Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT

1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA. Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G). A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed. Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values. Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR. Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous. 2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy. Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy. Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV. Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.

Caratterizzazione del soppressore tumorale miR-101 nel colon carcinoma

I microRNA sono una classe di piccole molecole di RNA non codificante che controllano la stabilità di numerosi RNA messaggeri, perciò sono considerati come “master regulator” dell’espressione genica. Ogni tumore è caratterizzato da un profilo di espressione alterato dei microRNA. Il miR-101 è un oncosoppressore represso nei tessuti tumorali ed è candidato come biomarcatore del cancro colon-rettale. È regolato da numerosi eventi fisiologici e patologici, come angiogenesi e carcinogenesi. Gli eventi molecolari coinvolti nella regolazione dell’espressione del miR-101 sono scarsamente conosciuti, poiché è trascritto da due loci genici non caratterizzati. L’obiettivo di questo lavoro è di caratterizzare i geni del miR-101 ed individuarne i regolatori molecolari coinvolti nella cancerogenesi colon-rettale.

Carcinoma polmonare non a piccole cellule T1aN0M0: Ruolo prognostico della Microvessel Density

Obiettivi. Valutare l’angiogenesi tumorale mediante la Microvessel density (MVD) come fattore predittivo di mortalità per tumore polmonare non a piccole cellule (NSCLC) pT1aN0M0 trattato chirurgicamente. Metodi. I dati demografici, clinici e istopatologici sono stati registrati per 82 pazienti (60 maschi, 22 femmine) sottoposti a resezione chirurgica in due diverse Chirurgie Toraciche tra gennaio 2002 e dicembre 2007 per tumori polmonari non a piccole cellule pT1AN0M0. La MVD è stata valutata mediante il conteggio visivo dei microvasi positivi alla colorazione immunoistochimica con anticorpo monoclonale anti-CD31 e definita come il numero medio di microvasi per 1 mm2 di campo ottico. Risultati. Sono state eseguite 59 lobectomie (72%) e 23 resezioni sublobari (28%). Reperti istopatologici: 43 adenocarcinomi (52%) e 39 neoplasie non- adenocarcinoma (48%) pT1aN0M0; MVD media: 161 (CD31/mm2); mediana: 148; range 50-365, cut-off=150. Una MVD elevata (> 150 CD31/mm2) è stata osservata in 40 pazienti (49%), una MVD ridotta ( ≤ 150 CD31/mm2 ) in 42 pazienti (51%). Sopravvivenze a 5 anni: 70 % e 95%, rispettivamente per il gruppo ad elevata MVD vs il gruppo a ridotta MVD con una p = 0,0041, statisticamente significativa. Il tipo di resezione chirurgica, il diametro del tumore, le principali comorbidità e l’istotipo nono sono stati fattori predittivi significativi di mortalità correlata alla malattia. La MVD è risultata essere superiore nel gruppo “Adenocarcinoma” (MVD mediana=180) rispetto al gruppo “Non-Adenocarcinoma (MVD mediana=125), con un test di Mann-Whitney statisticamente significativo (p < 0,0001). Nel gruppo “Adenocarcinoma” la sopravvivenza a 5 anni è stata del 66% e 93 %, rispettivamente per i pazienti con MVD elevata e ridotta (p = 0.043. Conclusioni. Il nostro studio ha mostrato che la Microvessel density valutata con la colorazione immunoistochimica per CD31 ha un valore prognostico rilevante nel carcinoma polmonare in stadio precoce pT1aN0M0.

Analisi fosfo-proteomica di campioni di tumore: il problema della stratificazione dei pazienti ed eterogeneità intra-tumorale nell'era della terapia personalizzata

Nella presente tesi indaghiamo la potenzialità di LCM e Reverse Phase Protein microarray negli studi clinici. Si analizza la possibilità di creare una bio banca con line cellular primarie, al fine di conseguire drug test di sensibilità prima di decidere il trattamento da somministrare ai singoli pazienti. Sono stati ottenuti profili proteomici da biopsie pre e post terapia. I risultati dimostrano che questa piattaforma mostra il meccanismo di resistenza acquisito durante la terapia biologica. Questo ci ha portato ad analizzare una possibile stratificazione per pazienti con mCRC . I dati hanno rivelato distinti pathway di attivazione tra metastasi resecabile e non resecabili. I risultati mostrano inoltre due potenziali bersagli farmacologici. Ma la valutazione dell'intero tumore tramite singole biopsie sembra essere un problema a causa dell’eterogeneità intratumorale a livello genomico. Abbiamo indagato questo problema a livello dell'architettura del segnale in campioni di mCRC e ccRCC . I risultati indicano una somiglianza complessiva nei profili proteomici all'interno dello stesso tumore. Considerando che una singola biopsia è rappresentativa di un intera lesione , abbiamo studiato la possibilità di creare linee di cellule primarie, per valutare il profilo molecolare di ogni paziente. Fino ad oggi non c'era un protocollo per creare linee cellulari immortalizzate senza alcuna variazione genetica . abbiamo cosiderato, però, l'approccio innovativo delle CRCs. Ad oggi , non è ancora chiaro se tali cellule mimino il profilo dei tessuti oppure I passaggi in vitro modifichino i loro pathways . Sulla base di un modello di topo , i nostri dati mostrano un profilo di proteomica simile tra le linee di cellule e tessuti di topo LCM. In conclusione, i nostri dati dimostrano l'utilità della piattaforma LCM / RPPA nella sperimentazione clinica e la possibilità di creare una bio - banca di linee cellulari primarie, per migliorare la decisione del trattamento.

Statistical mechanics formalism and methods for the analysis of real networks

This thesis provides a thoroughly theoretical background in network theory and shows novel applications to real problems and data. In the first chapter a general introduction to network ensembles is given, and the relations with “standard” equilibrium statistical mechanics are described. Moreover, an entropy measure is considered to analyze statistical properties of the integrated PPI-signalling-mRNA expression networks in different cases. In the second chapter multilayer networks are introduced to evaluate and quantify the correlations between real interdependent networks. Multiplex networks describing citation-collaboration interactions and patterns in colorectal cancer are presented. The last chapter is completely dedicated to control theory and its relation with network theory. We characterise how the structural controllability of a network is affected by the fraction of low in-degree and low out-degree nodes. Finally, we present a novel approach to the controllability of multiplex networks

Expressionsunterschiede verschiedener miRNAs bei chronisch-entzündlichen Darmerkrankungen

Chronisch-entzündliche Darmerkrankungen konfrontieren unsere heutige Gesellschaft mit hohen Inzidenzraten in der westlichen Welt und zunehmend steigenden Inzidenzraten im asiatischen Raum. Die Folgen für die Patienten sind eine starke Beeinträchtigung der Lebensqualität, mit sozialen und wirtschaftlichen Folgen sowie ein erhöhtes Risiko für die Entwicklung kolorektaler Karzinome. Durch die Entdeckung von 22 nt langen, regulierenden RNAs, auch genannt miRNAs, wurde ein neuer Baustein im Verständnis zellulärer Regelprozesse und der Differenzierung und Aktivierung von Antworten etwa des Immunsystems entdeckt. Somit stellt sich die Frage nach der Bedeutung von miRNAs im Rahmen von chronisch-entzündlichen Darmerkrankungen. Hierzu wurden in dieser Arbeit über ein miRNA-Array System 12 miRNAs als potentiell relevante Ziele identifiziert und an einem Kollektiv aus insgesamt 131 Patienten und 163 Biopsien aus dem Bereich des Darmes überprüft. Es zeigte sich hierbei, dass im Rahmen eines Morbus Crohn mit Befall des Dickdarms die miRNAs let-7d und miR-22 in gesteigerter Expression vorlagen. Da im terminalen Ileum eine gesonderte Immunsituation vorliegt, wurde dieser Bereich zusätzlich bei der Erkrankung Morbus Crohn untersucht. Es zeigten sich Expressionsveränderungen für die miRNAs miR-30e, miR-185, miR-374b und miR-424. Bei Patienten mit einer Colitis ulcerosa waren die miRNAs let-7d, miR-185 und miR-424 in ihrem Expressionsverhalten verändert. Zusätzlich konnte gezeigt werden, dass in Abhängigkeit vom Entzündungsgrad bei bestehender Colitis ulcerosa eine zunehmenden Überexpression der miRNAs let-7d, miR-185 und miR-424 erfolgte. Die miRNAs miR-18a und miR-185 wiesen unter Remissionsbedingungen Expressionsveränderungen auf und lassen somit den Verdacht eines protektiven Effektes aufkommen. Mit Hilfe von computerbasierten Datenbankanalysen konnten gemeinsam regulierenden miRNAs Proteine und Pathways zugeordnet werden, welche einen Zusammenhang mit bereits pathogenetisch bestätigten Signalwegen wie etwa dem nF-ĸB und MAPK-Signalweg nahelegen. Auch konnte herausgearbeitet werden, dass einige, der von diesen miRNAs regulierten Proteine, bereits in veröffentlichten Arbeiten als fehlreguliert festgestellt wurden, jedoch blieb die Ursache dieser Fehlregulation gänzlich unbekannt. Mit den in dieser Arbeit erhobenen Daten konnte gezeigt werden, dass eine Kongruenz der Befunde vorliegt, welche einen Zusammenhang der miRNA-Expression mit der Fehlregulation bestimmter Proteine nicht nur nahelegt, sondern darüber hinaus auch noch einige weitere potentielle Proteinziele für weitere Untersuchungen aufführt. Dazu ist es jedoch notwendig, die Relevanz der hier entdeckten, computerbasierten Proteine in zukünftigen Untersuchungen einer genauen Prüfung zu unterziehen.

Resezioni epatiche eco-guidate radicali ma conservative simultanee alle resezioni colo-rettali. Un approccio sicuro al cancro colorettale avanzato

Il cancro colorettale (CRC) rimane la prima causa di morte nei paesi occidentali.Dal 15% al 25% dei pazienti affetti da CRC presenta metastasi epatiche sincrone (CRLM) al momento della diagnosi.La resezione epatica radicale rimane l’unica terapia potenzialmente curativa in presenza di CRLM con una sopravvivenza a 5 anni compresa tra il 17% ed il 35% ed a 10 anni tra il 16% e il 23% rispettivamente. La tempistica ottimale per la resezione chirurgica in caso di presentazione sincrona di CRC è controversa.Questo studio intende dimostrare che le resezioni epatiche ecoguidate radicali ma conservative simultanee ad una resezione colorettale rappresentano una tecnica sicura ed efficace nei pazienti con CRC avanzato. 48 pazienti sono stati sottoposti ad una resezione simultanea colorettale ed epatica. L’età media +SD (range) era di 64,2+9,7 (38-84).Un solo paziente è deceduto entro 30 giorni. La mortalità post operatoria è stata complessivamente del 2,1%. Nove pazienti (18,8%) hanno sviluppato una o più complicanza ,4 (8,3%) di grado III-IV sec. Clavien-Dindo e 5 (10,4%) di grado I-II. La durata complessiva dell’intervento chirurgico simultaneo è stata di 486,6+144,0 (153-804) minuti.Questo studio conferma che le resezioni colorettali ed epatiche simultanee possono essere eseguite senza un significativo aumento della morbilità e mortalità perioperatorie, anche in pazienti sottoposti ad una resezione anteriore ultrabassa ed in quelli in cui sia indicato il clampaggio intermittente dell’ilo epatico. L’IOUS è efficace nel ridurre l’estensione della resezione epatica in pazienti sia con CRLM anche multiple e bilobari .Poichè le complicanze maggiori sono frequenti dopo resezioni epatiche maggiori simultanee, riducendo l’estensione della resezione del parenchima epatico si può avere un impatto favorevole sul decorso post operatorio.Le resezioni epatiche ecoguidate radicali ma conservative simultanee ad una resezione colorettale sono una tecnica sicura ed efficace in pazienti con carcinoma colorettale avanzato e andrebbero considerate l’opzione primaria in casi selezionati

Identifizierung neuer potentieller Biomarker für das Kolonkarzinom sowie dessen Vorstufen

Zur Detektion von neuen potentiellen Biomarkerkandidaten für das KRK sowie dessen Vorstufen wurde Blutplasma von gesunden Kontrollen und von Patienten mit kolorektalen Adenomen und Karzinomen untersucht. Mit Hilfe immunoaffinitäts-basierter Chromatographiesäulen wurde das Blutplasma mit einer automatisierten Methode von hoch und moderat abundanten Proteinen entfernt und daraufhin massenspektrometrisch analysiert. Hierfür wurde eine labelfreie quantitative und datenunabhängige Methode angewendet. Nach Auswertung der Daten kristallisierten sich mehrere potentielle Biomarkerkandidaten heraus, wobei das Augenmerk auf solchen Proteinen lag, die sowohl bei Adenompatienten als auch bei Karzinompatienten vermehrt im Plasma vorlagen. Nach Western Blot Experimenten und einer weiteren Validierung mittels eines ELISAs an einem größeren Patientenset zeigte sich, dass die beiden Proteine AMBP und CRP als potentielle Biomarker zur Detektion von Adenomen und Karzinomen herangezogen werden konnten. Receiver Operating Charakteristik (ROC)-Analysen ergaben einen AUC-Wert von 0,79 für AMBP und 0,77 für CRP für das Erkennen von Adenomen und Karzinomen. Dies resultierte in einer Sensitivität von 72,5% für AMBP bzw. 60% für CRP bei einer Spezifität von 80%. Durch die Kombination beider Markerproteine erhielt man sogar eine noch höhere Sensitivität von 80% bei 80% Spezifität für das Aufdecken kolorektaler Neoplasien. Ebenso zeigte sich in der histochemischen Analyse, dass AMBP direkt von intestinalen Epithelzellen exprimiert wird.

Longer-term assessment of trastuzumab-related cardiac adverse events in the Herceptin Adjuvant (HERA) trial

We investigated the incidence of cardiac adverse events in patients with early breast cancer in the Herceptin Adjuvant (HERA) trial who were treated with 1 year of trastuzumab after completion of (neo)adjuvant chemotherapy.

Effect of EpCAM, CD44, CD133 and CD166 expression on patient survival in tumours of the ampulla of Vater

Carcinomas of the Vaterian system are rare and presumably arise from pre-existing adenomas. According to the cancer stem cell (CSC) hypothesis, only a small subset of tumor cells has the ability to initiate and develop tumor growth. In colorectal cancer, CD44, CD133, CD166 and EpCAM have been proposed to represent CSC marker proteins and their expression has been shown to correlate with patient survival.

Pre- and posttransplant management of solid organ transplant recipients: risk-adjusted follow-up

Solid organ transplant recipients (SOTR) have an increased risk of skin cancer due to their long-term immunosuppressive state. As the number of these patients is increasing, as well as their life expectancy, it is important to discuss the screening and management of skin cancer in this group of patients. The role of the dermatologist, in collaboration with the transplant team, is important both before transplantation, where patients are screened for skin lesions and the individual risk for skin cancer development is assessed, and after transplantation. Posttransplant management consists of regular dermatological consultations (the frequency depends on different factors discussed below), where early skin cancer screening and management, as well as patient education on sun protective behavior is taught and enforced. Indeed, SOTR are very sensitive to sun damage due to their immunosuppressive state, leading to cumulative sun damage which results in field cancerization with numerous lesions such as in situ squamous cell carcinoma, actinic keratosis and Bowen's disease. These lesions should be recognized and treated as early as possible. Therapeutic options discussed will involve topical therapy, surgical management, adjustment of the patient's immunosuppressive therapy (i.e. reduction of immunosuppression and/or switch to mammalian target of rapamycin inhibitors) and chemoprevention with the retinoid acitretin, which reduces the recurrence rate of squamous cell carcinoma. The dermatological follow-up of SOTR should be integrated into the comprehensive posttransplant care.

Prognostic impact of lymph node ratio outperforms positive lymph nodes and lymph nodes harvested: a time-dependent analysis in mismatch repair-proficient and –deficient colrectal cancers

Objective: We compare the prognostic strength of the lymph node ratio (LNR), positive lymph nodes (+LNs) and collected lymph nodes (LNcoll) using a time-dependent analysis in colorectal cancer patients stratified by mismatch repair (MMR) status. Method: 580 stage III-IV patients were included. Multivariable Cox regression analysis and time-dependent receiver operating characteristic (tROC) curve analysis were performed. The Area under the Curve (AUC) over time was compared for the three features. Results were validated on a second cohort of 105 stage III-IV patients. Results: The AUC for the LNR was 0.71 and outperformed + LNs and LNcoll by 10–15 % in both MMR-proficient and deficient cancers. LNR and + LNs were both significant (p<0.0001) in multivariable analysis but the effect was considerably stronger for the LNR [LNR: HR=5.18 (95 % CI: 3.5–7.6); +LNs=1.06 (95 % CI: 1.04–1.08)]. Similar results were obtained for patients with >12 LNcoll. An optimal cut off score for LNR=0.231 was validated on the second cohort (p<0.001). Conclusion: The LNR outperforms the + LNs and LNcoll even in patients with >12 LNcoll. Its clinical value is not confounded by MMR status. A cut-of score of 0.231 may best stratify patients into prognostic subgroups and could be a basis for the future prospective analysis of the LNR.

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms. Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [(14)CO(2)]-liberation. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides (p<0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity (p<0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc (p<0.05) and ODC (p<0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARgamma-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.

Systemic prokinetic pharmacologic treatment for postoperative adynamic ileus following abdominal surgery in adults

BACKGROUND: Postoperative adynamic bowel atony interferes with recovery following abdominal surgery. Prokinetic pharmacologic drugs are widely used to accelerate postoperative recovery. OBJECTIVES: To evaluate the benefits and harms of systemic acting prokinetic drugs to treat postoperative adynamic ileus in patients undergoing abdominal surgery. SEARCH STRATEGY: Trials were identified by computerised searches of the Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, and the Cochrane Colorectal Cancer Group specialised register. The reference lists of included trials and review articles were tracked and authors contacted. SELECTION CRITERIA: Randomised controlled parallel-group trials (RCT) comparing the effect of systemically acting prokinetic drugs against placebo or no intervention. DATA COLLECTION AND ANALYSIS: Four reviewers independently extracted the data and assessed trial quality. Trial authors were contacted for additional information if needed. MAIN RESULTS: Thirty-nine RCTs met the inclusion criteria contributing a total of 4615 participants. Most trials enrolled a small number of patients and showed moderate to poor (reporting of) methodological quality, in particular regarding allocation concealment and intention-to-treat analysis. Fifteen systemic acting prokinetic drugs were investigated and ten comparisons could be summarized. Six RCTs support the effect of Alvimopan, a novel peripheral mu receptor antagonist. However, the trials do not meet reporting guidelines and the drug is still in an investigational stage. Erythromycin showed homogenous and consistent absence of effect across all included trials and outcomes. The evidence is insufficient to recommend the use of cholecystokinin-like drugs, cisapride, dopamine-antagonists, propranolol or vasopressin. Effects are either inconsistent across outcomes, or trials are too small and often of poor methodological quality. Cisapride has been withdrawn from the market due to adverse cardiac events in many countries. Intravenous lidocaine and neostigmine might show a potential effect, but more evidence on clinically relevant outcomes is needed. Heterogeneity among included trials was seen in 10 comparisons. No major adverse drug effects were evident. AUTHORS' CONCLUSIONS: Alvimopan may prove to be beneficial but proper judgement needs adherence to reporting standards. Further trials are needed on intravenous lidocaine and neostigmine. The remaining drugs can not be recommended due to lack of evidence or absence of effect.

T cell-mediated immunoregulation in the gastrointestinal tract

In the intestinal tract, only a single layer of epithelial cells separates innate and adaptive immune effector cells from a vast amount of antigens. Here, the immune system faces a considerable challenge in tolerating commensal flora and dietary antigens while preventing the dissemination of potential pathogens. Failure to tightly control immune reactions may result in detrimental inflammation. In this respect, 'conventional' regulatory CD4(+) T cells, including naturally occurring and adaptive CD4(+) CD25(+) Foxp3(+) T cells, Th3 and Tr1 cells, have recently been the focus of considerable attention. However, regulatory mechanisms in the intestinal mucosa are highly complex, including adaptations of nonhaematopoietic cells and innate immune cells as well as the presence of unconventional T cells with regulatory properties such as resident TCRgammadelta or TCRalphabeta CD8(+) intraepithelial lymphocytes. This review aims to summarize the currently available knowledge on conventional and unconventional regulatory T cell subsets (Tregs), with special emphasis on clinical data and the potential role or malfunctioning of Tregs in four major human gastrointestinal diseases, i.e. inflammatory bowel diseases, coeliac disease, food allergy and colorectal cancer. We conclude that the clinical data confirms some but not all of the findings derived from experimental animal models.