Дисертації з теми "Pancreatic neuroendocrine tumor"

京都大学
新制・論文博士
博士(医学)
乙第13418号
論医博第2226号
新制||医||1052(附属図書館)
京都大学大学院医学研究科医学専攻
(主査)教授 羽賀 博典, 教授 長船 健二, 教授 伊藤 貴浩
学位規則第4条第2項該当
Doctor of Medical Science
Kyoto University
DFAM

Neuroendocrine tumors (NET´s) are often characterized by overproduction of peptide hormones. In spite of pronounced clinical symptoms, the tumor lesions can be small and difficult to detect. The general aim of this thesis was to investigate, in vitro and in vivo, some of the potential monoamine pathways present in NET´s, using radiolabeled tracers for positron emission tomography (PET), with the intention to explore the value of PET-imaging in the management of NET´s.

We used the 11C-labeled serotonin precursor 5-hydroxy tryptophan (HTP) as the tracer for imaging of NET´s. More than 95% of the subjects displayed a high tracer uptake on PET and tumor detection rate with PET was higher in >50% of the patients compared both to computed tomography (CT) and somatostatin receptor scintigraphy (SRS). The primary tumor was imaged by PET in 84% (16/19), compared to 47% and 42% for SRS and CT, respectively. Tumor visibility was better with PET due to a higher tumor-to-background ratio and a better spatial resolution. There was a strong correlation (r = .907) between changes in urinary-5-hydroxy indole acetic acid and changes in transport rate of 11C-5-HTP during treatment, indicating the use of PET in treatment monitoring of NET´s.

Pretreatment with carbidopa decreased the urinary radioactivity concentration four-fold and significantly (p<0.001) increased the tumor tracer uptake. This greatly improved image interpretation and tumor lesion detection.

A screening for expression of monoamine pathways in NET´s revealed a high in vitro binding of the monoamineoxidase-A ligand harmine to tumor sections. PET examinations with 11C-harmine could visualize tumors in all patients, including non-functioning endocrine pancreatic tumors (EPT´s).

Finally, the in vitro turnover and in vivo distribution of the amino acids glutamate, glutamine and aspartate was investigated. Limited uptake in vivo indicates the lack of utility for these substances as PET-tracers for imaging and characterization of NET´s.

Introduction Endoscopic ultrasound (EUS) elastography is a recent ultrasound method used for the real-time visualization and evaluation of tissue elasticity. Qualitative and quantitative methods have been used, in particular in evaluation of pancreatic diseases and malignant lymph nodes, with interesting results regarding the accuracy and the differential diagnosis between malignant and benign masses. No consensus has been reached with regard to the superiority of different quantitative methods, but strain ratio and strain histogram (SH) remain the most used. SH corresponds to a graphical representation of the color distribution in a region of interest (ROI), and mean SH (mSH) value is a SH-derived quantitative measure of the global hardness in the evaluated ROI. Materials and methods The aim of the present study was to describe the different elastographic patterns of solid pancreatic masses (pancreatic adenocarcinoma, PA, and pancreatic neuroendocrine tumor, pNET) using the mSH value. A group of normal patients was enrolled to define the normal pancreatic elasticity. This is a prospective, observational, monocentric study. Experienced EUS examiners performed the endoscopic procedure and elastographic measures. Contrast enhancement was performed, and according to the European Federation Societies of Ultrasound in Medicine and Biology guidelines and recommendations, 2 phases were defined for CE-US and CEH-EUS of the pancreas: an early and/or arterial phase (starting from 10 to 30 seconds) and a late and/or venous phase (from 30 to 120 seconds). Enhancement was evaluated in arterial phase. When the lesion resulted hypovascular (hypo-enhanced), the aspect was defined as typical for adenocarcinoma while an hypervascular lesion (with strong arterial hyper-enhancement) was considered typical of neuroendocrine tumors. SH was calculated automatically by machine integrated software in a ROI manually selected by the operator. Four parameters were calculated using the histogram to quantify the elasticity of the pancreas: the mSH, the standard deviation of the histogram, the kurtosis and the skewness. Three different measures were performed by the same operator and the mean value of the previous described values was evaluated. After the elastographic measure a FNA was performed when a lesion was observed. We used the histology obtained by FNA or surgical specimens or the global assessment of cytology and imaging as reference standard for the diagnosis, except in case of normal examinations. Results A total of 88 patients were included: 9 normals, 45 PA, 9 pNET (5 grade 1, 4 grade2-3), 5 chronic pancreatitis, 20 others (IPMN, metastatic lesions, pancreatic cystoadenoma, etc). Only patients with normal pancreas, PA and pNET were included in the statistical analysis. Mean age was 64 (range: 19-88). All the patients presenting a pancreatic lesion, except one, received the contrast. All the patients with pNET presented a typical hypervascular enhancement. On the other hand, of patients with histological proven pancreatic adenocarcinoma, only 37 presented a typical hypoenhancement, while 5 presented an hypervascularisation and 2 patients an atypical enhancement (represented by a partial arterial enhancement). Regarding elastography, we obtained a statistically significant difference between the malignant lesions (p-Net grade 2/3 and adenocarcinoma) and normal pancreas, considering all the parameters evaluated (mSH, standard deviation, kurtosis and skewness). No differences were found in elastographic measurement between p-NET and adenocarcinoma, neither between p-NET grade 1 (that, considering the good prognosis and slow evolution, could be considered neither as benign neither as malign) and p-NET grade 2/3. At univariate analysis we found that all the elastographic parameters measured were capable to differentiate benign versus malign disease, with a cut-off calculated with ROC-curves of 43.250 for mSH, 44.63 for standard deviation, 1.5 for the skewness and 5 for kurtosis. At multivariate analysis only mSH resulted statistically significative to distinguish benign versus malign lesions. At a model constructed by logistic regression (9.8958 – 0.3170*moyenne + 0.2989*SD + 0.7935*kurtosis – 7.0642*skewness) a cut off of 0.57 was found to distinguish benign versus malign lesions. Conclusions Mean strain histogram seems able to differentiate pancreatic cancer from benign pancreas. EUS elastography could potentially be used in negative EUS–FNA cases, which represent up to 25% of patients with focal masses. The presence of a strong suspicion of pancreatic cancer is indicated when combining meanSH, standard deviation, skewness and kurtosis in a statistical model we obtain a cut-off>0.57.
Introduzione L’elastometria condotta tramite ecoendoscopia è una nuova tecnica utilizzata per la visualizzazione e la valutazione in real time dell’elasticità dei tessuti. È noto infatti che i tessuti patologici, quali ad esempio i tessuti tumorali, presentano un’elasticità diversa rispetto ai tessuti normali. Fino ad oggi sono state utilizzate tecniche di misurazione dell’elasticità sia di tipo qualitativo che di tipo quantitativo. Le misure qualitative sono però gravate da una scarsa riproducibilità. Per tale ragione esse sono state praticamente abbandonate afavore di misure quantitative. Non esiste un consenso circa la superiorità di un metodo quantitativo rispetto ad un altro, ma lo “strain ratio” (SR) e lo “strain histogram” restano le metodiche più utilizzate. Lo SH, utilizzato in questo studio, corrisponde a una rappresentazione grafica della distribuzione del colore in una determinata area di interesse, e il valore del mSH è una misura dell’elasticità globale in un’area di interesse prescelta, normalmente corrispondente alla lesione. L’accuratezza dell’elastometria è stata valutata nelle patologie pancreatiche e linfonodali, mostrando risulatati interessanti soprattutto nella diagnosi differenziale tra le masse benigne e le masse maligne. Materiali e metodi Lo scopo del nostro studio è la quantificazione del pattern elastografico nel pancreas normale, nell’adenocarcinoma pancreatico (AP) e nei tumori neuroendocrine (NET). Si tratta di uno studio prospettico, monocentrico e osservazionale. L’ecoendoscopia con contrasto è stata effettuata e, secondo le line guida della società Europea di ecografia, sono state individuate 2 fasi contrastografiche per lo studio del pancreas: la prima, dai 10 ai 30 secondi, denominata fase precoce o arteriosa e la seconda, tardiva o venosa, dai 30 ai 120 secondi. L’enhancement delle lesioni pancreatiche è stato valutato in fase arteriosa. All’ecoendoscopia con contrasto, l’aspetto tipico di AP è definito in presenza di ipo-enhancement, mentre l’aspetto tipèico in caso di NET è definite in presenza di iper-enhancement arterioso. Successivamente all’elastometria è stata eseguita un’elastografia. Lo SH è stato calcolato automaticamente da un software in un’area scelta manualmente dall’operatore. 4 parametri sono stati presi in considerazione: lo SH medio (Msh), la deviazione standard, la kurtosis e lo swekness. 3 misure differenti sono state effettuate dallo stesso operatore e il valore medio delle misure è stato preso in considerazione. Dopo la misura elastografica è stato eseguito il prelievo bioptico. Il risultato istologico ottenuto tramite biopsia è stato utilizzato come referenza per definire la natura della lesione, eccetto in caso di pancreas normale. Risultati Nello studio sono stati inclusi 88 pazienti: 9 pancreas normali, 9 NET, 45 AP, 5 pancreatiti croniche e 20 lesioni miste. Solo I pazienti con AP, NET e pancreas normale sono stati inclusi nell’analisi finale. Alla somministrazione di contrasto, i pazienti con NET hanno presentato un pattern ipervascolare dopo somministrazione di contrasto, mentre tra i pazienti con AP, solo 37 hanno presentato un pattern tipico ipovascolare, mentre 5 hanno presentato un pattern ipervascolare e 2 un enhancement atipico (arterioso parziale). Per ciò che riguarda l’elastografia, abbiamo ottenuto una differenza statisticamente significativa tra il pancreas normale e le lesioni maligne, per tutti i 4 parametri valutati (media, deviazione standard, kurtosis e skewness). Invece, non abbiamo trovato una differenza statisticamente significativa tra i NET e l’AP. All’analisi univariata abbiamo trovato che tutti i parametri erano in grado di differenziare il pancreas benigno dal pancreas maligno, con un cut-off calcolato mediante la curva ROC di 43.250 per mSH, 44.63 per la deviazione standard, 1.5 per lo skewness and 5 per la kurtosis. All’analisi multivariata, solo il mSH è risultato statisticamente significativo. Abbiamo costruito un modello statistico tramite regressione logistica (9.8958 – 0.3170*mSH + 0.2989*SD + 0.7935*kurtosis – 7.0642*skewness), individuando un cut off di 0.57 per distinguere il pancreas normale dale lesioni maligne. Conclusioni Il mSH sembra efficace nella differenziazione tra lesioni pancreatiche maligne e pancreas benigno. Proponiamo pertanto l’uso dell’elastometria, e del nostro modello statistico, per definire le lesioni da operare o da sorvegliare in caso di biopsia negativa. Riteniamo infatti che, pur in caso di biopsia negativa, un indice elastografico superiore a 0.57 sia indicativo di una lesione maligna e pertanto necessiti di una presa in carico chirurgica.

INTRODUCTION Pancreatic Neuroendocrine Tumours (PNETS) have increased in incidence over the past three decades. Treatment options currently include surgery, locoregional and systemic therapies, however the prognosis remains poor and biomarkers that accurately predict the clinical behavior of these tumours are lacking. Dysregulation of microRNAs (miRNAs) has recently been shown to play a role in the development of many cancers through post-transcriptional gene regulation, however, few studies have investigated the role of miRNAs as diagnostic or prognostic markers in PNETs. METHODS Patients undergoing resection of PNETs at our institutions between 1992 and 2014 were retrospectively included in this study. RNA was extracted from formalin-fixed, paraffin embedded PNET samples and microarray analysis performed and correlated with clinicopathological data. MicroRNAs with statistically significant differential expression between patients with locoregional disease only, versus those who developed metastases were subject to in-silico analysis using three target gene prediction databases. RESULTS 37 patients were included in the study. Patient subgroup DM had poorer overall survival (OS) as compared to subgroup L (p = 0.046). 506 miRNAs with differential expression between the ‘Distant metastasis’ group and ‘Locoregional’ group were identified. Of these, 265 miRNAs were downregulated, whilst 241 were upregulated. Four of these miRNAs were differentially expressed to statistical significance. These included miR-3653 which was upregulated and miR-4417, miR-574-3p and miR-664b-3p which were all downregulated. Only miRNA-3653 was identified by all three databases as having a potential PNET-related target; ATRX. CONCLUSION Higher expression of tumour miR-3653 was seen in the group of patients with metastatic disease compared to those with only locoregional disease. Several bioinformatic tools predicted transcriptional regulator ATRX as a possible target for miR-3653. Thus, it is possible that miR-3653 could be a biomarker for metastatic potential and consequently poorer prognosis in patients with PNETs. However, these conclusions cannot be made with certainty given the limitations of this study and further work beginning with validation is required.

Introduction: The lack of valid biomarkers represents a major unmet clinical need in pancreatic neuroendocrine neoplasms (PanNENs). Chromogranin A (CgA) is the most commonly measured PanNEN biomarker, despite relevant limitations related to variable sensitivity, poor specificity and lack of assay standardization. Therefore, novel biomarkers are needed to improve diagnosis, detect disease recurrence and assess treatment response. In this project, the role of CgA-derived fragments as PanNEN biomarkers was investigated, focusing on the N-terminal peptide vasostatin-1 (VS-1). A multianalyte biomarker, NETest, was also assessed as PanNEN biomarker. Given the role of CgA-derived fragments in tumor vascular biology, their tissue expression was correlated with pathological features in PanNENs. Finally, the role of CgA-derived fragments was investigated in the setting of pancreatic ductal adenocarcinoma (PDAC). Methods: The project was developed according to different tasks: 1) CgA-derived fragments and total-CgA were measured in patients who underwent surgery for PanNENs; 2) NETest was evaluated as biomarker evaluate the efficacy of surgical resection in patients with PanNENs; 3) The site of CgA cleavage was investigated and CgA-derived fragments tissue expression was assessed; 4) The role of CgA fragmentation in terms of prognosis was assessed in patients with PDAC. Results: Aim 1. VS-1 is significantly correlated with tumor size, aggressiveness features and risk of disease relapse in patients with localized non-functioning (NF) PanNENs. VS-1 is not affected by proton pump inhibitors treatment. Aims 2-3. VS-1 is able to early assess surgical effectiveness in patients with NF-PanNETs, especially in those with aggressive tumors. Total-CgA, VS-2 and pancreastatin are not useful in this context. Aim 4. NETest seems able to provide a proper evaluation of the initial postoperative disease status in setting of PanNENs. Aim 5. CgA processing occurs within neuroendocrine tumor cells. VS-1 tissue expression is a marker of tumor differentiation. Aim 6. C-terminal CgA cleavage is enhanced in PDAC and it is associated with poorer survival outcomes. Conclusions: VS-1 has been identified as a promising biomarker for PanNENs. CgA C-terminal fragmentation is enhanced in PDAC, whereas N-terminal cleavage seems more relevant in PanNENs. Whether CgA fragmentation in PanNENs is just a disease epiphenomenon or plays a role in tumor vascular biology remains an open point.
Introduzione: La mancanza di biomarcatori validi rappresenta un'importante esigenza clinica nell’ambito delle neoplasie neuroendocrine pancreatiche (PanNEN). La cromogranina A (CgA) è il biomarcatore neuroendocrino più comunemente utilizzato, nonostante le rilevanti limitazioni legate alla sensibilità variabile, alla scarsa specificità ed alla mancanza di standardizzazione del suo dosaggio. Sono pertanto necessari nuovi biomarcatori al fine migliorare la diagnosi, predire la ricorrenza di malattia e valutare la risposta ai trattamenti. In questo progetto abbiamo studiato il ruolo dei frammenti derivati dalla CgA come biomarcatori nelle PanNEN, concentrandoci sul peptide N-terminale chiamato vasostatina-1 (VS-1). Anche un biomarcatore multianalita, NETest, è stato valutato nell’ambito delle PanNEN. Inoltre, considerato il ruolo dei frammenti derivati dalla CgA nella biologia vascolare dei tumori, la loro espressione tissutale è stata correlata con caratteristiche patologiche di aggressività in campioni di PanNEN. Infine, il ruolo dei frammenti della CgA è stato studiato anche nel contesto dell'adenocarcinoma duttale pancreatico (PDAC). Metodi: Il progetto è stato sviluppato secondo diverse attività: 1) I frammenti derivati dalla CgA e la CgA totale sono stati misurati in pazienti sottoposti ad intervento chirurgico per PanNEN; 2) Il NETest è stato valutato come biomarcatore per predire l'efficacia della resezione chirurgica in pazienti con PanNEN; 3) Il sito di clivaggio della CgA così come l'espressione tissutale dei frammenti derivati dalla CgA sono stati oggetto di studio; 4) Il ruolo prognostico della frammentazione della CgA è stato valutato in pazienti con PDAC. Risultati: Obiettivo 1. I livelli plasmatici di VS-1 sono significativamente correlati con le dimensioni del tumore, le caratteristiche di aggressività e il rischio di recidiva di malattia in pazienti con PanNEN localizzate non funzionanti (NF). La VS-1 non è influenzata dal trattamento con inibitori di pompa protonica. Obiettivi 2-3. La VS-1 è in grado di valutare precocemente l'efficacia chirurgica nei pazienti con NF-PanNET, specialmente in quelli con tumori aggressivi. La CgA totale, la vasostatina-2 e la pancreastatina non sono risultati utili in questo contesto. Obiettivo 4. Il NETest sembra in grado di fornire precocemente una corretta valutazione dello stato di malattia dopo resezione chirurgica in pazienti affetti da PanNEN. Obiettivo 5. Il clivaggio della CgA avviene all'interno delle cellule tumorali neuroendocrine. Una elevata espressione tissutale di VS-1 sembra essere un marker di differenziazione del tumore. Obiettivo 6. La scissione della CgA nella regione C-terminale è incrementata nel PDAC ed è associata a peggiore prognosi. Conclusioni: La VS-1 è stato identificata come un promettente biomarcatore per i pazienti affetti da PanNEN. La frammentazione C-terminale della CgA è incrementata nel PDAC, mentre la scissione N-terminale sembra essere più rilevante nelle PanNEN. Se la frammentazione della CgA sia solo un epifenomeno o svolga un ruolo nella biologia vascolare delle PanNEN resta un punto aperto.

Scopo: identificare le alterazioni molecolari clinicamente rilevanti associate ai tumori neuroendocrini del pancreas (PanNETs). Materiali e Metodi: 204 pazienti sono stati studiati per la ricerca mutazionale in 8 geni frequentemente alterati in PanNETs utilizzando Tecnologia Ion Torrent. Tali geni si dividono tra MEN1, ATRX e DAXX coinvolti nel rimodellamento della cromatina, PIK3CA,TSC2,PTEN,MTOR implicati nel mTOR pathway e infine ATM presente come ruolo centrale nel mantenimento della stabilità e riparazione del DNA. Inoltre sono stati valutati: i) le variazioni del numero di copie (CNV) tramite dati CGH array disponibili in 90 casi, ii) l’allungamento alternativo dei telomeri (ALT) tramite FISH in un numero selezionato di casi iii) l’espressione proteica tramite immunoistochimica per Menina, ATRX, Daxx, Atm e Pten in 171PanNETs. Risultati: l’analisi mutazionale mirata tramite next generation sequencing di 204 PanNETs ha dimostrato la presenza di mutazioni in 112/204 (55%), mentre in 92 (45%) risultano non alterati. E’ stato inoltre rilevato i) le mutazioni a carico dei geni Daxx e ATRX risultano essere mutuamente esclusive e più frequentemente presenti pari a 53/204 (26%) e associate con mutazioni di MEN1 o ATM a loro volta mutuamente esclusive; ii) mutazioni DAXX/ATRX sono associate a un sottoinsieme di PanNETs che presentano perdite cromosomiche ; iii ) le mutazioni PTEN e TSC2 si escludono a vicenda e sono state trovate in 31/204 (15%), mentre l'attivazione mTOR è associato con una migliore sopravvivenza libera da malattia. Conclusioni : in questo studio abbiamo dimostrato che i PanNETs possono essere suddivisi tra due principali sottogruppi molecolari. Un primo gruppo mostra una simultanea perdita cromosomica nelle neoplasie con mutazioni a carico di MEN1 e la contemporanea alterazione nei geni DAXX , ATRX , PTEN e TSC2 . Il secondo gruppo di casi presenta mutazioni di MEN1 senza alterazioni cromosomiche rincorrenti il cui significato deve essere ancora scoperto.
Purpose: To identify clinically relevant molecular alterations associated to pancreatic endocrine tumors (PanNETs) discovered by recent exome sequencing data. Patients and Methods: A series of 204 PanNETs were explored for intragenic mutations in 8 genes reported as recurrently altered in PanNETs using Ion Torrent technology. The 8 genes were MEN1, ATM, ATRX, DAXX, PIK3CA, TSC2, PTEN, MTOR. Their relation with chromosomal copy number aberrations were assessed in 90 cases having CGH array data available, and in a selected number of cases alternative lengthening of telomeres (ALT) was investigated by FISH. A total of 171 PanNETs were also investigated by immunohistochemistry for Menin, Atrx, Daxx, Atm and Pten. Results: Our targetted next-generation mutational analysis of 204 PanNETs for three chromatin remodelling genes (MEN1, ATRX, DAXX), MTOR pathway genes (PTEN, TSC2, PIK3CA, MTOR) and the ATM DNA repair gene assessed that 112/204 (55%) PanNETS had mutations, while 92 (45%) lacked mutations, and: i) the mutually exclusive mutations in DAXX and ATRX chromatin remodelling genes were the most frequent, accounting for 53/204 (26%) cases, and were associated with MEN1 or ATM mutations that were in turn mutually exclusive; ii) DAXX/ATRX mutations are associated with a subset of PanNETS showing a peculiar pattern of recurrent chromosomal losses; iii) the mutually exclusive mutations in genes belonging to mTOR pathways were found in 31/204 (15%), and mTOR pathway activation is associated with shorter disease-free survival. Conclusions: We report that PanNET can be subdived into two main molecular subgroups. The first showing the peculiar simultaneous loss of twelve chromosomes, which includes the vast majority of neoplasms showing MEN1 mutations and concurrent mutations in DAXX, ATRX, PTEN and TSC2. The second group, where only a small number of cases shows MEN1 mutations, lacks recurrent chromosomal anomalies and the gene alterations responsible for their development remain to be discovered.

Introduzione: I tumori pancreatici neuroendocrini non funzionanti (NF-PNET) asintomatici ben differenziati sono un’entità sempre più spesso diagnosticata e la loro gestione è controversa vista la loro buona prognosi seppur eterogenea. Scopo: Scopo dello studio è stato quello di valutare la storia naturale dei NF-PNET sporadici asintomatici con diametro < 2 cm valutando il rapporto rischio-beneficio di una gestione conservativa. Metodi: Tra Gennaio 2000 e Giugno 2011, 46 pazienti con una diagnosi di NF-PNET < 2 cm sono stati inseriti in un programma di follow-up di almeno 18 mesi con imaging seriale in due centri di riferimento Risultati: I pazienti sono stati prevalentemente di sesso femminile (65%) con un’età mediana di 60 anni. I tumori erano principalmente localizzati a livello della testa del pancreas (52%) con un diametro mediano di 13 mm (9-15 mm). Dopo un follow-up mediano di 34 mesi (24-52 mesi) e una media di 4 imaging seriali (3-6), non si sono osservate metastasi a distanza o linfonodali. In 6 pazienti (13%) è stato osservato un aumento dimensionale del 20%. La crescita mediana complessiva tumorale è stata di 0.12 mm all’anno e non è stato identificato nessun fattore correlate al paziente o al tumore predittivo di crescita tumorale. Complessivamente, 8 pazienti (17%) sono stati sottoposti a intervento chirurgico dopo un tempo mediano di 41 mesi (27-58 mesi). Tutte le lesioni sottoposte a resezione sono state classificate come stadio I (n=7) o 2 (n=1) di grado 1, con assenza di metastasi linfonodali e invasione vascolare. Conclusioni: In pazienti selezionati una gestione conservativa di NF-PNET sporadici asintomatici < 2 cm è sicura. Necessitiamo di studi prospettici per validare questa politica di “wait and see”.
Introduction: Asymptomatic sporadic non-functioning well-differentiated pancreatic neuroendocrine tumors (NF-PNET) are increasingly diagnosed, and their management is controversial because of their overall good but heterogeneous prognosis. Objective: To assess the natural history of asymptomatic sporadic NF-PNETs smaller than 2 cm in size and the risk-benefit balance of non-operative management. Methods: From January 2000 to June 2011, 46 patients with proven AS-NF-PNET smaller than 2 cm in size were followed-up for at least 18 months with serial imaging in tertiary referral centers. Results: Patients were mainly female (65%), with a median age of 60 years. Tumors were mainly located in the pancreatic head (52%), with a median lesion size of 13 mm (9 –15). After a median follow-up of 34 months (24 –52) and an average of 4 (3– 6) serial imaging sessions, distant or nodal metastases appeared on the imaging in none of the patients. In 6 (13%) patients, a 20% increase in size was observed. Overall median tumor growth was 0.12 mm per year and neither patients nor tumor characteristics were found to be significant predictors of tumor growth. Overall, 8 patients (17%) underwent surgery after a median time from initial evaluation of 41 months (27–58); all resected lesions were ENETS T stage 1 (n=7) or 2 (n=1), grade 1, node negative, with neither vascular nor peripancreatic fat invasion. Conclusions: In selected patients, non-operative management of asymptomatic sporadic NF-PNET smaller than 2 cm in size is safe. Larger and prospective multicentric studies with long-term follow-up are now needed to validate this “wait and see” policy.

Introduzione: l’attuale diffusione e maggior accessibilità delle tecniche imaging negli ultimi anni hanno aumentato l’incidenza di riscontro di neoplasie endocrine non-funzionanti del pancreas (NF-PNETs) asintomatiche. I NF-PNETs incidentali (I-NF-PNETs) solitamente presentano diametro e stadio minori rispetto ai NF-PNETs sintomatici (S-NF-PNETs) ed il riscontro occasionale sembra rappresentare un fattore prognostico favorevole sia per lo stadio di malattia sia per il grading istologico. Vi è comunque una assenza di dati circa la gestione dei I-NF-PETs potenzialmente non aggressivi. Obiettivi:1) definire il comportamento biologico dei I-NF-PETs sottoposti a resezione chirurgica e 2) valutare una eventuale politica di Follow-Up nella gestione dei I-NF-PETS di stadio I. Metodi: sono stati inclusi nello studio tutti i pazienti con diagnosi confermata all’istologia di NF-PET sporadico sottoposti tra il 1990 ed il 2011 a resezione chirurgica presso il Dipartimento Chirurgico dell’Università di Verona ed il reparto di chirurgia dell’Opedale S. Cuore-Don Calabria di Negrar. E’ stata eseguita una valutazione comparativa delle caratteristiche demografiche, cliniche e patologiche tra I-NF-PETs e S-NF-PETs. E’ stata eseguita l’analisi statistica adeguata per identificare le differenze statisticamente significative del comportamento biologico dei I-NF-PETs versus gli S-NF-PETs. Risultati: Sono stati evidenziati 131 pazienti (42.8%) con diagnosi di I-NF-PET e 175 pazienti (57.2%) con diagnosi di S-NF-PET. Non è stata riscontrata differenza di sesso tra i due gruppi (p=0.752). L’età media è stata per i maschi: 62 anni (range 24-83) nei I-NF-PETs e 55 anni (range 17 – 78) per gli S-NF-PETs; per le femmine rispettivamente di 55 anni (range 35 – 72) e 53 anni (range 25 – 74), p= 0.223. Gli I-NF-PETs si sono riscontrati più frequentemente a livello del corpo-coda del pancreas (65 casi, 49.6%), mentre gli S-NF-PETs si sono localizzati maggiormente a livello del corpo-coda (56.6%) e della coda (38.3%) (p= <0.001). Si sono ottenuti margini di resezione microscopicamente liberi da malattia (R0) in 123 pazienti (93.9%) con I-NF-PET ed in 131 pazienti (74.9%) con S-NF-PET (p<0.001). Il diametro medio riscontrato è stato di 20 mm (range 7 – 120) per gli I-NF-PETs e di 35 mm (range 5 – 140) per gli S-NF-PETs; p= 0.016. Comunque gli I-NF-PETs di stadio I si sono riscontrati più frequentemente rispetto agli S-NF-PETs (p<0.001). Ugualmente, si è riscontrata una localizzazione linfonodale di malattia (N1) nel 44.6% dei pazienti (78 casi) con S-NF-PET rispetto al 20.6% (27 casi) dei pazienti con tumore incidentale; p<0.001. Un paziente con I-NF-PET allo stadio I ha dimostrato avere una malattia aggressiva. Questo caso inizialmente era stato classificato come benigno ed era stato sottoposto ad intervento di enucleazione (R0), ma è stata evidenziata la comparsa di metastasi epatiche a distanza di 28 mesi dall’intervento chirurgico. In questo caso, tuttavia si era evidenziata, alla imaging radiologica preoperatoria, una dilatazione del dotto di Wirsung condizionata dalla neoplasia ed alla immunoistochimica si era evidenziata una positività alla serotonina. Queste caratteristiche possono rappresentare nella maggior parte dei casi una tendenziale aggressività della neoplasia. Da settembre 2007 a settembre 2011 sono stati inoltre considerati 19 pazienti con diagnosi di I-NF-PET. In tutti i casi si trattava di NET-G1 con un diametro medio di 15 mm (range 9-20). In nessun caso si erano evidenziate caratteristiche radiologiche di potenziale aggressività neoplastica (tra cui la presenza di dilatazione del dotto di Wirsung). Il Follow-Up è stato condotto per tutti i pazienti con una mediana di 22 mesi (tange 6-48). Tutti i pazienti sono risultati vivi, asintomatici con neoplasia stabile senza evidenza di progressione di malattia. Conclusioni: questo studio dimostra che i pazienti con NF-PET di riscontro incidentale rappresentano circa il 40% dei NF-PETs resecabili e che la diagnosi di I-NF-PET è aumentata negli ultimi anni. La diagnosi incidentale sembra rappresentare un fattore prognostico importante sia per le caratteristiche istopatologiche di malattia sia in termini di sopravvivenza e ricorrenza di malattia dopo resezione. Tuttavia la chirurgia pancreatica evidenzia un alto indice di comorbidità post-operatorie e per le neoplasie incidentali del diametro < 20 mm senza caratteristiche radiologiche di sospetta aggressività, si potrebbe proporre il Follow-Up clinico-radiologico.
Introduction: the widespread use of imaging techniques allowed increasing incidentally detection of asymptomatic non-functioning PNETs (NF-PNETs). Incidental non-functioning PNETs (I-NF-PETs) are usually smaller and lower in stage than symptomatic NF-PNETs (S-NF-PETs) and incidental detection seems to be an important favourable prognostic factor even after accounting for tumor stage, grade and location. There is a complete lack of data as regards of the admitted correct management of asymptomatic patients with potentially benign NF-PET. Aims:1) to define the biological behaviour of I-NF-PETs who underwent surgical resection and 2) to evaluate a follow-up policy in the management of I-NF-PNETs at stage I. Methods: All patients with a pathologically confirmed diagnosis of sporadic NF-PETs who underwent resection at the Departments of Surgery of the University of Verona and of Ospedale “Sacro Cuore – Don Calabria” of Negrar between 1990 and 2011 were included. A comparison of demographic, clinical and pathological characteristics between I-NF-PETs and S-NF-PETs was made. Statistical analyses were performed to identify differences in biological behavior between I-NF-PETs and S-NF-PETs. Results: A total of 131 patients (42.8%) had diagnosis of I-NF-PETs and the remaining 175 patients (57.2%) had diagnosis of S-NF-PETs. No sex predilection was observed (p=0.752). The median patient age was for male: 62 years (range 24 – 83) and 55 (range 17 – 78) with I-NF-PET and S-NF-PET diagnosis respectively; for female was 55 years (range 35 – 72) and 53 (range 25 – 74) with I-NF-PET and S-NF-PET (p= 0.223) respectively. The most common location of I-NF-PETs was in the body-tail of the pancreas (65 cases, 49.6%), whereas S-NF-PETs were most commonly founding both in the body-tail (56.6%) and in the head of the pancreas (38.3%) (p= <0.001). Clear surgical margins (R0) were obtained in 123 patients (93.9%) with I-NF-PET and in 131 patients (74.9%) with S-NF-PET (p<0.001). Median tumor size was lesser for I-NF-PETs with a median of 20 mm (range 7 – 120), than S-NF-PETs (median 35 mm; range 5 – 140); p= 0.016). Therefore T1 incidental tumors were mostly found than symptomatic PETs (p<0.001). Equally lymph-node metastases (N1) were identified in 44.6% of patients with S-NF-PET (78 cases) versus a 20.6% of patients with incidental tumor (27 cases); p<0.001. One patient with I-NF-PET on stage I was found to have malignant disease; this patient initially was classified as benign and underwent enucleation with clear surgical margins (R0), but had liver disease recurrence after 28 months after surgical resection. In this case preoperative imaging evaluation demonstraded the main pancreatic duct (MPD) obstruction (> 5 mm) and a serotonin immunoreactivity at the immunohistochemical evaluation. From September 2007 to September 2011 a total of 19 patients with I-NF-PNET diagnosis were enrolled. All cases was classified as NET-G1 and median size was 15 mm (range 9 – 20). In all cases, no MPD obstruction was confirmed at preoperative imaging. All this patients refused surgical resection. Currently Follow-Up was available for all patients, with a median follow-up of 22 months (range 6 – 48). All Patients were alive, asymptomatic and with tumor stable in size and no evidence of progression disease. Conclusions: this study shows that patients with incidentally detected NF-PETs represent about 40% of resectable NF-PETs and frequency of incidental diagnosis was increasing in last years. Incidental detection seems to be an important favorable prognostic factor for histopathological features, patients overall survival and disease free survival. Anyway pancreatic surgery have a recognized high rate of perioperative morbidities and for < 20 mm and carefully selected pancreatic neuroendocrine “incidentalomas” a clinical-laboratory and radiographic surveillance might be possible.

Background & Aims:Fine-needle aspiration (FNA) of pancreatic neuroendocrine tumors (Pan-NENs) has been proposed to obtain the grading, using Ki-67 proliferation index calculation. Data on reliability of grading (G) and Ki-67 index calculations from FNA cell blocks are controversial as there are potential limitations. Methods:One hundred patients subjected to FNA for a presumed PanNEN and subsequent resection material were evaluated at a single institution. FNA was obtained with endoultrasonography or with a percutaneous approach. Ki-67 calculation was performed using WHO guidelines. A comparison be-tween cytology (c) and histology (h) was performed for grading, Ki-67 values and diagnostic rate. Results:The overall level of agreement for G (n=63) was moderate (Cohen’s k = 0.455, 95%CI from 0.219 to 0.691, p<0.001; r=0.430, 95%CI from 0.204 to 0.613, p<0.001). The overall sensitivity for G was 76.2%, whereas for G1, G2, and G3 it was 76.6%, 84.6% and 33.3% respectively. Mean in-dex values of cKi-67 and hKi-67 were 4.35 ± 9.5% and 5.26 ± 12% respectively (p=0.334). Spear-man’s r for Ki-67 was good and very good for the overall population and for PanNENs of tumor size ≤ 20 mm (respectively r=0.615, 95%CI from 0.434 to 0.749, p<0.001, and r=0.862, 95%CI from 0.718 to 0.935, p<0.001).The Bland-Altman plot showed an agreement between cKi-67 and hKi-67 assessment.The overall concordance rate for diagnosis was 100%. Conclusions:In the presence of a suspected PanNEN, FNA (especially obtained through endoultra-sonography) achieves an excellent diagnostic rate, with satisfactory and reliable results for grading and Ki-67 assessment, particularly for tumors ≤ 20 mm.

Cancer is characterised as a set of diseases that is involved in uncontrolled cell growth with the ability to invade or spread to the other part of the body. Carcinogenesis is recognized as a process through aggregation of genetic and epigenetic changes in normal cell that ultimately leading to unlimited growth proliferation and invasion. Pancreatic neuroendocrine tumour (PNET) is a rare tumour that arise from neuroendocrine gland, occurs in various part of the body. The prevalence rate of PNETs is near about 25–30 per 100,000 population in the United States and according to Surveillance Epidemiology and End Results (SEER), the incidence rate of PNETs increased five-fold from 1973 to 2011. PNETs comprises approximately 7% of all types of cancer in the pancreas. Normally, 5 years survival rate of PNETs near about 42%. PNETs is a heterogenous group of disorder with less 5 years survival rate due to lack of effective therapeutic options for patients with advanced stages, absence of symptomatology specially in case of non-functional PNETs and also to the phenomenon of chemoresistance, dependent on multiple mechanisms. Recent data shows that incidence rate of this tumour increases as a result of germline genetic mutations. Concerning to genetic change, it is very important to explain the differences that occurs at the level of chemoresistance. The treatment plan of the PNETs varies on type, location and aggressiveness of the tumour. Surgery is the only curative treatment in early stage but in advanced stages chemotherapy and radiotherapy are the most palliative treatment option of PNETs. Chemotherapy which is mainly based cisplatin combined with capecitabine and the response rate of treatment is near about 30%. Cisplatin is responsible for the formation of DNA adducts, leading to DNA damage, and induces generation of ROS, that consequently leads to oxidative stress, cell damage and death. Glutathione (GSH) plays an important role in the maintenance of intercellular redox balance and detoxification. Chemoresistance can be based on the alteration of the detoxification mechanisms and GSH system has been pointed as one of the most important. Cysteine is a rate limitant substrate for GSH synthesis, and xCT cyst(e)ine transporter is implicated in cancer severity and chemoresistance. The Hypothesis of the project is: the disruption of xCT and uptake of cysteine leads to the reversal of resistance to alkylating agents in pancreatic neuroendocrine tumours (PNETs). To accomplish the hypothesis we defined 3 aims: 1st aim will be to address the expression of xCT in PNETs cell lines, and the modulation of xCT expression by cysteine and cisplatin; 2nd aim will be focused on the effect of xCT inhibition in PNETs cell death, using erastin and sulfasalazine, and 3rd aim will be focused on the effect of new nanoformulations in order to disturb cysteine uptake (Sechry and Sechry@PUREG4-FA) and glutathione synthesis (BSO@PUREG4-FA). Our work allowed to reveal the role of xCT transporter and the role of cysteine in PNETs cell line resistance. This cell line showing different response patterns in cysteine transporters activity helped to reveal the differences of the transporter in chemoresistance mechanism. It also showed that besides xCT transporter other cysteine transporter such as EAAT3 also appeared to be involved in the dynamics of chemoresistance mechanism. This work was also important to undercover the effect of new nanoformulations in order to disturb cysteine uptake by using SeChry and GSH synthesis by using BSO in PNETs cell lines. SeChry, but not SeChry@PUREG4-FA, induced cell death in BON-1 cell lines. SeChry cytotoxicity can be selective for cancer cells and this was taken in consideration in our new strategy by using SeChry@PUREG4-FA, however the assay was not successful and new markers for targeted delivery must be investigated in PNETs. BSO@PUREG4-FA induced cell death in combination with platinum salts in PNETs cell lines. Possibly, the use of folate functionalised particles will help to bypass the critical step in the non-specific delivery of BSO to non-cancer cell. The targeted BSO delivery to cancer cells can be explored as a novel strategy in cancer therapeutics. Moreover, more assays with cancer and non-cancer cells must be done in order to determine if folate receptor is in fact a suitable target to delivery drugs to PNETS cells, and find new and more specific targets.

Pancreatic Neuroendocrine Tumors (PanNETs) are rare malignancies with a wide range of clinical-pathological characteristics and different prognosis with a challenging clinical management. They typically carry out modifications of tumour suppressor pathways and no oncogenic alterations have been found yet. The recent acquisitions on the biological behavior of these rare tumors, due to the analysis of large cohorts of patients with consistent follow up data, lead to a change in classification of PanNETs in recent years. Our group recently identified novel pathways (mTOR, chromatin-remodeling and DNA repair) involved in PanNETs tumorigenesis, demonstrating that 60% of PanNETs had mutations in one of the genes involved in these pathways. We performed whole genome sequencing of 102 primary G1-G2 PanNETs in order to dissect the genomic events that underpin their pathogenesis. Mutations in DAXX/ATRX, mTOR pathway and alterations in the regulation of telomere length mechanism resulted correlated to worse survival. We describe 5 mutational signatures, including a novel G:C>T:A Base-Excision-Repair-deficiency signature due to MUTYH inactivation. We uncover a larger than expected proportion of germline mutations in clinically sporadic PanNETs. These include previously unreported mutations in DNA repair genes MUTYH, CHEK2, and BRCA2, which together with MEN1 and VHL occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, commonly occurred in genes involved in 4 main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling, (including novel EWSR1 gene fusions), and telomere maintenance. Copy number analysis performed by SNP-array, identified 4 distinct groups characterized by different patterns of chromosomal losses and gains. The group identified by the presence of recurrent chromosomal losses, with recurrent loss of chromosome 11, where resides MEN1 locus, resulted enriched for DAXX/ATRX mutations and altered telomere length. In addition, gene expression analyses identified a subgroup of PanNETs associated with upregulation of hypoxia and HIF signalling. These results open novel scenario on the understanding of PanNETs biology and uncover potential prognostic biomarkers able to better predict and stratify the prognosis of patients and influence their clinical management.

RASSF1 è un gene soppressore tumorale, la cui ipermetilazione del promotore è stata associata alla patogenesi di diversi tipi di tumore, tra cui i tumori endocrini del pancreas (PET) e il più aggressivo adenocarcinoma pancreatico (PDAC). La perdita allelica della regione 3p21.3, in cui è compreso il locus di RASSF1, è inoltre un evento che è stato recentemente associato all’insorgenza dei PET. Tuttavia, studi precedenti non hanno fornito un prova sperimentale diretta dell’effettiva riduzione dell’espressione di RASSF1A, in relazione alla metilazione. In questo studio è stata condotta un’analisi approfondita dello stato di RASSF1 a livello genetico ed epigenetico al fine di definirne il ruolo come putativo marcatore per la diagnosi precoce di cancro del pancreas; inoltre, scopo di questo lavoro è stato anche quello di cercare di delineare chiaramente l’evento regolatorio associato alla trascrizione di RASSF1 in questi tipi di neoplasie. In particolare, abbiamo determinato in PET e PDAC: (i) lo stato di metilazione di RASSF1 utilizzando la PCR metilazione-specifica ed il sequenziamento del DNA (ii) l’espressione delle varianti di mRNA di RASSF1 tramite RT-PCR e immunofluorescenza (iii) il numero di copie del gene RASSF1 mediante l’impiego dell’ibridazione fluorescente in situ (FISH) su una larga casistica di tumori endocrini e metastasi. La nostra analisi sui PET ha indicato l’esistenza di un circuito regolatorio associato alla metilazione che comporta la downregolazione di RASSF1A e l’incremento di RASSF1C, evidenziando quindi la metilazione come un processo che regola finemente l’attività del gene RASSF1 nei PET. Per quanto riguarda il numero di copie del gene d’interesse, sono state trovate alterazioni del locus di RASSF1 nel 25% dei casi con perdite predominanti rispetto a guadagni (19% vs 6%). In particolare, la più alta proporzione di cellule monosomiche nei PET correlava con uno stadio più avanzato della malattia. Diversamente, nei PDAC la ridotta espressione di RASSF1A nei PET rispetto al tessuto normale non era invece associata ad alcun effetto inibitorio o regolatorio della metilazione sulla trascrizione di RASSF1A. Nonostante l’elevata frequenza di perdita del 3p riscontrata nei casi di PDAC a disposizione (52% di perdite e 20% di guadagni), l’analisi FISH ha evidenziato che il numero in eccesso di copie del centromero del cromosoma 3 risulta il miglior fattore prognostico per la sopravvivenza dei pazienti affetti da PDAC. In conclusione quindi il locus di RASSF1 è caratterizzato da cambiamenti a livello epigenetico e trascrizionale durante la tumorigenesi, ma la metilazione non può essere considerata un marker decisivo per PET e PDAC. D’altra parte, la downregolazione di RASSF1A e le alterazioni nel numero di copie del cromosoma 3p potrebbero rappresentare eventi rilevanti per la patogenesi e la progressione di entrambi i tipi di neoplasie pancreatiche.
RASSF1 is a tumour suppressor gene, whose promoter hypermethylation has been suggested as a major event in the pathogenesis of different tumors, including pancreatic endocrine tumor (PETs) and the more aggressive pancreatic ductal adenocarcinoma (PDAC). Furthermore, allelic losses at 3p21.3, which includes the locus of RASSF1, have been recently related to PET. Despite this, previous studies did not give a direct proof that RASSF1 reduced expression was related to its promoter methylation. In this study we conducted an exhaustive analysis of RASSF1 status at both genetic and epigenetic level in order to better define its role as a putative marker of pancreatic cancers as well as clearly depict RASSF1-associated transcription regulatory event in these malignancies. Specifically, we analyzed both in PET and PDAC: (i) the methylation status of RASSF1 by methylation-specific PCR and sequencing (ii) the expression of RASSF1 variants by quantitative RT-PCR and immunofluorescence (iii) RASSF1 DNA copy number by fluorescence in situ hybridization (FISH) in a larger series of tumor specimens. Our analysis dealing with PET indicated the existence in the RASSF1 locus of a methylation-associated relay circuitry down-regulating RASSF1A and boosting RASSF1C, thus highlighting methylation as a fine-tuning process of RASSF1 gene activity in PET. Looking at copy number status, RASSF1 locus alterations were found in 25% of PET cases, with losses predominant over gains (19% vs. 6%). Interestingly, the higher proportion of monosomic cells in PET was positively correlated with an advanced stage of disease. Otherwise, the lower RASSF1A expression found in PDAC with respect to normal pancreas was not associated with either inhibitory or tuning effect of methylation on RASSF1A transcription. Surprisingly, FISH analysis despite the high frequency of 3p loss among PDAC cases (52% of losses vs 20% of gains), highlighted the excess of CEP3 copy number as the best prognostic factor affecting survival of PDAC patients. We can conclude that the RASSF1 locus suffers of changes at epigenetic and transcriptional level during tumorigenesis but methylation cannot be considered a decisive marker lesion for PET and PDAC. On the other hand, down-regulation of RASSF1A and alterations in copy number of chromosome 3p may represent relevant events for the pathogenesis and progression of both types of tumors.

博士
長庚大學
臨床醫學研究所
106
Gastroenteropancreatic neuroendocrine tumors (GEP NET) is the second most prevalent malignancy in the gastrointestinal tract. The differences in epidemiological distribution of and genetic alterations in pancreatic NETs in Western and Eastern populations imply different pancreatic NET pathogenesis between patients of different ethnicities. Chromogranin A (CgA) expression generally correlates with activity of neuroendocrine cells. The immunohistochemical staining of CgA in tumor cells is the gold standard for diagnosis of NETs, however, circulatory CgA levels have been claimed to be a useful biomarker for the assessment of GEP NET, especially those of patients with GEP NETs in non-Western countries. Our study aimed to elucidate the significance of plasma CgA levels for GEP NET and perform a comprehensive genomic analysis to assess mutation frequencies and determine correlations between genetic alterations and clinical outcome in Taiwanese patients with pancreatic NETs. Our study showed CgA is a biomarker for GEP NETs with high sensitivity and specificity rates. The baseline plasma CgA levels predicted overall survival and change of series CgA predicted treatment response in Asian patients with GEP NET. The genetic profiles of Asian patients with pancreatic NETs were distinct from those of Caucasian patients. Asian patients with pancreatic NETs were more frequently mutated for genes of the mTOR and angiogenesis pathway.