Annual Activity Report 2017

Tzipora C. Falik-Zaccai

Prof. Tzipora C. Falik-Zaccai

Institute of Human Genetics – Galilee Medical Center, Nahariya, Israel

Faculty of Medicine in the Galilee, Bar Ilan University, Safed, Israel

We focus our scientific work on the identification of causative genes and genetic variants for rare genetic diseases that are common among the unique, highly consanguineous Galilee and Golan Heights populations. Our work provides the option for personalized medicine to individuals from different ethnic groups, religions and villages in northern Israel.

The work begins in the medical genetics clinics where patients are tested and characterized for any previously unrecorded genetic disorder, perhaps a very rare one. We have already discovered and reported disease-causing genes and genetic variants that have never been studied before, and we continue to do so with this highly unusual population. The disorders shed light on basic and important processes in which proteins take part, thus broadening our understanding of their contribution and importance to normal, healthy life.

We have recently identified and published two novel genes; PLAA is one gene that, in carrying a pathogenic variant, causes progressive leukoencephalopathy with severe mental retardation, spasticity and progressive microcephaly.

The second gene PPP1R13 was first reported by us as causing a novel fatal form of cardiocutaneous syndrome due to an uncontrolled inflammation in the heart of the affected babies. Since the identification of these two genes genetic counseling and prenatal diagnosis have been offered to family members, and population screening has been established for populations at risk, funded by the government. Our work has contributed significantly to the prevention of these two devastating diseases among the Moslem and Druze populations.

Omry Koren

Dr. Omry Koren

Polycystic ovary syndrome, diet and the microbiome

Working hypothesis and aims: We hypothesize that women with polycystic ovary syndrome (PCOS) have an altered microbiota compared to normal ovulatory women and that a low-carbohydrate diet will positively alter their gut microbiota leading to improvement in their overall symptoms and fertility. Therefore, we aim to characterize the microbiome of normal and overweight healthy, PCOS untreated, and PCOS diet-treated women and to test whether the diet intervention affects symptoms and health.

We further aim to perform fecal transplants from all study groups into germ-free mice in order to show a causal effect between the microbiome and health outcomes, as well as test the metabolomics profiles of each group. Outcomes of this research will likely lead to improvement of current treatments for PCOS in overweight as well as normal weight women, and to an understanding of the roles of gut microbiota composition in PCOS.

Background: The polycystic ovary syndrome (PCOS), consisting of anovulation, is the leading cause of infertility and affects up to 10% of reproductive age women. While PCOS has been extensively studied in the last two decades, the precise mechanisms leading to the clinical complex of PCOS have remained enigmatic to a large extent. It has been shown that diet and physical activity improve the state of women with PCOS and reduce the metabolic syndrome like characteristics (low grade inflammation and insulin resistance) and this is often the treatment. Studies in the new field of microbiome research focus on the composition of overall microorganisms in our body and their impacts on our health. Changes in the composition of the gut microbiota (dysbiosis) have been linked with different health states such as pregnancy, obesity, IBD, metabolic syndrome, etc. and have been associated with low grade inflammation and reduced insulin sensitivity. Since it has been shown that the microbiome is affected by both diet and host hormones and in return affects our health status, we believe there is a strong link between the gut microbiome composition, diet, and PCOS.

Methods: Our methods include diet intervention, collection of fecal samples, DNA extraction and PCR amplification, sequencing and identification of 16S rRNA sequences, statistical analyses, clinical measures, microbiota transfer experiments into germ-free mice, and metabolomics.

Results: While we are still in the process of recruitment and sample collection, we have to date tested and analyzed the microbiota of a sample group of control vs. PCOS subjects (PCOS subjects including a sample from before intervention and after the diet intervention), and found distinct differences between groups. With the analysis of the full study group we hope to show that these differences are statistically significant and perhaps detect additional differences between groups.

Evan Elliott

Dr. Evan Elliott

Autism biobank and personalized medicine program

Within the past year, the autism biobank and registry program has made various strides towards our long term goal of biomarker identification and patient stratification. Our team has started the process of building a registry of individuals diagnosed with autism who are willing to take part in future autism research programs, and to collect their blood, stool, and saliva samples for our autism biobank. This biobank will be open for all autism researchers. Our laboratory will perform microbiome and immune system characterization of these samples to perform stratification and to understand the biological basis of autism.

We have begun recruiting families with individuals diagnosed with autism, and currently have recruited approximately 25 families, including the collection of biological samples, which have been placed in our biobank. We are now continuing our recruitment through Ziv hospital in Safed, and are now starting long-term collaborations with autism treatment centers in autism, with the goal of recruiting at least another 100 families within the next year.

In our laboratory, in collaboration with Dr. Omry Koren, preliminary analysis of the microbiome of individuals with genetic predisposition to autism has revealed changes in bacteria that play a very important aspect in human health. Therefore, within the next several years, we will work to verify these results in the samples which we are recruiting through our registry and biobank. Considering the real possibility of using probiotics in patients, it is not unlikely that our research will lead directly to new diet regimens or probiotic treatment in a subset of individuals diagnosed with autism.

Meital Gal-Tanamy

Dr. Meital Gal-Tanamy

The interplay between epigenetic and genomic signatures of Hepatitis C virus in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) serves as a model of a diverse spectrum of cancers since it is induced by a number of well-known cancer causing agents, mainly Hepatitis C virus (HCV) and Hepatitis B virus (HBV). We explored the link between genome wide alterations in chromatin organization induced by HCV and HCV-specific genomic signature.

Epigenetic analysis unraveled both known and novel pathways that are dysregulated by the virus, such as Hepatic lipid metabolism, cell motility and cell cycle. These epigenetic alternations influence host gene expression and leave an “epigenetic signature” on the host chromatin that is not fully purged following virus eradication.

To explore the mutational signature we performed high-resolution target sequencing that enables the detection of low-frequency passenger mutations in 64 HCC biopsies from three trigger groups – HBV, HCV, and “other”. We identified novel distinct trigger-dependent regional mutations signatures. Inverse correlation between a high mutation rate and enrichment of chromatin modifications associated with active transcription provides a link between triggers and the cancer genome. Our study unveils novel trigger-specific mechanisms leading to cancer.

The evolutionary theory assumes that occurrence of mutations in cancer is random. However, recent studies suggest that passenger mutations are not randomly scattered in cancer genomes and that chromatin organization dictates mutation profiles. HCC is a unique case. Its origins are well recognized, which provides a unique opportunity to test the hypothesis that different etiological agents induce differential epigenetic profiles, and correspondingly differential rates of oncogenic mutations. We have identified a unique HCV-specific epigenetic profile that dictates a unique HCV-specific genomic profile in HCC. These mechanisms are implicated in an HCV “hit and run” scenario following HCV eradication that may elucidate the cause for the residual risk for HCC following HCV eradication. The results of this study may have implications to predict sensitivities to existing drugs according to etiology, or to design etiology-dependent prognostic assays for the early detection of HCC. Moreover, since epigenetic changes, in contrast to genetic changes, are potentially reversible, this research may open new avenues to utilize epigenetic modifying drugs to revert the oncogenic effects of chronic hepatitis infections and to prevent HCC. Importantly, our study provides a perspective into the mechanisms that shape etiology-specific mutational signature development in cancer.

Hava Gil-Henn

Dr. Hava Gil-Henn

Development of diagnostic, prognostic, and therapeutic tools to block breast cancer metastasis

While primary tumors can often be removed and are usually responsible for only a small percentage of cancer deaths, complications associated with far-reaching metastasis are the primary cause of mortality from cancer. Most treatments for cancer shrink or slow tumor growth, but no treatment that permanently eradicates metastasis exists at present. Accordingly, a better understanding of the molecular, cellular, and physiological conditions that help initiate metastasis would be valuable for identifying and improving outcomes of patients with metastatic disease.

Additionally, the standard treatment for many cancer patients today is the surgical removal of the primary tumor followed by systemic chemotherapy, which is meant to prevent metastatic recurrence. However, because only a certain percentage of these patients eventually relapse and develop metastatic disease, there is a significant subset of patients who are unnecessarily subjected to the acute and long-term side effects of chemotherapy. Our work aims to develop a method for identifying the tumors that have increased likelihood to disseminate and to tailor anti-metastatic therapeutic intervention that is specific to the particular patient.

Research in our laboratory uses a combination of molecular biology, cell biology, highresolution imaging, proteomics, genomics, structural biology, and in vivo models of cancer metastasis in order to elucidate the proteins and signaling pathways that contribute to the tissue invasiveness and metastatic potential of cancer cells. We are aiming to use the information gained in our studies in order to develop both diagnostic and prognostic tools for assessing the metastatic potential of specific tumors as well as new therapeutic tools to block cancer metastasis.

As part of this effort, we are now testing our novel approaches on patient tumor samples with the long-term vision of applying our findings from bench to bedside. We have now shown that several kinase inhibitors are able to significantly reduce in vivo breast cancer metastasis (Meirson et al., manuscript in preparation). We are now aiming to collaborate with oncologists in order to perform clinical trials in breast cancer patients using our approach.

Orly Avni

Dr. Orly Avni

The effects of social stress on immune regulation are imprinted on gut microbiota, and the implication of this for autoimmunity

There are many factors that contribute to the onset of autoimmune diseases, including a variable genetic contribution and, in most cases, a substantial environmental influence. Dys-biotic gut microbiota and psychological/social stress were both found to be associated with increased occurrence of autoimmune disorders. As we began to increasingly appreciate the bi-directional interactions between the microbiota and the immune system, and between the microbiota and the brain, we asked whether the effects of stress might indirectly increase the susceptibility to immune disorders by modulating the gut microbiota.

In a recent study, we have shown that chronic social stress is imprinted on the gut microbial composition and activity, which consequently stimulates mesenteric autoreactive CD4+ T-helper (Th) cells (a type of lymphocyte that can potentially attack other body tissues). Our results suggest that stress produces changes in growth and virulentassociated transcriptional patterns in selected stress-responsive indigenous bacterial communities. As a consequence, those CD4+ Th cells that began to recognize indigenous bacteria as intruders were transformed into more aggressive lymphocytes in order to attack them. Some of these sensitized bacterial-specific Th cells also possess a certain degree of selfreactivity, which may jeopardize the normal tolerance to healthy tissue and increase the risk in individuals with a genetic predisposition to develop autoimmune disorders.

Affiliate Researchers

Prof. Farid Nakhoul Single-nucleotide polymorphism (SNPs) in the autophagy-related gene 5 (ATG5) in patients with Diabetic Nephropathy & Retinopathy

The current study's aim is to evaluate whether or not a specific polymorphism (functionally silent difference) in the ATG5 gene is associated with diabetic renal and retinal diseases. To achieve this goal we intend to screen the ATG5 autophagy gene of diabetic (DM), diabetic nephropathy (DN) or diabetic retinopathy (DR) patients and explore whether these patients have specific variant SNP or region of SNPs in their ATG5 gene that differ from those in the ATG5 gene of healthy people.

Any day now, we should be receiving the final certification from the Helsinki Committee (medical research ethics committee) for this study. Meanwhile, we have prepared a list of potential patients from our outpatient clinics, for each one of the study groups (DM patients, DN patients and DR patients). This will expedite patient recruitment, once we receive certification.

We are also currently focusing on bio-informatics analysis in order to plan the necessary primers for coding single nucleotide polymorphisms (SNPs) of the ATG5 gene.

Furthermore, we are reviewing the various DNA extraction kits, to find the optimal kit for our needs, so we can start calibrating our experimental system.

Sincerely yours,
Prof. Farid Nakhoul
Director, Institute of Nephrology, Baruch Padeh Medical Center, Poriya

Prof. Jamal Zidan

Comparison of Human Epidermal Growth Factor Receptor 2 (HER2), Estrogen Receptor, Progesterone Receptor, and Ki67 expression in primary biopsy and in surgically resected tumor following neoadjuvant chemotherapy in breast cancer patients.

Neoadjuvant chemotherapy (NAC) in breast cancer patients is a relatively standard treatment for locally advanced and initially inoperable breast cancer. Following NAC lumpectomy or mastectomy is done. A core-needle biopsy (CNB) is usually performed before NAC. NAC is mainly based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2), and Ki67. Patients continue the same treatment after operation based on the same detective factors done in the CNB. According to the literature the expression levels of these detective factors can change after NAC.

In our study ER, PR, HER2, Ki67 levels were evaluated in breast cancer samples in CNB before NAC and in the same tumors after surgery. Surgery was done after finishing NAC. We used immunohistochemical tests for the detection and evaluation of these factors. As of today, 52 patients are evaluable: 54% are Jews and 46% Arabs. Mean age is 52 years. 56% are postmenopausal, 44% are premenopausal. Most tumors (94%) were invasive ductal carcinoma. Significant changes were found in ER, PR, HER2 and Ki67 levels before and after NAC (Table 1). This may affect the chemotherapy choice after operation.

Dr. Andrei Braester

Structural and Functional Characterization of Complement Components in Chronic Lymphocytic Leukemia Patients undergoing Immunotherapy.

Principal Investigator (PI): Dr. Andrei Braester, MD.

Director, Institute of Hematology, Galilee Medical Center Co-Investigator: Regina Michelis, PhD.

Researcher, Institute of Hematology, Galilee Medical Center

Background: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world. The therapeutic approach in CLL includes chemotherapeutic regimens and immunotherapy. Therapeutic monoclonal antibodies (clones of special white blood cells designed to target the leukemia) bring about anti-tumor effects through triggering complement-mediated cytotoxicity (CDC) and other mechanisms. The efficacy of CDC depends on several factors, including the availability and activity of the body’s complementary immune system. Preliminary results indicate differences in the patterns of complement complexes C4 and C5 in more than 50% of CLL patients, as well higher basal activity levels (indicated by C5b-9), as compared to healthy control subjects (HC).

Aims: To study the structure of circulating complement complexes and the importance of their structural integrity for complement activity during immunotherapy in patients with CLL.

Results: Differences in the patterns of complement C5 and C4 were found in some of the CLL patients. Specifically, the C5 complex, which exists as a single protein band in 14 HC and in some of the CLL patients (20 out of 36 patients), appeared in 44% of the patients as a double-band (16 out of 36 patients). Complement activity analysis (followed by the levels of C5b-9, the terminal product of complement activation) revealed higher basal levels of C5b-9 in the CLL patients with altered pattern of C5 (3946±758 S.E.M. ng/ml) compared with both HC (682±158 S.E.M. ng/ml) and with CLL patients presenting normal C5 pattern (2363±655 S.E.M. ng/ml). Complement activation by administering aggregated IgG was inversely correlated with basal activity, resulting in a modest increase of C5b-9 levels to 16758±2032 ng/ml in the CLL patients with altered C5 pattern, compared with both HC and CLL patients with normal C5 pattern, where C5b-9 levels increased to 23416±1879 and 20553±2424 ng/ml, respectively. The complement activation in the CLL patients with altered C5 was significantly lower compared with the activation observed in HC samples (p<0.03). Activation by administering Zymosan was similar in all subjects' groups.

The results obtained according to the stages of the project:

Stage 1 (structural integrity analysis): this stage is mostly completed, except for the collection of blood samples from CLL patients treated with RTX, which will start next month.

Stage 2 (subunit composition): this stage is in progress. However, due to technical problems, a new immunoprecipitation technique is required, which will be tested next month.

Stage 3 (correlation with activity): this stage is completed.

Summary and conclusions: The data indicate a possible link between the activation potential of the complement system in CLL patients and structural alterations in the C5 complex. As aggregated IgG activates the classical complement pathway while Zymosan mainly targets the alternative pathway, their observed difference in complement activation may indicate disturbance in the classical complement pathway in some of the patients. The exact mechanisms by which "modified" C5 distracts the complement activity needs further clarification. Yet, the appearance of "modified" C5 in CLL patients with disturbed complement activity bears the potential to develop a marker which will assist in identifying patients who are likely to be less responsive to future immunotherapy treatment due to compromised CDC. Development of such a marker may assist clinicians in refining and personalizing the immunotherapeutic approach, improving CDC and consequently the therapy results.

Dr. Armaly Zaher

Impact of Haptoglobin Genotype on Intravenous Iron Administration-induced Oxidative Stress and Inflammatory response in Patients with Chronic Kidney Disease (CKD)


Impact of L-carnitine Pretreatment on Intravenous Iron Administration-induced Oxidative Stress and Inflammatory Response in Patients with CKD

Background: Anemia is a common problem in CKD patients. It is attributed to decreased erythropoietin (EPO) production, low iron stores, and accompanying chronic inflammation. Therapy includes infusions of recombinant EPO to help boost production, and also iron replenishment. However, the latter induces oxidative stress and inflammation. Randomized, controlled studies suggested that L-carnitine supplementation might have positive effects on the response to EPO in long term hemodialysis patients. However, there is no evidence whether this approach would also be beneficial in earlier-stage CKD patients. Thus, the present study examined whether L-carnitine prevents intravenous iron replenishment (IVIR) induced oxidative stress and whether it improves response to EPO. In addition, we examined whether the haptoglobin phenotype affects iron-induced oxidative stress in CKD patients. Haptoglobin protects the body by binding to and neutralizing loose hemoglobin in the bloodstream, which can cause kidney damage. The haptoglobin gene comes in 2 forms Hp1 and Hp2. People with two copies of Hp2 produce a weaker form of haptoglobin.

Aims: Our hypothesis is that: 1) Intravenous administration of iron (IVIR) to CKD patients at early stages of the diseases (Stage 2-4) provokes oxidative stress and inflammation; 2) Prophylactic L-carnitine supplementation could prevent the IVIR-induced oxidative stress and inflammatory responses in these patients; and 3) we examined whether the haptoglobin phenotype affects iron-induced oxidative stress in CKD patients.

Methods: The current study included 32 anemic CKD patients (stages 2-4) that were divided into 2 subgroups: Group 1 consists of 16 patients who were given a weekly IVIR (Sodium ferric gluconate, [125 mg/100 ml] for 12 weeks. Group 2 consists of 16 patients who received the same IVIR regimen but also a weekly does of carnitine (20 mg/kg, IV) 30 minutes prior to IVIR administration, throughout the whole treatment period. Weekly blood samples were drawn before and after each IVIR for Hb, C-reactive protein (CRP), advanced oxidative protein products (AOPP), TBARS, neutrophil gelatinase-associated lipocalin (NGAL), Hp phenotype, in addition to routine complete blood count and biochemical analyses.

Results: Combined administration of IVIR and carnitine increased hemoglobin (Hb) levels more profoundly (+8%) than in those treated with IVIR alone (+13%). While IVIR alone induced oxidative and inflammatory responses, patients who received carnitine did not exhibit these adverse effects, as was evident by the absence of IVIR-induced elevation in CRP, NGAL, AOPP, and TBARS. The highest increase in AOPP was observed in Hp2-2 patients. Simultaneous administration of Carnitine with IVIR eliminated the IVIR-induced oxidative stress, as evident by preventing the elevations in AOPP and NGAL, preferentially in patients with Hp2-2 phenotype.

Conclusion: Our findings indicate that co-administration of carnitine with IVIR preferentially attenuates the adverse consequences of IVIR and suggests a role for carnitine therapy in these patients. Moreover, this study demonstrates that Hp2-2 is a significant risk factor for IVIR-induced oxidative stress in CKD patients.

Dr. Khaled Khazim and Dr. Cohen Idan

Genome-wide transcriptome profiling of circulating neutrophils in patients with end stage renal disease

Midterm progress report

In this research proposal we aimed to try and understand the underlying mechanism(s) of circulating blood neutrophils activation in ESRD patients using a full transcriptome analysis profiling. Our major goal was to try and uncover cellular mechanisms that will potentially lead to a better understanding of this process and may discover novel therapeutic targets for treatment or intervention. Additionally, by doing so, we hoped to discover a single "factor" of these cells that may be later used as a "biomarker" for assessing and monitoring atherosclerosis initiation and progression. We are happy to report that the mutual efforts of Dr. Idan Cohen, Din Richtert (the MD project student from the Faculty of Medicine) and I have already yielded 2-3 strong factors that play a pivotal role in neutrophils biology and can be used as potential biomarkers:

During this time, we optimized and perfected neutrophils isolation and RNA purification from donors’ blood and could see a 30-fold reduction in the expression of the Hypoxia-inducible factor 1-alpha (HIF-1a). In parallel we also could show 10-fold reduction in the expression of Sirtuin 1 (SIRT-1), which is known as a master regulator of longevity, metabolism, epigenetics and inflammation. Interestingly, without knowing its exact mechanism, stabilizing HIF-1α agent is currently in phase 2 and 3 clinical trials for the treatment of anemia in patients with ESRD. We are now almost ready for the last part of the suggested proposal, the genome wide transcriptome profiling and would be more than happy of any additional financial support or the extension of this grant.

Maya Wolf, MD Fetal and maternal hormonal and signaling molecule association with outcome of labor induction

A comparative evaluation of the correlation of fetal and maternal labor-associated hormones and signaling molecules with two methods of induction of labor in women with previous cesarean section


Maya Wolf, MD, Department of Obstetrics & Gynecology, Galilee Medical Center, and the Faculty of Medicine, Bar-Ilan University, Israel.

Jacob Bornstein, MD, MPA, Professor (PI) Department of Obstetrics & Gynecology, Galilee Medical center, and the Faculty of Medicine, Bar-Ilan University, Israel

Dr. Eilam Palzur – Research lab, Galilee Medical Center, Nahariya, Israel

Dr. Mona Shehada – the biochemistry lab, Galilee Medical Center, Nahariya, Israel

Intermediate summary of the progress of the research study

To date, 42 pregnant women at term admitted for induction of labor, who have undergone previous cesarean section and who have matched all inclusion study criteria, have been recruited for the research study. All the women signed informed consent forms. The study aims at a total of 60 women. The women underwent randomization by computer program, and were placed in one of two groups: those who undergo induction of labor by balloon catheter or those who undergo breast stimulation. From all women, blood for the analysis of hormone and signaling molecule levels affecting uterine activity (Oxytocin, PGF2 α, PGE2, Prolactin, Testosterone, Progesterone, Estradiol, and Cortisol) was drawn at time 0, 3 and 6 hours post catheter-balloon insertion or initiation of breast stimulation for labor induction. In addition, immediately after delivery, cord blood was tested for those hormone and signaling molecules levels, and for bilirubin and cord pH.

The samples are marked and have been preserved in the biochemistry laboratory for testing.

Enrollment is expected to be complete in two months. To ensure uniform laboratory conditions for the analysis of blood samples, we will process all study samples together.

Prof. Offer Amir

miRNA in Acute Coronary Event: pathogenesis and risk stratification

Below is a brief progress report on our study of miRNA and its role in acute coronary events. Our study is aimed at providing ways of using miRNA to predict the onset of cardiac arrest and to identify an individual’s risk level for experiencing such an event.

  1. The protocol for isolating blood components was optimized and adjusted to our experimental settings. Experimental settings of miRNA extraction and sample preparation for Next Generation Sequencing (NGS) were also optimized. A small pilot study was successfully performed.
  2. More than 300 patients were recruited to the study so far. Blood samples were collected and processed according to the protocol.
  3. miRNA from 72 platelets samples was isolated and prepared for NGS. miRNA were sequenced on the HiSeq Illumina machine by Genomic Unit in the Faculty of Medicine, Safed.
  4. Bioinformatics analysis following the NGS was performed, where differential expression analysis comparing all groups participating in the study was carried out using a web-based platform for small RNA Oasis and DeSeq package (R). Several miRNA where identified as differentially expressed.
  5. Further, we intend to validate our findings using larger patient cohorts and a different experimental technique. The technique we have chosen is quantitative Real Time PCR (qPCR). So far we have optimized all steps of this procedure and adjusted for our experimental settings. For instance, one of the main issues in performing miRNA qPCR is identifying molecules those expression levels remains stable across multiple samples which can serve as an internal reference. To identify such molecules suitable for our study, a NormFinder program was applied, with our NGS data used as an initial input.

Dr. Hadassah Goldberg & Dr. Rona Fell Isolation and characterization of potentially metastasizing tumor cells from primary breast cancer samples.”

This is an interim report concerning a study funded by the Dangoor Foundation, 2016-17. The study is aimed at isolating breast cancer cells that harbor the potential to form distant metastases from primary tumors. The study was designed to be executed in five steps:

  1. Collection of fresh samples from breast cancer tumors immediately after their surgical removal, in addition to a normal breast tissue sample, to be used as a control.
  2. Separation of each tissue sample into a single cell suspension.
  3. Depletion of the hematopoietic cells from the total cell mixture.
  4. Sorting the singe cell suspension into two sub-populations according to their metastatic potential, by using pre-defined biological markers.
  5. Performing a comparative gene expression analysis between the two cell-populations. The gene expression analysis results of the normal breast tissue will be used for subtraction of background normal breast values.

Since the study is based on fresh tumor samples, steps 1-4 must be performed immediately after the surgical removal of the tumor. Consequently, it is essential that the study platform is operative prior to initiation of sample collection. Up to now, we have completed the following steps:

  1. We constructed the method for tissue separation into a single cell suspension by acquiring designated gentleMACS columns and suspension kits.
  2. We acquired the means necessary for depletion of the hematopoietic cells from the total cell mixture, including MACS columns and an antibody cocktail (Lin depletion kit) conjugated to micro-beads. By using the MACS platform the hematopoietic cells will be captured by the magnet while the tumor (or normal breast) cells are washed through and collected. We have already performed preliminary tests to confirm the accuracy of the hematopoietic depletion method using lab materials.
  3. We have conducted a comprehensive survey of the literature in order to select the antibodies for separation of the metastasizing tumor cells which are identified by markers of the ‘epithelial to mesenchymal transition’ (EMT) process. The EMT is a hallmark of tumor cells with an invasive potential. Antibodies selection was also based on result of our recently completed study. The separation into two sub-populations will be done by the FACS technology, using the “Beckman Coulter MoFlo Astrios” located in the multidisciplinary unit of the Faculty of Medicine, Bar-Ilan University.

We already ordered the antibodies and expect them to arrive in a few days. Once this step is accomplished, we will start collecting tumor samples and perform the study.

We already coordinated the process of sample collection with the breast surgeon Dr. Assi Drobot and the pathologist expert Dr. Liat Appel from the Galilee Medical Center. Dr. Drobot has also agreed to present the study to the patients and ask them to sign an informed consent prior to their surgery.