Histopathological evaluation and risk factors related to the development of pouchitis in patients with ileal pouches for ulcerative colitis

OBJECTIVE: Many changes in mucosal morphology are observed following ileal pouch construction, including colonic metaplasia and dysplasia. Additionally, one rare but potential complication is the development of adenocarcinoma of the reservoir. The aim of this study was to evaluate the most frequently observed histopathological changes in ileal pouches and to correlate these changes with potential risk factors for complications. METHODS: A total of 41 patients were enrolled in the study and divided into the following three groups: a non-pouchitis group (group 1) (n = 20; 8 males; mean age: 47.5 years) demonstrating optimal outcome; a pouchitis without antibiotics group (group 2) (n = 14; 4 males; mean age: 47 years), containing individuals with pouchitis who did not receive treatment with antibiotics; and a pouchitis plus antibiotics group (group 3) (n = 7; 3 males; mean age: 41 years), containing those patients with pouchitis who were administered antibiotics. Ileal pouch endoscopy was performed, and tissue biopsy samples were collected for histopathological analysis. RESULTS: Colonic metaplasia was found in 15 (36.6%) of the 41 patients evaluated; of these, five (25%) were from group 1, eight (57.1%) were from group 2, and two (28.6%) were from group 3. However, no correlation was established between the presence of metaplasia and pouchitis (p = 0.17). and no differences in mucosal atrophy or the degree of chronic or acute inflammation were observed between groups 1, 2, and 3 (p > 0.45). Moreover, no dysplasia or neoplastic changes were detected. However, the degree of mucosal atrophy correlated well with the time of postoperative follow-up (p = 0.05). CONCLUSIONS: The degree of mucosal atrophy, the presence of colonic metaplasia, and the degree of acute or chronic inflammation do not appear to constitute risk factors for the development of pouchitis. Moreover, we observed that longer postoperative follow-up times were associated with greater degrees of mucosal atrophy.

Human papillomavirus genotypes distribution in 175 invasive cervical cancer cases from Brazil

Abstract Background Invasive cervical cancer is the second most common malignant tumor affecting Brazilian women. Knowledge on Human Papillomavirus (HPV) genotypes in invasive cervical cancer cases is crucial to guide the introduction and further evaluate the impact of new preventive strategies based on HPV. We aimed to provide updated comprehensive data about the HPV types’ distribution in patients with invasive cervical cancer. Methods Fresh tumor tissue samples of histologically confirmed invasive cervical cancer were collected from 175 women attending two cancer reference hospitals from São Paulo State: ICESP and Hospital de Câncer de Barretos. HPV detection and genotyping were performed by the Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton,USA). Results 170 out of 172 valid samples (99%) were HPV DNA positive. The most frequent types were HPV16 (77.6%), HPV18 (12.3%), HPV31 (8.8%), HPV33 (7.1%) and HPV35 (5.9%). Most infections (75%) were caused by individual HPV types. Women with adenocarcinoma were not younger than those with squamous cell carcinoma, as well, as women infected with HPV33 were older than those infected by other HPV types. Some differences between results obtained in the two hospitals were observed: higher overall prevalence of HPV16, absence of single infection by HPV31 and HPV45 was verified in HC-Barretos in comparison to ICESP patients. Conclusions To our knowledge, this is one of the largest studies made with fresh tumor tissues of invasive cervical cancer cases in Brazil. This study depicted a distinct HPV genotype distribution between two centers that may reflect the local epidemiology of HPV transmission among these populations. Due to the impact of these findings on cervical cancer preventive strategies, extension of this investigation to routine screening populations is warranted.

Placentation in the paca (Agouti paca L)

Background: The paca is a South American rodent with potential as a commercial food animal. We examined paca placenta as part of a wider effort to understand the reproductive biology of this species. Methods: Thirteen specimens between midgestation and term of pregnancy were studied by light and transmission electron microscopy. Results: The placenta is divided into several lobes separated by interlobular trophoblast. Maternal arterial channels and fetal veins are found at the centre of each lobe. In the labyrinth, maternal blood flows through trophoblast-lined lacunae in close proximity to the fetal capillaries. The interhaemal barrier is of the haemomonochorial type with a single layer of syncytiotrophoblast. Caveolae occur in the apical membrane of the syncytiotrophoblast and recesses in the basal membrane, but there is no evidence of transtrophoblastic channels. The interlobular areas consist of cords of syncytiotrophoblast defining maternal blood channels that drain the labyrinth. Yolk sac endoderm covers much of the fetal surface of the placenta. The subplacenta comprises cytotrophoblast and syncytiotrophoblast. There are dilated intercellular spaces between the cytotrophoblasts and lacunae lined by syncytiotrophoblast. In the junctional zone between subplacenta and decidua, there are nests of multinucleated giant cells with vacuolated cytoplasm. The entire placenta rests on a pedicle of maternal tissue. An inverted yolk sac placenta is also present. The presence of small vesicles and tubules in the apical membrane of the yolk sac endoderm and larger vesicles in the supranuclear region suggest that the yolk sac placenta participates in maternal-fetal transfer of protein. Conclusion: The paca placenta closely resembles that of other hystricomorph rodents. The lobulated structure allows for a larger exchange area and the development of precocial young

Long noncoding intronic RNAs are differentially expressed in primary and metastatic pancreatic cancer

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is known by its aggressiveness and lack of effective therapeutic options. Thus, improvement in current knowledge of molecular changes associated with pancreatic cancer is urgently needed to explore novel venues of diagnostics and treatment of this dismal disease. While there is mounting evidence that long noncoding RNAs (lncRNAs) transcribed from intronic and intergenic regions of the human genome may play different roles in the regulation of gene expression in normal and cancer cells, their expression pattern and biological relevance in pancreatic cancer is currently unknown. In the present work we investigated the relative abundance of a collection of lncRNAs in patients' pancreatic tissue samples aiming at identifying gene expression profiles correlated to pancreatic cancer and metastasis. Methods Custom 3,355-element spotted cDNA microarray interrogating protein-coding genes and putative lncRNA were used to obtain expression profiles from 38 clinical samples of tumor and non-tumor pancreatic tissues. Bioinformatics analyses were performed to characterize structure and conservation of lncRNAs expressed in pancreatic tissues, as well as to identify expression signatures correlated to tissue histology. Strand-specific reverse transcription followed by PCR and qRT-PCR were employed to determine strandedness of lncRNAs and to validate microarray results, respectively. Results We show that subsets of intronic/intergenic lncRNAs are expressed across tumor and non-tumor pancreatic tissue samples. Enrichment of promoter-associated chromatin marks and over-representation of conserved DNA elements and stable secondary structure predictions suggest that these transcripts are generated from independent transcriptional units and that at least a fraction is under evolutionary selection, and thus potentially functional. Statistically significant expression signatures comprising protein-coding mRNAs and lncRNAs that correlate to PDAC or to pancreatic cancer metastasis were identified. Interestingly, loci harboring intronic lncRNAs differentially expressed in PDAC metastases were enriched in genes associated to the MAPK pathway. Orientation-specific RT-PCR documented that intronic transcripts are expressed in sense, antisense or both orientations relative to protein-coding mRNAs. Differential expression of a subset of intronic lncRNAs (PPP3CB, MAP3K14 and DAPK1 loci) in metastatic samples was confirmed by Real-Time PCR. Conclusion Our findings reveal sets of intronic lncRNAs expressed in pancreatic tissues whose abundance is correlated to PDAC or metastasis, thus pointing to the potential relevance of this class of transcripts in biological processes related to malignant transformation and metastasis in pancreatic cancer.

Morphometric analysis of the placenta in the New World mouse Necromys lasiurus (Rodentia, Cricetidae): a comparison of placental development in cricetids and murids

Background: Stereology is an established method to extrapolate three-dimensional quantities from two-dimensional images. It was applied to placentation in the mouse, but not yet for other rodents. Herein, we provide the first study on quantitative placental development in a sigmodontine rodent species with relatively similar gestational time. Placental structure was also compared to the mouse, in order to evaluate similarities and differences in developmental patterns at the end of gestation. Methods: Fetal and placental tissues of Necromys lasiurus were collected and weighed at 3 different stages of gestation (early, mid and late gestation) for placental stereology. The total and relative volumes of placenta and of its main layers were investigated. Volume fractions of labyrinth components were quantified by the One Stop method in 31 placentae collected from different individuals, using the Mercator® software. Data generated at the end of gestation from N. lasiurus placentae were compared to those of Mus musculus domesticus obtained at the same stage. Results: A significant increase in the total absolute volumes of the placenta and its main layers occurred from early to mid-gestation, followed by a reduction near term, with the labyrinth layer becoming the most prominent area. Moreover, at the end of gestation, the total volume of the mouse placenta was significantly increased compared to that of N. lasiurus although the proportions of the labyrinth layer and junctional zones were similar. Analysis of the volume fractions of the components in the labyrinth indicated a significant increase in fetal vessels and sinusoidal giant cells, a decrease in labyrinthine trophoblast whereas the proportion of maternal blood space remained stable in the course of gestation. On the other hand, in the mouse, volume fractions of fetal vessels and sinusoidal giant cells decreased whereas the volume fraction of labyrinthine trophoblast increased compared to N. lasiurus placenta. Conclusions: Placental development differed between N. lasiurus and M. musculus domesticus. In particular, the low placental efficiency in N. lasiurus seemed to induce morphological optimization of fetomaternal exchanges. In conclusion, despite similar structural aspects of placentation in these species, the quantitative dynamics showed important differences.

Avaliação da expressão do gene MGMT nos tecidos normal e neoplásico de doentes com câncer colorretal

OBJETIVO: Avaliar a expressão tecidual do gene de reparo MGMT comparando a mucosa cólica normal e neoplásica em doentes com câncer colorretal. MÉTODOS: Foram estudados 44 portadores de adenocarcinoma colorretal confirmado por estudo histopatológico. Foram excluídos doentes suspeitos de pertencerem a famílias com câncer colorretal hereditário (HNPCC e PAF) e os portadores de câncer do reto médio e inferior submetidos a tratamento quimioradioterápico neoadjuvante. A expressão do gene MGMT foi avaliada pela técnica da reação de polimerase em cadeia em tempo real (RT-PCR). A comparação dos resultados encontrados para expressão do gene MGMT entre tecidos normais e neoplásicos foi feita pelo teste t de Student pareado, adotando-se nível de significância de 5% (p <0,05). RESULTADOS: A expressão tecidual do gene MGMT em todos os doentes foi menor no tecido neoplásico quando comparada a do tecido normal (p=0,002). CONCLUSÃO: O gene de reparo MGMT encontra-se menos expresso no tecido neoplásico quando comparados aos tecidos normais em portadores de CCR esporádico.

Resultados da gastrectomia D2 para o câncer gástrico: dissecção da cadeia linfática ou ressecção linfonodal múltipla?

RACIONAL: A literatura oriental é notável por apresentar taxas de sobrevida para o tratamento cirúrgico do adenocarcinoma gástrico superiores àquelas apresentadas nos países ocidentais. OBJETIVO: Analisar o resultado a longo prazo após a gastrectomia D2 por câncer gástrico. MÉTODOS: Duzentos e setenta e quatro pacientes foram submetidos à gastrectomia com dissecção linfonodal D2 como tratamento exclusivo. Os critérios de inclusão foram: 1) remoção dos linfonodos de acordo com dissecção linfática padronizada Japonesa, 2) operação potencialmente curativa descrita no prontuário como dissecção D2 ou mais linfonodos; 3) invasão tumoral da parede gástrica restrita ao órgão (T1-T3); 4) ausência de metástases à distância (N0-N2/M0); 5) mínimo de cinco anos de acompanhamento. Dados clinicopatológicos incluíam sexo, idade, localização do tumor, classificação de Borrmann do tumor macroscópico, o tipo de gastrectomia, as taxas de mortalidade, tipo histológico, classificação e estadiamento TNM de acordo com a UICC TNM 1997. RESULTADOS: Gastrectomia total foi realizada em 77 casos (28,1%) e subtotal em 197 (71,9%). O tumor foi localizado no terço superior em 28 casos (10,2%), no terço médio em 53 (19,3%), e no terço inferior em 182 (66,5%). Borrmann foi atribuído cinco casos (1,8%) como BI, 34 (12,4%) BII, 230 (84,0%) BIII e 16 (5,9%) BIV. Os tumores foram histologicamente classificados como Laurén tipo intestinal em 119 casos (43,4%) e como o tipo difuso em 155 (56,6%). De acordo com a UICC TNM foram câncer gástrico precoce (T1) foi diagnosticada em 68 casos (24,8%); 51 (18,6%) T2 e 155 (56,6%) T3. Nenhum envolvimento linfonodal (N0) foi observado em 129 casos (47,1%), enquanto 100 (36,5%) eram N1 (1-6 linfonodos) e 45 (16,4%) N2 (7-15 gânglios linfáticos). O número médio de linfonodos dissecados foi de 35. A sobrevida em cinco anos para os estádios de I a III B foi de 70,4%. CONCLUSÃO: Cirurgiões digestivos devem ser estimulados a realizarem gastrectomias D2 para não deixar de lado o único tratamento para adenocarcinoma gástrico que provou ser eficiente os dias atuais. Deve ser enfatizado que a padronização da dissecção linfática de acordo com a localização do tumor é mais importante do que apenas o número de gânglios removidos

MYC, FBXW7 and TP53 copy number variation and expression in Gastric Cancer

Abstract Background MYC deregulation is a common event in gastric carcinogenesis, usually as a consequence of gene amplification, chromosomal translocations, or posttranslational mechanisms. FBXW7 is a p53-controlled tumor-suppressor that plays a role in the regulation of cell cycle exit and reentry via MYC degradation. Methods We evaluated MYC, FBXW7, and TP53 copy number, mRNA levels, and protein expression in gastric cancer and paired non-neoplastic specimens from 33 patients and also in gastric adenocarcinoma cell lines. We also determined the invasion potential of the gastric cancer cell lines. Results MYC amplification was observed in 51.5% of gastric tumor samples. Deletion of one copy of FBXW7 and TP53 was observed in 45.5% and 21.2% of gastric tumors, respectively. MYC mRNA expression was significantly higher in tumors than in non-neoplastic samples. FBXW7 and TP53 mRNA expression was markedly lower in tumors than in paired non-neoplastic specimens. Moreover, deregulated MYC and FBXW7 mRNA expression was associated with the presence of lymph node metastasis and tumor stage III-IV. Additionally, MYC immunostaining was more frequently observed in intestinal-type than diffuse-type gastric cancers and was associated with MYC mRNA expression. In vitro studies showed that increased MYC and reduced FBXW7 expression is associated with a more invasive phenotype in gastric cancer cell lines. This result encouraged us to investigate the activity of the gelatinases MMP-2 and MMP-9 in both cell lines. Both gelatinases are synthesized predominantly by stromal cells rather than cancer cells, and it has been proposed that both contribute to cancer progression. We observed a significant increase in MMP-9 activity in ACP02 compared with ACP03 cells. These results confirmed that ACP02 cells have greater invasion capability than ACP03 cells. Conclusion In conclusion, FBXW7 and MYC mRNA may play a role in aggressive biologic behavior of gastric cancer cells and may be a useful indicator of poor prognosis. Furthermore, MYC is a candidate target for new therapies against gastric cancer.

Carcinoma de células escamosas em glândula mamária de gata doméstica

Background: In the feline species, 80% to 93% of neoplasias in the mammary gland are malignant, being the majority carcinomas. Among them, there is the mammary squamous cell carcinoma, which amounts to a very rare neoplasm in the domestic cat, with considerable potential for malignancy. This study aimed to report a case of squamous cell mammary carcinoma in the feline species. Case: A female cat, mixed breed, ten years old, presented history of skin lesion. The cat had been spayed two years before, but with previous administration of contraceptives. At the physical examination, it was observed ulcer between the caudal abdominal mammary glands. The occurrence of skin or mammary neoplasia was conceived. The following complementary tests were requested: complete blood count, serum biochemical profi le (renal and hepatic), chest radiographs, abdominal ultrasound, and incisional biopsy of the ulcerated region periphery, followed by classic histopathology. The lesion histopathology was compatible with squamous cell carcinoma of the mammary gland. Due to such a diagnosis, bilateral mastectomy was recommended. The material obtained during the surgical procedure was sent for anatomopathological analysis. Microscopically, surgical margins infi ltration and a regional lymph node were verifi ed. The owner was advised of the need for complementary therapies and medical monitoring of the cat. However, there was no return. It is noteworthy that the animal’s physical and laboratory examinations showed no neoplasia in other regions, being the squamous cell carcinoma of the mammary gland considered primary. Discussion: The malignant mammary neoplasia genesis in feline species, in general, seems to be related to steroid hormones. The ovariectomized females are less likely to develop the disease when compared to intact cats, but there is no protective effect of surgery on those spayed after two years of age regarding the appearance of the neoplasia. Thus, at the time the reported patient was ovariectomized, this effect no longer occurred. The synthetic progestins regularly used to prevent estrus increase by three times the risk of breast carcinomas onset. In humans, there is no clear defi nition of the etiology and pathogenesis of mammary squamous cell carcinoma. However, it has been suggested its association with extreme forms of squamous metaplasia present in pre-existing mammary adenocarcinoma, besides cysts, chronic infl ammations, abscesses and mammary gland adenofi bromas. In a hypothetical way, this etiology could also be related to the feline mammary carcinoma, although, for the case at issue, the exogenous and endogenous hormonal infl uence should not be excluded. It has been reported that mammary squamous cell carcinomas in cats are classifi ed in grades II and III (ie, moderately and poorly differentiated, respectively). Thus, they are considered tumors with more unfavorable prognosis. However, the monitoring of the clinical course, in order to evaluate possible recurrence of the neoplasia and metastases to distant sites, was not possible as the animal under discussion did not return. The squamous cell carcinoma is the most common skin tumor in feline species, despite the primary location in the mammary gland. It is, therefore, important to differentiate squamous cell carcinoma originated in the breast from histological types derived from skin. The description of this special and rare feline mammary carcinoma is important due to its particular characteristics and potential for malignancy.

MYC, FBXW7 and TP53 copy number variation and expression in gastric cancer

BACKGROUND: MYC deregulation is a common event in gastric carcinogenesis, usually as a consequence of gene amplification, chromosomal translocations, or posttranslational mechanisms. FBXW7 is a p53-controlled tumor-suppressor that plays a role in the regulation of cell cycle exit and reentry via MYC degradation. METHODS: We evaluated MYC, FBXW7, and TP53 copy number, mRNA levels, and protein expression in gastric cancer and paired non-neoplastic specimens from 33 patients and also in gastric adenocarcinoma cell lines. We also determined the invasion potential of the gastric cancer cell lines. RESULTS: MYC amplification was observed in 51.5% of gastric tumor samples. Deletion of one copy of FBXW7 and TP53 was observed in 45.5% and 21.2% of gastric tumors, respectively. MYC mRNA expression was significantly higher in tumors than in non-neoplastic samples. FBXW7 and TP53 mRNA expression was markedly lower in tumors than in paired non-neoplastic specimens. Moreover, deregulated MYC and FBXW7 mRNA expression was associated with the presence of lymph node metastasis and tumor stage III-IV. Additionally, MYC immunostaining was more frequently observed in intestinal-type than diffuse-type gastric cancers and was associated with MYC mRNA expression. In vitro studies showed that increased MYC and reduced FBXW7 expression is associated with a more invasive phenotype in gastric cancer cell lines. This result encouraged us to investigate the activity of the gelatinases MMP-2 and MMP-9 in both cell lines. Both gelatinases are synthesized predominantly by stromal cells rather than cancer cells, and it has been proposed that both contribute to cancer progression. We observed a significant increase in MMP-9 activity in ACP02 compared with ACP03 cells. These results confirmed that ACP02 cells have greater invasion capability than ACP03 cells. CONCLUSION: In conclusion, FBXW7 and MYC mRNA may play a role in aggressive biologic behavior of gastric cancer cells and may be a useful indicator of poor prognosis. Furthermore, MYC is a candidate target for new therapies against gastric cancer.

Metachronic malignant transformation of small bowel and rectal endometriosis in the same patient

[ES] Background: Malignant transformation of intestinal endometriosis is a rare event with an unknown rate of incidence. Metachronous progression of endometriosis to adenocarcinoma from two distant intestinal foci happening in the same patient has not been previously reported. Case presentation: We describe a case of metachronic transformation of ileal and rectal endometriosis into an adenocarcinoma occurring in a 45-year-old female without macroscopic pelvic involvement of her endometriosis. First, a right colectomy was performed due to intestinal obstruction by an ileal mass. Pathological examination revealed an ileal endometrioid adenocarcinoma and contiguous microscopic endometriotic foci. Twenty months later, a rectal mass was discovered. An endoscopic biopsy revealed an adenocarcinoma. En bloc anterior rectum resection, hysterectomy and bilateral salpingectomy were performed. A second endometrioid adenocarcinoma arising from a focus of endometriosis within the wall of the rectum was diagnosed. Conclusion: Intestinal endometriosis should be considered a premalignant condition in premenopausal women.

Biochemistry in healthy and neoplastic human tissues: metabolic alteration revealed by HR-MAS nuclear magnetic resonance spectroscopy

This thesis is focused on the metabolomic study of human cancer tissues by ex vivo High Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. This new technique allows for the acquisition of spectra directly on intact tissues (biopsy or surgery), and it has become very important for integrated metabonomics studies. The objective is to identify metabolites that can be used as markers for the discrimination of the different types of cancer, for the grading, and for the assessment of the evolution of the tumour. Furthermore, an attempt to recognize metabolites, that although involved in the metabolism of tumoral tissues in low concentration, can be important modulators of neoplastic proliferation, was performed. In addition, NMR data was integrated with statistical techniques in order to obtain semi-quantitative information about the metabolite markers. In the case of gliomas, the NMR study was correlated with gene expression of neoplastic tissues. Chapter 1 begins with a general description of a new “omics” study, the metabolomics. The study of metabolism can contribute significantly to biomedical research and, ultimately, to clinical medical practice. This rapidly developing discipline involves the study of the metabolome: the total repertoire of small molecules present in cells, tissues, organs, and biological fluids. Metabolomic approaches are becoming increasingly popular in disease diagnosis and will play an important role on improving our understanding of cancer mechanism. Chapter 2 addresses in more detail the basis of NMR Spectroscopy, presenting the new HR-MAS NMR tool, that is gaining importance in the examination of tumour tissues, and in the assessment of tumour grade. Some advanced chemometric methods were used in an attempt to enhance the interpretation and quantitative information of the HR-MAS NMR data are and presented in chapter 3. Chemometric methods seem to have a high potential in the study of human diseases, as it permits the extraction of new and relevant information from spectroscopic data, allowing a better interpretation of the results. Chapter 4 reports results obtained from HR-MAS NMR analyses performed on different brain tumours: medulloblastoma, meningioms and gliomas. The medulloblastoma study is a case report of primitive neuroectodermal tumor (PNET) localised in the cerebellar region by Magnetic Resonance Imaging (MRI) in a 3-year-old child. In vivo single voxel 1H MRS shows high specificity in detecting the main metabolic alterations in the primitive cerebellar lesion; which consist of very high amounts of the choline-containing compounds and of very low levels of creatine derivatives and N-acetylaspartate. Ex vivo HR-MAS NMR, performed at 9.4 Tesla on the neoplastic specimen collected during surgery, allows the unambiguous identification of several metabolites giving a more in-depth evaluation of the metabolic pattern of the lesion. The ex vivo HR-MAS NMR spectra show higher detail than that obtained in vivo. In addition, the spectroscopic data appear to correlate with some morphological features of the medulloblastoma. The present study shows that ex vivo HR-MAS 1H NMR is able to strongly improve the clinical possibility of in vivo MRS and can be used in conjunction with in vivo spectroscopy for clinical purposes. Three histological subtypes of meningiomas (meningothelial, fibrous and oncocytic) were analysed both by in vivo and ex vivo MRS experiments. The ex vivo HR-MAS investigations are very helpful for the assignment of the in vivo resonances of human meningiomas and for the validation of the quantification procedure of in vivo MR spectra. By using one- and two dimensional experiments, several metabolites in different histological subtypes of meningiomas, were identified. The spectroscopic data confirmed the presence of the typical metabolites of these benign neoplasms and, at the same time, that meningomas with different morphological characteristics have different metabolic profiles, particularly regarding macromolecules and lipids. The profile of total choline metabolites (tCho) and the expression of the Kennedy pathway genes in biopsies of human gliomas were also investigated using HR-MAS NMR, and microfluidic genomic cards. 1H HR-MAS spectra, allowed the resolution and relative quantification by LCModel of the resonances from choline (Cho), phosphorylcholine (PC) and glycerolphorylcholine (GPC), the three main components of the combined tCho peak observed in gliomas by in vivo 1H MRS spectroscopy. All glioma biopsies depicted an increase in tCho as calculated from the addition of Cho, PC and GPC HR-MAS resonances. However, the increase was constantly derived from augmented GPC in low grade NMR gliomas or increased PC content in the high grade gliomas, respectively. This circumstance allowed the unambiguous discrimination of high and low grade gliomas by 1H HR-MAS, which could not be achieved by calculating the tCho/Cr ratio commonly used by in vivo 1H MR spectroscopy. The expression of the genes involved in choline metabolism was investigated in the same biopsies. The present findings offer a convenient procedure to classify accurately glioma grade using 1H HR-MAS, providing in addition the genetic background for the alterations of choline metabolism observed in high and low gliomas grade. Chapter 5 reports the study on human gastrointestinal tract (stomach and colon) neoplasms. The human healthy gastric mucosa, and the characteristics of the biochemical profile of human gastric adenocarcinoma in comparison with that of healthy gastric mucosa were analyzed using ex vivo HR-MAS NMR. Healthy human mucosa is mainly characterized by the presence of small metabolites (more than 50 identified) and macromolecules. The adenocarcinoma spectra were dominated by the presence of signals due to triglycerides, that are usually very low in healthy gastric mucosa. The use of spin-echo experiments enable us to detect some metabolites in the unhealthy tissues and to determine their variation with respect to the healthy ones. Then, the ex vivo HR-MAS NMR analysis was applied to human gastric tissue, to obtain information on the molecular steps involved in the gastric carcinogenesis. A microscopic investigation was also carried out in order to identify and locate the lipids in the cellular and extra-cellular environments. Correlation of the morphological changes detected by transmission (TEM) and scanning (SEM) electron microscopy, with the metabolic profile of gastric mucosa in healthy, gastric atrophy autoimmune diseases (AAG), Helicobacter pylori-related gastritis and adenocarcinoma subjects, were obtained. These ultrastructural studies of AAG and gastric adenocarcinoma revealed lipid intra- and extra-cellularly accumulation associated with a severe prenecrotic hypoxia and mitochondrial degeneration. A deep insight into the metabolic profile of human healthy and neoplastic colon tissues was gained using ex vivo HR-MAS NMR spectroscopy in combination with multivariate methods: Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). The NMR spectra of healthy tissues highlight different metabolic profiles with respect to those of neoplastic and microscopically normal colon specimens (these last obtained at least 15 cm far from the adenocarcinoma). Furthermore, metabolic variations are detected not only for neoplastic tissues with different histological diagnosis, but also for those classified identical by histological analysis. These findings suggest that the same subclass of colon carcinoma is characterized, at a certain degree, by metabolic heterogeneity. The statistical multivariate approach applied to the NMR data is crucial in order to find metabolic markers of the neoplastic state of colon tissues, and to correctly classify the samples. Significant different levels of choline containing compounds, taurine and myoinositol, were observed. Chapter 6 deals with the metabolic profile of normal and tumoral renal human tissues obtained by ex vivo HR-MAS NMR. The spectra of human normal cortex and medulla show the presence of differently distributed osmolytes as markers of physiological renal condition. The marked decrease or disappearance of these metabolites and the high lipid content (triglycerides and cholesteryl esters) is typical of clear cell renal carcinoma (RCC), while papillary RCC is characterized by the absence of lipids and very high amounts of taurine. This research is a contribution to the biochemical classification of renal neoplastic pathologies, especially for RCCs, which can be evaluated by in vivo MRS for clinical purposes. Moreover, these data help to gain a better knowledge of the molecular processes envolved in the onset of renal carcinogenesis.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.

Identificazione di bersagli terapeutici e realizzazione di tecnologie innovative in oncologia ortopedica

Controlled delivery of anticancer drugs through osteotropic nanoparticles (NP) is a novel approach for the adjuvant therapy of osteolytic bone metastases. Doxorubicin (DXR) is widely used in chemotherapy, although its activity is restricted by dose-dependent cardiotoxicity and marrow toxicity. However, its efficacy can be improved when specific targeting at the tumor site is obtained. The aim of this study was to obtain osteotropic biodegradable NP by nanoprecipitation of a copolymer between poly(D,L-lactide-co-glycolide) (PLGA) and an osteotropic bisphosphonate, sodium alendronate (ALE). NP were subsequently characterised for their chemical-physical properties, biocompatibility, and the ability to inhibit osteoclast-mediated bone resorption, and then loaded with DXR. The effectiveness of NP-loaded DXR was investigated through in vitro and in vivo experiments, and compared to that of free DXR. For the in vitro analysis, six human cell lines were used as a representative panel of bone tumors, including breast and renal adenocarcinoma, osteosarcoma and neuroblastoma. The in vitro uptake and the inhibition of tumor cell proliferation were verified. To analyse the in vivo activity of NP-loaded DXR, osteolytic bone metastases were induced through the intratibial inoculation in BALB/c-nu/nu mice of a human breast cancer cell line, followed by the intraperitoneal administration of the free or NP-loaded DXR. In vitro, aAll of the cell lines were able to uptake both free and NP-loaded drug, and their proliferation was inhibited up to 80% after incubation either with free or NP-loaded DXR. In addition, in vivo experiments showed that NP-loaded DXR were also able to reduce the incidence of bone metastases, not only in comparison with untreated mice, but also with free DXR-treated mice. In conclusion, this research demonstrated an improvement in the therapeutic effect of the antineoplastic drug DXR, when loaded to bone-targeted NP conjugated with ALE. Osteotropic PLGA-ALE NP are suitable to be loaded with DXR and offer as a valuable tool for a tissue specific treatment of skeletal metastases.

Tumori primitivi multipli del polmone. Profilo clinico e biologico in pazienti affetti da neoplasie polmonari multiple. Analisi di elementi clinici e marcatori biologici come possibili fattori di differenziazione dei tumori polmonari multipli

Background: MPLC represents a diagnostic challenge. Topic of the discussion is how to distinguish these patients as a metastatic or a multifocal disease. While in case of the different histology there are less doubt on the opposite in case of same histology is mandatory to investigate on other clinical features to rule out this question. Matherials and Methods: A retrospective review identified all patients treated surgically for a presumed diagnosis of SPLC. Pre-operative staging was obtained with Total CT scan and fluoro-deoxy positron emission tomography and mediastinoscopy. Patients with nodes interest or extra-thoracic location were excluded from this study. Epidermal growth factor receptor (EGFR) expression with complete immunohistochemical analisis was evaluated. Survival was estimated using Kaplan-Meyer method, and clinical features were estimated using a long-rank test or Cox proportional hazards model for categorical and continuous variable, respectively. Results: According to American College Chest Physician, 18 patients underwent to surgical resection for a diagnosis of MPLC. Of these, 8 patients had 3 or more nodules while 10 patients had less than 3 nodules. Pathologic examination demonstrated that 13/18(70%) of patients with multiple histological types was Adenocarcinoma, 2/18(10%) Squamous carcinoma, 2/18(10%) large cell carcinoma and 1/18(5%) Adenosquamosu carcinoma. Expression of EGFR has been evaluated in all nodules: in 7 patients of 18 (38%) the percentage of expression of each nodule resulted different. Conclusions: MPLC represent a multifocal disease where interactions of clinical informations with biological studies reinforce the diagnosis. EGFR could contribute to differentiate the nodules. However, further researches are necessary to validate this hypothesis.