Neuroendocrine tumors (NETs) are a rare type of malignancy, however, their incidence and prevalence have markedly increased over the past several decades. Gastroenteropancreatic neuroendocrine tumors (GEP-NETs), NETs of the gastrointestinal (GI) tract, account for the majority of these tumors. Data from the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute (NCI), as well as from multiple cancer registries in Canada and Europe, indicate that the small intestine is one of the most common primary sites of GEP-NET development in Caucasian populations. Smaller studies from Asian countries, however, report a significantly lower proportion of small intestinal NETs (SI-NETs) in those countries’ populations. Interestingly, this finding persists in a study of Asians living in the United States, which, therefore, may suggest that genetics, and not environment, play a predominant role in driving this difference. Multiple studies exploring the genetics of SI-NETs in Caucasians have been published and have identified several genetic alterations implicated in tumorigenesis. However, the most consistently reported finding is chromosome 18 loss of heterozygosity (chr18 LOH), which has been detected in 61-78% of SI-NETs. Unfortunately, these studies have yet to be performed in a strictly Asian cohort. The proposed study, therefore, is a multicenter, cross-sectional study that will compare the proportion of SI-NETs with chr18 LOH in Asian versus Caucasian populations by performing a fluorescence in-situ hybridization (FISH) analysis of SI-NET tissue from both Asian and Caucasian samples. Overall, the results of this study may help to identify possible mechanisms driving the difference in proportion of SI-NETs between Asians and Caucasians, which in turn, may help to better characterize the genetic events leading into SI-NET tumorigenesis.

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

IFN-α has been applied in medical treatment of various neuroendocrine (NE) tumors, either alone or combination with somatostatin analogues. They can improve clinical symptoms in 50-70% of patients but a significant tumor reduction is only observed in 5-15% patients. Vasostatin (vaso) is believed to be an angiogenic inhibitor. The aim of this study is to evaluate the feasibility to use IFN-α and vasostatin gene therapy in NE tumors. We constructed plasmid vectors carrying human IFN-α2 (hIFN-α2) gene and human vasostatin gene, which were transfected into BON I cell to obtain stable gene-expressing cell lines. We found that in animal tumor model and cell experiments gene transfer of vasostatin caused a faster cell growth and tumor development via down-regulation of the tumor suppressor gene and p27. Cell adhesion, spreading, migration and invasion ability were increased in vaso-expressing BON I cells. Transfecting chicken vinculin could reverse the malignant behavior and restored expression of tumor suppressor genes. Moreover, vinculin knockdown could result in a faster cell growth and an increased colony formation. Condition medium taken from hIFN-α2-expressing BON I cells showed significant antiproliferative effects both on the NE tumor cells, BON I and LCC18, and the endothelial cells, PAE. It also suppressed cell adhesion and cell invasion and inhibited angiogenesis on CAM assay. Mice implanted with a mixture of WT BON cells and hIFN-α2-expressing BON cells (1:1) demonstrated significantly lower tumor incidence and longer tumor doubling time. Furthermore, long-acting IFN-α2b (PEGIntron®) demonstrated a better anti-tumor effect in contrast with IFN-α2b (IntronA®). Intratumoral injection of hIFN-α2 plasmids significantly inhibited NE tumor growth and caused tumor regression. We concluded that gene transfer of vasostatin into BON I cells might cause an enhanced malignant tumor behavior. Therefore, vasostatin therapy can not be recommended for patients with NE tumors. Vinculin might play an important role in NE tumor development and growth regulation. Gene therapy by using plasmid DNA carrying hIFN-α2 gene is feasible and promising in NE tumors.

Texture analysis derived from computed tomography (CT) might be able to provide clinically relevant imaging biomarkers and might be associated with histopathological features in tumors. The present study sought to elucidate the possible associations between texture features derived from CT images with proliferation index Ki-67 and grading in pulmonary neuroendocrine tumors. Overall, 38 patients (n = 22 females, 58%) with a mean age of 60.8 ± 15.2 years were included into this retrospective study. The texture analysis was performed using the free available Mazda software. All tumors were histopathologically confirmed. In discrimination analysis, “S(1,1)SumEntrp” was significantly different between typical and atypical carcinoids (mean 1.74 ± 0.11 versus 1.79 ± 0.14, p = 0.007). The correlation analysis revealed a moderate positive association between Ki-67 index with the first order parameter kurtosis (r = 0.66, p = 0.001). Several other texture features were associated with the Ki-67 index, the highest correlation coefficient showed “S(4,4)InvDfMom” (r = 0.59, p = 0.004). Several texture features derived from CT were associated with the proliferation index Ki-67 and might therefore be a valuable novel biomarker in pulmonary neuroendocrine tumors. “Sumentrp” might be a promising parameter to aid in the discrimination between typical and atypical carcinoids.

Small intestinal neuroendocrine tumor (SI-NET) is the most common small bowel tumor in Europe and USA, with an annual incidence of around 0.3-1.3/100000 persons. SI-NETs are the most common type of gastroenteropancreatic NETs (GEP-NETs), and they are known for their ability to produce hormones such as tachykinins and serotonin, as well as for their favorable long-term prognosis in comparison to gastrointestinal adenocarcinoma. The overall aim of the thesis was to investigate unknown or unclear aspects of SI-NET disease, in connection with prognosis, treatment and follow-up. Paper I confirmed several known negative prognostic factors and also showed, for the first time, that para-aortal lymph node metastases and peritoneal carcinomatosis were associated with worse survival by multivariable analyses. Locoregional surgery was associated with a low post-operative mortality, and a prolonged long-term survival by multivariable analysis. In Paper II we continued to investigate peritoneal carcinomatosis and found it be a risk factor not only for death, but also for emergency re-surgery. Furthermore, genetic analyses of samples from primary tumors in patients with and without peritoneal carcinomatosis showed a difference in the DNA between these two groups. In Paper III the outcome after liver surgery and/or radiofrequency ablation of liver metastases was investigated. To summarize, no difference in survival was seen in patients treated with surgery/radiofrequency ablation in comparison with matched controls. However, a superior radiological response of liver metasases and lower U-5-HIAA values were seen in patients subjected to liver surgery and/or radiofrequency ablation compared to matched controls. Paper IV compared ultrasonography, computed tomography and 11C-5HTP-PET in the follow-up after radiofrequency ablation of NET liver metastases. The study concluded that 11C-5HTP-PET depicted all residual tumors after RFA and that it, if used, should be combined with computed tomography for easier interpretation, as RFA areas are not clearly distinguishable with 11C-5HTP-PET alone. Paper V studied gallstone complications after somatostatin analog treatment in SI-NET patients, and concluded that there was a rather high risk to be subjected to a cholecystectomy due to biliary colic, cholecystitis, cholangitis or pancreatitis after primary surgery in somatostatin analog treated patients.

VIPomas are rare pancreatic endocrine tumors (PETs), detected in 1 in 10 million people per year and usually present as a constellation of well-defined clinical features characterized by watery diarrhea, hypokalemia, and achlorhydria (WDHA). Theses tumors secrete an excess of vasoactive intestinal peptide (VIP) and are typically diagnosed only after they have metastasized liver, lymph nodes and lungs (60% to 80%). The diagnosis is confirmed by identifying hyper secretion of VIP in a setting of the localized pancreatic tumor. Symptomatic pancreatic VIPomas are usually solitary, more than 3 cm in diameter, and occur in the tail of pancreas in 75 percent of patients. We demonstrate a rare glimpse at an unusual small size of VIPoma at its earliest clinical presentation.

Einleitung: Neuroendokrine Tumoren (NET) des gastroenteropankreatischen Systems entstammen den Zellen des diffusen neuroendokrinen Systems und sind seltene Tumoren mit einer steigenden Inzidenz. Die Beurteilung der Dignität von NET, allein mit Hilfe histologischer Kriterien, ist problematisch. Die WHO-Klassifikation von 2000 unterscheidet nicht nur histomorphologisch in gut und wenig differenziert, beurteilt werden auch Tumorgröße, Angioinvasion, Invasion der Musculuaris propria, hormonales Tumorsyndrom, Proliferationsindex (Ki67) sowie vorhandene Metastasen. Die neue WHO-Klassifikation von 2010 nutzt ein standardisiertes Grading-System, in dem nach Proliferationsindex in G1 (Ki67≤2%) niedriggradig maligne, G2 (3-20%) intermediärgradig maligne und G3 (>20%) hochgradig maligne, neuroendokrine Neoplasien (NEN) eingeteilt wird. Das Tumormikroenvironment gastroenteropankreatischer NET, im Hinblick auf die Tumor-Klassifikationen von 2000 und 2010, ist bisher nicht untersucht. Methoden: In meiner Arbeit wurden Paraffinblöcke von 55 Patienten immunhistochemisch untersucht: 32 Dünndarm-, 13 kolorektale und 10 Pankreas-NET. Als Tumormarker wurden Chromogranin, CD56, und S100 beurteilt. Marker für das Tumorstroma waren α-SMA und Desmin, Endothelzellmarker waren CD34 und vWF, und Immunzellmarker waren CD3 für T-Lymphozyten, CD20 für B-Lymphozyten und CD68 für Makrophagen. Kim1p galt als Marker für fibrohistiozytäre Zellen, der CC-Chemokinrezeptor-2 (CCR2) wurde exemplarisch für die Zytokinrezeptoren gefärbt. Die Auswertung erfolgte computergestützt. Die Immunzellen wurden quantitativ ausgewertet. Ergebnisse: In meiner Arbeit konnte ich zeigen, dass NET mit zunehmender Malignität weniger Stromazellen aufweisen. Auch die o.g. Tumormarker nahmen, wenn vorhanden, ab. Fibrohistiozytäre Zellen, die dendritische Zellen beinhalten, stellten die häufigsten Immunzellen dar, gefolgt von CD3+ T-Zellen. Im Vergleich der Organlokalisationen waren CD68+ Makrophagen in kolorektalen NET signifikant häufiger als in Dünndarm-NET. Nach der WHO-Klassifikation (2000) waren in den NET der WHO-Gruppe 2 und 3 signifikant mehr Kim1p+ Zellen als in der WHO-Gruppe 1 nachweisbar. CD3+ T-Zellen waren signifikant häufiger in NET der WHO-Gruppe 3 im Vergleich zur WHO-Gruppe 2. CD68+ Makrophagen waren in der WHO-Gruppe 3 im Vergleich zu den WHO-Gruppen 1 und 2 signifikant häufiger vorhanden. Die dargestellten Unterschiede zeigten sich auch für die neue Klassifikation (2010), allerdings mit vermindertem Signifikanzniveau. In NET der WHO-Gruppe 3 traten die Immunzellen in engen Kontakt zu den Tumorzellen, bei geringerem Tumorstroma. Zusammenfassung: Es bestehen deutliche Unterschiede in der Zusammensetzung des Tumormikroenvironments der NET, abhängig von der Malignität. Meine Ergebnisse weisen auf einen möglichen Zusammenhang zwischen der Anzahl an Immunzellen und der Differenzierung hin. Ob dies relevant für eine maligne Transformation ist, bleibt zu klären.

Le carcinome médullaire de la thyroïde (CMT) est une tumeur neuroendocrine rare, se développant à partir des cellules sécrétant la calcitonine. Cette tumeur survient dans un contexte familial dans un tiers des cas. Toutes les formes germinales et près de 40% des formes sporadiques sont causées par une mutation ponctuelle activatrice de l’oncogène RET, codant pour un récepteur membranaire à activité tyrosine kinase. Les événements oncogéniques à l’origine des formes sporadiques non mutées RET restent mal définis, à l’exception de mutations activatrices des oncogènes RAS découvertes récemment.Le pronostic péjoratif du CMT est essentiellement lié à un envahissement ganglionnaire précoce. A ce titre, la chirurgie initiale est souvent insuffisante et les formes métastatiques ont longtemps été considérées en impasse thérapeutique. L’avènement récent des inhibiteurs séléctifs de tyrosine kinases (ITK) a apporté un nouvel élan à la prise en charge des tumeurs réfractaires, certains d’entre eux incluant dans leur spectre d’action le récepteur RET. Mais l’optimisation de ces traitements requiert une connaissance préalable des mécanismes moléculaires sous-jacents au développement tumoral.Dans ce contexte et en nous appuyant sur une collection importante de prélèvements humains, nous avons cherché à approfondir la decription du ‘paysage génomique’ du CMT.Dans un premier temps, nous avons évalué les anomalies structurales ponctuelles et chromosomiques présentées par les CMT. Nous avons montré, par optimisation de méthodes de séquençage, que les mutations des gènes RET et RAS interviennent dans plus de 96% des cas et que ces évènements sont mutuellement exclusifs. Ces mutations permettent de distinguer plusieurs groupes d’agressivité et de réponse aux traitements par ITK. Nous avons également observé - par technique d’hybridation génomique comparative - des anomalies de grande ampleur récurrentes dans cette pathologie : les délétions du bras court du chromosome 1 et des chromosomes entiers 4 et 22 apparaissent comme étant des évènements précoces et indépendants de la tumorigenèse du CMT.Dans un second temps, nous avons déterminé - par approche de type biopuce - les profils d’expression de microARN dans les CMT. Certains de ces régulateurs post-transcriptionnels majeurs semblent liés au caractère invasif de la tumeur, et notamment les miR-21, miR-199 et miR-129. Nous avons également démontré le potentiel d’utilisation des microARN miR-21 et miR-199 en tant que biomarqueurs circulants du CMT. L’impact fonctionnel des formes précurseurs mir-21 et mir-129 a ensuite été évalué par transfection dans les modèles cellulaires TT et MZ-CRC1.Les observations ainsi obtenues offrent de nombreuses perspectives d’études. Elles permettent la définition de marqueurs tissulaires distinguant a priori les tumeurs métastatiques et/ou réfractaires aux thérapies. Enfin, elles mettent en lumière de nouvelles pistes pour la découverte de cibles thérapeutiques additionnelles dans cette pathologie. / Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor, arising from calcitonin-secreting cells. This cancer occurs in a family context in a third of cases. All inherited forms and nearly 40% of sporadic forms are caused by activating point-mutations in the RET oncogene, coding for a tyrosine-kinase receptor. Other oncogenic events causing sporadic cases remain unclear, but activating mutations of RAS oncogenes have been discovered recently.Prognosis of MTC is essentially linked to early lymph node occurrence. Initial surgery of metastatic forms is often insufficient and patients are considered in therapeutic dead-end. The recent advent of selective tyrosine-kinase inhibitors (TKIs) has brought a new impetus to the management of refractory tumors, some of them targeting the RET receptor. Optimization of these treatments require improving knowledge of the underlying molecular mechanisms of tumor development.In this context and helped by a large collection of human specimens, we have sought to deepen the description of genomic landscape of MTC.At first, we evaluated the structural and chromosomal abnormalities presented by MTC. We showed, by optimizing sequencing methods, that RET and RAS mutations are involved in over 96% of the cases, these events are mutually exclusives. These mutations can distinguish several groups of aggressiveness and of response to TKI treatments. We also observed, by comparative genomic hybridization techniques, recurrent abnormalities such as deletion of the short arm of chromosome 1 and loss of entire chromosomes 4 and 22. These losses appear to be early events of tumorigenesis MTC.In a second step, we determined - by a microarray approach – the microRNA expression profile of MTC. Some of these post-transcriptional regulators seem related to tumor invasiveness, such as miR-21, miR-199 and miR-129. We demonstrated the potential of microRNAs miR-21 and miR-199 as circulating diagnosis biomarkers of MTC. The functional impact of the precursor forms mir-21 and mir-129 was then evaluated by transfection in TT and MZ- CRC1 cellular models.Observations obtained pave the way for a lot of new potential studies. They allow the definition of tissue biomarkers distinguishing metastatic forms or refractory patients. Finally, they highlight new pathways for the discovery of additional therapeutic targets in this disease.

<p>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.</p><p>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. </p><p>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.</p><p>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).</p><p>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. </p>

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 &gt;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&lt;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.