Tumor Tissue Fragments Research Articles

Real-Time GFP Intravital Imaging of the Differences in Cellular and Angiogenic Behavior of Subcutaneous and Orthotopic Nude-Mouse Models of Human PC-3 Prostate Cancer.

There are two major types of mouse xenograft models of cancer: subcutaneous implantation and orthotopic implantation. Subcutaneous transplant models are widely used with both cancer cell lines and human-tumor specimens. Recently, subcutaneous models of patient tumors, termed patient-derived xenographs (PDX) have become highly popular and have acquired such names as "Avatar" and "Xenopatients." However, such s.c. models rarely metastasize and are therefore not patient-like. In contrast, orthotopic models have the capability to metastasize. If intact fragments of tumor tissue are implanted by surgical orthotopic implantation (SOI), the metastatic potential can match that of the donor patient. The present study images in real time, using green fluorescent protein (GFP) expression, the very different tumor behavior at the orthotopic and subcutaneous sites of human prostate cancer PC-3 in athymic nude mice. By day-2 after tumor implantation, the orthotopic tumor is already highly vascularized and the cancer cells have begun to migrate out of the tumor. In contrast, the subcutaneous tumor only begins to be vascularized by day-3 and cells do not migrate from the tumor. Angiogenesis is much more extensive in the orthotopic tumor throughout the 2-week observation period. The orthotopic PC-3-GFP tumor progresses very rapidly and distinct metastasis have appeared in lymph nodes by day-3 which rapidly appear in many areas of the abdominal cavity including portal lymph nodes by day-7. At day-14, no invasion or metastasis was observed with the s.c. tumor even when the animal was extensively explored. These results explain why orthotopic tumors mimimc clinical metastatic tumors in nude mice and why subcutaneous tumors do not. J. Cell. Biochem. 117: 2546-2551, 2016. © 2016 Wiley Periodicals, Inc.

A multi-site cutting device implements efficiently the divide-and-conquer strategy in tumor sampling.

We recently showed that in order to detect intra-tumor heterogeneity a Divide-and-Conquer (DAC) strategy of tumor sampling outperforms current routine protocols. This paper is a continuation of this work, but here we focus on DAC implementation in the Pathology Laboratory. In particular, we describe a new simple method that makes use of a cutting grid device and is applied to clear cell renal cell carcinomas for DAC implementation. This method assures a thorough sampling of large surgical specimens, facilitates the demonstration of intratumor heterogeneity, and saves time to pathologists in the daily practice. The method involves the following steps: 1. Thin slicing of the tumor (by hand or machine), 2. Application of a cutting grid to the slices ( e.g., a French fry cutter), resulting in multiple tissue cubes with fixed position within the slice, 3. Selection of tissue cubes for analysis, and finally, 4. Inclusion of selected cubes into a cassette for histological processing (with about eight tissue fragments within each cassette). Thus, using our approach in a 10 cm in-diameter-tumor we generate 80 tumor tissue fragments placed in 10 cassettes and, notably, in a tenth of time. Eighty samples obtained across all the regions of the tumor will assure a much higher performance in detecting intratumor heterogeneity, as proved recently with synthetic data.

Abstract 5002: Anti-tumor activity of a novel anti-human PD-1 antibody BGB-A317 in mouse models

Abstract Background: Programmed death receptor-1 (PD-1) is a co-inhibitory immune checkpoint molecule. The engagement of PD-1 by its ligands negatively regulates T cell function and induces immune tolerance in both normal and pathological conditions, especially in cancer. BGB-A317 is a novel humanized IgG4 anti-PD-1 antibody under clinical development. The anti-tumor activity of BGB-A317 was evaluated in mouse models and reported here. Material and methods: NOD/SCID mice transplanted with human peripheral blood monocyte (PBL-SCID models) were employed to examine the anti-tumor activity and pharmacokinetics of BGB-A317. In these models, human PBMCs (4-5×106) from healthy donors were co-injected with human tumor cells (A431, 2.5×106) or primary tumor tissue fragments derived from cancer patients (BCCO-028 and BCLU-054, 3×3×3 mm3) subcutaneously in the right flank of the mice. BCCO-028 is human colon cancer and BCLU-054 is human non-small cell lung cancer (NSCLC). The mice were then treated with BGB-A317 or PBS intra-peritoneally once a week from the day of tumor implantation. The tumor infiltrated lymphocytes and PD-L1 expression were examined by immunohistochemistry staining and flow cytometry. Results: In A431 model, 3 doses of BGB-A317, 1, 3, 10mg/kg, were examined. All three doses of BGB-A317 induced significant tumor growth inhibition (TGI) on day 29 (78%, 89%, and 89%, respectively) (p < 0.001 vs. vehicle treatment). Both AUC0-168h and Cmax of BGB-A317 after single dose (day 1, 1st dose) or at steady state (day 29, 5th dose) increased proportionally from 1 to 10 mg/kg. There was about 2-fold accumulation in drug levels (AUC0-168h and Cmax) at the steady state compared to those after single dose. Increased lymphocyte infiltration and PD-L1 expression were observed in BGB-A317 treated groups. In BCCO-028 model, BGB-A317 at 10 mg/kg QW induced significant tumor growth inhibition, with 83% TGI (p < 0.05 vs. vehicle treatment) on day 36. In BCLU-054 model, BGB-A317 at 10 mg/kg QW induced significant tumor growth inhibition, with 45% of TGI (p < 0.05 vs. vehicle treatment) on day 23. BGB-A317 treatment had no significant impact on animal body weight throughout all of the studies. Conclusions: BGB-A317 demonstrated significant anti-tumor activity in 3 human cancer xenograft PBL-SCID mouse models, including A431 human epidermoid carcinoma, BCCO-028 colon cancer, and BCLU-054 NSCLC models. Citation Format: Xiaomin Song, Zhiyu Tang, Tong Zhang, Mingming Guo, Wenfeng Gong, Yong Liu, Ningning Zhang, Yilu Zhang, Yanjuan Zhang, Jie Ma, Jing Song, Kang Li, Lai Wang. Anti-tumor activity of a novel anti-human PD-1 antibody BGB-A317 in mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5002.

A multi-site cutting device implements efficiently the divide-and-conquer strategy in tumor sampling

We recently showed that in order to detect intra-tumor heterogeneity a Divide-and-Conquer (DAC) strategy of tumor sampling outperforms current routine protocols. This paper is a continuation of this work, but here we focus on DAC implementation in the Pathology Laboratory. In particular, we describe a new simple method that makes use of a cutting grid device and is applied to clear cell renal cell carcinomas for DAC implementation. This method assures a thorough sampling of large surgical specimens, facilitates the demonstration of intratumor heterogeneity, and saves time to pathologists in the daily practice. The method involves the following steps: 1. Thin slicing of the tumor (by hand or machine), 2. Application of a cutting grid to the slices ( e.g., a French fry cutter), resulting in multiple tissue cubes with fixed position within the slice, 3. Selection of tissue cubes for analysis, and finally, 4. Inclusion of selected cubes into a cassette for histological processing (with about eight tissue fragments within each cassette). Thus, using our approach in a 10 cm in-diameter-tumor we generate 80 tumor tissue fragments placed in 10 cassettes and, notably, in a tenth of time. Eighty samples obtained across all the regions of the tumor will assure a much higher performance in detecting intratumor heterogeneity, as proved recently with synthetic data.

A multi-site cutting device implements efficiently the divide-and-conquer strategy in tumor sampling

We recently showed that in order to detect intra-tumor heterogeneity a Divide-and-Conquer (DAC) strategy of tumor sampling outperforms current routine protocols. This paper is a continuation of this work, but here we focus on DAC implementation in the Pathology Laboratory. In particular, we describe a new simple method that makes use of a cutting grid device and is applied to clear cell renal cell carcinomas for DAC implementation. This method assures a thorough sampling of large surgical specimens, facilitates the demonstration of intratumor heterogeneity, and saves time to pathologists in the daily practice. The method involves the following steps: 1. Thin slicing of the tumor (by hand or machine), 2. Application of a cutting grid to the slices (e.g., a French fry cutter), resulting in multiple tissue cubes with fixed position within the slice, 3. Selection of tissue cubes for analysis, and finally, 4. Inclusion of selected cubes into a cassette for histological processing (with about eight tissue fragments within each cassette). Thus, using our approach in a 10 cm in-diameter-tumor we generate 80 tumor tissue fragments placed in 10 cassettes and, notably, in a tenth of time. Eighty samples obtained across all the regions of the tumor will assure a much higher performance in detecting intratumor heterogeneity, as proved recently with synthetic data.

Aberrant expression of serotonin receptors in an aldosterone- and cortisol-producing adenoma

Aberrant expression of serotonin receptors in an aldosterone- and cortisol-producing adenoma Introduction Aberrant expression of serotonin receptors has been described to be involved in the pathophysiology of both aldosterone-producing and cortisol-producing adrenal adenomas. Case report A 46-year-old woman was referred for evaluation of severe hypertension associated with hypokalaemia. Primary aldosteronism with concurrent subclinical Cushing’s syndrome was diagnosed. A CT-scan identified a lesion of 4 cm in the right adrenal gland and a second lesion of 1 cm in the left adrenal gland, both displaying benign imaging phenotype. Following dexamethasone suppression, we explored the potential aberrant expression of serotonin receptors in the adrenal cortex by intravenous administration of metoclopramide, a serotonin type 4 receptor (5-HT4-R) agonist. This led to abnormal cortisol increase and physiological response of aldosterone in peripheral blood. Adrenal venous sampling was then performed in basal conditions and after metoclopramide stimulation. An increase in cortisol level was observed in the left adrenal vein after stimulation. 131I-19-Iodocholesterol scintigraphy showed intense radiotracer uptake in the right adrenal mass and weak uptake in the contralateral mass. Right laparoscopic adrenalectomy was performed. Histological examination revealed an adrenocortical adenoma. In vitro, cultured adrenocortical cells derived from the tumoural tissue exhibited aldosterone and cortisol co-secretion. Administration of 5-HT or metoclopramide to the cells induced a dose-dependent increase in cortisol production. These effects were inhibited by concomitant administration of the 5-HT4-R antagonist GR113808. Incubation of tumour tissue fragments with 5-HT induced a significant increase in aldosterone production which was abrogated in the presence of GR113808. These data were suggestive of aberrant expression of 5HT4-R in the tumoural tissue. Conclusion We report a rare case of an aldosterone- and cortisol-co-producing adenoma in a patient with severe hypertension and bilateral adrenal masses exhibiting an abnormal plasma cortisol response to metoclopramide. In vitro studies revealed enhanced sensitivity of the tumour tissue to 5-HT indicative of illicit expression of 5-HT4 receptors.

Direct preparation protocol to obtain mitotic chromosomes from canine mammary tumors.

Currently, mammary neoplasms in female canines are a serious problem in veterinary clinics. In addition, the canine species is an excellent disease model for human oncology because of the biological and genetic similarities between the species. Cytogenetics has allowed further study of the characterization of neoplasms in canines. We hypothesized that the use of a direct preparation protocol for mitotic chromosome analysis would provide a simple and low cost protocol for use in all laboratories. The objective of this method is to display in a few hours of dividing cells just like the time of collection since cell division in tissue can be obtained. Ten female canines with the spontaneous occurrence of mammary neoplasia were used to test a pioneering direct preparation protocol to obtain mitotic chromosomes. The excised breast tumor tissue fragments were subjected to the protocol consisting of treatment with colchicine, treatment with hypotonic solution, and fixation. Mitotic chromosomes were absent in cell suspensions of only two samples among the 10 materials analyzed, based on the analysis of five blades for each preparation obtained. So, the cell suspension obtained allowed for the observation of eight tissue samples viable for cytogenetic analysis, five of which had excellent numbers of mitotic chromosomes. However, the technique was unsuccessful in producing high-quality cell suspensions because of inadequate condensation and scattering of chromosomes. While adjustments to methodological procedures are needed, this protocol represents a low cost and simplified method to study the cytogenetics of canine tumors.

Establishment of human patient-derived endometrial cancer xenografts in NOD scid gamma mice for the study of invasion and metastasis.

ObjectiveMost endometrial cancers are detected early and have a good prognosis, while some endometrial cancers are highly invasive, metastasize early, and respond suboptimally to therapy. Currently, appropriate model systems to study the aggressive nature of these tumors are lacking. The objective of this study was to establish a mouse xenograft model of endometrial tumors derived from patients in order to study the biological aggressive characteristics that underlie invasion and metastasis.MethodsEndometrial tumor tissue fragments (1.5 mm×1.5 mm) from patients undergoing surgery, were transplanted under the renal capsule of NOD scid gamma mice. After 6–8 weeks, tumors were excised and serially transplanted into additional mice for propagation. Immunohistochemical analysis of the tumors was done for various tumor markers.ResultsFour cases of different subtypes of endometrial cancer were grown and propagated in mice. Three of the four tumor cases invaded into the kidneys and to adjacent organs. While all tumors exhibited minimal to no staining for estrogen receptor α, progesterone receptor staining was observed for tumor grafts. In addition, levels and localization of E-cadherin, cytokeratin and vimentin varied depending on subtype. Finally, all tumor xenografts stained positively for urokinase plasminogen activator while 3 tumor xenografts, which showed invasive characteristics, stained positively for urokinase plasminogen activator receptor.ConclusionEndometrial tumors transplanted under the renal capsule exhibit growth, invasion and local spread. These tumors can be propagated and used to study aggressive endometrial cancer.

Glioma Tissue Obtained by Modern Ultrasonic Aspiration with a Simple Sterile Suction Trap for Primary Cell Culture and Pathological Evaluation

Objectives: Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Methods: Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Results: Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Conclusion: Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

Abstract C271: FAK inhibitor defactinib (VS-6063) enhances the efficacy of paclitaxel and preferentially targets ovarian cancer stem cells.

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors and plays important roles in tumorigenesis. Amplification and overexpression of FAK have been observed in aggressive human cancers including ovarian and breast cancers. Defactinib is a potent, selective, and orally active FAK inhibitor with demonstrated tolerability and preliminary clinical activity as a single agent in a Phase 1 clinical trial. In an ongoing Phase 1/1b clinical trial (NCT01778803), we have demonstrated that defactinib can be combined with paclitaxel and have observed preliminary signs of clinical activity. We report here preclinical data supporting the clinical testing of defactinib in combination with paclitaxel in patients with ovarian cancer. In human ovarian cancer cell lines TOV-21G and OV-7, defactinib was found to enhance the efficacy of paclitaxel. Combination Index analyses demonstrated synergistic inhibition of tumor cell proliferation/survival with combination of defactinib and paclitaxel. We find that FAK inhibitors preferentially target breast and ovarian cancer stem cells (CSCs) relative to bulk tumor cells as evidenced by a diminished proportion of CSCs in multiple orthogonal CSC assays. In direct contrast, the standard-of-care cytotoxic agents, paclitaxel and carboplatin, increased the percentage of CSCs, indicating that these agents do not effectively target CSCs. Importantly, combination of FAK inhibitors with cytotoxic agents attenuated the enrichment of CSCs induced by the cytotoxic agents. We subsequently determined if defactinib reduces CSCs in tumor specimens from ovarian cancer patients who were previously treated with taxane and platinum. Ex vivo treatment of ovarian tumor tissue fragments with defactinib reduced the percentage of CSCs as assessed by CD24hi/CD117hi CSC markers and the Aldefluor assay. To assess drug effects on tumor-initiating capability, the ‘gold standard’ for CSC activity, TOV-21G cells were pretreated in vitro with the FAK inhibitor, paclitaxel or the combination of both agents and 1,000 of the resulting cells were injected into immunodeficient mice. The FAK inhibitor alone or in combination with paclitaxel prevented tumor initiation, while paclitaxel alone did not reduce tumor initiation, suggesting that treatment with a FAK inhibitor could effectively eliminate tumor initiating CSCs. In summary, our data indicate that defactinib preferentially targets cancer stem cells and enhances the activity of paclitaxel in preclinical models of ovarian cancer. These results provide additional support for the current clinical development of defactinib in combination with paclitaxel for the treatment of ovarian cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C271. Citation Format: Christian M. Vidal, Vihren N. Kolev, Quentin G. Wright, Winnie F. Tam, David T. Weaver, Mahesh V. Padval, Qunli Xu, Jonathan A. Pachter. FAK inhibitor defactinib (VS-6063) enhances the efficacy of paclitaxel and preferentially targets ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C271.

Abstract 5123: In vitro culture for characterization of circulating GFP-expressing tumor cells in orthotopic lung cancer models .

Abstract In the present study circulating tumor cells (CTCs) were isolated from H460-RFP orthotopic human lung cancer model in mice. In vitro culture of CTCs has enabled us comparison of the chemosensitivity of tumor cells from primary H460 culture and of CTCs solated from blood grown in vitro. Cell line H460-RFP human lung cancer cells were grown to 70-80% confluence. Primary tumor were grown after subcutaneous injection of cultured cells. Fragments of tumor tissue (1 mm × 1 mm) were implanted into the lungs of nude mice. At 14 days, the mice were sacrificied and blood was taken by cardiac puncture for CTC-analysis. The blood (0.5 ml), obtained by cardiac puncture, was put into an EDTA tube (BD) and the CTC cells were enriched by immunomagnetic separation targeted to eptihelial antigens. The enriched fraction or whole blood was put into culture. Cell growth was then monitored by fluorescence microscopy. The isolation and culture of the fluorescent CTC will enable their further characterization. Citation Format: Katarína Kološtová, Marián Liberko, Eva Hroncová, Robert M. Hoffman, Vladimír Bobek. In vitro culture for characterization of circulating GFP-expressing tumor cells in orthotopic lung cancer models . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5123. doi:10.1158/1538-7445.AM2013-5123

Abstract 3853: Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma.

Abstract Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that includes two entities ALK+ and ALK- ALCL based on chromosomal translocations of the anaplastic lymphoma kinase (ALK). ALK- ALCL have very poor clinical outcome with conventional chemotherapy. Lack of representative cell lines and/or models has compromised the development of successful therapy. Toward this end, we report the generation and characterization of a group of novel ALCL tumor grafts. We implanted subcutaneously (s.c.) tumor tissue fragments, fresh or viable cryopreserved, from 11 cases of primary systemic ALCL (3 ALK+ and 8 ALK-) in six- to eight-week old severe immunocombined immunodeficiency NOD Cg-Prkdcscid/Il2rgtm1wjl/SzJ (NSG) mice. A single leukaemic ALCL sample was injected i.v. or s.c. Six cases led to successful lymphoma growth within 6-12 weeks as subcutaneous solid spheroid masses constituted by a uniform population of large pleomorphic cells CD30-positive. All tumor grafts were re-implanted in recipient animals, in same cases up to 21 consecutive implants with an analogous time growth rate. Two cases, in the same way of the original human neoplasia, were early associated with concomitant enlargement of multiple organs. All tumor graft lines eventually showed disseminate disease with involvement of distant lymph nodes and of spleen (with homig in peri-arteriolar spaces), liver (initially observed within the sinusoidal spaces) and kidney. The morphological and immunohistochemical (IHC) evaluation (expression of CD30, cytotoxic proteins and ALK) of the serially propagated ALCL tumor grafts demonstrated a high concordance between primary and derived animal tumors which showed highly stable profile along different passages from the early generation (T1) until the final ones (up to T16). The clonal relationship with primary and corresponding tumor grafts was confirmed by gene TCR rearrangement analyses and by ALK FISH studies. In addition we interrogated the model using gene expression profiling and single nucleotide polymorphism which established a close relationship among primary and derived tumors. Massive parallel sequencing on mRNA transcripts defines the presence of two novel chimeras involving NF1-MAZ and TRAF1-ALK1, which confirmes the evidence of ALK signaling and pinpoint a role of RAS in ALK- ALCL. The tumor grafts displayed, also, therapeutic responses with conventional therapy (CHOP) which reflected that seen in the donor patients: initial partial responses followed invariably by clinical relapses. To overcome this refractory we assayed the sensitivity of two tumorgraft lines, generated from ALK+ ALCL, to a new selective ALK inhibitor (CEP28122) that led a rapid and stable response. This study demonstrated the key role of these libraries of tumor grafts, especially in developing and testing new therapeutic regimens in refractory ALCL patients. Citation Format: Fabrizio Tabbo’, Antonella Barreca, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Francesco Abate, Michela Boi, Maria Todaro, Cristina Abele, Domenico Novero, Alberto Zamo’, Marco Chilosi, Maurilio Ponzoni, Mangeng Cheng, Bruce Ruggeri, Pierpaolo Piccaluga, Stefano Pileri, Enrico Tiacci, Brunangelo Falini, Lenny Shultz, Roberto Piva, Enzo Medico, Francesco Bertoni, Raul Rabadan, Giorgio Inghirami. Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3853. doi:10.1158/1538-7445.AM2013-3853

Orthotopic xenografts of RCC retain histological, immunophenotypic and genetic features of tumours in patients.

Renal cell carcinoma (RCC) is an aggressive malignancy with limited responsiveness to existing treatments. In vivo models of human cancer, including RCC, are critical for developing more effective therapies. Unfortunately, current RCC models do not accurately represent relevant properties of the human disease. The goal of this study was to develop clinically relevant animal models of RCC for preclinical investigations. We transplanted intact human tumour tissue fragments orthotopically in immunodeficient mice. The xenografts were validated by comparing the morphological, phenotypic and genetic characteristics of the kidney tumour tissues before and after implantation. Twenty kidney tumours were transplanted into mice. Successful tumour growth was detected in 19 cases (95%). The histopathological and immunophenotypic features of the xenografts and those of the original tumours largely overlapped in all cases. Evaluation of genetic alterations in a subset of 10 cases demonstrated that the grafts largely retained the genetic features of the pre-implantation RCC tissues. Indeed, primary tumours and corresponding grafts displayed identical VHL mutations. Moreover, an identical pattern of DNA copy amplification or loss was observed in 6/10 cases (60%). In summary, orthotopic engrafting of RCC tissue fragments can be successfully used to generate animal models that closely resemble RCC in patients. These models will be invaluable for in vivo preclinical drug testing and for deeper understanding of kidney carcinogenesis. The raw data of the SNP array analysis has been submitted to the GEO database (Accession No. GSE29062).

The activity of selected glycosidases in salivary gland tumors.

The monitoring of the patients after salivary gland tumors surgery is an important clinical issue. Still imperfect diagnostic procedures also remain a challenge for searching new sensitive and specific biomarkers of neoplastic processes in salivary glands. The aim of the presented study was an the assessment of the activity of HEX, with its isoforms HEX-A and HEX-B, GLU, GAL, MAN and FUC in salivary gland tumor tissues in comparison to a healthy salivary gland tissues taken during autopsy. A group of 42 patients with benign and malignant salivary gland tumors, aged 25-65 were examined. Fragments of salivary gland tumor tissue, fragments of healthy tissue removed during autopsy, blood serum and saliva were collected from patients with salivary gland tumors and healthy volunteers. In salivary gland tumor tissue the activity of HEX, HEX-A, HEX-B, GAL, FUC was considerably higher than in comparison to healthy salivary gland tissue and ascending trend of activity of GLU, MAN was also noticed. The activity of all lysosomal exoglycosidases in blood serum in patients with salivary gland tumors was considerably higher in comparison to healthy volunteers blood serum. The considerably higher activity of HEX, HEX-A, GLU, GAL, MAN, FUC and descending trend of activity of HEX-B were noticed in saliva of patients with salivary gland tumors in comparison to healthy volunteers. The assessment of HEX in blood serum and saliva of patients with salivary gland tumor can be possibly used in diagnostics and monitoring of salivary glands tumors.

Abstract 4197: Mouse xenograft models of human soft tissue sarcomas

Abstract Sarcoma represents a class of tumors arising from mesenchymal tissue. New approaches that target sarcoma-specific molecular alterations hold a promise of more effective therapies. Currently available sarcoma models do not accurately resemble relevant properties of the human disease and very often are inadequate for predicting the efficacy of investigational drugs in preclinical settings. Thus, in order to test novel targeted therapies, it is essential to develop in vivo models of sarcomas. To this end, we have created a panel of well-characterized xenografts of primary human sarcomas that represent a spectrum of histotypes. Specifically, we transplanted intact human sarcomas under the skin of immunodeficient mice. We then compared the morphologic, phenotypic, and genetic characteristics of the tumor tissues before and after implantation in recipient mice. To date, we have implanted into Nu/Nu recipients tumor tissue fragments from 32 gastrointestinal stromal tumors (GIST) 10 dedifferentiated liposarcomas and 6 leiomyosarcomas. Successful tumor growth was detected in 86% cases of GIST, 44% of liposarcomas and 33% of leiomyosarcomas. Upon serial transplantation, 35% of GISTs, 44% of liposarcomas and 33% of leiomyosarcomas have grown beyond passage F3. Histological analysis of the primary tumors and corresponding xenografts revealed that morphology and immunophenotype (c-Kit status for GISTs, MDM2 for liposarcomas, Smooth Muscle Actin for leiomyosarcomas) was retained throughout all passages. Moreover, all major genetic abnormalities present in the primary tumors were retained in the xenografts, as revealed by genome-wide Single Nucleotide Polymorphism (SNP) array analyses. Our results show that this approach can be used to generate animal models that faithfully reproduce characteristics of human intact tumors. These models can be used for in vivo preclinical drug testing, diagnostic and prognostic molecular markers discovery, and for identification of molecular mechanisms of drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4197.

Circulating tumour tissue fragments in patients with pulmonary metastasis of clear cell renal cell carcinoma

Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the 'seed and soil' hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF-A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF-A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p < 0.001, Fisher's exact test). In patients with tumours that, in retrospect, were not of the VEGF-A-expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF-A-induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis.

Use of cholesterol-rich nanoparticles that bind to lipoprotein receptors as a vehicle to paclitaxel in the treatment of breast cancer: pharmacokinetics, tumor uptake and a pilot clinical study

In animal experiments paclitaxel oleate associated with a cholesterol-rich nanoemulsion concentrated in the neoplastic tissues and showed reduced toxicity and increased antitumor activity compared with paclitaxel-Cremophor EL. Here, a clinical study was performed in breast cancer patients to evaluate the tumoral uptake, pharmacokinetics and toxicity of paclitaxel associated to nanoemulsions. Twenty-four hours before mastectomy [(3)H]-paclitaxel oleate associated with [(14)C]-cholesteryl oleate-nanoemulsion or [(3)H]-paclitaxel in Cremophor EL were injected into five patients for collection of blood samples and fragments of tumor and normal breast tissue. A pilot clinical study of paclitaxel-nanoemulsion administered at 3-week intervals was performed in four breast cancer patients with refractory advanced disease at 175 and 220 mg/m(2) dose levels. T (1/2) of paclitaxel oleate associated to the nanoemulsion was greater than that of paclitaxel (t (1/2) = 15.4 +/- 4.7 and 3.5 +/- 0.80 h). Uptake of the [(14)C]-cholesteryl ester nanoemulsion and [(3)H]-paclitaxel oleate by breast malignant tissue was threefold greater than the normal breast tissue and toxicity was minimal at the two dose levels. Our results suggest that the paclitaxel-nanoemulsion preparation can be advantageous for use in the treatment of breast cancer because the pharmacokinetic parameters are improved, the drug is concentrated in the neoplastic tissue and the toxicity of paclitaxel is reduced.

Clinicopathological Characteristics and Non-Adhesive Organ Culture of Insulinomas

Insulinoma is a very rare type of islet cell tumour, but nevertheless the most common endocrine tumour of the pancreas. We aimed at reviewing our clinical experience with this tumour type and to assess whether organ culture could be obtained from surgically resected insulinoma material. All patients with insulinomas (6 men and 10 women) referred to Haukeland University Hospital between 1986 and 2006 were included in the study. Median age of onset was 53 years (range 21-74). Biochemical diagnosis was established during a 72 h fast test. Imaging and localization of the tumours were performed with intra-operative ultrasonography, endoscopic ultrasonography, CT-scan and/or transcutaneous ultrasonography. For six patients, organ cultures were set up from tumour tissue fragments. The annual incidence of insulinoma was 0.8 per million. The patients generally presented with non-specific, episodic symptoms, which often were mistaken for cardiovascular, neurological or diabetic disease and in some cases delayed the diagnosis with several years. Two patients had diabetes prior to the diagnosis of insulinoma. Patient weight gain was probably due to increased food intake, compensating for the hypoglycemia. Intra-operative ultrasonography detected all tumours correctly, whereas 73% were detected by endoscopic ultrasonography and 38% by CT scan. Five insulinomas were located in the head, eight in the body and three in the tail of the pancreas. All were removed by open-access surgery, eleven cases by resection and five by enucleation. One tumour was malignant with liver metastases and two patients had tumours defined as borderline. Insulinoma tissue fragments developed into spheroids during the first week of culturing and insulin secretion into the media was demonstrated. Insulinomas are rare and diagnostically challenging tumours. Intra-operative ultrasonography was superior to other imaging modalities to locate the lesion. In organ culture, insulinomas readily form spheroids which may be used to yield insight into beta-cell biology.

Transient infection of freshly isolated human colorectal tumor cells by reovirus T3D intermediate subviral particles

Reovirus T3D preferentially kills tumor cells expressing Ras oncogenes and has shown great promise as an anticancer agent in various preclinical tumor models. Here, we investigated whether reovirus can infect and kill tumor cell cultures and tissue fragments isolated from resected human colorectal tumors, and whether this was affected by the presence of endogenous oncogenic KRAS. Tissue fragments and single-cell populations isolated from human colorectal tumor biopsies were infected with reovirus virions or with intermediate subviral particles (ISVPs). Reovirus virions were capable of infecting neither single-cell tumor cell populations nor small fragments of intact viable tumor tissue. However, infection of tumor cells with ISVPs resulted in transient viral protein synthesis, irrespective of the presence of oncogenic KRAS, but this did not lead to the production of infectious virus particles, and tumor cell viability was largely unaffected. ISVPs failed to infect intact tissue fragments. Thermolysin treatment of tumor tissue liberated single cells from the tissue and allowed infection with ISVPs, but this did not result in the production of infectious virus particles. Immunohistochemistry on tissue microarrays showed that junction adhesion molecule 1, the major cellular reovirus receptor, was improperly localized in the cytoplasm of colorectal tumor cells and was expressed at very low levels in liver metastases. This may contribute to the observed resistance of tumor cells to reovirus T3D virions. We conclude that infection of human colorectal tumor cells by reovirus T3D requires processing of virions to ISVPs, but that oncolysis is prevented by a tumor cell response that aborts viral protein synthesis and the generation of infectious viral particles, irrespective of KRAS mutation status.

Sinonasal-type hemangiopericytoma: a clinicopathologic analysis of 6 cases

To study the clinicopathologic features, histologic diagnosis and differential diagnosis of sinonasal-type of hemangiopericytoma (SNTHPC). The clinical, radiographic and pathologic findings of 6 cases of SNTHPC were analyzed. Immunohistochemistry and electron microscopy were performed on selected examples. Amongst the 6 patients studied, 4 were males and 2 were females. The age of patients ranged from 56 to 71 years (mean = 60.5 years old). The commonest clinical presentation was nasal obstruction and/or epistaxis. Other symptoms could include increased nasal secretion, eyeball pain, decreased visual acuity, increased tear secretion and headache. The tumor involved nasal cavity and/or paranasal sinuses. Gross examination showed polypoid tumor masses, brownish fleshy tissue or whitish tumor tissue fragments. Histologically, the tumor showed a mixture of diffuse, fascicular, storiform, reticulated and whorled growth patterns. The tumor cells were spindle-shaped and possessed clear to eosinophilic cytoplasm. Mitotic figures were rarely seen. The intervening vasculature was characteristically thin-walled, with focal hyalinization changes and rarely the staghorn pattern. Immunohistochemical study showed that the tumor cells expressed vimentin (6/6), smooth muscle actin (5/6) and CD34 (3/6). Electron microscopy demonstrated the presence of intracytoplasmic myofilaments. The tumor cells were linked together by primitive cell junctions. In general, the histologic diagnosis of SNTHPC was difficult, and only 1 case had the correct initial pathologic diagnosis made. Follow-up data were available in 5 patients and 2 of them had local recurrences. SNTHPC is a low to intermediate grade soft tissue tumor with pericytes differentiation. Correct diagnosis relies on detailed pathologic assessment and application of ancillary investigations.