A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science, Johannesburg 2017 / The high mortality rates of pancreatic ductal adenocarcinoma (PDAC) are largely attributed to a delayed diagnosis, of which in advanced disease, patients are unable to receive surgical resection with curative intent. Clinical presentations and genetic features shared between PDAC and other pancreatic conditions such as chronic pancreatitis (CP) are insufficient to facilitate the disease and often lead to diagnostic uncertainty at an early stage. The purpose of this study was to develop sensitive and specific non-invasive markers to aid in the detection and disease monitoring of PDAC. Here, circulating cell-free DNA (cfDNA) isolated from plasma samples of patients with PDAC (n= 155) and two control groups consisting of patients with either CP (n= 46) or critical limb ischemia (CLI) (n= 88) revealed significant differences in measured concentrations between the three patient groups (p= 0.006-Kruskal-Wallis test).When two groups were compared with each other using the Wilcoxon rank-sum test, observable differences were seen between the two pancreatic diseases: PDAC and CP (p= 0.002), and between the two controls: CP and the CLI groups (p= 0.007). A strong association was also observed in elevated cfDNA levels of CLI patients with HIV (p= 0.03), indicating a poor prognosis for patients. Results from methylationspecific PCR (MSP) in age-matched patient samples showed promoter methylation to account for the loss of Smad4 in late-stage PDAC; with an observed association with overall increasing cfDNA levels (p= 0.03).This study indicates the potential clinical utility of cfDNA as a non-invasive tool to predict disease progression both quantitatively and qualitatively, as well as to trace epigenetic changes in tumour markers associated with PDAC. Further investigation to identify hypermethylated genes in cfDNA for the early detection of PDAC is warranted. / XL2018

No description available.

Diabetes melito ou intolerância à glicose estão presentes em até 80% dos pacientes com adenocarcinoma de pâncreas. Portadores desta neoplasia têm resistência à insulina e alteração na secreção de insulina em resposta à glicose, o que pode levar ao aparecimento ou piora de diabetes. Para identificar genes diferencialmente expressos, que podem representar fatores diabetogênicos produzidos pelo adenocarcinoma de pâncreas, utilizou-se a comparação de microarranjos de oligonucleotídeos hibridizados com RNA complementar (cRNA) de tumores pancreáticos de pacientes com e sem diabetes melito no pré-operatório. Uma lâmina foi hibridizada com cRNA de dois pacientes portadores de diabetes melito, e outra com cRNA de dois pacientes com tolerância normal à glicose pelo teste oral. Considerando a expressão ajustada para os controles internos dos microarranjos, 293 genes estavam duas ou mais vezes mais expressos na lâmina dos portadores de diabetes melito; destes, 25 genes estavam pelo menos cinco vezes mais expressos. Duzentos e noventa e sete genes estavam pelo menos duas vezes mais expressos na lâmina dos pacientes com tolerância normal à glicose, dos quais 54 genes estavam cinco ou mais vezes mais expressos nestes indivíduos. Dos genes mais expressos nos tumores dos indivíduos portadores de diabetes melito, três deles, FAM3D, do inglês Family with Sequence Similarity number 3 member D, neuropeptídeo Y (NPY), e proteína de ligação do cálcio S100A8, foram estudados por reação em cadeia da polimerase em tempo real. A expressão do FAM3D foi 4070 (1000-37588) nas amostras de tumores de pacientes com diabetes melito, contra 109 (10-1112) nas de pacientes não-diabéticos (com intolerância à glicose ou com tolerância normal à glicose) (p<0,05). A expressão do NPY foi 0,46 (0,19-0,91) nos tumores dos portadores de diabetes, contra 0,32 (0,21- 0,58) nos tumores dos não-diabéticos (p = NS). Quanto à expressão de S100A8, foi 0,52 (0,27-0,60) nos tumores dos diabéticos, e 0,34 (0,16-1,44) nos não-diabéticos. Estudo imunohistoquímico mostrou que o FAM3D está expresso no núcleo e no citoplasma de células de tumores pancreáticos, tanto de indivíduos com diabetes melito quanto de não-diabéticos, assim como no citoplasma de células de ilhotas pancreáticas e de células ductais normais do pâncreas. Concluímos que o FAM3D é uma proteína expressa em tecido pancreático normal e tumoral, e que existe maior conteúdo do mRNA do FAM3D nos adenocarcinomas de pâncreas de portadores de diabetes melito do que nos de não-diabéticos. / Pancreatic ductal adenocarcinoma is closely related to diabetes mellitus; up to 80% of pancreas adenocarcinoma patients have diabetes or impaired glucose tolerance. Pancreas adenocarcinoma patients have both insulin resistance and altered insulin secretion in response to glucose, and impaired glucose metabolism has been reported in muscle of tumor patients, involving glycogen metabolism and post-receptor insulin signaling. But despite progress in research about this issue, precise mechanisms responsible for the interaction of pancreatic adenocarcinoma and diabetes mellitus remain unknown. The aim of this study was to identify differentially expressed genes between pancreas adenocarcinoma of patients who had and who did not have diabetes mellitus before surgery. Clinical and laboratorial data of 33 patients with pancreatic adenocarcinoma were evaluated, and tumor gene expression was analyzed by microarray method between two patients who had diabetes mellitus and two who did not have glycemic homeostasis impairment, and later used quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in twelve tumor fragments mRNA to confirm obtained data. Pancreatic adenocarcinoma patients who had diabetes mellitus had higher HOMA-IR (p < 0.05) and a trend to lower HOMA-beta indexes than non-diabetic patients. icroarray revealed 293 genes twice more expressed in the pool of diabetic patients as compared to the pool of normal glucose tolerance patients. Of these, 25 were five times more expressed in diabetic patients? pancreatic adenocarcinomas. Three genes were chosen for RT-qPCR: Family with Sequence Similarity number 3 member D (FAM3D), neuropeptide Y (NPY), and calcium-binding protein S100A8. FAM3D expression was 4070 (1000-37588) in diabetic patients tumors versus 109 (10-1112) in non-diabetic (impaired glucose and normal glucose tolerance) patients? tumors (p<0.05). NPY expression was 0.46 (0.19- 0.91) in diabetic patients and 0.32 (0.21-0.58) in non-diabetic patients? tumors (p=NS). Calcium-binding protein S100A8 expression was 0.52 (0.27-0.60) in diabetic and 0.34 (0.16-1.44) in non-diabetic patients (p=NS). Immunohistochemistry revealed that FAM3D protein was expressed in pancreatic adenocarcinoma cells in a diffuse nuclear and cytoplasmic pattern. It was also expressed in the cytoplasm of islets of Langerhans and normal pancreatic ducts cells. The present study indicates that cytokine-like FAM3D protein is expressed in normal and tumoral pancreatic tissue, and that FAM3D mRNA content is higher in pancreatic adenocarcinoma in diabetic than in non-diabetic patients.

Estudo retrospectivo de 119 pacientes com diagnóstico de adenocarcinoma ductal invasivo no estádio clínico III tratadas com quimioterapia neoadjuvante (FEC), mastectomia e adjuvância. Destas, 85 optaram por reconstrução mamária imediata (RMI) com retalho transverso músculo-cutâneo de reto-abdominal e 34, não. Com seguimento médio de 52,7 meses avaliou-se o tempo de hospitalização, a inter-relação com a adjuvância, recidiva local, o tempo livre de doença e o tempo total de sobrevida, concluindo-se que, apesar de aumentar o tempo de hospitalização, a RMI não interfere com os demais ítens, podendo ser indicada para pacientes portadoras de carcinoma mamário em estádio clínico avançado / A retrospective study with 119 patients diagnosed with invasive ductal adenocarcinoma of the breast treated with neoadjuvant chemotherapy (FEC), mastectomy and adjuvant therapy. Eight-five patients chose immediate breast reconstruction (IBR) with transverse rectus abdominis myocutaneous flap and, 34 did not do it. The mean follow-up was 52.7 months. Length of stay, adjuvant therapy interrelation, local recurrence, disease-free survival and overall survival were evaluated. It was concluded that despite a longer stay, IBR did not interfere with any of the other factors analyzed and may be indicated for patients with advanced breast disease

L'adenocarcinoma ductal de pàncrees és un càncer molt agressiu que actualment representa la quarta causa de mort per càncer als països occidentals. Les teràpies clàssiques, basades en la resecció quirúrgica, la radioteràpia i el tractament amb quimioteràpics com la gemcitabina, no són efectives en la gran majoria del pacients. En aquests darrers anys s'està estudiant l'aplicació de la teràpia gènica com a teràpia alternativa o adjuvant per al tractament d'aquesta neoplàsia. Una aproximació important és la que es basa en la transferència del gen de la timidina quinasa del virus Herpes simplex tipus 1 (TK) i l'administració de la pro-droga ganciclovir (GCV). Un dels atractius que presenta aquest sistema TK/GCV és que disposa d'un mecanisme amplificador de la mort cel·lular, que va més enllà d'eliminar la cèl·lula tumoral modificada genèticament amb el gen TK i que es coneix com l'efecte adjacent. S'ha proposat, que aquest efecte podria ser degut al trànsit dels metabòlits tòxics del GCV a través dels canals intercel·lulars que formen les unions gap.En aquesta tesi hem realitzat una caracterització de l'expressió de les molécules constitutives de les unions gap, les connexines, en l'adenocarcinoma de pàncrees; i hem estudiat el seu paper en l'eficàcia de dues estratègies terapèutiques basades en l'administració de compostos anàlegs de nucleòsids: el sistema suïcida TK/GCV i el quimioteràpic gemcitabina. S'ha estudiat també la possible contribució de l'E-cadherina, element clau de les unions adherents epitelials, en l'efecte citotòxic d'aquestes teràpies i amb especial èmfasi en el sistema TK/GCV.

Estudo retrospectivo de 119 pacientes com diagnóstico de adenocarcinoma ductal invasivo no estádio clínico III tratadas com quimioterapia neoadjuvante (FEC), mastectomia e adjuvância. Destas, 85 optaram por reconstrução mamária imediata (RMI) com retalho transverso músculo-cutâneo de reto-abdominal e 34, não. Com seguimento médio de 52,7 meses avaliou-se o tempo de hospitalização, a inter-relação com a adjuvância, recidiva local, o tempo livre de doença e o tempo total de sobrevida, concluindo-se que, apesar de aumentar o tempo de hospitalização, a RMI não interfere com os demais ítens, podendo ser indicada para pacientes portadoras de carcinoma mamário em estádio clínico avançado / A retrospective study with 119 patients diagnosed with invasive ductal adenocarcinoma of the breast treated with neoadjuvant chemotherapy (FEC), mastectomy and adjuvant therapy. Eight-five patients chose immediate breast reconstruction (IBR) with transverse rectus abdominis myocutaneous flap and, 34 did not do it. The mean follow-up was 52.7 months. Length of stay, adjuvant therapy interrelation, local recurrence, disease-free survival and overall survival were evaluated. It was concluded that despite a longer stay, IBR did not interfere with any of the other factors analyzed and may be indicated for patients with advanced breast disease

Diabetes melito ou intolerância à glicose estão presentes em até 80% dos pacientes com adenocarcinoma de pâncreas. Portadores desta neoplasia têm resistência à insulina e alteração na secreção de insulina em resposta à glicose, o que pode levar ao aparecimento ou piora de diabetes. Para identificar genes diferencialmente expressos, que podem representar fatores diabetogênicos produzidos pelo adenocarcinoma de pâncreas, utilizou-se a comparação de microarranjos de oligonucleotídeos hibridizados com RNA complementar (cRNA) de tumores pancreáticos de pacientes com e sem diabetes melito no pré-operatório. Uma lâmina foi hibridizada com cRNA de dois pacientes portadores de diabetes melito, e outra com cRNA de dois pacientes com tolerância normal à glicose pelo teste oral. Considerando a expressão ajustada para os controles internos dos microarranjos, 293 genes estavam duas ou mais vezes mais expressos na lâmina dos portadores de diabetes melito; destes, 25 genes estavam pelo menos cinco vezes mais expressos. Duzentos e noventa e sete genes estavam pelo menos duas vezes mais expressos na lâmina dos pacientes com tolerância normal à glicose, dos quais 54 genes estavam cinco ou mais vezes mais expressos nestes indivíduos. Dos genes mais expressos nos tumores dos indivíduos portadores de diabetes melito, três deles, FAM3D, do inglês Family with Sequence Similarity number 3 member D, neuropeptídeo Y (NPY), e proteína de ligação do cálcio S100A8, foram estudados por reação em cadeia da polimerase em tempo real. A expressão do FAM3D foi 4070 (1000-37588) nas amostras de tumores de pacientes com diabetes melito, contra 109 (10-1112) nas de pacientes não-diabéticos (com intolerância à glicose ou com tolerância normal à glicose) (p<0,05). A expressão do NPY foi 0,46 (0,19-0,91) nos tumores dos portadores de diabetes, contra 0,32 (0,21- 0,58) nos tumores dos não-diabéticos (p = NS). Quanto à expressão de S100A8, foi 0,52 (0,27-0,60) nos tumores dos diabéticos, e 0,34 (0,16-1,44) nos não-diabéticos. Estudo imunohistoquímico mostrou que o FAM3D está expresso no núcleo e no citoplasma de células de tumores pancreáticos, tanto de indivíduos com diabetes melito quanto de não-diabéticos, assim como no citoplasma de células de ilhotas pancreáticas e de células ductais normais do pâncreas. Concluímos que o FAM3D é uma proteína expressa em tecido pancreático normal e tumoral, e que existe maior conteúdo do mRNA do FAM3D nos adenocarcinomas de pâncreas de portadores de diabetes melito do que nos de não-diabéticos. / Pancreatic ductal adenocarcinoma is closely related to diabetes mellitus; up to 80% of pancreas adenocarcinoma patients have diabetes or impaired glucose tolerance. Pancreas adenocarcinoma patients have both insulin resistance and altered insulin secretion in response to glucose, and impaired glucose metabolism has been reported in muscle of tumor patients, involving glycogen metabolism and post-receptor insulin signaling. But despite progress in research about this issue, precise mechanisms responsible for the interaction of pancreatic adenocarcinoma and diabetes mellitus remain unknown. The aim of this study was to identify differentially expressed genes between pancreas adenocarcinoma of patients who had and who did not have diabetes mellitus before surgery. Clinical and laboratorial data of 33 patients with pancreatic adenocarcinoma were evaluated, and tumor gene expression was analyzed by microarray method between two patients who had diabetes mellitus and two who did not have glycemic homeostasis impairment, and later used quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in twelve tumor fragments mRNA to confirm obtained data. Pancreatic adenocarcinoma patients who had diabetes mellitus had higher HOMA-IR (p < 0.05) and a trend to lower HOMA-beta indexes than non-diabetic patients. icroarray revealed 293 genes twice more expressed in the pool of diabetic patients as compared to the pool of normal glucose tolerance patients. Of these, 25 were five times more expressed in diabetic patients? pancreatic adenocarcinomas. Three genes were chosen for RT-qPCR: Family with Sequence Similarity number 3 member D (FAM3D), neuropeptide Y (NPY), and calcium-binding protein S100A8. FAM3D expression was 4070 (1000-37588) in diabetic patients tumors versus 109 (10-1112) in non-diabetic (impaired glucose and normal glucose tolerance) patients? tumors (p<0.05). NPY expression was 0.46 (0.19- 0.91) in diabetic patients and 0.32 (0.21-0.58) in non-diabetic patients? tumors (p=NS). Calcium-binding protein S100A8 expression was 0.52 (0.27-0.60) in diabetic and 0.34 (0.16-1.44) in non-diabetic patients (p=NS). Immunohistochemistry revealed that FAM3D protein was expressed in pancreatic adenocarcinoma cells in a diffuse nuclear and cytoplasmic pattern. It was also expressed in the cytoplasm of islets of Langerhans and normal pancreatic ducts cells. The present study indicates that cytokine-like FAM3D protein is expressed in normal and tumoral pancreatic tissue, and that FAM3D mRNA content is higher in pancreatic adenocarcinoma in diabetic than in non-diabetic patients.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in the United States, with an average five-year survival rate of just 6.7%. One unifying aspect of PDAC is mutational activation of the KRAS oncogene, which occurs in over 90% of PDAC. Therefore, inhibiting KRAS function is likely an effective therapeutic strategy for this disease, and current research in our lab and others is focused on identifying downstream effectors of KRAS signaling that may be therapeutic targets. miRNAs are powerful regulators of gene expression that can behave as oncogenes or tumor suppressors. Dysregulation of miRNA expression is commonly observed in human tumors, including PDAC. The mir-17~92 cluster of miRNAs is an established oncogene in a variety of tumor contexts, and members of the mir-17~92 cluster are upregulated in PDAC, but their role has not been explored in vivo. This dissertation encompasses two studies exploring the role of miRNAs in pancreatic tumorigenesis. In Chapter II, I demonstrate that deletion of the mir-17~92 cluster impairs PDAC precursor lesion formation and maintenance, and correlates with reduced ERK signaling in these lesions. mir-17~92 deficient tumors and cell lines are also less invasive, which I attribute to the loss of the miR-19 family of miRNAs. In Chapter III, I find that Dicer heterozygosity inhibits PDAC metastasis, and that this phenotype is attributable to an increased sensitivity to anoikis. Ongoing experiments will determine whether shifts in particular miRNA signatures between cell lines can be attributed to this phenotype. Together these findings illustrate the importance of miRNA biogenesis, and the mir-17~92 cluster in particular, in supporting PDAC development and progression.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Changes in the microenvironment have been implicated in the transition of pre-invasive ductal carcinoma in-situ (DCIS) to invasive breast cancer. Normal myoepithelial cells have a tumour suppressor phenotype but they are altered in DCIS and ultimately lost with transition to invasive cancer. A consistent change in DCIS is up-regulation of the integrin αvβ6 in myoepithelial cells. Preliminary observations identified a correlation between myopeithelial αvβ6 and an increased peri-ductal inflammatory infiltrate. The hypothesis of this study is that the altered myoepithelial phenotype influences the peri-ductal inflammatory environment, which in turn mediates a pro-apoptotic effect on myoepithelial cells contributing to their loss. To investigate this, the inflammatory infiltrate was characterised in a series of DCIS tissue in relation to αvβ6 status. This demonstrated significantly higher levels of CD4+ve and FOXP3+ve T cells around αvβ6+ve DCIS ducts compared to αvβ6-ve ducts (P=<0.01), suggesting an increase in Treg cells. In-vivo, Matrigel plugs containing injected into the flanks of female C57/Blk6 normal mice generated influx of higher levels of CD4+ve cells (p=0.005) and FOXP3+ T cells (p=0.007) in the presence of αvβ6+ve myoepithelial cells compared to αvβ6-ve cells, supporting the findings in human tissue samples. Since Treg cells produce TRAIL that can induce apoptosis, we investigated the influence of αvβ6 on myoepithelial cells on the levels of TRAIL in T cells and the hypothesis that αvβ6-positive myoepithelial ells may be more susceptible to TRAIL-induced apoptosis, leading to loss of the myoepithelial barrier. Firstly, levels of TRAIL in Jurkat and primary T cell populations co-cultured with β4 (ii) or β6 myoepithelial cells were measured. This demonstrated a higher level of TRAIL in primary T cells co-cultured β6 myoepithelial cells compared to those co-cultured with β4 myoepithelial cells. β6+ve and β6-ve myoepithelial cells were exposed to TRAIL, and this demonstrated that TRAIL enhanced apoptosis, measured by cleaved PARP, in β6+ve cells. Furthermore, these cells showed loss of the anti-apoptotic protein Galectin-7, and knockdown of Galectin-7 in normal β6-ve myoepithelial cells rendered them more susceptible to TRAIL-induced apoptosis. In DCIS tissues, an inverse relationship between αvβ6 and Galectin-7 in myoepithelial cells was demonstrated, and Cytokine Array analysis showed that αvβ6+ve myoepithelial cells express higher levels of IL-16, which has a role in Treg cell recruitment. Taken together these results suggest that expression of αvβ6 by myoepithelial cells in DCIS generates a tumour-promoter peri-ductal inflammatory infiltrate through altered cytokine release, is associated with reduced galectin-7 expression and enhances myoepithelial cell apoptosis in response to TRAIL. This provides a potential mechanism by which myoepithelial cells may be lost during evolution of DCIS and so contribute to progression to invasive disease.