, or endophthalmitis) of early therapeutic deep anterior lamellar keratoplasty (DALK) for active Acanthamoeba keratitis (AK) poorly responsive to medical treatment. Methods: Retrospective, noncomparative case series of 11 patients (11 eyes) affected by active AK poorly responsive to medical treatment who underwent early therapeutic DALK. Surgery was performed in all cases within 30 to 60 days from the onset of symptoms. Corneal ulcer depth was less than 300 μm in all cases. A 3-drug combination (chlorhexidine gluconate, propamidine isethionate, and neomycin sulfate) was the antiamoebic protocol used preoperatively and postoperatively. Cannula big bubble and "layer-by-layer" manual dissection techniques were performed. Eradication of infection, episodes of rejection, postoperative endothelial cell density, and the best spectacle-corrected visual acuity were evaluated. Histologic examination of surgical margins was performed, and margin clearance was assessed. Mean follow-up was approximately 2 years. Results: Four descemetic DALK and 7 predescemetic DALK were performed. One small Descemet membrane rupture occurred. Peripheral surgical margins were free of infection in all cases. Deep surgical margins not free from infection were found in 2 cases. However, no episode of infection recurrence was observed. The postoperative average best spectacle-corrected visual acuity was 0.8 (range, 0.6-1.0). No case of rejection was recorded. Conclusions: Early therapeutic DALK could be considered a new approach to eradicate active infection in AK cases poorly responsive to medical treatment, with significant ulcer in the optical zone. Further studies are needed to validate this new indication for DALK