It was previously demonstrated that miR-199a was down-regulated in testicular germ cell tumor (TGCT) partly caused by hypermethylation of its promoter. More detailed analyses showed that miR-199a-5p, one of its two derivatives, suppressed TGCT invasiveness and proliferation via directing targeting PODXL and MAFB. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this project we identified DNMT3A, the de novo methyltransferase, as a direct target of miR-199a-3p using a 3’-UTR reporter assay. In NT2 (NTera 2) and HT (Hs 1.Tes) cells, miR-199a-3p regulated the expression of endogenous DNMT3A (both DNMT3A1 and DNMT3A2 isoforms), especially DNMT3A2 isoform. In clinical samples, the expression of DNMT3A2 and miR-199a-3p were reciprocally regulated. However, DNMT3A did not regulate miR-199a expression. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation partly through targeting DNMT3A. MiR-199a-3p could restore the expression of APC and MGMT via de-methylation in their promoters. Our studies demonstrated the dysregulation of miR-199a-3p in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potential therapeutic use of synthetic miR-199a-3p oligonucleotides as effective demethylation agent in the treatment of TGCT. However, since DNMT3A expression did not regulate miR-199a expression, the mechanism of promoter DNA hypermethylation of miR-199a in TGCT needs further investigation. / MiR-199a is encoded by two loci in the human genome, namely, miR-199a-1 on chromosome 19 and miR-199a-2 on chromosome 1. Another microRNA, miR-214, also locates on chromosome 1. Previous study revealed that it is co-transcribed with miR-199a-2, which is directed by miR-199a-2 promoter. However, the biological significance of the co-expression of miR-199a and miR-214 remains largely unknown. In this project, it was determined that miR-199a and miR-214 were concordantly expressed in TGCT. Silencing of DNMT1 increased the expression of miR-199a and miR-214, accompanied by de-methylation in the promoters of miR-199a-1/2. Overexpression of TP53 down-regulated the expression of DNMT1 and increased the expression of mature miR-199-3p/5p and miR-214. In addition, silencing of PSMD10 up-regulated the expression of TP53, while miR-214 over-expression resulted in PSMD10 down-regulation and TP53 up-regulation. Collectively, our findings highlighted a miR-199a/miR-214/PSMD10/TP53/DNMT1 self-regulatory network, which caused the down-regulation of miR-199a, miR-214 and TP53, as well as the up-regulation of DNMT1 and PSMD10 in TGCT. These observations partly explain the mechanism of promoter DNA hypermethylation in miR-199a in TGCT. They also suggest a potential therapeutic approach by targeting the miR-199a/miR-214/PSMD10/TP53/DNMT1 regulatory network in the treatment of TGCT. / 先前的研究證實miR-199a在睾丸生殖細胞腫瘤 (簡稱睾丸癌) 中是低表達的，部分歸因於其啟動子區域過度甲基化。對其功能研究發現miR-199a能抑制睾丸癌細胞的生長，侵襲和轉移，且miR-199a的抑癌屬性應歸功於它的兩個衍生物之一miR-199a-5p。然而，miR-199a的另一個衍生物miR-199a-3p在睾丸癌中的生物學功能仍然在很大程度上是未知的。此研究中，DNMT3A被鑒定為miR-199a-3p的直接靶定目標。在NT2和HT細胞中，miR-199a-3p能調控內源性DNMT3A(DNMT3A1和DNMT3A2)的表達水準，尤其是DNMT3A2。在臨床樣本中，DNMT3A2的表達水準與miR-199a-3p的表達水準呈負相關。但DNMT3A並不能調控miR-199a的表達水準。進一步研究顯示過表達miR-199a-3p能減少APC和MGMT啟動子區域甲基化而恢復其表達水準。研究證實異常表達的miR-199-3p可能在睾丸癌的癌變過程中發揮作用，並提出一個潛在的治療方案，即使用miR-199a -3p作為有效的去甲基化藥劑治療睾丸癌。然而睾丸癌中導致miR-199a啟動子區域過度甲基化的機制有待進一步研究。 / 在人類基因組中，miR-199a-1(位於19號染色體)和miR-199a-2(位於1號染色體)都編碼miR-199a。同時miR -214也位於1號染色體，研究表明miR-214與miR-199a-2由miR-199a-2啟動子介導共同轉錄，但miR-199a和miR- 214共同表達的生物學意義仍未知。此研究中，miR-199a和miR-214在睾丸癌中的表達呈現一致性。沉默DNMT1後miR-199a和miR-214的表達水準顯著提高，並伴隨著miR-199a-1/2啟動子區域的DNA去甲基化。在NT2細胞中。過表達TP53能下調DNMT1的表達水準，同時上調miR-199-3p/5p和miR- 214的表達水準。此外，過表達miR -214能導致PSMD10表達水準的下調以及TP53表達水準的上調。綜上所述，我們提出一個miR-199a/miR-214/PSMD10/TP53/DNMT1自我調控網路，此調控通路能引起睾丸癌中miR-199a，miR-214和TP53表達水準的下調，以及DNMT1和PSMD10表達水準的上調，且部分解釋睾丸癌中miR-199a啟動子區域過度甲基化的機制，同時該調控網路可作為治療睾丸癌的一個潛在靶點。 / Chen, Bifeng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 103-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 20, December, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Introduction: The role of positron emission tomography (PET) with 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) is currently under evaluation in urologic oncology. The aim of the present study was to investigate the use of [18F]FDG positron emission tomography ([18F]FDG-PET) in the detection and treatment control of malignant germ cell tumors compared to computed tomography (CT). Materials and Methods: Thirty-two PET studies and CT scans were carried out in 23 patients with histologically proven germ cell tumors (10 seminomas, 12 non-seminomatous germ cell tumors (NSGCT), 1 unclassified serologic recurrent disease) Lugano stage I–III. The scans were done either after initial diagnosis (n = 21) and/or within 3–45 days after chemotherapy was completed (n = 11). PET and CT were validated either by histology (n = 7) or clinical follow-up of 6–11 months after the last PET study has been performed (n = 16). Sensitivity, specificity, accuracy, positive and negative predictive values were determined for PET and CT. Differences between PET and CT for parameters of diagnostic value were evaluated by =χ2 test. Results: Although not statistically significant, the sensitivity, accuracy and negative predictive value were higher for PET than for CT with respect to the detection of metastatic infradiaphragmatic and supradiaphragmatic lesions after initial diagnosis. The specificity and positive predictive value of PET and CT were comparable. After chemotherapy, PET was found to be significantly superior in specificity and accuracy compared to CT with respect to infradiaphragmatic lesions (p < 0.05). False-positive PET findings in supradiaphragmatic lesions after chemotherapy occurred in the case of inflammatory processes and resulted in a loss of specificity and accuracy compared to CT (p < 0.05). Conclusions: These preliminary results demonstrate [18F]FDG-PET to be a useful diagnostic tool for the initial staging and treatment control in patients with germ cell tumors. Possible advantages compared to CT, however, are as yet not clearly defined. The possibility of false-positive PET findings due to reactive supradiaphragmatic inflammatory processes early after chemotherapy have to be considered. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Introduction: The role of positron emission tomography (PET) with 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) is currently under evaluation in urologic oncology. The aim of the present study was to investigate the use of [18F]FDG positron emission tomography ([18F]FDG-PET) in the detection and treatment control of malignant germ cell tumors compared to computed tomography (CT). Materials and Methods: Thirty-two PET studies and CT scans were carried out in 23 patients with histologically proven germ cell tumors (10 seminomas, 12 non-seminomatous germ cell tumors (NSGCT), 1 unclassified serologic recurrent disease) Lugano stage I–III. The scans were done either after initial diagnosis (n = 21) and/or within 3–45 days after chemotherapy was completed (n = 11). PET and CT were validated either by histology (n = 7) or clinical follow-up of 6–11 months after the last PET study has been performed (n = 16). Sensitivity, specificity, accuracy, positive and negative predictive values were determined for PET and CT. Differences between PET and CT for parameters of diagnostic value were evaluated by =χ2 test. Results: Although not statistically significant, the sensitivity, accuracy and negative predictive value were higher for PET than for CT with respect to the detection of metastatic infradiaphragmatic and supradiaphragmatic lesions after initial diagnosis. The specificity and positive predictive value of PET and CT were comparable. After chemotherapy, PET was found to be significantly superior in specificity and accuracy compared to CT with respect to infradiaphragmatic lesions (p < 0.05). False-positive PET findings in supradiaphragmatic lesions after chemotherapy occurred in the case of inflammatory processes and resulted in a loss of specificity and accuracy compared to CT (p < 0.05). Conclusions: These preliminary results demonstrate [18F]FDG-PET to be a useful diagnostic tool for the initial staging and treatment control in patients with germ cell tumors. Possible advantages compared to CT, however, are as yet not clearly defined. The possibility of false-positive PET findings due to reactive supradiaphragmatic inflammatory processes early after chemotherapy have to be considered. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Welche Ursachen führen bei Keimzelltumoren zu Spätrezidiven? Sind es ungünstige Tumorkonstellationen, Behandlungsfehler oder individuelle Faktoren, die zu Spätrezidiven von Keimzelltumoren (KZT) führen? Ziel der Untersuchung war es, diese Fragen zu beantworten, um Patienten zu identifizieren, für die sich daraus Konsequenzen in der Therapie und Verlaufskontrolle ergeben. Indem wir Spätrezidive nach 4 Jahren auswerteten, wollten wir die besonderen Merkmale dieser Patientengruppe herausarbeiten. Unter 759 erfassten Patienten sahen wir 165 Frührezidive (< 2 Jahre), 92 Rezidive nach 2 Jahren und 73 Spätrezidive mehr als 4 Jahre nach der Initialdiagnose. Unsere statistische Auswertung bezieht sich auf die Spätrezidive mit einer mittleren Beobachtungszeit von 143 Monaten. Die initiale Konstellation der Tumormarker spielt eine bedeutende Rolle. 66% der Patienten hatten sowohl AFP als auch ß-HCG erhöht (gegenüber 47% bei den Frührezidiven und 33% im Gesamtkollektiv). Das gonadal reine Embryonalzellkarzinom (EZK) führt häufiger zu Spätrezidiven als andere Keimzelltumoren. Risikobehaftet sind höhere Stadien, von denen sich unter den Spätrezidiven 85% fanden; initial aber nur 57%. Demgegenüber ist poor-prognosis nach IGCCCG kein Risikofaktor. Mehr als die Hälfte der spätrezidivierten Patienten hatte bereits vorher mindestens ein Rezidiv gehabt. Eine initial von der heutigen Leitlinie abweichende Behandlung war bei 40% der Spätrezidive zu finden. Gründe hierfür waren eine falsche Histologie, die ungenaue Stadienzuordnung oder eine fehlende bzw. inkomplette Residualtumorresektion. Eine günstige Prognose haben die Patienten, bei denen eine Metastasenresektion vorgenommen werden kann. Die Heilungsrate (NED) war bei den operierten Spätrezidiven (70%) deutlich hoher als denjenigen, die zusätzlich chemotherapiert (46%) oder lediglich mit Chemotherapie behandelt worden waren (36%). Als Grund hierfür, ist die Früherkennung und noch vorhandene Operabilität des Befundes anzunehmen. Alle Seminom-Spätrezidive mit initialer Radiatio haben von der Chemotherapie profitiert. Bis auf einen Todesfall in der Chemotherapie wurden alle geheilt. Spätrezidive von KZT sollten als besondere Entität definiert werden. Krankheitsverlauf, Behandlung und Nachsorge weichen von dem üblichen Muster ab. Nur Seminome oder chemonaive Patienten sind zytostatisch zu behandeln. Bei den Nichtseminomen, die bereits initial oder im Frührezidiv eine Chemotherapie erhalten haben, sollte wenn möglich, eine operative Behandlung erwogen werden. Patienten mit initial erhöhten Tumormarkern (AFP + ß-HCG), reinem gonadalem EZK, höheren Krankheitsstadien oder einem Rezidiv innerhalb der ersten 4 Jahre haben eine ungünstige Prognose und erfordern eine jährliche lebenslange spezielle Nachsorge. Die Nachsorge sollte neben der klinischen Untersuchung und AFP-Bestimmung ein Schnittbildverfahren des Thorax und Retroperitoneums beinhalten. / What are the reasons for late relapse of testicular germ cell tumor? Do unfavourable tumor constellations, mismanaged treatment or individual factors; lead to the late relapse of testicular germ cell tumor? The aim of this investigation was to find an answer to this question and to identify the patients, who would benefit from modified treatments and follow-up modalities. By researching the late relapses diagnosed at least 4 years later, we wanted to identify the special characteristics of this group of patients. Among the 759 patients with testis cancer we found 165 early relapses (