Floryne O. Buishand

Evaluation of clinico-pathological criteria and the Ki67 index as prognostic indicators in canine insulinoma

The establishment of reliable prognostic biomarkers for canine insulinoma are warranted to facilitate optimal patient management. Using univariate and multivariate analyses, the present study evaluated the prognostic power of several clinical and histopathological criteria, as well as the Ki67 index, in 26 dogs with insulinoma. On univariate analysis, stromal fibrosis within these tumours was found to be significantly predictive for survival, while tumour size, the Ki67 index, and TNM (Tumour, Node, Metastasis) stage were significant in the prognosis of both disease-free interval and survival time. On multivariate analysis, tumour size retained predictive power for disease-free intervals, while the Ki67 index proved prognostically significant for both the disease-free interval and overall survival time. This study demonstrates that, in addition to known factors such as tumour size and stage, Ki67 can act as a biomarker of insulinoma that can be used to predict clinical outcome.

Expression of insulin-like growth factor-1 by canine insulinomas and their metastases

The long-term prognosis after surgical resection of canine insulinoma is poor. Signs of hypoglycaemia often recur soon after surgery because tumour tissue has only been resected partially and/or functional (micro-)metastases were present. Using quantitative real-time PCR, the expression of 16 target genes was compared between primary canine insulinomas and their corresponding metastases. There was significantly higher expression of genes encoding for growth hormone (GH) and insulin-like growth factor-1 (IGF-1) in metastases compared to their primary tumours. Immunohistochemical examination of proteins of the GH-IGF-1 axis revealed expression of GH, IGF-1 and GH receptor (GHR) in both primary insulinomas and metastases. Immunohistochemical staining for IGF-1 was significantly higher in metastases compared to primary tumours.

Gene expression profiling of primary canine insulinomas and their metastases

The gene expression profile of 10 primary canine insulinomas was compared with that of their accompanying metastases using microarray analysis and quantitative real time-PCR. Analysis of microarray data revealed 84 genes that were differentially expressed between primary insulinomas and their metastases, along with 243 genes differentially expressed between a low-metastatic and a high-metastatic subset of primary insulinomas. The genes differently expressed between primary insulinomas and their metastases clustered together in nine signalling pathways. Comparing the low-metastatic to the high-metastatic subset of primary insulinomas, 26 pathways appeared to be significantly influenced. The acinar enzymes pancreatic lipase (PNLIP) and chymotrypsinogen B1 (CTRB1) were amongst the most down-regulated genes in the malignant group of primary insulinomas and in metastases. Immunofluorescence demonstrated co-localisation of insulin and PNLIP in tumour cells. Different subsets of canine insulinomas can be identified on the basis of their gene expression profile. Canine insulinomas appear to contain amphicrine cells, which exhibit both endocrine and exocrine cell features.

Use of a bipolar vessel‐sealing device in resection of canine insulinoma

OBJECTIVES To describe partial pancreatectomy using a bipolar vessel-sealing device (BVSD) and compare this novel technique to the conventional suture-fracture (SF) method for canine insulinoma. METHODS Pre-, intra- and postoperative data of eight dogs with insulinoma, which underwent resection using the BVSD (LigaSure V), were prospectively collected and compared with those of eight randomly selected case-matched patients that underwent resection using the conventional SF technique. RESULTS Mean surgical time was significantly (P=0·022) shorter in the BVSD (107 ± 9 minutes) than in the SF (135 ± 22 minutes) group. The BVSD technique was negatively associated with surgical time and duration of the hospitalisation period. Neither technique caused intraoperative complications, such as bleeding, collateral damage to adjacent tissues or problems with sealing or suturing the pancreatic tissue. Three dogs in the SF group and none in the BVSD group developed postoperative clinical signs associated with pancreatitis. CLINICAL SIGNIFICANCE BVSD is a safe and viable alternative to conventional methods of pancreatectomy for canine insulinoma. It provides the possibility to remove insulinomas in the pancreatic limbs and corpus with relative ease. BVSD pancreatectomy in dogs with insulinoma significantly decreases operative and hospitalisation times and is not associated with more clinical complications than SF pancreatectomy.

Evaluation of prognostic indicators using validated canine insulinoma tissue microarrays.

Tissue microarray (TMA) technology allows analysis of multiple tumour samples simultaneously on a single slide. The aim of the present study was to develop and assess a TMA containing 32 primary canine insulinomas and 13 insulinoma metastases. The results of histopathological and immunohistochemical analyses of triplicate core biopsies were compared with those of individual tissue sections using weighted κ statistics. Inter-observer agreement of TMA immunohistochemistry scores were assessed for chromogranin A (CgA), insulin, growth hormone (GH), growth hormone receptor (GHR) and Ki67 index, as well as the prognostic utility of clinicopathological, histopathological and immunohistochemical criteria. There was substantial agreement of scores for histopathological parameters (κ = 0.64-0.70) and a substantial to near-perfect agreement for homogenous immunohistochemical parameters (κ = 0.69-1.00). Except for GH, which demonstrated heterogeneous staining, there was good to excellent inter-observer agreement for all other immunohistochemical staining scores (intra-class correlation coefficients: 0.70-1.00). On univariate analysis, the presence of nuclear atypia was significantly predictive of disease-free intervals (DFIs) for canine insulinoma, while tumour size, TNM stage, necrosis and Ki67 index were significant in terms of prognosis, with respect to both DFI and survival time. On multivariate analysis, tumour size and Ki67 index retained predictive power for survival time, as did tumour size for DFI. This study confirms the applicability of TMA technology for evaluation of canine insulinoma.

Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas

The long-term prognosis after surgical resection of malignant insulinoma (INS) is poor. Novel adjuvant therapies, specifically targeting cancer stem cells (CSCs), are warranted. Therefore, the goal of this study was to characterize and target putative INS CSCs. Using fluorescence-activated cell sorting, human INS cell line CM and pancreatic carcinoid cell line BON1 were screened for the presence of stem cell-associated markers. CD90, CD166, and GD2 were identified as potential CSC markers. Only CD90(+) INS cells had an increased tumor-initiating potential in athymic nude mice. Anti-CD90 monoclonal antibodies decreased the viability and metastatic potential of injected cells in a zebrafish embryo INS xenograft model. Primary INS stained positive for CD90 by immunohistochemistry, however also intratumoral fibroblasts and vascular endothelium showed positive staining. The results of this study suggest that anti-CD90 monoclonals form a potential novel adjuvant therapeutic modality by targeting either INS cells directly, or by targeting the INS microenvironment.

Notch pathway inhibition targets chemoresistant insulinoma cancer stem cells

Insulinomas (INS) are the most common neuroendocrine pancreatic tumours in humans and dogs. The long-term prognosis for malignant INS is still poor due to a low success rate of the current treatment modalities, particularly chemotherapy. A better understanding of the molecular processes underlying the development and progression of INS is required to develop novel targeted therapies. Cancer stem cells (CSCs) are thought to be critical for the engraftment and chemoresistance of many tumours, including INS. This study was aimed to characterise and target INS CSCs in order to develop novel targeted therapies. Highly invasive and tumourigenic human and canine INS CSC-like cells were successfully isolated. These cells expressed stem cell markers (OCT4, SOX9, SOX2, CD133 and CD34), exhibited greater resistance to 5-fluorouracil (5-FU) and demonstrated a more invasive and tumourigenic phenotype in vivo compared to bulk INS cells. Here, we demonstrated that Notch-signalling-related genes (NOTCH2 and HES1) were overexpressed in INS CSC-like cells. Protein analysis showed an active NOTCH2-HES1 signalling in INS cell lines, especially in cells resistant to 5-FU. Inhibition of the Notch pathway, using a gamma secretase inhibitor (GSI), enhanced the sensitivity of INS CSC-like cells to 5-FU. When used in combination GSI and 5-FU, the clonogenicity in vitro and the tumourigenicity in vivo of INS CSC-like cells were significantly reduced. These findings suggested that the combined strategy of Notch signalling inhibition and 5-FU synergistically attenuated enriched INS CSC populations, providing a rationale for future therapeutic exploitation.

Utility of contrast-enhanced computed tomography in the evaluation of canine insulinoma location

Abstract Objectives: To determine 1) the sensitivity of contrast-enhanced CT (CECT) for detection of primary canine insulinomas and metastases 2) the sensitivity of CECT to locate canine insulinomas within the pancreas and 3) the CECT attenuation pattern of canine insulinomas and post-contrast phase in which insulinomas have the best visibility. Methods: A retrospective review was performed of the medical records of 27 canine insulinoma patients. Simultaneous occurrence of blood glucose < 3.5 mmol/L (reference interval: 4.2–5.8 mmol/L) and plasma insulin > 10 mIU/L (reference interval: 1.4–24.5 mIU/L) were considered diagnostic for insulinoma. The dogs had a mean age of 9.0 ± 1.7 (SD) years and comprised 11 males and 17 females. Results: Using CECT-scans, 26/27 insulinomas were successfully detected. However, CECT-scans predicted the correct location of insulinomas within the pancreas in only 14/27 dogs. In 9/13 inaccurately located insulinoma cases, the location error was major. There was no significant difference between triple, double and single-phase CECT-scans with location accuracies of 54%, 50% and 50%, respectively. Also, there was no specific post-contrast phase in which insulinomas could be visualised best. Detection of lymph node metastases with CECT-scans had a sensitivity of 67% (10/15 lymph node metastases). Detection of liver metastases had a sensitivity of 75% (6/8 liver metastases). This study highlights that major location errors mainly occurred if single- or double-phase CECT-scans were used (6/9 cases). Conclusion: It is suggested that triple-phase CECT-scans have superior outcome over single- or double-phase CECT-scans in pre-operative imaging of canine insulinomas.

Trichostatin A preferentially reverses the upregulation of gene expression levels induced by gain of chromosome 7 in colorectal cancer cell lines

Epithelial cancers are defined by a tumor‐specific distribution of chromosomal aneuploidies that are maintained when cells metastasize and are conserved in cell lines derived from primary tumors. Correlations between genomic copy number and gene expression have been observed for different tumors including, colorectal (CRC), breast, and pancreatic cancer. These ploidy‐driven transcriptional deregulations are characterized by low‐level expression changes of most genes on the affected chromosomes. The emergence of these aberrations at an early stage of tumorigenesis and the strong selection for the maintenance of these aneuploidies suggest that aneuploidy‐dependent transcriptional deregulations might contribute to cellular transformation and maintenance of the malignant phenotype. The histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) has anticancer effects and is well known to lead to large‐scale gene‐expression changes. Here we assessed if TSA could disrupt the aneuploidy‐driven gene expression in the aneuploid colon cancer cell line SW480 and the artificially generated aneuploid cell line DLD‐1 + 7. We found that TSA increases transcriptional activity throughout the genome, yet inhibits aneuploidy‐induced gene‐expression changes on chromosome 7. Among the TSA affected genes on chromosome 7, we identified potential CRC oncogenes. These experiments represent the first attempt to explain how histone acetylation affects aneuploidy‐driven gene‐expression changes.

Identification of Novel Targets for Lung Cancer Therapy Using an Induced Pluripotent Stem Cell Model

RATIONALE Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. OBJECTIVES To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. METHODS Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. RESULTS The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. CONCLUSIONS Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy.