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תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Tel Hashomer Medical Research Infrastructure and Services Ltd. BioMarkers for Human Diseases Diagnostic and Treatment Assessment Contact : Sylvie Luria PhD. Technology Transfer and Business & Development Manager Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Cell: 052-6667277 [email protected] http://research.sheba.co.il/e/ Trophoblast Markers for Application in non-invasive Prenatal Diagnosis 1 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Non-invasive prenatal diagnosis based on the isolation of rare fetal cells from maternal blood has been the source of anticipation over the last few decades. However, two major obstacles have hampered the development of a robust protocol that can be applied in the clinical laboratory: First, the fetal cells - although proven unequivocally to be present in maternal blood - are very rare and can be found at a frequency of 1 fetal cell to106-107 nucleated maternal blood cells. In addition, specific fetal cell markers have yet to be identified, thus accurate identification is not yet possible among the immense background of maternal blood cells. Trophoblasts present an attractive target cell type for non-invasive prenatal diagnosis, as they can be isolated from maternal blood early in the first trimester, are distinguishable from maternal blood cells due to their unique structure, and are absent in normal adult blood. Trophoblast cells have been isolated from maternal blood by several different methods related to surface antigen expression, for example HLA-G, and cell size. Development of methods for isolation, identification and diagnosis of rare fetal cells in maternal peripheral blood has been our ongoing research focus for the last decade. We have developed a method enabling placental trophoblast cells derived from maternal blood to proliferate under in-vitro culture conditions. We have recently identified novel trophoblast markers, thereby improving fetal cell detection. The combination of these two aspects of our research data is the basis of our current efforts to advance the system towards metaphase chromosome analysis and the analysis of fetal DNA markers for genetic disease diagnosis. 2 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Transcriptional Signatures of Multiple Sclerosis for Disease Management Multiple Sclerosis (MS) is a devastating autoimmune disease that frequently occurs in young adults between 20-40 years of age, is more prevalent in females, and has a characteristic geographical distribution. The estimated prevalence in USA is 120/100,000, (250,000 to 350,000 cases) and in Israel the prevalence is about 40/100,000. MS disease is characterized by CNS inflammation which is mediated by autoreactive activated lymphocytes against myelin proteins, leading to myelin destruction and axonal loss. The disease preferentially attacks white matter throughout the central nerve system (CNS), which results in deterioration of motor and sensory skills, as well as coordination and cognitive impairment. The research performed in the Sheba MS Center focus on the understanding of the pathophysiology and the etiology of the disease and the development of tools for diagnosis, disease assessment and prognosis, prediction outcome in relapsing–remitting multiple sclerosis and drug personalization. During the last years research programs on differential expression patterns in MS using DNA microarray technology were conducted. The goal of these studies was to establish a subset of genes that are critical in diagnosing patient's clinical state, predicting clinical outcome and assess responsiveness to medication(s). Gene expression profiles were established for the following unmet needs: Diagnosis of Multiple Sclerosis using gene expression pattern analysis Gene expression signature predicts clinical outcome in RRMS 3 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Human Serum markers for Bone Formation and Bone Maladies Bone markers are useful tools for the management of bone diseases, because they provide information that is different but complementary to bone mineral density (BMD) measurement. Several biochemical markers of bone formation and bone resorption have been developed for variety of bone diseases including bone development, Paget disease and osteoporosis, and for their potential use in cancer patients whose disease has metastasized to bone. Problems in the measurement and interpretation of bone marker values are still hampering the optimal utility of this clinical tool. Most of these difficulties originate from problems related to the handling and control of analytical and biological variability of bone marker measurements. In this context, certain applications of bone marker testing may have different requirements for their variation in appearance, modifications, applications and more. Bone markers can be measured in blood and urine and fall into two categories: (a) enzymes or other proteins secreted by osteoblasts or osteoclasts; and (b) substances produced during the formation or breakdown of Type I collagen, the primary protein forming the organic matrix in bone. The collagens that form fibrils are the most abundant components of most connective tissue and provide the three-dimensional scaffold that maintains tissue integrity. Collagen type I is the major ECM protein of the bone, comprising 90% of its protein, and is highly expressed during bone formation. Collagen type I and other filbrillar collagens are first synthesized and secreted into the ECM in the form of soluble precursor, procollagen. Proteolytic processing of procollagen N- and C- propeptides by specific N- and C-proteinase lead to the production of mature collagen monomer capable of forming fibrils spontaneously. The C-propeptide is cleavages by bone morphogenetic protein-1 (BMP-1). In their search for bone formation markers, researchers have focused on metabolites and enzymes specific to osteoblast activity, mainly those involved in collagen type I synthesis and mineralization, but each was found to have disadvantages. We have found highly conserved ECM markers which serve as specific adaptor that stimulates filbrillar collagen formation and therefore may reflect regulatory processes of bone formation and bone maladies. The markers have modified appearance in the serum. The markers are expressed in differentiated osteoblast and regulate the basic metabolic process of bone matrix formation. Their appearance should reflect changes in bone metabolic balance. We have observed highly correlated data suggested their implication in monitoring and assessment of bone metastases in cancer patients. 4 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Gene expression signature for prediction of metastasis to the central nervous system Metastasis is the major cause of morbidity and mortality in human cancer. Metastases to the central nervous system (CNS) are especially debilitating and are associated with extremely short life expectancy. In adults, lung cancer is the most common primary tumor to spread to the CNS. Prophylactic cranial irradiation and/or intrathecal chemotherapy for high risk patients in certain diseases (e.g. leukemias, small cell lung cancer) prevents CNS relapse. Presently, however, there are no biological or clinical parameters that identify patients with localized non-small cell lung cancer (NSCLC, the most common subtype of lung cancer) at high risk for CNS metastasis. We identified a risk score based on the expression of three genes in primary NSCLC that predict the risk of brain metastases. An early diagnosis and vigorous treatment of the brain metastasis, while only rarely curative, may lead to a useful remission of the neurological symptoms and may both enhance the quality of a patient’s life and prolong survival. Central nervous system metastases are diagnosed after their primary cancer has been diagnosed and treated. Thus the brain metastasis market is characterized by a high level of unmet need, large patient potential and consequently high commercial value. In particular, there is a need to identify patients with localized cancer that are at high risk for developing brain metastasis. 5 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Immunoassay s for Monitoring Patient’s Response to Biological Drugs Biologic agents have revolutionized the medical management of many diseases. Since their introduction over 20 years ago they continue to provide safe and effective treatment alternatives for a wide variety of diseases. Their use continues to expand as current agents find new indications and novel agents are introduced. Experience has demonstrated that these agents, although highly effective, are capable of a wide range of unusual and atypical reactions, some of which can be life-threatening. As older biologic agents go off patent, subtle production alterations have the potential to cause immune reactions or antibody formation in previously relatively nonimmunogenic agents. Although antibody formation is reported for all recombinant proteins, the clinical consequence is highly variable. Many antibodies have no clinical effect, while others have adverse effects ranging from loss of efficacy to life-threatening disease, as in the case of Pure red cell aplasia (PRCA. For example, Adalimumab, a fully humanized monoclonal antibody that binds TNF-α, was approved for use in rheumatoid arthritis rheumatoid arthritis as well as for other inflammatory diseases such as psoriasis, Crohn disease, ulcerative colitis, and Wegener granulomatosis Anti-TNFα monoclonal antibodies are an important biologic class of drugs used for treatment of Crohn's disease, Ulcerative colitis and rheumatoid arthritis. Like other biologics, Anti-TNF agents can elicit the generation of anti-drug antibodies by the recipient patient. These antibodies can hasten the drug clearance and neutralize its effect. There is growing need for reliable methods for measurement of anti-drug antibodies in patients receiving anti-TNF agents and other biologics. However, available ELISA methods are limited by technical caveats causing many false negative and positive results We have developed a novel ELISA based assay using alternative binding of the drug to the ELISA plate with or without splicing of the drug to its fragments by using a detection antibody directed against a specific antibody chain. This assay can be used for detecting anti-drug antibodies in serum or other body fluids of patients for all used biological drugs.. The assay development has been completed, calibrated and validated in over 200 anti-TNF treated patient samples and more than 100 control sera. The assay is now routinely used for clinical follow-up of patients treated by the anti-TNF agents Infliximab and Adalimumab in Israel. 6 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 BioMarkers for Human Cancer, Diagnostic, Treatment and Treatment Assessment A vast research activity in cancer biology: Gene expression profiling of cancer: Hematopoietic malignancies, lung cancer, brain tumors and neuroblastoma Developmental biology and cancer Epigenetics of cancer Pharmacogenomics for diagnosis, prognosis and tailored therapy of cancer Childhood leukemia genomics Adoptive immunotherapy for malignant melanoma P53 regulation of gene expression Involvement of nuclear envelope proteins in regulation of global gene expression Molecular basis of neuronal tumors Genomics of lung cancer Clinical research for development of new anti cancer therapies - collaboration with pharmaceuticals companies 7 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Diagnostic Platform for Prediction of Warfarin Dosing Warfarin is the most commonly prescribed anticoagulant drug for the prophylaxis and treatment of venous and arterial thromboembolic disorders. The effectiveness and safety of warfarin is critically dependent on maintaining the prothrombin time, expressed as the international normalized ratio (INR), within the therapeutic range (INR=2-3). A patient is at increased risk of bleeding when INR is above the upper limit of the therapeutic range and is at risk of thromboembolic events when INR falls below it. Polymorphism of major genetic markers for warfarin (coumadin) dosing prediction and patients' management are the focus of this invention. The known genetic determinants include several functional and common polymorphisms in CYP2C9 and VKORC1 genes, which explain the low-end of warfarin dosing range and mostly occur in patients of Caucasian and Chinese origins. We identified a new VKORC1 polymorphism that is specifically indicative of the high dose requirements and is dominant over the dose-reducing effect of the known CYP2C9 and VKORC1 markers. This marker is significantly overrepresented in Jews of Ethiopian origin, in Ashkenazis. We believe that it is also common in Afro-American and Indian populations. This information prompts the development of a more inclusive and universal diagnostic reagent that can improve the individualized warfarin dosing regimen. 8 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Methionine Synthase as a Diagnostic Tool for Autoimmune Diseases Autoimmune diseases are diseases where the target of the disease is "self" or "self antigen". Among the many types of autoimmune diseases, there are a number of diseases that are believed to involve T cell immunity directed to self antigens, including, for example, Systemic lupus erythematosus (SLE), multiple sclerosis (MS), antiphospholipid syndrome (APS), type I diabetes, and rheumatoid arthritis (RA). The diagnosis of autoimmune diseases is considered problematic since immune tests such as autoantibodies are frequently inconclusive and the clinical diseases are generally not specific. Some highly specific antibodies such as Sm (Smith antigen, a non-histone nuclear protein) and anti-P-ribosomal exist for SLE, however, (these antibodies are relatively rare. These antibodies are very specific for SLE but occur only in 10-20% of patients. There is a need to find pathogenic antibodies that bind to important cellular components and impair function. When a patient is diagnosed with an autoimmune disease such as APS or SLE, the choice of appropriate therapeutic interventions would be considerably facilitated by diagnostic and prognostic indicators that accurately reflect the current severity of the disease, predict future severity, and monitor response to therapy. We have discovered that in autoimmune serum patients there are antibodies to to methionine synthase (MTR) in a test sample of an individual comprising: a) contacting a test sample from the individual with at least one capture molecule; and b) detecting binding of the antibody to MTR in the test sample to said capture molecule, thereby detecting the presence of the antibody to MTR in the test sample. 9 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Functional variant of KLOTHO modifies breast cancer risk in BRCA1 mutations carriers The Klotho protein is a 1014-amino acid single pass transmembrane protein. Several activities of klotho have been described to date, including retention of the calcium channel TRPV5, activation of FoxO forkhead transcription factors, binding to fibroblast growth factor receptors (FGFR) 1-4 and acting as a co-receptor for FGF23. Klotho is also a potent inhibitor of the insulin receptor (IR) and the IGF-1 receptor (IGF-1R). Since the IGF-1 and the insulin pathways play important roles in breast cancer pathogenesis, we have recently studied klotho expression and activities in human breast cancer. High klotho expression was found in normal breast samples, but very low expression occurs in ductal carcinoma in situ (DCIS) and invasive breast cancer. Moreover, over-expression of klotho reduced proliferation and inhibited ligand-dependent activation of the IGF-1 and insulin pathways in breast cancer cells. These data suggest that klotho can behave as a potential tumor suppressor in human breast cancer. A functional variant of klotho, termed KL-VS, contains six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S. The presence of phenylalanine at a position equivalent to position 352 in the human KLOTHO gene is highly conserved among species and its substitution to valine may alter the excretion and enzymatic activity of the protein. Homozygosity for this variant was under-represented in elderly compared with newborns in various populations and was also identified as an independent risk factor for early-onset coronary artery disease. We have recently identified klotho as a potent tumor suppressor gene in breast cancer. We observed that in Ashkenazi Jewish women who are BRCA1 mutation carriers, presence of the KLOTHO V allele resulted in increased breast cancer risk and the diagnosis of breast cancer was made at a significantly younger age. This observation is supported by studies in breast cancer cells, which indicated reduced growth inhibitory activity of the klotho variant compared to wild type klotho. The biologic interaction between klotho and BRCA1 has not been elucidated yet. Klotho is a potent inhibitor of the IGF-1 pathway. Recent data indicate an association between mutations in BRCA1 and BRCA2 and the IGF-1 pathway in breast cancer. Levels of the IGF-1R are increased in breast cancer of BRCA1 mutation carriers, also, IGF-1 levels are increased in cancers from carriers of BRCA1 and BRCA2 mutations. In addition, intact BRCA1 protein can lower IGF-1R levels in breast cancer cells. Klotho V allele shows reduced excretion and enzymatic activity. Thus, a possible explanation of our observation is the combination of increased activation of the IGF-1 pathway due to BRCA1 mutation and reduced inhibition by klotho. No association between the klotho variant and breast cancer was noted among non-carriers of BRCA1 or BRCA2 mutations. 10 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Photo – Active backbone for Photodynamic Therapy and Imaging Early detection of cancer is a key to reducing mortality, because it allows treating malignant tumors in the initial stages more efficiently. However, small tumors (<2 mm) known to be pre-invasive are difficult to diagnose because of limitation in sensitivity and resolution of conventional imaging techniques such as X-ray, CT, ultrasound and magnetic resonance imaging (MRI). There is a need for selective and specific contrast agents allowing increased sensitivity of imaging methods in detection of early-stage disease. Oncology therapeutics research has focused on developing compounds that selectively target neoplastic processes with the hope that such agents will inhibit cancer cells proliferation and spare normal cells. Cancer targeting based on receptor-specific pathways for both diagnostic and therapeutic purposes is a novel and promising trend in oncology. This trend has permitted to develop molecules that make available tumor detection/treatment with high sensitivity and specificity. In the past decade, there has been increasing evidence for variety of peptide receptors overexpressed in cancer cells in comparison to normal tissue. The best data has been provided for somatostatin receptors (SSTr) present in neuroendocrine tumors. Somatostatin is a small peptide, a natural hormone that regulates release of several physiologically important biomolecules. Somatostatin analogs are the first class of receptor binding peptides having gained routine clinical application in oncology for treatment and diagnostic of cancer. We have discovered a stable ligand that binds somatostatin receptors on cancer cells. This molecule was labelled with a fluorescent moiety demonstrated high binding specificity to somatostatin receptors on cancer cells - in vitro as well as on tumors models in animals. Selective tumor uptake compared to normal tissues was demonstrated in mouse xenograft models of human lung and colon carcinomas. After administration, the agent is removed from normal tissues, being retained in the tumors for at least 72 hours, thus providing significant tumor-to-normal tissue ratio. The labelled conjugate emits green fluorescence under illumination with blue light and can be easily detected by fluorescence imaging techniques. 11 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Prediction of Brain Metastasis by Gene Expression Signatures Metastasis is the major cause of morbidity and mortality in human cancer. Metastases to the central nervous system (CNS) are especially debilitating and are associated with extremely short life expectancy. In adults, lung cancer is the most common primary tumor to spread to the CNS. Prophylactic cranial irradiation and/or intrathecal chemotherapy for high risk patients in certain diseases (e.g. leukemias, small cell lung cancer) prevents CNS relapse. Presently, however, there are no biological or clinical parameters that identify patients with localized non-small cell lung cancer (NSCLC, the most common subtype of lung cancer) at high risk for CNS metastasis. An early diagnosis and vigorous treatment of the brain metastasis, while only rarely curative, may lead to a useful remission of the neurological symptoms and may both enhance the quality of a patient’s life and prolong survival. Central nervous system metastases are diagnosed after their primary cancer has been diagnosed and treated. We have identified a risk score based on the expression of three genes in primary NSCLC that predict the risk of brain metastases. This risk score may be used for identification of NSCLC Patients that may benefit from prophylactic therapy to the CNS. Prediction of brain metastasis in NSCLC patients with high risk score at the time of diagnosis may be eligible for prophylactic CNS directed therapy. Brain metastases from unknown primary neoplasms are most likely to be from a primary lung cancer (72 per cent), followed in frequency by breast cancer, colon carcinoma, and melanoma. The unique gene expression signature that we have identified will be analyzed for its applicability in these types of cancer as well. 12 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Novel Personalized Treatment and marker for Cancer Apoptosis is a fundamental mechanism of cell death that can be engaged by a range of cellular insults. The p53 onco-suppressor and its downstream effector genes have a wellestablished role in protecting against cancer development. The human p53 gene is mutated in about 50% of human cancers. In addition, p53 is intrinsically unstable and mutations usually reduce further its stability and inhibit the onco-suppressing function of p53. Various types of stress activate the tumor suppression activity of p53, mostly at post-translational level, by a series of modifications, including phosphorylation among others. The maintenance of p53 trans-activation activity is important for p53 apoptotic function. P53 apoptotic induced pathway is important in the cellular response to genotoxic drug induced damage. Chemoresistance of cancer cells, due to inhibition of apoptotic response, is the major reason for the failure of anticancer therapies. In this regard, p53 phosphorylation at serine 46 (Ser46), which is a late event after DNA damage, was shown to be a necessary step for inducing apoptosis in response to severe DNA damage. HIPK2 regulates p53apoptotic function via serine-46 (Ser46) phosphorylation and activation of p53 is a key determinant in colon, ovarian and breast cancer cell death. HIPK2 is an important regulator of p53 activity in response to a chemotherapeutic drug. Our results suggest that different drug-activated pathways may regulate HIPK2 and that HIPK2/p53Ser46 deregulation that is involved in chemo-resistance. Moreover, our discovery demonstrates that loss of tumor suppressor activity in response to absence of HIPK2 that phosphorylates p53 at Ser46 could be reversed by zinc supplementation in vitro and in vivo. In addition, in the presence of zinc the structure of p53 undergoes conformational changes thereby becoming accessible to other known Ser46 kinases, such as, p38, PKCδ, and DYRK. Understanding the cellular models of drug resistance has been pivotal in unraveling the main effectors of resistance chemotherapy at the molecular level (i.e. intracellular drug inactivation, detoxifying systems, defects in DNA repair, apoptosis evasion, membrane transporters and cell adhesion). Our study revealed a major mechanism that cancer cells acquire chemo-resistance and have hypothesized the feasibility of an alternative approach. We have designed both in vitro and in vivo systems to demonstrate the use of specific agent to prevent and to treat chemo-resistance cancer cells. Our clinical studies have demonstrated that up to 40% of breast cancer patients with WTp53 have low levels of HIPK2 and are prone to chemo-resistance. 13