Panayiotis Loukopoulos

Genome‐wide array‐based comparative genomic hybridization analysis of pancreatic adenocarcinoma: Identification of genetic indicators that predict patient outcome

We analyzed the subchromosomal numerical aberrations of 44 surgically resected pancreatic adenocarcinomas by array‐based comparative genomic hybridization. The aberration profile ranged widely between cases, suggesting the presence of multiple or complementary mechanisms of evolution in pancreatic cancer, and was associated with lymph node metastasis and venous or serosal invasion. A large number of small loci, previously uncharacterized in pancreatic cancer, showed non‐random loss or gain. Frequent losses at 1p36, 4p16, 7q36, 9q34, 11p15, 11q13, 14q32‐33, 16p13, 17p11‐13, 17q11‐25, 18q21‐tel, 19p13, 21q22 and 22q11‐12, and gains at 1q25, 2p16, 2q21‐37, 3q25, 5p14, 5q11‐13, 7q21, 7p22, 8p22, 8q21‐23, 10q21, 12p13, 13q22, 15q13‐22 and 18q11 were identified. Sixteen loci were amplified recurrently. We identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Gain of LUNX, HCK, E2F1 and DNMT3b at 20q11, loss of p73 at 1p36 and gain of PPM1D at 17q23 independently predicted patient outcome. Expression profiling of amplified genes identified Smurf1 and TRRAP at 7q22.1, BCAS1 at 20q13.2‐3, and VCL at 10q22.1 as potential novel oncogenes. Our results contribute to a complete description of genomic structural aberrations and the identification of potential therapeutic targets and genetic indicators that predict patient outcome in pancreatic adenocarcinoma. (Cancer Sci 2007; 98: 392–400)

Orthotopic Transplantation Models of Pancreatic Adenocarcinoma Derived From Cell Lines and Primary Tumors and Displaying Varying Metastatic Activity

Objective: To establish a series of clinically relevant orthotopic transplantation models of human pancreatic adenocarcinoma from both cell lines and primary tumors under uniform experimental conditions. Methods: Ten pancreatic cancer cell lines and 12 primary tumors were orthotopically transplanted in SCID mice. The cell lines and xenografts were characterized for K-ras, BRAF, p53, p16, and DPC4 aberrations employing direct sequencing, immunohistochemistry, and Western blotting. Results: All xenografts showed high intrapancreatic tumorigenicity and extensive local tumor growth, and each showed a unique behavioral and genetic profile. Tumor characteristics were retained during serial passaging. The cell line–derived xenografts represented the entire expected range of histologic differentiation. Although the overall metastatic rate was moderate to high, the metastatic pattern varied; 4 cell lines showed a high metastatic rate to the liver. The primary tumor–derived xenografts retained their similarity to the corresponding original donor tumors with regard to histologic presentation and biologic behavior. K-ras, p53, p16, and DPC4 aberrations were revealed in 80%, 70%, 50%, and 40% of cell lines and 100%, 33%, 75%, and 58% of primary tumor derived xenografts, respectively. No BRAF mutations were present. The metastatic behavior of the xenografts was significantly associated with the degree of histologic differentiation, number of genes altered, and p53 status. Conclusions: The new models reflected the wide range of patho-biological features and genetic alterations that characterize human pancreatic cancer and may be used collectively or selectively as a markedly improved in vivo tool for preclinical and molecular studies of pancreatic cancer.

Matrix metalloproteinase-2 and-9 involvement in canine tumors

Matrix metalloproteinases (MMPs) are a family of enzymes implicated in the degradation and remodeling of extracellular matrix and in vascularization. They are also involved in pathologic processes such as tumor invasion and metastasis in experimental cancer models and in human malignancies. We used gelatin zymography and immunohistochemistry to determine whether MMP-2 and MMP-9 are present in canine tumors and normal tissues and whether MMP production correlates with clinicopathologic parameters of prognostic importance. High levels of pro-MMP-9, pro-MMP-2, and active MMP-2 were detected in most canine tumors. Significantly higher MMP levels were measured in canine tumors than in nontumors, malignancies had higher MMP levels than benign tumors, and sarcomas had higher active MMP-2 than carcinomas. Cartilaginous tumors produced higher MMP levels than did nonsarcomatous malignancies, benign tumors, and normal tissues, and significantly greater MMP-2 than osteosarcomas and fibrosarcomas. Pro-MMP-9 production correlated with the histologic grade of osteosarcomas. The 62-kd form of active MMP-2 was detected only in high-grade, p53-positive, metastatic malignancies. Zymography proved to be a sensitive and quantitative technique for the assessment of MMP presence but has the limitation of requiring fresh tissue; immunohistochemistry is qualitative and comparatively insensitive but could be of value in archival studies. MMP presence was shown in a range of canine tumors, and their link to tumor type and grade was demonstrated for the first time. This study will allow a substantially improved evaluation of veterinary cancer patients and provides baseline information necessary for the design of clinical trials targeting MMPs.

Clinical and Pathologic Relevance of p53 Index in Canine Osseous Tumors

The clinicopathologic value of the immunohistochemical (IHC) expression of p53 protein was evaluated in 167 canine osseous tumors. p53 staining frequency and intensity in tumor cells was expressed as a p53 index. p53 index was significantly higher in osteosarcomas than in other sarcomas, chondrosarcoma, multilobular tumor of bone, and tumors initially misdiagnosed as osteosarcomas as well as in appendicular versus axial and in distal versus proximal osteosarcomas. A strong correlation is demonstrated between the p53 index and a range of clinicopathologic parameters in osteosarcoma, including the tumor site, histologic grade and score, mitotic index, degree of tumor necrosis, and pleomorphism. Chondroblastic osteosarcomas had significantly higher and telangiectatic osteosarcomas significantly lower p53 index than did osteosarcomas belonging to other histopathologic subtypes, a fact that tends to reinforce the perception of these osteosarcomas as distinct clinicopathologic entities. Entire males had higher p53 index than did neutered males. p53 index was higher in Rottweilers than in Great Danes and Terriers, confirming breed susceptibilities to osteosarcoma. p53 index showed no association with age, primary or secondary site status, or the presence of metastases or other tumor types. Biopsy samples had a higher p53 index than did postmortem samples, either because of differences in sample processing or the possibility that p53 overexpression is more evident at the earlier stages of osteosarcoma pathogenesis, presumably represented by the biopsy material. IHC examination for p53 and the derived index has the potential to be used as an additional diagnostic tool and prognostic indicator for osseous tumors.

Isolation of Mycobacterium avium subspecies paratuberculosis from Ugandan cattle and strain differentiation using optimised DNA typing techniques

BackgroundThe occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda.ResultsTwenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2).ConclusionsThere is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.

Effect of locally injected autologous platelet-rich plasma on second intention wound healing of acute full-thickness skin defects in dogs

OBJECTIVES Second intention wound healing may be impaired by wound and host factors and thus more advanced therapies are required for a fast and satisfactory outcome. Platelet-rich plasma (PRP), rich in growth factors and cytokines essential for tissue repair, could improve wound healing. The purpose of this experimental study was to evaluate the effect of locally injected autologous PRP on second intention healing of acute full-thickness skin defects in dogs. METHODS Three 2 x 2 cm full-thickness skin defects were created bilaterally on the dorsolateral area of the trunk of six Beagle dogs. The wounds of one randomly selected side received PRP treatment, whereas the contralateral wounds were left untreated (controls). Wound healing was evaluated by planimetry, laser-Doppler flowmetry measurements of tissue perfusion, and histologically. RESULTS The rate of wound healing did not differ significantly between the two groups. Tissue perfusion was significantly higher in the PRP-treated group (p = 0.008) compared to controls on day 10. Histological evaluation revealed a trend towards greater collagen production and a significantly better collagen orientation (p = 0.019) in PRP-treated wounds on day 20. CLINICAL SIGNIFICANCE Locally injected autologous PRP does not accelerate the healing process, but increases tissue perfusion and may promote the formation of organized collagen bundles in acute full-thickness skin defects in dogs.

Uterine leiomyosarcoma and pyometra in a dog

A seven-year-old entire female, mixed-breed dog, weighing 19 kg, was presented with a 2-day history of abdominal distension, reduced appetite and general dullness. Abdominal palpation revealed a large mass. Radiography showed a large mass occupying the left mid-abdominal area and convoluted loops of tubular fluid opacity occupying the right mid-abdominal area. Ultrasonography revealed a large heterogeneous mass with an anechoic area and some hyperechoic foci, indicative of calcification, in the mid-abdominal area. Furthermore, hypoechoic areas were found in the middle and caudal abdominal area and were presumed to be the fluid-filled uterine horns. At laparotomy, a 10·5×14·5-cm firm mass was found in the uterine body, while the uterine horns were filled with a thick red-brownish exudate; ovariohysterectomy was subsequently performed. A diagnosis of uterine leiomyosarcoma associated with pyometra was established by histopathology and immunohistochemistry.

Foreign body-associated intestinal pyogranuloma resulting in intestinal obstruction in four dogs.

Intestinal obstruction resulting from an intramural foreign body-associated pyogranuloma was diagnosed in four dogs. Vomiting and weight loss were the main clinical signs. On physical examination, a mass in the abdomen was detected in three dogs. Abdominal radiography demonstrated the presence of soft tissue opacity in three of the dogs and gas-filled dilated intestinal loops in all four dogs. Abdominal ultrasonography showed hyperkinetic fluid-filled dilated intestinal loops and a hypoechoic small intestinal mass in all the dogs. Exploratory coeliotomy confirmed the presence of a jejunal mass, which was removed by resection and anastomosis in all the dogs. In one of the dogs a linear foreign body was also found cranial to the mass and was removed through a separate enterotomy incision. The lesions were diagnosed as foreign body-associated intestinal pyogranulomas on histological examination. Three dogs recovered without complications, but the fourth showed signs of septic peritonitis four days after surgery and was euthanased at the owner’s request. The other three dogs remained disease-free 12 to 42 months after surgery.

Cytological versus histopathological diagnosis in canine osteosarcoma

SIR, – The role of cytology in the diagnosis of primary bone tumours is less established and perhaps not comprehensively studied compared with the diagnosis of soft tissue tumours. We evaluated the diagnostic accuracy of the cytological examination of canine osteosarcoma, comparing the cytological

Prevalence and spectrum of Johne’s disease lesions in cattle slaughtered at two abattoirs in Kampala, Uganda

This study was conducted to determine the prevalence and characteristics of Johne’s disease (JD) lesions in Ugandan cattle slaughtered at two of the main abattoirs in Kampala. Ileocaecal junction and the associated lymph nodes of 1,022 cattle were examined for gross and microscopic lesions, followed by Ziehl Neelsen staining of the tissues bearing lesions. Confirmation of Mycobacterium avium subsp. paratuberculosis infection was done in some of the tissues using culture and IS900 PCR. The lesions were then described, characterised and tabulated. Characteristic Johne’s disease granulomas were found in 4.7% of the samples examined, derived from Zebu, Ankole longhorn, Friesian breeds of cattle and their crosses. Lesions were found both in the lymph nodes and ileocaecal junction mucosa. The lesions tended to be more severe in the lymph node than in the mucosa. There were also some unique and atypical lesions found in association with Johne’s disease granulomas. The diagnostic values of various gross lesions and criteria of lesion classifications and diagnosis are revisited and discussed based on the findings of this study. The prevalence of Johne’s disease lesions among slaughtered cattle in Kampala’s two abattoirs indicates that the disease is well established in the cattle population in the country. The diverse manifestations in lesions of JD need to be considered when making histological diagnosis in tissues where the disease is suspected.