Download Trophoblast Markers for Application in non

‫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