1 Kalra et al. BMC Cancer (2015) 15:376 DOI /s RESEARCH ARTICLE Open Access Mesothelioma patient derived tumor xenografts with defined BAP1 mutations that mimic the molecular characteristics of human malignant mesothelioma Neetu Kalra 1, Jingli Zhang 1, Anish Thomas 1, Liqiang Xi 2, Mitchell Cheung 3, Jacqueline Talarchek 3, Sandra Burkett 4, Maria G Tsokos 5, Yuanbin Chen 1, Mark Raffeld 2, Markku Miettinen 2, Ira Pastan 6, Joseph R Testa 3 and Raffit Hassan 1* Abstract Background: The development and evaluation of new therapeutic approaches for malignant mesothelioma has been sparse due, in part, to lack of suitable tumor models. Methods: We established primary mesothelioma cultures from pleural and ascitic fluids of five patients with advanced mesothelioma. Electron microscopy and immunohistochemistry (IHC) confirmed their mesothelial origin. Patient derived xenografts were generated by injecting the cells in nude or SCID mice, and malignant potential of the cells was analyzed by soft agar colony assay. Molecular profiles of the primary patient tumors, early passage cell cultures, and patient derived xenografts were assessed using mutational analysis, fluorescence in situ hybridization (FISH) analysis and IHC. Results: Primary cultures from all five tumors exhibited morphologic and IHC features consistent to those of mesothelioma cells. Mutations of BAP1 and CDKN2A were each detected in four tumors. BAP1 mutation was associated with the lack of expression of BAP1 protein. Three cell cultures, all of which were derived from BAP1 mutant primary tumors, exhibited anchorage independent growth and also formed tumors in mice, suggesting that BAP1 loss may enhance tumor growth in vivo. Both early passage cell cultures and mouse xenograft tumors harbored BAP1 mutations and CDKN2A deletions identical to those found in the corresponding primary patient tumors. Conclusions: The mesothelioma patient derived tumor xenografts with mutational alterations that mimic those observed in patient tumors which we established can be used for preclinical development of novel drug regimens and for studying the functional aspects of BAP1 biology in mesothelioma. Keywords: Mesothelioma, Malignant effusions, BAP1, CDKN2A, Patient derived tumor xenografts Background Malignant mesothelioma is an asbestos-related aggressive tumor with poor prognosis, occurring in the mesothelial lining of pleural or peritoneal cavities. Malignant mesothelioma is a deadly and clinically challenging disease due to its low incidence, resistance to most chemotherapies, and complexity of tumor anatomy. Despite multimodality therapy, the median overall survival is less than a year for * Correspondence: 1 Thoracic and GI Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Full list of author information is available at the end of the article patients with pleural tumors, and the 5-year survival rate is less than 15% for peritoneal mesothelioma . The development and evaluation of new therapeutic approaches for malignant mesothelioma has been sparse due, in part, to lack of suitable tumor models. Efforts to generate clinically relevant tumor models have focused on two main approaches in the recent years: generation of primary tumor-derived cell lines and of mouse models. Primary tumor-derived cell lines have adapted to growth outside a natural tumor microenvironment and due to selective processes associated with long term culturing will develop genetic changes that are distinct 2015 Kalra et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
2 Kalra et al. BMC Cancer (2015) 15:376 Page 2 of 11 from the genetic stress imposed on tumors in patients [2-4]. Therefore even when propagated in vivo, they may not be suitable for preclinical testing of anti-cancer drugs or for studying the underlying gene expression associated with novel drug responses. Mouse models generated via engraftment of primary human tumors into immunecompromised mouse models have become increasingly popular for preclinical testing of anticancer drugs. However their usefulness depends upon the preservation of biological and morphological characteristics of the primary tumors . Many of the currently available mesothelioma cell lines do not form tumors in mice, and others have been propagated in culture for many passages, leading to various cytogenetic changes. Thus, these lines often do not show much similarity with the original tumors . The most common genetic alterations associated with mesothelioma, including CDKN2A deletions and NF2 mutations, have been known for about two decades [7-9]. More recently, mutations in the BAP1 tumor suppressor gene have been observed in 20-25% of mesothelioma tumor samples [10,11]. BAP1, a nuclear ubiquitin hydrolase, plays an important role in various cellular processes including cell proliferation, DNA repair and regulation of gene expression at the chromatin level . This study describes molecular and immunohistochemical characterization of five primary mesothelioma cell lines. By comparing mutational and immunohistochemical profiles between primary cell cultures, and patient derived xenografts, we report the stability of both the genetic profile and protein expression in the xenografts, highlighting their potential for exploring genetic changes associated with responses to established and novel drugs. Methods Pathological examination of the original tumor specimens All patients whose samples were utilized for this study were enrolled in Institutional Review Board approved protocols at the Center for Cancer Research, National Cancer Institute. All patients provided written informed consent which allowed the storage and use of body fluids, tumor samples and data that were collected for future research. Tumor samples obtained from five patients at the time of diagnosis or at the time of debulking surgery were evaluated by a pathologist to establish the diagnosis and characterize the subtype of mesothelioma. Establishment of early-passage mesothelioma cell cultures Early passage primary mesothelioma cell cultures were isolated from ascites or pleural fluid obtained from mesothelioma patients at the National Cancer Institute. The ascites or pleural fluid ( ml) was centrifuged at 1000 rpm at room temperature for 3 minutes; the cell pellets were washed twice with phosphate buffered saline (PBS), and red blood cells were removed using a BD Pharm Lyse -Lysing Buffer kit (BD Bioscience, NJ), according to the manufacturer s instructions, and washed again two times with PBS. The cells were then resuspended in RPMI 1640 (Invitrogen, CA) supplemented with 2 mm glutamine, 100 units penicillin-streptomycin, and 1 mm sodium pyruvate (each from Invitrogen, CA) plus 20% fetal bovine serum (FBS) (Lonza, MD). The cells were seeded into 175 ml culture flasks at a density of cells/ml. After incubating at 37 C in a humidified, 5% CO 2 atmosphere overnight, the medium containing non-adherent cells was replaced with fresh medium. The cultures were maintained by changing the medium depending upon the growth of the cells. To authenticate these cell lines for future use by us or other investigators we performed Short Tandem Repeats (STR) analysis of these cells. Immunohistochemistry Cells were detached using trypsin-edta and then washed and centrifuged. The cell pellets were fixed in formalin and embedded in paraffin. Tumor sections were prepared, and immunohistochemical studies were carried out for the mesothelial markers calretinin, WT1, CK5/6, and mesothelin and BAP1 using specific antibodies (Santa Cruz Biotechnology, TX). All immunostaining was carried out using an automated Ventana system (Ventana Medical Systems, AZ) using their UltraView polymer based detection system. IHC staining was scored semiquantitatively as follows: negative (less than 5% of cells stained), 1+ positive (5-50%), and 2+ positive (50-100%). Electron microscopy Cells from all five cell lines were washed in PBS, fixed in PBS-buffered 2.5% glutaraldehyde (Sigma Chemicals, MO), postfixed in 0.5% osmium tetroxide and embedded into Spurr s epoxy resin (Ladd Research Industries, VT). Ultrathin sections were stained with uranyl acetate-lead citrate and viewed in a Phillips CM10 transmission electron microscope. Spectral karyotyping and fluorescent in situ hybridization Spectral karyotyping was performed according to the manufacturer s protocol using 24-color human SKY paint probes (Applied Spectral Imaging, CA). Fluorescent in Situ Hybridization (FISH) analysis was carried out using a CDKN2A probe (Abbott Molecular, IL) encompassing the overlapping genes encoding p16 INK4a and p14 ARF. Spectral images of the hybridized metaphases were acquired using a SD301 SpectraCubeTM system (Applied Spectral Imaging Inc., CA) mounted on top of an epi-fluorescence microscope Axioplan 2 (Zeiss). Images were analyzed using Spectral Imaging 6.0 acquisition software (Applied Spectral Imaging Inc., CA). At least 10 SKY hybridized metaphases were analyzed in this experiment. FISH
3 Kalra et al. BMC Cancer (2015) 15:376 Page 3 of 11 analyses were performed under an Axioplan 2 (Zeiss) fluorescence microscope coupled with a CCD camera (ASI) and images were captured with FISH view 5.5 software (Applied Spectral Imaging Inc., Vista, CA). Mutation analysis Genomic DNA was extracted from early passage cells using a DNA isolation kit (Mo-Bio Laboratories, CA). PCR was done using exon specific primers for the entire coding region of BAP1, the tyrosine kinase domain of the EGFR,  and exons 2 9 oftp53. Polymerase Chain Reaction (PCR) products were purified and subjected to Sanger sequencing on an ABI PRISM sequencing apparatus (ABI Prism 310 Genetic Analyser, Applied Biosystems, NY). In addition, a panel of hotspot mutations in 7 genes (AKT1, BRAF, EBRR2, EGFR, KRAS, NRAS,andPIK3CA) was analyzed with pyrosequencing for single-nucleotide variants (SNVs) or with fragment analysis for insertions/deletions (indels). Briefly PCR amplification was initially performed with primers flanking the mutation hotspot under co-amplification at lower denaturation temperature (COLD)-PCR conditions and was followed by targeted pyrosequencing on a PyroMark Q24 instrument (Qiagen) or by capillary electrophoresis using a Genetic Analyzer 3130xl (Life Technologies) [14-20]. Flow cytometry Mesothelin expression on early and late passage primary cell cultures were evaluated by flow cytometry using the anti-mesothelin primary antibody (MN) as previously described . Western blotting Monolayers of confluent cells were washed twice in PBS and then lysed in 1 Cell Lysis Buffer supplemented with 1 mm phenylmethylsulfonylfluoride (Cell Signaling Technology, MA). Fifty micrograms of total protein were subjected to SDS-polyacrylamide gel electrophoresis (Invitrogen, CA) for each cell line followed by immunoblotting with mouse monoclonal E-cadherin, N-cadherin and vimentin antibodies (BD Bioscience, NJ) and BAP1 antibody (Santa Cruz Biotechnology, CA) (1:1,000 in 5% blocking reagent in Tris-buffered saline/tween-20) overnight at 4 C. The following day, blots were incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology; 1:1,000 dilution) for 1 hour at 25 C. Signals were visualized with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, NJ) on X-ray film (Eastman Kodak, NY). Soft agar colony assay Twenty thousand cells were suspended in 0.3% low melting agarose (Lonza, Rockland, ME) in RPMI containing 20% FBS. This suspension was overlaid onto a solid layer of 0.6% agarose in a 6-well plate. The cells were treated with fresh RPMI containing 20% FBS every other day. After 2 3 weeks, depending upon the growth, the cells were fixed with methanol, stained with 0.02% crystal violet, and photographed at 10 magnification. In vivo xenograft studies All animal experiments were performed in accordance with NIH guidelines and approved by the NCI Animal Care and Use Committee. Cultured cells ( cells) were injected subcutaneously into the dorsal side of three nude or SCID mice. The animals were examined every week for the development of tumors. All animal care was done in accordance with institutional guidelines. The single tumor for each primary cell was excised and fixed in 10% formalin to process for routine histopathological examination and to perform immunohistochemical staining for the earlier mentioned markers. Results Primary mesothelioma cultures established from malignant effusions of patients with mesothelioma Table 1 summarizes characteristics, diagnosis and course of treatment of patients with malignant mesothelioma from whom the primary cultures were derived. Four of the five tumors had epithelial histology and one was predominantly epithelial with focal sarcomatous features. All tumors were positive (15-100% of tumor cells) for mesothelioma-related markers mesothelin and calretinin by IHC. Four of the tumors were also positive for the cytokeratin 5/6. Morphologic features, electron microscopy and immunohistochemical staining of primary cell cultures are indicative of their being of mesothelial origin Primary cultures from all five tumors exhibited morphologic and immunohistochemical features consistent to those of mesothelial cells. All primary cultures except NCI-Meso17 grew as adherent monolayers characterized by polygonal epithelial-type cells that tended to group together as clusters in a colony-like formation. In addition to polygonal epithelial cells, NCI-Meso17 cells also included spindle like cells, which became the predominant cell population as the cells were maintained in culture. Cell blocks were prepared from early-passage ( 5 passages) mesothelioma cells and sections were evaluated by hematoxylin and eosin (H&E) and IHC (Figure 1 and Table 1). IHC revealed high levels of expression of mesothelin (2+ to 3+), and WT1 and calretinin (3+) in three samples (NCI-Meso16, NCI-Meso19, NCI-Meso21). NCI-Meso17, which was derived from a patient who had biphasic disease, was negative for most of the above markers, but retained a few WT-1 positive cells. NCI- Meso18, which was derived from an epithelioid mesothelioma, retained moderate expression of calretinin and minimal expression of mesothelin and WT1.
4 Kalra et al. BMC Cancer (2015) 15:376 Page 4 of 11 Table 1 Clinical and pathological characteristics of patients from which primary cultures were established Cell lines Sex Age (dx) Ethnicity Diagnosis Patient tumor TNM stage Treatment Survival from diagnosis Histology Positive markers NCI-Meso16 M 72 Caucasian Pleural Epithelioid Mesothelin: 2+, 30%, Calretinin, WT1, CK5/ 6(focal) NCI-Meso17 F 63 Caucasian Peritoneal Predominantly epitheliod with focal sarcomatous features Mesothelin: 2+, 15%, WT1, CK5/6, CA125 NCI-Meso18 M 60 Caucasian Pleural Epithelioid Mesothelin: 3+, 100%, Calretinin, CK5/6, WT1 NCI-Meso19 M 19 African Pleural Epithelioid Mesothelin: 2+, 100%, Calretinin NCI-Meso21 M 66 Caucasian Pleural Epithelioid Mesothelin: 3+, 75%, WT1, calretinin, CK5/6 ct4n1m0 (IV) Cisplatin + Pemetrexed (6 cycles) with SS1P (first two cycles) NA Debulking + IP Cisplatin + doxorubicin IP Pemetrexed + IV Cisplatin IP Taxol Debulking + IP mitomycin + doxorubicin phase II investigationsl agent ct3n0m0 (III) Cisplatin + Pemetrexed EPP Cisplatin + Pemetrexed Gemcitabine Navelbine phase II investigational agent T4N3MX (IV) Died before any specific treatment T4 (IV) Cisplatin + Pemetrexed (6 cycles) with SS1P (first two cycles) 8 months 8 years 34 months 3 months 13 months NA, not applicable; IP, intraperitoneal; IV, intravenous; EPP, extra-pleural pneumonectomy. Patient tumor mesothelin expression is reported based on the intensity of staining (1+ to 3+) and percentage of cells stained (1-100%). Electron microscopy analysis further supported the mesothelial origin of these cells, as the long irregular and branched microvilli characteristic of mesothelioma cells were present in all the cell cultures (Figure 1). We also noticed the presence of intracytoplasmic intermediate filaments in all the primary cells and glycogen bodies in some of them. Tonofilaments could also be seen in one of the cell cultures (NCI-Meso21). Thus, the electron microscopy data strongly indicated that the primary cell cultures are of mesothelioma origin. The cell morphology was retained throughout the 30 passages we tested, and we performed STR analysis at different passages in order to eliminate the possibility of cross contamination among cell cultures (Additional file 1). STR analysis showed only modest changes in DNA fingerprinting profiles between primary tumors and the corresponding cell cultures . We also assessed cell surface expression of mesothelin by flow cytometry in early and late passage cells (Additional file 2). Primary cells which expressed mesothelin at early passages (NCI-Meso16, NCI-Meso18, NCI-Meso19, and NCI-Meso21) continued to express mesothelin in late passage cultures. NCI- Meso17 which did not express mesothelin in early passages remained mesothelin negative in late passages. Following thawing, cryopreserved cells could be propagated in culture without a noticeable change in growth and morphology. In all subsequent experiments, cells were used between the third to sixth passages in culture. Cytogenetic analysis reveals abnormal karyotype in all five primary cultures Spectral karyotyping of early passage cell cultures uncovered multiple structural and numerical chromosome abnormalities in all five cultures as summarized
5 Kalra et al. BMC Cancer (2015) 15:376 Page 5 of 11 Figure 1 Characteristics of the primary mesothelioma cell cultures. Left panel: Hematoxylin and eosin staining of primary cell cultures. Middle panels: Representative immunohistochemical stains of mesothelioma cell culture pellets that were formalin-fixed and paraffin-embedded. NCI-Meso16, NCI-Meso19 and NCI-Meso21 cells were strongly and diffusely positive for mesothelin, calretinin and WT1. NCI-Meso18 cells expressed primarily calretinin in a smaller percentage of cells, whereas NCI-Meso17 cells showed a small percentage of WT-1 positive cells. Right panel: Electron microscopic images of primary tumor cells which exhibit numerous long and complex surface villi (thick arrows) that are characteristic of mesothelioma cells. The thin arrow in the electron microscopic image of the NCI-Meso21 cells points to condensed aggregates of intermediate filaments indicative of tonofilamens, shown in detail at a higher magnification in the inset. in Additional file 3. The more frequent chromosomal rearrangements included clonal rearrangement or deletions of chromosomes 1 (4/5 cell cultures), 3 (3/5), 9 (4/5) and 22 (2/5). The karyotype analysis showed a very high number of structural abnormalities in NCI-Meso19, a cell culture derived from a mesothelioma of a young patient harboring a somatic TP53 mutation. These results are consistent with earlier reports showing frequent alterations of 1p, 3p, 9p, and 22 in human mesothelioma specimens and derived cell lines . To assess the stability of karyotypic changes, we evaluated karyotypes of early and late passage cells. This revealed only minor karyotypic changes between passages which are summarized in Additional file 3. Epithelial-mesothelial markers are present in all five primary cell cultures All five cell cultures expressed N-cadherin, consistent with their mesothelial origin as described previously . E-cadherin, an epithelial marker, was also expressed in all cultures except NCI-Meso17, which was derived from the patient who had biphasic disease. NCI-Meso17 had high expression of the mesenchymal marker, vimentin (Additional file 4). Frequent BAP1 mutations in primary cell cultures Mutation analysis of early passage cells revealed BAP1 mutations in 4 of 5 cell cultures (Figure 2A and Table 2). NCI-Meso16 cells had a splice site mutation in intron 4 of BAP1. NCI-Meso17 and NCI-Meso21 had frame shift mutations at the intron15/exon16 junction and in exon 13 of BAP1 gene, respectively (Table 2 and Figure 2A). NCI-Meso18 had a large BAP1 deletion. We found a clear correlation between BAP1 mutation and the absence of BAP1 protein by IHC (Figure 2B). For western blot analysis, we used the mesothelioma cell lines NCI- H28 (harboring a BAP1 nonsense mutation) and NCI- H2052 (wild type for BAP1) as positive and negative controls, respectively, for the presence of a BAP1 protein (Figure 2C). BAP1 protein expression detected by western blot was preserved after serial passages with late passage cells showing similar expression patterns as early
6 Kalra et al. BMC Cancer (2015) 15:376 Page 6 of 11 Figure 2 BAP1 alterations in primary mesothelioma cells. Chromatograms showing BAP1 mutations (A). Arrow in the top panel indicates the location of mutation. NCI-Meso16 cells had a splice site mutation in intron 4 of BAP1. NCI-Meso17 and NCI-Meso21 had frame shift mutations at the intron15/exon16 junction and in exon 13 of BAP1 gene, respectively. Immunohistochemistry (B) showing lack of expression of BAP1 protein in cells with mutant BAP1. Only NCI-Meso19 cells which were BAP1 wild-type expressed the BAP1 protein. Western blot (C) showing the expression of BAP1 in primary mesothelioma cells. For western blot, mesothelioma cell lines NCI-H28 and NCI-H2052 were used as controls for mutant and wild-type BAP1 respectively. A faint BAP1 band was observed for NCI-Meso17 cells, which exhibits a 5-bp deletion in exon 13. Western blot showing (D) the expression of BAP1 in late passage primary mesothelioma cells. The faint BAP1 band which was observed for NCI-Meso17 cells in early passage was absent in late passage. Table 2 Mutations in early passage malignant mesothelioma primary cell cultures Cell lines CDKN2A deletion BAP1 NF2 TP53 EGFR KRAS NRAS BRAF PIK3 CA ERBB2 AKT1 NCI-Meso16 wt Intron 4 spice site mutation Wt Wt Wt Wt Wt Wt Wt Wt Wt c.256-2a > G, Hm NCI-Meso17 Hm Deletion of CAGAT at intron Wt Wt Wt Wt Wt Wt Wt Wt Wt 15/exon 16 junction. c _1985delCAGAT NCI-Meso18 Hm Large deletion Wt Wt Wt Wt Wt Wt Wt Wt Wt NCI-Meso19 Hm Wt Large deletion *P322S Wt Wt Wt Wt Wt Wt Wt NCI-Meso21 Hz Homozygous 20 bp deletion Wt Wt Wt Wt Wt Wt Wt Wt Wt in exon 13. c. 1302_1321delTGGGCAA CTGTCAGTGCTGC All 5 cell lines were analyzed for mutations in BAP1 whole gene, P53 exons 2 9, EGFR Exons 18 24, KRAS and NRAS Codon 12, 13 and 61, BRAF , PIK3CA Exon 9 and 20, ERBB2 insertion in Exon 20 and AKT1 codon 17. *TP53 heterozygous mis-sense mutation in codon 322 (P322S, Exon 9), polymorphism codon 72 (P72R, Exon 4). Hm, homozygous. Hz, heterozygous. Wt, wild-type.
7 Kalra et al. BMC Cancer (2015) 15:376 Page 7 of 11 passage cells (Figure 2D). NCI-Meso17 cells which exhibits a 5-bp deletion in exon 13 demonstrated a faint BAP1 band in early passages which was not detected in late passage cells. Frequent deletions of CDKN2A in primary cell cultures We screened cell cultures for deletions of the CDKN2A locus in chromosome 9p21 by FISH analysis on primary cell cultures. Four of the samples were found to have homozygous deletions of CDKN2A (Table 2), consistent with rates reported in malignant mesothelioma cells [7,24-27]. The percentage of nuclei with a homozygous deletion pattern was more than 60% in four cultures. One culture had primarily wild type cells, with less than 10% of cells showing a homozygous deletion of CDKN2A. Mutations in other genes tested We also performed sequence analysis of NF2 and TP53, and hot spot mutation screening for several oncogenes, as summarized in Table 2. A large deletion in NF2 and a missense mutation in exon 8 (P322S) of the TP53 gene were found in one of the primary cultures (Table 2). We did not see mutations in any of the other genes tested. In vitro soft agar colony growth of mesothelioma cells correlates with tumorigenic potential in vivo A soft agar colony assay was performed to examine anchorage independent growth. Three of the five cultures (NCI- Meso16, NCI-Meso17 and NCI-Meso21) formed colonies within 3 weeks (Figure 3A). Cells that formed colonies in the in vitro assay also formed visible tumors in mice within 3 8 weeks. Notably, all three of these cell cultures had BAP1 mutations. Tumor growth varied among the cell cultures, with a tumor volume of 100 mm 3 achieved between 40 to 80 days after inoculation (Figure 3A). Early passage mesothelioma cells and their tumor xenografts retain molecular features of human malignant mesothelioma The expression of mesothelioma specific immunohistochemical markers in primary cell cultures were preserved in the tumor sections from mouse xenografts (Figure 3B). Furthermore, the xenografts lacked BAP1 expression, as shown by the absence of nuclear staining, similar to the corresponding primary cells. This finding was further supported by detection of the identical BAP1 mutations in tumor xenografts, and the corresponding primary tumor samples and primary cell cultures (Table 3). In addition, xenograft tumors exhibited the same CDKN2A deletion status as in primary cell cultures, except in one case where most but not all cells had a homozygous deletion of CDKN2A, whereas the reverse was true in the corresponding primary cell culture. This suggests that the tumor cells with loss of CDKN2A had a selective growth advantage in vivo. The two remaining tumor samples completely matched with their primary cultures for their CDKN2A deletion status, further confirming that primary cells obtained from patient s malignant effusions have the same genetic alterations as in the corresponding primary human tumor. Discussion There is a need to develop better preclinical tumor models to evaluate new therapeutic approaches for mesothelioma. Patient derived tumor xenograft models are increasingly being recognized as a robust approach for evaluating the efficacy of novel therapeutic agents, analyzing the process of tumor progression at the cellular and molecular level, and for the identification of new therapeutic targets. However, their utility depends heavily on preservation and stability of biological and morphological characteristics of the primary tumors. Confirmation of this stability is crucial in order to reliably identify molecular responses to treatment in xenografts which can be extrapolated back to patients. In this report, we describe the establishment of primary mesothelioma cultures and patient derived tumor xenografts with mutational alterations that recapitulate those in the original patient tumors. We isolated malignant mesothelioma cells from the ascites or pleural effusion of five mesothelioma patients and grew them successfully in culture. Furthermore, we performed detailed morphologic and molecular characterization of early passage cultures of these cells and patient derived tumor xenografts in nude mice and assessed their malignant potential in vitro. Using a number of techniques, we demonstrated that these cells were of mesothelial origin and are indeed malignant. Four of the cell lines had BAP1 mutations. We also showed a strong correlation between the mutation and the absence of expression of BAP1 protein. Three out of five cells, all of which were derived from BAP1 mutant primary tumors, exhibited anchorage independent growth and also formed tumors in vivo, suggesting that BAP1 loss may enhance tumor growth in vivo. Both early passage cell cultures and mouse xenograft tumors harbored the BAP1 mutations and CDKN2A deletions identical to those observed in the corresponding primary tumor. Given the similarities to the primary tumor, these models offer an opportunity in mesothelioma to study efficacy of novel therapeutic agents and to identify molecular responses to treatment. We believe these models would also enable further studies into functional aspects of BAP1, which is mutated in nearly a quarter of all mesotheliomas. We also found a strong correlation between the ability of these cells to grow in an anchorage independent fashion in vitro and to induce tumors in vivo, similar to other work . These features, along with the stable nature of these cell cultures, suggest that these early passage cells
8 Kalra et al. BMC Cancer (2015) 15:376 Page 8 of 11 Figure 3 Colony-forming ability and tumorigenicity of primary mesothelioma cells. Colony formation in vitro in 0.3% soft agar (A left panel). Colonies could be seen for NCI-Meso16, NCI-Meso17 and NCI-Meso21 cell cultures, each of which showed BAP1 mutations. Tumorigenic potential of these cell cultures was also investigated by subcutaneous injection of cells with matrigel in nude or SCID mice. Representative photographs of subcutaneous tumors derived following injection of NCI-Meso16, NCI-Meso17 and NCI-Meso21 cells is shown (A middle panel) along with the corresponding tumor growth curves (A right panel). Immunohistochemical staining for different markers showed the same pattern in tumor xenografts as in the matching primary cell cultures (B).
9 Kalra et al. BMC Cancer (2015) 15:376 Page 9 of 11 Table 3 Comparison of patient tumors with primary cell culture and with patient derived xenograft in terms of BAP1 mutation and CDKN2A deletion BAP1 mutation NCI-Meso16 (splice site mutation in Intron 4) NCI-Meso17 (5 bp deletion in intron 15- Exon 16 junction) NCI-Meso18 (large deletion) Patient tumor Primary cell culture Mouse xenograft CDKN2A deletion by FISH Patient tumor Present Present Present NCI-Meso16 (wt) Homozygous deleted cells some wt cells Present Present Present NCI-Meso17 (Homozygous deletion) Sample not available large deletion Does not form tumor NCI-Meso19 (Wt) Wt Wt Does not form tumor NCI-Meso21 (20 bp deletion in Exon 13) Wt- wild-type. Sample not available NCI-Meso18 (Homozygous deletion) NCI-Meso19 (Homozygous deletion) Present Present NCI-Meso21 (Heterozygous deletion) Primary cell culture Mostly wt. Some homozygous deleted cells Present Present Present Sample not available Present Mouse xenograft Mostly wt. Some homozygous deleted cells Does not form tumor Present Present Does not form tumor Sample not available Present Present would be useful for functional and preclinical studies. It is noteworthy that the cell cultures that formed colonies in vitro and tumors in nude mice were cell lines that had BAP1 mutations. Previous studies of BAP1 have shown that BAP1 loss promotes colony-forming ability of mesothelioma cells, and that re-expression of BAP1 in BAP1- deficient markedly decreases colony-formation . Combined with the fact that these same cell cultures were also tumorigenic suggests that BAP1 loss may also enhance tumor growth in vivo. Some BAP1 missense mutations have been shown to affect the ubiquitin hydrolase activity of its protein product; however, splice site mutations cause exon-skipping leading to aberrant, out-of-frame transcripts . A recent study has suggested that BAP1 inactivation is more closely associated with the epithelioid subtype of malignant pleural mesothelioma . Consistent with this possibility, 3 of the 4 cell cultures harboring BAP1 mutations in our study had epithelioid histology. Since the p16 INK4a /p14 ARF proteins encoded by the CDKN2A locus are essential for normal cell cycle control, FISH analysis of this locus can be useful for the diagnosis of early-stage mesotheliomas of epithelial type . Our finding of homozygous CDKN2A deletions in four of five cultures helps confirm the malignant nature of these cells. Recent experimental work has documented the importance of this locus to mesothelioma carcinogenesis. These studies have shown that mice deficient for either p16 Ink4a or p19 Arf, the murine homolog of human p14 ARF, have increased susceptibility to asbestos-induced mesothelioma and that inactivation of both p16 Ink4a or p19 Arf cooperate to accelerate asbestos-induced tumorigenesis . Karyotypic analysis revealed recurrent abnormalities in the short arm of chromosome 1, consistent with a deletion hotspot previously implicated in mesothelioma [31,32]. Deletions of chromosome arms 3p, 9p and 22q, which include the tumor suppressor genes BAP1, CDKN2A, and NF2, respectively, have all been linked to mesothelioma. We also identified variable rearrangements of chromosome 10 in all our primary cultures, a finding that to our knowledge has not been reported earlier for malignant mesothelioma. Thus, investigations of the functional significance of these genomic hotspots, along with the mutational studies, may lead to the identification of molecular targets for the treatment of this disease. Conclusions In summary, we describe the establishment of primary mesothelioma cultures and patient derived tumor xenografts with features that mirror the primary tumors. The primary cell cultures derived from ascitic or pleural fluids of patients with mesothelioma are highly tumorigenic and maintain the histologic and molecular features of the original tumors when grown in murine models. To our knowledge this is the first report that compares the characteristics of the patient s tumors with the primary cells and mouse xenograft at the genetic level in malignant mesothelioma. Given that these primary cultures and patient derived tumor xenograft models recapitulate phenotypic and genetic features of the original primary mesotheliomas, they should prove useful for preclinical studies of novel drug regimens and for functional studies of BAP1 biology in mesothelioma.
10 Kalra et al. BMC Cancer (2015) 15:376 Page 10 of 11 Additional files Additional file 1: Short tandem repeat analysis of 5 mesothelioma primary cells. Additional file 2: Flow cytometry showing cell surface expression of mesothelin in early and late passage cells. Cells were incubated with the anti-mesothelin mab MN, conjugated with R-PE or isotype control antibody. Results are shown in terms of histogram plots for each cell line where the area under the blue line depicts the binding of MN antibody and the area under the red line shows the binding of isotype control antibody. Additional file 3: Table S2. Karyotyping of early and late passage primary mesothelioma cell lines. Additional file 4: Western blot images showing the expression E-cadherin, N-cadherin and vimentin in primary cell cultures. Abbreviations COLD: Co-amplification at lower denaturation temperature; FISH: Fluorescence in situ hybridization; IHC: Immunohistochemistry; PBS: Phosphate buffered saline; PCR: Polymerase Chain Reaction; STR: Short Tandem Repeats; SNVs: Single-nucleotide variants. Competing interests The authors declare that they have no competing interest. Authors contributions RH and JRT conceived and directed the project. NK, JZ, LX, MC, JT, SB, MGT, and YC carried out experimental works. NK, JZ, AT, LX, MC, JT, SB, MGT, YC, MR, MM, IP, JRT, and RH analyzed data. NK, AT, YC, SB, MGT, JRT, and RH wrote the paper. All authors read and approved the final manuscript. Acknowledgements This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research; additional partial support was provided by NCI R01 CA (J.R.T) and the Mesothelioma Applied Research Foundation (M.C.). The authors have no conflicts of interest. Author details 1 Thoracic and GI Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2 Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 3 Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. 4 Molecular Cytogenetics Core Facility, National Cancer Institute, Frederick, MD 21702, USA. 5 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. 6 Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Received: 13 October 2014 Accepted: 24 April 2015 References 1. Thomas A, Hassan R. Immunotherapies for non-small-cell lung cancer and mesothelioma. Lancet Oncol. 2012;13(7):e Orengo AM, Spoletini L, Procopio A, Favoni RE, De Cupis A, Ardizzoni A, et al. Establishment of four new mesothelioma cell lines: characterization by ultrastructural and immunophenotypic analysis. Eur Respir J. 1999;13(3): Usami N, Fukui T, Kondo M, Taniguchi T, Yokoyama T, Mori S, et al. Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients. Cancer Sci. 2006;97(5): Kobayashi M, Takeuchi T, Ohtsuki Y. Establishment of three novel human malignant pleural mesothelioma cell lines: morphological and cytogenetical studies and EGFR mutation status. Anticancer Res. 2008;28(1A): Shimosato Y, Kameya T, Najai K, Hirohashi S, Koide T, Hayashi H, et al. Transplantation of human tumors in nude mice. J Natl Cancer Inst. 1976;56(6): Relan V, Morrison L, Parsonson K, Clarke BE, Duhig EE, Windsor MN, et al. Phenotypes and karyotypes of human malignant mesothelioma cell lines. PLoS One. 2013;8(3):e Cheng JQ, Jhanwar SC, Klein WM, Bell DW, Lee WC, Altomare DA, et al. p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma. Cancer Res. 1994;54(21): Bianchi AB, Mitsunaga SI, Cheng JQ, Klein WM, Jhanwar SC, Seizinger B, et al. High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas. Proc Natl Acad Sci U S A. 1995;92(24): Sekido Y, Pass HI, Bader S, Mew DJ, Christman MF, Gazdar AF, et al. Neurofibromatosis type 2 (NF2) gene is somatically mutated in mesothelioma but not in lung cancer. Cancer Res. 1995;55(6): Bott M, Brevet M, Taylor BS, Shimizu S, Ito T, Wang L, et al. The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma. Nat Genet. 2011;43(7): Testa JR, Cheung M, Pei J, Below JE, Tan Y, Sementino E, et al. Germline BAP1 mutations predispose to malignant mesothelioma. Nat Genet. 2011;43(10): Ventii KH, Devi NS, Friedrich KL, Chernova TA, Tighiouart M, Van Meir EG, et al. BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Res. 2008;68(17): Kalra N, Ashai A, Xi L, Zhang J, Avital I, Raffeld M, et al. Patients with peritoneal mesothelioma lack epidermal growth factor receptor tyrosine kinase mutations that would make them sensitive to tyrosine kinase inhibitors. Oncol Rep. 2012;27(6): Mohamedali A, Lea NC, Feakins RM, Raj K, Mufti GJ, Kocher HM. AKT1 (E17K) mutation in pancreatic cancer. Technol Cancer Res Treat. 2008;7(5): Xi L, Arons E, Navarro W, Calvo KR, Stetler-Stevenson M, Raffeld M, et al. Both variant and IGHV4-34-expressing hairy cell leukemia lack the BRAF V600E mutation. Blood. 2012;119(4): Su Z, Dias-Santagata D, Duke M, Hutchinson K, Lin YL, Borger DR, et al. A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell lung cancer. J Mol Diagn. 2011;13(1): Pan Q, Pao W, Ladanyi M. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn. 2005;7(3): Chowdhuri SR, Xi L, Pham TH, Hanson J, Rodriguez-Canales J, Berman A, et al. EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection. Mod Pathol. 2012;25(4): Nosho K, Kawasaki T, Ohnishi M, Suemoto Y, Kirkner GJ, Zepf D, et al. PIK3CA mutation in colorectal cancer: relationship with genetic and epigenetic alterations. Neoplasia. 2008;10(6): Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med. 2008;14(5): Zhang J, Qiu S, Zhang Y, Merino M, Fetsch P, Avital I, et al. Loss of mesothelin expression by mesothelioma cells grown in vitro determines sensitivity to anti-mesothelin immunotoxin SS1P. Anticancer Res. 2012;32(12): Parson W, Kirchebner R, Mühlmann R, Renner K, Kofler A, Schmidt S, et al. Cancer cell line identification by short tandem repeat profiling: power and limitations. FASEB J. 2005;19(3): Flejter WL, Li FP, Antman KH, Testa JR. Recurring loss involving chromosomes 1, 3, and 22 in malignant mesothelioma: possible sites of tumor suppressor genes. Genes Chromosomes Cancer. 1989;1(2): Xio S, Li D, Vijg J, Sugarbaker DJ, Corson JM, Fletcher JA. Codeletion of p15 and p16 in primary malignant mesothelioma. Oncogene. 1995;11(3): Kratzke RA, Otterson GA, Lincoln CE, Ewing S, Oie H, Geradts J, et al. Immunohistochemical analysis of the p16ink4 cyclin-dependent kinase inhibitor in malignant mesothelioma. J Natl Cancer Inst. 1995;87(24): Illei PB, Rusch VW, Zakowski MF, Ladanyi M. Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas. Clin Cancer Res. 2003;9(6): Pass HI, Stevens EJ, Oie H, Tsokos MG, Abati AD, Fetsch PA, et al. Characteristics of nine newly derived mesothelioma cell lines. Ann Thorac Surg. 1995;59(4): Yoshikawa Y, Sato A, Tsujimura T, Emi M, Morinaga T, Fukuoka K, et al. Frequent inactivation of the BAP1 gene in epithelioid-type malignant mesothelioma. Cancer Sci. 2012;103(5):
11 Kalra et al. BMC Cancer (2015) 15:376 Page 11 of Chung CT, Santos Gda C, Hwang DM, Ludovski O, Pintilie M, Squire JA, et al. FISH assay development for the detection of p16/cdkn2a deletion in malignant pleural mesothelioma. J Clin Pathol. 2010;63(7): Altomare DA, Menges CW, Xu J, Pei J, Zhang L, Tadevosyan A, et al. Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis. PLoS One. 2011;6(4):e Lee WC, Balsara B, Liu Z, Jhanwar SC, Testa JR. Loss of heterozygosity analysis defines a critical region in chromosome 1p22 commonly deleted in human malignant mesothelioma. Cancer Res. 1996;56(19): Tsujimura T, Torii I, Sato A, Song M, Fukuoka K, Hasegawa S, et al. Pathological and molecular biological approaches to early mesothelioma. Int J Clin Oncol. 2012;17(1):40 7. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color figure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at
The Use of Kinase Inhibitors: Translational Lab Results Targeting Specific Cell Signaling Pathways for the Treatment of Malignant Peritoneal Mesothelioma Sheelu Varghese, Ph.D. H. Richard Alexander, M.D.
Non Small Cell Lung Cancer: Scientific Discoveries and the Pursuit of Progress Lung Cancer Accounts for 14% of All New Cancer Diagnoses in the United States 1 Lung cancer is the second most common malignancy
White paper Evaluation of BRAF (V600E) Mutation by Immunohistochemical Staining with anti-braf V600E (VE1) Antibody: A Comparison with Sanger Sequencing 2 Evaluation of BRAF (V600E) Mutation by Immunohistochemical
Targeted Therapy What the Surgeon Needs to Know AATS Focus in Thoracic Surgery 2014 David R. Jones, M.D. Professor & Chief, Thoracic Surgery Memorial Sloan Kettering Cancer Center I have no disclosures
Quantitative Telomerase Detection Kit (QTD Kit) Catalog No. MT3010, MT3011, MT3012 For Research Use Only. Not for use in diagnostic procedures 1 Table of Contents 1. Introduction Background Product Overview
- PNA BRAF Mutation Detection Kit Catalog Number KA2102 50 tests/kit Version: 01 Intended for research use only www.abnova.com Introduction and Background Intended use The PNA BRAF Mutation Detection Kit
Seattle Montreal IAP September 2006 Case Presentations Allen M. Gown, M.D. Medical Director and Chief Pathologist PhenoPath Laboratories Clinical Professor of Pathology University of British Columbia Case
Corporate Medical Policy Molecular Analysis for Targeted Therapy for Non-Small Cell Lung File Name: Origination: Last CAP Review: Next CAP Review: Last Review: molecular_analysis_for_targeted_therapy_for_non_small_cell_lung_cancer
Appendix 2 Molecular Biology Core Curriculum Websites and Other Resources Chapter 1 - The Molecular Basis of Cancer 1. Inside Cancer http://www.insidecancer.org/ From the Dolan DNA Learning Center Cold
Effects of Herceptin on circulating tumor cells in HER2 positive early breast cancer J.-L. Zhang, Q. Yao, J.-H. Chen,Y. Wang, H. Wang, Q. Fan, R. Ling, J. Yi and L. Wang Xijing Hospital Vascular Endocrine
Update on Clinical Trials and Foundation Funded Grants Mary Hesdorffer, MS, APRN-BC Medical Liaison Meso Foundation www.curemeso.org Delivering the Diagnosis Delivering the Diagnosis Day 1 Taking control
DNA STAINING REAGENTS KIT STOCK NO. PI-STAIN Table of Contents: Intended Use... 2 Background and Principle... 2 Reagents and Materials Provided... 2 Reagents and Materials that may be Required, but are
Case based applications part III Los Angeles Society Of Pathologists January 25, 2014 Sanja Dacic, MD, PhD University of Pittsburgh Medical Center 1 CASE 1 A 44-year-old woman with multiple lung nodules.
Metastatic breast cancer Immunostain Update: Diagnosis of metastatic breast carcinoma, emphasizing distinction from GYN primary 1/3 of breast cancer patients will show metastasis 1 st presentation or 20-30
[ID]FIGS1[/ID] [ID]FIGS2[/ID] [ID]FIGS3[/ID] [ID]FIGS4[/ID] [ID]FIGS5[/ID] [ID]FIGS6[/ID] [ID]TBLS1[/ID] [ID]TBLS2[/ID] SUPPLEMENTAL MATERIAL Wolf et al., http://www.jem.org/cgi/content/full/jem.20101470/dc1
Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied
Product Manual Pure-IP Western Blot Detection Kit Catalog Number PRB-5002 20 blots FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction The technique of immunoprecipitation (IP) is used
Targeted Therapies in Lung Cancer I Edited by: Simona Carnio Thoracic Oncology Division - St Luigi Hospital Orbassano (TO) - Italy Silvia Novello Department of Oncology - University of Torino - Italy Why
CancerStemCell Markers in Lung Cancers: Proofsof ConceptsandSome Reservations Lourdes Cortes-Dericks, PhD University of Hamburg Hamburg, Germany Lung cancer: highest death rate and poorest patient survival
HER2 Testing in Breast Cancer GAIL H. VANCE, M.D. AGT MEETING JUNE 13, 2014 LOUISVILLE, KENTUCKY No Conflict of Interest to Report Human Epidermal Growth Factor Receptor 2-HER2 Human epidermal growth factor
292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 email@example.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.
Haematopoietic Chimerism Analysis after Allogeneic Stem Cell Transplantation Dr Ros Ganderton, Ms Kate Parratt, Dr Debbie Richardson, Dr Kim Orchard and Dr Liz Hodges Departments of Molecular Pathology
Important: Please read these instructions carefully The viability of these cells is warranted for 30 days from date of shipment when specified reagents and growth conditions are followed as described in
Protein kinase C alpha expression and resistance to neo-adjuvant gemcitabine-containing chemotherapy in non-small cell lung cancer Dan Vogl Lay Abstract Early stage non-small cell lung cancer can be cured
microrna 2 micrornas Non protein coding, endogenous RNAs of 21-22nt length Evolutionarily conserved Regulate gene expression by binding complementary regions at 3 regions of target mrnas Act as negative
BAP1 germline mutations A new Cutaneous Nevus Melanoma Syndrome Thomas Wiesner Disclosure Listed as co-inventor US patent application US 61/463,389 BAP1 mutational analysis in determining susceptibility
Dr. Daniel Duvall, Medical Officer Center for Medicare, Hospital and Ambulatory Policy Group Centers for Medicare and Medicaid Services 7500 Security Boulevard Baltimore, Maryland 21244 Dr. Edith Hambrick,
Monoclonal Antibodies in Cancer Ralph Schwall, PhD Associate Director, Translational Oncology Genentech, Inc. Disclaimer I had nothing to do with Herceptin Using lessons learned in new antibody projects
PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 8 Recombinant DNA Technology The Role of Recombinant DNA Technology in Biotechnology Biotechnology
MALIGNANT PLEURAL FLUIDS AID MESOTHELIOMA CELL GROWTH AND PROPAGATION HUI MIN CHEAH PhD Candidate School of Medicine and Pharmacology University of Western Australia Lung Institute of Western Australia
ALCHEMIST (Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing Trials) 3 Integrated Trials Testing Targeted Therapy in Early Stage Lung Cancer Part of NCI s Precision Medicine Effort in
Vol. 0, 6759 6763, October 5, 004 Clinical Cancer Research 6759 Perspective Evaluating the Efficiency of Targeted Designs for Randomized Clinical Trials Richard Simon and Aboubakar Maitournam Biometric
Genomic Medicine The Future of Cancer Care Shayma Master Kazmi, M.D. Medical Oncology/Hematology Cancer Treatment Centers of America Personalized Medicine Personalized health care is a broad term for interventions
Mabsolus-2015-UK:Mise en page 1 03/07/15 14:13 Page1 Services provider Department of MABSolys from conception to validation MAB Solut is involved at each stage of your project Creation of antibodies Production
Chromatin Immunoprecipitation A) Prepare a yeast culture (see the Galactose Induction Protocol for details). 1) Start a small culture (e.g. 2 ml) in YEPD or selective media from a single colony. 2) Spin
for lung cancer Outcomes in advanced lung cancer have seen meaningful improvement in the past decade thanks to new precision drug therapies. Because tumors usually develop resistance to the drugs, scientists
Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR) Goals of the lab: 1. To understand how DNA s chemical properties can be exploited for purification 2. To learn practical applications of DNA purification
Overview of Mesothelioma Raffit Hassan, M.D. Mesothelioma Is an aggressive tumor that arises from the serosal surfaces of body cavities In 1960, Wagner reported increased incidence of mesothelioma in asbestos
Pharmacogenomic markers in EGFR-targeted therapy of lung cancer Rafal Dziadziuszko, MD, PhD University of Colorado Cancer Center, Aurora, CO, USA Medical University of Gdansk, Poland EMEA Workshop on Biomarkers,
Cytology : first alert of mesothelioma? Professor B. Weynand, UCL Yvoir, Belgium Introduction 3 cavities with the same embryologic origin the mesoderme Pleura Exudates Pleura Peritoneum Pericardium 22%
DATA SUPPLEMENT EXPRESSION OF TUMOR-DERIVED VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IS ASSOCIATED WITH OUTCOME IN EARLY SQUAMOUS CELL CARCINOMA OF THE LUNG María J. Pajares et al. PATIENTS
Brochure More information from http://www.researchandmarkets.com/reports/296370/ Future Oncology: Technology, Products, Market and Service Opportunities Description: Future Oncology is an analytical newsletter
Future Directions in Clinical Research Karen Kelly, MD Associate Director for Clinical Research UC Davis Cancer Center Outline 1. Status of Cancer Treatment 2. Overview of Clinical Research at UCDCC 3.
Nuevas tecnologías basadas en biomarcadores para oncología Simposio ASEBIO 14 de marzo 2013, PCB Jose Jimeno, MD, PhD Co-Founder / Vice Chairman Pangaea Biotech SL Barcelona, Spain PANGAEA BIOTECH BUSINESS
Pleurectomy/decortication, hyperthermic pleural lavage with povidone-iodine and systemic chemotherapy in malignant pleural mesothelioma. A 10-year experience. L Lang-Lazdunski, A Bille, S Marshall, R Lal,
Successes and Limitations of Targeted Cancer Therapy in Lung Cancer Kenichi Suda a, b Tetsuya Mitsudomi a a Division of Thoracic Surgery, Department of Surgery, Kinki University Faculty of Medicine, Osaka-Sayama,
July 2013 Edition Vol. 7, Issue 7 Breast and Lung Cancer Biomarker Research at ASCO: Changing Treatment Patterns By Julie Katz, MPH, MPhil Biomarkers played a prominent role in the research presented in
Ganetespib and Docetaxel in NSCLC Suresh S. Ramalingam, MD Associate Professor Director, Division of Medical Oncology Emory University Winship Cancer Institute 1 Role of Docetaxel in Advanced NSCLC Docetaxel
1 page Overview 21 Oct Tuesday 1500 on REGISTRATION 1800 Welcome Reception & Cocktails at the Cape Town International Conference Centre (CTICC) 22 Oct Wednesday 0730 REGISTRATION 0830 OPENING 0900 PLENARY
Supplemental Text/Tables PCR Amplification and Sequencing PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Foster City, CA). Each PCR reaction contained 20 ng genomic
THE His Tag Antibody, mab, Mouse Cat. No. A00186 Technical Manual No. TM0243 Update date 01052011 I Description.... 1 II Key Features. 2 III Storage 2 IV Applications.... 2 V Examples - ELISA..... 2 VI
Major Advances in Cancer Prevention, Diagnosis and Treatment~ Why Mesothelioma Leads the Way H. Richard Alexander, Jr., M.D. Department of Surgery and The Greenebaum Cancer Center University of Maryland
Mesothelioma 2014 Paul Baas Department of Thoracic Oncology The Netherlands Cancer Institute Amsterdam Disclosures Grants from Pfizer and Roche Advisor for MSD and Verastem Once upon a time. http://amlbenzene.net/diseases-asbestos.htm
The Need for a PARP in vivo Pharmacodynamic Assay Jay George, Ph.D., Chief Scientific Officer, Trevigen, Inc., Gaithersburg, MD For further infomation, please contact: William Booth, Ph.D. Tel: +44 (0)1235
PNAClamp BRAF Mutation Detection Kit For in vitro diagnostic use Instruction manual for product # PNAC-2001 Version 1.2 Store at -20 C. Instruction Version: Ver. 1.2 Date of Revision: 2012 February 1 /
PR110 G-Biosciences 1-800-628-7730 1-314-991-6034 firstname.lastname@example.org A Geno Technology, Inc. (USA) brand name Dot Blot Analysis Teacher s Guidebook (Cat. # BE 502) think proteins! think G-Biosciences
EARLY ONLINE RELEASE Note: This article was posted on the Archives Web site as an Early Online Release. Early Online Release articles have been peer reviewed, copyedited, and reviewed by the authors. Additional
Gebruik van predictieve markers voor targeted therapy in de algemene praktijk Gerrit A. Meijer, MD, PhD Professor of Pathology Chair of the Department of Pathology VU Universtiy Medical Center Amsterdam
Gene mutation and molecular medicine Chapter 15 Lecture Objectives What Are Mutations? How Are DNA Molecules and Mutations Analyzed? How Do Defective Proteins Lead to Diseases? What DNA Changes Lead to
بسم هللا الرحمن الرحيم Updates in Mesothelioma By Samieh Amer, MD Professor of Cardiothoracic Surgery Faculty of Medicine, Cairo University History Wagner and his colleagues (1960) 33 cases of mesothelioma
Advances in Treatment of Malignant Pleural Mesothelioma: A Reason for Hope Daniel H. Sterman, M.D. Associate Professor of Medicine and Surgery Co-Director, PENN Mesothelioma and Pleural Program University
Provisional Translation (as of January 27, 2014)* November 15, 2013 Pharmaceuticals and Bio-products Subcommittees, Science Board Summary of Discussion on Non-clinical Pharmacology Studies on Anticancer
Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN
http://www.springer.com/3-540-22006-2 1 Molecular Pathology Laboratory of the Future Christopher A. Moskaluk 1.1 The Past The integration of laboratory analysis with human medicine has traditionally been
Biotechnology Chapter 20: Biotechnology: DNA Technology & Genomics The BIG Questions How can we use our knowledge of DNA to: o Diagnose disease or defect? o Cure disease or defect? o Change/improve organisms?
Forensic DNA Testing Terminology ABI 310 Genetic Analyzer a capillary electrophoresis instrument used by forensic DNA laboratories to separate short tandem repeat (STR) loci on the basis of their size.
Supporting Information Kondo et al. 1.173/pnas.787415 SI Methods Conventional and Quantitative RT-PCR. Total RNA was extracted from cultured ES cells or ES-derived cells using the RNeasy Minikit (Qiagen).
Protocol 1101-1088 Phase I study of intra-pleural administration of GL-ONC1 in patients with malignant pleural effusion: primary, metastases and mesothelioma Principal Investigator: Valerie W. Rusch, MD,
IFU022 v140202 Research Use Only Instructions For Use Part III Data Analysis for Ion Torrent Sequencing MANUFACTURER: Multiplicom N.V. Galileilaan 18 2845 Niel Belgium Revision date: August 21, 2014 Page
El papel de la anatomía patológica en oncología: presente y futuro The role of pathological anatomy in oncology: present and future Federico Rojo Fundación Jiménez Díaz, Madrid All patients with same diagnosis:
Technical Manual ECL Western Blotting Substrate INSTRUCTIONS FOR USE OF PRODUCTS W1001 AND W1015. PRINTED IN USA. 6/09 ECL Western Blotting Substrate All technical literature is available on the Internet
Chromosomal analysis for prenatal samples, peripheral blood, bone marrow biopsy samples, various types of lymphomas, and solid tumors FISH (fluorescence in situ hybridization) assays and many probes that
Lectures 26 and 27 recombinant DNA technology I. oal of genetics A. historically - easy to isolate total DNA - difficult to isolate individual gene B. recombinant DNA technology C. why get the gene? 1.
User Manual Ordering information: (for detailed kit content see Table 2) EdU Flow Cytometry Kits for 50 assays: Product number EdU Used fluorescent dye BCK-FC488-50 10 mg 6-FAM Azide BCK-FC555-50 10 mg
Protocol for Western Blotting Materials Materials used on Day 3 Protease inhibitor stock: 1 μg/μl pepstatin A in DMSO 200 μm leupeptin in OG Buffer 200 mm PMSF: Freshly made. Ex) 34.8 mg PMSF in 1 ml isopropanol
An update in Mesothelioma and Thymoma Management ASCO 2014 update Khalid Alsaleh King Saud University FRCPC, FACP, MSc Mesothelioma Outline Outline Presented By Marjorie Zauderer at 2014 ASCO Annual Meeting
Shristi Pandey Genomics and Medicine Winter 2011 Prof. Doug Brutlag Chronic Myeloid Leukemia: A look into how genomics is changing the way we treat Cancer. Until the late 1990s, nearly all treatment methods
Stem Cell Research Serum- and feeder-free media StemPro hesc SFM Human Embryonic Stem Cell Culture Medium Stem Cell Research StemPro hesc SFM a fully defined, serum- and feederfree medium (SFM) specially