Patent Application
20120058108
The present invention relates to the fields of genetics and oncology and provides methods for detecting tumors as well as methods for treating patients and predicting the prognosis to a patient.
Specifically, the present invention relates to a method of demonstrating the malignant character of a tumor or cell subpopulation in a subject, to a method of predicting a prognosis, to a method of treating a subject having a tumor with NAV3 copy number change and with over expression of at least one gene or gene product selected from specific lists, and to a method of selecting a treatment to a subject. The present invention also relates to uses of NAV3 gene or gene product and at least one gene and/or gene product selected from specific lists for demonstrating the malignant character of a tumor or cell subpopulation, for predicting a prognosis to a subject, for selecting a treatment to a subject, and for cancer therapy in a subject having a tumor with NAV3 copy number change. Furthermore, the present invention also relates to a use of an antagonist, antibody or inhibitory molecule of at least one gene and/or gene product selected from specific lists for cancer therapy in a subject.
Still, the present invention relates to a diagnostic kit comprising tools for detecting NAV3 copy number change in a biological sample and tools for detecting over expression of at least one gene or gene product selected from specific lists in a biological sample. The present invention also relates to a use of a diagnostic kit of the invention for demonstrating the malignant character of a tumor or cell subpopulation, for predicting a prognosis to a subject with a colorectal tumor, brain tumor or tumor of epidermal keratinocytes and for selecting a treatment to a subject with a colorectal tumor, brain tumor or tumor of epidermal keratinocytes.
Cancer progression is characterized by the development of chromosomal instability, aneuploidy, and a series of acquired genetic aberrations that affect genes important for cell growth and survival. A single gene may affect several critical pathways and contribute to the conversion of a normal cell to a cancer cell, involving a stepwise process requiring the activation of oncogenes and inactivation of tumor-suppressor genes. Recent technology has enabled the identification of even rare mutations that contribute to the development of cancer.
Colorectal cancer (CRC) development via benign precursor lesions, adenomatous polyps, along with the accumulation of genetic and epigenetic changes is one of the best-known examples of multistep carcinogenesis. Recent evidence also suggests that tissue microenvironment is of profound importance for the growth potential and spread of tumour cells (Kim B. G. et al. 2006, Nature 441: 1015-9; Reuter J. A. et al. 2009, Cancer Cell 15: 477-88) and such an effect may be driven by chronic inflammation. CRC is known to be linked to inflammatory bowel disease (IBD).
The overwhelming majority of colorectal cancers display one of the two major genomic instability phenotypes, microsatellite instability (MSI) or chromosomal instability (CIN), also called microsatellite stable (MSS). About 85% of CRC are related to MSS and exhibit aneuploidy and loss of heterozygosity (LOH), and adenomatous polyposis coli (APC) or beta-catenin mutations are the most common early molecular aberrations in this phenotype. These mutations lead to aberrant Wnt pathway activation, which is thought to initiate colon adenoma formation. Several signaling pathways are involved in the development of sporadic colon cancers. Thus, it takes several years or decades to develop colon cancer even if mutations of the adenomatous polyposis coli (APC) gene are present. Activation of KRAS is needed, too, for adenoma progression to carcinomas as a second step (Phelps R. A. et al. 2009, Cell 37: 623-34). It has also been shown that the loss of functional APC induces aneuploidy in vivo through transient tetraploidy (Caldwell C. M. et al. 2007, J Cell Biol 178: 1109-20), which may enhance fitness of cells containing broken or rearranged chromosomes.
We have previously shown that chromosome 12q21 aberrations, specifically allelic loss of neuron navigator 3 (NAV3) gene, associate with several subtypes of cutaneous T-cell lymphoma (CTCL) (Karenko L. et al. 2005, Cancer Res 65: 8101-10; Hahtola S et al. 2008, J Invest Dermatology 128: 2304-9; WO2008059112 A1) and CTCL-associated lung cancers (Hahtola S et al. 2008, Genes Chromosomes Cancer 47: 107-17). More recently, NAV3 mutations or copy number changes, respectively, have been reported in melanoma (Bleeker F. et al. 2008, Human Mutation 29: E451-59), pancreatic cancer (Bleeker F. et al. 2009, Hum Mutat 30 (2): E451-9) and in human glioblastomas (Nord H. et al. 2009, Neuro Oncol Mar20). In addition, NAV3 has been found to be the only gene that was significantly differently expressed (down-regulated) in adrenal carcinoma compared to adrenocortical adenomas (Soon P. H. et al. 2009, ERC). Within the cancer gene landscape, NAV3 belongs to the “hill type” candidate cancer (CAN) genes commonly mutated in human breast and colon cancer (Wood L. et al. 2007, Science 318: 1108-13).
It is considered increasingly important to concentrate research efforts on the identification of pathways affected by genetic aberrations, and thereby providing further tools for diagnosing diseases and for designing specific pharmaceuticals for individualized medicine. Now, we have found surprising and previously unknown correlations of NAV3 aberrations with specific pathways and genes related thereto. Thus, we provide novel targets for cancer treatment and novel means and methods for diagnosing or following up cancer.
Novel biomarkers or combinations of biomarkers for providing more effective and early diagnosis of potentially aggressive tumors as well as identifying tumors susceptible to targeted therapies are warranted. The present invention provides a specific solution for predicting or identifying tumor and carcinoma progression. The present invention also discloses a tool for evaluating clinical aggressiveness of tumors and patient survival. Furthermore, the invention provides new therapeutic targets for carcinoma therapy.
The object of the invention is thus to provide novel methods and means for demonstrating the malignant character of a tumor and staging and monitoring a cancer, such methods and means allowing an accurate diagnosis of the disease.
Other objects of the invention are to provide novel methods and means for predicting tumor initiation or tumor progression as well as predicting a prognosis for a patient, selecting a treatment and treating a patient having a cancer. The methods and means of the present invention are specific and reliable and they utilize personalized medicine. The methods and means allow a well targeted therapeutic intervention, which may be life-saving.
Still another object of the invention is to provide novel biomarkers and combinations of biomarkers useful in detecting the cancer as well as in prognosis assessments.
Demonstrating NAV3 aberrations, linked to lymph node metastasis and to relevant inflammation and cell proliferation pathways, will provide a relatively simple and affordable tool to monitor cancer, specifically a cancer of colorectum, brain or epidermal keratinocytes. NAV3 regulates the expression of other genes and signaling pathways that have an effect in the malignant trans-formation and/or in the biological behavior of the malignant tumor. Therefore, NAV3 affected pathways as well as different combinations thereof are also targets for the design of anti-cancer therapies.
NAV3 copy number changes, especially NAV3 deletion, is directly involved in early carcinogenesis and opens up novel possibilities for more accurate diagnostics but also for rational therapeutic approaches.
The present invention relates to a method of demonstrating the malignant character of a tumor or cell subpopulation in a subject, the method comprising:
i) determining NAV3 copy number change in a biological sample from the subject; and
ii) determining over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin, and those listed in tables 8-12, in the biological sample or another biological sample from the subject;
iii) demonstrating the malignant character of a tumor or cell subpopulation in a subject, when both NAV3 copy number change and over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 are present in the sample(s) from the subject.
The present invention further relates to a method of treating a subject having a tumor with NAV3 copy number change and with over expression of at least one gene or gene product selected from IL23R, GnRHR, betacatenin and those listed in tables 8-12, comprising a step, wherein at least one gene or gene product with over expression is affected.
The present invention further relates to a method of predicting a prognosis comprising:
i) determining NAV3 copy number change in a biological sample from a subject;
ii) determining over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12, in the biological sample or another biological sample from the subject; and
iii) predicting a prognosis to the subject having both NAV3 copy number change and over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 in the sample(s).
The present invention further relates to a method of selecting a treatment to a subject, comprising:
i) determining NAV3 copy number change in a biological sample from the subject;
ii) determining over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12, in the biological sample or another biological sample from the subject; and
iii) selecting a treatment to the subject having both NAV3 copy number change and over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 in the sample(s).
The present invention further relates to a use of NAV3 gene or gene product and at least one gene and/or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 for demonstrating the malignant character of a tumor or cell subpopulation with NAV3 copy number change and with over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12, in a subject.
The present invention further relates to a use of NAV3 gene or gene product and at least one gene and/or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 for predicting a prognosis to a subject.
The present invention further relates to a use of NAV3 gene or gene product and at least one gene and/or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 for selecting a treatment to a subject.
The present invention further relates to a use of at least one gene and/or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 for cancer therapy in a subject having a tumor with NAV3 copy number change.
The present invention further relates to a use of an antagonist, anti-body or inhibitory molecule of at least one gene and/or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 for cancer therapy in a subject having a tumor with NAV3 copy number change and with over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12.
The present invention further relates to a diagnostic kit comprising tools for detecting NAV3 copy number change in a biological sample and tools for detecting over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 in a biological sample.
The present invention further relates to a use of a diagnostic kit of the invention for demonstrating the malignant character of a tumor or cell subpopulation.
The present invention further relates to a use of a diagnostic kit of the invention for predicting a prognosis to a subject with a colorectal tumor, brain tumor or tumor of epidermal keratinocytes.
Still, the present invention further relates to a use of a diagnostic kit of the invention for selecting a treatment to a subject with a colorectal tumor, brain tumor or tumor of epidermal keratinocytes.
In the following the invention will be described in greater detail by means of preferred embodiments with reference to the attached drawings, in which
We now report that NAV3 copy number changes are frequently found in MSS type CRC, in colon adenomas, in brain tumors, in epidermal keratinocytes and in several established cell lines. NAV3 aberrations correlated with chromosome 12 polysomy and with lymph node involvement. Furthermore, specific siRNA gene silencing of NAV3 and expression array analyses revealed at least two important target molecules for NAV3.
All of the up-regulated genes or their gene products listed in the present application can be utilized in the development of novel diagnostic principles. In addition to determining NAV3 copy number, expression of at least one gene or gene product is studied in the methods of the present invention. As used herein the expression “gene product” refers to an mRNA, protein or to any product achieved from the gene. In a specific embodiment of the invention, the gene product is a protein.
For proteins that are secretory, methods to test their increased level in patient blood can be used as a diagnostic method. For non-secretory proteins, immunohistochemistry will be the most appropriate method to be used.
Diagnostic methods can be carried out in vitro, in vivo or ex vivo. In a specific embodiment of the invention, the method is in vitro method.
In a specific embodiment of the invention, a diagnostic kit of the invention is for demonstrating the malignant character of a tumor or cell subpopulation. Tools for detecting NAV3 copy number change and/or over expression of any gene or gene product may comprise suitable probes, primers or antibodies.
In a specific embodiment of the invention, the tumor is a colorectal tumor, brain tumor or tumor of epidermal ceratinocytes.
Most primary brain tumors originate from glia (gliomas) such as astrocytes (astrocytomas), oligodendrocytes (oligodendrogliomas), or ependymal cells (ependymoma). There are also mixed forms, with both an astrocytic and an oligodendroglial cell component. These are called mixed gliomas or oligoastrocytomas. Also, mixed glio-neuronal tumors (tumors displaying a neuronal, as well as a glial component, e.g. gangliogliomas, disembryoplastic neuroepithelia) tumors) and tumors originating from neuronal cells (e.g. gangliocytoma, central gangliocytoma) can be encountered. Other varieties of primary brain tumors include: primitive neuroectodermal tumors (PNET, e.g. medulloblastoma, medulloepithelioma, neuroblastoma, retinoblastoma, ependymoblastoma), tumors of the pineal parenchyma (e.g. pineocytoma, pineoblastoma), ependymal cell tumors, choroid plexus tumors, neuroepithelial tumors of uncertain origin (e.g. gliomatosis cerebri, astroblastoma), etc. In a specific embodiment of the invention the brain tumor is a glioma, i.e. glioblastoma.
As used herein the expression “tumor” refers to an abnormal mass of tissue due to abnormal excess of cells divisions or lack of normal cell death.
In a specific embodiment of the invention, the tumor is a benign tumor or a malignant tumor, i.e. not cancerous tumor or cancerous tumor. The expression “adenoma” refers to a noncancerous tumor. In a specific embodiment of the invention, the tumor is a carcinoma. Carcinomas are malignant tumors derived from epithelial cells.
Methods of the present invention demonstrate the malignant character of a tumor. Malignant character refers to a malignant tumor, i.e. cancerous tumor. Malignant tumors may be aggressive i.e. fast growing and possibly metastasizing. Any “cell subpopulation”, referring to a restricted population of cells, can be studied for malignant characters. The cell subpopulation may locate within the tissue-infiltrating inflammatory cells.
In the present invention, a biological sample can be any suitable tissue sample, such as biopsy from the tissue or lymph node or a metastatic tumor lesion in any body organ or whole blood. The biological sample may also be urine, stool or other body excretion. The biological sample can be, if necessary, pretreated in a suitable manner known to those skilled in the art.
The present invention also relates to a prediction of a prognosis. In a specific embodiment of the invention, a presence of a tumor with NAV3 copy number change and with over expression of at least one gene or gene product selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 is associated with lymph node metastases, high grade malignancy and/or poor survival. In a specific embodiment of the invention the prognosis is poor. “Poor prognosis” refers to a high expectancy of metastasis or tumor spread, unresponsiveness to state-of-the-art treatment modalities and/or poor survival. In a patient with predicted poor survival, the life expectancy is less than in a corresponding comparative patient group.
In the clinical settings, the capability to diagnose precisely not only the type of a tumor but the genetic, functional and immunologic changes that are present in that particular tumor must be enhanced. For more detailed therapy, the choice of therapeutic molecules or antibodies can then be tailored to that particular tumor. In other words, personalized medicine is needed with a combination of at least two drugs targeting different molecules.
As a whole, the present invention reveals several exploitable results that can be further used in the development of effective novel therapeutic principles and diagnostics. The finding of up-regulation of the genes, which are up-regulated in the NAV3 deficiency and are involved in malignant transformation, can be used in selecting rational therapy for a given cancer.
The decision which of the therapeutic alternatives should be used, i.e. a selection of a treatment, is based on the testing of the malignant tumor for NAV3 copy number changes (deletion of the NAV3 gene leading to decreased expression of NAV3) and for the upregulation of any one of the genes or gene products selected from IL23R, GnRHR, beta-catenin, those listed in tables 8-12 and the IL23/IL23R as well as the GnRHR pathways.
In a specific embodiment of the invention, the method of treatment further comprises a step, wherein the gene(s) or gene product(s) selected from IL23R, GnRHR, beta-catenin and those listed in tables 8-12 is affected by underexpressing or inactivating the gene(s) or gene product(s) or decreasing amount of the gene product(s).
In a specific embodiment of the invention the method of treatment further comprises a step, wherein a gene or gene product of NAV3 is affected.
In a specific embodiment of the invention, the gene or gene product of NAV3 is affected by overexpressing or activating the gene or gene product or increasing amount of the gene product. In a specific embodiment of the invention, the gene(s) and/or gene product(s), i.e. selected from NAV3, IL23R, GnRHR, beta-catenin and those listed in tables 8-12, is activated, inactivated, overexpressed or underexpressed, or amount of the gene product is increased or decreased. In a specific embodiment of the invention, at least GnRH and/or JAK/STAT signalling pathway is affected.
Any therapeutic molecule or molecules can be used for affecting the target gene products. In a specific embodiment of the invention, an antagonist, antibody or inhibitory molecule is used for affecting at least one gene product. “An inhibitory molecule” refers to any molecule (e.g. small inhibitory RNA molecule or antibody), which inhibits or reduces the action of a target. In a specific embodiment of the invention, the inhibitory molecule is siRNA. The up-regulation of the membrane associated proteins is of special interest, as antibody mediated therapy is presently widely used and the methods to generate humanized target specific therapeutic antibodies for target molecules are readily available.
A method of treatment may be in a form of a conventional medicament or may be given as gene therapy. In a specific embodiment of the invention, the method of treatment is gene therapy.
In therapy, restoration of the normal function of a gene can be used. This may be reached by enhancing the expression of functionally homologous genes, by introducing an intact gene or by using an altered form of the gene or antisense oligonucleotide against a gene in any technique presently available for gene therapy to prevent the progression of a proliferating disease. In particular, tumor cell growth may be slowed down or even stopped by such therapy. Such techniques include the ex vivo and in situ therapy methods, the former comprising transducing or transfecting an intact or altered gene (or its functional domains) in a recombinant or peptide form or as antisense oligonucleotides or in a vector to the patient, the latter comprising inserting the altered gene or oligonucleotide into a carrier, which is then introduced into the patient. Depending on the disease to be treated, a transient cure or a permanent cure may be achieved. Alternatively, monoclonal or humanized antibodies or peptides binding to a target protein can be used to suppress the function of the protein and thus tumor cell growth may be slowed down or even stopped. Anti-bodies against a target protein could also be used to carry other agents, such as cytotoxic substances, to the cancer cells over-expressing the gene. Such agents could then be used to kill specifically the cancer cells.
A treatment may target a gene or gene product of NAV3 and/or any gene(s) or gene product(s) listed in the present application. However, any other gene(s) or gene product(s) along the GnRH and Jak/Stat pathway may also be used as targets for therapy.
A therapeutic molecule such as an antagonist, antibody or inhibitory molecule may be administered alone or in combination with other agents or compositions. In addition to any therapeutic molecule, a pharmaceutical composition administered to a patient may also comprise any other therapeutically effective agents, pharmaceutically acceptable carriers, buffers, excipients, adjuvants, antiseptics, filling, stabilising or thickening agents, and/or any components normally found in corresponding products.
The pharmaceutical composition may be in any form, such as solid, semisolid or liquid form, suitable for administration. The administration of a medicament may for example be conducted through an oral or inhalable administration or through an intratumoral, intramuscular, intra-arterial or intravenous injection.
The subject for diagnosis, treatment or any other method or use of the invention is a human or an animal. In a specific embodiment of the invention, the subject is a human. In a specific embodiment of the invention, the subject has a colorectal adenoma or colorectal cancer. In another specific embodiment of the invention, the subject has a colorectal tumor, brain tumor or tumor of epidermal keratinocytes.
NAV3 (neuronal navigator 3, also called POMFIL1) is a spliced gene (40 exons) located in c12q21.1 and expressed in brain tissue, activated T cells, placenta, colon, and in certain cancer cell lines. The amino acid sequence of NAV3 is well conserved among different species, indicating that NAV3 plays a fundamental role in cellular processes. Amino acid sequence homology suggests that NAV3 participates in cell signaling and tumor suppression. NAV3 is the human homolog of unc-53 protein of C. Elegans, a critical mediator of cell migration. Also, NAV3 homologues NAV1 and NAV2 bind to intracellular filaments and regulate cell expansion and migration.
Accumulating evidence suggests that disruption of the NAV3 gene contributes to cancer progression. The NAV3 transcript expression is downregulated in 40% of primary neuroblastomas (Coy J F et al. 2002, Gene 290 (1-2):73-94). NAV3 is also deleted or translocated in several subtypes of cutaneous T-cell lymphoma (CTCL) (Karenko L. et al. 2005, Cancer Res 65(18):8101-10; Hahtola S. et al. 2008, J Invest Dermatol 128(9):2304-9, Vermeer M. H. et al. 2008, Cancer Res 68(8):2689-98). We have also identified NAV3 gene copy number changes (deletions/amplifications) in cancers of epithelial origin (Hahtola S et al. 2008, J Invest Dermatol 128(9):2304-9; Hahtola S et al. 2008, Genes Chromosomes Cancer 47(2):107-17; WO 2008059112 (A1)), and allelic NAV3 deletions in colorectal cancer (Sipila L et al. 2008, EJC supplements 6 (12) 118).
In a specific embodiment of the invention, NAV3 copy number change is caused by a deletion, amplification or translocation of NAV3 gene or major part of it. The copy number changes of NAV3 gene can be determined in haploid, diploid and/or polyploid cells.
“Deletion” refers to an absence of any fragment(s) of a gene, which absence adversely affects the function of the gene. “Deletion” also refers to the absence of a whole gene or the absence of a chromosomal fragment containing the gene. In a specific embodiment of the invention, NAV3 deletion is a total or partial deletion of NAV3.
“Amplification” refers to an insertion of any fragment(s) of a gene in the presence of at least one copy of that gene. “Amplification” may also refer to the amplification of a whole gene or a chromosomal fragment containing the gene. In a specific embodiment of the invention, NAV3 amplification is a total or partial amplification of NAV3. Amplification of a gene can for example be detected by a copy number change of the gene.
“Translocation” refers to a transfer of chromosomal regions between non-homologous chromosomes. NAV3 may be translocated partly, totally or as a part of the larger chromosomal fragment comprising NAV3.
According to the method of the present invention, the presence or absence of gene copy number change can be detected from a biological sample by any known detection method suitable for detecting deletions or amplifications. Such methods are easily recognized by those skilled in the art and include fluorescence in situ hybridisations, such as multi-colour fluorescence in situ hybridisations, multi-fluor in situ-hybridisation (MFISH), spectral karyotyping (SKY), Combined binary ratio labelling (COBRA), colour changing karyotyping (CCK). In comparative genomic hybridization (CGH) the genetic changes are classified as DNA gains and losses. CGH reveals a characteristic pattern that includes aberrations at chromosomal and subchromosomal levels. The conventional G-banding techniques can also be used in cases were the coarse detection of gains or losses is regarded as sufficient. Preferable methods are those suitable for use in clinical laboratories.
Any known detection method suitable for detecting a gene expression of any gene or NAV3 copy number, i.e. methods based on detecting the copy number of the gene (or DNA) and/or those based on detecting the gene expression products (mRNA or protein) can be used in the methods of the present invention. Such methods are easily recognized by those skilled in the art and include conventional polymerase chain reaction (PCR)-methods, RT-PCR, in situ hybridisations, such as FISH, mRNA in situ hybridisation, Northern analysis, Southern and Western analyses, immunohistochemistry, and other immunoassays, such as ELISA. Preferable methods are those suitable for use in routine clinical laboratories.
LOH analysis may also be utilized for detecting gene copy number changes. As used herein the expression “loss of heterozygosity (LOH)” refers to the loss of a single parent's contribution to part of the cell's genome. LOH can be considered as an event to unmask a mutant allele of a gene which may play a role in suppressing tumor formation. Thus, LOH is an important marker for tumor initiation or progression. LOH in cancers can be identified by the presence of heterozygosity at a genetic locus in germline DNA and the absence of heterozygosity at the same locus in the tumor cells.
In a specific embodiment of the invention, NAV3 copy number change is confirmed by FISH, LOH, CGH, sequencing analysis, immunohistochemistry, PCR, qPCR or tissue microarray.
Markers suitable for detecting a gene expression or gene copy number changes include any biological markers such as microsatellite markers, SNP-markers, any probes, primers or antibodies associated with a target gene.
Results of the present study also confirm that aberrations of NAV3 are common in various tumors and cancers. We used three different methods to look for NAV3 copy number changes; LOH, array-CGH and FISH. With the FISH method, we observed cells with the copy number changes of chromosome 12 and the NAV3 gene in a substantial number of cases with colorectal carcinomas (CRC). Importantly, NAV3 deletion was also detected in 23% of adenoma samples. However, the findings in all cases showed a heterogenous population of tumour cells consisting mainly of cells with normal diploid character and abnormal cells with a variable number of chromosome 12 and/or NAV3 label. In this setting, an allelic deletion or amplification of the gene differs from e.g. the situation with mutations in tumor suppressor genes in homogeneous tumors. It is important to note that this type of copy number aberrations can be observed only with the FISH method, which analyses individual cells, but in most cases not with array-CGH or with LOH, two methods that require that over 40% of the cells in the sample to show the same type of aberration. Our findings from an unselected clinical series of tissue samples are underscored by the fact that similar chromosome 12 polysomy and NAV3 copy number changes were observed also in three established CRC cell lines of the MSS type and in one cell line of the MSI type.
In the cases of overt carcinoma, the cases with NAV3 aberration clearly showed more lymph node metastases.
In the present study, in the adenomatous polyps, NAV3 loss was more often seen in cases that by classical criteria (size, differentiation) indicated higher risk for subsequent malignant transformation.
To understand the effects of NAV3 downregulation on gene expression profiles and to characterize the effects of NAV3 deletion in vitro, we silenced NAV3 expression with RNA interference in normal colon epithelial cells, an established glioblastoma cell line, a newly derived glioblastoma cell line, and primary human keratinocytes devoid of human papilloma virus infection. Gene expression profiles at several time points after transfection were analyzed using Agilent dual-color 4×44K microarray. With this approach, NAV3 gene silencing led to the consistent upregulation of 39 genes in colon cells, whereas 28 genes were upregulated in the glial cells. Among these were IL23 and GnRH receptors, and PathwayExpress analyses suggested that the GnRH and Jak-Stat signaling pathways are targeted by NAV3. When the results from all cell lines were combined, 49 genes, including GnRHR and IL23R were upregulated in 7 of 10 samples, at least once in each celltype. Altogether, 17 genes including the gonadotropin releasing hormone receptor (GnRHR) and interleukin 23 receptor (IL23R) were upregulated in all eight experiments with colon and glial cells.
The siRNA silencing of NAV3 mimics the in vivo found allelic deletion and thus, the upregulation of the two receptor molecules, GnRHR and IL23R, known to be involved both in oncogenic processes as well as in inflammation/immune reactions, has potential importance both for the initiation of the malignant process as well as determining the biological behaviour of the malignant cells.
Importantly, in the MSS tumors, up-regulation of IL23R immunoreactivity correlated with Dukes' staging (p<0.001) and lymph node metastases (p<0.001), while nuclear beta-catenin correlated with only lymph node metastases (p=0.045). Logrank test identified up-regulated IL23R immunoreactivity as an unfavorable marker (p=0.009).
Furthermore, NAV3 depletion linked tissue inflammation to tumor growth by upregulating the inflammatory mediators IL23R, vanin 3, and CYSLTR2. Our findings thus suggest that NAV3 copy number changes are directly involved in early carcinogenesis since in a microenvironment of inflammation, up-regulated IL23R gives the malignant cell a growth advantage.
GnRHR is an autocrine gonadotropin hormone receptor that stimulates the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH) by pituitary gonadotrope cells (Yeung C M et al. 2005, Mol Hum Reprod 11(11):837-42. In addition to the pituitary gonadotrope cells, GnRHR is known to be expressed on the surface of lymphocytes, breast, ovary, and prostate cells. A multitude of recent reports demonstrate that the GnRH-GnRHR axis is active also in extra-pituitary cells, and upregulation of GnRHR has been observed in many cancers, especially in carcinomas (Harrison G S et al. 2004, Endocr Relat Cancer 11(4):725-48).
GnRHR activation is thought to lead first to the activation of immediate early genes (1E) that affect the beta-catenin and the T-cell factor (TCF) pathways (Salisbury T B et al. 2008, Mol Endocrinol 22(6):1295-303). Beta-catenin, a member of the canonical Wnt signaling pathway, also plays an essential role in transducing the GnRH signal (Salisbury T B et al. 2008, Mol Endocrinol 22(6):1295-303) through the inactivation of glycogen synthase kinase-3, which regulates beta-catenin degradation (Gardner S et al. 2009, Neuroendocrinology 89(3):241-51). On the other hand, several reports demonstrate that the increased expression levels of beta-catenins affect lymph node metastasis of tumors (Buhmeida A et al. 2008, APMIS 116(1):1-9).
In our patient material, nuclear beta-catenin expression was surprisingly found to associate with NAV3 aberrations and to correlate with lymph node metastases. Also, our identification of GnRH as a NAV3-regulated target molecule represents a candidate pathway for carcinoma prevention and therapy.
The identification of IL-23R is linked to JAK-STAT signaling and supports our hypothesis that the pro-inflammatory tissue microenvironment affects the growth potential and spread of tumor cells. The IL-23R ligand IL-23 is secreted by activated inflammatory cells, such as macrophages and dendritic cells. The IL-23R heterodimer is composed of an IL-12 receptor beta 1 (IL-12Rbeta1) and an IL-23R (related to IL-12Rbeta2) chain (Beyer B M et al. 2008, J Mol Biol 382(4):942-55); the latter of which was upregulated in NAV3 silenced cells. JAK2 and STAT3 both associate with IL-23R. JAK2 phosphorylates the receptor in response to IL-23 and activated STAT3 dimers translocate to the nucleus and bind to IL-23R in a ligand-dependent manner. The JAK/STAT pathway is also activated by mutations in upstream genes and has been shown to be constitutively activated in a number of human malignancies (Constantinescu S N et al. 2008, Trends Biochem Sci 33(3):122-31; Li W X. 2008, Trends Cell Biol 18(11):545-51).
Recent accumulating evidence suggests that IL-23 may redirect cytotoxic T-cell responses to tumor cells toward proinflammatory effector pathways, which nourish the tumor instead of fighting it (Langowski J L et al. 2006, Nature 442(7101):461-5). Consequently, IL-23R-expressing tumor cells are likely to gain a growth advantage and persist even in the presence of tumor-specific T cells. IL-23R has also been linked to inflammatory bowel disease (IBD). In children with early Crohn's disease, mucosal levels of IL12p40 and IL23R mRNA have been shown to be significantly higher than in late disease (Kugathasan S et al. 2007, Gut 56(12):1696-705). Interestingly, IBD has been associated with an increased risk of CRC and other cancers (Lakatos P L et al. 2008, World J Gastroenterol 14(25):3937-47; Hemminki K et al. 2008, Int J Cancer 123(6):1417-21).
The communication between tissue inflammatory cells and epithelial cells during cancer initiation was shown through the loss of Smad4-dependent signaling in T cells, leading to spontaneous epithelial cancers in the gastrointestinal tract of mice (Kim B G et al. 2006, Nature 441(7096):1015-9). The Smad 4 −/− T cells produced IL-6, which activates the JAK-STAT signaling pathway like the IL23/IL-23R complex. L-6/STAT3 signaling has also recently been shown to have a pivotal role in tumor formation in mouse models of colitis associated cancer (Grivennikov S et al. 2009, Cancer Cell 15(2):103-13).
In addition to GNRHR and 123R, five genes linked to carcinogenesis (ARL11, SMR3B, FAM107A, MFSD2 and BCLB6) and two genes linked to inflammation (CYSLTR2 and VNN3) were shown to be upregulated by NAV3 silencing in the present study. Polymorphisms in the MYCL1 LD region, including MFSD2, have been recently shown to affect lung cancer survival rates although MFSD2 genes show ethnic differences in allelic frequencies (Spinola M et al. 2007, Lung Cancer 55(3):271-7). It has been recently shown that epithelial vanin-1 controls inflammation-driven carcinogenesis in the colitis-associated colon cancer mouse model (Poyet et al, 2009), and both vanin-1 and vanin-3 expression levels are enhanced by proinflammatory cytokines in psoriasis skin cells (Jansen P A et al. 2009, J Invest Dermatol Mar 26). In this context, it is of interest to note that both psoriasis and celiac patient groups have an increased risk of cancer (Grulich A E and Vajdic C M. 2005, Pathology 37(6):409-19). The upregulation of the membrane associated protein cluster and their downstream targets are of special interest.
In the present invention, at least one gene or gene product is selected for methods, means or uses. In a specific embodiment of the invention, the gene(s) selected from IL23R, GnRHR, beta-catenin, and those listed in tables 8-12, codes for a protein, which is a membrane protein, a protein regulating cellular processes, or a purine nucleotide binding protein. In one specific embodiment of the invention, the gene or gene product is selected from a group consisting of ARL11, SMR3B, FAM107A, MFSD2, BCLB6, CYSLTR2, VNN3, GNGT1, DNER, GnRHR, IL23R, beta-catenin, JAK1 and JAK3. In another specific embodiment of the invention, the gene or gene product is IL23R or/and GnRHR.
In one specific embodiment of the invention, the gene or gene product combination is at least IL23R, GNRHR and beta-catenin. In a specific embodiment of the invention, the gene or gene product combination is IL23R, GNRHR and beta-catenin.
In one specific embodiment of the invention, the gene or gene product combination is all 49 genes or gene products listed in table 9. In another specific embodiment of the invention, the gene or gene product combination is all 17 genes or gene products listed in table 8. In one specific embodiment of the invention, the gene or gene product combination is all 14 genes or gene products listed in table 10. In one specific embodiment of the invention, the gene or gene product combination is all genes or gene products listed in table 11. In one specific embodiment of the invention, the gene or gene product combination is all genes or gene products listed in table 12.
The following examples are given for further illustration of the invention. It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims.
Surgical biopsy samples from 59 patients (61 CRC and 10 adenoma samples), operated on CRC at Mikkeli Central Hospital were studied by FISH, LOH and immunohistochemistry. The study was approved by the Ethical Review Board of Mikkeli Central Hospital and by The National Authority for Medicolegal Affairs. Histology of the formalin-fixed paraffin-embedded tissue samples was verified by an experienced pathologist (MH) and tumours, adenomas or normal mucosa were microdissected to get pure normal or at least 50% ratio of carcinoma or adenoma tissue. Paraffin embedded sections were cut at 50 μm thickness and nuclei were isolated for FISH analysis and DNA was purified for LOH analysis following standard protocols (Hyytinen E et al. 1994, Cytometry 16: 93-96; Isola J. et al. 1994, Am. J. Pathol 145: 1301-1308). All adenomas were MSS while 14 of the 56 carcinomas had high-degree MSI. Mononucleotide repeat markers BAT25 and BAT26 from the Bethesda panel (Boland et al., 1998, Cancer Res 58: 5248-5257), supplemented with five dinucleotide repeat markers (D12S1684, D12S326, D12S1708, D18S474, and D9S167), were used to determine the microsatellite instability (MSI) status. Samples with at least two unstable markers were considered to have MSI, whereas those with one unstable marker or none were regarded microsatellite-stable (MSS).
The following parameters were included in the statistical analyses: tumor grade, tumor stage by Dukes, and by TNM classification as defined by American Cancer Society (www.cancer.org), presence of lymph node metastases, follow-up time (months) and clinical outcome (alive, died of other reason or died of disease).
Samples for the FISH assay were prepared from 119 brain tumor cases. Cases consisted of 55 astrocytomas, 20 oligodendrogliomas, 13 ependymomas, 18 medulloblastomas, and 13 neuroblastomas. All tissue samples had been processed by routine formalin fixation and embedded in paraffin.
Paraffin embedded sections were cut at 50 μm thickness and nuclei were isolated for FISH analysis following standard protocols (Hyytinen E et al. 1994, Cytometry 16: 93-96; Isola J. et al. 1994, Am. J. Pathol 145: 1301-1308).
Cells, Cell Lines and Cell Cultures CRL-1541 and CRL-1539 normal colon cells (ATCC, Manassas, Va., USA), the glioblastoma cell line A172 derived from grade 4 glioblastoma, and primary epidermal keratinocytes (all from ATCC, Manassas, Va., USA) were grown as instructed by ATCC for no more than 30 passages. Also colorectal cancer cell lines CCL-228 (SW480), CCL-230 (SW403), CCL-248 (T84), RKO, LIM1215, and HCA7 (ATCC, Manassas, Va., USA) were grown as instructed by ATCC. Among the CRC cell lines, RKO, LIM1215, and HCA7 are known to be mismatch repair deficient and have MSI.
Fresh cells from a grade 4 multiform glioblastoma extracted perioperatively were used to derive the cell line 0205. Tumor tissue was mechanically dissociated, and cells were cultured in DMEM without added serum, supplemented with nutrient mixture F12 (Sigma-Aldrich, ST Louis, Mo., USA), Fungizone 2.5 μg/μl (Apothecon, NJ, USA), B27 (Gibco/Invitrogen, Carlsbad, Calif., USA), epidermal growth factor (EGF) 20 ng/ml (Sigma-Aldrich, ST Louis, Va., USA), basic fibroblast growth factor (bFGF) 40 ng/ml (Sigma-Aldrich, ST Louis, Va., USA), penicillin-streptomycin 100 U/ml, (Biowhittaker, Verviers, Belgium) and Glutamax (1×) (Gibco/Invitrogen, Carlsbad, Calif., USA). After five passages, a clonal cell population emerged from the bulk preparation, and aliquots were frozen. For all subsequent experiments, fresh aliquots were thawed, and only cells culture less than 20 passages were used. Neuronal markers (glial fibrillary acidic protein and neuronal class beta-tubulin) expression was assessed by immunostaining. Harvesting of cells was approved by the Ethical committee of the Helsinki and Uusimaa hospital district, and written informed consent was acquired from patients.
Cell lines CRL-1541, CRL-1539 and A172 were studied for NAV3 copy number changes with multicolor FISH as previously described (Karenko L et al. 2005, Cancer Res 65:8101-10). FISH analysis was also performed for the tumor tissue from which the 0205 cell line was established.
Furthermore, NAV3-specific FISH assay was performed on nuclei isolated from patient samples (61 CRC and 10 adenoma) and on metaphase chromosomes of colon carcinoma cell lines (CCL-228, CCL-230, CCL-248, RKO, LIM1215 and HCA7). Bacterial artificial chromosome (BAC) clones specific to NAV3 DNA (RP11-36P3 and RP11-136F16; Research Genetics Inc., Huntsville, Ala., USA) and the chromosome 12 centromere probe (pA12H8; American Type Cell Culture) were used and labelled either with Alexa 594-5-dUTP and Alexa 488-5-dUTP (Invitrogen), respectively (patient samples) or with dioxigenin or biotin (metaphases from cell lines). The detailed methods for probe labelling, the preparation of slides for the FISH assay, and the use of arm-specific MFISH are shown below.
Also, NAV3-specific FISH assay was performed on nuclei isolated from samples of 119 brain tumor cases. The detailed methods for probe labelling, the preparation of slides for the FISH assay, and the use of arm-specific MFISH are shown below.
Probe labeling. For analyzing patient samples, two bacterial artificial chromosome (BAC) clones specific to NAV3 DNA (RP11-36P3 and RP11-136F16; Research Genetics Inc., Huntsville, Ala., USA) and the chromosome 12 centromere probe (pA12H8; American Type Cell Culture) were labeled with Alexa 594-5-dUTP and Alexa 488-5-dUTP (Invitrogen), respectively, using nick translation. BAC and centromere probes were mixed together with human COT-1 DNA (Invitrogen), precipitated and diluted into hybridization buffer (15% w/v dextran sulphate, 70% formamide in 2×SSC, pH 7.0). For analyzing cell lines NAV3 specific BAC-probes (see above) were prepared and labeled with digoxigenin and centromere 12-specific probe was prepared and labeled with biotin or dUTP conjugated with fluorescein isothiocyanate (FITC) by nick translation as described previously (Karenko et al. 2005) or similarly with Diethylaminocoumarin-5-dUTP (DEAC, Perkin Elmer Life and Analytical Sciences, Boston, Mass., USA).
FISH methods. Nuclei slides were pretreated with 1 M sodium thiocyanate at +80° C. for 5 minutes, washed three times with 2×SSC, treated with 50% glycerol, 0.1×SSC at +90° C. for 6 minutes, washed with 2×SSC for 3 minutes and with distilled water three times for 2 minutes. Slides were digested with proteinase K (Sigma; 8 μg/ml in 20 mM Tris-HCl, pH 7.5, 2 mM CaCl2) at +37° C. for 8 minutes. After dehydration and air drying, probe mix was added, slides were denatured for 6 min at +85° C. and hybridized for 48 hr at +37° C. Slides were washed three times with 1.5 M Urea, 0.1×SSC at +47° C. for 10 minutes, with 0.1×SSC for 10 minutes at +47° C., three times with PBS, 0.1% NP-40 at room temperature, rinsed with distilled water, air dried and mounted in Vectashield Mounting Medium with 4″,6-diamino-2 phenylindole dihydrochloride (DAPI; Vector).
FISH slides of patient samples were evaluated using Olympus BX51 microscope (Tokyo, Japan) equipped with a 60× oil immersion objective and a triple bandpass filter for simultaneous detection of Alexa488, Alexa594 and DAPI (Chroma Technology Corp., Brattleboro, Vt., USA).
Conventional metaphase slides were prepared for FISH as previously described (Abdel-Rahman W. M. et al. 2001, Proc Natl Acad Sci USA 98: 2538-43). The hybridization of the probes and detection of digoxigenin-labeled NAV3-probes and biotin labeled centromere 12-specific probes were performed with avidin-FITC (Vector laboratories, Burlingame, Calif., USA) and sheep anti-digoxigenin conjugated with rhodamine (Roche, Mannheim, Germany) as described previously (Karenko et al. 2005). The armspecific MFISH was performed with XaCyte kit (Metasystems GmbH, Altlusheim, Germany) as recommended by the manufacturer). The hybridized metaphases were analyzed with epifluorescence microscope (Axioplan Imaging 2, Zeiss, GmbH, Jena, Germany equipped with a CCD-camera), and MFISH-program module (Isis, Metasystems, Altlussheim, Germany) either manually or using an automatic capturing facility (Metafer, Metasystems, Altlussheim, Germany).
The FISH slides from patient-derived colorectal and brain tumor samples were analyzed, blinded to sample identity, by two independent analyzers. Results are indicated as the percentage of abnormal cells in a total number of 200 counted cell nuclei. Cells with two chromosome 12 signals (diploid cells) and only one signal for NAV3 and cells with more than two chromosome 12 signals but with a lesser number of NAV3 signals (e.g. three chromosome 12 centromere signals and one NAV3 signal) were interpreted as cells with NAV3-deletion. Cells showing more frequent NAV3-signals than centromere 12-signals were interpreted as cells with NAV3-amplification. A sample was considered to be NAV3 aberrant if the percentage of interphase cells showing amplification was more than 7% or percentage of interphase cells showing deletion was more than 4% (calculated from normal distribution of normal sample results as average +3 standard deviations). For the cell lines CCL-228, CCL-230, CCL-248, RKO, LIM1215 and HCA7 ten to 47 metaphases were analyzed for NAV3 and centromere 12, and 6 to 11 metaphases were analyzed for arm-MFISH.
NAV3 LOH assay was performed on colorectal tumor samples as previously described (Hahtola S et al. 2008, J Invest Dermatology 128: 2304-9). In addition, A/G polymorphism (rs1852464) within exon 19 of the NAV3 gene showing up to 0.493 heterozygosity in the Caucasians/Europeans was used in the SNuPE reaction.
DNA samples were first PCR amplified using primers rs1852464F 5′ CCTGCTATTTTCATCTTTCAAGC 3′ (SEQ ID NO:1) and rs1852464R 5′ GGCTGGGATGCTGTTTGAG 3′ (SEQ ID NO:2) to yield a 130 bp PCR fragment containing the A/G polymorphism. The PCR product was subsequently purified by Exonuclease I (10 U/μl) and SAP (Shrimp alkaline phosphatase, 2 U/μl) (ExoSAP-IT, Amersham Biosciences) and PCR Extension was performed using a fluorescently labeled extension primer 5′ GATGCTGTTTGAGCGCATCATGCTGGGCCC 3′ (SEQ ID NO:3) and nucleotide mix containing ddCTP. Extension products were 43 bp or 49 bp depending on whether G or A was present in the template and they were separated and results analyzed as previously described (Hahtola et al. 2008, J Invest Dermatology 128: 2304-9).
A sample was scored as showing LOH, if one of the alleles in the tumor sample had 40% or more decreased signal at rs1852464 or, in the event of constitutional homozygosity for this marker, at the flanking microsatellite markers compared to its matching normal (Cleton-Jansen A. M. et al. 2001, Cancer Res 61: 1171-7).
Corresponding NAV3 Abnormalities are Found in Colorectal Adenomas and Carcinomas Arising in the Same Patient when Using FISH or LOH Methods
Comparison of the NAV3 copy number between the adenoma and carcinoma samples of a given patient, obtained at the same surgical operation in all but two cases, revealed that NAV3 deletion was frequently detectable by FISH technique in the adenoma stage, too (
Cells with NAV3 copy number changes were detected in 40% of MSS-type colorectal carcinoma samples; cells with either NAV3 deletion or amplification were detected in 15% of samples while 15% samples showed only NAV3 deletion and 10% had solely low level NAV3 amplification (three to five copies). Cells with NAV3 deletion were also detected in 12.5% of MSI-type samples and in 23% of adenoma samples. In addition, cells with chromosome 12 polysomy, most often three or five copies, were detected in 70% of MSS-type colorectal carcinoma samples, in 50% of MSI-type samples and in 31% of adenoma samples. The FISH results are illustrated in
NAV3 copy number changes in colon carcinoma cells were confirmed by LOH assay. LOH was detected in 21% of MSS carcinomas and in 18% of adenoma samples. The results of each marker are shown in Table 1.
NAV3 Copy Number Changes or Translocations, as Demonstrated with FISH, are Found in Established CRC Cell Lines
Six different established CRC cell lines (CCL-228, CCL-230, CCL-248, RKO, LIM1215 and HCA7) and two normal colon cell lines (CRL-1541 and CRL-1539) were analyzed with NAV3-specific FISH (Table 2). The normal colon cells CRL-1541 and CRL-1539 did not show aberrant signals for NAV3 in relation to chromosome 12 centromere signals but in CRL-1539, 8% of the cells were tetrasomic for both of these signals. Three cell lines (SW403, RKO, T84) showed a notable portion of metaphases with loss of NAV3 (SW-403 and T84 90% or more and RKO more than 40% of metaphases; Table 2 and
NAV3 Aberrations of Colorectal Samples Associate with Chromosome 12 Polysomy and with Lymph Node Metastasis
NAV3 aberrations of colorectal samples correlated with chromosome 12 polysomy and lymph node metastasis with statistical significance (Table 3). Moreover, chromosome 12 polysomy occurred more often in tumors with high grade malignancy (Table 3).
1) ns = not statistically significant
2) correlation analysed for the MSS patient group.
2) Chi-square test, exact, two sided
3) log-rankt test
Altogether 119 brain tumor cases of which 55 astrocytomas, 20 oligodendrogliomas, 13 ependymomas, 18 medulloblastomas, and 13 neuroblastomas were analyzed for NAV3 gene copy number changes and chromosome 12 polysomy using FISH (see Table 4). Cut-off levels for NAV3 deletion, NAV3 amplification, and chromosome 12 polysomy were 4%, 7%, and 10%, respectively.
Both deletion and amplification of NAV3 were detected in astocytomas; 20% (11 out of 55) of the studied cases showed NAV3 deletion and 11% (6 out of 55) amplification. Chromosome 12 polysomy was observed in 45% (25 out of 55) of the cases. Statistical analysis using Kaplan-Meier test showed a tendency of NAV3 amplification predicting better outcome of the disease than deletion or normal copy number of NAV3.
Studied oligodendroglioma cases showed NAV3 deletion in 15% (3 out of 20) and amplification in 30% (6 out of 20). Polysomy of chromosome 12 was detected in 43% (10 out of 23) of the cases. Chromosome 12 polysomy seemed to predict poorer outcome of the disease when analyzed with Kaplan-Meyer test.
Ependymomas showed deletion of NAV3 in 31% (4 out of 13) of the studied cases, NAV3 amplification in 54% (7 out of 13) and chromosome 12 polysomy in 69% (9 out of 13). Statistical analysis indicated that NAV3 amplification and chromosome 12 polysomy predict poorer survival.
Deletion of NAV3 was observed in 11% (2 out of 18) of studied medulloblastoma cases, amplification of NAV3 in 39% (7 out of 18) and polysomy of chromosome 12 in 61% (11 out of 18). Statistical analysis using Kaplan-Meier test implicated that NAV3 deletion predict poorer survival and NAV3 amplification, in contrast, predicted better outlook than normal copy number of NAV3.
No NAV3 deletion was detected in studied neuroblastoma cases but 69% (9 out of 13) showed NAV3 amplification and 77% (10 out of 13) chromosome 12 polysomy. In Kaplan-Meier test NAV3 amplification and chromosome 12 polysomy seemed to predict poorer survival.
Glial tumors graduses 1, 2, and 3 glial were grouped into one class and compared to gradus 4 glial tumors concerning NAV3 aberrations (Table 5). Analysis showed that better differentiated lower graduses 1, 2, and 3 contained more NAV3 amplification then gradus 4 glial tumors. NAV3 amplification was observed in 27% (13 out of 48) of gradus 1, 2, and 3 glial tumors contrast to 5% (2 out of 38) in gradus 4 gliomas. On the contrary, gradus 4 glial tumors contained more NAV3 deletion than graduses 1, 2, and 3. NAV3 deletion was observed in 18% (7 out of 38) of gradus 4 glial tumors contrast to 15% (7 out of 48) in gradus 1, 2, and 3 tumors.
DNA was extracted from 50 micrometer paraffin embedded tissue sections by standard protocol. Reference-DNA was extracted from blood pooled at the Finnish Red Cross from 4 healthy males and females. DNA was then digested, labeled and hybridized to a 244K oligonucleotide array according to the manufacturer's (Agilent Technologies, Santa Clara, USA) protocol. Samples were scanned with a DNA microarray scanner and analyzed using Feature Extraction and CGH Analytics software (Agilent Technologies, Santa Clara, USA). Analysis was performed using the z-score and a 1 Mb moving average window. Log 2-values under +/−0.4 were not considered aberrant. Three colon carcinoma cell-lines and two colon carcinoma tumour samples were analyzed using this method.
Array-CGH studies were performed on two samples from the patient material and on three established CRC cell lines CLL-230, CLL-248 and CLL-228. Array-CGH data demonstrated a deletion in 12q21 spanning the NAV3 locus in one of the patient samples, thus confirming the FISH results (this patient sample had 41% NAV3 deleted cells by the FISH method) (
The NAV3-gene was silenced with pooled siRNA oligos (On-Target SMART pool, Dharmacon, Chicago, Ill., USA) The specific sequences of the oligos were as follows: 5′-GGACUUAACCUAUAUACUA-3′ (SEQ ID NO:4) (Exon 12), 5″-GAGAGGGUCUUCAGAUGUA-3″ (SEQ ID NO:5) (Exon 38-39), 5″-CAGGGAGCCUCUAAUUUAA-3″ (SEQ ID NO:6) (Exon 7), and 5′-GCUGUUAGCUCAGAUAUUU-3′ (SEQ ID NO:7) (Exon 30-31).
Glioblastoma cells A172, the primary epidermal keratinocytes, and the normal colon cell lines CRL-1541 and CRL-1539 were cultured in 6-well plates to 70% confluence, and thereafter transfected with 200 μmol of NAV3 siRNA pool or scrambled control siRNA (Dharmacon, IL, USA), using Dharmafect1 transfection reagent (Dharmacon). Glioblastoma cells 0205 were induced to grow as an adherent monolayer by altering the above mentioned culture conditions as follows: bFGF and EGF were withdrawn and the medium was supplemented with 10% FCS for 4 days before transfection. For the transfection of one 6-plate well, the siRNA was diluted to 1 μM in 100 μl of 1× siRNA buffer (received from manufacturer) and mixed with an equal amount of normal growth medium. Four microliters of transfection reagent was incubated for 5 minutes at room temperature with 196 μl of growth media. The siRNA dilution was added and incubated for 20 minutes at room temperature before addition to the cells in 1600 μl of growth media. Six hours post transfection, the transfection medium was replaced with normal growth medium. The viability of the cells was monitored 48 hours after transfection with trypan blue staining. All cell types showed over 70% viability, and in addition, only adherent cells were used for RNA preparation.
To identify genes regulated by NAV3, changes in gene expression profiles induced by NAV3-targeted RNAi were measured with Agilent 4×44K dual-color microrrays and oligonucleotide probes (Biochip center, Biomedicum Helsinki, http://www.helsinki.fi/biochipcenter/). For this analysis, total RNA samples from the siRNA-transfected cells were obtained 6, 24, and 48 hours post-transfection (CRL 1541: 6 and 48 h, CRL 1539: 6 and 24 h, A172: 6 and 48 h, 0205: 6 and 48 h, primary epidermal keratinocytes (PEK): 24 and 48 h).
Cells were lysed in 350 μl of RLT buffer (Qiagen), RNA was purified with the RNeasy Micro or Mini kit (depending on the amount of cells) (Qiagen, Hilden, Germany) and stored at −70° C. The total RNA obtained from the NAV3-silenced cells was hybridized with the corresponding time point samples from the same cells transfected with scrambled oligonucleotides. Before hybridization, the quality of the RNA samples was assessed with the 2100 Bioanalyzer (Agilent Technologies).
Microarray data from the two normal colon cell lines CRL-1541 and CRL-1539, and data from glioblastoma cells and primary epidermal keratinocytes transfected with the NAV3 silencing oligos or with the control oligos were analyzed. Probe intensities were background corrected and normalized with LOWESS using Agilent Feature Extractor 9.1.3.1. For each sample, fold changes within each sample were computed, and 200 probes with the highest expression and 200 probes with the lowest expression were considered as differentially expressed. Genes that were consistently differentially expressed in all normal colon cell lines and tumor samples were used for computational pathway analysis using PathwayExpress (Draghici S. et al. 2007, Genome Res 17(10): 1537-45). In this method, expression values of differentially expressed genes are used to compute an impact factor and an associated p-value for signalling pathways in the KEGG database [REFE: PMID: 18477636], taking into account the topology of the pathways.
With this method, a gene coding a protein located upstream of a pathway has more numerical impact than downstream proteins.
In addition, a novel Gene Ontology (GO) based clustering method was used to reveal gene clusters that share a common biological function or location in the cell (Ovaska K et al. 2008, BioData Min 1(1):11). Distances between genes are computed using the Lin semantic similarity measure (Lin. Proceedings of the 15th International Conference on Machine Learning 1998: 296-304) based on GO annotations. Genes having similar GO annotations have shorter distances from each other and are clustered closely. The clusters, together with expression values, are visualized using a heat map and dendrograms.
qRT-PCR
Efficient NAV3-silencing and the upregulation of IL23R and GnRHR were confirmed by Light Cycler qRT-PCR and RNA in situ hybridization.
For the quantitative RT-PCR, total RNA was reverse transcribed with Revert Aid™ First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to manufacturer's instructions. To prepare standard curves, serial dilutions of cDNA from A172 cells were made. The reactions were performed with a LightCycler480™ apparatus using the LC 480 SYBR Green 1 master kit (Roche Diagnostics, Mannheim, Germany). Briefly, the DNA was denatured in 95° C. for 5 minutes, then 45 cycles of 95° C., 58° C., and 72° C. were run for 10, 15, and 10 seconds respectively, followed by melting curve analysis according to the manufacturer's guidelines. After background adjustment, the fit point method was used to determine the crossing-point value as previously described (Linja M J et al. 2004, Clin Cancer Res 10(3):1032-40). For the normalization of the expression levels, the expression of the TATA-binding protein (TBP) was measured as described (Linja M J et al. 2004, Clin Cancer Res 10(3):1032-40). The relative expression level was obtained by dividing the values for NAV3 with the TBP value. In addition to the melting curve analysis, the PCR products were separated by 1.5% agarose gel electrophoresis to ensure the right product size. The upregulation of IL23R and GnRHR was also confirmed by qRT PCR, increasing the annealing temperature to 60° C. All primers used in the reactions are listed in Table 6.
To assess knockdown efficiency of the NAV3 gene, we performed NAV3-targeted RNA in situ hybridization of the siRNA-transfected cells of cell lines A172 and 0205. The expression of NAV3 was compared to untransfected cells and cells transfected with the scrambled oligos. Briefly, NAV3 mRNA transcripts were detected with a probe recognizing exons 37-39, with overnight hybridization at 45° C.
mRNA In Situ Hybridization for NAV3 mRNA
The RNA probes were generated based on specific exon locations of NAV3 gene exerting following oligonucleotides (exon 37-39: antisense 5″-TTGGCTGCTTCTTGGAGTTT-3″ (SEQ ID NO: 14), sense 5′-CACCAAATCTAGAGCTGCATCA-3′ (SEQ ID NO: 15)). The resulting PCR products were cloned in pCR®II-TOPO® vector (Invitrogen). RNA probes were labeled using the DIG RNA labelling Kit (SP6/T7, Roche) according to the manufacturer's instructions.
The cell lines A172 and 0205 were cultured on sterile glass slides (LAB-TEK II chamber Slide w/Cover RS Glass Slide, Nalge Nunc International) and fixed in 4% paraformaldehyde (MERCK)—PBS for 15 min at room temperature. The slides were rinsed with PBS and refixed in 1% PFA-PBS (7 min, +4-8° C.) followed by rewashing in PBS. Slides were acetylated in 0.1 M triethanolamine (TEA, Riedel-de Haan) pH 8.0-0.25% acetic anhydride (MERCK) and prehybridized with 4×SSC-50% deionized formamide. In situ hybridization was performed in a humidified chamber overnight at 45° C. using hybridization solution containing denatured DIG-RNA antisense probe (4 ng/μl) or sense probe (19 ng/μl). The posthybridization slides were washed with prewarmed buffers at 37° C. water bath (1×10 min, 1×5 min 2×SSC, and 2×5 min 1×SSC) and the excess of RNA was removed with RNase (20 μg/ml RNase A, Sigma; 500 mM NaCl; 10 mM Tris pH 8.0; 1 mM EDTA) for 30 min at 37° C.). After being washed, the hybridized probes were detected immunologically by anti-DIG-alkaline phosphatase Fab diluted 1:1000 in blocking solution. The slides were washed and incubated overnight in humidified chamber with color substrate solution (1:50 NBT/BCIP Stock Solution (Roche) in detection buffer containing 0.002 mM fresh levamisole (Vector Laboratories)). To terminate the reaction slides were washed, rinsed briefly in water and counterstained with 0.1% Kernchrot (1×5 min, Gurr, BDH Chemicals). All reagents and instruments were either treated with diethyl pyrocarbonate (DEPC) or were RNase free.
To identify in vivo relevant target genes of NAV3, we studied the gene expression profiles of NAV3-silenced normal colon cells CRL-1541 and CRL-1539 (with normal NAV gene copy numbers). In these experiments we identified, among 39 genes constantly upregulated in the NAV3-silenced normal colon cells, the upregulation of two key receptors involved in carcinogenesis. Firstly, the gonadotropin releasing hormone receptor (GnRHR) pathway was identified using PathwayExpress to be statistically significantly enriched (multiple hypothesis corrected p-value <0.001), and this result was driven by the upregulation of GNRHR, transcript variant 1, with fold changes 4.7-32.4. Secondly, the Jak-STAT pathway was identified to be marginally statistically significant (corrected p-value <0.058) and was affected by upregulation of interleukin 23 receptor (IL23R), with fold changes 3.4-14.01. The upregulation of these receptors was confirmed by qRT-PCR in selected samples. See
With multicolor FISH, the number of NAV3 signals was compared to the number of centromere 12 signals, and cells thereafter defined to have either normal, amplified, or deleted NAV3 signals. The studied cell lines CRL-1541, CRL-1539, 0205 and A172 were shown to consist mainly of cells without NAV3 aberrations (Table 7) and were therefore suitable for NAV3 silencing studies. The efficient silencing of NAV3 was confirmed by qRT-PCR and RNA in situ hybridization (
When the gene expression profiles of all NAV3-silenced CRL-1541 and CRL-1539 normal colon cells, the glioblastoma cell line A172 derived from grade 4 glioblastoma, and primary epidermal keratinocytes (all from ATCC, Manassas, Va., USA) were evaluated, we identified 17 genes upregulated in all experiments (Table 8). Of these, the upregulation of two key receptors involved in carcinogenesis were noted. First, the gonadotropin releasing hormone receptor (GnRHR) pathway was identified using PathwayExpress to be statistically significantly enriched (multiple hypothesis corrected p-value <0.001). This result was driven by the upregulation of GNRHR transcript variant 1 which had a median fold change of 13.7. Second, the Jak-STAT pathway was identified to be marginally statistically significantly altered (corrected p-value <0.058) by upregulation of the interleukin 23 receptor (IL23R), which had a median fold change of 7.2. The upregulation of these receptors was confirmed by qRT-PCR in selected samples. We also decided to include the gene expression profiles of NAV3-silenced primary human keratinocytes (PEK) in the analysis. We then identified 52 genes, including GnRHR and IL23R that were upregulated in 7 of the 10 experiments performed with all three cell types (Table 9).
Homo sapiens chromosome 12 open reading frame 42
Homo sapiens gonadotropin-releasing hormone
Homo sapiens ADP-ribosylation factor-like 11
Homo sapiens P25 mRNA, complete cds, [AF334588]
Homo sapiens clone DNA147309 YPLR6490
Homo sapiens hypothetical protein FLJ33641
Homo sapiens cDNA clone UI-H-EU0-azt-m-22-0-UI
Homo sapiens POM121-like protein, mRNA (cDNA
Homo sapiens clone F22H myosin-reactive
Homo sapiens interleukin 23 receptor (IL23R),
Homo sapiens vanin 3 (VNN3), transcript variant 1,
Homo sapiens cDNA FLJ38732 fis, clone
Homo sapiens hypothetical gene supported by
Homo sapiens cDNA FLJ11405 fis, clone HEMBA1000769. [AK021467]
Homo sapiens chromosome 12 open reading frame 42 (C12orf42), mRNA
Homo sapiens P25 mRNA, complete cds. [AF334588]
Homo sapiens gonadotropin-releasing hormone receptor (GNRHR), transcript
Homo sapiens cDNA FLJ38732 fis, clone KIDNE2010750. [AK096051]
Homo sapiens clone F22H myosin-reactive immunoglobulin heavy chain
Homo sapiens clone DNA147309 YPLR6490 (UNQ6490) mRNA, complete cds.
Homo sapiens cat eye syndrome chromosome region, candidate 1 (CECR1),
Homo sapiens olfactory receptor, family 2, subfamily W, member 3 (OR2W3),
Homo sapiens glycerol kinase (GK), transcript variant 1, mRNA [NM_203391]
Homo sapiens ADP-ribosylation factor-like 11 (ARL11), mRNA
Homo sapiens tumor protein D52-like 3 (TPD52L3), transcript variant 1,
Homo sapiens hypothetical protein FLJ33641 (FLJ33641), mRNA
Homo sapiens zinc finger, DHHC-type containing 15 (ZDHHC15), mRNA
Homo sapiens POM121-like protein, mRNA (cDNA clone IMAGE:40053742).
Homo sapiens chloride channel, calcium activated, family member 3
Homo sapiens interleukin 23 receptor (IL23R), mRNA [NM_144701]
Homo sapiens vanin 3 (VNN3), transcript variant 1, mRNA [NM_018399]
Homo sapiens opioid receptor, kappa 1 (OPRK1), mRNA [NM_000912]
Homo sapiens hypothetical gene supported by BC006119,
Homo sapiens cysteinyl leukotriene receptor 2 (CYSLTR2), mRNA
Homo sapiens submaxillary gland androgen regulated protein 3 homolog B
Homo sapiens guanine nucleotide binding protein (G protein), gamma transducing
Homo sapiens evolutionary related interleukin-1beta converting enzyme
Homo sapiens family with sequence similarity 107, member A (FAM107A),
Homo sapiens zinc finger, DHHC-type containing 15 (ZDHHC15),
Homo sapiens coiled-coil domain containing 62 (CCDC62), transcript
Homo sapiens cDNA FLJ37906 fis, clone COLON2004318. [AK095225]
Homo sapiens B-cell CLL/lymphoma 6, member B (zinc finger protein)
Homo sapiens zinc finger and SCAN domain containing 4 (ZSCAN4),
Homo sapiens PRO2610 mRNA, complete cds. [AF119887]
H. sapiens mRNA for alu elements. [X92185]
Homo sapiens chromosome 19 open reading frame 30 (C19orf30), mRNA
Gene ontology based clustering was performed to identify biological processes affected by NAV3 depletion. To increase the number of genes for GO analysis, we performed clustering on the 52 genes that were up-regulated in 7 of 10 experiments. From this group, we identified four GO clusters (
Gene products are clustered (grouped) based on their Gene Ontology (GO) annotations. Genes that have similar annotations belong to the same cluster. The distance between gene products is defined using information-theoretical semantic similarity measures.
In the Table 10 below, the most informative (most specific) common GO terms of each cluster are displayed. All gene products in the cluster are annotated with the GO term mentioned. A GO term is more informative if it α-curs rarely in the GO annotations of the whole genome. The priori column contains the a priori probability p that a random gene product is annotated with the given GO term. Information is defined as log2p. Generally, clusters with (1) a large number of gene products and (2) common GO terms with high information content are the most interesting.
In addition to the membrane cluster genes IL23R and GnRHR discussed previously, other genes in the membrane cluster (
The nucleus cluster included the FAM107A gene, also called TU3A, which has been reported to be epigenetically silenced in a variety of cancers. In addition, we identified the BCLB6 gene, a transcription factor-coding gene, upregulated by glial cell line-derived neurotrophic factor. The complete list of genes in each cluster is given in
When the colon and glioma cells were treated as specific groups, we identified 39 genes consistently upregulated in the NAV3-silenced normal colon cells and 28 genes upregulated in the glial cells (Tables 11 and 12). DNER is a transmembrane protein carrying extracellular EGF-like repeats and an atypical Notch ligand, and which was upregulated only in NAV3 silenced colon cells (Table 11).
Homo sapiens gonadotropin-releasing
Homo sapiens hypothetical protein
Homo sapiens aquaporin 10 (AQP10)
Homo sapiens delta-notch-like EGF
Homo sapiens opioid receptor, kappa 1
Homo sapiens galactose-3-O-
Homo sapiens ADP-ribosylation factor-like 11 (ARL11)
Homo sapiens ankyrin repeat domain 45 (ANKRD45), mRNA
Homo sapiens glycerol kinase (GK),
Homo sapiens family with sequence similarity 107,
Homo sapiens B-cell CLL/lymphoma 6,
Homo sapiens chromosome 12 open
Homo sapiens cDNA FLJ14490 fis,
Homo sapiens retinal short chain
Homo sapiens POM121-like protein
Homo sapiens clone F22H
Homo sapiens zinc finger protein 224
Homo sapiens cat eye syndrome chromosome region,
Homo sapiens tumor protein D52-like 3
Homo sapiens vanin 3 (VNN3), transcript
Homo sapiens coiled-coil domain containing
H. sapiens mRNA for alu elements. [X92185]
Homo sapiens chloride channel, calcium activated,
Homo sapiens chromosome 12 open reading
Homo sapiens cDNA FLJ11405 fis,
Homo sapiens gonadotropin-releasing hormone
Homo sapiens P25 mRNA, complete cds,
Homo sapiens POM121-like protein,
Homo sapiens cDNA FLJ38732 fis,
Homo sapiens cDNA clone
Homo sapiens vanin 3 (VNN3), transcript
Homo sapiens cysteinyl leukotriene
Homo sapiens clone F22H myosin-reactive
Homo sapiens hypothetical gene supported
Homo sapiens chromosome 19 open reading
Homo sapiens zinc finger and SCAN domain
Homo sapiens sperm associated antigen
Homo sapiens clone DNA147309 YPLR6490
Homo sapiens ADP-ribosylation factor-
Homo sapiens interleukin 23 receptor
Homo sapiens hypothetical protein
IL23R and beta-catenin expression were studied by immunohistochemistry in tissue microarrays prepared from paraffin-embedded colon biopsies. From each patient, paired samples from the histologically normal colon and two samples from the colon tumour were included in the array. Also, from 10 patients paired samples of an adenomatous lesion and another sample from the normal colon, were included. Altogether 57 patients, 43 MSS tumour samples, 14 MSI tumour samples, 14 adenoma samples and 57 corresponding normal colon samples were included in the tissue microarrays.
Immunohistochemistry was carried out by using the avidin-biotinperoxidase complex tehnique (Vectastain Elite ABC kit [mouse IgG], Vector laboratories, Burlingame, Calif., USA) with DAB as chromogenic substrate and Mayer's hematoxylin as counterstain. Following standard deparaffinization, endogenous peroxidase activity was blocked in 3% H2O2 in PBS for 10 min and the sections were pretreated in a +95° C. water bath in citrate buffer (DakoCytomation, Glostrup, Denmark) for 20 min. The slides were then incubated with mouse monoclonal antibodies against IL23R(R&D MAB14001; diluted in 1:30, slides pretreated with 1% trypsin at 37° C. 30 min) or against beta-catenin (Zymed CAT-5H10, Invitrogen, Carlsbad, Calif. 92008 U.S.A; diluted in 1:250, slide pretreatment at +95° C. for 30 min in DAKO Target Retrieval Solution S1699, pH 6-6, 2) at +4° C. overnight. DAB was used as colourigenic substrate for IL23R while VECTOR NovaRED substrate kit SK-4800 was used for betacatenin.
For statistical analyses, the immunolabelling result was scored as follows: for IL23R immunoreactivity no staining (score 0), weak positive staining (score 1), clearly positive staining (score 2), strongly positive staining (score 3; see also
Correlations between categorical variables were analyzed using the chi-square test, or when not valid, the Fisher's exact test. Survival analyses were performed using death caused by colon cancer as the primary endpoint. Follow-up times were obtained from Statistics Finland, a government agency. SPSS version 15.0 software (SPSS, IL, USA) was used.
Expression of IL23R or Nuclear Beta-Catenin in NAV3 Aberrant MSS Tumors Associates with Dukes Staging and Lymph Node Metastases, and Expression of IL23R Associates with Poor Prognosis
Immunohistochemical detection of beta-catenin (linked to the GnRH pathway) and IL23R expression were then performed on a tissue microarray including 43 MSS (including also 8 adenoma samples of the same patients) and 14 MSI patient samples (tumour and corresponding normal colon epithelium). In all normal colon samples of these cases, the beta-catenin staining was always membranous and no or only weak IL23R expression (grade 1) was found except for three cases (
In normal epithelial cells beta-catenin is known to locate in cell membranes but changes its distribution toward the nucleus and cytoplasm in carcinoma samples. In the representative MSS tumour samples (duplicate samples of each), nuclear beta-catenin was found in 10/43 cases and up-regulation of IL23R-immunoreactivity (scores 2-3) in 27/43 cases. The nuclear beta-catenin expression correlated with lymph node metastases. Up-regulated IL23R-immunoreactivity in tissue array samples strongly correlated with Dukes staging (see Table 3 in Example 2, NAV3 aberrations associate with chromosome 12 polysomy and with lymph node metastasis) and also with lymph node metastases (see Table 3 in Example 2). Tumor samples with allelic NAV3 deletions showed most frequently clearly up-regulated IL23R reactivity (
In a Kaplan-Meier survival analysis (
For 11 MSS type CRC patients, it was possible to compare the IL23R, beta-catenin and NAV3 FISH results in the adenoma and tumour samples. NAV3 copy number changes, together with elevated IL23R expression (moderate or strong immunostaining) and/or nuclear beta-catenin were present in 3 of the adenoma samples and elevated IL23R expression alone in two additional adenomas (data not shown). In the corresponding tumour samples, the finding was similar except for two samples where no NAV3 aberration was found despite the upregulated IL23R expression. In both of these latter cases, the corresponding adenoma lesion had shown chromosome 12 polysomy, however.
GnRHR immunostaining was performed in a LabVision immunostainer (Labvision, CA, USA) following antigen retrieval using Tris-EDTA buffer, pH 9.0 in a microwave oven for 24 minutes at 900 watts followed by cooling for 20 minutes at room temperature. GnRHR was detected with mouse monoclonal antibody (diluted 1:10, Abcam, Cambridge, UK) and a polymer based detection system (Envision, K5007, DakoCytomation) with diaminobenzidine (DAB) as the chromogen (
Two representative cases of colorectal tissue sections (paired samples of histologically normal colon and the colon tumor) and two cases of astrocytoma (glioblastoma) prepared from paraffin-embedded colon biopsies of patients with colorectal cancer or astrocytoma were included. For these samples, immunohistochemistry was carried out as previously described using the anti-GnRHR antibody and mouse monoclonal antibody against IL23R(R&D MAB14001; diluted 1:30, slides pretreated with 1% trypsin at 37° C. for 30 min) and avidin-biotin-peroxidase complex detection (Vectastain Elite ABC kit [mouse IgG], Vector Laboratories, Burlingame, Calif., USA) with DAB as the chromogenic substrate.
Confirmation of IL23 and GnRHR Protein Upregulation in NAV3 Silenced 0205 cells, and CRC and Glioblastoma Tissue Samples
Upregulation of the GnRHR protein levels in NAV3-silenced 0205 cells was demonstrated by immunostaining. GnRHR staining was higher in NAV3-silenced cells than wild type cells and siRNA scrambled control transfected cells (
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FI09/50838 | 10/19/2009 | WO | 00 | 11/23/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61196761 | Oct 2008 | US |
